Novel strategies for immune therapy of arthritis - Towards sustained disease remission - About the cover Het tweeluik “Amistad entrega felicidad” van Angeles Nieto (“Vriendschap brengt geluk”).

Copyright © 2006 by Future Rheumatology for chapter 2 Copyright © 2002 by Arthritis and Rheumatism for chapter 3 Copyright © 2006 by PLoS ONE for chapter 4 Copyright © 2008 by Blood for chapter 6 Copyright © 2008 by Autoimmunity for chapter 7 Copyright © 2009 by S. Roord for all other chapters All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means without prior permission of the author.

Cover Angeles Nieto Layout and Printing Gildeprint Drukkerijen, Enschede, The Netherlands ISBN 978-94-901-2206-5

Address of correspondence Sarah Teklenburg-Roord, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Department of Pediatric Immunology, PO Box 85090, 3508 AB Utrecht, The Netherlands. [email protected]. Novel strategies for immune therapy of arthritis - Towards sustained disease remission -

Nieuwe strategieën voor immuun therapie van arthritis - Op weg naar blijvende ziekte remissie - (met een samenvatting in het Nederlands)

Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. J.C. Stoof, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 9 april 2009 des middags te 2.30 uur

door Sarah Theresa Anne Roord geboren op 23 juni 1974 te Utrecht Promotoren: Prof. dr. B.J. Prakken Prof. dr. W. Kuis

Co-promotoren: Dr. N. Wulffraat Dr. F. van Wijk

The printing of this thesis was supported by Abbott BV, BV, BD Biosciences, Boehringer Ingelheim, Eijkman Graduate Fonds, GlaxoSmithKline BV, J.E. Jurriaanse Stichting, Pfizer BV, Phadia BV, Reumafonds (Dutch Arthritis Association), Roche Nederland BV, Schering Plough BV, Stichting Kind en Afweer, Wyeth Pharmaceuticals, ZonMW. Voor mijn ouders

TABLE OF CONTENTS

Chapter 1 General introduction 9

PART 1 Tolerance induction by antigen specific immunomodulation Chapter 2 Present and future of combination therapy of autoimmune diseases 29

Chapter 3 Inhibition of adjuvant-induced arthritis by interleukin-10-driven 41 regulatory cells induced via nasal administration of a peptide analog of an arthritis-related heat-shock protein 60 epitope

Chapter 4 Modulation of T cell function by combination of epitope specific 65 and low dose anticytokine therapy controls autoimmune arthritis

PART 2 Tolerance induction by autologous bone marrow transplantation Chapter 5 Autologous bone marrow transplantation specifically 87 suppresses auto and IL-17 production in proteoglycan induced arthritis

Chapter 6 Autologous bone marrow transplantation in autoimmune 109 arthritis restores immune homeostasis through CD4+CD25+ Foxp3+ regulatory T cells

Chapter 7 Regulatory T cells in autologous stem cell transplantation 131 for autoimmune disease

Chapter 8 Summary and general discussion 147

Nederlandse samenvatting 169 Dankwoord 179 Curriculum Vitae 187 List of publications 191 List of abbreviations 195 Colour Figures 199

1General introduction regel 1 PART 1: Tolerance induction of arthritis by antigen specific regel 2 immunomodulation regel 3 regel 4 Current treatment of rheumatological diseases: non-specific regel 5 regel 6 Juvenile Idiopathic Arthritis (JIA) and (RA) are among the most common regel 7 autoimmune diseases worldwide1. They are characterized by chronic, systemic inflammatory regel 8 episodes that target synovial joints and have a relapsing-remitting course. In recent years, regel 9 extensive research in this field has focused on in depth analysis of the underlying mechanisms regel 10 involved in the disease processes. The diseases seem to consist of an initial T cell directed regel 11 stage, followed by self-reverberating cycles of non-specific inflammation, eventually leading to regel 12 joint damage. Likely, local antigens in joints and synovium play a role in this process, however regel 13 the ‘one’ disease-triggering antigen has not been found yet, and probably does not exist either. regel 14 Treatment of JIA and RA can be a major challenge and those diseases are known to lead to regel 15 significant morbidity and even mortality. regel 16 Treatment of JIA and RA has thus far mainly focused on the second inflammatory stage regel 17 of disease. Induction of disease remission can be achieved by the early use of powerful regel 18 immunosuppressants and cytostatic drugs, such as high dose corticosteroids and regel 19 (MTX)2;3. Unfortunately, persistent disease remission is still exceptional and regel 20 even then progression of joint damage is hard to prevent. In addition, due to the fact that they regel 21 induce a generalized and non-specific inhibition of immune response and inflammation, these regel 22 agents can have considerable side effects. regel 23 regel 24 Modulation of the immune response by targeting molecules that are involved regel 25 1 Chapter in disease pathogenesis- the innate immune response regel 26 10 Since conventional treatments mainly focus on the general inflammatory pathogenic regel 27 component of the disease, they do not focus on specific components that play a role in disease regel 28 pathogenesis. In this respect, recently introduced biologics have contributed an important regel 29 step forward, targeting for example cytokines that play a role in disease pathogenesis in the regel 30 innate immune response (Table 1)1;4;5. Several biological agents have been identified that regel 31 can block pro-inflammatory cytokines. ®, a soluble TNF receptor , regel 32 ®, a chimeric anti α TNF antibody, and ®, a recombinant human anti regel 33 TNFα antibody, can target TNFα; ®, an IL-1 receptor antagonist, neutralizes IL-1; and regel 34 MRA® (a humanized IL-6 receptor antibody) targets IL-6. The use of these agents has shown regel 35 clinical efficacy in several clinical trials in JIA and RA patients and they are currently approved regel 36 for treatment of RA and/or JIA1;4. However, unfortunately, persistent disease remission can only be achieved as long as the drugs are administered and only then progression of joint regel 1 damage can be prevented. Therefore, it does not lead to a permanent remission of disease or regel 2 a cure. In addition, due to the fact that most of these agents induce a generalized inhibition of regel 3 immune response and inflammation, they can cause considerable immunodepression, leading regel 4 to possibly severe side effects, such as malignancies and infections, including tuberculosis, regel 5 and the induction of other autoimmune diseases4;6-10. regel 6 regel 7 Table 1. Selection of cytokine targets in RA and JIA therapy regel 8 regel 9 Targeted molecule Agents Drug name regel 10 TNFα * regel 11 -Chimeric MAB (mouse x human) Infliximab® regel 12 -Humanized murine CDR3-engrafted MAB CDP571 -Human MAB D2E7 (Adalimumab®) regel 13 -PEG-linked Fab fragment CDP870 regel 14 -Recombinant human methionyl TNF binding protein TNFbp regel 15 PEGylated dimer * Soluble TNFR:Fc Fusion Proteins regel 16 -Recombinant soluble p75 TNFR-Fc fusion protein Etanercept® regel 17 -Recombinant soluble p55 TNFR-Fc fusion protein Lenercept® regel 18 -Pegylated Truncated p55 TNF-R regel 19 -PEG soluble TNF Receptor Type 1 Pegsunercept® regel 20 IL-1 rHuIL-1 receptor antagonist Anakinra® rHuIL-1 receptor inhibitor regel 21 IL- 6 Humanized anti IL-6 receptor MAB Atlizumab (MRA)® regel 22 Anti IL-6 MAB B-E8 regel 23 IL-15 Anti IL-15 MAB Hu-MAX IL-15/AMG-714 regel 24 IL-12 Anti IL-12 MAB ABT-874/J-695 General introduction General IFNγ rHuIFNγ regel 25 IFNβ rHuIFNβ 11 regel 26 IL-11 rHuIL-11 regel 27 IL-10 rHuIL-10 regel 28 IL-4 rHuIL-4 Current biological agents and agents in development and/or suggested for treatment of human arthritis. regel 29 Agents currently in use in clinical practice are depicted in bold face. MAB=. regel 30 TNFR=TNF receptor. rHu=recombinant human. regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 Modulation of the immune response by targeting molecules that are involved regel 2 in disease pathogenesis- the adaptive immune response regel 3 Other attempts have focused on several arms of the specific, adaptive immune response. regel 4 Table 2 summarizes a selection of the efforts targeting the adaptive immune response in regel 5 autoimmune diseases, such as RA, JIA, Multiple Sclerosis (MS) and type 1 diabetes. Those regel 6 attempts range from inhibition of leukocytes trafficking into the joint by targeting adhesion regel 7 molecules, to depletion of all CD3+ and CD4+ T cells or B cells. Other efforts target only regel 8 pathogenic T cells, e.g. cells expressing OX-40, or target molecules that are involved in CD4+ regel 9 signaling pathways, such as co stimulatory molecules CD28, CD80, and CTLA411;12. Lastly, regel 10 some attempts aim to modulate only the response to a pathogenic antigen present at the regel 11 site of inflammation by either immunization to this antigen or mucosal tolerance induction regel 12 to such an antigen. Some of these attempts have led to encouraging results in clinical trials, regel 13 and as a consequence some drugs have become readily available on the market. ®, regel 14 a recombinant CTLA-4Ig fusion protein that blocks the co stimulatory signal mediated by regel 15 the CD28-CD80/86 pathway, and ®, an anti CD20 MAB, are currently in use for RA. regel 16 ®, an anti α4 monoclonal antibody (MAB), is being used for adult MS regel 17 patients. regel 18 On the contrary, the first attempts that focused on targeting CD4+ T cells showed that, in order regel 19 to achieve therapeutic levels within the joints, the required dose would lead to an unacceptably regel 20 profound CD4 lymphopenia with the subsequent risk of severe immune suppression and have regel 21 therefore been abandoned as a therapeutic strategy in RA13-16. OKTcdr4a, a non depleting regel 22 ‘humanized’ anti CD4 MAB led to more promising results through an immediate, though regel 23 transient, clinical benefit17. regel 24 That targeting an immune receptor involved in T cell signaling does not come without risk was regel 25 1 Chapter recently unfortunately shown by a phase I clinical trial using a novel super agonist anti CD28 regel 26 12 monoclonal antibody (TGN1412, TeGenero®). The super agonist antibody was administered to regel 27 six healthy volunteers and led to a cytokine storm with subsequent multi organ failure18. regel 28 Despite this dramatic failure, altogether, immune therapy directed against this category of regel 29 molecules represents a conceptual step forward due to their targeted mechanism of action, regel 30 as they aim to modulate certain aspects of adaptive immunity rather than suppress an regel 31 individual pathway (i.e. a cytokine), but caution with regards to both expected and unexpected regel 32 lymphodepletion and other side effects is warranted. regel 33 regel 34 regel 35 regel 36 Table 2. Therapy specifically aimed at the adaptive immune response in autoimmune regel 1 diseases regel 2 regel 3 Treatment type Examples regel 4 A. Therapy against leukocyte trafficking into site of regel 5 inflammation regel 6 1. Therapy against adhesion molecules Anti ICAM-1 MAB Anti α4 integrin MAB (Natalizumab®) regel 7 B. Therapy against CD4+ T cells regel 8 1. Depleting anti CD4 MABs regel 9 Murine antibodies B-F5 regel 10 Chimeric antibodies cM-T412 regel 11 Humanized antibodies 4162W94 regel 12 2. Non depleting anti CD4 MABs OKTcdr4a regel 13 regel 14 3. Therapy against accessory molecules of T cell activation CTLA4-Ig (Abatacept®) LEA29Y regel 15 CD2 antagonist (®) regel 16 Anti CD40 MAB regel 17 Anti CD28 MAB (TeGenero®) regel 18 C. Therapy specifically aimed at disease triggering CD4+ T cells regel 19 1. Therapy against surface markers on pathogenic T cells Anti OX-40 MAB regel 20 OX-40 immunotoxin regel 21 regel 22 2. T cell vaccination Vβ17 Vβ3/Vβ4/Vβ17 regel 23 AV11 66-80 regel 24

3. Antigen specific immunomodulation introduction General regel 25 - Immunization to antigen MBP 83-99 DiaPep277 13 regel 26 GAD65 regel 27 regel 28 - Mucosal tolerance induction dnaJP1 Collagen type II regel 29 Insulin regel 30 D. Therapy aimed at B cells regel 31 1. Anti CD20 MAB Anti CD20 MAB (Rituximab®) regel 32 E. Therapy aimed at CD3+ T cells 1. Anti CD3 MAB OKT3 regel 33 145 2C11 regel 34 Agents currently in use in clinical practice are depicted in bold face. regel 35 regel 36 regel 1 An elegant approach: antigen specific tolerance induction regel 2 The induction of tolerance to a disease trigger or antigen present at the site of inflammation is regel 3 called antigen specific tolerance induction. The induction of antigen specific immune tolerance regel 4 would ideally spare the patient generalized immune suppression and could be expected to regel 5 provide a long-lasting effect, maybe even a cure, devoid of side effects. Antigen specific regel 6 peripheral T cell tolerance can be induced through administration of a relevant antigen via regel 7 the nasal or oral route. In several experimental autoimmune models, oral administration of regel 8 the disease triggering auto antigen led to considerable suppression of disease activity, which regel 9 could be equaled and sometimes even surpassed by the nasal administration of antigen19;20. regel 10 For the induction of antigen specific tolerance, antigens with pathogenic relevance will need to regel 11 be identified. Candidate antigens need to be of relevance to the generation and/or modulation of regel 12 the autoimmune response and they need to be recognized by a sizeable portion of the patient regel 13 population. The search for ‘the one disease triggering antigen’ however, has thus far not been regel 14 successful and attempts at inducing tolerance to candidates in this respect, such as chicken regel 15 and bovine type II collagen and human cartilage glycoprotein 39 (HCgp39), major constituents regel 16 of articular cartilage, were encouraging but not convincing21-24. Likely to be more successful regel 17 would be to move away from attempting to find the ‘one disease-triggering antigen’ but focus regel 18 on ‘key players’ at the site of inflammation, that play a role in disease perpetuation. Heat Shock regel 19 Proteins (HSPs) are such antigens. HSPs are present in all cells and are up regulated during regel 20 stress. As ubiquitous and bacterial derived products, HSP-derived peptides are perceived as regel 21 a “danger” signal and elicit a default pro-inflammatory physiologic response, which involves regel 22 both the adaptive and the innate arms of the immune system25-27. Such response contributes regel 23 to the clearing of a possible pathogen invasion, but also induces, through cellular stress, regel 24 increased availability of self-HSP derived peptides. These peptides then form a new target for regel 25 1 Chapter the immune system and induce self-perpetuating cycles of inflammation, fueled by the self- regel 26 14 antigens and self-reactive T cells. Others and we have shown previously, that HSP peptides are regel 27 recognized by T cells with regulatory function, which are then capable of preventing further regel 28 tissue damage28. If such regulatory function is impaired, loops of inflammation continue and regel 29 autoimmune arthritis prevails29-31. regel 30 regel 31 Induction of Regulatory T cells: a principal aim in the quest for a cure for regel 32 arthritis regel 33 Due to the pro-inflammatory character of RA and JIA, modulation of pathogenic T cells from regel 34 a pro-inflammatory to a regulatory phenotype is a principal aim in the quest for a cure32-36. regel 35 Indeed, many studies in experimental models of autoimmune disease have shown that regel 36 regulatory T cells (Treg) play an important role in limiting autoimmunity and in recovery from disease and phenotypical and functional characteristics for Treg have been identified37. regel 1 They can roughly be divided into two subtypes: naturally occurring Tregs (nTregs) and Tregs regel 2 induced in the periphery (iTregs). NTregs emerge during T cell development in the thymus regel 3 and account for 5-10% of peripheral CD4+ T cells 37. They constitutively co-express CD4, the α regel 4 chain of the IL-2 receptor CD25, CTL-associated antigen 4 (CTLA-4) and glucocorticoid–induced regel 5 receptor (GITR). However, at present their most distinctive marker is regel 6 transcription factor Foxp338, which is also known to play a key role in their development. The regel 7 nomenclature for mouse vs. human forkhead-box (FOX) transcription factors has been clearly regel 8 set: genes that encode for human transcription factors are spelled in capital letters (FOXP3), regel 9 whereas in genes that encode for mouse transcription factors, only the first letter is capitalized regel 10 (Foxp3)39. Recently it has become clear, that FOXP3 is not exclusively present on human Tregs regel 11 but becomes transiently up regulated on activated human effector T cells as well; however as regel 12 such, it still remains an exclusive marker of Tregs in the mouse40. The suppressive function of regel 13 nTregs consists of the suppression of proliferation of effector T cells through a cell-cell contact regel 14 dependent mechanism and/or through the production of suppressive cytokines such as IL- regel 15 10 and TGFβ. The second main group of Tregs is induced in the periphery (iTregs) and can regel 16 be divided up into several subtypes, of which Tr1, Th3 cells and induced or adaptive Foxp3+ regel 17 Tregs are the best characterized. They are generally induced after encounter of antigen in anti- regel 18 inflammatory/inhibitory environments, for example by mucosal tolerance induction, and they regel 19 exhibit their suppressive function through the production of cytokines, such as IL-10 (Tr1) and regel 20 TGFβ (Th3)32;41-43. regel 21 regel 22 Antigen specific immunomodulation by HSP peptides in experimental arthritis regel 23 (Adjuvant Arthritis) regel 24 Animal models are often employed to gain information on pathogenesis as well as novel introduction General regel 25 venues of therapy for arthritis. The role of HSPs in antigen specific immunomodulation has 15 regel 26 become clear in the Adjuvant Arthritis (AA) model. This is one of the most commonly employed regel 27 experimental models of arthritis and has close histopathological resemblance to RA and JIA. It regel 28 can be induced in susceptible Lewis rats by two immunizations of heat killed Mycobacterium regel 29 Tuberculosis (Mt) on day 0 and day 21 and induces a self-remitting form of arthritis (Figure regel 30 1). It is a T cell dependent disease that can be passively transferred by a T cell clone that is regel 31 specific for the 180-188 amino acid sequence of mycobacterial HSP6044. regel 32 In AA, nasal administration of the 15-mer mycobacterial HSP60 176-190 peptide (containing regel 33 the core 180-188 sequence), prior to the induction of disease, is able to delay the onset and regel 34 decrease the severity of arthritis45. The nasal administration of the core peptide 180-188 regel 35 after the induction of AA is also able to improve disease severity46 and in this thesis we aim to regel 36 further improve this effect.      regel 1   regel 2  regel 3  regel 4  regel 5 regel 6  regel 7      regel 8  regel 9  regel 10 Figure 1. Adjuvant Arthritis  regel 11 Two injections of Mt in CFA in susceptible Lewis rats lead to a self-remitting form of arthritis regel 12  regel 13 Antigen specific immunomodulation by HSP peptides in clinical trials regel 14 The promising results of antigen specific immunomodulation by HSP in experimental disease  regel 15 models of arthritis as well as type I diabetes warranted subsequent clinical trials in human regel 16 disease 45-47. In a Phase I/IIa clinical trial, Albani et al. administered a dnaJ derived peptide, regel 17 dnaJP1, orally to 15 patients with early, active RA and focused on the immunological changes  regel 18 48 induced by the treatment . Interestingly, immunological analysis showed that this treatment regel 19 was able to induce immune deviation from pro-inflammatory to modulatory T cell responses.  regel 20 Recently, Albani/Koffeman et al. completed a double blind placebo controlled pilot phase II  regel 21 clinical trial with dnaJP1 (results submitted for publication). This study focused on safety and regel 22 clinical efficacy  of the drug.  It involved  160  patients  who received dnaJP1  or placebo orally    regel 23 once per day for six months. The dnaJP1 peptide treatment showed encouraging clinical and  regel 24 immunological effects, suggesting that induction of immune tolerance to an inflammatory

Chapter 1 Chapter  regel 25 ubiquitous antigen may indeed translate into clinical improvement of the disease. regel 26 16 In several placebo controlled clinical  trials in type I diabetes   in adults and children,   the s.c.   regel 27 administration of DiaPep277, an immunogenic peptide of HSP60, was safe and well tolerated, regel 28 while the effects on preservation of insulin production differed. Despite a beneficial effect on regel 29 preservation of C-peptide levels in most trials over time due to DiaPep277 treatment, only              regel 30 one group describes concomitant preservation of endogenous insulin secretion up to 18 regel 31 months of treatment 49;50. The other  studies  did not show a  beneficial  effect on HbA1c  levels    regel 32 nor on daily insulin  requirement  51-53. Interestingly,  the treatment  did induce  immune  deviation;    regel 33 DiaPep277 treatment led to the production of IL-10 and a decrease in auto antigen specific T  regel 34 cell proliferation in the patients in which the treatment led to β cell preservation54. regel 35  regel 36                                      

         Room for improvement: Combination therapy of anticytokine therapy and regel 1  antigen specific immunomodulation regel 2  Thus, antigen specific immunomodulation has proven to be a promising approach to treatment regel 3 regel 4  of arthritis, since it shows immune deviation to a regulatory/immunosuppressive phenotype as well as clinical improvement in both arthritis and type I diabetes. However, there is room for regel 5  further enhancement of the clinical effect. A combination of treatment strategies with different regel 6      and complementary mechanisms of action is likely to be more successful than the above regel 7  described treatment methods on their own. By acting complementarily or synergistically, regel 8 they can enhance efficacy. Cost and toxicity can be decreased, due to the opportunity of regel 9  administering lower amounts of drugs than when they are given separately. Due to the pro- regel 10  inflammatory environment that predominates during active arthritis, a dual strategy targeting regel 11  this pro-inflammatory environment prior to attempting to modulate the immune response in regel 12  an antigen specific manner may be especially promising. This initial therapy may be aimed at regel 13 neutralizing pro-inflammatory cytokines and/or pro-inflammatory T cells present at the site regel 14  of inflammation, prior to antigen specific tolerance induction. Previous work has shown that regel 15  TNFα may be a particularly interesting target in this respect. Besides clinical improvement of regel 16  disease, anti TNFα also has a beneficial effect on the function of the T cells, both effector and regel 17 regel 18  regulatory cells. Several studies have shown that anti TNFα therapy can improve Treg function and numbers in RA patients55-57. Also, anti TNFα therapy was shown to induce a shift to a more regel 19  anti-inflammatory cytokine profile in PBMCs (Peripheral Blood Mononuclear Cells) and T cells regel 20    of RA patients58. regel 21               Through these interactions the design of an optimal biologic therapy based on the combination regel 22 regel 23  of anticytokine and T cell epitope specific approaches may become more easily accessible and will be a focus of the first part of this thesis. regel 24  introduction General regel 25            17 regel 26  regel 27 regel 28  regel 29              regel 30           regel 31            regel 32 regel 33  regel 34  regel 35             regel 36                          

  regel 1 PART II: Induction of tolerance by autologous Bone Marrow regel 2 Transplantation regel 3 regel 4 Autologous Bone Marrow Transplantation for severe, treatment resistant regel 5 disease regel 6 Despite the progress in current treatment methods and the addition of biologicals to the regel 7 treatment regimens, some RA and JIA patients remain unresponsive to treatment. For those regel 8 severely ill patients, autologous Bone Marrow Transplantation (aBMT) has shown to be the last regel 9 resort. The results of aBMT in treatment-resistant JIA and other autoimmune diseases (AID) regel 10 are remarkably good59-61. It induces drug free disease remission in a majority of JIA patients regel 11 during a follow-up of 12-60 months post transplantation62;63. However, as the assumed genetic regel 12 predisposition of the patient does not change in an autologous BMT setting, it is questionable regel 13 whether the achieved disease remission will be ever lasting. regel 14 regel 15 Working mechanism of ABMT regel 16 The underlying working mechanism of aBMT in JIA and other AID is still largely unknown, while regel 17 unraveling it is of great importance, since understanding its mechanism of action may help regel 18 to device therapies with similar efficacy without the need of undergoing severe conditioning regel 19 that precedes aBMT. It has been hypothesized that in aBMT, due to the stringent conditioning regel 20 regimen leading to severe aplasia, auto reactive immune cells are depleted in the host. In regel 21 addition, an environment is created by this severe immune ablation in which, after rescue by regel 22 autologous BM, renewed acquisition of self tolerance can be acquired for an extended period regel 23 of time60;61;64;65. Regulatory T cells may guide this self-tolerance induction. Indeed, in previous regel 24 work in JIA patients undergoing aBMT, de Kleer et al. showed that aBMT induces a restoration regel 25 1 Chapter of the frequency of CD4+CD25brightFOXP3+ Tregs from severely reduced levels before aBMT regel 26 18 to normal levels after. The study found that two mechanisms were induced by aBMT; on one regel 27 hand (preferential) homeostatic proliferation of Tregs during the lymphopenic phase of the regel 28 reconstitution of the immune system and on the other hand renewed thymopoiesis of naive regel 29 Treg. Furthermore, a deviation of auto reactive T cells from a pro-inflammatory phenotype regel 30 before aBMT, to a tolerant phenotype after aBMT was seen66. Foxp3+ Tregs were also found regel 31 to play a role in the resetting of the immune system post aBMT in Experimental Autoimmune regel 32 Encephalomyelitis (EAE), a model for Multiple Sclerosis (MS), and in Systemic Lupus regel 33 Erythematosus (SLE) patients59;67. regel 34 Thus an important role for Tregs in tolerance induction post aBMT has been established in regel 35 autoimmune diseases (AID). However, disease relapses may and probably will occur in time regel 36 when this self-tolerance is broken again. Indeed, some patients that underwent aBMT have shown to get a severe disease relapse after years of drug free disease remission, albeit some regel 1 with a restored sensitivity to previously ineffective therapies68-70. regel 2 Despite this recent insight in the role for Tregs in tolerance induction, many questions regarding regel 3 working mechanism of aBMT and optimal composition of the graft remain unanswered. Some regel 4 for example still argue that the effect of aBMT is merely attributable to the severe aplasia regel 5 induced by the strict conditioning regimen that precedes transplantation. Furthermore, regel 6 traditionally the graft is depleted of T cells, either by CD34+ selection or by depletion of T regel 7 cells. The rationale for T cell depletion of the graft is to deplete auto reactive T cells that are regel 8 present in the autologous graft; however in theory simultaneously Tregs may be depleted as regel 9 well. Indeed a large, multi center placebo controlled trial recently showed no clinical benefit of regel 10 T cell depletion71. Lastly, in most experimental settings in which aBMT has been explored thus regel 11 far, BM from healthy donor animals was used, whereas in the clinical setting, autologous BM is regel 12 harvested from patients that are in an active disease state. regel 13 Thus the need has arisen for a clinically relevant experimental arthritis model, to further regel 14 explore the working mechanism of aBMT, to answer questions regarding the optimal BM graft, regel 15 and to develop strategies that may help to improve the efficacy and prevent late relapses of regel 16 the disease. regel 17 regel 18 Experimental model: Proteoglycan Induced Arthritis regel 19 Earlier pioneering work in animal models by van Bekkum et al., provided the experimental regel 20 basis for the treatment of AID by autologous BMT. They successfully treated Adjuvant Arthritis regel 21 in Buffalo rats by aBMT and defined conditioning regimens and levels of T cell depletion72. Also regel 22 in Collagen Induced Arthritis (CIA), an antibody mediated mouse model, a decrease of arthritis regel 23 occurred after irradiation and BMT73. This work has provided pivotal insight, however, in order regel 24 to study the maintenance and the break of tolerance as seen in humans in the long term, we introduction General regel 25 wanted to turn to a model that is mediated by both T and B cells and that is relapsing and 19 regel 26 remitting, as is the human disease. regel 27 Proteoglycan Induced Arthritis (PGIA) fits both these criteria. It can be induced in retired BALB/c regel 28 mice by two injections of human cartilage PG in the adjuvant DDA. This causes a progressive regel 29 polyarthritis that is initiated by a cross recognition of mouse PG by CD4+ T cells, while also B regel 30 cells are crucial for the development of disease74;75. PGIA is extensively studied, has clinical, regel 31 immunological and histopathological resemblance to human arthritis and has a chronic regel 32 relapsing remitting course (Figure 2). Therefore, PGIA can serve as a useful model to unravel regel 33 remaining questions regarding aBMT and develop strategies to further improve outcome and regel 34 will be employed as such in the second part of this thesis. regel 35 regel 36 regel 1   regel 2 regel 3 regel 4 regel 5 regel 6 regel 7    regel 8 regel 9  regel 10 Figure 2. Proteoglycan Induced arthritis regel 11 Two injections of PG i.p. lead to a relapsing-remitting form of arthritis  regel 12 regel 13 Aim of the study regel 14 Conventional treatment of JIA and RA consists of non specific suppression of disease. This regel 15 type of treatment needs to be administered continuously to remain effective and does not regel 16 lead to a cure. Consequently, the generalized and prolonged immunosuppression may lead to  regel 17 severe side effects. regel 18 In the first part of this thesis we attempt to optimize the current treatment methods by regel 19 searching for strategies that focus more specifically on the disease process. One approach regel 20 to achieve this can be antigen specific immunomodulation. By inducing tolerance to antigens regel 21 present at the site of inflammation, the rest of the immune system can be bypassed, thus                 regel 22 decreasing the unnecessary side effects. Furthermore, this should be an active process that regel 23 leads to a specific change in the antigen-specific immune response. In theory, such a change regel 24 in immune reactivity may ultimately lead to a re-establishment of immune tolerance and regel 25 1 Chapter thus, ultimately, to a cure. Indeed, promising results of antigen specific  immunomodulation      regel 26 20 have been obtained in experimental arthritis models, in which mucosal administration of  regel 27 the disease triggering agent leads to suppression of disease and tolerance induction to the regel 28 disease trigger.  However,  especially  with regards  to  the clinical suppression of disease,  there     regel 29 is still room for improvement.  regel 30 We first attempted to improve the clinical effect of mucosal tolerance induction in the Adjuvant             regel 31 Arthritis model by exploring the use of an altered peptide ligand of the wild type epitope 180- regel 32 188. In altered peptide ligands a single amino acid has been replaced by another  amino  acid,    regel 33 which leads to a change of T cell activation that may in turn result in deletion, activation or regel 34 suppression of T cells specific for the wild type epitope.  regel 35 Another aspect that is of importance for antigen specific therapy is the issue of timing. That is;  regel 36 generally the effect of mucosal tolerance induction is better the earlier in the disease process                                  

  the intervention is performed. However, in patients, treatment is often not initiated until a regel 1 phase in which disease is at a more advanced stage at which inflammation has reached a regel 2 threshold, beyond which an environment is created in which antigen specific modulation of regel 3 the immune response may be more difficult to achieve. Therefore, we aimed to enhance this regel 4 effect by attempting to reset the situation to a phase comparable to the situation early in the regel 5 disease course. We attempted this in experimental arthritis by shutting down the predominant regel 6 pro-inflammatory response by antagonizing a pro-inflammatory cytokine, TNF, that plays an regel 7 important role in the disease process, thus creating an environment in which antigen specific regel 8 tolerance has a greater chance of being induced. regel 9 regel 10 The second part of the thesis focuses on improving treatment of patients, who remain resistant regel 11 to the above described treatment methods. For those severely ill patients, autologous Bone regel 12 Marrow Transplantation (aBMT) has been a last resort and has thus far been the only treatment regel 13 capable of inducing a lasting, and symptom free disease remission in a majority regel 14 of patients. The working mechanism of BMT is still largely unknown, but it would be of great regel 15 importance to understand the mechanisms underlying this success. It can then be applied regel 16 to develop alternative strategies, aiming at specific immunomodulation in order to restore regel 17 the immune balance long term, without the need for high impact treatment such as severe regel 18 lymphodepletion and aBMT. regel 19 regel 20 In both types of intervention, we were particularly interested in the role of the balance between regel 21 regulation (regulatory T cells) and activation (Th1, Th17 – type responses) and how to actively regel 22 induce and maintain this tolerant environment, thus providing the basis for a possible cure. regel 23 regel 24

General introduction General regel 25 Outline of the thesis 21 regel 26 regel 27 In the first part of the thesis we show that antigen specific tolerance can be induced in regel 28 experimental arthritis (Adjuvant Arthritis) by nasal administration of an HSP60 peptide, 180- regel 29 188, that plays a pathological role in disease. The clinical improvement can be enhanced regel 30 by replacement of the native peptide by an altered peptide ligand, in which a single peptide regel 31 (leucine) at position 183 is replaced by alanine (alanine 183). regel 32 In chapter 4, we further improve the clinical effect of nasal administration of the native 180- regel 33 188 peptide by first shutting down the dominant pro-inflammatory environment by anti TNFα regel 34 therapy. regel 35 regel 36 regel 1 In chapter 5 and 6, we show that conditioning followed by aBMT in experimental arthritis can regel 2 lead to significant disease suppression and unravel the underlying mechanism of aBMT, by regel 3 investigating the effect of different types of donor BM on disease outcome and by assessing regel 4 the repopulation of Tregs after aBMT. regel 5 Altogether, this thesis aims to identify methods to more specifically obtain long lasting disease regel 6 remission in chronic arthritis. regel 7 regel 8 regel 9 Reference List regel 10 regel 11 1. Goldblatt F, Isenberg DA. New therapies for rheumatoid arthritis. Clin.Exp.Immunol. regel 12 2005;140:195-204. regel 13 2. Fries JF. Current treatment paradigms in rheumatoid arthritis. Rheumatology.(Oxford) regel 14 2000;39 Suppl 1:30-35. regel 15 3. van der Kooij SM, Allaart CF, Dijkmans BA, Breedveld FC. Innovative treatment strategies for regel 16 patients with rheumatoid arthritis. Curr.Opin.Rheumatol. 2008;20:287-294. regel 17 4. Taylor PC. Anti-tumor necrosis factor therapies. Curr.Opin.Rheumatol. 2001;13:164-169. regel 18 5. Castro-Rueda H, Kavanaugh A. Biologic therapy for early rheumatoid arthritis: the latest regel 19 evidence. Curr.Opin.Rheumatol. 2008;20:314-319. regel 20 6. Bloom BJ. Development of diabetes mellitus during etanercept therapy in a child with regel 21 systemic-onset juvenile rheumatoid arthritis. Arthritis Rheum. 2000;43:2606-2608. regel 22 7. Dekker L, Armbrust W, Rademaker CM et al. Safety of anti-TNFalpha therapy in children with regel 23 juvenile idiopathic arthritis. Clin.Exp.Rheumatol. 2004;22:252-258. regel 24 8. Keane J, Gershon S, Wise RP et al. Tuberculosis associated with infliximab, a tumor necrosis regel 25 1 Chapter factor alpha-neutralizing agent. N.Engl.J.Med. 2001;345:1098-1104. regel 26 22 9. Lepore L, Marchetti F, Facchini S, Leone V, Ventura A. Drug-induced systemic lupus regel 27 erythematosus associated with etanercept therapy in a child with juvenile idiopathic arthritis. regel 28 Clin.Exp.Rheumatol. 2003;21:276-277. regel 29 10. Maini RN, Taylor PC. Anti-cytokine therapy for rheumatoid arthritis. Annu.Rev.Med. regel 30 2000;51:207-229. regel 31 11. Moreland LW, Alten R, Van den BF et al. Costimulatory blockade in patients with rheumatoid regel 32 arthritis: a pilot, dose-finding, double-blind, placebo-controlled clinical trial evaluating CTLA- regel 33 4Ig and LEA29Y eighty-five days after the first infusion. Arthritis Rheum. 2002;46:1470- regel 34 1479. regel 35 12. Buch MH, Vital EM, Emery P. Abatacept in the treatment of rheumatoid arthritis. Arthritis Res. regel 36 Ther. 2008;10 Suppl 1:S5. 13. Tak PP, van der Lubbe PA, Cauli A et al. Reduction of synovial inflammation after anti-CD4 regel 1 monoclonal antibody treatment in early rheumatoid arthritis. Arthritis Rheum. 1995;38:1457- regel 2 1465. regel 3 14. van der Lubbe PA, Dijkmans BA, Markusse HM, Nassander U, Breedveld FC. A randomized, regel 4 double-blind, placebo-controlled study of CD4 monoclonal antibody therapy in early regel 5 rheumatoid arthritis. Arthritis Rheum. 1995;38:1097-1106. regel 6 15. Choy EH, Pitzalis C, Cauli A et al. Percentage of anti-CD4 monoclonal antibody-coated regel 7 lymphocytes in the rheumatoid joint is associated with clinical improvement. Implications for regel 8 the development of immunotherapeutic dosing regimens. Arthritis Rheum. 1996;39:52-56. regel 9 16. Choy EH, Connolly DJ, Rapson N et al. Pharmacokinetic, pharmacodynamic and clinical regel 10 effects of a humanized IgG1 anti-CD4 monoclonal antibody in the peripheral blood and regel 11 synovial fluid of rheumatoid arthritis patients. Rheumatology.(Oxford) 2000;39:1139-1146. regel 12 17. Schulze-Koops H, Davis LS, Haverty TP, Wacholtz MC, Lipsky PE. Reduction of Th1 cell activity regel 13 in the peripheral circulation of patients with rheumatoid arthritis after treatment with a non- regel 14 depleting humanized monoclonal antibody to CD4. J.Rheumatol. 1998;25:2065-2076. regel 15 18. Suntharalingam G, Perry MR, Ward S et al. Cytokine storm in a phase 1 trial of the anti-CD28 regel 16 monoclonal antibody TGN1412. N.Engl.J.Med. 2006;355:1018-1028. regel 17 19. Weiner HL. Oral tolerance for the treatment of autoimmune diseases. Annu.Rev.Med. regel 18 1997;48:341-351. regel 19 20. Wauben MH. Immunological mechanisms involved in experimental peptide of regel 20 T-cell-mediated diseases. Crit Rev.Immunol. 2000;20:451-469. regel 21 21. McKown KM, Carbone LD, Kaplan SB et al. Lack of efficacy of oral bovine type II collagen added regel 22 to existing therapy in rheumatoid arthritis. Arthritis Rheum. 1999;42:1204-1208. regel 23 22. Barnett ML, Kremer JM, St Clair EW et al. Treatment of rheumatoid arthritis with oral type II regel 24 collagen. Results of a multicenter, double-blind, placebo-controlled trial. Arthritis Rheum. introduction General regel 25 1998;41:290-297. 23 regel 26 23. Choy EH, Scott DL, Kingsley GH et al. Control of rheumatoid arthritis by oral tolerance. Arthritis regel 27 Rheum. 2001;44:1993-1997. regel 28 24. Keystone EC. Abandoned therapies and unpublished trials in rheumatoid arthritis. Curr.Opin. regel 29 Rheumatol. 2003;15:253-258. regel 30 25. Albani S. Infection and molecular mimicry in autoimmune diseases of childhood. Clin.Exp. regel 31 Rheumatol. 1994;12 Suppl 10:S35-S41. regel 32 26. Albani S, Ravelli A, Massa M et al. Immune responses to the Escherichia coli dnaJ heat shock regel 33 protein in juvenile rheumatoid arthritis and their correlation with disease activity. J.Pediatr. regel 34 1994;124:561-565. regel 35 regel 36 regel 1 27. Albani S, Keystone EC, Nelson JL et al. Positive selection in autoimmunity: abnormal immune regel 2 responses to a bacterial dnaJ antigenic determinant in patients with early rheumatoid regel 3 arthritis. Nat.Med. 1995;1:448-452. regel 4 28. Zanin-Zhorov A, Cahalon L, Tal G et al. Heat shock protein 60 enhances CD4 CD25 regulatory T regel 5 cell function via innate TLR2 signaling. J.Clin.Invest 2006;116:2022-2032. regel 6 29. de Kleer IM, Kamphuis SM, Rijkers GT et al. The spontaneous remission of juvenile idiopathic regel 7 arthritis is characterized by CD30+ T cells directed to human heat-shock protein 60 capable regel 8 of producing the regulatory cytokine interleukin-10. Arthritis Rheum. 2003;48:2001-2010. regel 9 30. Prakken AB, Van Eden W, Rijkers GT et al. Autoreactivity to human heat-shock protein 60 regel 10 predicts disease remission in oligoarticular juvenile rheumatoid arthritis. Arthritis Rheum. regel 11 1996;39:1826-1832. regel 12 31. Quintana FJ, Cohen IR. DNA vaccines coding for heat-shock proteins (HSPs): tools for the regel 13 activation of HSP-specific regulatory T cells. Expert.Opin.Biol.Ther. 2005;5:545-554. regel 14 32. Baecher-Allan C, Viglietta V, Hafler DA. Human CD4+CD25+ regulatory T cells. Semin.Immunol. regel 15 2004;16:89-98. regel 16 33. Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G. Ex vivo isolation and characterization regel 17 of CD4(+)CD25(+) T cells with regulatory properties from human blood. J.Exp.Med. regel 18 2001;193:1303-1310. regel 19 34. Jonuleit H, Schmitt E, Stassen M et al. Identification and functional characterization of regel 20 human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood. regel 21 J.Exp.Med. 2001;193:1285-1294. regel 22 35. Sakaguchi S. The origin of FOXP3-expressing CD4+ regulatory T cells: thymus or periphery. regel 23 J.Clin.Invest 2003;112:1310-1312. regel 24 36. Walker MR, Kasprowicz DJ, Gersuk VH et al. Induction of FoxP3 and acquisition of T regulatory regel 25 1 Chapter activity by stimulated human CD4+. J.Clin.Invest 2003;112:1437-1443. regel 26 24 37. Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in regel 27 immunological tolerance to self and non-self. Nat.Immunol. 2005;6:345-352. regel 28 38. Zheng Y, Rudensky AY. Foxp3 in control of the regulatory T cell lineage. Nat.Immunol. regel 29 2007;8:457-462. regel 30 39. Coffer PJ, Burgering BM. Forkhead-box transcription factors and their role in the immune regel 31 system. Nat.Rev.Immunol. 2004;4:889-899. regel 32 40. Allan SE, Crome SQ, Crellin NK et al. Activation-induced FOXP3 in human T effector cells does regel 33 not suppress proliferation or cytokine production. Int.Immunol. 2007;19:345-354. regel 34 41. Bach JF, Francois BJ. Regulatory T cells under scrutiny. Nat.Rev.Immunol. 2003;3:189-198. regel 35 42. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nat.Rev.Immunol. regel 36 2003;3:253-257. 43. Maloy KJ, Powrie F. Regulatory T cells in the control of immune pathology. Nat.Immunol. regel 1 2001;2:816-822. regel 2 44. Van Eden W, Thole JE, Van Der ZR et al. Cloning of the mycobacterial epitope recognized by T regel 3 lymphocytes in adjuvant arthritis. Nature 1988;331:171-173. regel 4 45. Prakken BJ, Van Der ZR, Anderton SM et al. Peptide-induced nasal tolerance for a mycobacterial regel 5 heat shock protein 60 T cell epitope in rats suppresses both adjuvant arthritis and regel 6 nonmicrobially induced experimental arthritis. Proc.Natl.Acad.Sci.U.S.A 1997;94:3284-3289. regel 7 46. Prakken BJ, Roord S, van Kooten PJ et al. Inhibition of adjuvant-induced arthritis by interleukin- regel 8 10-driven regulatory cells induced via nasal administration of a peptide analog of an arthritis- regel 9 related heat-shock protein 60 T cell epitope. Arthritis Rheum. 2002;46:1937-1946. regel 10 47. Elias D, Cohen IR. Peptide therapy for diabetes in NOD mice. Lancet 1994;343:704-706. regel 11 48. Prakken BJ, Samodal R, Le TD et al. Epitope-specific immunotherapy induces immune regel 12 deviation of proinflammatory T cells in rheumatoid arthritis. Proc.Natl.Acad.Sci.U.S.A regel 13 2004;101:4228-4233. regel 14 49. Raz I, Elias D, Avron A et al. Beta-cell function in new-onset type 1 diabetes and regel 15 immunomodulation with a heat-shock protein peptide (DiaPep277): a randomised, double- regel 16 blind, phase II trial. Lancet 2001;358:1749-1753. regel 17 50. Raz I, Avron A, Tamir M et al. Treatment of new-onset type 1 diabetes with peptide DiaPep277 regel 18 is safe and associated with preserved beta-cell function: extension of a randomized, double- regel 19 blind, phase II trial. Diabetes Metab Res.Rev. 2007;23:292-298. regel 20 51. Huurman VA, Decochez K, Mathieu C, Cohen IR, Roep BO. Therapy with the hsp60 peptide regel 21 DiaPep277 in C-peptide positive type 1 diabetes patients. Diabetes Metab Res.Rev. regel 22 2007;23:269-275. regel 23 52. Lazar L, Ofan R, Weintrob N et al. Heat-shock protein peptide DiaPep277 treatment in children regel 24 with newly diagnosed type 1 diabetes: a randomised, double-blind phase II study. Diabetes introduction General regel 25 Metab Res.Rev. 2007;23:286-291. 25 regel 26 53. Schloot NC, Meierhoff G, Lengyel C et al. Effect of heat shock protein peptide DiaPep277 regel 27 on beta-cell function in paediatric and adult patients with recent-onset diabetes mellitus type regel 28 1: two prospective, randomized, double-blind phase II trials. Diabetes Metab Res.Rev. regel 29 2007;23:276-285. regel 30 54. Huurman VA, van der Meide PE, Duinkerken G et al. Immunological efficacy of heat shock regel 31 protein 60 peptide DiaPep277 therapy in clinical type I diabetes. Clin.Exp.Immunol. regel 32 2008;152:488-497. regel 33 55. Ehrenstein MR, Evans JG, Singh A et al. Compromised function of regulatory T cells in regel 34 rheumatoid arthritis and reversal by anti-TNFalpha therapy. J.Exp.Med. 2004;200:277-285. regel 35 regel 36 regel 1 56. Toubi E, Kessel A, Mahmudov Z et al. Increased spontaneous apoptosis of CD4+CD25+ T cells regel 2 in patients with active rheumatoid arthritis is reduced by infliximab. Ann.N.Y.Acad.Sci. regel 3 2005;1051:506-514. regel 4 57. Valencia X, Stephens G, Goldbach-Mansky R et al. TNF downmodulates the function of human regel 5 CD4+CD25hi T-regulatory cells. Blood 2006;108:253-261. regel 6 58. Schuerwegh AJ, Van Offel JF, Stevens WJ, Bridts CH, De Clerck LS. Influence of therapy with regel 7 chimeric monoclonal tumour necrosis factor-alpha antibodies on intracellular cytokine regel 8 profiles of T lymphocytes and monocytes in rheumatoid arthritis patients. Rheumatology. regel 9 (Oxford) 2003;42:541-548. regel 10 59. Alexander T, Thiel A, Rosen O et al. Depletion of autoreactive immunological memory followed regel 11 by autologous hematopoietic stem cell transplantation in patients with refractory SLE induces regel 12 long-term remission through de novo generation of a juvenile and tolerant immune system. regel 13 Blood 2008 regel 14 60. Mancardi G, Saccardi R. Autologous haematopoietic stem-cell transplantation in multiple regel 15 sclerosis. Lancet Neurol. 2008;7:626-636. regel 16 61. Sykes M, Nikolic B. Treatment of severe autoimmune disease by stem-cell transplantation. regel 17 Nature 2005;435:620-627. regel 18 62. De K, I, Brinkman DM, Ferster A et al. Autologous stem cell transplantation for refractory regel 19 juvenile idiopathic arthritis: analysis of clinical effects, mortality, and transplant related regel 20 morbidity. Ann.Rheum.Dis. 2004;63:1318-1326. regel 21 63. Wulffraat NM, Sanders LA, Kuis W. Autologous hemopoietic stem-cell transplantation for regel 22 children with refractory autoimmune disease. Curr.Rheumatol.Rep. 2000;2:316-323. regel 23 64. Touraine JL, Roncarolo MG, Raudrant D et al. Induction of transplantation tolerance in humans regel 24 using fetal cell transplants. Transplant.Proc. 2005;37:65-66. regel 25 1 Chapter 65. Dazzi F, van Laar JM, Cope A, Tyndall A. Cell therapy for autoimmune diseases. Arthritis Res. regel 26 26 Ther. 2007;9:206. regel 27 66. De K, I, Vastert B, Klein M et al. Autologous stem cell transplantation for autoimmunity induces regel 28 immunologic self-tolerance by reprogramming autoreactive T cells and restoring the regel 29 CD4+CD25+ immune regulatory network. Blood 2006;107:1696-1702. regel 30 67. Herrmann MM, Gaertner S, Stadelmann C et al. Tolerance induction by bone marrow regel 31 transplantation in a multiple sclerosis model. Blood 2005;106:1875-1883. regel 32 68. Brinkman DM, De K, I, Ten CR et al. Autologous stem cell transplantation in children with severe regel 33 progressive systemic or polyarticular juvenile idiopathic arthritis: long-term follow-up of a regel 34 prospective clinical trial. Arthritis Rheum. 2007;56:2410-2421. regel 35 69. Rabusin M, Andolina M, Maximova N. Haematopoietic SCT in autoimmune diseases in children: regel 36 rationale and new perspectives. Bone Marrow Transplant. 2008;41 Suppl 2:S96-S99. 70. Passweg J, Tyndall A. Autologous stem cell transplantation in autoimmune diseases. Semin. regel 1 Hematol. 2007;44:278-285. regel 2 71. Moore J, Brooks P, Milliken S et al. A pilot randomized trial comparing CD34-selected versus regel 3 unmanipulated hemopoietic stem cell transplantation for severe, refractory rheumatoid regel 4 arthritis. Arthritis Rheum. 2002;46:2301-2309. regel 5 72. van Bekkum DW. Experimental basis for the treatment of autoimmune diseases with regel 6 autologous hematopoietic stem cell transplantation. Bone Marrow Transplant. 2003;32 Suppl regel 7 1:S37-S39. regel 8 73. Morgan ME, Flierman R, van Duivenvoorde LM et al. Effective treatment of collagen-induced regel 9 arthritis by adoptive transfer of CD25+ regulatory T cells. Arthritis Rheum. 2005;52:2212- regel 10 2221. regel 11 74. Glant TT, Finnegan A, Mikecz K. Proteoglycan-induced arthritis: immune regulation, cellular regel 12 mechanisms, and genetics. Crit Rev.Immunol. 2003;23:199-250. regel 13 75. Glant TT, Mikecz K. Proteoglycan aggrecan-induced arthritis: a murine autoimmune model of regel 14 rheumatoid arthritis. Methods Mol.Med. 2004;102:313-338. regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24

General introduction General regel 25 27 regel 26 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 1 Chapter regel 26 28 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Present and future of combination therapy of autoimmune2 diseases

Sarah T.A. Roord1, Berent J. Prakken1, Salvatore Albani2

1 Department of Pediatric Immunology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, the Netherlands. 2 Department of Pediatrics, University of California San Diego, La Jolla, CA, U.S.A.

Future Rheumatology 2006; 1(6): 649-655. regel 1 Introduction regel 2 regel 3 Treatment regimens for rheumatological diseases have changed considerably over the past regel 4 20 years. To date, treatment of rheumatoid arthritis (RA) mainly focused on the inflammatory regel 5 pathogenic component of the disease. Significant progress in the induction of disease remission regel 6 has been achieved by the use of powerful immunosuppressants and cytostatic drugs, such regel 7 as corticosteroids, methotrexate (MTX) and in the early stage of the disease1;2. regel 8 Biologics have recently been added for treatment resistant disease, more specifically targeting regel 9 components that play a role in the disease pathogenesis3;4. Unfortunately, persistent disease regel 10 remission can only be achieved as long as the drugs are administered, and only then can regel 11 progression of joint damage be prevented. In addition, since most of these agents induce a regel 12 generalized and nonspecific inhibition of immune response and inflammation, they can cause regel 13 considerable immunodepression, leading to complications that prompted US FDA to formulate regel 14 black-box warnings for some of these products. A more specific modulation of the immune regel 15 response could theoretically overcome these pitfalls. Some of these novel strategies are in the regel 16 clinical development stage. regel 17 The combination of treatment strategies with different and complementary mechanisms regel 18 of action is likely to be more successful and is thus being introduced into the standard of regel 19 practice; by acting complementarily or synergistically, they can enhance efficacy. Cost and regel 20 toxicity can be decreased, owing to the opportunity to administer lower amounts of drugs than regel 21 when they are given separately. Combination of nonbiologics, currently approved biologics regel 22 and novel biologics will be discussed in this editorial. A dual therapeutic strategy targeting the regel 23 dominating proinflammatory milieu as well as the more specific immune response is, in our regel 24 view, especially promising. Induction of specific immune tolerance may provide a long-lasting regel 25 2 Chapter disease remission, enabling changes in dosing and scheduling of those drugs with the least regel 26 30 convenient safety profile, leading certainly to a more individualized approach to medicine and regel 27 opening the possibility of identifying a strategy leading to long- lasting remission. regel 28 regel 29 Combination of nonbiologics regel 30 It has become common practice, that patients are started on disease modifying anti rheumatic regel 31 drugs (DMARDs) therapy early in the course of their disease. In order to achieve synergy regel 32 without subsequent increase in toxicity, long-term remission and slowing of radiological regel 33 damage, the combination of several DMARDS with different mechanisms of action was tested regel 34 and found to be effective in some combinations5;6. For instance, in two open-label randomized regel 35 trials in patients with early RA, the combination of MTX, sulfasalazine, hydroxychloroquine regel 36 (HCQ) and prednisone demonstrated greater clinical improvement and significantly less radiographic progression compared with a single DMARD with or without prednisone2;7. Many regel 1 other combinations are used in common practice, with a general direct correlation between regel 2 increased clinical efficacy and overlapping undesired effects, which limit the use of such regel 3 combinations. regel 4 The underlying reason for the synergistic effect of these DMARDs is not fully known at present. regel 5 This effect may be partly due to the influence on each other’s ; for example, regel 6 HCQ leads to slower clearance and uptake with a greater area under the curve for MTX in regel 7 patients taking the combination of HCQ and MTX8. The synergy may be also mechanistic and regel 8 affect both the adaptive and innate arms of the immune response. For instance, MTX, as a regel 9 folate inhibitor, acts on rapidly proliferating cells, which are purportedly composing the pool regel 10 of effector cells that may fuel autoimmune inflammation. Conversely, HCQ is an inhibitor regel 11 of intracellular processes involved in antigen presentation, thus potentially affecting the regel 12 repertoire of autoantigens available to effector cells9. regel 13 Unfortunately, a proportion of patients still do not respond sufficiently to DMARD therapy, and regel 14 complementary approaches are needed. Major disadvantages of the use of DMARDs are that regel 15 they are not specific (and therefore lead to side effects) and that they must be administered regel 16 continuously to retain efficacy. regel 17 regel 18 Combination of currently approved biologics regel 19 More success with fewer side effects can be expected by more specifically targeting pro- regel 20 inflammatory cytokines that are known to play a role in the autoimmune process, such as regel 21 tumor necrosis factor (TNF)α, interleuking (IL)-1 or IL-6. Several biological agents have been regel 22 identified that can block these cytokines: regel 23 • Enbrel (Etanercept®), a soluble TNF receptor fusion protein, targets TNFα; regel 24 • Remicade (Infliximab®), a chimeric anti TNFα antibody, targets TNFα; diseases autoimmune of therapy Combination regel 25 • Humira (Adalimumab®), a recombinant human anti TNFα antibody, targets TNFα; 31 regel 26 • Kineret (Anakinra®), an IL-1 receptor antagonist (IL-1RA), neutralizes IL-1; regel 27 • MRA® (a humanized IL-6 receptor antibody) targets IL-6. regel 28 The use of these agents has shown clinical efficacy in several clinical trials, but the fact that regel 29 the effects are only temporary, their cost is high and severe side effects may occur, such as regel 30 an increase in serious infections and possibly lymphomas due to anti TNFα treatment, is regel 31 hampering their success3;10. regel 32 The potential for additive or synergistic effects of two biologics has been tested in the treatment regel 33 of RA by adding Kineret to treatment with Enbrel. However, no additional clinical effect was regel 34 demonstrated, whereas an increased safety risk did become apparent, due to higher incidence regel 35 of serious infections, injection site reactions and neutropenia11. Therefore, the combination of regel 36 two anticytokine biologics is not recommended. regel 1 Conversely, a field with great potential is the combination of biologics that target different regel 2 immunopathogenic pathways; therefore exploiting a potential complementarity in mechanism regel 3 of action. However, these approaches are at the initial stage of clinical testing to ascertain both regel 4 efficacy and tolerability. regel 5 regel 6 Combination of nonbiologics with currently approved biologics regel 7 Promising results were obtained when combining MTX with a biological agent. The combination regel 8 of MTX with anti TNFα or IL-1RA treatment is found to improve the clinical outcome significantly, regel 9 compared to MTX alone as well as anti TNFα treatment alone12-20. The combination treatments regel 10 led to a decrease in disease activity, reduction of disability, an increase in remission rates regel 11 and even a decrease in progression of joint damage. Unfortunately, the exact mechanism regel 12 underlying this synergistic effect is not currently known, but may be explained in part by regel 13 a decreased amount of neutralizing antibodies against TNF antagonists21. Furthermore, regel 14 knowledge on the influence of MTX on T cells and the inflammatory process has been regel 15 expanding in recent years and may also provide some explanation for the combinatory regel 16 effect of MTX with anticytokine treatment. MTX promotes the release of the endogenous anti- regel 17 inflammatory mediator adenosine, presumably through its capacity to increase intracellular regel 18 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) concentrations22. Adenosine has a regel 19 central role in regulation of inflammatory responses, one of which is the contribution to the regel 20 resolution of inflammation, both by down-regulating macrophage activation and by advancing regel 21 T-helper (Th)2 versus Th1 cell development23. Direct evidence for the promotion of a more anti- regel 22 inflammatory environment by MTX was gained in early RA patients, where a decrease in T-cell- regel 23 derived TNFα was observed, together with an increase in IL-10-producing T cells24. In in vitro regel 24 experiments MTX was demonstrated to increase IL-10 and IL-4 gene transcription as well as regel 25 2 Chapter decrease IFNγ gene transcription25. Therefore the combination of blocking proinflammatory regel 26 32 cytokines by anticytokine biologics with the promotion of a more anti-inflammatory regel 27 environment by MTX may work synergistically. regel 28 Hence, an important step towards obtaining disease remission and slowing of radiological regel 29 damage has been obtained by combining several DMARDs or combining MTX and anticytokine regel 30 biologics. However, the effects only last while the drugs are administered. regel 31 regel 32 Combination of currently approved biologics with novel biologics regel 33 The induction of specific immune tolerance would ideally spare the patient generalized immune regel 34 suppression and could be expected to provide a long-lasting effect (and maybe even a cure) regel 35 that is devoid of side effects. The induction of immune tolerance requires the identification regel 36 of the appropriate target. Efforts at inducing tolerance independent of a specific antigenic target are mainly based on molecules interfering with either T (CD3 or CD80/CTLA-4) or B cells regel 1 (CD20/CD22). These attempts have led to very encouraging results and some of these drugs regel 2 are already available on the market. In recent onset Type I diabetes, two humanized anti CD3 regel 3 monoclonal antibodies were able to maintain residual β cell function better than placebo or regel 4 a control group, as shown by increased C peptide responses and decreased insulin needs. regel 5 Short-term treatment even produced lasting effects for up to 2 years26;27. Orencia (Abatacept®), regel 6 a recombinant Cytotoxic T-lymphocyte antigen (CTLA)4- immunoglobulin (Ig) fusion protein, regel 7 blocks the costimulatory signal required for T cell activation by competing with CD28 for CD80 regel 8 and CD86 binding. It demonstrated effectiveness in active RA patients, in improvement of regel 9 signs and symptoms of disease, physical function and the quality of life over a period of 12 regel 10 months in two Phase II studies28;29. In a Phase III randomized trial, it also demonstrated clinical regel 11 and functional benefits in nonresponders to TNFα therapy30. Although the role of B cells in regel 12 RA is not fully understood, selective depletion of B cells by a monoclonal antibody against regel 13 CD20, Rituxan (Rituximab®), led to sustained clinical improvement in an open label study31. In regel 14 a subsequent randomized, double-blind, controlled study in 161 RA patients, treatment with regel 15 Rituxan, alone or in combination with cyclophosphamide or MTX, led to significant improvement regel 16 of disease symptoms32. This category of molecules represents a significant step forward with regel 17 their mechanism of action, because in most cases they aim to modulate certain aspects of regel 18 adaptive immunity rather than suppressing an individual pathway (i.e., a cytokine). regel 19 Another approach would be induction of tolerance using an antigen. However, the search for regel 20 ‘the one disease triggering antigen’ has not been successful to date, and attempts to induce regel 21 tolerance to candidates in this respect, such as chicken and bovine Type II collagen and regel 22 human cartilage glycoprotein (HCgp)39, major constituents of articular cartilage, were also regel 23 not encouraging33-36. In fact, we feel that the focus of antigen specific therapy should move regel 24 away from the one disease-triggering antigen and should focus mainly on key players at the diseases autoimmune of therapy Combination regel 25 site of inflammation, which play a role in disease perpetuation. Heat Shock Proteins (HSPs) 33 regel 26 are such cells. HSPs are present in all cells and are up regulated during stress. As ubiquitous regel 27 and bacterial derived products, HSP-derived peptides are perceived as a danger signal and regel 28 elicit a default proinflammatory physiological response, which involves both the adaptive regel 29 and the innate arms of the immune system37-39. Such a response contributes to the clearing regel 30 of a possible pathogen invasion, but also induces increased availability of self-HSP-derived regel 31 peptides through cellular stress. These peptides then form a new target for the immune regel 32 system and induce self-perpetuating cycles of inflammation, fueled by the self-antigens and regel 33 self-reactive T cells. We have previously demonstrated, that HSP peptides are recognized by regel 34 T cells with regulatory function, which are then capable of preventing further tissue damage. regel 35 If such regulatory function is impaired, loops of inflammation continue and autoimmune regel 36 arthritis prevails40;41. regel 1 Promising results of antigen specific immunomodulation by HSP in experimental disease models regel 2 of arthritis and Type I diabetes warranted subsequent clinical trials in human disease42;43. In regel 3 RA, we have recently reported the results of a Phase I/IIa clinical trial with a dnaJ-derived regel 4 peptide, dnaJP1, administered orally to 15 patients with early, active disease44. Interestingly, regel 5 with this treatment, we were able to induce immune deviation from proinflammatory to regel 6 modulatory T cell responses, leading to significant reduction in TNFα and IFNγ production regel 7 and an increase in IL-10 and IL-4. These effects were mediated via restoration of function of regel 8 CD4CD25bright regulatory T cells (Treg), producing IL-10 and expressing FOXP3. Recently, we regel 9 completed the Phase II clinical trial with dnaJP1 (submitted). This study focused on safety and regel 10 clinical efficacy of the drug. It involved 160 patients who received dnaJP1 or placebo orally regel 11 once per day for 6 months. The dnaJP1 peptide treatment showed encouraging clinical and regel 12 immunological effects, suggesting that induction of immune tolerance to an inflammatory regel 13 ubiquitous antigen may translate into clinical improvement of the disease. regel 14 Due to safety and specificity in mechanism of action, epitope specific immunotherapy has the regel 15 profile of an ideal “work with” approach. As such, it could exploit synergy and complementarity regel 16 in mechanisms of action with both biologics and more traditional DMARDs. regel 17 We have obtained the first results in the combination of anti TNFα therapy and antigen specific regel 18 immunomodulation to an HSP60 peptide in an experimental form of arthritis: Adjuvant Arthritis regel 19 (AA). AA is a T cell dependent disease that can be passively transferred by a T cell clone that is regel 20 specific for the 180-188 amino acid sequence of mycobacterial HSP6045;46. In previous studies regel 21 we demonstrated that nasal administration of peptide 180-188 after the induction of AA is regel 22 mildly effective43. Interestingly, by giving a single low dose of Enbrel before mucosal tolerance regel 23 induction to HSP60 peptide 180-188, significant suppression of arthritis was seen to the same regel 24 extent as a full course of Enbrel therapy47. This implies that lower doses of anti TNFα can be regel 25 2 Chapter given, resulting in lower cost and less long-term side effects. Interestingly, two distinctly regel 26 34 different immunological mechanisms were at the basis of equivalent clinical suppression regel 27 of arthritis when comparing full dose Enbrel therapy with the combination treatment of anti regel 28 TNFα and 180-188 peptide. Where anti TNFα treatment induced mainly immune suppression, regel 29 combination treatment was able to induce active modulation through induction of IL-10 regel 30 production by effector T cells as well as the induction of CD4+CD25+ regulatory T cells, again regel 31 producing IL-10 and expressing Foxp3. regel 32 Similarly promising results were recently published by Bresson et al. in experimental diabetes regel 33 48. By combining anti CD3 and intranasal proinsulin peptide treatment, recent onset diabetes regel 34 could be reversed more potently than when anti CD3 or the peptide was given alone. This regel 35 combination treatment induced Tregs; the level of CD4+CD25+FOXP3+ cells increased and regel 36 insulin specific production of IL-10, TGFβ and IL-4 by Tregs was enhanced. The Tregs were capable of suppressing autoaggressive CD8+ responses in vitro. Furthermore, adoptive regel 1 transfer of the peptide specific Tregs suppressed disease in recent onset diabetic recipients to regel 2 the same extent as in the donors. regel 3 The reason the induction of tolerance is facilitated by the combination with anti TNFα and anti regel 4 CD3, may be partly because both have been demonstrated to create a tolerogenic environment. regel 5 Several studies have shown that anti TNFα treatment can improve Treg function and numbers regel 6 in RA patients49-51. Additionally, anti TNFα treatment was shown to induce a shift to a more regel 7 anti-inflammatory cytokine profile in peripheral blood mononuclear cells (PBMCs) and T cells regel 8 of RA patients52. In non-obese diabetic mice, it was demonstrated that anti CD3 treatment led regel 9 to a decrease in the amount of autoaggressive T cells and an expansion of CD4+CD25+ cells in regel 10 draining lymph nodes. Disease suppression was mediated through production of TGFβ53. regel 11 As aforementioned, epitope specific immunotherapy also has the potential to act in synergy regel 12 with more traditional DMARDs. In our phase II trial with dnaJP1 in human RA, we unexpectedly regel 13 obtained the first results of combination treatment of a DMARD and mucosal tolerance induction. regel 14 Interestingly, the clinical effect of dnaJP1 was clearly enhanced in patients using HCQ. HCQ is regel 15 traditionally an antimalarial drug that, due to its immunomodulatory effects, is also used in regel 16 the treatment of RA. The explanation of the enhanced effect of dnaJP1 tolerance induction regel 17 due to HCQ, may be partly because it is known to decrease TNFα and IL-6 production. HCQ’s regel 18 main effect is exerted through blockade of the processing of proteins by antigen presenting regel 19 cells (APCs)9;54;55. This creates an environment of low self-presentation of proteins, whereby regel 20 the dnaJP1 peptide might be more easily presented, as it does not need to be processed in regel 21 order to be presented by APC. In this way, such peptides may have a greater impact on the regel 22 regulatory immune system. regel 23 Combination of MTX with antigen specific tolerance induction may also be beneficial. MTX regel 24 administration may create a better milieu for antigen specific immunomodulation, by creating diseases autoimmune of therapy Combination regel 25 an anti-inflammatory environment and maybe even a more tolerogenic microenvironment via 35 regel 26 its action on rapidly proliferating effector T cells and the decrease in TNFα production23;24. regel 27 regel 28 Conclusion and Future Perspectives regel 29 The combination of different treatments, especially of DMARDs or anticytokine biologics with regel 30 novel biologics, appears to be effective through synergistic, as well as complementary, working regel 31 mechanisms. These approaches may implicate important changes in the future management regel 32 of autoimmunity. regel 33 Immune tolerance may still be the ultimate objective in the treatment of autoimmune diseases. regel 34 The combination treatment approach may exploit the strengths of complementary drugs and regel 35 reduce the chances of developing side effects. This approach, which is associated with a more regel 36 regel 1 focused and effective approach toward modulation of adaptive immunity, may render true regel 2 immune tolerization attainable. regel 3 Secondly, the increasing knowledge of the pathophysiology of disease may enable the choice regel 4 of different associations of drugs, for example, based on genetic and pathological patterns. This regel 5 may lead to the identification of subgroups of patients who may be more prone to responding regel 6 to certain cocktails of drugs; thereby providing an important step towards individualized regel 7 medicine. regel 8 Lastly, the complementarities in mechanisms of action and the diverse potency of various regel 9 DMARDs and biologics may eventually lead to a progressive treatment design, where various regel 10 drugs may be used at different times. In such a protocol, DMARDs and anticytokine biologics regel 11 can be applied to induce disease remission, followed by epitope specific therapy in order to regel 12 maintain it. regel 13 regel 14 regel 15 Reference List regel 16 regel 17 1. Fries JF. Current treatment paradigms in rheumatoid arthritis. Rheumatology.(Oxford) regel 18 2000;39 Suppl 1:30-35. regel 19 2. Mottonen T, Hannonen P, Leirisalo-Repo M et al. Comparison of combination therapy with regel 20 single-drug therapy in early rheumatoid arthritis: a randomised trial. FIN-RACo trial group. regel 21 Lancet 1999;353:1568-1573. regel 22 3. Goldblatt F, Isenberg DA. New therapies for rheumatoid arthritis. Clin.Exp.Immunol. regel 23 2005;140:195-204. regel 24 4. Taylor PC. Anti-tumor necrosis factor therapies. Curr.Opin.Rheumatol. 2001;13:164-169. regel 25 2 Chapter 5. Choy EH. Two is better than one? Combination therapy in rheumatoid arthritis. Rheumatology. regel 26 36 (Oxford) 2004;43:1205-1207. regel 27 6. Goekoop YP, Allaart CF, Breedveld FC, Dijkmans BA. Combination therapy in rheumatoid regel 28 arthritis. Curr.Opin.Rheumatol. 2001;13:177-183. regel 29 7. Calguneri M, Pay S, Caliskaner Z et al. Combination therapy versus monotherapy for the regel 30 treatment of patients with rheumatoid arthritis. Clin.Exp.Rheumatol. 1999;17:699-704. regel 31 8. Carmichael SJ, Beal J, Day RO, Tett SE. Combination therapy with methotrexate and regel 32 hydroxychloroquine for rheumatoid arthritis increases exposure to methotrexate. regel 33 J.Rheumatol. 2002;29:2077-2083. regel 34 9. Ziegler HK, Unanue ER. Decrease in macrophage antigen catabolism caused by ammonia regel 35 and chloroquine is associated with inhibition of antigen presentation to T cells. Proc.Natl.Acad. regel 36 Sci.U.S.A 1982;79:175-178. 10. Choy E. Clinical trial outcome of anti-tumour necrosis factor alpha therapy in rheumatic regel 1 arthritis. Cytokine 2004;28:158-161. regel 2 11. Genovese MC, Cohen S, Moreland L et al. Combination therapy with etanercept and anakinra regel 3 in the treatment of patients with rheumatoid arthritis who have been treated unsuccessfully regel 4 with methotrexate. Arthritis Rheum. 2004;50:1412-1419. regel 5 12. Klareskog L, van der HD, de Jager JP et al. Therapeutic effect of the combination of etanercept regel 6 and methotrexate compared with each treatment alone in patients with rheumatoid arthritis: regel 7 double-blind randomised controlled trial. Lancet 2004;363:675-681. regel 8 13. Quinn MA, Conaghan PG, O’Connor PJ et al. Very early treatment with infliximab in addition regel 9 to methotrexate in early, poor-prognosis rheumatoid arthritis reduces magnetic resonance regel 10 imaging evidence of synovitis and damage, with sustained benefit after infliximab withdrawal: regel 11 results from a twelve-month randomized, double-blind, placebo-controlled trial. Arthritis regel 12 Rheum. 2005;52:27-35. regel 13 14. Cohen S, Hurd E, Cush J et al. Treatment of rheumatoid arthritis with anakinra, a recombinant regel 14 human interleukin-1 receptor antagonist, in combination with methotrexate: results of a regel 15 twenty-four-week, multicenter, randomized, double-blind, placebo-controlled trial. Arthritis regel 16 Rheum. 2002;46:614-624. regel 17 15. Weinblatt ME, Keystone EC, Furst DE et al. Adalimumab, a fully human anti-tumor necrosis regel 18 factor alpha monoclonal antibody, for the treatment of rheumatoid arthritis in patients taking regel 19 concomitant methotrexate: the ARMADA trial. Arthritis Rheum. 2003;48:35-45. regel 20 16. Maini R, St Clair EW, Breedveld F et al. Infliximab (chimeric anti-tumour necrosis factor regel 21 alpha monoclonal antibody) versus placebo in rheumatoid arthritis patients receiving regel 22 concomitant methotrexate: a randomised phase III trial. ATTRACT Study Group. Lancet regel 23 1999;354:1932-1939. regel 24 17. Weinblatt ME, Kremer JM, Bankhurst AD et al. A trial of etanercept, a recombinant tumor diseases autoimmune of therapy Combination regel 25 necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving 37 regel 26 methotrexate. N.Engl.J.Med. 1999;340:253-259. regel 27 18. Lipsky PE, van der Heijde DM, St Clair EW et al. Infliximab and methotrexate in the treatment of regel 28 rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant regel 29 Therapy Study Group. N.Engl.J.Med. 2000;343:1594-1602. regel 30 19. Breedveld FC, Weisman MH, Kavanaugh AF et al. The PREMIER study: A multicenter, regel 31 randomized, double-blind clinical trial of combination therapy with adalimumab plus regel 32 methotrexate versus methotrexate alone or adalimumab alone in patients with early, regel 33 aggressive rheumatoid arthritis who had not had previous methotrexate treatment. Arthritis regel 34 Rheum. 2006;54:26-37. regel 35 regel 36 regel 1 20. van der HD, Klareskog L, Rodriguez-Valverde V et al. Comparison of etanercept and methotrexate, regel 2 alone and combined, in the treatment of rheumatoid arthritis: two-year clinical and radiographic regel 3 results from the TEMPO study, a double-blind, randomized trial. Arthritis Rheum. 2006;54:1063- regel 4 1074. regel 5 21. Maini RN, Breedveld FC, Kalden JR et al. Therapeutic efficacy of multiple intravenous infusions regel 6 of anti-tumor necrosis factor alpha monoclonal antibody combined with low-dose weekly regel 7 methotrexate in rheumatoid arthritis. Arthritis Rheum. 1998;41:1552-1563. regel 8 22. Cronstein BN. Low-dose methotrexate: a mainstay in the treatment of rheumatoid arthritis. regel 9 Pharmacol.Rev. 2005;57:163-172. regel 10 23. Hasko G, Cronstein BN. Adenosine: an endogenous regulator of innate immunity. Trends regel 11 Immunol. 2004;25:33-39. regel 12 24. Rudwaleit M, Yin Z, Siegert S et al. Response to methotrexate in early rheumatoid arthritis is regel 13 associated with a decrease of T cell derived tumour necrosis factor alpha, increase of regel 14 interleukin 10, and predicted by the initial concentration of interleukin 4. Ann.Rheum.Dis. regel 15 2000;59:311-314. regel 16 25. Constantin A, Loubet-Lescoulie P, Lambert N et al. Antiinflammatory and immunoregulatory regel 17 action of methotrexate in the treatment of rheumatoid arthritis: evidence of increased regel 18 interleukin-4 and interleukin-10 gene expression demonstrated in vitro by competitive regel 19 reverse transcriptase-polymerase chain reaction. Arthritis Rheum. 1998;41:48-57. regel 20 26. Herold KC, Gitelman SE, Masharani U et al. A single course of anti-CD3 monoclonal antibody regel 21 hOKT3gamma1(Ala-Ala) results in improvement in C-peptide responses and clinical regel 22 parameters for at least 2 years after onset of type 1 diabetes. Diabetes 2005;54:1763-1769. regel 23 27. Keymeulen B, Vandemeulebroucke E, Ziegler AG et al. Insulin needs after CD3-antibody regel 24 therapy in new-onset type 1 diabetes. N.Engl.J.Med. 2005;352:2598-2608. regel 25 2 Chapter 28. Moreland LW, Alten R, Van den BF et al. Costimulatory blockade in patients with rheumatoid regel 26 38 arthritis: a pilot, dose-finding, double-blind, placebo-controlled clinical trial evaluating CTLA- regel 27 4Ig and LEA29Y eighty-five days after the first infusion. Arthritis Rheum. 2002;46:1470- regel 28 1479. regel 29 29. Kremer JM, Westhovens R, Leon M et al. Treatment of rheumatoid arthritis by selective regel 30 inhibition of T-cell activation with fusion protein CTLA4Ig. N.Engl.J.Med. 2003;349:1907-1915. regel 31 30. Genovese MC, Becker JC, Schiff M et al. Abatacept for rheumatoid arthritis refractory to tumor regel 32 necrosis factor alpha inhibition. N.Engl.J.Med. 2005;353:1114-1123. regel 33 31. Edwards JC, Cambridge G. Sustained improvement in rheumatoid arthritis following a protocol regel 34 designed to deplete B lymphocytes. Rheumatology.(Oxford) 2001;40:205-211. regel 35 32. Edwards JC, Szczepanski L, Szechinski J et al. Efficacy of B-cell-targeted therapy with regel 36 rituximab in patients with rheumatoid arthritis. N.Engl.J.Med. 2004;350:2572-2581. 33. McKown KM, Carbone LD, Kaplan SB et al. Lack of efficacy of oral bovine type II collagen added regel 1 to existing therapy in rheumatoid arthritis. Arthritis Rheum. 1999;42:1204-1208. regel 2 34. Barnett ML, Kremer JM, St Clair EW et al. Treatment of rheumatoid arthritis with oral type II regel 3 collagen. Results of a multicenter, double-blind, placebo-controlled trial. Arthritis Rheum. regel 4 1998;41:290-297. regel 5 35. Choy EH, Scott DL, Kingsley GH et al. Control of rheumatoid arthritis by oral tolerance. Arthritis regel 6 Rheum. 2001;44:1993-1997. regel 7 36. Keystone EC. Abandoned therapies and unpublished trials in rheumatoid arthritis. Curr.Opin. regel 8 Rheumatol. 2003;15:253-258. regel 9 37. Albani S. Infection and molecular mimicry in autoimmune diseases of childhood. Clin.Exp. regel 10 Rheumatol. 1994;12 Suppl 10:S35-S41. regel 11 38. Albani S, Ravelli A, Massa M et al. Immune responses to the Escherichia coli dnaJ heat shock regel 12 protein in juvenile rheumatoid arthritis and their correlation with disease activity. J.Pediatr. regel 13 1994;124:561-565. regel 14 39. Albani S, Keystone EC, Nelson JL et al. Positive selection in autoimmunity: abnormal immune regel 15 responses to a bacterial dnaJ antigenic determinant in patients with early rheumatoid regel 16 arthritis. Nat.Med. 1995;1:448-452. regel 17 40. de Kleer IM, Kamphuis SM, Rijkers GT et al. The spontaneous remission of juvenile idiopathic regel 18 arthritis is characterized by CD30+ T cells directed to human heat-shock protein 60 capable regel 19 of producing the regulatory cytokine interleukin-10. Arthritis Rheum. 2003;48:2001-2010. regel 20 41. Prakken AB, Van Eden W, Rijkers GT et al. Autoreactivity to human heat-shock protein 60 regel 21 predicts disease remission in oligoarticular juvenile rheumatoid arthritis. Arthritis Rheum. regel 22 1996;39:1826-1832. regel 23 42. Prakken BJ, Van Der ZR, Anderton SM et al. Peptide-induced nasal tolerance for a mycobacterial regel 24 heat shock protein 60 T cell epitope in rats suppresses both adjuvant arthritis and diseases autoimmune of therapy Combination regel 25 nonmicrobially induced experimental arthritis. Proc.Natl.Acad.Sci.U.S.A 1997;94:3284-3289. 39 regel 26 43. Prakken BJ, Roord S, van Kooten PJ et al. Inhibition of adjuvant-induced arthritis by interleukin- regel 27 10-driven regulatory cells induced via nasal administration of a peptide analog of an arthritis- regel 28 related heat-shock protein 60 T cell epitope. Arthritis Rheum. 2002;46:1937-1946. regel 29 44. Prakken BJ, Samodal R, Le TD et al. Epitope-specific immunotherapy induces immune regel 30 deviation of proinflammatory T cells in rheumatoid arthritis. Proc.Natl.Acad.Sci.U.S.A regel 31 2004;101:4228-4233. regel 32 45. Van Eden W, Holoshitz J, Nevo Z et al. Arthritis induced by a T-lymphocyte clone that responds regel 33 to Mycobacterium tuberculosis and to cartilage proteoglycans. Proc.Natl.Acad.Sci.U.S.A regel 34 1985;82:5117-5120. regel 35 regel 36 regel 1 46. Van Eden W, Thole JE, Van Der ZR et al. Cloning of the mycobacterial epitope recognized by T regel 2 lymphocytes in adjuvant arthritis. Nature 1988;331:171-173. regel 3 47. Roord ST, Zonneveld-Huijssoon E, Le T et al. Modulation of T cell function by combination regel 4 of epitope specific and low dose anticytokine therapy controls autoimmune arthritis. PLoS. regel 5 ONE. 2006;1:e87. regel 6 48. Bresson D, Togher L, Rodrigo E et al. Anti-CD3 and nasal proinsulin combination therapy regel 7 enhances remission from recent-onset autoimmune diabetes by inducing Tregs. J.Clin.Invest regel 8 2006;116:1371-1381. regel 9 49. Ehrenstein MR, Evans JG, Singh A et al. Compromised function of regulatory T cells in regel 10 rheumatoid arthritis and reversal by anti-TNFalpha therapy. J.Exp.Med. 2004;200:277-285. regel 11 50. Valencia X, Stephens G, Goldbach-Mansky R et al. TNF downmodulates the function of human regel 12 CD4+CD25hi T-regulatory cells. Blood 2006;108:253-261. regel 13 51. Toubi E, Kessel A, Mahmudov Z et al. Increased spontaneous apoptosis of CD4+CD25+ T cells regel 14 in patients with active rheumatoid arthritis is reduced by infliximab. Ann.N.Y.Acad.Sci. regel 15 2005;1051:506-514. regel 16 52. Schuerwegh AJ, Van Offel JF, Stevens WJ, Bridts CH, De Clerck LS. Influence of therapy with regel 17 chimeric monoclonal tumour necrosis factor-alpha antibodies on intracellular cytokine regel 18 profiles of T lymphocytes and monocytes in rheumatoid arthritis patients. Rheumatology. regel 19 (Oxford) 2003;42:541-548. regel 20 53. Belghith M, Bluestone JA, Barriot S et al. TGF-beta-dependent mechanisms mediate restoration regel 21 of self-tolerance induced by antibodies to CD3 in overt autoimmune diabetes. Nat.Med. regel 22 2003;9:1202-1208. regel 23 54. Savarino A, Boelaert JR, Cassone A, Majori G, Cauda R. Effects of chloroquine on viral infections: regel 24 an old drug against today’s diseases? Lancet Infect.Dis. 2003;3:722-727. regel 25 2 Chapter 55. van den Borne BE, Dijkmans BA, de Rooij HH, le CS, Verweij CL. Chloroquine and regel 26 40 hydroxychloroquine equally affect tumor necrosis factor-alpha, , and - regel 27 gamma production by peripheral blood mononuclear cells. J.Rheumatol. 1997;24:55-60. regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 Inhibition of adjuvant-induced arthritis by interleukin-10- regel 12 regel 13 driven regulatory cells induced via nasal administration of regel 14 regel 15 3 regel 16 a peptide analog of an arthritis-related heat-shock protein regel 17 60 T cell epitope regel 18 regel 19 regel 20 regel 21 regel 22 Berent J. Prakken1,2, Sarah Roord1,2, Peter J.S. van Kooten3, Josée P.A. Wagenaar3, regel 23 Wil­lem van Eden3, Salvatore Albani1,4, Marca H.M. Wauben3. regel 24 regel 25 regel 26 1Department of Pediatrics, University of California San Diego, La Jolla, CA, USA regel 27 2Department of Immunology, Wilhelmina Children’s Hospital, regel 28 University Medical Center Utrecht, the Netherlands regel 29 3Department of Infecti­ous Disea­ses & Immunology, Faculty of Veterinary­ Medicine, regel 30 Utrecht University, The Ne­ther­lands. regel 31 4Androclus Therapeutics, La Jolla, CA, USA regel 32 regel 33 regel 34 regel 35 Arthritis and Rheumatism 2002; 46(7):1937-46. regel 36 regel 1 ABSTRACT regel 2 regel 3 Objective regel 4 To prevent and treat experimental arthritis via nasal administration of an altered peptide ligand regel 5 (APL) from the major arthritogenic epitope in adjuvant-induced arthritis (AIA) and to explore regel 6 the mechanisms involved. regel 7 regel 8 Methods regel 9 Peptides were administered nasally before and after induction of arthritis. Splenocytes and regel 10 lymph node cells draining both the site of inflammation and the site of tolerance induction regel 11 were used for cell transfer and were studied for antigen specific T cell characteristics. In regel 12 addition, attempts were made to stop T cell tolerance in vitro, using anticytokine antibodies. regel 13 regel 14 Results regel 15 Nasal administration of a modulatory APL of the heat shock protein 60 (Hsp60) 180-188 T regel 16 cell epitope, alanine 183, had a suppressive effect in AIA that far exceeded that of the wild regel 17 type epitope. In addition to its effectiveness in preventing AIA, alanine 183 may be effective regel 18 in the treatment of ongoing AIA. The protective effect of alanine 183 can be passively regel 19 transferred using activated splenocytes. Nasal administration of alanine 183 did not lead regel 20 to detectable T cell proliferation or interleukin-2 (IL-2) production in mandibular lymph node regel 21 cells, while transforming growth factor β (TGFβ), IL-10, and IL-4 were readily produced. regel 22 Likewise, after nasally induced tolerance, followed by induction of arthritis, inguinal lymph regel 23 node cells produced IL-4, TGFβ and IL-10. After neutralizing in vitro the individual cytokines with regel 24 anticytokine antibodies, only blocking of IL-10 production led to a reversal of tolerance, at the regel 25 3 Chapter site of tolerance induction and the site of inflammation. regel 26 42 regel 27 Conclusion regel 28 Nasal administration of an APL of Hsp60 180-188 induces highly effective protection against regel 29 AIA through generation of regulatory cells that produce IL-4, TGFβ, and IL-10, whereas the regel 30 induced tolerance is driven mainly by production of IL-10. regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 INTRODUCTION regel 1 regel 2 Antigen specific peripheral T cell tolerance can be induced through oral or nasal administration regel 3 of a relevant antigen1;2. In several experimental autoimmune models, oral administration of regel 4 the disease-triggering autoantigen led to considerable suppression of disease activity3-5. In regel 5 comparison with orally induced tolerance, nasally induced tolerance has been shown to be regel 6 equally or, in the case of a peptide antigen, even more effective in suppressing experimental regel 7 autoimmune diseases1;6-9. regel 8 Previously, we described nasally induced tolerance for an arthritis-related heat shock protein regel 9 60 (Hsp60) T cell epitope in adjuvant induced arthritis (AIA)10, an experimental arthritis regel 10 model with close histopathologic resemblance to rheumatoid arthritis. AIA can be passively regel 11 transferred by a single T cell clone, obtained from AIA rats, recognizing the 180-188 amino acid regel 12 sequence of the myco­bac­teri­al Hsp6011;12. Nasal administration of the 15-mer mycobacterial regel 13 Hsp60 176-190 peptide (which contains the core 180-188 sequence) prior to the induction of regel 14 AIA, delayed the onset and reduced the severity of arthritis10. The arthritis-suppressive effect regel 15 of Hsp60 176-190 was not complete, however, and a minority of the rats developed arthritis regel 16 despite the treatment. regel 17 We questioned whether use of a peptide analog of the wild-type Hsp60 180-188 peptide could regel 18 enhance the arthritis-suppressive effect. We previously defined such a modulatory altered regel 19 13 regel 20 peptide ligand of Hsp60 180-188, peptide 180-188183L→A(alanine 183) . Alanine 183 can effectively inhibit the proliferative A2b response in vitro, and prevents AIA upon immunization regel 21 in vivo. In addition to the modulatory effect on A2b, the leucine-to-alanine substitution at regel 22 position 183 resulted in a higher major histocompatibility complex (MHC)-binding affinity for regel 23 rat RT1.Bl compared with that of the original peptide14. regel 24 We now demonstrate that nasally administered of alanine 183 suppresses AIA much more analog a peptide by arthritis adjuvant of Inhibition regel 25 efficiently than does the wild-type epitope. Importantly, nasal administration of alanine 183 43 regel 26 was effective not only in the prevention of AIA but also in the treatment of ongoing arthritis. regel 27 This protective effect can be passively transferred with activated spleen cells, which indicates regel 28 induction of an active T cell regulatory mechanism rather than clonal deletion of arthritogenic T regel 29 cells. Analysis of cytokine production by T cells generated after nasally induced tolerance with regel 30 alanine 183 showed upregulation of interleukin-10 (IL-10), IL-4 and transforming growth factor regel 31 β (TGFβ) production. However, we provide evidence that the tolerance is driven mainly by IL-10 regel 32 production, suggesting that nasal administration of alanine 183 induces induction in vivo of regel 33 so-called type 1 T regulatory cells (Tr1)15. regel 34 regel 35 regel 36 regel 1 MATERIALS AND METHODS regel 2 regel 3 Animals regel 4 Male inbred Lewis rats (RT1Bl) were obtained from the University of Limburg (Maastricht, The regel 5 Netherlands), and from Harlan (Indianapolis, IN). Rats were 6-9 weeks old at the start of each regel 6 experiment. regel 7 regel 8 Antigens and adjuvants regel 9 Heat-killed Mycobacterium tuberculosis (Mt, strain H37Ra) was obtained from Difco (Detroit, regel 10 MI). The purified recombi­nant Hsp60 of Mycobacterium bovis bacillus Calmette-Guérin (identi­ regel 11 cal to M tuberculosis Hsp60) was purified as described previously12. Freund’s Incomplete regel 12 Adjuvant (IFA; Difco) was used as adjuvant. The peptides used in this study were prepared regel 13 in large quanti­ties by standard solid-phase 9-fluorenylmethoxycarbonyl chemistry. Peptides regel 14 were obtained as C-termi­nal amides. All peptides were analyzed and purified by reverse-phase regel 15 high-performance liquid chromatography and checked by fast atom-bombardment mass regel 16 spectrometry. regel 17 The following peptides were used: M tuberculosis Hsp60 180-188, 176-190, alanine 183 and regel 18 ovalbumin (OVA) 323-339. Hsp60 180-188 contains the M tuberculosis Hsp60 sequence 180- regel 19 188 (TFGLQLELT), which is also included in M tuberculosis Hsp60 sequence 176-190). Hsp60 regel 20 180-188 is the core sequence recognized by the arthritogenic T cell clone A2b. Hsp60 176-190 regel 21 is a dominant T cell epitope found after AIA and after immunization with mycobacte­rial Hsp6016. regel 22 Alanine 183 is a modulatory peptide analog of Hsp60 180-188, containing an alanine residue regel 23 at position 183 instead of the leucine residue present in the native sequence13. OVA 323-339 regel 24 (ISQAVHAAHAEINEAGR) contains a RT1.Bl binding T cell epitope recognized by the OVA-specific regel 25 3 Chapter T cell clone 1/C11.P7 17. Peptide myelin basic protein 72-85 (QKSQRSQDENPV) was biotinylated regel 26 44 during synthesis and used as marker peptide in the MHC-peptide binding assays17. regel 27 regel 28 Tissue culture reagents regel 29 Iscove’s modified Dulbecco’s medium supplemented with 5% fetal calf serum (FCS), 2mM regel 30 L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin (all from GIBCO BRL, Grand regel 31 Island, NY) and 5 x 10-5 M 2-mercaptoethanol was used as culture medium. regel 32 regel 33 regel 34 regel 35 regel 36 MHC-peptide binding assay regel 1 Lewis rat MHC class II molecules were purified from the Z1a T cell clone through affinity regel 2 chromatography using the OX-6 monoclonal antibody, as desribed previously17. The MHC regel 3 binding affinity of the peptides was determined by using a competition assay on isolated regel 4 rat MHC class II RT1.Bl molecules, essentially as described previously17. Briefly, detergent- regel 5 solubilized MHC class II molecules (3 µM) were incubated with 100 nM of biotinylated marker regel 6 peptide (72-85) and nonbiotinylated competitor peptides (dose range 0-128 μM) for 48 hours regel 7 at room temperature in the presence of a protease inhibitor mix. The MHC-peptide complexes regel 8 were analyzed using nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. regel 9 Following Western Blotting (ECL GST Wester blotting detection ; Amersham, Arlington regel 10 Heights, IL), the biotinylated peptides were visualized on preflashed Hyperfilm (Amersham) regel 11 through enhanced chemiluminescence (Amersham). regel 12 regel 13 T cell proliferation assay regel 14 Primed lymph node cells and splenocytes were cultured­ in triplicate (200µl per well) in 96- regel 15 well, round-bottomed plates (Costar, Cambridge, MA) at 2 x 105 cells per well, with or without regel 16 antigen. Lymph node cells and splenocytes were tested for proliferation to individual peptides regel 17 at 2, 10 and 20 µg/ml. Concanavalin A (ConA; 2.5 µg/ml) was used as a positive control for regel 18 T cell prolifera­tion. Cultures were incubated for 96 h at 37o C in a humidified atmosphere of regel 19 3 3 regel 20 5% CO2 and pulsed for the final 16 hours with H-thymidine ( H-TdR; Amersham) 0.4 µCi/well, specific activity 1 Ci/mmol. TdR uptake was measured using a liquid scintillation beta counter. regel 21 The magnitude of the proliferative res­ponse was expressed as stimulati­on index (SI): the mean regel 22 counts per minute (cpm) of cells cultured with antigen, divided by the mean cpm of cells regel 23 cultured with medium alone. regel 24 Inhibition of adjuvant arthritis by a peptide analog a peptide by arthritis adjuvant of Inhibition regel 25 Cytokine neutralization assays 45 regel 26 Lymph node cells were cultured as described above, in the presence of the following regel 27 anticytokine antibodies: anti-pan TGFβ (polyclonal Rabbit; R&D systems, Minneapolis, MN), regel 28 anti-IL-4 (polyclonal rabbit, Biosource, Floris, Belgium), anti-IL-10 (polyclonal rabbit, Biosource) regel 29 or anti–Interferonγ (polyclonal Rabbit, Biosource). All neutralizing antibodies were used regel 30 at a concentration of 5 μg/ml according to the manufacturers’ instructions (R&D Systems; regel 31 Biosource). Proliferation was measured as described above. regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 Determination of IL-2 production regel 2 IL-2 in culture supernatants was determined by the ability to stimulate proliferation of the regel 3 IL-2-dependent CTLL-16 line. Briefly, supernatants were collected after culturing lymph regel 4 node cells or spleen cells for 24 hours with or without antigen. Supernatants were serially regel 5 diluted in 96-well, round-bottomed plates in culture medium supplemented with 10% FCS. regel 6 2 x 104 CTLL-16 cells were added to each well, and the plates were incubated for 24 hours at regel 7 370C. Subsequently, cultures were pulsed for 16 hours with 3H-TdR, and TdR incorporation was regel 8 measured. regel 9 regel 10 Determination of TGFß production regel 11 TGFß was measured in culture supernatants of lymph node cells or splenocytes after 48 hours of regel 12 culture with or without antigen, using a capture enzyme-linked immunosorbent assay (ELISA) regel 13 as previously described18, with minor modifications. Briefly, 96-well, flat-bottomed ELISA regel 14 microtiter plates (Costar) were coated for 1 hour at room temperature with 100 μl monoclonal regel 15 mouse anti-human TGF-β1,2,3 antibody (Genzyme, Cambridge, MA). After coating, plates were regel 16 washed 3 times with wash buffer (phosphate buffered saline [PBS] containing 0.005% Tween regel 17 20), blocked for 2 hours at room temperature with 100 μl/well PBS containing 0.05% Tween 20 regel 18 and 1% bovine serum albumine (BSA; Sigma), and washed again 3 times. Culture supernatants regel 19 (100 μl) were added, serially diluted in blocking buffer (PBS with 0.05% Tween and 1% BSA), regel 20 and incubated overnight at 4oC. The plates were washed 3 times and incubated for 2 hours regel 21 at room temperature with 100 μl of biotinylated chicken anti-human TGFβ1 (2 μg/ml; R&D regel 22 Systems). The plates were washed 4 times and 1:40,000 diluted streptavidin-horseradish regel 23 peroxidase (HRP) in blocking buffer (100 μl/well) was added for 1 hour at room temperature. regel 24 The plates were washed 4 times with tap water and incubated at room temperature with 100 regel 25 3 Chapter μl/well HRP substrate solution (Genzyme) before absorbency was read at 450 nm on an ELISA regel 26 46 reader. regel 27 regel 28 Intracellular cytokine staining regel 29 Lymph node cells were cultured for 24 or 48 hours with medium or antigen. During the last regel 30 4 hours of culture 1μM monensin (GolgiStop; Pharmingen, San Diego, CA) was added to the regel 31 cultures. Viable cells were harvested, incubated for 20 minutes on ice in blocking buffer (PBS regel 32 with 10% normal rat serum and 0.02% 1M sodium azide), and subsequently stained for 20 regel 33 minutes on ice with phycoerythrin (PE) or fluorescein isothyocyanate (FITC)- conjugated anti- regel 34 rat CD4 (clone OX-35, mouse IgG2a,κ; Pharmingen). The cells were washed twice in staining regel 35 buffer (PBS containing 2% FCS and 0.02% 1M sodium azide) and resuspended in 100 μl of regel 36 fixation buffer (Cytofix/Cytoperm; .Pharmingen) for 20 minutes on ice. The fixed cells were washed twice in permeabilization buffer (Perm/Wash; Pharmingen), resuspended in 100 μl of regel 1 permeabilization buffer, and stained with the following conjugated monoclonal antibodies: PE- regel 2 conjugated anti-rat IL-4 (clone OX-81, mouse IgG1,κ; Pharmingen), PE-conjugated anti-rat IL-10 regel 3 (clone A5-4, IgG2b; Pharmingen) and with FITC-conjugated anti-rat IFNγ (clone DB.1, mouse regel 4 IgG1; Harlan). regel 5 Finally, the cells were washed twice and resuspended in staining buffer. PE- or FITC-conjugated regel 6 anti-mouse IgG1 (clone MOPC-21; Pharmingen) and PE-conjugated mouse IgG2b (Caltag, South regel 7 San Francisco, CA) were used as isotype controls. As a second specificity control, fixed and regel 8 permeabilized cells were incubated for 30 minutes with non conjugated anticytokine antibody regel 9 (the same clone as the conjugated anticytokine antibody) prior to the actual staining with regel 10 the conjugated anticytokine antibody. This preincubation procedure blocked the staining to regel 11 background level. Stained cells were analyzed on a FACScan cytometer (Becton Dickinson, regel 12 Mountain View, CA). At least 5,000 events were acquired from each sample and subsequently regel 13 analyzed with Lysis II and CellQuest software (Becton Dickinson). regel 14 regel 15 Peptide immunotherapy protocols regel 16 Pretreatment (vaccination) protocol regel 17 Rats were lightly anesthetized using ether, and 100 μg of peptide dissolved in PBS was regel 18 administered nasally by micropipette, in a total volume of 10 μl (5 μl per nostril; peptide regel 19 concentra­ti­on 10 mg/ml). Peptide was administered on day -15, -11, -7 and -3 prece­ding regel 20 induction of arthritis. regel 21 Treatment protocol regel 22 As treatment, the same dose, 100 μg of peptide dissolved in 10 μl PBS, was administered regel 23 nasally. Treatment was started when >50% of the rats demonstrated weight loss (i.e., at the regel 24 time of onset of clinical arthritis). Nasal administration of peptide was repeated 3 times, at analog a peptide by arthritis adjuvant of Inhibition regel 25 3-day intervals. 47 regel 26 regel 27 Induction and clinical assessment of AIA regel 28 AIA was induced by administering 0.5 mg M tuberculosis suspended in 100 μl of IFA as a single regel 29 intradermal injection in the base of the tail. Rats were examined daily, in a blinded manner, for regel 30 clinical signs of arthritis. The severity of arthritis was determined by assessing weight loss regel 31 and scoring each paw (0-4 scale) based on the degree of swelling, erythema and deformation­ regel 32 of the joints (maximum arthritis score 16)19. regel 33 regel 34 regel 35 regel 36 regel 1 Adoptive transfer of protection regel 2 Donor rats were treated nasally with alanine 183 or OVA 323-339, 100 μg of peptide regel 3 dissolved in 10 μl PBS, 4 times with 3-4-day intervals. Splenocytes were harvested 3 days regel 4 after administration of the last dose, and lymphocytes were isolated by a Ficoll gradient. In regel 5 order to activate T cells, the cells (5x106/ml) were cultured for 48 hours in medium with 2% regel 6 normal rat serum and 2 μg/ml ConA (according to a method adapted from Miller et al20). After regel 7 48 hours, viable cells were harvested using a Ficoll gradient and 1x108 cells were injected regel 8 intraveneously into naïve recipient rats. Simultaneously, recipient rats were immunized with regel 9 0.5 mg M Tuberculosis in IFA to induce AIA. regel 10 regel 11 Statistical Analysis regel 12 The Mann-Whitney U-Test was used to compare arthritis scores and weight loss between the regel 13 different groups. regel 14 regel 15 regel 16 RESULTS regel 17 regel 18 Effects of nasal administration of Hsp60 peptides 176-190 and 180-188, and regel 19 the peptide analog alanine 183 in the prevention of AIA regel 20 In a previous study we showed that nasal administration of Hsp60 peptide 176-190 delays the regel 21 onset and moderately suppresses the severity of arthritis in AIA and in avridine arthritis10. The regel 22 first goal of the current study was to attempt to increase the suppressive effect in AIA using regel 23 alanine 183, a modulatory peptide analogue of the Hsp60 180-188 core epitope recognized by regel 24 the arthritogenic T cell clone A2b. Figure 1 shows the MHC class II (RT1.Bl) binding affinities regel 25 3 Chapter of the peptides used in this study. Peptides Hsp60 176-190 and OVA 323-339 are strong RT1. regel 26 48 Bl binders, while the Hsp60 180-188 core epitope is an intermediate binder. The alanine-to- regel 27 leucine substitution in Hsp60 180-188, peptide alanine 183, resulted in an increased MHC regel 28 binding affinity. regel 29 Peptides Hsp60 176-190, 180-188, alanine 183 and OVA 323-339 were given nasally on days regel 30 -15, -11, -7 and -3 prior to induction of AIA. On day 0, rats were immunized with M Tuberculosis regel 31 to induce arthritis. The results of a representative experiment are shown in Figure 2. Rats that regel 32 were nasally exposed to Hsp60 180-188 showed a delayed onset of arthritis (mean time of regel 33 onset in Hsp60 180-1880-treated rats 14 days after immunization compared with 12 days regel 34 after immunization in OVA 323-339-treated rats), and had a lower arthritis score (mean± SEM regel 35 maximum score 5.0± 2.0 for Hsp60 180-188 treated rats versus 8.2±2.1 for control rats regel 36 treated with OVA 323-339). A similar, modes arthritis suppressive effect was demonstrated with Hsp60 176-190 (mean time of onset day 14, mean ± SEM maximum arthritis score 5.2±1.8). regel 1 The differences in mean maximum arthritis scores between the 3 groups (Hsp60 180-188, regel 2 Hsp60 176-190 and OVA 323-339) did not reach statistical significance in this experiment. regel 3 The effect of nasal administration of alanine 183, however, was much more pronounced (mean regel 4 time of onset day 17; mean ± SEM maximum arthritis score 1.0 ± 0.7). The difference in the regel 5 mean maximum arthritis score compared with that of each of the other groups was significant regel 6 (P < 0.05). Weight curves (a sensitive measure of physical well-being) are shown in Figure 2. regel 7 The mean ± SEM weight gain on day 21 (the day of the maximum arthritis score) compared regel 8 with that on day 10 (before the onset of arthritis; see above) was 18 ± 9 grams for rats regel 9 treated with alanine 183, compared with mean weight loss of 13 ± 15 grams in rats treated regel 10 with Hsp60 180-188 18 ± 19 grams in rats treated with Hsp60 176-190 and 48 ± 11 grams regel 11 in rats treated with OVA 323-339 (P<0.05). This experiment was repeated 3 times with similar regel 12 results. regel 13 regel 14   regel 15 regel 16 regel 17  regel 18 regel 19 regel 20  regel 21 regel 22 regel 23 regel 24

 analog a peptide by arthritis adjuvant of Inhibition regel 25 49 regel 26 regel 27 regel 28  regel 29  regel 30 Figure 1. regel 31 Binding of heat shock protein 60 (Hsp60) 180-188, alanine 183, Hsp60 176-190, and ovalbumin (OVA) regel 32  323-339 to rat major histocompatibility complex (MHC) class II (RT1.Bl) molecules. Detergent-solubilized regel 33 MHC class II molecules were incubated with the biotinylated marker peptide myelin basic protein 72-85 regel 34 and unlabeled competitor peptides (dose range 0-128 μM). Peptide binding was analyzed by Western regel 35 blotting as described in Materials and Methods. regel 36 regel 1  regel 2 regel 3  regel 4   regel 5  regel 6  regel 7  regel 8   regel 9  regel 10  regel 11 regel 12  regel 13        regel 14  regel 15  regel 16  regel 17  regel 18    regel 19  regel 20   regel 21  regel 22  regel 23  regel 24   regel 25 3 Chapter 

regel 26 50   regel 27  regel 28        regel 29  regel 30 Figure 2. regel 31 Modulation of adjuvant induced arthritis (AIA) development, as assessed by arthritis score regel 32 and body weight change, after nasal administration of mycobacterial Hsp60 peptides 176- regel 33 190, 180-188, alanine 183, or OVA 323-339. Rats were treated on days –15, -11, -7, and –3. regel 34 On day 0, rats were immunized with 0.5 mg Mycobacterium tuberculosis in 100 μl Freund’s regel 35 incomplete adjuvant to induce AIA. There were 5 rats in each treatment group. Values are the regel 36 mean and SEM. See Figure 1 for other definitions. Effects of nasally administered alanine 183 in the treatment of AIA regel 1 Most successful immune interventions in AIA have used preventive designs. Succesful regel 2 treatment after the onset of AIA is much more difficult. We investigated whether nasally regel 3 administered alanine 183 could also be used to treat ongoing arthritis. Rats were immunized regel 4 with M Tuberculosis to induce AIA were weighed daily, beginning on day 8. Treatment was regel 5 started on day 12 after arthritis induction (i.e., at the time of onset of clinical arthritis), when regel 6 > 50% of rats had lost weight. Treatment was also administered on days 15, 18 and 22. The regel 7 results of a representative experiment (1 of 3) are shown in Figure 3. regel 8 Treatment with alanine 183 had a significant (P < 0.05) arthritis suppressive effect (mean regel 9 ± SEM maximum arthritis score 2.0 ± 1.5, compared with 7.3 ± 2.2 in controls treated with regel 10 PBS) (Figure 3). Treatment with OVA 323-339 did not influence the course of disease (mean regel 11 ± SEM maximum arthritis score 6.6 ± 1.6). There was no difference in the incidence of regel 12 arthritis between treatment groups (4 of 5 alanine 183-treated rats; 4 of 5 PBS-treated rats; 5 regel 13 of 5 OVA-treated rats). Figure 3 shows the weight curves of rats in the same experiment. After regel 14 treatment was started, rats receiving alanine 183 regained the weight they previously lost, regel 15 whereas control rats receiving OVA 323-339 or PBS kept loosing weight until day 27. Again, the regel 16 differences in weight change between alanine 183 group and the 2 control groups reached regel 17 statistical significance (P < 0.05). These results show that nasal administration of alanine 183 regel 18 can interfere in an ongoing arthritis process. regel 19 regel 20 Passive transfer of protection induced by nasally administered alanine 183 regel 21 To determine whether the protective effect of nasally administered alanine 183 was mediated regel 22 via active immune regulation, we investigated whether the protection was passively regel 23 transferable. In this experiment, donor rats were treated nasally with either alanine 183 or OVA regel 24 323-339, as described above. Three days after the final dose was administered, splenocytes analog a peptide by arthritis adjuvant of Inhibition regel 25 were harvested, and activated in vitro with Con A. After 48 hours, viable cells were injected 51 regel 26 intraveneously into naïve recipient rats. Simultaneously, the recipient rats were immunized regel 27 with M Tuberculosis in IFA to induce AIA. The results of 1 of 3 representative experiments are regel 28 shown in Figure 4. regel 29 After passive transfer, rats that received activated splenocytes from alanine 183-treated regel 30 donor rats had a significantly lower mean maximum arthritis score compared with recipient regel 31 rats receiving activated splenocytes from OVA 323-339 treated donor rats (mean ± SEM 1.1 regel 32 ± 0.7 versus 8.2 ± 2.1; P < 0.05). Likewise, the alanine 183-treated recipient rats had a regel 33 more favorable weight curve than did the OVA 323-339-treated recipient rats (Figure 4). These regel 34 findings indicate that the protection found after nasal administration of alanine 183 is cell- regel 35 mediated, ruling out clonal deletion of the arthritogenic A2b-like cells as the main mechanism regel 36 of protection.   regel 1  regel 2  regel 3  regel 4   regel 5   regel 6   regel 7  regel 8    regel 9  regel 10   regel 11  regel 12         regel 13  regel 14         regel 15   regel 16    regel 17   regel 18   regel 19    regel 20    regel 21    regel 22    regel 23   regel 24   regel 25 3 Chapter

   regel 26 52  regel 27 

        regel 28   regel 29 Figure 3.      regel 30 Effects of treatment of ongoing adjuvant induced arthritis (AIA) with nasally administered alanine 183  regel 31 or ovalbumin (OVA) 323-339, as assessed by arthritis score and body weight change. On day 0, rats   regel 32 were immunized with 0.5 mg Mycobacterium tuberculosis in Freund’s incomplete adjuvant to induce AIA. regel 33 Starting on day 8, rats were weighed daily. Treatment was started when >50% of the rats had lost weight regel 34 (i.e., onset of clinical arthritis). At that time, rats were randomized into 3 treatment groups: no treatment,  regel 35 nasal OVA 323-339, or nasal alanine 183 (n=5 in each treatment group). Treatment was repeated 3 times regel 36 at 3-day intervals. Values are the mean and SEM. PBS = phosphate buffered saline.  regel 1 regel 2

 regel 3 regel 4 regel 5   regel 6 regel 7   regel 8 regel 9  regel 10  regel 11 regel 12

 regel 13        regel 14  regel 15  regel 16  regel 17

 regel 18  regel 19  regel 20 regel 21   regel 22   regel 23  regel 24

Inhibition of adjuvant arthritis by a peptide analog a peptide by arthritis adjuvant of Inhibition regel 25  53 regel 26  regel 27  regel 28       regel 29  regel 30 regel 31  Figure 4. regel 32 Adoptive transfer of protection induced by nasal administration of alanine 183, as assessed by arthritis regel 33 score and body weight change. Donor rats were given 4 nasal treatments with alanine 183 or ovalbumin regel 34 (OVA) 323-339 (n=5 in each treatment group). Values are the mean and SEM. AIA= adjuvant induced regel 35 arthritis. regel 36 regel 1 Lymphocyte proliferation and IL-2 production after nasal administration of regel 2 peptides regel 3 To analyze the T cell response induced by nasal administration of peptide, we first measured regel 4 antigen specific T cell proliferation and IL-2 production. Rats were treated nasally with either regel 5 alanine 183 or OVA 323-339. Three days after the final dose was administered, splenocytes and regel 6 mandibular lymph node cells were harvested and restimulated with peptide. IL-2 production in regel 7 supernatants of cells was measured after 24 hours of culture, and proliferation was measured regel 8 after 96 hours of culture. The results of a representative experiment (1 of 4) are shown in Table regel 9 1. After nasal administration of alanine 183, no antigen specific proliferation (SI < 2), or IL-2 regel 10 production (SI < 2) could be detected in mandibular lymph node cells and splenocytes. regel 11 regel 12 Table 1. Lymphocyte proliferation and IL-2 production after nasal administration of peptides regel 13 regel 14 Cell In vivo treatment and Proliferation IL-2 regel 15 source in vitro stimulation (S.I.) (S.I.) regel 16 Spleen Alanine 183 1.2 1.1 regel 17 MLN Alanine 183 1.4 <1 Spleen OVA 323-339 1.1 1.8 regel 18 MLN OVA 323-339 2.8 1.3 regel 19 regel 20 Rats were treated on days -15,-11,-7 and -3 prior to the experiment. Cells from mandibular lymph regel 21 nodes (MLNs; 3 rats) and splenocytes (3 rats) were cultured with or without antigen. OVA = ovalbumin; regel 22 stimulation index (SI) = the mean cpm of cells cultured with antigen, divided by the mean cpm of cells regel 23 cultured with medium alone. regel 24 regel 25 3 Chapter Antigen induced cytokine production after nasally induced tolerance regel 26 54 Next, we analyzed the antigen specific production of TGFβ, IL-4, IL-10 and IFNγ induced by regel 27 nasal immunization with peptide. Rats were treated nasally with either alanine 183 or OVA regel 28 323-339, as described above. Three days after the final dose was administered, mandibular regel 29 lymph node cells were harvested and cultured with alanine 183 or OVA 323-339. In a second regel 30 set of experiments, nasal induction of tolerance was followed by arthritis induction with using regel 31 M Tuberculosis. In these experiments, both mandibular and inguinal lymph node cells were regel 32 harvested 10 days after induction of arthritis and cultured with Hsp60 176-190, alanine 183, regel 33 OVA 323-339, or medium alone. regel 34 regel 35 regel 36 TGFß production regel 1 After 48 hours of culture, supernatants were collected, and TGFβ production was determined regel 2 using a capture ELISA. The results of a representative experiment (1 of 4) are shown in Table regel 3 2. After nasal administration of both alanine 183 and OVA 323-339, mandibular lymph node regel 4 cells produced TGFβ in an antigen specific manner. However, if nasally induced tolerance was regel 5 followed by induction of arthritis, inguinal lymph node cells from rats treated with OVA 323-339 regel 6 produced no detectable levels of TGFβ. In contrast, inguinal lymph node cells from rats treated regel 7 with alanine 183 did produce TGFβ after in vitro stimulation with both alanine 183 and the wild- regel 8 type epitope Hsp60 180-188. In all studies, Con A was used as a positive control for cytokine regel 9 production. No significant differences in cytokine production between the 2 treatment groups regel 10 was found following Con A activation. regel 11 regel 12 Table 2. Production of transforming growth factor β (TGFβ) after nasal administration of regel 13 peptides regel 14 regel 15 Cell Source In vivo treatment Induction of AIA In vitro stimulation Production of TGFβ regel 16 (pg/ml) regel 17 MLN Alanine 183 No Alanine 183 276 regel 18 MLN OVA 323-339 No OVA 323-339 3732 regel 19 ILN Alanine 183 Yes Alanine 183 72 ILN OVA 323-339 Yes OVA 323-339 *Below regel 20 ILN Alanine 183 Yes 180-188 103 regel 21 ILN OVA 323-339 Yes 180-188 *Below regel 22 Rats were treated on days -15,-11,-7 and -3 prior to the experiment. At day 0, mandibular lymph node regel 23 cells (MLNs; 3 rats) were isolated and cultu­red with or without antigen. In a second set of experiments, regel 24 nasally induced tolerance was followed by induction of adjuvant induced arthritis (AIA). Ten days after analog a peptide by arthritis adjuvant of Inhibition regel 25 induction of AIA, inguinal lymph node cells (ILNs; 3 rats) were isolated and cultured with or without 55 regel 26 antigen. TGFβ = transforming growth factor β; OVA = ovalbumin. *Below the assay’s limit of detection. regel 27 regel 28 Intracellular detection of IL-4, IL-10, and IFNγ regel 29 To measure intracellular cytokine production of IL-4, IL-10 and IFNγ, mandibular lymph regel 30 node cells were isolated after nasal induction of tolerance and cultured with alanine 183 or regel 31 medium alone. After 48 hours, viable cells were harvested and stained for surface markers regel 32 and intracellular cytokines. The results are shown in Figure 5. Antigen specific intracellular regel 33 cytokine production was expressed as the percentage of double-positive cells (both CD4+ and regel 34 cytokines) in samples cultured with antigen minus the percentage of double-positive cells in regel 35 samples cultured with medium alone. After nasal induction of tolerance with alanine 183 and regel 36 regel 1 in vitro activation with alanine 183, a significant number of CD4+ mandibular lymph node cells regel 2 produced IL-10 (mean ± SEM 9.9 ± 4.7%; P < 0.05 compared with OVA 323-339-restimulated regel 3 cells), and IL-4 (mean ± SEM 2.5 ± 1.1%; P < 0.05 compared with OVA 323-339-restimulated regel 4 cells), but did not produce IFNγ (Figure 5A). As an additional control we used mandibular regel 5 lymph node cells from rats treated nasally wih OVA 323-339. In vitro activation of those cell regel 6 with alanine 183 did not lead to antigen specific cytokine production (Figure 5A). regel 7 When nasal induction of tolerance was followed by induction of arthritis, the same pattern of regel 8 cytokine production was observed in inguinal lymph node cells (ILNs), not only upon in vitro regel 9 restimulation with alanine 183 but also upon in vitro stimulation with Hsp60 176-190 (figure regel 10 5B). Again, ILN cells from rats treated with OVA 323-339 were used as controls and did not regel 11 show significant cytokine production following in vitro activation with either alanine 183 or regel 12 Hsp60 176-190 (Figure 5B). Thus, nasally induced tolerance using alanine 183 leads to an regel 13 antigen specific increase of IL-10, IL-4 and TGFβ production in draining lymph nodes both at regel 14 the site of tolerance induction and at the site of inflammation. regel 15 regel 16 Effects of cytokine neutralization on T cell responsiveness after tolerance regel 17 induction regel 18 To unravel the role of the different cytokines in the regulation of tolerance, we blocked the regel 19 effect of cytokines in vitro using anti-cytokine antibodies. As a readout system, we used regel 20 antigen specific lymphocyte proliferation toward Hsp60 176-190. At the site of tolerance regel 21 induction (mandibular lymph node cells), we tested the effect of anticytokine antibodies on regel 22 the specific lymphocyte proliferation for Hsp60 176-190 after nasal induction of tolerance regel 23 followed by induction of arthritis (Figure 6). Both anti IL-10 and anti TGFb led to restoration of regel 24 the proliferative response to Hsp60 176-190 in mandibular lymph node cells from rats treated regel 25 3 Chapter with alanine 183. regel 26 56 Similarly, we tested the effect of anticytokine antibodies on the specific lymphocyte regel 27 proliferation for peptide Hsp60 176-190 at the stie of inflammation, the inguinal lymph node regel 28 cells (Figure 6). Again, anti-IL-10 significantly enhanced the response to Hsp60 176-190 in regel 29 rats treated with alanine 183, but other anticytokines did not influence the proliferative regel 30 response. In contrast, in rats treated with OVA 323-339, none of the anticytokines influenced regel 31 the proliferative response to Hsp60 176-190 in ILN cells. regel 32 regel 33 regel 34 regel 35 regel 36    regel 1   regel 2 regel 3    regel 4 regel 5 regel 6 regel 7    regel 8 regel 9 regel 10 A regel 11 regel 12      regel 13 regel 14 regel 15    regel 16  regel 17 regel 18    regel 19 regel 20 regel 21 B regel 22 Figure 5. regel 23 Intracellular cytokine production of interleukin-4 (IL-4), IL-10, and interferon-γ (IFNγ) after nasal regel 24 induction of tolerance with alanine 183. A. Experiments with mandibular lymph node cells (MLNs). MLNs analog a peptide by arthritis adjuvant of Inhibition regel 25 were isolated from alanine 183-treated rats and cultured with alanine 183 or medium alone. After 48 57 regel 26 hours, viable cells were harvested and stained for surface markers and intracellular cytokines. As a regel 27 regel 28 control, MLNs were obtained from ovalbumin (OVA) 323-339-treated rats and cultured in a similar manner, with alanine 183 or medium alone. B. Experiments with inguinal lymph node cells (ILNs). Nasal regel 29 induction of tolerance with either alanine 183 or OVA 323-339 was followed by induction of arthritis with regel 30 Mycobacterium tuberculosis. Ten days after immunization, ILNs were isolated and cultured with heat regel 31 shock protein 60 176-190 or medium. After 48 hours, viable cells were harvested and stained for surface regel 32 markers and intracellular cytokines. Antigen specific intracellular cytokine production was expressed regel 33 as the percentage of double-positive cells for both CD4 and cytokine in cells cultured with antigens, regel 34 subtracted by the percentage of double-positive cells in cell samples cultured with medium alone. The regel 35 gates for analysis were set on the isotype controls of the different antibodies. regel 36 regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13  regel 14  Figure 6. regel 15 Effects of anticytokine antibodies on the proliferative response to 176-190 in mandibular lymph node regel 16 cells (MLNs) and inguinal lymph node cells (ILNs) after nasal induction of tolerance with alanine 183  regel 17 followed by induction of arthritis with Mycobacterium tuberculosis. Anti-IL-10 = anti- interleukin-10; regel 18 anti-TGF = anti-transforming growth factor. Stimulation index (SI) = the mean cpm of cells cultured with regel 19 antigen, divided by the mean cpm of cells cultured with medium alone. regel 20  regel 21             regel 22                 regel 23 regel 24  regel 25 3 Chapter regel 26 58 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36

    DISCUSSION regel 1 regel 2 We previously reported that in the experimental model of AIA, nasal administration of regel 3 mycobacterial Hsp60 peptide 176-190 induces antigen specific T cell tolerance and a moderate regel 4 suppression of arthritis10. In the present study we show that peptide analog alanine 183 has regel 5 an arthritis suppressive effect that is superior to that of the wild-type epitope, and that this regel 6 analog peptide can even be used to treat ongoing arthritis. regel 7 Other studies have shown that T cell epitope analogs can selectively change the phenotype of regel 8 the T cell response and thereby can interfere in T cell-mediated autoimmune diseases13;21;22. regel 9 Alanine 183 is a previously defined modulatory analog peptide of the Hsp60 180-188 epitope regel 10 recognized by the arthritogenic T cell clone A2b, and its affinity for rat MHC class II is higher regel 11 13;14 regel 12  than that of the wild type epitope . The strong arthritis suppressive effect of alanine 183 seen after nasal administration is not, however, the consequence of its increased MHC-binding regel 13  affinity, because the longer wild type epitope Hsp60 176-190, which has an even higher MHC- regel 14  binding affinity for rat MHC class II than does alanine 183, is less effective in preventing regel 15 arthritis10;23. regel 16  Although results of previous studies suggested otherwise24, the finding that the tolerance- regel 17 inducing capacity of a given peptide can not be explained solely by its MHC binding affinity is regel 18 in line with a more recent study in experimental autoimmune encephalomyelitis (EAE)25. This regel 19  indicates that apart from its increased MHC-binding affinity, Alanine 183 must have additional regel 20            characteristics that contribute to the strong arthritis-suppressive effect. In EAE, it has been regel 21 regel 22                shown that immunization with T cell epitope analogs can lead to activation of a different population of cells capable of inducing bystander suppression26;27. This could indicate that regel 23  nasal administration of alanine 183 does not induce a phenotypical and functional change in regel 24 A2b-like cells, but, instead, induces a new population of alanine 183-specific cells. analog a peptide by arthritis adjuvant of Inhibition regel 25 In the second part of the study, we analyzed the mechanism of nasal induction of tolerance 59 regel 26 using alanine 183. First, we were able to passively transfer protection from animals treated regel 27 nasally with alanine 183 to untreated naive animals via activated spleen cells. This result regel 28 indicates that the protection is an active cell mediated process and rules out the possibility regel 29 of clonal deletion of the arthritogenic A2b-like cells as the main mechanism of nasal induction regel 30 of tolerance. In support of this evidence, we found no indications for increased apoptosis as regel 31 measured by TUNEL assay and annexin V staining (data not shown). regel 32 Finally, we showed that after nasal administration of peptide and in vitro antigen specific regel 33 restimulation, mandibular lymph node cells neither proliferated nor produced significant regel 34 levels of IL-2 and IFNγ, while TGFβ, IL-4, and, at a high level, IL-10 could be detected. When regel 35 nasal induction of tolerance was followed by induction of arthritis, a similar cytokine pattern regel 36

    regel 1 was demonstrated in lymph nodes draining the site of disease induction. Interestingly, this regel 2 cytokine pattern was found not only upon in vitro restimulation with alanine 183 but also upon regel 3 restimulation with the wild type epitope, Hsp60 176-190. Neutralization of the effects of these regel 4 cytokines with anticytokine antibodies in vitro showed that both anti-IL-10 and anti-TGFβ could regel 5 reverse the suppressed Hsp60 176-190 response in mandibular lymph node cells. However, regel 6 at the site of inflammation (the inguinal lymph node cells), only anti-IL-10 could reverse this regel 7 state of tolerance. These findings suggest that although TGFβ is certainly involved, IL-10 is of regel 8 decisive importance for the maintenance of tolerance. This does not necessarily mean that regel 9 the in vivo maintenance of tolerance is completely dependent on IL-10. In a similar model of regel 10 nasal induction of tolerance in BALB/c mice, both local and systemic administration of anti- regel 11 IL-10 failed to abrogate nasal tolerance28. regel 12 Similar to oral tolerance, 3 distinct mechanisms (e.g. clonal deletion, clonal anergy, and active regel 13 suppression) of nasal tolerance have been proposed9;10;25;29-32. Previous studies attributed regel 14 suppression to a switch to Th2 or Th3 cytokines33;34, but other studies failed to reveal such a regel 15 change 25;35 or found the switch to be independent of tolerance induction28. The cytokine profile regel 16 of T cells generated after nasal induction of tolerance with alanine 183 also showed Th2 and regel 17 Th3 characteristics, namely production of IL-10, IL-4 and TGFβ. However, we demonstrated that regel 18 only IL-10 plays an important role in the maintenance tolerance at the site of inflammation. regel 19 Previously, Groux et al showed in a transgenic mouse system that chronic activation of CD4+ regel 20 T cells in the presence of IL-10 resulted in the generation of Tr1 cells15. Such in vitro-induced regel 21 Tr1 cells are characterized by a low proliferative capacity and secretion of IL-10 and TGF-β. The regel 22 low proliferative response and the cytokine profile, with high levels of IL-10 found after nasal regel 23 induction of tolerance with alanine 183, showed a remarkable resemblance to the pattern of regel 24 such Tr1 cells, suggesting the induction of Tr1-like cells in vivo. The critical importance of IL- regel 25 3 Chapter 10 in the regulation of autoimmune diseases was also shown in studies of EAE and collagen regel 26 60 induced arthritis (CIA), in which IL-10 deficient mice developed more severe disease36 than regel 27 did IL-4 knockout or wild-type mice, whereas IL-10 transgenic mice appeared to be resistant regel 28 to CIA37. regel 29 These findings and our present data suggest that IL-10 plays a dominant role in the regulation regel 30 of peripheral tolerance and in nasally induced tolerance. However, the strong protective regel 31 effects of the regulatory T cells in vivo should not be attributed solely to IL-10. Other soluble regel 32 mediators, such as TGFβ and IL-4, or direct cell-cell contact may ultimately determine the regel 33 clinical efficacy. regel 34 regel 35 regel 36 Reference List regel 1 regel 2 1. Metzler B, Wraith DC. Inhibition of experimental autoimmune encephalomyelitis by inhalation regel 3 but not oral administration of the encephalitogenic peptide: influence of MHC binding affinity. regel 4 Int.Immunol. 1993;5:1159-1165. regel 5 2. Hoyne GF, O’Hehir RE, Wraith DC, Thomas WR, Lamb JR. Inhibition of T cell and antibody regel 6 responses to house dust mite allergen by inhalation of the dominant T cell epitope in naive regel 7 and sensitized mice. J.Exp.Med. 1993;178:1783-1788. regel 8 3. Higgins PJ, Weiner HL. Suppression of experimental autoimmune encephalomyelitis by oral regel 9 administration of myelin basic protein and its fragments. J.Immunol. 1988;140:440-445. regel 10 4. Thompson HS, Staines NA. Gastric administration of type II collagen delays the onset and regel 11 severity of collagen-induced arthritis in rats. Clin.Exp.Immunol. 1986;64:581-586. regel 12 5. Zhang ZJ, Davidson L, Eisenbarth G, Weiner HL. Suppression of diabetes in nonobese diabetic regel 13 mice by oral administration of porcine insulin. Proc.Natl.Acad.Sci.U.S.A 1991;88:10252- regel 14 10256. regel 15 6. Daniel D, Wegmann DR. Protection of nonobese diabetic mice from diabetes by intranasal or regel 16 subcutaneous administration of insulin peptide B-(9-23). Proc.Natl.Acad.Sci.U.S.A regel 17 1996;93:956-960. regel 18 7. Joosten LA, Coenen-de Roo CJ, Helsen MM et al. Induction of tolerance with intranasal regel 19 administration of human cartilage gp-39 in DBA/1 mice: amelioration of clinical, histologic, regel 20 and radiologic signs of type II collagen-induced arthritis. Arthritis Rheum. 2000;43:645-655. regel 21 8. Staines NA, Harper N, Ward FJ et al. Mucosal tolerance and suppression of collagen-induced regel 22 arthritis (CIA) induced by nasal inhalation of synthetic peptide 184-198 of bovine type II regel 23 collagen (CII) expressing a dominant T cell epitope. Clin.Exp.Immunol. 1996;103:368-375. regel 24 9. Wauben MH. Immunological mechanisms involved in experimental peptide immunotherapy of analog a peptide by arthritis adjuvant of Inhibition regel 25 T-cell-mediated diseases. Crit Rev.Immunol. 2000;20:451-469. 61 regel 26 10. Prakken BJ, Van Der ZR, Anderton SM et al. Peptide-induced nasal tolerance for a mycobacterial regel 27 heat shock protein 60 T cell epitope in rats suppresses both adjuvant arthritis and regel 28 nonmicrobially induced experimental arthritis. Proc.Natl.Acad.Sci.U.S.A 1997;94:3284-3289. regel 29 11. Holoshitz J, Naparstek Y, Ben-Nun A, Cohen IR. Lines of T lymphocytes induce or vaccinate regel 30 against autoimmune arthritis. Science 1983;219:56-58. regel 31 12. Van Eden W, Thole JE, Van Der ZR et al. Cloning of the mycobacterial epitope recognized by T regel 32 lymphocytes in adjuvant arthritis. Nature 1988;331:171-173. regel 33 13. Wauben MH, Boog CJ, Van Der ZR et al. Disease inhibition by major histocompatibility complex regel 34 binding peptide analogues of disease-associated epitopes: more than blocking alone. J.Exp. regel 35 Med. 1992;176:667-677. regel 36 regel 1 14. Wauben MH, Van Der ZR, Joosten I et al. A peptide variant of an arthritis-related T cell epitope regel 2 induces T cells that recognize this epitope as a synthetic peptide but not in its naturally regel 3 processed form. J.Immunol. 1993;150:5722-5730. regel 4 15. Groux H, O’Garra A, Bigler M et al. A CD4+ T-cell subset inhibits antigen-specific T-cell responses regel 5 and prevents colitis. Nature 1997;389:737-742. regel 6 16. Anderton SM, Van Der ZR, Noordzij A, van EW. Differential mycobacterial 65-kDa heat shock regel 7 protein T cell epitope recognition after adjuvant arthritis-inducing or protective immunization regel 8 protocols. J.Immunol. 1994;152:3656-3664. regel 9 17. Joosten I, Wauben MH, Holewijn MC et al. Direct binding of autoimmune disease related T cell regel 10 epitopes to purified Lewis rat MHC class II molecules. Int.Immunol. 1994;6:751-759. regel 11 18. Miller A, al-Sabbagh A, Santos LM, Das MP, Weiner HL. Epitopes of myelin basic protein that regel 12 trigger TGF-beta release after oral tolerization are distinct from encephalitogenic epitopes and regel 13 mediate epitope-driven bystander suppression. J.Immunol. 1993;151:7307-7315. regel 14 19. Trentham DE, Townes AS, Kang AH. Autoimmunity to type II collagen an experimental model of regel 15 arthritis. J.Exp.Med. 1977;146:857-868. regel 16 20. Miller A, Lider O, Roberts AB, Sporn MB, Weiner HL. Suppressor T cells generated by oral regel 17 tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by regel 18 the release of transforming growth factor beta after antigen-specific triggering. Proc.Natl. regel 19 Acad.Sci.U.S.A 1992;89:421-425. regel 20 21. Das MP, Nicholson LB, Greer JM, Kuchroo VK. Autopathogenic T helper cell type 1 (Th1) and regel 21 protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin regel 22 proteolipid protein. J.Exp.Med. 1997;186:867-876. regel 23 22. Nicholson LB, Waldner H, Carrizosa AM et al. Heteroclitic proliferative responses and changes regel 24 in cytokine profile induced by altered peptides: implications for autoimmunity. Proc.Natl. regel 25 3 Chapter Acad.Sci.U.S.A 1998;95:264-269. regel 26 62 23. Wauben MH, van der KM, Grosfeld-Stulemeyer MC, Joosten I. Definition of an extended MHC regel 27 class II-peptide binding motif for the autoimmune disease-associated Lewis rat RT1.BL regel 28 molecule. Int.Immunol. 1997;9:281-290. regel 29 24. Liu GY, Wraith DC. Affinity for class II MHC determines the extent to which soluble peptides regel 30 tolerize autoreactive T cells in naive and primed adult mice--implications for autoimmunity. regel 31 Int.Immunol. 1995;7:1255-1263. regel 32 25. Anderton SM, Wraith DC. Hierarchy in the ability of T cell epitopes to induce peripheral tolerance regel 33 to antigens from myelin. Eur.J.Immunol. 1998;28:1251-1261. regel 34 26. Nicholson LB, Murtaza A, Hafler BP, Sette A, Kuchroo VK. A T cell receptor antagonist peptide regel 35 induces T cells that mediate bystander suppression and prevent autoimmune regel 36 encephalomyelitis induced with multiple myelin antigens. Proc.Natl.Acad.Sci.U.S.A 1997;94:9279-9284. 27. Smeltz RB, Wauben MH, Wolf NA, Swanborg RH. Critical requirement for aspartic acid at regel 1 position 82 of myelin basic protein 73-86 for recruitment of V beta 8.2+ T cells and regel 2 encephalitogenicity in the Lewis rat. J.Immunol. 1999;162:829-836. regel 3 28. Wolvers DA, van der Cammen MJ, Kraal G. Mucosal tolerance is associated with, but regel 4 independent of, up-regulation Th2 responses. Immunology 1997;92:328-333. regel 5 29. Chen Y, Inobe J, Marks R et al. Peripheral deletion of antigen-reactive T cells in oral tolerance. regel 6 Nature 1995;376:177-180. regel 7 30. Weiner HL. The mucosal milieu creates tolerogenic dendritic cells and T(R)1 and T(H)3 regel 8 regulatory cells. Nat.Immunol. 2001;2:671-672. regel 9 31. Bayrak S, Mitchison NA. Bystander suppression of murine collagen-induced arthritis by long- regel 10 term nasal administration of a self type II collagen peptide. Clin.Exp.Immunol. 1998;113:92- regel 11 95. regel 12 32. Chen Y, Kuchroo VK, Inobe J, Hafler DA, Weiner HL. Regulatory T cell clones induced by oral regel 13 tolerance: suppression of autoimmune encephalomyelitis. Science 1994;265:1237-1240. regel 14 33. Myers LK, Seyer JM, Stuart JM, Kang AH. Suppression of murine collagen-induced arthritis by regel 15 nasal administration of collagen. Immunology 1997;90:161-164. regel 16 34. Tian J, Atkinson MA, Clare-Salzler M et al. Nasal administration of glutamate decarboxylase regel 17 (GAD65) peptides induces Th2 responses and prevents murine insulin-dependent diabetes. regel 18 J.Exp.Med. 1996;183:1561-1567. regel 19 35. Hoyne GF, Jarnicki AG, Thomas WR, Lamb JR. Characterization of the specificity and duration regel 20 of T cell tolerance to intranasally administered peptides in mice: a role for intramolecular regel 21 epitope suppression. Int.Immunol. 1997;9:1165-1173. regel 22 36. Bettelli E, Das MP, Howard ED et al. IL-10 is critical in the regulation of autoimmune regel 23 encephalomyelitis as demonstrated by studies of IL-10- and IL-4-deficient and transgenic regel 24 mice. J.Immunol. 1998;161:3299-3306. analog a peptide by arthritis adjuvant of Inhibition regel 25 37. Johansson AC, Hansson AS, Nandakumar KS, Backlund J, Holmdahl R. IL-10-deficient B10.Q 63 regel 26 mice develop more severe collagen-induced arthritis, but are protected from arthritis induced regel 27 with anti-type II collagen antibodies. J.Immunol. 2001;167:3505-3512. regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 3 Chapter regel 26 64 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Modulation of T cell function by combination of epitope specific and low4 dose anticytokine therapy controls autoimmune arthritis

Sarah T.A. Roord1,2,3,5, Evelien Zonneveld-Huijssoon3,5, Tho Le1,2,5, Gisella Puga Yung1,2,5, Eva Koffeman1,2,5, Arash Ronaghy1, Negar Ghahramani4, Paola Lanza4, Rosario Billetta4, Berent J. Prakken3,5 and Salvatore Albani1,2,4,5.

Departments of Medicine1 and Pediatrics2, University of California San Diego, La Jolla, CA, USA 3Department of Pediatric Immunology, University Medical Center Utrecht, Wilhelmina Children’s Hospital, Utrecht, The Netherlands 4Androclus Therapeutics, San Diego, CA, USA 5IACOPO project, EUREKA Institute for Translational Medicine, Siracusa, Italy

PLoS ONE. 2006 Dec 20;1:e87. regel 1 ABSTRACT regel 2 regel 3 Innate and adaptive immunity contribute to the pathogenesis of autoimmune arthritis by regel 4 generating and maintaining inflammation, which leads to tissue damage. Current biological regel 5 therapies target innate immunity, eminently by interfering with single pro-inflammatory regel 6 cytokine pathways. This approach has shown excellent efficacy in a good proportion of regel 7 patients with Rheumatoid Arthritis (RA), but is limited by cost and side effects. Adaptive regel 8 immunity, particularly T cells with a regulatory function, plays a fundamental role in controlling regel 9 inflammation in physiologic conditions. A growing body of evidence suggests that modulation regel 10 of T cell function is impaired in autoimmunity. Restoration of such function could be of regel 11 significant therapeutic value. We have recently demonstrated that epitope specific therapy regel 12 can restore modulation of T cell function in RA patients. Here, we tested the hypothesis that regel 13 a combination of anti-cytokine and epitope specific immunotherapy may facilitate the control regel 14 of autoimmune inflammation by generating active T cell regulation. This novel combination regel 15 of mucosal tolerization to a pathogenic T cell epitope and single low dose anti-TNFα was regel 16 as therapeutically effective as full dose anti-TNFα treatment. Analysis of the underlying regel 17 immunological mechanisms showed induction of T cell immune deviation. regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 4 Chapter regel 26 66 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 INTRODUCTION regel 1 regel 2 Much progress has recently been achieved on our knowledge of the immunological and regel 3 molecular mechanisms, which lead to amplification, and perpetuation of autoimmune regel 4 inflammation. This progress has been translated into a generation of biologic therapeutic agents regel 5 that target pro-inflammatory cytokines, with the aim of interfering with their mechanism of regel 6 action. This approach is destined to progressively complement and, in some cases, replace regel 7 currently used immunosuppressive and anti-inflammatory therapies. Despite their success1;2, regel 8 current anti-cytokine approaches remain hampered with limitations associated eminently regel 9 with generalized immunosuppression and subsequent increased occurrence of malignancies regel 10 and infectious diseases, in particular tuberculosis3-6. regel 11 Conceptually, therapeutic intervention focused on modulation of T cell function leads to the regel 12 promise of higher specificity and lower toxicity7-16. This objective has for long remained a regel 13 challenge in humans, particularly due to the difficulty of identifying means of intervention that regel 14 could affect T cell function in a specific fashion. regel 15 In a Phase I/IIa trial, we have recently described immunological effects of epitope specific regel 16 immunotherapy in a group of patients with rheumatoid arthritis. The epitope employed was regel 17 derived from the heat shock protein (HSP) dnaJ. We have proposed a central role for HSP- regel 18 specific T cell responses in the physiologic mechanisms of modulation of inflammation17-20. We regel 19 have also suggested impairment of such modulation as one of the mechanisms of amplification regel 20 of autoimmune inflammation21-24. Our treatment sought to restore such control by inducing regel 21 mucosal tolerization to a peptide with a potential pathogenic, not necessarily etiologic role25. regel 22 Immunological effects of the treatment consisted of immunodeviation from pro-inflammatory regel 23 to tolerogenic type T cell responses to the peptide employed in the treatment. Restoration of regel 24 regulatory T cell activity was also observed. therapy specific epitope and anticytokine of therapy Combination regel 25 Effects of anti cytokine therapy on T cell function, both effector and regulatory, have been 67 regel 26 suggested13;26-28. These interactions are relevant for many different reasons, including regel 27 ultimately the design of an optimal biologic therapy based on the combination of anti-cytokine regel 28 and T cell epitope specific approaches. regel 29 The work presented here lays the foundation for this strategy by exploring clinical and regel 30 immunological effects of the combination of epitope specific T cell and anti-cytokine therapy. regel 31 We employed for this purpose Adjuvant Arthritis (AA). This is an experimental form of arthritis regel 32 that is T cell dependent and can be passively transferred by a T cell clone that is specific for the regel 33 180-188 amino acid sequence of mycobacterial HSP6029;30. In previous studies we showed that regel 34 nasal administration of a 15-mer peptide (176-190) encompassing this arthritogenic epitope regel 35 leads to T cell tolerance31 and can prevent AA. Treatment with nasal administration of peptide regel 36 regel 1 180-188 after the induction of AA is mildly effective. Here, we compared immunological and regel 2 clinical effects of different dose regimens, namely full dose anti-TNFα, which is known to be regel 3 effective32, mucosal tolerization to the peptide alone, anti-TNFα at one third of the effective regel 4 dose, and the combination of low dose anti-TNFα and epitope specific therapy. regel 5 We found that the combination of low dose anti-TNFα associated with mucosal tolerization regel 6 to the arthritogenic T cell epitope led to a significant reduction of arthritis clinically as well regel 7 as histologically, to a degree entirely comparable with what was achieved with full dose anti- regel 8 TNFα. Interestingly, treatment regimens differed for their influence on immune responses. regel 9 Indeed, combination therapy induced T cells with a regulatory phenotype, consisting of regel 10 CD4+CD25+ cells producing IL-10 and expressing FOXP3. Combination treatment also induced regel 11 immune deviation in CD4+CD25- cells, which were found producing IL-10, as well as IL-4. Such regel 12 changes were not present in the full dose anti-TNFα therapy group. regel 13 Our data provide a compelling rationale for testing the combination of anti-cytokine and epitope regel 14 specific immunotherapy in human autoimmune disease. regel 15 regel 16 regel 17 METHODS regel 18 regel 19 Animals regel 20 Male inbred Lewis rats (RT1B) were obtained from Harlan (Indianapolis). Rats were 6-9 weeks regel 21 old at the start of each experiment. regel 22 regel 23 Antigens and adjuvants regel 24 Heat killed Mycobacterium tuberculosis (Mt, strain H37Ra) was obtained from Difco (Detroit, regel 25 4 Chapter MI). Incomplete Freund’s Adjuvant (IFA; Difco, Detroit, MI) was used as adjuvant. The peptide regel 26 68 used in this study was prepared in large quantities by standard solid phase Fmoc chemistry. It regel 27 was obtained as COOH terminal amide and was analyzed and purified by reverse-phase HPLC. regel 28 The following peptide was used: Mycobacterium tuberculosis HSP60 180-188, containing regel 29 Mycobacterium tuberculosis HSP60 sequence 180-188 (TFGLQLELT). 180-188 is recognized regel 30 by the arthritogenic T cell clone A2b and is a dominant T cell epitope found after Adjuvant regel 31 Arthritis (AA) and after immunization with mycobacterial HSP60. regel 32 regel 33 Induction and Clinical Assessment of Experimental Arthritis regel 34 Rats were lightly anesthetized using isoflurane and AA was induced by a single intradermal regel 35 (i.d.) injection in the base of the tail with 0.1 mg Mycobacterium tuberculosis (Mt) suspended regel 36 in 100 µl of IFA (Complete Freund’s Adjuvant; CFA). Rats were examined daily for clinical signs of arthritis in a blinded set-up. Severity of arthritis was assessed by scoring each paw from regel 1 zero to four based on degree of swelling, erythema and deformation of the joints. Thus the regel 2 maximum score was 16. On day 23 after the induction of arthritis the rats were sacrificed by regel 3 CO2 inhalation, after which mandibular lymph nodes (MLN), Inguinal Lymph nodes (ILN), regel 4 spleen and hind limb joints were collected. regel 5 regel 6 Immunotherapy Protocols regel 7 Rats were lightly anesthetized using metofane for all nasal treatments or isoflurane for all regel 8 subcutaneous treatments. Etanercept (Enbrel®, Wyeth) was administered subcutaneously regel 9 (s.c.) at a concentration of 0.3 mg/kg per rat dissolved in 100 µl PBS using a 25-gauge needle. regel 10 This was done on day 9 after the induction of arthritis with Mt. Some rats in control groups regel 11 received additional Etanercept on day 11 and 13. 100 µg of peptide dissolved in PBS was regel 12 administered nasally in a total volume of 10 µl (5 µl per nostril, peptide concentration 10 mg/ regel 13 ml) using a micropipette. This was done on day 10, 13, 16, 19 after arthritis induction with Mt. regel 14 regel 15 Adoptive transfer regel 16 MLN, ILN and spleen of 2-3 rats per group after combination treatment with Etanercept and regel 17 180-188 were harvested on day 23 after the induction of arthritis. Cells were cultured in vitro regel 18 with 2.5 µg/ml con A for 48 hours. Subsequently, 13x106 MLN, 11x106 ILN and 11x106 spleen regel 19 cells were injected i.v. into the tail vein of rats one week after induction of arthritis with Mt. regel 20 Rats were subsequently examined daily for clinical signs of arthritis in a blinded set-up as regel 21 described previously. regel 22 regel 23 Histological assessment of hind limb joints regel 24 Hind limb joints were collected on day 23 after the induction of arthritis, after the rats were therapy specific epitope and anticytokine of therapy Combination regel 25 sacrificed by CO2 inhalation. Formalin-fixed tissues were decalcified, and glass slides stained 69 regel 26 with H&E and Safranin O (for cartilage) were prepared and evaluated by standard methodology regel 27 (Comparative Biosciences, Inc.). The pathologist examined all of the submitted tissue sections regel 28 in a blinded fashion by light microscopy and scored for inflammation of the synovium, pannus regel 29 formation, cartilage damage, inflammation of the bone marrow and periostal proliferation. regel 30 Each of these parameters was scored for 10 days, severity from 0 (normal) to 4 (severe). A regel 31 cumulative score was given based upon the sum of all of the parameters measured. regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 Intracellular Cytokine Staining regel 2 MLN cells were cultured for 72 hours with medium alone or antigen. During the last 4 hours regel 3 of culture 1 M monensin (GolgiStop®, Pharmingen, San Diego, CA) was added. Viable cells regel 4 were harvested and washed with FACS blocking buffer (PBS with 10% FBS) and 0.03% 1 M regel 5 sodium azide) and subsequently stained for 30 minutes on ice in 100 µl of blocking buffer regel 6 with the following conjugated monoclonal antibodies for extracellular antigens: PE, FITC regel 7 or CY-conjugated anti-rat CD4 (clone OX-35, mouse IgG2a), FITC-conjugated anti-rat CD25 regel 8 (clone OX-39, mouse IgG1) (BD Pharmingen, San Diego CA). The cells were washed twice in regel 9 staining buffer (PBS containing 3% FBS and 0.03% 1M sodium azide) and resuspended in regel 10 100 µl fixation buffer (Cytofix/Cytoperm®, BD Pharmingen, San Diego, CA) for 20 minutes on regel 11 ice. The cells were washed twice in permeabilization buffer (Perm/Wash®, Pharmingen, San regel 12 Diego CA) and resuspended in 100 µl permeabilization buffer and stained with the following regel 13 conjugated monoclonal antibodies: PE-conjugated anti-rat IL-4 (clone OX-81, mouse IgG1), regel 14 PE-conjugated anti-rat IL-10 (clone A5-4, mouse IgG2b), PE-conjugated anti-rat TNFα (clone regel 15 TN3.-19.12, hamster IgG) and PE-conjugated anti-mouse CTLA-4 (anti CD152) (clone UC10- regel 16 4F10-11, armenian hamster IgG, group 1κ) (all antibodies from Pharmingen, San Diego, CA). regel 17 The appropriate isotype controls were used. Finally, the cells were washed twice, resuspended regel 18 in staining buffer, and transferred to FACS tubes for analysis. Stained cells were analyzed on regel 19 a FACStar Plus cytometer (Becton and Dickinson). At least 5.000 events were acquired from regel 20 each sample and subsequently analyzed with Lysis II software. regel 21 regel 22 Sorting of CD4+CD25+ and CD4+CD25- after magnetic bead separation regel 23 MLN were incubated for 15 hours with medium or antigen. Viable cells were harvested and first regel 24 the cell suspensions were depleted of monocytes, phagocytes, NK cells and B cells by magnetic regel 25 4 Chapter bead separation using the CELLection®Biotin Binder kit (Dynal A.S. Oslo, Norway). In brief, cells regel 26 70 were incubated with the following monoclonal antibodies: biotin mouse anti rat mononuclear regel 27 phagocyte, (C17, Pharmingen), biotin mouse anti rat CD161a (10/78, Pharmingen) and biotin regel 28 mouse anti rat CD45RA (OX-33, Caltag Laboratories). Positive selection was performed using regel 29 streptavidin coated magnetic Dynabeads using the Dynal Magnetic Particle Concentrator. regel 30 The thus obtained cells were washed in FACS blocking buffer and stained extracellularly with regel 31 anti rat CD4 and anti rat CD25. Subsequently, cells were sorted by FACS (FACS Vantage, Beckton regel 32 Dickinson San Jose, CA) into CD4+CD25+ and CD4+CD25- cells. regel 33 regel 34 regel 35 regel 36 Real Time Quantitative PCR (Taqman) regel 1 MLN were incubated for 15 hours with medium or antigen. Cells were sorted into CD4+CD25+ regel 2 cells and CD4+CD25- cells as described above, resuspended in Lysis buffer (Qiagen,Valencia, regel 3 CA) and frozen at –80 °C until analysis. mRNA was extracted from sorted cells by using regel 4 RNeasy Mini Kit (Qiagen). mRNA was then reverse-transcribed into cDNA with an oligo dT regel 5 primer (Oligo(dT)12-18, Invitrogen). Subsequently, single stranded cDNA was amplified with regel 6 the cytokine specific forward and reverse primer sets for GAPDH (housekeeping gene), IL- regel 7 10, TNFαand FOXP3. mRNA levels were determined by Real Time Quantitative PCR on an ABI regel 8 PRISM® 7000 thermal cycler (Perkin Elmer). The following combinations of primers and probes regel 9 were used: IL-10 Forward 5’GCC TGG CTC AGC ACT GCT AT 3’, IL-10 Reverse 5’CGG ATG GAA TGG regel 10 CCT TTG 3’, IL-10 Probe-FAM 5’ TTG CCT GCT CTT ACT GGC TGG AGT GAA 3’. TNFα Forward 5’ACA regel 11 AGG CTG CCC CGA CTA C 3’, TNFα Reverse 5’TCC TGG TAT GAA ATG GCA AAC C 3’, TNFα Probe-JOE regel 12 5’TGC TCC TCA CCC ACA CCG TCA GC 3’. FOXP3 Forward 5’CCA TTG GTT CAC ACG CAT GT 3’, FOXP3 regel 13 Reverse 5’TGG CGG ATG GCA TTC TTC 3’, FOXP3 Probe-JOE 5’CGC CTA CTT CAG AAA CCA CCC 3’. regel 14 GAPDH Forward 5’TGA CTC TAC CCA CGG CAA GTT 3’, GAPDH Reverse 5’TTC CCG TTG ATG ACC AGC TT regel 15 3’, GAPDH Probe-FAM 5’ACG GCA CAG TCA AGG CTG AGA ATG G 3’. regel 16 To quantify the amount of mRNA for the different target genes the standard curve method was regel 17 used33.The relative amounts of target gene and GAPDH were quantified by a linear extrapolation regel 18 of the Ct values using the equation to the line obtained from the standard curve of the respective regel 19 target genes. Data were normalized for target gene expression, which was obtained by dividing regel 20 the relative quantity of target gene for each sample divided by the relative quantity of GAPDH regel 21 for the same sample. The final read outs are expressed as induction index (arbitrary units) regel 22 defined as stimulated subtracted by reference condition,i.e. only media culture. regel 23 regel 24 Statistical analysis therapy specific epitope and anticytokine of therapy Combination regel 25 A two tailed, paired t-test was performed to compare clinical scores on day 23 and to compare 71 regel 26 Area under the arthritis score curve. Kolmogorov-Smirnov Statistics were applied for statistical regel 27 analysis of FACS histograms. regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 RESULTS regel 2 regel 3 Combination of epitope specific therapy and a single low dose of Etanercept regel 4 (Enbrel®) has clinical efficacy comparable to full dose Etanercept in controlling regel 5 Adjuvant Arthritis regel 6 Lewis rats were immunized with 100 µg Mt to induce AA and randomly divided into 5 treatment regel 7 groups: i) no treatment; ii) three doses of Etanercept s.c., equivalent in this model to a full regel 8 course of Etanercept treatment; iii) one dose of Etanercept s.c.; iv) four nasal administrations regel 9 of HSP60 peptide 180-188; v) combination of one dose of Etanercept s.c. followed by four nasal regel 10 treatments with HSP60 peptide 180-188. Three independent experiments were performed, regel 11 with 6 rats per treatment group. The lowest effective dose of Etanercept was determined in regel 12 preliminary experiments (not shown). Two different parameters were employed to measure regel 13 clinical outcomes, in order to ascertain full evaluation of the effects of the various treatment regel 14 regimens: i) mean arthritis scores on day 23 (the day of maximum arthritis severity); ii) area regel 15 under the curve of the corresponding arthritis score curves, thus taking into consideration the regel 16 whole time course of the treatment. regel 17 A significant reduction of AA mean arthritis scores on day 23 (p=0.0004) was achieved with regel 18 epitope specific and low single dose Etanercept combination treatment as well as with a full regel 19 course of Etanercept therapy (p=0.004) compared to no treatment. Similarly, when assessing regel 20 the areas under the curve (AUC) of the corresponding arthritis score curves, a significant regel 21 decrease of AA was seen after epitope specific and low single dose Etanercept combination regel 22 treatment (p=0.02 vs. no treatment). Comparable disease control was achieved in the full regel 23 dose Etanercept treatment group (p=0.03 vs. no treatment). regel 24 One dose of Etanercept alone on day 9 was able to suppress arthritis only temporarily; however, regel 25 4 Chapter as expected, after day 17 the arthritis revived (Day 23 p=0.3, AUC p=0.1 vs. no treatment). regel 26 72 Treatment with HSP60 peptide 180-188 alone showed a trend towards reduction of arthritis, regel 27 without achievement of statistically significant differences (Day 23 p=0.07, AUC p=0.26 vs. regel 28 no treatment). Combination of treatment with an irrelevant peptide, derived from Ovalbumin, regel 29 and low dose Etanercept lacked efficacy in suppressing arthritis, thus confirming the epitope regel 30 specificity of the treatment (Figure 1 and Table 1). regel 31 Hence, regardless of the outcome parameter employed, combination of epitope specific and regel 32 low single dose Etanercept therapy enabled complete clinical control of the arthritic process regel 33 to a degree statistically comparable with full dose Etanercept, a therapeutic regimen known regel 34 to fully control AA. regel 35 regel 36        regel 1  regel 2   regel 3  regel 4  regel 5   regel 6   regel 7  regel 8  regel 9  regel 10  regel 11  regel 12  Figure 1. Combination therapy of Etanercept with mycobacterial heat shock protein 60 regel 13  regel 14  (HSP60) 180-188 led to significant reduction of Adjuvant Arthritis (AA). regel 15  Arthritis was induced on day 0 and day 21 with Complete Freund’s Adjuvant (CFA). On day 9, rats were randomly divided  into  five  treatment  groups:  three doses  of Etanercept   s.c. on  day  9, 11, 13 (equivalent regel 16 to a full course of Etanercept treatment); one dose of Etanercept on day 9; four doses of mycobacterial regel 17  HSP60 peptide 180-188 on day 10, 13, 16, 19; combination treatment of one dose of Etanercept s.c. regel 18  on day 9 followed by 180-188 nasally on day 10, 13, 16, 19; or no treatment (PBS). Arthritis scores regel 19 were assessed every other day from day 8 onward. N=15-18 rats per treatment group. Shown are mean regel 20 arthritis scores. regel 21 regel 22 Epitope specific and low single dose Etanercept combination therapy leads to regel 23 a decrease of damage in the hind limb joints regel 24 Combination therapy of anticytokine and epitope specific therapy specific epitope and anticytokine of therapy Combination regel 25  Next we investigated if clinical control of AA with combination therapy was matched in the same treatment groups by a corresponding decrease in joint destruction by the arthritic process. 73 regel 26 Hind limb joints were collected on day 23 after the induction of arthritis and scored for severity regel 27 of inflammation in the synovium, pannus formation, cartilage damage, inflammation of the regel 28 bone marrow and periostal proliferation, with a maximum total score of 20. regel 29 Epitope specific and single low dose Etanercept combination therapy led to a significant regel 30 improvement of the histological score in the joints (p=0.014 vs. untreated). Similarly, full regel 31 course of anti-TNFα therapy led to a significant decrease of histological damage (p=0.001 vs. regel 32 no treatment). Single dose of Etanercept did not lead to significant improvement (p=0.214) regel 33 (Figure 2). regel 34 regel 35 regel 36 regel 1 Table 1 Combination treatment as well as a full course Etanercept treatment led to significant regel 2 reduction of arthritis on day 23, when maximum score of disease is reached, as well as a regel 3 significant reduction of the area under the arthritis score curve (AUC), representative of the regel 4 whole treatment period. regel 5 regel 6 TREATMENT GROUPS Mean arthritis Arthritis score on day 23 Area under the AUC vs. no treat- regel 7 score on day 23 vs. no treatment (p-value) Curve (AUC) ment (p-value) regel 8 Etanercept 3x 5.8 0.004 42.639 0.03 Etanercept 1x 8.3 n.s. 48.858 n.s. regel 9 180-188 4x 7.1 n.s. 54.726 n.s. regel 10 Etanercept 1x + 180- 4.9 0.0004 40.396 0.02 regel 11 188 4x regel 12 Etanercept 1x + 9.0 n.s. 79.597 n.s. OVA 4x regel 13 No treatment 9.8 77.74 8 regel 14 regel 15 Statistical analysis was performed by the paired t-test. Different treatment groups were set out against regel 16 no treatment. regel 17  regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 4 Chapter regel 26 74 regel 27 regel 28   regel 29  Figure 2. Combination therapy as well as a full course of Etanercept treatment led to reduction regel 30 of histological damage in the ankle joints.  regel 31 Joints were harvested on day 23 after the induction of arthritis. Formalin-fixed tissues were decalcified, regel 32 and glass slides stained with H&E were prepared. Submitted tissue sections were examined by light regel 33  microscopy and scored for severity of inflammation of the synovium, pannus formation, cartilage regel 34 damage, bone marrow inflammation and periostal proliferation, with a maximum score of 4 per parameter. regel 35 N=3-4 per treatment group. H&E staining is shown of one rat per treatment, representative for the whole regel 36 treatment group. A: Combination therapy; B: Etanercept 1x; C: Peptide Mt. 180-188 4x monotherapy; D: No treatment; E: Etanercept 3x. (For colour image see addendum)

  Epitope specific and single low dose Etanercept combination therapy induces regel 1 immune deviation of CD4+ T cells regel 2 We then analyzed the immune mechanism responsible for the clinical effects of the treatments regel 3 tested. We focused in this part of our analysis on defining qualitatively CD4+ mediated T cell regel 4 responses to the inciting antigen. The rationale behind this strategy was to identify qualitative regel 5 changes in cytokine responses induced by the treatment. To this end, we measured cytokine regel 6 production and surface marker expression of CD4+ T cells present in the Mandibular regel 7 Lymphnodes (MLN), the draining site of the nasal mucosa where T cell immune deviation may regel 8 be induced. MLN were isolated on day 23 after arthritis induction and cultured with HSP60 regel 9 peptide 180-188 or media. It has to be noted that HSP60 peptide 180-188 is the major immune regel 10 dominant epitope following induction of AA (due to the presence of mycobacterial HSP65 in regel 11 CFA) and thus can act as an important surrogate parameter for (immune) therapy in AA34;35. regel 12 After 72 hours viable cells were harvested and stained for surface markers and intracellular regel 13 cytokines and analyzed by FACS. The results showed differences between treatment groups in regel 14 the immune mechanisms underlying sometimes comparable clinical efficacy. In fact, only the regel 15 epitope specific/low dose Etanercept combination treatment group showed clear indication of regel 16 T cell immune deviation, as indicated by the significant difference compared to the untreated regel 17 group as well as the Etanercept groups in the increased production of IL-10 regulatory cytokine regel 18 (MFI 11.84, isotype control 7.69, p<0.001). Expression of CTLA-4, a marker of T cells with regel 19 regulatory function, also significantly increased when compared both to untreated and regel 20 Etanercept groups (MFI 18.16, isotype control 7.10, p<0.001). An increase in IL-4 production regel 21 was seen after the combination treatment as well as after the full course of Etanercept therapy regel 22 (MFI 8.20 and 10.19, isotype control 6.96, p<0.001) (Figure 3). regel 23 These data are, in our opinion, intriguing as they show differences in underlying immunological regel 24 mechanism between two clinically equally effective treatments. Indeed, the marked increases therapy specific epitope and anticytokine of therapy Combination regel 25 in IL-10 production and CTLA-4 expression following combination therapy were both strongly 75 regel 26 suggestive of restored modulation of T cell function. regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 Figure 3. The combination therapy of Etanercept and HSP60 180-188 led to an antigen specific regel 19 increase of IL-10 and IL-4 production and up regulation of CTLA-4 expression in CD4+ T cells in regel 20 draining Mandibular Lymphnodes (MLN). regel 21 MLN were harvested on day 23 after the induction of arthritis. Cells were cultured for 72 hours with regel 22 medium or antigen. Intracellular production of IL-4, IL-10, and expression of CTLA-4 were measured by regel 23 FACS. Depicted are Mean Fluorescence Indexes (MFI) of MLN cells cultured with mycobacterial HSP60 regel 24 peptide 180-188, of cells gated on CD4. Results are representative of one experiment. (For colour image regel 25 4 Chapter see addendum) regel 26 76 regel 27 Enhancement of CD4+CD25+ regulatory T cell (Treg) function by epitope regel 28 specific/low dose anticytokine combination therapy regel 29 In this part of the project, we addressed the questions on: i) whether certain aspects of regel 30 regulatory T cell (Treg) function were affected by the combination therapy; ii) whether such regel 31 induction would affect immune deviation in effector T cells; iii) whether differences in these regel 32 parameters between full dose Etanercept and epitope specific and low single dose Etanercept regel 33 combination therapy could be found. regel 34 To this end, we chose to measure by real time PCR (TaqMan) expression of two functional regel 35 markers of Treg function: IL-10 and FOXP3. FOXP3 is a forkhead transcription factor whose regel 36 expression is deemed crucial for Treg function. IL-10 is considered among the most important soluble mediators for regulatory T cell function. We also measured expression of TNFα, to evaluate if the different therapeutic regimens had a direct effect on the inflammatory response regel 1 of effector CD4+CD25- cells. regel 2 CD4+CD25+ cells were studied, a category of Treg that appears functionally impaired in RA regel 3 and whose efficiency might not be entirely restored by full dose anti-TNFα treatment26. regel 4 CD4+CD25+ and CD4+CD25- MLN cells were isolated on day 23 after arthritis induction and regel 5 cultured with HSP60 peptide 180-188 or media. After 15 hours viable cells were harvested, regel 6 stained for CD4 and CD25 and sorted by FACS. Subsequently mRNA was extracted from sorted regel 7 CD4+CD25+ and CD4+CD25- cells and levels of FOXP3, IL-10 and TNFα measured by Real Time regel 8 Quantitative PCR. Figure 4 shows the ratio of the induction index (cytokine/transcription factor regel 9 divided by housekeeping gene GAPDH) of stimulation with HSP60 peptide 180-188 less the regel 10 background value. regel 11  regel 12  regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24

Combination therapy of anticytokine and epitope specific therapy specific epitope and anticytokine of therapy Combination regel 25 77 regel 26 regel 27 regel 28   regel 29 Figure 4. Combination therapy led to an increase in FOXP3 and IL-10 gene transcription in regel 30  regel 31 CD4+CD25+ cells,  whereas  it also   led to an increase  in IL-10 transcription   in CD4+CD25-  cells.  regel 32 TNFα transcription was abolished by the combination therapy  as well  as by a full course  of Etanercept treatment. regel 33  Results are expressed as the induction index (marker/housekeeping gene GAPDH) of HSP60 peptide 180- regel 34  188 stimulation subtracted by media alone as measured by Real Time Quantitative PCR. IL-10 production regel 35 Combination therapy vs. Etanercept treatment p=0.002. N= 2-4 per treatment. regel 36

     regel 1 When FOXP3 expression was measured, a significant increase was found both in the    regel 2  combination and full dose anti-TNFα groups, underscoring likely effects of both therapeutic   regel 3   regimens on some Treg functions, in accordance with recent findings in Rheumatoid Arthritis   regel 4 patients26. Cytokine mediated Treg function however might reportedly not be affected by anti-  regel 5 TNFα therapy, and indeed, when IL-10 expression by CD4+CD25+ cells was measured there    regel 6 was a significant increase (p=0.002) only in the combination therapy group. Interestingly, regel 7  combination therapy and, to a lesser degree, full dose Etanercept, also induced immune  regel 8 deviation of CD4+CD25- effector cells, with higher production of IL-10 consistent with what  regel 9 shown in the FACS analysis. As expected, FOXP3 expression was not induced in CD4+CD25-   regel 10 cells and TNFα expression was abolished by the combination treatment as well as by full dose regel 11 Etanercept.   regel 12               These data provide evidence for enhancement of Treg function by epitope specific and low  regel 13 dose Etanercept combination therapy. Enhanced or restored function of Treg led to immune  regel 14 deviation in effector CD4 cells, with production of IL-10. These immunological changes  regel 15 correlated with the changes in the clinical picture induced by the treatment.  regel 16  regel 17 Adoptive transfer of MLN T cells obtained from animals treated with epitope  regel 18 specific/low dose Etanercept combination therapy was able to treat full blown  regel 19 autoimmune arthritis regel 20 The purpose of this experiment was to evaluate whether the effects of the combination regel 21 therapy on T cells could induce clinical amelioration upon adoptive transfer into sick animals. regel 22 We employed T cells from spleen, Inguinal Lymphnodes (ILN) and MLN after combination regel 23 treatment with Etanercept and 180-188 on day 23 after the induction of arthritis, cultured regel 24 them for 48 hours with con A and subsequently injected them i.v. into the tail vein of rats one regel 25 4 Chapter week after arthritis induction with Mt. regel 26 78 Interestingly, only T cells derived from MLN of animals treated with the combination therapy regel 27 were able to significantly reduce (p=0.0305) clinical symptoms measured as mean arthritis regel 28 score, when transferred into animals in which disease had been induced (Figure 5-A). Spleen regel 29 cells from animals treated with combination therapy transferred to diseased animals failed regel 30 to exert an efficient control of the disease process (Figure 5-B) as measured using percent of regel 31 clinical amelioration. ILN were able to induce a good level of clinical amelioration (Figure 5-B), regel 32 however differences with no treatment control group did not reach statistical significance regel 33 (Figure 5-A). regel 34 Hence, epitope specific mucosal tolerization acts presumably on a population of T cells that   regel 35 resides in the lymph nodes draining the mucosa where the tolerization occurs (MLN) and to regel 36 a lesser degree ILN. The effect of the treatment on T cells is lasting enough to allow efficient control of the disease process by adoptive transfer in animals in which AA was induced.    regel 1     regel 2     regel 3  

 regel 4 regel 5    regel 6

  regel 7 regel 8 

 regel 9  regel 10

  regel 11               regel 12   regel 13  Figure 5. Adoptive transfer of Mandibular Lymph node (MLN) T cells from combination therapy regel 14  treated animals led to significant reduction of Adjuvant Arthritis (AA) in diseased animals, regel 15 measured as mean arthritis score as well as percentage of disease amelioration. regel 16 A. Adoptive transfer of T cells from combination therapy groups. Adoptive Transfer Groups received regel 17 11x106 Inguinal lymph node (ILN) cells, 13x106 MLN cells, or 11x106 spleen cells. Data represent Mean + regel 18 SD. Disease induction and scoring was performed as described in the legend to Figure 1. regel 19 B. Percentage of clinical amelioration for each treatment group in AA rats. The Area Under the Curve regel 20 (AUC) of each individual treatment group was used to score the Clinical Amelioration (CA) of the distinct regel 21 treatment groups. AUC was calculated using the curves originated by scoring the disease for the different regel 22 treatment groups and plotted as percentage of CA with respect to the non-treated group. The non-treated regel 23 group was considered as having an average percentage of disease = 100%. Formula is as follows: CA = regel 24 100 - %AUC. therapy specific epitope and anticytokine of therapy Combination regel 25 79 regel 26 regel 27 DISCUSSION regel 28 regel 29 Recent years have witnessed a dramatic progress in our ability to understand mechanisms regel 30 of autoimmune inflammation and to translate such understanding into novel therapeutic regel 31 approaches. Particularly remarkable is the success of therapies aimed at interfering with regel 32 the pro-inflammatory role played by certain cytokines, in particular TNFα. The broadening regel 33  of clinical applications employing anti-TNFα therapy has led, however, to two interesting  regel 34 developments in clinical immunology, including: i) the recognition of significant generalized regel 35 immune suppression in treated patients, with a sizable increase in onset or relapse of certain regel 36 regel 1 infectious diseases and neoplasias; ii) the need to understand in depth the effects of the regel 2 treatment on the immune system. regel 3 In fact, the effects of anti-TNFα treatment on cytokine production and immunoregulation are regel 4 still largely unknown and sometimes contradicting. Schotte et al. described the reduction regel 5 of the number of PBMC producing the pro-inflammatory cytokines TNFα, IFNγ and IL-1 after regel 6 Etanercept therapy, whereas the number of IL-10 producing PBMC remained the same, possibly regel 7 indicating an immune suppression rather than active immunomodulation due to Etanercept27. regel 8 Sieper and colleagues on the other hand investigated the effects of the treatment on the T regel 9 cell population and postulated that neutralization of peripheral TNFα by Etanercept does not regel 10 lead to a down regulation of the ability to produce TNFα or IFNγ by T cells, but rather to an up regel 11 regulation, possibly due to a counter regulatory mechanism28. Ehrenstein et al.26 found that regel 12 Treg function in RA is impaired, and that treatment with Infliximab, a monoclonal antibody regel 13 directed against TNFα, restored it only partially. Namely, Treg mechanisms based on cell- regel 14 to-cell contact were restored by Infliximab treatment, while Treg mechanisms relying on regel 15 soluble mediators such as IL-10 remained ineffective despite the treatment. A recent elegant regel 16 study by Valencia et al. added important insight into the role of TNFα on T regulatory cells. regel 17 They showed that CD4CD25bright T regulatory cells constitutively express the TNF receptor II. regel 18 An environment with high levels of circulating TNF led to up regulation of the TNF receptor II, regel 19 which down regulated both the quantity as well as the quality of FOXP3+ T regulatory cells. regel 20 Additionally they showed that CD4CD25bright cells of patients with active RA expressed high regel 21 levels of TNF receptor II, reduced levels of FOXP3 and were poor suppressors, which could be regel 22 reversed by anti- TNFα treatment13. These studies, at times contradicting with respect to some regel 23 mechanisms, underscored that short-term treatment with anti-TNFα may partially restore a regel 24 more tolerogenic microenvironment, which could be instrumental for the induction of immune regel 25 4 Chapter tolerance with epitope specific immune therapy. regel 26 80 Intervention on T cell mediated adaptive immunity would be, in theory, ideal, given the regel 27 possibility of focusing the approach on one or more possible antigens involved in the disease regel 28 process, thus sparing the patient generalized immune suppression. Progress is therefore regel 29 needed in the area of modulation, rather than suppression, of T cells. The most important regel 30 conceptual development may, however, be the fact that the search for the one inciting and regel 31 still unidentified antigen should be replaced by approaches targeting mechanisms of control regel 32 of self-reverberating T cell mediated inflammation. This would realistically shift the focus from regel 33 etiology to pathogenesis based immune modulation. regel 34 A considerable body of evidence, to which we contributed21;36;37, supports the concept that regel 35 peptides derived from heat shock proteins (HSP) may play a role in amplification of autoimmune regel 36 inflammation. As ubiquitous and bacterial derived products, HSP-derived peptides are in fact perceived as a “danger” signal and elicit a default pro-inflammatory physiologic response. regel 1 Such response contributes to clearing a possible pathogen invasion but also induces, regel 2 through cellular stress, increased availability of self-HSP derived peptides. These peptides regel 3 are recognized by T cells with regulatory function. Such function is impaired in autoimmune regel 4 arthritis17;18. regel 5 We have recently reported the results from a Phase I/IIa clinical trial in Rheumatoid Arthritis25. regel 6 The objective of our clinical intervention was to restore natural mechanisms of immune regel 7 modulation by exploiting the ability of the mucosal route in inducing tolerization to a HSP- regel 8 derived peptide, which we previously described as part of the pro-inflammatory mechanisms regel 9 of RA pathogenesis21. Interestingly, we were able to induce in treated patients immune deviation regel 10 from pro-inflammatory to modulatory T cell responses, leading to significant reduction in TNFα regel 11 and IFNγ production and increase in IL-10 and IL-4. These effects were mediated via restoration regel 12 of function of CD4CD25bright Treg, producing IL-10 and expressing FOXP325. regel 13 The study reported here addresses the questions on whether epitope specific and anticytokine regel 14 therapy can be complementary, and if such synergy may be advantageous in order to exploit regel 15 modulation of adaptive immunity while reducing generalized immune suppression, costs and regel 16 side effects. In order to explore the concept, we chose AA, a T cell, HSP-dependent model of RA, regel 17 which can be treated with full dose Etanercept. We have previously shown in AA that mucosal regel 18 tolerization to the inciting peptide leads to immune deviation31;38. regel 19 Combination of epitope specific and anti-cytokine therapy induced full clinical control of AA, to regel 20 a degree comparable to full dose Etanercept and significantly better than the other treatment regel 21 regimens, including low dose Etanercept or epitope specific therapy alone. The comparable regel 22 clinical efficacy achieved by combination treatment as well as full course Etanercept was regel 23 obtained through distinctly different immune mechanisms in both effector T cells as well as regel 24 regulatory T cells. therapy specific epitope and anticytokine of therapy Combination regel 25 In effector CD4 cells, the combination therapy induced immune deviation while full dose 81 regel 26 Etanercept appeared to be eminently suppressive. Combination therapy led to an increased regel 27 production of IL-10, which was not found in the other treatment regimens, including full dose regel 28 Etanercept. Both treatments induced suppression of TNFα production and an increase of IL-4 regel 29 production, which may indicate the presence of a T Helper 2 type tolerogenic mechanism regel 30 complementing the main effects of the therapy. regel 31 Increasing evidence is, however, shifting the focus of modulation of adaptive immunity from regel 32 effector to regulatory T cells39-43. Recent progress in Molecular Immunology has enabled the regel 33 identification of phenotypical and functional characteristics for these T cells, such as co- regel 34 expression of CD4, CD25 and CTLA-4, as well as production of certain regulatory cytokines. regel 35 Several mechanisms of actions have been proposed for Treg, based either on release of regel 36 regel 1 cytokines with a tolerogenic function (eminently IL-10), or based on direct cell-to-cell contact regel 2 by the use of receptors and pathways not yet fully elucidated7;39;44-46. regel 3 When we sought to analyze the effects of combination therapy and full dose Etanercept regel 4 therapy, it appeared evident that different functions of regulatory T cells were affected by regel 5 the two treatments. Both treatments significantly increased the expression of the forkhead regel 6 transcription factor FOXP3, a functional marker of Treg, which act eminently by cell-to-cell regel 7 contact. A similar observation was described in human RA by Ehrenstein et al.26. However, regel 8 CTLA-4 expression and IL-10 production were induced only by combination therapy regimens, regel 9 and not by anti-TNFα treatment alone, again in agreement with what was found by Ehrenstein. regel 10 Here we provide evidence that restoration of such function can be achieved by adding epitope regel 11 specific immunotherapy to low dose Etanercept. regel 12 Undoubtedly, anti-TNFα therapy creates an environment in which epitope specific regel 13 immunomodulation can be induced more efficiently. Further evidence for this concept was regel 14 recently obtained by Bresson et al., who showed that combination of peptide therapy with regel 15 anti CD3 enhanced the clinical improvement in experimental diabetes compared to anti- regel 16 inflammatory therapy alone, also through the induction of CD25+FOXP3+ Tregs, as well as regel 17 insulin specific Tregs producing IL-10 and TGFβ47. regel 18 Recently, it was elegantly shown by Zanin-Zhorov et al, that HSP60 peptides enhance regel 19 CD4+CD25+ regulatory T cell function via TLR2 signaling48, thereby providing an additional regel 20 explanation for the regulatory effects observed after combination therapy. regel 21 Further underscoring the profound immunological differences in mechanisms of action regel 22 between full dose Etanercept and combination therapy, only cells derived from MLN of regel 23 animals treated with combination therapy were able to control disease when transferred into regel 24 sick animals. Cells with regulatory properties have been recently described as residing in the regel 25 4 Chapter MLN49. regel 26 82 This work lays the foundation for a swift translation of this novel immunotherapeutic concept regel 27 in human Rheumatoid Arthritis. The implications, should this approach succeed, range from regel 28 increasing the range of success and utilization of epitope specific immunotherapy, to reducing regel 29 significantly the costs and undesirable effects of current first generation biologics. regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Reference List regel 1 regel 2 1. Olsen NJ, Stein CM. New drugs for rheumatoid arthritis. N.Engl.J.Med. 2004;350:2167-2179. regel 3 2. Taylor PC. Anti-tumor necrosis factor therapies. Curr.Opin.Rheumatol. 2001;13:164-169. regel 4 3. Banchereau J, Pascual V, Palucka AK. Autoimmunity through cytokine-induced dendritic cell regel 5 activation. Immunity. 2004;20:539-550. regel 6 4. Bloom BJ. Development of diabetes mellitus during etanercept therapy in a child with regel 7 systemic-onset juvenile rheumatoid arthritis. Arthritis Rheum. 2000;43:2606-2608. regel 8 5. Dekker L, Armbrust W, Rademaker CM et al. Safety of anti-TNFalpha therapy in children with regel 9 juvenile idiopathic arthritis. Clin.Exp.Rheumatol. 2004;22:252-258. regel 10 6. Keane J, Gershon S, Wise RP et al. Tuberculosis associated with infliximab, a tumor necrosis regel 11 factor alpha-neutralizing agent. N.Engl.J.Med. 2001;345:1098-1104. regel 12 7. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nat.Rev.Immunol. regel 13 2003;3:253-257. regel 14 8. Bluestone JA, Boehmer H. Regulatory T cells. Semin.Immunol. 2006;18:77. regel 15 9. Cope AP, Feldmann M. Emerging approaches for the therapy of autoimmune and chronic regel 16 inflammatory disease. Curr.Opin.Immunol. 2004;16:780-786. regel 17 10. Larche M. Peptide immunotherapy. Immunol.Allergy Clin.North Am. 2006;26:321-332. regel 18 11. Masteller EL, Tang Q, Bluestone JA. Antigen-specific regulatory T cells -- ex vivo expansion and regel 19 therapeutic potential. Semin.Immunol. 2006;18:103-110. regel 20 12. Tarzi M, Klunker S, Texier C et al. Induction of interleukin-10 and suppressor of cytokine regel 21 signalling-3 gene expression following peptide immunotherapy. Clin.Exp.Allergy 2006;36:465- regel 22 474. regel 23 13. Valencia X, Stephens G, Goldbach-Mansky R et al. TNF downmodulates the function of human regel 24 CD4+CD25hi T-regulatory cells. Blood 2006;108:253-261. therapy specific epitope and anticytokine of therapy Combination regel 25 14. Viglietta V, Baecher-Allan C, Weiner HL, Hafler DA. Loss of functional suppression by CD4+CD25+ 83 regel 26 regulatory T cells in patients with multiple sclerosis. J.Exp.Med. 2004;199:971-979. regel 27 15. Anderson PO, Manzo BA, Sundstedt A et al. Persistent antigenic stimulation alters the regel 28 transcription program in T cells, resulting in antigen-specific tolerance. Eur.J.Immunol. regel 29 2006;36:1374-1385. regel 30 16. Garrood T, Pitzalis C. Targeting the inflamed synovium: the quest for specificity. Arthritis regel 31 Rheum. 2006;54:1055-1060. regel 32 17. de Kleer IM, Kamphuis SM, Rijkers GT et al. The spontaneous remission of juvenile idiopathic regel 33 arthritis is characterized by CD30+ T cells directed to human heat-shock protein 60 capable regel 34 of producing the regulatory cytokine interleukin-10. Arthritis Rheum. 2003;48:2001-2010. regel 35 regel 36 regel 1 18. Prakken AB, Van Eden W, Rijkers GT et al. Autoreactivity to human heat-shock protein 60 regel 2 predicts disease remission in oligoarticular juvenile rheumatoid arthritis. Arthritis Rheum. regel 3 1996;39:1826-1832. regel 4 19. Van Eden W, Van Der ZR, Paul AG et al. Do heat shock proteins control the balance of T-cell regel 5 regulation in inflammatory diseases? Immunol.Today 1998;19:303-307. regel 6 20. Kamphuis S, Kuis W, de Jager W et al. Tolerogenic immune responses to novel T-cell epitopes regel 7 from heat-shock protein 60 in juvenile idiopathic arthritis. Lancet 2005;366:50-56. regel 8 21. Albani S, Keystone EC, Nelson JL et al. Positive selection in autoimmunity: abnormal immune regel 9 responses to a bacterial dnaJ antigenic determinant in patients with early rheumatoid regel 10 arthritis. Nat.Med. 1995;1:448-452. regel 11 22. Albani S, Carson DA. A multistep molecular mimicry hypothesis for the pathogenesis of regel 12 rheumatoid arthritis. Immunol.Today 1996;17:466-470. regel 13 23. La Cava A, Nelson JL, Ollier WE et al. Genetic bias in immune responses to a cassette shared regel 14 by different microorganisms in patients with rheumatoid arthritis. J.Clin.Invest 1997;100:658- regel 15 663. regel 16 24. Prakken BJ, Carson DA, Albani S. T cell repertoire formation and molecular mimicry in regel 17 rheumatoid arthritis. Curr.Dir.Autoimmun. 2001;3:51-63. regel 18 25. Prakken BJ, Samodal R, Le TD et al. Epitope-specific immunotherapy induces immune regel 19 deviation of proinflammatory T cells in rheumatoid arthritis. Proc.Natl.Acad.Sci.U.S.A regel 20 2004;101:4228-4233. regel 21 26. Ehrenstein MR, Evans JG, Singh A et al. Compromised function of regulatory T cells in regel 22 rheumatoid arthritis and reversal by anti-TNFalpha therapy. J.Exp.Med. 2004;200:277-285. regel 23 27. Schotte H, Schluter B, Willeke P et al. Long-term treatment with etanercept significantly regel 24 reduces the number of proinflammatory cytokine-secreting peripheral blood mononuclear regel 25 4 Chapter cells in patients with rheumatoid arthritis. Rheumatology.(Oxford) 2004;43:960-964. regel 26 84 28. Zou J, Rudwaleit M, Brandt J et al. Up regulation of the production of tumour necrosis factor regel 27 alpha and by T cells in ankylosing spondylitis during treatment with regel 28 etanercept. Ann.Rheum.Dis. 2003;62:561-564. regel 29 29. Van Eden W, Holoshitz J, Nevo Z et al. Arthritis induced by a T-lymphocyte clone that responds regel 30 to Mycobacterium tuberculosis and to cartilage proteoglycans. Proc.Natl.Acad.Sci.U.S.A regel 31 1985;82:5117-5120. regel 32 30. Van Eden W, Thole JE, Van Der ZR et al. Cloning of the mycobacterial epitope recognized by T regel 33 lymphocytes in adjuvant arthritis. Nature 1988;331:171-173. regel 34 31. Prakken BJ, Van Der ZR, Anderton SM et al. Peptide-induced nasal tolerance for a mycobacterial regel 35 heat shock protein 60 T cell epitope in rats suppresses both adjuvant arthritis and regel 36 nonmicrobially induced experimental arthritis. Proc.Natl.Acad.Sci.U.S.A 1997;94:3284-3289. 32. Bendele AM, McComb J, Gould T et al. Effects of PEGylated soluble tumor necrosis factor regel 1 receptor type I (PEG sTNF-RI) alone and in combination with methotrexate in adjuvant arthritic regel 2 rats. Clin.Exp.Rheumatol. 1999;17:553-560. regel 3 33. Johnson MR, Wang K, Smith JB, Heslin MJ, Diasio RB. Quantitation of dihydropyrimidine regel 4 dehydrogenase expression by real-time reverse transcription polymerase chain reaction. regel 5 Anal.Biochem. 2000;278:175-184. regel 6 34. Prakken BJ, Roord S, Ronaghy A et al. Heat shock protein 60 and adjuvant arthritis: a model for regel 7 T cell regulation in human arthritis. Springer Semin.Immunopathol. 2003;25:47-63. regel 8 35. Anderton SM, van der ZR, Prakken B, Noordzij A, van Eden W. Activation of T cells recognizing regel 9 self 60-kD heat shock protein can protect against experimental arthritis. J.Exp.Med. regel 10 1995;181:943-952. regel 11 36. Albani S. Infection and molecular mimicry in autoimmune diseases of childhood. Clin.Exp. regel 12 Rheumatol. 1994;12 Suppl 10:S35-S41. regel 13 37. Albani S, Ravelli A, Massa M et al. Immune responses to the Escherichia coli dnaJ heat shock regel 14 protein in juvenile rheumatoid arthritis and their correlation with disease activity. J.Pediatr. regel 15 1994;124:561-565. regel 16 38. Prakken BJ, Roord S, van Kooten PJ et al. Inhibition of adjuvant-induced arthritis by interleukin- regel 17 10-driven regulatory cells induced via nasal administration of a peptide analog of an arthritis- regel 18 related heat-shock protein 60 T cell epitope. Arthritis Rheum. 2002;46:1937-1946. regel 19 39. Baecher-Allan C, Viglietta V, Hafler DA. Human CD4+CD25+ regulatory T cells. Semin.Immunol. regel 20 2004;16:89-98. regel 21 40. Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G. Ex vivo isolation and characterization regel 22 of CD4(+)CD25(+) T cells with regulatory properties from human blood. J.Exp.Med. regel 23 2001;193:1303-1310. regel 24 41. Jonuleit H, Schmitt E, Stassen M et al. Identification and functional characterization of therapy specific epitope and anticytokine of therapy Combination regel 25 human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood. 85 regel 26 J.Exp.Med. 2001;193:1285-1294. regel 27 42. Sakaguchi S. The origin of FOXP3-expressing CD4+ regulatory T cells: thymus or periphery. regel 28 J.Clin.Invest 2003;112:1310-1312. regel 29 43. Walker MR, Kasprowicz DJ, Gersuk VH et al. Induction of FoxP3 and acquisition of T regulatory regel 30 activity by stimulated human CD4+. J.Clin.Invest 2003;112:1437-1443. regel 31 44. Bach JF, Francois BJ. Regulatory T cells under scrutiny. Nat.Rev.Immunol. 2003;3:189-198. regel 32 45. Maloy KJ, Powrie F. Regulatory T cells in the control of immune pathology. Nat.Immunol. regel 33 2001;2:816-822. regel 34 regel 35 regel 36 regel 1 46. de K, I, Vastert B, Klein M et al. Autologous stem cell transplantation for autoimmunity induces regel 2 immunologic self-tolerance by reprogramming autoreactive T cells and restoring the regel 3 CD4+CD25+ immune regulatory network. Blood 2006;107:1696-1702. regel 4 47. Bresson D, Togher L, Rodrigo E et al. Anti-CD3 and nasal proinsulin combination therapy regel 5 enhances remission from recent-onset autoimmune diabetes by inducing Tregs. J.Clin.Invest regel 6 2006;116:1371-1381. regel 7 48. Zanin-Zhorov A, Cahalon L, Tal G et al. Heat shock protein 60 enhances CD4 CD25 regulatory T regel 8 cell function via innate TLR2 signaling. J.Clin.Invest 2006;116:2022-2032. regel 9 49. Nolte-’t Hoen EN, Wagenaar-Hilbers JP, Boot EP et al. Identification of a CD4(+)CD25(+) T cell regel 10 subset committed in vivo to suppress antigen-specific T cell responses without additional regel 11 stimulation. Eur.J.Immunol. 2004;34:3016-3027. regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 4 Chapter regel 26 86 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Autologous bone marrow transplantation specifically suppresses autoantibody5 and IL-17 production in proteoglycan induced arthritis

Sarah T.A. Roord1, Femke van Wijk1, Evelien Zonneveld-Huijssoon1, Nathalie van der Meij1, Wilco de Jager1, Henk Rozemuller1, Anton Martens1, Femke Broere2, Nico Wulffraat1, Tibor Glant3, Berent J. Prakken1.

1University Medical Center Utrecht, Wilhelmina Children’s Hospital, Utrecht, The Netherlands. 2Department of Infecti­ous Disea­ses & Immunology, Faculty of Veterinary­ Medicine, Utrecht University, The Ne­ther­lands. 3Rush University Medical Center, Chicago, Illinois, USA.

Submitted for publication regel 1 ABSTRACT regel 2 regel 3 In patients with treatment resistant Juvenile Idiopathic Arthritis (JIA), autologous Bone Marrow regel 4 Transplantation (aBMT) leads to drug free disease remission in a majority of patients. Earlier, regel 5 we found a role for CD4+CD25+Foxp3+ Tregs in restoration of the immune balance after aBMT regel 6 in Proteoglycan Induced Arthritis (PGIA). We now further unravelled the immune mechanism regel 7 of aBMT and the role of the composition of the BM graft. We found that aBMT induces a rapid regel 8 decrease of arthritis followed by stabilization of disease at a level below its severity before regel 9 aBMT. The effect was not attributable to the conditioning regimen alone and couldn’t be broken regel 10 by a re-boost with the disease-triggering antigen, indicating restoration of immune tolerance. regel 11 Interestingly, the most sustained improvement of disease occurred in animals that received regel 12 BM from sick syngeneic donors, of which T cells were not removed. Importantly, aBMT led regel 13 to a strong and specific decrease in auto antibody and IL-17 production. Thus, aBMT induces regel 14 sustained disease remission in PGIA, which is not achieved by immune suppression alone. The regel 15 specific inhibition of IL-17 and auto antibody production provides new clues for the mechanism regel 16 of action of aBMT and may help to develop therapies aimed at restoring immune tolerance in regel 17 arthritis. regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 5 Chapter regel 26 88 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 INTRODUCTION regel 1 regel 2 Juvenile Idiopathic Arthritis (JIA) and Rheumatoid Arthritis (RA) are common autoimmune regel 3 diseases, which often lead to major disability. The treatment of JIA and RA is often a challenge. regel 4 The introduction of immunotherapy with biological agents, aiming at molecular processes regel 5 that are at the basis of disease has great potential, as underscored by the success of regel 6 therapies that target cytokines such as TNFα1;2. However, such treatment fails to bring on a regel 7 sustained medication-free remission of the disease. This prompts continuous administration regel 8 in order to keep disease from relapsing, thus increasing the long-term risks for side effects. regel 9 Furthermore, unfortunately, some patients fail to respond to conventional treatment or regel 10 become unresponsive in the course of time3;4. In JIA, for these severely ill patients, autologous regel 11 bone marrow transplantation (aBMT) has proven to be an effective last resort. It induces drug regel 12 free disease remission in a majority of patients during a follow-up of up to 80 months post regel 13 transplantation5-7. However, despite these promising results, in due time some patients get regel 14 a severe disease relapse, albeit some with a restored sensitivity to previously ineffective regel 15 therapies5;8;9. The drug free disease remission achieved by aBMT in the majority of patients, regel 16 suggests that aBMT can, at least temporarily, restore immune tolerance in JIA. regel 17 Unravelling the mechanism of aBMT in autoimmune diseases such as JIA remains a challenge, regel 18 since understanding its mechanism of action may help to device therapies with similar regel 19 efficacy without the need of undergoing aBMT. It has been hypothesized that in aBMT, due regel 20 to the stringent conditioning regimen leading to severe aplasia, auto-reactive immune cells regel 21 are depleted in the host. In addition, an environment is created in which, after rescue by regel 22 autologous BM, renewed acquisition of self tolerance can be acquired for an extended period regel 23 of time10-13. Some however argue that the effect of aBMT is merely attributable to severe aplasia regel 24 due to the strict immunoablation that precedes transplantation. In both cases, it can be argued production IL-17 and antibody auto suppresses aBMT regel 25 that relapses are bound to take place at some point in time. Therefore, the need has arisen for 89 regel 26 further in depth analysis of what takes place during disease remission after aBMT, preferably regel 27 in a system that resembles the clinical situation. regel 28 In animal models, the stage was set by van Bekkum et al., providing the experimental basis for regel 29 the treatment of autoimmune diseases by aBMT. He demonstrated that Adjuvant Arthritis (AA) regel 30 in Buffalo rats can be treated by aBMT and defined conditioning regimens and levels of T cell regel 31 depletion14. In Collagen Induced Arthritis (CIA), an antibody mediated model in mice, a decrease regel 32 of arthritis occurred after irradiation and BMT, however a rapid relapse occurred, that could in regel 33 turn be prevented by the addition of CD4+CD25+ Tregs to the graft15. Both models have provided regel 34 pivotal insight in the model of aBMT. However, essential information is still lacking. First of all, regel 35 in both models, mostly BM from healthy syngeneic donor animals was used. Yet, the clinical regel 36 regel 1 situation is essentially different, as human BM is harvested from patients that are in an active regel 2 disease state. Therefore we wanted to study aBMT in a model in which BM is harvested from regel 3 animals that are sick at the time of BM harvest. Secondly, the role of T cell depletion of the graft regel 4 is still controversial16. It aims to delete harmful auto-reactive T cells, however theoretically regel 5 might eliminate beneficial Tregs as well, while we earlier showed that just those Tregs might regel 6 be involved in establishing disease remission in patients with JIA after aBMT17. Lastly, unlike regel 7 human arthritis, both AA and CIA are monophasic disease models. To fully understand the regel 8 mechanism behind the induction, maintenance and the break of tolerance as seen in humans regel 9 in the long term, we wanted to turn to a model that is relapsing and remitting. regel 10 For these purposes, we turned to the Proteoglycan Induced Arthritis (PGIA) model. PGIA can be regel 11 induced in female retired BALB/c mice by two injections of human cartilage PG in the adjuvant regel 12 DDA. This causes a progressive poly-arthritis that is initiated by a cross recognition of mouse regel 13 PG by Th1 (T helper type 1) cells, but is also mediated by B cells. PGIA is extensively studied, regel 14 has clinical, immunological and histopathological resemblance to human arthritis and has a regel 15 chronic relapsing remitting course18;19. regel 16 We previously established a role for CD4+CD25+Foxp3+ Tregs in the maintenance of tolerance regel 17 after aBMT20. We now aimed to determine whether aBMT can lead to sustained disease regel 18 remission in this model, what role of the composition of the graft is and, finally, how aBMT may regel 19 affect key pro-inflammatory pathways in PGIA. In this study we show that in PGIA, irradiation regel 20 followed by aBMT induces a rapid decrease of arthritis followed by stabilization of disease at a regel 21 level below its severity before aBMT. The effect is not attributable to the conditioning regimen regel 22 alone and cannot be broken by a boost with the disease-triggering antigen. Interestingly, regel 23 the longest sustained improvement of disease occurs in animals that received BM from sick regel 24 syngeneic donors, without T cell depletion. The effect may (in part) be mediated by a decrease regel 25 5 Chapter in auto antibody production and a suppression of IL-17 production, a cytokine known to play a regel 26 90 role in causing and sustaining tissue damage in arthritis21. regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 MATERIALS AND METHODS regel 1 regel 2 Antigens, immunization, and assessment of arthritis regel 3 Mice regel 4 Female retired BALB/c mice were obtained from Charles River (Sulzfeld, Germany) and served regel 5 as recipients of aBMT. Male donor BALB/c mice of 7 weeks of age were also obtained from regel 6 Charles River and served as donors for aBMT. regel 7 The mice were kept in the animal facility of the University of Utrecht under regular conditions regel 8 until aBMT, after which they were housed under sterile conditions: in filter top cages and regel 9 receiving distilled drinking water containing the antibiotic ciprofloxacin (100 µg/ml). The regel 10 experiments were approved by the Animal Experiments Committee of the University of regel 11 Utrecht. regel 12 regel 13 Proteoglycan (PG) preparation regel 14 PG was purified from human articular cartilage, removed during knee joint replacement regel 15 surgery, by 4 M guanidinium chloride extraction and the human GAG side chains were depleted regel 16 by digestion with chondroitinase ABC as described previously19;22. The Institute of Infectious regel 17 Diseases and Immunology, of the Faculty of Veterinary Medicine in Utrecht kindly provided regel 18 the PG. regel 19 regel 20 Induction and assessment of arthritis regel 21 Arthritis was induced in BALB/c mice by two i.p. injections of 0.4 mg human deglycosylated regel 22 PG in 2 mg of the synthetic adjuvant Dimethyl Dioctadecyl Ammonium Bromide (DDA, Sigma regel 23 Aldrich, Zwijndrecht, The Netherlands) on day 0 and 21. The onset and severity of arthritis regel 24 were assessed every other day in a blinded fashion by a visual scoring system as described production IL-17 and antibody auto suppresses aBMT regel 25 previously19;23. In brief, the degree of joint swelling, redness and deformation of each paw 91 regel 26 (scored from 0-4) was determined in order to express a total arthritis score, with a maximum regel 27 severity index of 16 per animal. In accordance with the Animal Experiment Committee, mice regel 28 were sacrificed when they achieved a total score of 12, or exhibited a cumulative weight loss regel 29 of 20% or a weight loss of more than 15% within 3 days. regel 30 regel 31 Treatment protocols regel 32 Bone Marrow Transplantation (BMT) regel 33 On day 35 after the first injection of PG in DDA, recipient mice that had become arthritic were regel 34 conditioned by a lethal dose of 7.5 Gy total body irradiation. Subsequently BMT was performed regel 35 by i.v. injection of 2x106 BM cells from syngeneic donor animals that were at the same stage regel 36 regel 1 of disease. As the composition of pseudo autologous and autologous BM is identical, the term regel 2 autologous BMT (aBMT) is used for both throughout this paper. regel 3 BM suspensions were prepared as follows: donor mice were sacrificed and BM was harvested regel 4 by flushing tibia and femur with Iscove’s Modified Dulbecco’s Medium (IMDM) containing 2% regel 5 fetal calf serum (FCS), 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin regel 6 (all from Gibco BRL, Grand Island, NY). 2x106 cells were resuspended in 200 µl 0.2% BSA before regel 7 injection i.v. Sick untreated mice were used as controls. regel 8 Recipient mice were assigned to 4 different transplantation groups: 1) Sick syngeneic animals regel 9 at the same stage of disease, graft un-manipulated; 2) Sick syngeneic animals at the same regel 10 stage of disease, T cells depleted from the graft; 3) Healthy syngeneic animals, graft un- regel 11 manipulated; 4) Conditioning alone: healthy syngeneic animals, radio protective dose of BM regel 12 (3x105 cells). Sick untreated mice were used as controls. regel 13 T cells were depleted from the donor graft by a single round of magnetic depletion using anti regel 14 mouse CD4 and anti mouse CD8 MACS micro beads and an LD column (both Myltenyi Biotec). regel 15 The remaining T cell depleted BM graft contained 0.67% (CD3+) T cells. regel 16 regel 17 Reboost with PG regel 18 On day 70 after the first injection with PG in DDA, animals that had undergone aBMT (N=6) regel 19 and animals that had not undergone aBMT (N=3), received a boost of 400 µg PG i.p. Arthritis regel 20 scores were assessed 3 times a week and the clinical response after the boost was compared regel 21 to animals that had received the same treatment (BMT vs. no BMT) but had not received a regel 22 boost. Results were depicted as percentage increase from the day of the boost. regel 23 regel 24 In vitro assays regel 25 5 Chapter T cell proliferation to PG and a major epitope of PG during the repopulation phase regel 26 92 On day 42, 55 and 81 after the first PG/DDA injection, mice were sacrificed and spleens and regel 27 popliteal and inguinal lymph nodes (LN) were harvested. Cells were mashed through a 70 µm regel 28 cell strainer (BD Biosciences, San Jose, CA), washed, and cultured in IMDM supplemented with regel 29 10% FCS, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin and 5 x10-5 M regel 30 2-mercaptoethanol (culture medium) for 120 hrs. They were either cultured in the medium regel 31 alone or in the presence of 1 µg/ml soluble anti CD3 (clone 145-2C11, BD Pharmingen, San regel 32 Diego, CA), 10 µg/ml PG or 50 µg/ml p70 (70ATEGRVRVNSAYQDK84), a major arthritogenic regel 33 epitope of PG24;25 (both kindly provided by the Division of Immunology of the Faculty of regel 34 Veterinary Medicine, University of Utrecht). During the last 16-18 hours 1 µCi 3H (3H-TdR; regel 35 Amersham, Buckinghamshire, U.K.) was added per well and 3H uptake was measured using regel 36 a liquid scintillation beta counter. Proliferative responses were calculated as the mean 3H incorporation (cpm) of triplicate wells. Proliferative responses were depicted as stimulation regel 1 index (SI): proliferation due to stimulus divided by proliferation in medium alone. regel 2 regel 3 Antibody response to PG during the repopulation phase regel 4 On day 42, 55 and 81 after the first PG/DDA injection, mice were sacrificed and blood was regel 5 drawn by cardiac puncture and collected in SSTII clotting tubes (BD biosciences), then spun regel 6 down at 3000 rpm for 10 minutes. Serum was collected and frozen at -80°C until analysis. regel 7 An ELISA was performed to measure PG specific Antibodies (Abs) as described previously26. regel 8 ELISA 96 well plates were coated overnight with human PG (0.5 µg protein/well) and then regel 9 blocked with 1.5% fat free milk in PBS. Next, the sera were added in increasing dilutions and regel 10 isotypes of PG specific Abs were determined using peroxidase conjugated mAbs to mouse regel 11 IgG1 and IgG2a (BD Biosciences) as secondary Abs. Serum Ab levels were calculated relative regel 12 to mouse serum Ig fractions of pooled sera of control animals. regel 13 regel 14 Cytokine production during repopulation post aBMT regel 15 On day 42, 55 and 81 after the first PG/DDA injection, mice were sacrificed and spleens and regel 16 popliteal and inguinal lymph nodes (LN) were harvested. Cells were mashed through a 70 regel 17 µm cell strainer (BD Biosciences, San Jose, CA), washed, and cultured in culture medium for regel 18 96 hrs, in the presence of 1 µg/ml soluble anti CD3 (clone 145-2C11, BD Pharmingen, San regel 19 Diego, CA), 10 µg/ml PG or 50 µg/ml p70 (70ATEGRVRVNSAYQDK84), a major arthritogenic regel 20 epitope of PG. Supernatants were harvested and frozen at –80°C until analysis. Cytokine regel 21 profiles were measured using a mouse cytokine multiplex kit (Biorad, CA, USA) according to regel 22 the manufacturer’s instruction. Analysis was performed using the Bio-plex manager software regel 23 v4.1 (Biorad). regel 24 aBMT suppresses auto antibody and IL-17 production IL-17 and antibody auto suppresses aBMT regel 25 Histological analysis of hind limb joints 93 regel 26 On the final day of the experiment, animals were sacrificed, hind limbs harvested and fixed regel 27 in formalin. Next, they were decalcified in equal parts of 6.8% Na-Formiat and 36,8% Formic regel 28 acid in distilled water and glass slides stained with H&E were prepared. A pathologist from the regel 29 Faculty of Veterinary Medicine, Utrecht, The Netherlands examined them. regel 30 regel 31 Statistical analysis regel 32 Data analysis was performed by SPSS 12.0 software. regel 33 regel 34 regel 35 regel 36 regel 1 RESULTS regel 2 regel 3 aBMT suppresses arthritis severity in PGIA regel 4 First, we investigated whether aBMT can lead to disease amelioration in PGIA and, if so, whether regel 5 the same effect can be established by conditioning alone. Arthritis was induced on day 0 and regel 6 day 21 by an injection of PG in DDA. From day 21 onward, the arthritis score was assessed regel 7 every other day by a visual scoring system of redness, swelling and deformities of the paws. regel 8 On day 38 conditioning was performed by a lethal dose of 7.5 Gy total body irradiation followed regel 9 by autologous Bone Marrow Transplantation. As ‘autologous’ Bone Marrow, we used BM from regel 10 sick syngeneic donor animals that were at the same stage of disease. As controls we used regel 11 arthritic mice that did not receive treatment and mice that underwent conditioning without regel 12 BMT. The control group for conditioning alone received a low, radio-protective dose of BM in regel 13 order to prevent the animals from dying from the lethal dose of irradiation. The arthritis score regel 14 on the day of transplantation was set to 100%. regel 15 As shown in Figure 1A, two injections of PG in DDA in BALB/c mice led to a severe chronic regel 16 arthritis, which became relapsing and remitting in time. When conditioning was applied, regel 17 followed by a full dose of BM, this led to rapid, significant and long lasting clinical improvement regel 18 (BMT; mean score 39% +/- 8 SEM on day 85 vs. no treatment score 200% +/-133 SEM p<0.05). regel 19 Some degree of relapse of arthritis occurred in all animals, but the arthritis scores remained regel 20 significantly lower than before aBMT. Furthermore, the Area under the Curve (AUC) ofthe regel 21 arthritis scores, which gains insight in the treatment effect throughout the whole disease regel 22 period, was also significantly lower after aBMT (mean AUC aBMT 2280 vs. no treatment 17470, regel 23 p<0.05). regel 24 We questioned whether the effects observed were attributable to the immune suppression regel 25 5 Chapter induced by the conditioning regime or whether BM rescue was critical for the observed regel 26 94 effective protection. Conditioning alone followed by a radio protective dose of BM indeed also regel 27 led to strong initial clinical improvement comparable to or even stronger than observed in mice regel 28 undergoing aBMT. However, a striking difference was observed after the initial phase. Whereas regel 29 mice receiving aBMT retained stable low arthritis scores, mice treated with conditioning alone regel 30 developed a relapse after the first improvement phase which led to a much more severe regel 31 and sustained arthritis, even more severe than before transplantation (conditioning alone; regel 32 score 121% +/-16 SEM on day 85 p 0.08 vs. no treatment). The AUC of the conditioning alone regel 33 scores was low (Mean AUC 2690, p<0.05 compared to no treatment), suggesting significant regel 34 improvement due to conditioning alone, however this is most likely caused by the initial rapid regel 35 improvement, which is counteracted in the end by the severe relapse, thus therefore we do regel 36 not consider this to be clinically relevant (Figure 1B). Altogether, aBMT is an effective way to control arthritis in PGIA, and this effect is not due to the conditioning regime alone.     

 regel 1 A   regel 2  regel 3 regel 4  regel 5 regel 6  regel 7 regel 8             regel 9

 regel 10             regel 11 B  regel 12    regel 13   regel 14  regel 15                     regel 16    regel 17   regel 18                 regel 19  

 regel 20   regel 21            regel 22  Figure 1. regel 23 A. Conditioning followed by autologous Bone Marrow Transplantation leads to a rapid and regel 24 sustained  decrease  in the severity  of  arthritis       production IL-17 and antibody auto suppresses aBMT regel 25 Arthritis was induced by two injections of PG in DDA on day 0 and day 21. On day 38 mice received a 95 regel 26                     lethal irradiation dose of 7.5 Gy followed by i.v. injection of 2x106 autologous BM cells from sick syngeneic regel 27  donor animals (N= 5). Control animals did not receive conditioning, nor aBMT (N= 8). Arthritis scores at regel 28  day 38 were set to 100% and the subsequent clinical effect was expressed as a percentage of the score regel 29  at the time of transplantation. For clarity, scores are shown every 4-5 days. regel 30 regel 31 B. Conditioning without aBMT leads to temporary disease amelioration followed by regel 32 exacerbation of arthritis  regel 33 Arthritis was induced by two injections of PG in DDA on day 0 and day 21. On day 38 mice received a regel 34 lethal irradiation dose of 7.5 Gy followed by i.v. injection of 2x106 autologous BM cells from sick syngeneic regel 35 donor animals (N= 5). As a control, N=4 animals received 7.5 Gy irradiation followed by a radio protective regel 36 dose of BM (3x105 cells), in order to survive the irradiation.

  regel 1 Sick BM of which T cells were not depleted leads to best clinical improvement regel 2 Next we set out to address the question whether the composition and the source of the BM may regel 3 influence the efficacy of aBMT. First, we wanted to establish whether depletion of (potentially regel 4 auto-reactive) T cells in the BM graft might influence the clinical outcome. Also, we wanted regel 5 to assess the difference between BMT from sick mice (which reflects the clinical setting in regel 6 JIA and RA patients undergoing aBMT) and BMT from healthy donors (reflecting a setting for regel 7 allogeneic transplantation). Thus, we studied the effect on the course of PGIA of three types of regel 8 donor BM: 1) an un-manipulated graft from sick syngeneic mice; 2) a T cell depleted graft from regel 9 sick syngeneic mice; 3) an un-manipulated graft from healthy syngeneic animals. As controls, regel 10 sick untreated mice were studied. regel 11 In line with the previous experiments, arthritis was induced by an injection of PG in DDA on regel 12 day 0 and day 21. On day 38, the mean arthritis score of all recipient mice was 5.7 (+/- 0.9 regel 13 SEM) and there were no significant differences in absolute arthritis scores between the regel 14 different treatment groups. The arthritis scores on day of aBMT were again set to 100%. In all regel 15 three treatment groups a rapid decrease of arthritis was seen after aBMT. However, in mice regel 16 that received BM from healthy donors (Healthy/no manipulation) or mice that received T cell regel 17 depleted BM (Sick/T cell depleted), the rapid clinical improvement was followed by relapses, regel 18 such that in time there was no significant clinical improvement compared to untreated animals regel 19 (p 0.30 (Healthy/no manipulation score) and p 0.08 (Sick/T cell depleted) vs. no treatment on regel 20 day 85). When assessing the AUC, the T cell depleted BM had a mean AUC of 4120 (p 0.05 vs. regel 21 no treatment) and Healthy BM had a mean AUC of 6590 (p 0.12 vs. no treatment). Thus, sick regel 22 BM was more effective than healthy BM in inducing a sustained clinical improvement and T regel 23 cell depletion of the sick BM graft was not able to further enhance the clinical efficacy of the regel 24 procedure (Figure 2). regel 25 5 Chapter regel 26 96 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36  

  regel 1   regel 2  regel 3

 regel 4 regel 5

 regel 6 regel 7  regel 8  regel 9             regel 10  regel 11 regel 12 Figure 2. BM from sick syngeneic donor animals leads to the best clinical improvement in PGIA.  regel 13 Arthritis was induced by two injections of PG in DDA on day 0 and day 21. On day 38 mice received a regel 14 lethal  irradiation  dose  of  7.5 Gy followed  by  transplantation    of 3 types  of  donor  BM:  1)  Sick  syngeneic  regel 15 animals that were at the same stage of disease, graft un-manipulated (N=5); 2) Sick syngeneic animals regel 16 that were at the same stage of disease, T cells removed  from the graft (N=5); 3) Healthy  syngeneic  regel 17 animals, graft un-manipulated (N=5). Arthritis scores at day 38 were   set to  100% and  the subsequent   regel 18 clinical effect was expressed as a percentage of the score at the time of transplantation. regel 19  regel 20 Autologous BMT leads to long-term restoration of the immune balance regel 21  Then, we questioned whether the clinical improvement induced by BMT could induce tolerance regel 22 to subsequent disease challenge. For this purpose, PGIA animals that had undergone aBMT regel 23 and animals that had not undergone aBMT, received a boost of 400 µg PG i.p. on day 70 after regel 24 the first injection with PG in DDA. Arthritis scores on the day of the boost were set to 100%. Data production IL-17 and antibody auto suppresses aBMT regel 25 are represented as the percentage of increase in arthritis after the boost compared to animals 97 regel 26 in the same treatment group that had not received a boost. regel 27 As shown in Figure 3, a boost given to PGIA animals that had not been transplanted led to an regel 28 impressive increase in arthritis (PGIA with boost 269% compared to PGIA without a boost). regel 29 Interestingly, animals that had undergone aBMT were resistant to exacerbation of disease regel 30 after the boost (BMT with boost 93% compared to transplanted animals without a boost). regel 31 These results indicate that aBMT in the PGIA model led to long-term restoration of the immune regel 32 balance, in which a state of resistance to disease induction by the disease-triggering antigen regel 33 was achieved. regel 34   regel 35 regel 36 

regel 1   regel 2   regel 3 regel 4  regel 5 regel 6  regel 7  regel 8

 regel 9   regel 10  regel 11      regel 12  regel 13 Figure 3. Transplanted animals are protected from disease enhancement by a re-boost with  regel 14 the disease-triggering agent.  regel 15 PGIA was induced by PG/DDA on day 0 and 21. On day 38 mice received 7.5 Gy irradiation and BM from  regel 16 sick syngeneic donor animals. On day 70, animals that had undergone aBMT (N=6) and animals that regel 17 had not (N=3) received a boost with 400 µg PG i.p. Results are depicted as percentage increase from regel 18 the day after the boost.  regel 19  regel 20 Protective   effect  of  aBMT  on histological   damage   in  joints       regel 21 Next, we wanted to know whether the clinical improvement after aBMT was also reflected in regel 22 the joint histopathology. Thus, we set out to determine the effect of aBMT on local histological regel 23 damage in the ankle, compared to PGIA animals and healthy controls. For this purpose, ankle regel 24 joints were harvested on the last day of the experiment, and after decalcification an H&E regel 25 5 Chapter stain was performed. Figure 4A shows that after aBMT there was only slight metaplasia of the regel 26 98 synovial membrane and that cartilage was not lost. On the other hand, in PGIA animals there regel 27 was clear loss of articular cartilage and necrosis of synovial cells while infiltrates were present regel 28 (Figure 4B). As a control, the histological picture of a healthy control animal is shown (Figure regel 29 4C). regel 30 These results indicate that aBMT led to less damage in local joints, weeks after transplantation, regel 31 compared to untreated animals. Whether damage had been reversed or whether the damage regel 32 halted due to aBMT, remains to be determined. regel 33 regel 34 regel 35 regel 36

        regel 1 regel 2 regel 3 regel 4 regel 5 regel 6   regel 7     regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17   regel 18  Figure 4. Less histological  damage after aBMT in hind limb joints regel 19    H&E staining was performed on decalcified joints, harvested on the last day of the experiment. regel 20   A. Ankle of a BMT mouse. Slight metaplasia of the synovial membrane (arrows) with some proliferation in regel 21                the articular cavity. Focal presence of articular cartilage. regel 22      B. Ankle of a PGIA mouse. Loss of articular cartilage with fibrin in the articular cavity (arrow) and necrosis regel 23                            of synovial cells, mild non-suppurative infiltrates. regel 24

 production IL-17 and antibody auto suppresses aBMT C. Ankle of a control mouse. No changes of the joint. regel 25   (For colour image see addendum) 99 regel 26   regel 27  Repopulation of antigen specific T cells and auto antibody production following regel 28 aBMT regel 29 Even though aBMT was able to induce long-term improvement compared to the level of disease regel 30 before transplantation, some degree of relapse of disease did occur. We thus wanted to assess regel 31 if, and if so, how, aBMT may affect the auto-antigenic adaptive immune response. We measured regel 32 antigen specific proliferation and antibody response to the disease-triggering antigen during regel 33 the repopulation post aBMT. On day 42, 55, and 81 after the first injection of PG/DDA, local regel 34 draining LN and spleen cells were cultured in the presence of PG and peptide p70, a major regel 35 epitope of PG (AA 70-84). Proliferative responses were measured by 3H incorporation and IgG1 regel 36  (a Th2 mediated antibody) and IgG2a (a Th1 mediated antibody) responses were measured  by ELISA.      regel 1 Early on during the repopulation, on day 42, draining LN did not contain enough cells to     regel 2 measure proliferation, but we were able to assess proliferation in spleen on that day, and in   regel 3 both organs from day 55 on. As can be seen in Table 1, overall, antigen specific proliferative   regel 4 responses were low and we did not find any clear differences in the response to PG and the regel 5   PG peptide during the repopulation phase post aBMT compared to untransplanted animals.  regel 6 Interestingly, we only found some antigen specific proliferation in transplanted animals; on   regel 7 day 55 to the PG peptide in the local draining LN (SI 1.9) and on day 81 in spleen to whole PG (SI regel 8 1.7). Proliferation to anti CD3 occurred in both transplanted as well as untransplanted animals         regel 9 (results not shown).   regel 10 With regards to the antibody responses, we found that after aBMT, the antibody responses regel 11 to PG were diminished compared to control animals. In time, thus after a longer period after  regel 12 the PG injection, Ab responses diminished in both transplanted and untransplanted animals,  regel 13 as can be expected. We did not find a polarization towards either a Th1 or Th2 response when                  regel 14 assessing the antibody response (Figure 5). Hence, transplantation led to a decrease in  regel 15 antigen specific proliferation and auto antibody production. regel 16  regel 17 Table 1. No antigen specific proliferation to the disease-triggering antigen in LN and spleen  regel 18 during repopulation after aBMT  regel 19 regel 20 Spleen LN  regel 21 PG PG peptide PG PG peptide regel 22 BMT Day 42 1,2 1,2 N.A. N.A. regel 23 Day 55 1,4 1,4 1,0 1,9 regel 24 Day 81 1,7 1,5 1,4 0,8 regel 25 5 Chapter PGIA Day 42 1,0 1,1 0,8 N.A. regel 26 100 Day 55 1,2 0,9 1,1 1,1 Day 81 1 1,1 1,1 1,2 regel 27 regel 28 On day 42, 55 and 81 after the first PG/DDA injection, mice were sacrificed and spleens and popliteal and regel 29 inguinal lymph nodes (LN) were harvested. Cells were cultured for 120 hours in the presence of PG or PG regel 30 peptide p70, a major epitope (amino acids 70-84) of PG, and during the last 16-18 hours in the presence regel 31 of 3H. Proliferative responses were represented as Stimulation Index (SI): mean 3H incorporation (cpm) regel 32 in presence of stimulus divided by medium alone. N.A.= not available. regel 33 regel 34 regel 35 regel 36

  

  regel 1    regel 2    regel 3 regel 4   regel 5  regel 6   regel 7

  regel 8       regel 9   regel 10 Figure 5. Decreased PG specific antibody response post aBMT  regel 11 On day 42, 55 and 81 after the first PG/DDA injection, mice were sacrificed and spleens and popliteal regel 12 regel 13 and  inguinal  lymph  nodes  (LN)  were  harvested.   PG specific  IgG1 and  IgG2a responses  were  measured     in supernatants by ELISA. regel 14 regel 15 Suppression of IL-17 production during repopulation post aBMT regel 16  Thus, no clear differences in T cell proliferation were found between the different groups regel 17 regel 18  which could be due to the overall low proliferation observed. We next questioned whether the observed clinical improvement was reflected in the cytokine production during repopulation. regel 19  On day 42, 55, and 81 after the first injection of PG/DDA, spleen cells were cultured in the regel 20 presence of soluble anti CD3, PG, or PG peptide and the production of IL-4, IL-10, IL-17, Tumor regel 21 Necrosis Factor-α (TNFα) and Interferon-γ (IFNγ) was measured in the supernatants by regel 22 Multiplex Immuno Assay. We were not able to obtain enough local draining LN cells during regel 23 repopulation post aBMT, so the data are restricted to the spleen cells. regel 24 Following aBMT we observed a non-significant decrease in both non-specific (CD3) and production IL-17 and antibody auto suppresses aBMT regel 25 antigen-specific (whole PG and the major peptide epitope) production of the pro-inflammatory 101 regel 26 cytokines TNFα and IFNγ. A similar trend was seen of a decrease of IL-10 production in regel 27 cultures after aBMT, while CD3-induced production of IL-4 was increased at day 55, at the regel 28 time of stabilisation of disease, but not at the earlier and later time points (Figure 6A). The far regel 29 most striking observation was however on IL-17 production. We were particularly interested regel 30 in the production of IL-17, since it is now considered a major driving force in autoimmunity. regel 31 For example, in arthritis it plays a critical role in disease induction as well as formation of regel 32 tissue damage21. We found extremely high levels of IL-17 production in arthritic animals in the regel 33 PGIA model upon stimulation with anti CD3 (Figure 6B). Post aBMT, the IL-17 production upon regel 34 stimulation with PG and the major PG epitope was almost completely lost (not detectable at regel 35 day 42 and 55 post aBMT, and on day 81 0.38 ng/ml after PG stimulation and 2.38 ng/ml after regel 36

  regel 1 PG peptide stimulation), while production of CD3-induced IL-17 was significantly suppressed at regel 2 all time points after aBMT (Figure 6B). Thus, aBMT led to a strong and sustained suppression regel 3  of systemic IL-17 production. regel 4 regel 5  regel 6     regel 7  regel 8  regel 9  regel 10  regel 11  regel 12         regel 13 regel 14  regel 15       regel 16 regel 17 regel 18 regel 19   regel 20    regel 21   regel 22  regel 23 

regel 24   regel 25 5 Chapter regel 26 102  regel 27     regel 28  regel 29 Figure 6. Suppression of IL-17 production post aBMT regel 30  On day 42, 55 and 81 after the first PG/DDA injection, mice were sacrificed and spleens were harvested. regel 31 Cells were cultured for 96 hrs in the presence of anti CD3 and the production of IL-4, IL-10, IL-17, IFNγ and regel 32 TNFα was measured in supernatants by Multiplex Immuno Assay.        regel 33 A. Geometric mean values are depicted in color scheme according to legend. regel 34 B. Mean values are depicted.  regel 35 (For colour image see addendum) regel 36  

  DISCUSSION regel 1 regel 2 Autologous BMT can lead to drug free disease remission in a majority of severely ill, treatment regel 3 resistant, JIA patients5;6. This is an important phenomenon as none of the other currently regel 4 available therapies is capable of achieving this. Therefore it is important to better understand regel 5 which immunological mechanism accounts for the effects of aBMT and study it’s mechanism regel 6 of action in an experimental model. PGIA is a very suitable model for this purpose for several regel 7 reasons. It has a close resemblance to human disease, in that it is a remitting-relapsing regel 8 disease, that is both B and T cell dependent, and that it has many histopathological similarities regel 9 to RA and JIA18;22. regel 10 PGIA can indeed serve as a useful model for aBMT. Conditioning followed by aBMT leads to regel 11 rapid amelioration of disease. Thereafter, the degree of the arthritis never returns to the level regel 12 before aBMT, but stabilizes at a lower level. Earlier, we showed that the stabilization of disease regel 13 is regulated by CD4+CD25+Foxp3+ Tregs20. Here we show that the arthritis suppressive effect regel 14 of conditioning and aBMT cannot be attributed to immune suppression alone, as conditioning regel 15 alone does not obtain the same clinical effect. On the contrary; despite initial improvement regel 16 shortly after irradiation, in the long run the arthritis returns in a severe form, to an extent that regel 17 is even more severe than before aBMT. regel 18 Whereas in other models BM from healthy donor animals was applied15, we found that arthritis regel 19 can best be suppressed with BM from animals that were at the same active stage of disease, regel 20 while replacing sick BM by healthy BM leads to a less sustained suppression of disease. These regel 21 observations are of particular interest due to the link with the clinical situation, as in JIA and regel 22 RA patients, autologous BM is harvested during a phase of active disease. Since BM is part of regel 23 the lymphocyte recirculation network and serves as a reservoir for (memory) T lymphocytes regel 24 and even preferentially for regulatory T cells (Tregs)27;28, it can be hypothesized that a memory production IL-17 and antibody auto suppresses aBMT regel 25 -PG specific- T cell pool resides in the BM of sick animals. After transplantation of the BM into 103 regel 26 an irradiated recipient, possibly the auto reactive T cells are ‘re-educated’ to become tolerant regel 27 Tregs in the lymphopenic environment17. Recently we found a role for Foxp3+CD4+CD25+ regel 28 Tregs in the second, stabilization phase of PGIA after aBMT20. Our present findings shed new regel 29 light on this observation and suggest that next to ‘naturally occurring’ Foxp3+ Tregs, possibly regel 30 also re-educated auto antigen specific Tregs originating from the graft may play a role. regel 31 This may be underlined by the observation that T cells do not need to be depleted from the BM regel 32 in order to have a better clinical effect. The rationale for depleting T cells from the graft stems regel 33 from the idea of eliminating potentially auto reactive T cells. However, in a pilot multi-centre regel 34 randomised trial in 33 RA patients, when CD34+ selection was performed as a way of depleting regel 35 T cells, no additional effect on time to relapse nor disease remission was achieved compared regel 36 regel 1 to unmanipulated grafts16. In addition, in JIA patients depletion of T cells from the graft in fact regel 2 even led to a major complication, Macrophage Activation Syndrome (MAS). Due to the lack of regel 3 control by T cells, macrophages could become overly activated and extremely destructive regel 4 leading to the clinical syndrome of MAS6. Larger studies are needed to better decide on the regel 5 usefulness of T cell depletion from the BM graft, while also preventing damaging complications regel 6 of MAS. Our present data suggest a positive effect of T cells in the graft, possibly due to the regel 7 presence and/or induction of auto-antigen specific Tregs. regel 8 Other than most current therapies, aBMT can lead to at least a temporary resetting of the regel 9 immune balance in patients with JIA and RA. Recently, we showed also in PGIA that a restoration regel 10 of the immune balance takes place at the time of clinical stabilisation of the disease at a level regel 11 below the severity before aBMT. During the early phase of clinical improvement, CD4+CD25+T regel 12 cells increased, but these cells were not suppressors and did not up regulate Foxp3. Only regel 13 at the time of clinical stabilisation of disease, the CD4+CD25+ T cells became functionally regel 14 suppressive Foxp3+ Tregs, suggesting an active role for these Tregs in the restoration of the regel 15 immune balance20. Based on these results, we hypothesized here that this active stabilisation regel 16 of disease might indicate protection from re-induction of arthritis. Indeed, we now show that regel 17 transplanted animals are protected against enhancement of disease after reimmunization regel 18 with the disease-triggering antigen. This further underscores the fact that aBMT leads to a regel 19 prolonged restoration of the immune balance. regel 20 As Foxp3+ Tregs seem to come in play later during the stabilisation phase of the disease, they regel 21 cannot be responsible for the recovery in the early phase of the disease following aBMT. We regel 22 now show a striking suppression of IL-17 production that is already present early post aBMT. regel 23 IL-17 has received much attention in recent years, due to its important role in the induction regel 24 and sustainment of tissue damage in several autoimmune diseases21. Th17 cells, producing regel 25 5 Chapter IL-17, have been established as a separate lineage of Thelper cells, distinct from conventional regel 26 104 Th1 and Th2 subsets. Th17 cells have been shown to sustain acute synovitis and turn it into a regel 27 chronic destructive process. The presence and function of Th17 cells has been established in regel 28 human JIA and RA, as well as several experimental arthritis models, such as CIA and Adjuvant regel 29 Arthritis29;30. Its role in PGIA has not been studied yet. Here, we show very high levels of IL-17 regel 30 production in sick PGIA animals, while IL-17 production is significantly decreased at several regel 31 time points post aBMT. In addition to the role we previously found for CD4+CD25+Foxp3+ regel 32 Tregs in the second phase of restoration of the immune balance post aBMT, here we found that regel 33 suppression of IL-17 likely plays an important role in the first phase of disease suppression regel 34 by aBMT. It can be hypothesized that this strong reduction of the pro-inflammatory response regel 35 is essential for the success of the next phase: the achievement of lasting immune tolerance. regel 36 Furthermore, the suppression of IL-17 may also be at least in part an explanation for the beneficial effect seen on histological damage in the joints post aBMT. Despite the disease remitting capacity of the aBMT approach, arthritis – though of less regel 1 severe disease – does reappear and therefore the search remains for strategies to further regel 2 improve the outcome. Antigen specific immunomodulation may be an elegant candidate for regel 3 this purpose. For example, mucosal administration of the disease-triggering antigen has been regel 4 shown to selectively modulate the reaction of the immune system to the disease trigger from regel 5 a pro-inflammatory to a tolerant or regulatory response. Due to its specificity, it promises to regel 6 have less severe side effects than the methods currently in use, and may possibly lead to regel 7 sustained immune tolerance31;32. We investigated whether we could detect antigen specific regel 8 proliferation to PG, which would be a prerequisite to be able to modulate the response. regel 9 Indeed, despite overall low levels of proliferation post aBMT, we found some proliferation to regel 10 the disease-triggering antigen in transplanted animals, both in LN as in spleen. Therefore, regel 11 the relatively ‘empty’ immune system due to the irradiation may provide a unique window regel 12 for achieving antigen specific immune modulation. The modulatory capacity of mucosal regel 13 administration of PG in this model was recently demonstrated by Broere et al. They were able regel 14 to suppress PGIA by mucosal administration of PG prior to disease induction, both orally and regel 15 nasally, and the protective effect was mediated by regulatory T cells26. It is of interest whether regel 16 this approach is also effective post aBMT and whether the tolerogenic environment can be regel 17 enhanced by it. In this way –in theory- the now observed disease exacerbations after years of regel 18 remission possibly could be prevented. Also, it could lead to the development of other forms regel 19 of combination therapy that without the need of aBMT may yield a similar immunological and regel 20 clinical effect. For example, in order to modulate the immune response in an antigen specific regel 21 way, a combination therapy aimed at first shutting down the pro-inflammatory environment regel 22 can be attempted. We previously indeed were able to suppress Adjuvant Arthritis by preceding regel 23 mucosal antigen specific immunotherapy by anti TNFα therapy33. Also, in a diabetes model, regel 24 combination of an anti-CD3ε-specific antibody and i.n. proinsulin peptide reversed recent- production IL-17 and antibody auto suppresses aBMT regel 25 onset diabetes in 2 murine diabetes models with much higher efficacy than with monotherapy 105 regel 26 with anti-CD3 or antigen alone34. regel 27 Altogether, we show here that aBMT leads to a restored immune balance and sustained disease regel 28 remission, which cannot be broken by the original disease trigger. It opens up opportunities to regel 29 further unravel the mechanism of immune tolerance post aBMT and investigate how to sustain regel 30 remission e.g. by active modulation of the immune response in an antigen specific manner. regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 Reference List regel 2 regel 3 1. Castro-Rueda H, Kavanaugh A. Biologic therapy for early rheumatoid arthritis: the latest regel 4 evidence. Curr.Opin.Rheumatol. 2008;20:314-319. regel 5 2. van der Kooij SM, Allaart CF, Dijkmans BA, Breedveld FC. Innovative treatment strategies for regel 6 patients with rheumatoid arthritis. Curr.Opin.Rheumatol. 2008;20:287-294. regel 7 3. Furst DE, Breedveld FC, Kalden JR et al. Updated consensus statement on biological agents for regel 8 the treatment of rheumatic diseases, 2007. Ann.Rheum.Dis. 2007;66 Suppl 3:iii2-22. regel 9 4. Gartlehner G, Hansen RA, Jonas BL, Thieda P, Lohr KN. Biologics for the treatment of juvenile regel 10 idiopathic arthritis: a systematic review and critical analysis of the evidence. Clin.Rheumatol. regel 11 2008;27:67-76. regel 12 5. Brinkman DM, De K, I, Ten CR et al. Autologous stem cell transplantation in children with severe regel 13 progressive systemic or polyarticular juvenile idiopathic arthritis: long-term follow-up of a regel 14 prospective clinical trial. Arthritis Rheum. 2007;56:2410-2421. regel 15 6. De K, I, Brinkman DM, Ferster A et al. Autologous stem cell transplantation for refractory regel 16 juvenile idiopathic arthritis: analysis of clinical effects, mortality, and transplant related regel 17 morbidity. Ann.Rheum.Dis. 2004;63:1318-1326. regel 18 7. Wulffraat NM, Sanders LA, Kuis W. Autologous hemopoietic stem-cell transplantation for regel 19 children with refractory autoimmune disease. Curr.Rheumatol.Rep. 2000;2:316-323. regel 20 8. Passweg J, Tyndall A. Autologous stem cell transplantation in autoimmune diseases. Semin. regel 21 Hematol. 2007;44:278-285. regel 22 9. Rabusin M, Andolina M, Maximova N. Haematopoietic SCT in autoimmune diseases in children: regel 23 rationale and new perspectives. 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Effective treatment of collagen-induced regel 36 arthritis by adoptive transfer of CD25+ regulatory T cells. Arthritis Rheum. 2005;52:2212- 2221. 16. Moore J, Brooks P, Milliken S et al. A pilot randomized trial comparing CD34-selected versus regel 1 unmanipulated hemopoietic stem cell transplantation for severe, refractory rheumatoid regel 2 arthritis. Arthritis Rheum. 2002;46:2301-2309. regel 3 17. De K, I, Vastert B, Klein M et al. Autologous stem cell transplantation for autoimmunity induces regel 4 immunologic self-tolerance by reprogramming autoreactive T cells and restoring the regel 5 CD4+CD25+ immune regulatory network. Blood 2006;107:1696-1702. regel 6 18. Glant TT, Finnegan A, Mikecz K. Proteoglycan-induced arthritis: immune regulation, cellular regel 7 mechanisms, and genetics. Crit Rev.Immunol. 2003;23:199-250. regel 8 19. Glant TT, Mikecz K. Proteoglycan aggrecan-induced arthritis: a murine autoimmune model of regel 9 rheumatoid arthritis. Methods Mol.Med. 2004;102:313-338. regel 10 20. Roord ST, de JW, Boon L et al. Autologous bone marrow transplantation in autoimmune arthritis regel 11 restores immune homeostasis through CD4+CD25+Foxp3+ regulatory T cells. Blood regel 12 2008;111:5233-5241. regel 13 21. Steinman L. A brief history of T(H)17, the first major revision in the T(H)1/T(H)2 hypothesis of regel 14 T cell-mediated tissue damage. Nat.Med. 2007;13:139-145. regel 15 22. Glant TT, Cs-Szabo G, Nagase H, Jacobs JJ, Mikecz K. Progressive polyarthritis induced in regel 16 BALB/c mice by aggrecan from normal and osteoarthritic human cartilage. Arthritis Rheum. regel 17 1998;41:1007-1018. regel 18 23. Hanyecz A, Berlo SE, Szanto S et al. Achievement of a synergistic adjuvant effect on arthritis regel 19 induction by activation of innate immunity and forcing the immune response toward the Th1 regel 20 phenotype. Arthritis Rheum. 2004;50:1665-1676. regel 21 24. Buzas EI, Brennan FR, Mikecz K et al. A proteoglycan (aggrecan)-specific T cell hybridoma regel 22 induces arthritis in BALB/c mice. J.Immunol. 1995;155:2679-2687. regel 23 25. Glant TT, Buzas EI, Finnegan A et al. Critical roles of glycosaminoglycan side chains of cartilage regel 24 proteoglycan (aggrecan) in antigen recognition and presentation. J.Immunol. 1998;160:3812- production IL-17 and antibody auto suppresses aBMT regel 25 3819. 107 regel 26 26. Broere F, Wieten L, Klein Koerkamp EI et al. Oral or nasal antigen induces regulatory T cells regel 27 that suppress arthritis and proliferation of arthritogenic T cells in joint draining lymph nodes. regel 28 J.Immunol. 2008;181:899-906. regel 29 27. Feuerer M, Beckhove P, Bai L et al. Therapy of human tumors in NOD/SCID mice with patient- regel 30 derived reactivated memory T cells from bone marrow. Nat.Med. 2001;7:452-458. regel 31 28. Zou L, Barnett B, Safah H et al. || one marrow is a reservoir for CD4+CD25+ regulatory T cells regel 32 that traffic through CXCL12/CXCR4 signals. Cancer Res. 2004;64:8451-8455. regel 33 29. Lubberts E. IL-17/Th17 targeting: on the road to prevent chronic destructive arthritis? Cytokine regel 34 2008;41:84-91. regel 35 regel 36 regel 1 30. Nistala K, Moncrieffe H, Newton KR et al. Interleukin-17-producing T cells are enriched in the regel 2 joints of children with arthritis, but have a reciprocal relationship to regulatory T cell numbers. regel 3 Arthritis Rheum. 2008;58:875-887. regel 4 31. Larche M, Wraith DC. Peptide-based therapeutic vaccines for allergic and autoimmune regel 5 diseases. Nat.Med. 2005;11:S69-S76. regel 6 32. Weiner HL. Oral tolerance for the treatment of autoimmune diseases. Annu.Rev.Med. regel 7 1997;48:341-351. regel 8 33. Roord ST, Zonneveld-Huijssoon E, Le T et al. Modulation of T cell function by combination regel 9 of epitope specific and low dose anticytokine therapy controls autoimmune arthritis. PLoS. regel 10 ONE. 2006;1:e87. regel 11 34. Bresson D, Togher L, Rodrigo E et al. Anti-CD3 and nasal proinsulin combination therapy regel 12 enhances remission from recent-onset autoimmune diabetes by inducing Tregs. J.Clin.Invest regel 13 2006;116:1371-1381. regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 5 Chapter regel 26 108 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Autologous bone marrow transplantation in autoimmune arthritis restores6 immune homeostasis through CD4+CD25+Foxp3+ regulatory T cells

Sarah T.A. Roord1, Wilco de Jager1, Louis Boon2, Nico Wulffraat1, Anton Martens3, Berent Prakken1*, Femke van Wijk1*.

* BP and FvW contributed equally to this manuscript

1Department of Pediatric Immunology, University Medical Center Utrecht, Wilhelmina Children’s Hospital, The Netherlands 2Bioceros B.V., Utrecht, The Netherlands 3Department of Hematology, University Medical Center Utrecht, Wilhelmina Children’s Hospital, Utrecht, The Netherlands

Blood. 2008 May 15;111(10):5233-41. regel 1 ABSTRACT regel 2 regel 3 Despite the earlier use of potent immunosuppressive or cytostatic drugs and the recent regel 4 emergence of biologicals as treatment for human autoimmune diseases (AID), some patients regel 5 still remain unresponsive to treatment. To those severely ill patients autologous bone marrow regel 6 transplantation (aBMT) is applied as a last resource, leading to disease remission in a majority regel 7 of patients. The underlying mechanism of action of aBMT is still largely unknown. regel 8 Here, we showed that regulatory T cells (Tregs) play a role in the natural disease course of regel 9 proteoglycan induced arthritis (PGIA) and in disease remission by aBMT. aBMT led to an initial regel 10 phase of rapid disease improvement corresponding with a relative increase in CD4+CD25+ regel 11 T cells. At this time the CD4+CD25+ cells did not yet show an increase in Foxp3 expression regel 12 and showed less potent suppression. After this initial improvement, disease relapsed but regel 13 stabilized at a level below the severity before aBMT. This second phase was actively regulated regel 14 by potently suppressive CD4+CD25+Foxp3+ Tregs. regel 15 This work provided further insight into the role of Tregs in restoration of the immune balance by regel 16 aBMT and can open the way to explore therapeutic interventions to further improve treatment regel 17 of AID and disease relapses. regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 6 Chapter regel 26 110 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 INTRODUCTION regel 1 regel 2 Human autoimmune diseases (AIDs) are often difficult to treat and the cause of major regel 3 disability. In recent years, treatment has become increasingly more aggressive, with the regel 4 earlier use of potent immunosuppressive or cytostatic drugs1-3. These methods are based on regel 5 a generalized and non-specific inhibition of immune response and inflammation, thus having regel 6 considerable side effects, and they are often not curative. In autoimmune arthritis, such as regel 7 reumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA), considerable progress has been regel 8 made by the introduction of immunotherapy with biological agents, which aims to interfere regel 9 with the molecular processes that are involved in the immune-mediated pathogenesis of regel 10 AID4-12. Despite the progress in treatment methods, some patients are still unresponsive. For regel 11 those severely ill children, autologous bone marrow transplantation (aBMT) has been the last regel 12 resource. The results of aBMT in JIA and other AIDs are remarkably good, since it induces drug- regel 13 free disease remission in a majority of JIA patients during a follow up of 12 to 60 months post regel 14 transplantation13;14. Obviously, it is questionable whether the achieved disease remission will regel 15 be long lasting, as the assumed genetic predisposition does not change in an autologous BMT regel 16 setting. Indeed, recently, this problem has arisen, since some of the patients who underwent regel 17 aBMT, have experienced severe disease relapse after many years of full disease remission (N. regel 18 Wulffraat, personal communication), suggesting that the arthritis-suppressive effect of aBMT regel 19 is not indefinite. regel 20 The underlying mechanism of action of aBMT in AID is still largely unknown. It has been regel 21 hypothesized that due to the stringent conditioning regimen, leading to severe aplasia, an regel 22 environment is created where renewed acquisition of self tolerance can be acquired after regel 23 rescue by autologous BM15;16. This self tolerance may be guided by regulatory T cells (Tregs), regel 24 and relapses may and probably will occur in time when this self tolerance is broken again. Tregs through homeostasis immune induces aBMT regel 25 Several types of Tregs have been identified to date, of which CD4+CD25+Foxp3+ Tregs have 111 regel 26 have been studied most extensively in the BMT setting (e.g. for their role in Graft versus Host regel 27 Disease)17. regel 28 Indeed, in previous work in JIA patients undergoing aBMT, we showed that aBMT induces a regel 29 restoration of the frequency of CD4+CD25brightFoxp3+ Tregs from severely reduced levels regel 30 before aBMT to normal levels after. The study suggested that 2 mechanisms may be in place: regel 31 on one hand (preferential) homeostatic proliferation of Tregs during the lymphopenic phase regel 32 of the reconstitution of the immune system and on the other hand renewed thymopoiesis of regel 33 naive Treg. Furthermore, a deviation of autoreactive T cells from a proinflammatory phenotype regel 34 before aBMT to a tolerant phenotype after aBMT was seen18. To further explore the mechanism regel 35 of aBMT, and develop strategies that may help to improve the efficacy and prevent late relapses regel 36 of the disease, it is necessary to turn to an experimental arthritis model. regel 1 Earlier pioneering work in animal models by van Bekkum provided the experimental basis for regel 2 the treatment of AID by autologous BMT. He succesfully treated adjuvant arthritis in Buffalo regel 3 rats by aBMT and defined conditioning regimens and levels of T cell depletion19. In addition, in regel 4 collagen induced arthritis, an antibody-mediated mouse model, a decrease of arthritis occured regel 5 after irradiation and BMT20. This work has provided pivotal insight; however, in order to study regel 6 the maintenance and the break of tolerance as seen in humans in the long term, we wanted to regel 7 turn to a model that is mediated by both T and B cells and that is relapsing and remitting, as is regel 8 the human disease. regel 9 Proteoglycan induced arthritis (PGIA) fits both these criteria. It can be induced in retired BALB/c regel 10 mice by two injections of human cartilage PG in the adjuvant DDA. This causes a progressive regel 11 polyarthritis that is initiated by a cross recognition of mouse PG by CD4+ T cells, however, regel 12 B cells are also crucial for the development of disease21;22. PGIA is extensively studied; has regel 13 clinical, immunologic and histopathological resemblance to human arthritis; and has a chronic regel 14 relapsing remitting course. We recently succeeded in setting up an aBMT model in PGIA (S.T.A.R. regel 15 et al, manuscript in preparation). The role of CD4+CD25+ Tregs in the natural disease course regel 16 as well as after aBMThas not yet been studied, and was a focus of the work presented here. regel 17 In this study we showed a role for Tregs in the natural disease course of PGIA, as well as a regel 18 role for Tregs in the restoration of the immune homeostasis after aBMT. We found that Tregs regel 19 were crucial for the development of PGIA, while after autologous BMT, a rapid decrease of regel 20 arthritis was observed, which could be (partially) reversed by in vivo depletion of Tregs. regel 21 Interestingly, the initial phase of clinical improvement corresponded with a relative increase regel 22 in CD4+CD25+ T cells, but not yet with an increase in Foxp3 expression in those cells, nor with regel 23 in vitro suppressive capacity. However, at a later stage, when disease activity stabilized, the regel 24 newly reinstated immune balance was regulated by potently suppressive CD4+CD25+Foxp3+ regel 25 6 Chapter Tregs. regel 26 112 regel 27 regel 28 METHODS regel 29 regel 30 Antigens, immunization, and assessment of arthritis regel 31 Mice regel 32 Female retired BALB/c mice were obtained from Charles River (Sulzfeld, Germany) and served regel 33 as recpients of autologous bone marrow. Male donor BALB/c mice of 7 weeks of age were also regel 34 obtained from Charles River and served as donors for autologous bone marrow. regel 35 The mice were kept in the animal facility of the Utrecht University under regular conditions until regel 36 aBMT, after which they were housed under sterile conditions in filter top cages and recieved distilled drinking water containing the antibiotic ciprofloxacin (100 µg/ml). The experiments regel 1 were approved by the Animal Experiment Committee of the Faculty of Veterinary Medicine regel 2 (Utrecht University). regel 3 regel 4 Proteoglycan preparation regel 5 Proteoglycan (PG) was purified from human articular cartilage and removed during knee joint regel 6 replacement surgery by 4 M guanidinium chloride extraction, and the human GAG side chains regel 7 were depleted by digestion with chondroitinase ABC as described previously22;23. The PG was regel 8 kindly provided by the Faculty of Veterinary Medicine in Utrecht, the Netherlands. regel 9 regel 10 Induction and assessment of arthritis regel 11 Arthritis was induced in BALB/c mice by two intraperitoneal injections of 0.4 mg human regel 12 deglycosylated PG in 2 mg of the synthetic adjuvant dimethyl dioctadecyl ammonium bromide regel 13 (DDA; Sigma Aldrich, Zwijndrecht, The Netherlands) on day 0 and 21. The onset and severity regel 14 of arthritis were assessed 3 times a week in a blinded fashion by a visual scoring system regel 15 as described previously22;24. In brief, the degree of joint swelling, redness and deformation regel 16 of each paw (scored from 0-4) was determined in order to express a total arthritis score, regel 17 with a maximum severity index of 16 per animal. In accordance with the Animal Experiment regel 18 Committee, mice were sacrificed when they achieved a total score of 12, or exhibited a regel 19 cumulative weight loss of 20% or a weight loss of more than 15% within 3 days. regel 20 regel 21 Treatment protocols regel 22 Bone Marrow Transplantation regel 23 On day 35 after the first injection of PG in DDA, recipient mice that had become arthritic were regel 24 conditioned by 7.5 Gy irradiation. Subsequently bone marrow transplantation (BMT) was Tregs through homeostasis immune induces aBMT regel 25 performed by intravenous injection of 2x106 BM cells from syngeneic donor animals that were 113 regel 26 at the same stage of disease. As the composition of pseudoautologous and autologous BM is regel 27 identical, the term autologous BMT (aBMT) is used for both throughout this paper. regel 28 BM suspensions were prepared as follows: donor mice were sacrificed and BM was harvested regel 29 by flushing tibia and femur with Iscove’s modified Dulbecco’s medium (IMDM) containing 2% regel 30 fetal calf serum (FCS), 2 mM L-glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin regel 31 (all from Gibco BRL, Grand Island, NY). Celles (2x106) were resuspended in 200 µl 0.2% BSA regel 32 before injection intravenously. Sick untreated mice were used as controls. regel 33 regel 34 regel 35 regel 36 regel 1 Anti CD25 administration regel 2 To assess the role of CD4+CD25+ Tregs in the natural course of PGIA, anti CD25 was regel 3 administered to animals that had recieved 2 PG injections on day 0 and 21, but had not yet regel 4 developed arthritis. Two doses of 200-250 µg of the anti CD25 Ab PC61 (kindly provided by L. regel 5 Boon) were administered on days 35 and 42 after the initial PG injection. regel 6 To assess the role of CD4+CD25+ Tregs in the disease course following aBMT, 250 or 200 µg of regel 7 the anti CD25 mAb PC61 was administered to BALB/c mice undergoing aBMT on the day of BMT regel 8 (day 35) and on day 42, and the dose dependent effect was assessed. regel 9 regel 10 In vitro assays regel 11 Repopulation white blood cell count regel 12 On a weekly basis following aBMT, blood was drawn from the tail vein and collected in heparin- regel 13 containing tubes. Subsequently, the white blood cell (WBC) count was measured on a Coulter regel 14 cell counter (Beckman Coulter, Hialeah, FL). regel 15 regel 16 Repopulation of CD3+, CD4+, CD4+CD25+ cells in peripheral blood, spleen and/or lymph nodes regel 17 by FACS analysis regel 18 To assess repopulation in peripheral blood, on a weekly basis following aBMT, blood was regel 19 drawn from the tail and collected in heparin-containing tubes. Erythrocytes were lysed regel 20 during 15 minutes in the dark in fluorescence activated cell sorting (FACS) lysing solution (BD regel 21 Pharmingen, San Diego, CA). regel 22 To assess repopulation in spleen and lymph nodes (LNs), mice were sacrificed on day 42, regel 23 day 55, and day 81, and cells were mashed through a 70 µm cell strainer (BD Biosciences, regel 24 San Jose, CA). Repopulation of cells was assessed by FACS analysis and FACS staining was regel 25 6 Chapter performed as follows: cells were washed twice in FACS buffer (PBS + 2% FCS). Non-specific regel 26 114 staining was limited by incubating the cells for 15 min. in 2% Normal Rat Serum (NRS). regel 27 Subsequently cells were stained with one or more of the following surface marker antibodies: regel 28 anti mouse CD3ε PercP (clone 145-2C11), CD4 PercP (RM4-5), CD25 FITC (PC61.5), CD44 FITC regel 29 (Pgp-1, Ly-24, IM7), CD45RB PE (16A) respectively (all from BD Pharmingen) for 30 minutes at regel 30 4°C. Unbound antibodies were washed away by 2 washes with FACS buffer. For Foxp3 analysis, regel 31 cells were incubated in Fix/perm concentrate diluted 1:4 in Fix/perm diluent (eBioscience) regel 32 overnight. The next day, they were washed twice in Perm buffer (eBioscience) and blocked regel 33 in 2% NRS. Subsequently, the cells were stained with Foxp3 APC (clone FJK-16s, eBioscience) regel 34 for 30 minutes at 4°C. Then they were washed twice in Perm buffer and finally resuspended in regel 35 FACS buffer to be acquired right away on a FACS Calibur machine. regel 36 Suppression assays regel 1 On day 42 and day 55 after the first PG/DDA injection, mice were sacrificed and spleens were regel 2 harvested. Cells were mashed through a 70 µm cell strainer (BD Biosciences) and stained regel 3 by anti mouse CD4 PercP and anti mouse CD25 APC (clone RM4-5 and PC61 respectively; regel 4 both BD Pharmingen). CD4+CD25- cells (10.000) were sorted by FACS Aria directly into a 96 regel 5 well plate and CD4+CD25+ sorted cells were added at a ratio of 1:5 and 1:1. A CD4 depleted regel 6 fraction of spleen cells (using anti mouse CD4 micro beads and a single run on an LS column regel 7 [MACS; Miltenyi Biotec, Auburn, CA}) was irradiated at 35 Gy and served as a source for antigen regel 8 presenting cells (APCs). CD4+CD25+ and CD4+CD25- cells were cultured in IMDM supplemented regel 9 with 10% FCS, 2 mM L-glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5 x10- regel 10 5 M 2-mercaptoethanol in the presence of 50.000 APC’s and 1 µg/ml soluble anti CD3 (clone regel 11 145-2C11, BD Pharmingen) for 120 hrs. During the last 16-18 hours 1 µCi (0.037 MBq) 3H regel 12 (3H-TdR; GE health care, Little Chalfont, United Kingdom) was added per well and 3H uptake regel 13 was measured using a liquid scintillation beta counter. Proliferative responses were calculated regel 14 as the mean 3H incorporation (cpm) of triplicate wells. regel 15 regel 16 Statistical analysis regel 17 Differences in clinical scores were determined by a Student t test. A P value less than .05 was regel 18 considered significant. regel 19 regel 20 regel 21 RESULTS regel 22 regel 23 CD25+ T cells actively suppress arthritis in animals that are resistant to the regel 24 induction of PGIA despite a proper immunization protocol Tregs through homeostasis immune induces aBMT regel 25 The PGIA model has been described extensively by Glant et al and Glant and Mikecz21;22 as a 115 regel 26 relapsing remitting chronic disease. By 2 injections of human PG in the adjuvant DDA, arthritis regel 27 is induced in BALB/c mice within a few days after the second injection. However, a small regel 28 percentage of animals don’t develop arthritis within the first few days, although eventually regel 29 nearly 100% of animals develop the disease. The reason for this delay in disease induction regel 30 in some animals is not yet known. Either the injection of antigen in those animals may be regel 31 insufficient to induce disease efficiently or regulatory mechanisms may actively play a role regel 32 in suppressing disease. To assess whether CD25+ Tregs played a role, we tested the effect of regel 33 depletion of those cells by administration of an anti-CD25 mAb in vivo to animals that were regel 34 tolerant to the induction of PGIA despite 2 injections of PG in DDA. regel 35 regel 36 regel 1 Arthritis was induced on day 0 and day 21 by an injection of PG in DDA adjuvant. From day regel 2 21 onward, the arthritis score was assessed 3 times a week by a visual scoring system of regel 3 redness, swelling and deformities of the paws. On day 35, animals that were resistant to regel 4 arthritis induction received an intraperitoneal injection of the anti-CD25 mAb PC61, followed regel 5 by a second injection on day 42. regel 6 Remarkably, in the animals that did not develop arthritis despite a proper immunization regel 7 protocol, CD25 depletion was able to rapidly induce an even more severe arthritis, apparent regel 8 after just one dose of the CD25-depleting mAb (Figure 1). This indicates that active suppression regel 9 of arthritis by CD25+ Tregs is vital for the tolerance to disease induction. regel 10 regel 11   regel 12  regel 13   regel 14  regel 15  regel 16  regel 17  regel 18   regel 19  regel 20  regel 21  regel 22            regel 23  regel 24 regel 25 6 Chapter  regel 26 116  regel 27 Figure 1. Depletion of CD25+ cells in animals resistant to arthritis induction leads to a rapid  regel 28 induction of disease.  regel 29 Arthritis was induced by 2 injections of PG in DDA on day 0 and day 21. On day 35, animals that had not  regel 30 developed arthritis by then received 2 injections of the anti-CD25 mAb PC61 with a 1-week interval, and  regel 31 mean arthritis scores are shown (+ SEM; N=9). regel 32 regel 33 regel 34 regel 35 regel 36

  Depletion of CD4+CD25+ T cells interferes with long-term disease remission regel 1 after aBMT treatment regel 2 These data suggested that presence or absence of CD4+CD25+ T cells could influence the regel 3 disease severity in PGIA, thus providing a rationale for further studying the role of these regel 4 cells in PGIA after aBMT. Next, we wanted to assess the effect of aBMT in PGIA and the role regel 5 of CD4+CD25+ Tregs in the clinical effect seen after aBMT. We recently showed that PGIA is regel 6 inhibited by aBMT (S.T.A.R. et al, manuscript in preparation). regel 7 Arthritis was induced according to the protocol. On day 35, the mean arthritis score of all regel 8 recipient mice was 6.2, and animals resistant to arthritis induction were excluded. On this regel 9 day conditioning was performed by a lethal 7.5 Gy irradiation dose followed by bone marrow regel 10  transplant from sick syngeneic animals that were at the same stage of disease. Sick untreated regel 11   mice were used as controls. regel 12   As shown in Figure 2A, conditioning followed by aBMT on day 35 led to a rapid improvement regel 13  in disease scores, which reached its maximum effect by day 42. Fairly rapidly after aBMT, regel 14  relapses occurred in the animals. Interestingly, however, shortly after the initial relapse, by regel 15  day 55, the arthritis stabilized at a degree that was significantly lower than the arthritis of regel 16  untreated animals (P=.006). As a consequence, after aBMT, despite the relapse, the severity regel 17   of arthritis never returned to the level of before aBMT. regel 18  To assess whether CD25+ Tregs play a role in the long-term disease remission after aBMT, regel 19  those cells were depleted in vivo by administration of an anti-CD25 mAb at 2 different doses regel 20  (250 µg and 200 µg) on days 35 and 42. regel 21            Administration of anti-CD25 on the day of aBMT did not influence the initial arthritis suppressive regel 22  effect, independent of the dose of anti-CD25 given (Figure 2B). However, administration of anti- regel 23 CD25 one week after aBMT during the initial reconstitution phase had dramatic effects. After regel 24 anti-CD25 treatment, the clinical improvement of aBMT was rapidly lost in a dose dependent Tregs through homeostasis immune induces aBMT regel 25  fashion. After a dose of 250 g, the arthritis worsened to a level far beyond the arthritis severity 117 regel 26  µ of sick untreated animals. The effect of the lower anti-CD25 dose (200 g) was similar, though regel 27  µ less powerful. Altogether, these results imply a role for CD4+CD25+ Tregs in disease remission regel 28  after aBMT, albeit not in causing the initial clinical improvement, but during the subsequent regel 29  phase of clinical stabilization of the disease at a lower level than before aBMT. regel 30  regel 31 regel 32 regel 33 regel 34 regel 35 regel 36

    

             regel 1              regel 2   regel 3    regel 4     regel 5    regel 6      regel 7                regel 8  regel 9              regel 10 regel 11               regel 12     regel 13    regel 14   regel 15  regel 16  regel 17    regel 18  regel 19   regel 20   regel 21   regel 22  regel 23   Figure 2. Conditioning followed by autologous bone marrow transplantation leads to a rapid regel 24  decrease in the severity of arthritis: the clinical effect is reduced by depletion of CD25+ Chapter 6 Chapter regel 25  cells. regel 26 118 (A) The effect of aBMT in PGIA. Arthritis was induced by 2 injections of PG in DDA on day 0 and day 21. regel 27 On day 35, mice received a lethal irradiation dose of 7.5 Gy followed by autologous BMT from syngeneic  regel 28 donor animals at the same stage of the disease. Mean arthritis scores are shown (+ SEM; N=17).  regel 29 (B) The effect of anti-CD25 treatment after aBMT. Following aBMT on day 35, the anti-CD25 mAb PC61 was  regel 30 administered on day 35 and day 42 at 2 different doses: 250 µg (N=2) or 200 µg (N=6). Mean arthritis regel 31  scores are shown (+ SEM).  regel 32   regel 33 Steap increase in CD4+CD25+ T cells in peripheral blood shortly after aBMT regel 34 To get insight into the repopulation of CD4+CD25+ T cells post aBMT, blood was drawn on a regel 35 weekly basis. From day 50 onward, the white blood cell (WBC) count was assessed and from regel 36 day 43 onward, repopulation of CD3+, CD4+ and CD4+CD25+ T cells was measured in peripheral blood lymphocytes (PBLs) by FACS analysis. As to be expected, irradiation and aBMT led to an initial decrease in the WBC count, which recovered by day 57, at which time the WBC count had regel 1 returned to levels before aBMT (Figure 3A). CD3+ T cells increased initially on day 42, followed regel 2 by a clear decrease that lasted until the end of the experiment. As shown in Figure 3B, on day regel 3 42 the percentage of CD4+ cells of the lymphocyte population increased slightly, while within regel 4 this population, there was a clear steap relative increase in CD4+CD25+ cells. Thus, although regel 5 the WBC count after BMT treatment had recovered by day 57 in this model, CD3+, CD4+ and regel 6 CD4+CD25+ lymphocytes did not fully recover yet by the end of the observation period (43 regel 7 days after aBMT). Intriguingly, shortly after aBMT there was a relative increase in CD4+CD25+ regel 8 cells coinciding with the time of maximum clinical improvement. We next questioned whether regel 9 those CD4+CD25+ T cells were true Treg that may have been involved in the early recovery regel 10 following aBMT. regel 11 regel 12

            regel 13     regel 14    regel 15    regel 16   

 regel 17   regel 18 

  regel 19  regel 20                 regel 21

              regel 22      regel 23    regel 24  aBMT induces immune homeostasis through Tregs through homeostasis immune induces aBMT   regel 25

  119 regel 26    regel 27   regel 28                 regel 29     regel 30 Figure 3. Peripheral blood repopulation of total white blood cells (WBC), and CD3+, CD4+, and regel 31  CD4+CD25+ T cells after BMT. regel 32  (A) Repopulation of WBCs and T lymphocytes in PBLs. After aBMT, blood was drawn from the tail vein of regel 33  the mice on a weekly basis and the WBC count was determined on a Coulter cell counter as well as the regel 34  repopulation of the percentage of CD3+ T cells by FACS, shown (+ SEM). regel 35 (B) Repopulation of CD4+ and CD4+CD25+ T cells in PBLs. FACS analysis on PBLs was performed on regel 36 repopulation of the percentage of CD4+ and CD4+CD25+ T cells, shown (+ SEM).

  regel 1 CD4+CD25+Foxp3+ Tregs in spleen and local lymph nodes reach a maximum regel 2 relative increase at the time of clinical stabilization of disease after aBMT regel 3 Obviously, the presence of CD4 and CD25 on T cells is not a satisfactory characterization of regel 4 Tregs, while also the peripheral blood may not be the best tissue to monitor reacquired immune regel 5 tolerance following aBMT. We therefore in subsequent experiments assessed the presence of regel 6 CD4+CD25+ Tregs locally in draining lymph nodes (LNs) and spleen, by applying Foxp3 as an regel 7 additional marker. regel 8 For this purpose, 3 mice per group were sacrificed before aBMT and on day 42, day 55 and regel 9 day 81. By FACS analysis, the percentage of CD25+ and CD25+Foxp3+ T cells within the CD4+ regel 10 population was measured as well as the percentage of Foxp3+ cells within the CD4+CD25+ T regel 11 cell population. As can be seen in Figure 4A, after aBMT there was a clear increase by day 42 regel 12 of CD25+ cells within the CD4+ lymphocyte population in both LNs and spleen. This paralelled regel 13 the increase in CD4+CD25+ T cells in peripheral blood documented before. The percentage of regel 14 Foxp3+ cells within the CD4+ T cells was, however, not increased. Within the CD4+CD25+ T cell regel 15 population, even a relative decrease of Foxp3+ cells was found (Figure 4B). This indicates that regel 16 at this early time point post BMT not all CD4+CD25+ T cells display a regulatory phenotype. regel 17 By day 55, a peak in CD25 expression was reached, now also corresponding with an increase regel 18 and peak in Foxp3 expression within the CD4+ T cells, especially in the LNs (Figure 4A). regel 19 Accordingly, the relative expression of Foxp3 within the CD4+CD25+ T cell population also regel 20 showed an increase (Figure 4B). These results indicate that by day 55 most of the CD4+CD25+ regel 21 T cells (re)gained a regulatory phenotype. regel 22 Thus, at the time of maximum clinical improvement, there is a marked relative increase in regel 23 CD4+CD25+ T cells not only in PBLs, but also in LNs and spleen. A large proportion of these regel 24 cells however do not express Foxp3, while two weeks later, at the time of clinical stabilization, regel 25 6 Chapter most CD25+ cells do express Foxp3. Combined with the time-dependent effects of blocking regel 26 120 CD25, shown in Figure 2B, this suggests that at day 55 indeed CD4+CD25+ T cells have (re) regel 27 gained a regulatory phenotype and may be involved in the clinical stabilisation of the disease. regel 28 To further investigate whether these CD25+ T cells are indeed Tregs, their functionality was regel 29 tested in vitro in a classical suppression assay. regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36  

                                                 

         

                                            

          regel 1              regel 2           regel 3 

          regel 4          regel 5             regel 6     

   regel 7       regel 8 

               regel 9                      regel 10         regel 11  

        regel 12

       regel 13              regel 14    

     regel 15              regel 16             regel 17      regel 18   regel 19      regel 20         regel 21       regel 22  regel 23      regel 24         Tregs through homeostasis immune induces aBMT   regel 25    

  121 regel 26            regel 27   regel 28    regel 29  Figure 4. After aBMT, the early relative increase in CD4+CD25+ T cells in spleen and lymph   nodes is initially not accompanied by an increase in Foxp3-expressing cells. regel 30          Repopulation of CD4+CD25+Foxp3+ cells in spleen and lymph nodes (LNs) regel 31  (A) Before BMT and on days 35, 42, 55, and 81, N=3 animals were sacrificed per group, and FACS regel 32  analysis was performed on the relative amount of CD25+ and CD25+Foxp3+ cells within the CD4+ T cell regel 33  population, shown (+ SEM). regel 34  (B) The relative amount of Foxp3+ cells within the CD4+CD25+ T cell compartment was determined by regel 35 FACS, shown (+ SEM).  regel 36    

  regel 1 The suppressive capacity of the CD4+CD25+ cells is reduced at the time of regel 2 initial clinical improvement regel 3 Next, we assessed the functionality of the CD4+CD25+ T cells in vitro during the repopulation regel 4 phase after aBMT. CD4+CD25+ Tregs are in general anergic in vitro and should be capable of regel 5 suppressing proliferation of CD4+CD25- T effector (Teff) cells in a contact dependent manner regel 6 25. For this purpose, on day 42 and day 55 spleens were harvested from 3 mice per group; regel 7 CD4+CD25+ T cells and CD4+CD25- T cells were FACS sorted and the suppressive capacity regel 8 of CD4+CD25+ T cells on the proliferation of CD4+CD25- T cells was tested in a classical regel 9 suppression assay. Figure 5A shows that on day 42, the CD4+CD25+ cells were less potent regel 10 suppressors at a 1:5 ratio (mean 25.9% suppression vs 79.5% suppression by control animals). regel 11 The suppression at a 1:5 ratio was fully restored by day 55. When added at a 5 times higher regel 12 ratio (Tsuppressor/Teff 1:1) CD4+CD25+ T cells were able to suppress effector T cells at both regel 13 time points similar to normal controls. regel 14 In addition, at day 42, CD4+CD25+ T cells were significantly less anergic to stimulation by regel 15 anti CD3 (P=.01) (Figure 4B), while at day 55 CD4+CD25+ cells were again fully anergic. Thus, regel 16 altogether the lack of anergy and less potent suppressive capacity at day 42 underscore regel 17 that at this time the CD4+CD25+ cells can not be considered full T regulatory cells. These regel 18 observations match the lack of Foxp3 expression in CD4+CD25+ cells during the first phase regel 19 of immune reconstitution (day 42). This lack of Foxp3 expression had restored by day 55, the regel 20 time of clinical stabilisation of disease, as had the functionality of the Tregs. regel 21 regel 22 The initial repopulation phase is dominated by memory Foxp3+ T cells, but this regel 23 is reversed during the second phase, when naive Foxp3+ T cells arise regel 24 Lastly, we wanted to assess whether the repopulating CD4+Foxp3+ cells were of a naive or regel 25 6 Chapter memory phenotype. Therefore, FACS analysis was performed on spleens and LN cells prior to regel 26 122 aBMT and at days 42, 55 and 81. The percentage of CD44high (memory) and CD45RBhigh (naive) regel 27 cells was determined within the CD4+ T cell and CD4+Foxp3+ T cell population. regel 28 Prior to aBMT, both in spleen and LNs, the ratio between memory and naive cells was dominated regel 29 by naive cells (Figure 6A). Shortly after aBMT this ratio changed, due to a steap relative increase regel 30 in memory cells corresponding with a relative decrease in naive cells. This predominance in regel 31 memory cells persisted until day 55, after which the contribution of the naive cell population regel 32 emerged, and was back to levels it had before aBMT by day 81 (Figure 6A). Memory and naive regel 33 CD4+Foxp3+ cells showed a similar trend, with a relative increase in memory Tregs and a regel 34 relative decrease of naive Tregs shortly after aBMT. Naïve Foxp3 cells recuperated faster (by regel 35 day 55) compared to the overall naive cell population (day 81). regel 36   All together, T cells during the initial repopulation phase were of a memory phenotype, but regel 1 regel 2 during the second phase, naive cells came up, first in the Foxp3+ Treg population. Thus the emergence of naive Tregs from the thymus coincided with the time of clinical stabilization. regel 3 regel 4 regel 5    regel 6         regel 7     regel 8       regel 9

     regel 10  

  regel 11      regel 12       regel 13      regel 14  regel 15    regel 16        regel 17       regel 18      regel 19  

   regel 20    regel 21    regel 22      regel 23    regel 24       aBMT induces immune homeostasis through Tregs through homeostasis immune induces aBMT regel 25  123 regel 26  Figure 5. Suppressive capacity of CD4+CD25+ T cells is reduced at day 42, but is recovered regel 27 by day 55. regel 28  (A) Suppressive capacity of CD4+CD25+ T cells. On day 42 and 55 after the first injection of PG/DDA regel 29 spleens were harvested. CD4+CD25+ and CD4+CD25- T cells were FACS sorted, and the suppressive regel 30 capacity of CD4+CD25+ cells on the proliferation of CD4+CD25- Teffector cells was tested in the presence regel 31 of APCs and anti-CD3. Shown is percent suppression of 3 mice per group (+ SEM). regel 32 (B) Anergy of CD4+CD25+ T cells. Sorted CD4+CD25+ T cells were stimulated with 1 μg/ml soluble anti regel 33 CD3 for 96 hours and proliferation was measured by 3H incorporation. Three mice per group were used. regel 34 Shown are stimulation indexes (SIs) of stimulation by anti-CD3 divided by culture in medium (+ SEM). regel 35 regel 36

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     regel 1   A        regel 2          regel 3            regel 4         regel 5        regel 6          regel 7         regel 8          regel 9           regel 10           regel 11        regel 12       regel 13         regel 14       regel 15           regel 16         regel 17     regel 18          regel 19  regel 20 B    regel 21 

  regel 22            regel 23           regel 24       regel 25 6 Chapter        regel 26 124     regel 27            regel 28             regel 29    regel 30       regel 31        regel 32        regel 33         regel 34               regel 35             regel 36        

       regel 1        Figure 6. The dominance of memory CD4+ and CD4+Foxp3+ T cells over naïve T cells shortly    regel 2     post BMT starts to reverse by day 55.          regel 3   Before BMT and on days 42, 55, and 81 spleens and LNs were harvested from sacrificed animals (N=3       high high regel 4   per group), and by FACS analysis the percentage of CD44 (memory) and CD45RB (naive) cells (A)     hi hi regel 5   as well as CD44 Foxp3+ (memory Tregs) and CD45RB Foxp3+ (naive Tregs) were determined within the       regel 6   CD4+ T cell population (B). Error bars represent SEM.       regel 7               regel 8   regel 9      DISCUSSION           regel 10        ABMT is an effective last treatment option for severy ill children suffering from therapy regel 11       regel 12   resistant JIA. It leads to drug-free disease remission in a majority of patients during a follow-up     13;14 regel 13   of 12-60 months after transplantation . Previous in vitro work in patients undergoing aBMT       bright regel 14   showed a restoration of the frequency of CD4+CD25 Foxp3+ T cells from severely reduced     regel 15     levels before aBMT to normal levels after, suggesting a role for Tregs in disease remission after     18 regel 16         aBMT . A role for Tregs in BMT was also found in an antibody-mediated experimental arthritis   regel 17   model, in which the addition of CD4+CD25+Tregs to the BM graft was found to have additional         20 regel 18  clinical effect . In this study, we aimed to explore whether CD4+CD25+Foxp3+ Tregs are  crucial for the mechanism of action of aBMT. For this purpose we turned to the relapsing regel 19    remitting experimental arthritis model PGIA. regel 20  PGIA can serve as a valid model for aBMT in arthritis, as irradiation followed by aBMT with BM regel 21           from syngeneic animals at the same stage of disease, leads to a lasting clinical improvement regel 22     regel 23  (S.T.A.R. et al, manuscript in preparation). We now show in this paper that CD4+CD25+Foxp3+            regel 24   Treg cells are crucial for the establishment of the renewed immune tolerance after aBMT. First,    Tregs through homeostasis immune induces aBMT regel 25  in vivo depletion of CD25+ cells by the anti-CD25 mAb PC61 shortly after aBMT led to a dose      dependent enhancement of disease. Second, the clinical stabilisation of arthritis corresponded 125 regel 26            regel 27  with a rise in CD4+CD25+Foxp3+ T cells that have increased in vitro suppressive capacity. At a     regel 28  closer look, the correlation between disease improvement and Tregs was less straightforward           then initially presumed. We found that the clinical improvement after aBMT could be split up regel 29     regel 30   into 2 phases: an initial rapid disease remission, followed by a second phase, consisting of a       relapse but stabilizing long-term at a degree of arthritis that was significantly less severe than regel 31       before aBMT. regel 32       The initial clinical improvement correlated with a relative increase in CD4+CD25+ T cells in regel 33               regel 34     PBLs, LNs, and spleen, again suggesting a critical role for Tregs. However, at this early time    regel 35    point, a large proportion of CD4+CD25+ T cells did not have a classical Treg phenotype, since            regel 36  they did not express the transcription factor Foxp3, nor were they potent suppressors in vitro.    

  regel 1 In fact, they were less anergic than their potently suppressive counterparts, which may explain regel 2 their decrease in functionality. regel 3 Thus, in the early phase after aBMT, at the time of maximum clinical improvement, CD4+CD25+ regel 4 cells mainly consisted of highly proliferative T cells, probably in a state of homeostatic regel 5 proliferation during lymphopenia after irradiation. The absence of disease at that time may regel 6 be explained by the relatively empty immune system after conditioning, where the lack of regel 7 inflammation and inflammatory Teff cells may help to explain the clinical picture. regel 8 This initial stage was followed by a phase of relapse and subsequent long-term stabilization of regel 9 disease. In contrast to the early phase, in this later stage, in vitro data pointed to a crucial role regel 10 for Tregs. At this stage, the CD4+ cells reached a peak in CD25 as well as Foxp3 expression, and regel 11 those cells now were anergic to nonspecific stimulation and had regained potent suppressive regel 12 capacity in vitro. The consequent clinical stabilization was long lasting, since it persisted until regel 13 the end of the experiment, and therefore suggested the establishment of a renewed immune regel 14 balance. regel 15 The exact nature and origin of the regulatory T cells that emerge post BMT has yet to be regel 16 determined. To date several types of Tregs have been identified25-27. They may be of donor or host regel 17 origin and may be naturally occurring Tregs, educated in the thymus, or peripherally induced regel 18 Tregs, educated by the encounter of disease triggering antigens in a lymphopenic environment. regel 19 Our data indicate that during the initial phase, the repopulating cells, including Foxp3+ cells, regel 20 were mainly of a memory phenotype. However, this phenotype did not yet determine their regel 21 origin. They may be “host” cells that have preferentially survived irradiation, as Tregs have regel 22 been described to preferentially survive irradiation in another experimental model28. On the regel 23 other hand, memory cells have been shown to preferentially home to the BM, and therefore regel 24 could be of “donor” origin as well29;30. Either way, at the time of clinical stabilization, the regel 25 6 Chapter contribution of naive Tregs came into play, indicating generation of novel naturally occuring regel 26 126 Tregs, which were clearly important in restoring the immune balance. Further experiments are regel 27 needed to determine whether cell therapy with Tregs may be of additional therapeutical value, regel 28 for example by addition of Tregs to the BM graft. regel 29 Lastly, we established a role for CD4+CD25+ Tregs in the natural disease course of PGIA that had regel 30 not yet been established previously21. In animals that initially were resistant to PGIA induction, regel 31 despite a proper immunization protocol, in vivo depletion of Tregs by the administration of regel 32 anti-CD25 led to a rapid and dramatic induction of arthritis. These data are in line with similar regel 33 observations in experimental autoimmune encephalomyelitis (EAE) as well as antigen induced regel 34 arthritis, where anti-CD25 administration led to enhancement of disease31-34. Furthermore, regel 35 CD25 depletion was found to inhibit the natural recovery from EAE, as was CD25 depletion regel 36 after recovery from EAE able to remove the resistance to reinduction of EAE observed in this model35. In our PGIA model, we hereby showed that not insufficient immunization but active regel 1 suppression of arthritis by CD25+ Tregs was responsible for the initial tolerance to disease regel 2 induction observed in a proportion of animals. regel 3 The establishment of a prominent role for Tregs in restoration of the immune balance by aBMT regel 4 provided here is a conceptual step forward. It may have important therapeutical translational regel 5 implications for improving clinical outcome by, for example, adding Tregs to the BM graft. regel 6 regel 7 regel 8 Reference List regel 9 regel 10 1. Ilowite NT. Current treatment of juvenile rheumatoid arthritis. Pediatrics 2002;109:109-115. regel 11 2. Onel KB. Advances in the medical treatment of juvenile rheumatoid arthritis. Curr.Opin.Pediatr. regel 12 2000;12:72-75. regel 13 3. Wulffraat NM, Kuis W. Treatment of refractory juvenile idiopathic arthritis. J.Rheumatol. regel 14 2001;28:929-931. regel 15 4. Haapasaari J, Kautiainen H, Hannula S, Pohjankoski H, Hakala M. Good results from combining regel 16 etanercept to prevailing DMARD therapy in refractory juvenile idiopathic arthritis. Clin.Exp. regel 17 Rheumatol. 2002;20:867-870. regel 18 5. Kietz DA, Pepmueller PH, Moore TL. Therapeutic use of etanercept in polyarticular course regel 19 juvenile idiopathic arthritis over a two year period. Ann.Rheum.Dis. 2002;61:171-173. regel 20 6. Lahdenne P, Vahasalo P, Honkanen V. Infliximab or etanercept in the treatment of children with regel 21 refractory juvenile idiopathic arthritis: an open label study. Ann.Rheum.Dis. 2003;62:245- regel 22 247. regel 23 7. Lovell DJ, Giannini EH, Reiff A et al. Etanercept in children with polyarticular juvenile rheumatoid regel 24 arthritis. Pediatric Rheumatology Collaborative Study Group. N.Engl.J.Med. 2000;342:763- Tregs through homeostasis immune induces aBMT regel 25 769. 127 regel 26 8. Lovell DJ, Giannini EH, Reiff A et al. Long-term efficacy and safety of etanercept in children regel 27 with polyarticular-course juvenile rheumatoid arthritis: interim results from an ongoing regel 28 multicenter, open-label, extended-treatment trial. Arthritis Rheum. 2003;48:218-226. regel 29 9. Quartier P, Taupin P, Bourdeaut F et al. Efficacy of etanercept for the treatment of juvenile regel 30 idiopathic arthritis according to the onset type. Arthritis Rheum. 2003;48:1093-1101. regel 31 10. Russo RA, Katsicas MM, Zelazko M. Etanercept in systemic juvenile idiopathic arthritis. Clin. regel 32 Exp.Rheumatol. 2002;20:723-726. regel 33 11. Schmeling H, Mathony K, John V et al. A combination of etanercept and methotrexate for the regel 34 treatment of refractory juvenile idiopathic arthritis: a pilot study. Ann.Rheum.Dis. 2001;60:410- regel 35 412. regel 36 regel 1 12. Takei S, Groh D, Bernstein B et al. Safety and efficacy of high dose etanercept in treatment of regel 2 juvenile rheumatoid arthritis. J.Rheumatol. 2001;28:1677-1680. regel 3 13. De K, I, Brinkman DM, Ferster A et al. Autologous stem cell transplantation for refractory regel 4 juvenile idiopathic arthritis: analysis of clinical effects, mortality, and transplant related regel 5 morbidity. Ann.Rheum.Dis. 2004;63:1318-1326. regel 6 14. Wulffraat NM, Sanders LA, Kuis W. Autologous hemopoietic stem-cell transplantation for regel 7 children with refractory autoimmune disease. Curr.Rheumatol.Rep. 2000;2:316-323. regel 8 15. Sykes M, Nikolic B. Treatment of severe autoimmune disease by stem-cell transplantation. regel 9 Nature 2005;435:620-627. regel 10 16. Touraine JL, Roncarolo MG, Raudrant D et al. Induction of transplantation tolerance in humans regel 11 using fetal cell transplants. Transplant.Proc. 2005;37:65-66. regel 12 17. Le NT, Chao N. Regulating regulatory T cells. Bone Marrow Transplant. 2007;39:1-9. regel 13 18. De K, I, Vastert B, Klein M et al. Autologous stem cell transplantation for autoimmunity induces regel 14 immunologic self-tolerance by reprogramming autoreactive T cells and restoring the regel 15 CD4+CD25+ immune regulatory network. Blood 2006;107:1696-1702. regel 16 19. van Bekkum DW. Experimental basis for the treatment of autoimmune diseases with regel 17 autologous hematopoietic stem cell transplantation. Bone Marrow Transplant. 2003;32 Suppl regel 18 1:S37-S39. regel 19 20. Morgan ME, Flierman R, van Duivenvoorde LM et al. Effective treatment of collagen-induced regel 20 arthritis by adoptive transfer of CD25+ regulatory T cells. Arthritis Rheum. 2005;52:2212- regel 21 2221. regel 22 21. Glant TT, Finnegan A, Mikecz K. Proteoglycan-induced arthritis: immune regulation, cellular regel 23 mechanisms, and genetics. Crit Rev.Immunol. 2003;23:199-250. regel 24 22. Glant TT, Mikecz K. Proteoglycan aggrecan-induced arthritis: a murine autoimmune model of regel 25 6 Chapter rheumatoid arthritis. Methods Mol.Med. 2004;102:313-338. regel 26 128 23. Glant TT, Cs-Szabo G, Nagase H, Jacobs JJ, Mikecz K. Progressive polyarthritis induced in regel 27 BALB/c mice by aggrecan from normal and osteoarthritic human cartilage. Arthritis Rheum. regel 28 1998;41:1007-1018. regel 29 24. Hanyecz A, Berlo SE, Szanto S et al. Achievement of a synergistic adjuvant effect on arthritis regel 30 induction by activation of innate immunity and forcing the immune response toward the Th1 regel 31 phenotype. Arthritis Rheum. 2004;50:1665-1676. regel 32 25. Bluestone JA, Abbas AK. Natural versus adaptive regulatory T cells. Nat.Rev.Immunol. regel 33 2003;3:253-257. regel 34 26. Bach JF, Francois BJ. Regulatory T cells under scrutiny. Nat.Rev.Immunol. 2003;3:189-198. regel 35 27. Bluestone JA, Boehmer H. Regulatory T cells. Semin.Immunol. 2006;18:77. regel 36 28. Benard A, Ceredig R, Rolink AG. Regulatory T cells control autoimmunity following syngeneic regel 1 bone marrow transplantation. Eur.J.Immunol. 2006;36:2324-2335. regel 2 29. Feuerer M, Beckhove P, Bai L et al. Therapy of human tumors in NOD/SCID mice with patient- regel 3 derived reactivated memory T cells from bone marrow. Nat.Med. 2001;7:452-458. regel 4 30. Zou L, Barnett B, Safah H et al. Bone marrow is a reservoir for CD4+CD25+ regulatory T cells regel 5 that traffic through CXCL12/CXCR4 signals. Cancer Res. 2004;64:8451-8455. regel 6 31. Montero E, Nussbaum G, Kaye JF et al. Regulation of experimental autoimmune regel 7 encephalomyelitis by CD4+, CD25+ and CD8+ T cells: analysis using depleting antibodies. regel 8 J.Autoimmun. 2004;23:1-7. regel 9 32. Zhang X, Koldzic DN, Izikson L et al. IL-10 is involved in the suppression of experimental regel 10 autoimmune encephalomyelitis by CD25+CD4+ regulatory T cells. Int.Immunol. 2004;16:249- regel 11 256. regel 12 33. Frey O, Petrow PK, Gajda M et al. The role of regulatory T cells in antigen-induced arthritis: regel 13 aggravation of arthritis after depletion and amelioration after transfer of CD4+CD25+ T cells. regel 14 Arthritis Res.Ther. 2005;7:R291-R301. regel 15 34. Stephens LA, Gray D, Anderton SM. CD4+CD25+ regulatory T cells limit the risk of autoimmune regel 16 disease arising from T cell receptor crossreactivity. Proc.Natl.Acad.Sci.U.S.A 2005;102:17418- regel 17 17423. regel 18 35. McGeachy MJ, Stephens LA, Anderton SM. Natural recovery and protection from autoimmune regel 19 encephalomyelitis: contribution of CD4+CD25+ regulatory cells within the central nervous regel 20 system. J.Immunol. 2005;175:3025-3032. regel 21 regel 22 regel 23 regel 24

aBMT induces immune homeostasis through Tregs through homeostasis immune induces aBMT regel 25 129 regel 26 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 6 Chapter regel 26 130 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Regulatory T cells in autologous stem cell transplantation for7 autoimmune disease

F. van Wijk, S. T. Roord, B. Vastert, I. de Kleer, N. Wulffraat, and B. J. Prakken

Department of Pediatric Immunology, University Medical Center Utrecht, Wilhelmina Children’s Hospital, Utrecht, the Netherlands

Autoimmunity. 2008 Oct 28:1 regel 1 ABSTRACT regel 2 regel 3 Since a decade autologous stem cell transplantation (ASCT) is successfully performed to regel 4 treat patients with severe autoimmune disease. However, the mechanism of action of this regel 5 intervention remains largely unknown. Scarce data from animal studies and human clinical regel 6 trials indicate that, besides extensive immune ablation, restoration of regulatory immune regel 7 networks is of critical importance. This review focuses on the role of naturally occurring and regel 8 induced regulatory T cells in controlling immune reconstitution and restoration of immune regel 9 tolerance and in preventing relapses of disease following ASCT. regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 7 Chapter regel 26 132 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 INTRODUCTION regel 1 regel 2 Standard therapeutic strategies for autoimmune disorders are based on generalized regel 3 immunosuppression and immunomodulation. This non-specific inhibition of immune reactivity regel 4 and inflammation results in considerable side effects, and does not lead to a permanent regel 5 cure of the disease. Although in the past decade considerable progress has been made by regel 6 the introduction of immunotherapy with biological agents, some patients do not respond or regel 7 relapse despite continuing therapy. Theoretical considerations, animal data and anecdotal regel 8 evidence suggested some time ago that intensive immune-ablation followed by autologous regel 9 stem cell transplantation (ASCT) could re-establish normal immune reactivity in patients with regel 10 severe autoimmune disease. Meanwhile, ASCT has been shown to have the potential to induce regel 11 dramatic and long-term improvements in a range of autoimmune disorders. Originally, the regel 12 rationale of this strategy is based on the deletion of the auto-aggressive lymphocyte population regel 13 and the hypothesis that the newly development of lymphocyte populations may lead to regel 14 self-tolerance. However, the exact mechanisms remain largely unknown. Data on immune regel 15 regulation following ACST in humans are scarce due to practical reasons and also relatively few regel 16 data from experimental autoimmune models for ASCT are available. The current understanding regel 17 on the mechanisms that govern immune regulation during immune reconstitution is partly regel 18 derived from the (experimental) allogenic SCT setting. Altogether, present findings suggest regel 19 that regulatory T cells (Treg) are of crucial importance for immune regulation in allogeneic SCT1 regel 20 and possibly also in ASCT. In this review we will discuss the latest insights on potential roles regel 21 of Treg in the processes of immune reconstitution and re-establishment of immune tolerance regel 22 following ACST. regel 23 regel 24 Regulatory T cells transplantation cell in stem T cells Regulatory regel 25 Many studies in experimental models of autoimmune disease have demonstrated the 133 regel 26 importance of Treg in limiting the development of auto-aggressive immune responses and regel 27 autoimmune tissue damage and in promoting recovery from disease2. Accordingly, it has been regel 28 proposed that failure of Treg to maintain peripheral tolerance may be the underlying cause regel 29 of many autoimmune diseases in humans. Potential, non-mutually exclusive, mechanisms regel 30 include deficient generation of Treg, altered suppressive function of Treg and resistance to regel 31 suppression of effector T cells in the target tissue. Recent data from our group and others regel 32 indicate that intensive immunoablation followed by ACST may represent a mean to restore regel 33 effective Treg development and function. regel 34 regel 35 regel 36 regel 1 Background information on regulatory T cells regel 2 T cells with a regulatory capacity (both CD4 and CD8) were first described in the 1970s, when regel 3 they were referred to as ‘suppressor’ T cells3, but they fell out of favour by the late 1980s. In regel 4 the past decade a wave of research has rehabilitated the old concept of suppressor cells and regel 5 has brought important new insights into the field of immune tolerance. Several subsets of Treg regel 6 have been described and a distinction has been made between Treg that emerge during T cell regel 7 development in the thymus (naturally occurring Treg), and Treg that develop following antigen regel 8 encounter in the periphery (adaptive or induced Treg). Further subdivisions of these subsets regel 9 are based on the expression of surface markers and the production of cytokines. Phenotypic regel 10 markers, rather than cytokine profiles, have been used to define a subset of naturally occurring regel 11 Treg, the CD4+CD25+ Treg. These T cells, first described by Sakaguchi et al2,4, originate from the regel 12 thymus and account for around 5 to 10% of peripheral CD4+ T cells. They constitutively express regel 13 high levels of CD25 (IL-2α receptor), CTLA-4, and glucocorticoid-induced tumor necrosis factor regel 14 receptor (GITR). However, at present, the most distinctive marker is the transcription factor regel 15 Foxp3, which is predominantly expressed in CD4+CD25+ Treg and has a key function in their regel 16 development and function5. The CD4+CD25+ T cell repertoire is biased to recognize ‘self’ and regel 17 therefore this Treg subtype has been mainly associated with controlling self tolerance4,6. regel 18 Treg expressing Foxp3 are crucial in preventing auto-reactivity during the development regel 19 of the peripheral immune system, illustrated by mutations in the human gene encoding regel 20 Foxp3 that result in a fatal autoimmune disease known as IPEX (immune dysregulation, regel 21 polyendocrinopathy, enteropathy, X-linked) syndrome. In the scurfy mouse, a spontaneous regel 22 defect in the analogous mouse foxp3 gene induces a comparable phenotype. Recently it has regel 23 been shown that Foxp3+ Treg also play an important role in restraining autoimmunity and regel 24 maintaining homeostasis throughout life7 as complete depletion of Foxp3+ Treg in healthy adult regel 25 7 Chapter mice resulted in a fatal lymphoproliferative disorder, similar as seen in scurfy mice. Naturally regel 26 134 occurring Treg exert their regulatory activity via cell-cell contact-dependent mechanisms and/ regel 27 or soluble factors such as TGFβ and IL-108. regel 28 Adaptive or induced Treg arise from naïve precursors in the periphery after antigen-recognition regel 29 and can be differentiated in vitro and in vivo. Under certain conditions these cells can acquire regel 30 Foxp3 expression and become yet phenotypically and functionally indistinguishable from regel 31 naturally occurring Treg9. In addition, several inducible CD4+ Treg subsets have been identified regel 32 that do not express Foxp3. The best-known examples are Tr1 10 and Th3 cells11 that can be regel 33 induced in vivo following mucosal antigen application. They migrate to inflamed tissue and regel 34 peripheral sites and function in a bystander fashion through the release of suppressive regel 35 cytokines (IL-10 and TGFβ). regel 36 Mechanisms of immune tolerance induction following autologous stem cell regel 1 transplantation regel 2 One of the original concepts underlying ACST in autoimmune disease is the eradication of auto- regel 3 aggressive effector and memory T cells. High-dose conditioning indeed ablates to a very large regel 4 extent the patients’ existing immune repertoire, including pathogenic T cell clones12. However, regel 5 elimination of host T cells is never complete and there is a reasonable risk of persisting regel 6 auto reactive T cells after conditioning. In addition, auto reactive T cells may be re-infused regel 7 with the graft. In experimental models of autologous bone marrow (BM) transplantation regel 8 lymphocytes present in the graft have been shown to contribute to relapses of autoimmune regel 9 disease13,14. To minimize this risk in humans, T cells are often depleted from the auto-graft regel 10 before infusion but this may also have important disadvantages. Extensive T cell depletion regel 11 of the auto-graft delays engraftment, enhances the risk of infections, and is associated with regel 12 the life-threatening complication macrophage activation syndrome15. Hence, it remains to regel 13 be determined whether aggressive conditioning and T cell depleting regimens have better regel 14 efficacy in re-establishing immune tolerance. Importantly, despite the expected occurrence regel 15 of auto reactive T cells, ASCT induces stable remission in a substantial proportion of patients regel 16 with severe autoimmune disease16. This is not seen with other strong immunosuppressive regel 17 treatments. Therefore it can be hypothesized that there must be a significant contribution regel 18 from altered immune reconstitution after ASCT to the induction of long-term clinical remission. regel 19 The radical lymphodepletion in combination with the instant reduction in inflammation caused regel 20 by the extensive immunosuppressive conditioning regimen may open a window for re- regel 21 establishment of normal immunity and immune tolerance. The unique characteristics of Treg regel 22 under conditions of immune reconstitution may contribute to this process. regel 23 regel 24 Regulatory T cells control homeostatic proliferation transplantation cell in stem T cells Regulatory regel 25 During the early phase of immune reconstitution, residual T cells in the host and from the graft 135 regel 26 rapidly expand to reconstitute the T cell pool, a process termed homeostatic proliferation. In regel 27 the lymphopenic state, MHC recognition and IL-7 are required for proliferation of naïve T cells, regel 28 whereas proliferation of memory CD4+ T cells requires either IL-7 or MHC. Self-antigens and regel 29 probably also gut associated commensal organisms drive the MHC-dependent expansion of regel 30 the T cell pool, and the resulting repertoire is limited in diversity and prone in skewing17. The regel 31 peripheral T cell responses that occur during homeostatic proliferation are both quantitatively regel 32 and qualitatively distinct from normal responses. All CD4+ T cells rapidly acquire an activated regel 33 or memory phenotype and it has been recently shown that mice CD4+ T cells are able to regel 34 spontaneously differentiate into autoimmune-associated Th17 cells when transferred to a regel 35 lymphopenic host18. Together these data indicate that due to the massive proliferation and regel 36 regel 1 activation of auto reactive T cells, lymphopenia represents an immunologically delicate regel 2 environment that provides fertile ground for the possible loss of self-tolerance19. Lymphopenia- regel 3 associated autoimmune manifestations have indeed been described both in experimental regel 4 models and humans20. At the same time, most lymphopenic hosts do not develop autoimmunity regel 5 and even in individuals with a pre-existing autoimmune disease relatively few relapses occur regel 6 during the phase of homeostatic proliferation following ASCT. One of the explanations may be that regel 7 additional triggers such as immune activation caused by infection or the presence of inflamed regel 8 tissue are required for the loss of self-tolerance during immune reconstitution18,21. Another regel 9 important factor that is thought to regulate the process of homeostatic proliferation and the regel 10 preservation of self-tolerance is the presence of Treg. Indeed, studies in mice have established regel 11 that CD4+CD25+ Treg control homeostatic proliferation itself by affecting T cell proliferation regel 12 22,23, the survival of cells that have undergone proliferation and the functional differentiation regel 13 into cytokine producing effector/memory T cells24. Furthermore the CD4+CD25+/naïve CD4 T regel 14 cell ratio has been identified as one of the key factors that determine the homeostatic plateau of regel 15 CD4+ T cells25. In the absence of CD4+CD25+ Treg, acute homeostatic proliferation can induce regel 16 autoimmunity26, which confirms the contribution of Treg in preservation of self-tolerance regel 17 during homeostatic proliferation. Together, these observations (all derived from experimental regel 18 animal models) indicate that CD4+CD25+ Treg play an essential role in guiding and regulating regel 19 the process of immune reconstitution and thus may be involved in the restoration of immune regel 20 tolerance following ASCT for autoimmunity. regel 21 regel 22 Regulatory T cells are involved in preventing relapses of autoimmune disease following ASCT regel 23 In the context of autoimmunity and ASCT, an important question is whether the regulatory regel 24 networks that arise during immune reconstitution can prevent relapses of pre-existing regel 25 7 Chapter autoimmune disease. That the Treg network may play a role in preventing disease flares regel 26 136 following ASCT has been demonstrated in animal models of autoimmune disease. High regel 27 numbers of CD25+CD4+ T cells and elevated levels of Foxp3 mRNA were found in rats that regel 28 were prevented from EAE relapse following BMT27. In a model of collagen-induced arthritis regel 29 infusion of CD4+CD25+ Treg with the syngenic BM graft reduced subsequent disease regel 30 relapses28. Furthermore, CD4+CD25+ Treg depletion immediately after pseudo-autologous BM regel 31 transplantation can provoke instant recurrence of joint inflammation in a model of proteoglycan regel 32 induced arthritis (S. Roord et al., article in preparation). For obvious reasons very little data are regel 33 available from human studies. We have recently shown that in patients with systemic Juvenile regel 34 Idiopathic Arthritis (JIA) circulating CD4+CD25+ Treg numbers, expressing high levels of Foxp3 regel 35 mRNA, are restored by ASCT29. Even after long-term follow-up the frequency of CD4+CD25+ regel 36 Treg was increased in all but one patient compared to pre-treatment levels. The recovery of the Treg repertoire could be attributed to both homeostatic proliferation of pre-existing Treg and regel 1 the newly occurrence of thymus-derived Treg. This will be further discussed below. regel 2 regel 3 Reconstitution of the regulatory T cell pool following ASCT regel 4 A critical issue for better understanding of the role of Treg in ASCT is how reconstitution of Treg regel 5 is regulated. In essence, Treg reconstitute via the same pathways as described for CD4+ T cells. regel 6 Gavin et al. were the first to demonstrate that mouse CD4+CD25+ T cells were quite capable regel 7 of homeostatic expansion in a lymphopenic environment despite their unresponsiveness to regel 8 strong antigenic stimuli in vitro and in vivo30. Kinetics and dependence on MHC class II were regel 9 shown to be similar to naïve CD4+CD25- T cells. It has also been suggested that there may regel 10 even be preferential expansion of Treg during homeostatic proliferation29,31, but this remains regel 11 controversial. The origin of the proliferating Treg may be diverse. They may (selectively) have regel 12 survived conditioning31,32, or may have been infused with the graft. Furthermore conversion regel 13 of mice CD4+CD25- T cells into functional CD4+CD25+ Treg during lymphopenia-induced regel 14 proliferation has been demonstrated33,34. These findings conflict with the more recent regel 15 observation that Foxp3-negative CD4 T cells do not convert into Foxp3+ T cells after transfer regel 16 to lymphopenic hosts35. regel 17 It is important to realize that collection and interpretation of data on Treg induction and regel 18 expansion is hampered by differences between mice and men and the lack of definitive Treg regel 19 markers. In mice, the Treg pool seems to be accurately defined by the expression of both CD25 regel 20 and Foxp3, whereas in humans these markers are not conclusive36. Furthermore, activated T regel 21 cells (which are abundantly present during homeostatic proliferation) also express high levels regel 22 of CD25 (mice and humans) and possibly even Foxp3 (only shown in humans)37. This makes regel 23 it hard to distinguish Treg from activated T cells and may explain conflicting data. In addition, regel 24 both in mice and men, very little is known about the mechanisms of induction, phenotype, and transplantation cell in stem T cells Regulatory regel 25 functional capacities of peripherally-generated Foxp3-expressing T cells. Finally, the low CD4 137 regel 26 T cell numbers found in the early phase of immune reconstitution limit the use of functional regel 27 assays. These assays are indispensable to confirm the identity and determine the regulatory regel 28 capacities of presumed Treg. Further studies in humans and experimental models are needed regel 29 to reveal the mechanisms of Treg (subset) development and expansion in lymphopenic regel 30 but also ‘normal’ conditions. In this context the role of the thymus in re-generating the Treg regel 31 compartment is following ASCT is of clear importance. regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 The role of the thymus in (regulatory) T cell reconstitution regel 2 After the initial phase of proliferating pre-existing T cells, naïve T cells start to appear in the regel 3 blood three to nine months after ASCT. These cells are derived from the thymus and are regel 4 required for the complete reconstitution of T cell immunity. Naïve CD4+CD25+ T cells are regel 5 also generated in the thymus and appear in the same time period. We showed in a cohort of regel 6 systemic JIA patients that these cells contain extremely high levels of Foxp3, about 10 times regel 7 higher than levels found in CD4+CD25+ T cells of healthy children. Furthermore, a correlation regel 8 was found between relapse of disease (90% of all relapses occurred in the first nine months of regel 9 ASCT) and the recovery of naïve thymus-derived Treg that reached a maximum at nine months. regel 10 These findings indicate that for long-term remission new generation of naturally occurring Treg regel 11 may be essential. This is supported by recent data suggesting that in autoimmune disease regel 12 there may be a shift in the homeostatic composition of Treg subsets due to a reduced thymus- regel 13 dependent de novo generation of Treg38. So another reason of tolerance induction following regel 14 ASCT might be the restored thymic output of Treg. regel 15 Besides the production of thymus-derived Treg, the de novo generation of the T cell repertoire regel 16 in general is thought to promote tolerance. Increased thymopoiesis found following ASCT might regel 17 contribute to the normalization of naïve T cell homeostasis12. In addition, after ASCT a broader regel 18 clonal diversity and extensive renewal of clonal specificities is found compared to pre-therapy, regel 19 which clearly indicates a qualitative change in the naïve T cell repertoire12. This qualitative regel 20 change is confirmed by data from our group demonstrating that ACST induces the generation of regel 21 auto reactive T cells with a tolerant phenotype in contrast to the pro-inflammatory phenotype regel 22 of auto reactive T cells found before treatment29. regel 23 Thymopoiesis is age-dependent and becomes limited after childhood due to thymic involution. regel 24 Although adult thymic output has been recently shown to be more substantial than initially regel 25 7 Chapter thought, there is a clear quantitative decline with age. Given the importance of the thymus in regel 26 138 the complete recovery of a broad T cell repertoire and the restoration of immune homeostasis regel 27 this may have strong implications for the clinical success of ACST. Indeed, children have been regel 28 shown to recover faster from ASCT than adults39. More extensive studies are required to reveal regel 29 the involvement of the thymus in the clinical efficacy of ACST and to explore the possibility of regel 30 therapeutic interventions that are able to increase the thymic output. regel 31 regel 32 Environmental factors influence the development and function of Treg regel 33 As discussed above, the long-term suppression of autoimmune symptoms following ACST regel 34 is associated with profound qualitative immunological changes including the de novo regel 35 generation of the T cell/Treg compartment. Of course the result is highly influenced by the regel 36 immune environment. Conversion or de novo generation of Treg is believed to occur only in a tolerogenic environment. Also the function of Treg is highly dependent on the local milieu, regel 1 which is illustrated by the inability of Treg to suppress chronic autoimmune inflammation. In regel 2 the synovial fluid of RA patients and in the cerebrospinal fluid of MS patients, both representing regel 3 the site of inflammation, an enrichment of Foxp3+ Treg is found 40-42 and these Treg seem to regel 4 exhibit normal intrinsic suppressive capacity. In these patients inflammation persists despite regel 5 the presence of high Treg numbers in the affected tissue, suggesting that Treg are unable to regel 6 suppress ongoing disease. The local inflammatory environment is thought to be responsible regel 7 for this situation. It has been shown for example that the inflammatory cytokines TNFα and IL-6 regel 8 can temporarily impair the peripheral generation and function of Treg43-45 and, probably more regel 9 importantly, render auto aggressive T cells resistant to Treg-mediated regulation44,46. These regel 10 findings indicate that Treg may only become efficient suppressors when the environmental regel 11 load of inflammatory mediators and the presence and activity of effector T cells are below a regel 12 certain threshold. Therefore the instant reduction in inflammation caused by the extensive regel 13 immunosuppressive conditioning regimen during ASCT may be of critical importance for regel 14 the restoration of a functional Treg network. At present it is unclear whether remaining regel 15 inflammatory seats hamper the induction of long-term remission but it is apparent that this regel 16 may represent one of the major risk factors for relapses. regel 17 regel 18 Relapses after establishment of long-term tolerance regel 19 While long-term remission can be achieved in autoimmune patients undergoing ACST, there regel 20 are indications that relapses may occur years after ASCT. Recently, two cases of late relapses regel 21 were reported in children that were treated with ASCT for refractory systemic and polyarticular regel 22 JIA (N. Wulffraat, personal communication). Recurrence of the disease was established seven regel 23 and eight years after ASCT. This suggests that although the immunological clock is reset by regel 24 ASCT, it cannot definitively prevent disease recurrence. This may be not surprising as the transplantation cell in stem T cells Regulatory regel 25 genetic make-up of these patients is not altered by the treatment and thus the pre-disposition 139 regel 26 for the development of an autoimmune disease remains. In time, multiple triggers from the regel 27 environment, such as infections, will add up and regulatory immune networks may become regel 28 misbalanced again. Finally, a threshold may be reached that initiates the development of regel 29 chronic inflammation in these susceptible individuals. regel 30 The appearance of autoimmune symptoms following a very long remission period could be regel 31 considered to be an unrelated de novo event. However, in the case of the two JIA patients, regel 32 exactly the same joints were affected before and (almost a decade) after ASCT. This regel 33 phenomenon of recurrence of disease in the same joints is commonly observed in JIA patients regel 34 that show relapses after a long drug-free and disease-free period induced by conventional regel 35 therapies47. These observations point to an important role of the local tissue environment regel 36     regel 1 in the recurrence of autoimmune disease. One reason may be that the synovium has been  regel 2  irreversibly altered as a consequence of chronic inflammation and tissue damage, making     regel 3 it susceptible to environmental triggers such as infections. In addition, ongoing (low) level  regel 4  inflammation and release of self-antigens may play a role. It is unclear whether, or to what       regel 5  extent, self-antigen depots reside after clearance of the inflammatory milieu. The presence of      regel 6 self-antigens might of course fuel the activation of auto aggressive T cells. On the other hand,           regel 7 one may also speculate that these self-antigens (such as heat shock proteins) are crucial in  regel 8  the re-establishment of specific tolerance and the induction of Treg. More research is required    regel 9   to elucidate tissue-specific immune responses following ASCT.  regel 10    regel 11 Conclusion and Future perspectives  regel 12 All together, the success of ASCT for autoimmunity is not only based on the loss of auto reactive     regel 13 T cell clones, but also on the complete re-assignment of imbalanced cellular and soluble         regel 14  networks, including Treg (for an overview see Figure 1). The Treg network plays an essential     regel 15 role during the phase of immune reconstitution in controlling homeostatic proliferation and     regel 16 the preservation of self-tolerance. Therefore Treg represent an attractive target for future       regel 17 therapeutic strategies in the context of ASCT. The period of immune re-establishment following  regel 18  ASCT may create a ‘window of opportunity’ for immunotherapy and the induction of permanent   regel 19 tolerance. It could, for example, be beneficial to facilitate the restoration of regulatory immune   regel 20 networks by the infusion of Foxp3+ Treg (together with the graft, or at later time-points). Several  regel 21 protocols have been set up to purify and expand these Treg ex vivo48,49 and to obtain sufficient   regel 22 numbers of cells. One of the disadvantages of Foxp3+ Treg may be their a-specific mode of regel 23 action, increasing the risks of side effects. Therefore, the induction of Treg that specifically regel 24 recognize self-antigens might represent a more potent strategy50. In this context, mucosal regel 25 7 Chapter application of self-antigens is explored as a therapy for autoimmune disease51,52 and several regel 26 140 candidate antigens have been identified53. The antigen-specific induction of tolerance may be regel 27 particularly effective following ASCT, due to the tolerogenic environment and reconstituting regel 28 immune system. Future mechanistic and clinical studies will provide further insights in the regel 29 possibilities of immunotherapy in ASCT. regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36      regel 1  regel 2      regel 3  regel 4        regel 5             regel 6     regel 7  regel 8      regel 9    regel 10  regel 11      regel 12     regel 13      regel 14         regel 15      regel 16  regel 17   regel 18    regel 19   regel 20 Figure 1. Overview in time of putative mechanisms of ASCT in the restoration of immune regel 21  tolerance. regel 22 regel 23 regel 24 Reference List transplantation cell in stem T cells Regulatory regel 25 141 regel 26 1. Salomon, B. L., M. Sudres, and J. L. Cohen. 2006. Regulatory T cells in graft-versus-host regel 27 disease. Springer Semin Immunopathol 28:25-29. regel 28 2. Sakaguchi, S. 2005. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in regel 29 immunological tolerance to self and non-self. Nat Immunol 6:345-352. regel 30 3. Katz, S. I., D. Parker, G. Sommer, and J. L. Turk. 1974. Suppressor cells in normal immunisation regel 31 as a basic homeostatic phenomenon. Nature 248:612-614. regel 32 4. Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995. Immunologic self-tolerance regel 33 maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). 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Effective treatment of relapsing experimental regel 23 autoimmune encephalomyelitis with pseudoautologous bone marrow transplantation. Bone regel 24 Marrow Transplant 18:1029-1034. regel 25 7 Chapter 14. van Bekkum, D. W. 2000. Conditioning regimens for the treatment of experimental arthritis regel 26 142 with autologous bone marrow transplantation. Bone Marrow Transplant 25:357-364. regel 27 15. De Kleer, I. M., D. M. Brinkman, A. Ferster, M. Abinun, P. Quartier, J. Van Der Net, R. Ten Cate, L. R. regel 28 Wedderburn, G. Horneff, J. Oppermann, F. Zintl, H. E. Foster, A. M. Prieur, A. Fasth, M. A. Van regel 29 Rossum, W. Kuis, and N. M. Wulffraat. 2004. Autologous stem cell transplantation for refractory regel 30 juvenile idiopathic arthritis: analysis of clinical effects, mortality, and transplant related regel 31 morbidity. Ann Rheum Dis 63:1318-1326. regel 32 16. Gratwohl, A., J. Passweg, C. Bocelli-Tyndall, A. Fassas, J. M. van Laar, D. Farge, M. Andolina, R. regel 33 Arnold, E. Carreras, J. Finke, I. Kotter, T. Kozak, I. Lisukov, B. Lowenberg, A. Marmont, J. Moore, regel 34 R. Saccardi, J. A. Snowden, F. van den Hoogen, N. M. Wulffraat, X. W. Zhao, and A. Tyndall. 2005. regel 35 Autologous hematopoietic stem cell transplantation for autoimmune diseases. Bone Marrow regel 36 Transplant 35:869-879. 17. Mackall, C. L., C. V. Bare, L. A. Granger, S. O. Sharrow, J. A. Titus, and R. E. Gress. 1996. Thymic- regel 1 independent T cell regeneration occurs via antigen-driven expansion of peripheral T cells regel 2 resulting in a repertoire that is limited in diversity and prone to skewing. J Immunol 156:4609- regel 3 4616. regel 4 18. Hirota, K., M. Hashimoto, H. Yoshitomi, S. Tanaka, T. Nomura, T. Yamaguchi, Y. Iwakura, N. regel 5 Sakaguchi, and S. Sakaguchi. 2007. T cell self-reactivity forms a cytokine milieu for regel 6 spontaneous development of IL-17+ Th cells that cause autoimmune arthritis. J Exp Med regel 7 204:41-47. regel 8 19. Baccala, R., and A. N. Theofilopoulos. 2005. The new paradigm of T-cell homeostatic regel 9 proliferation-induced autoimmunity. Trends Immunol 26:5-8. regel 10 20. Theofilopoulos, A. N., W. Dummer, and D. H. Kono. 2001. T cell homeostasis and systemic regel 11 autoimmunity. J Clin Invest 108:335-340. regel 12 21. Krupica, T., Jr., T. J. Fry, and C. L. Mackall. 2006. Autoimmunity during lymphopenia: a two-hit regel 13 model. Clin Immunol 120:121-128. regel 14 22. Annacker, O., R. Pimenta-Araujo, O. Burlen-Defranoux, T. C. Barbosa, A. Cumano, and A. Bandeira. regel 15 2001. CD25+ CD4+ T cells regulate the expansion of peripheral CD4 T cells through the regel 16 production of IL-10. J Immunol 166:3008-3018. regel 17 23. Hagen, K. A., C. T. Moses, E. F. Drasler, K. M. Podetz-Pedersen, S. C. Jameson, and A. Khoruts. regel 18 2004. A role for CD28 in lymphopenia-induced proliferation of CD4 T cells. J Immunol 173:3909- regel 19 3915. regel 20 24. Shen, S., Y. Ding, C. E. Tadokoro, D. Olivares-Villagomez, M. Camps-Ramirez, M. A. Curotto de regel 21 Lafaille, and J. J. Lafaille. 2005. Control of homeostatic proliferation by regulatory T cells. J Clin regel 22 Invest 115:3517-3526. regel 23 25. Almeida, A. R., N. Legrand, M. Papiernik, and A. A. Freitas. 2002. Homeostasis of peripheral regel 24 CD4+ T cells: IL-2R alpha and IL-2 shape a population of regulatory cells that controls CD4+ T transplantation cell in stem T cells Regulatory regel 25 cell numbers. J Immunol 169:4850-4860. 143 regel 26 26. McHugh, R. S., and E. M. Shevach. 2002. Cutting edge: depletion of CD4+CD25+ regulatory regel 27 T cells is necessary, but not sufficient, for induction of organ-specific autoimmune disease. J regel 28 Immunol 168:5979-5983. regel 29 27. Herrmann, M. M., S. Gaertner, C. Stadelmann, J. van den Brandt, R. Boscke, W. Budach, H. regel 30 M. Reichardt, and R. Weissert. 2005. Tolerance induction by bone marrow transplantation in a regel 31 multiple sclerosis model. Blood 106:1875-1883. regel 32 28. Morgan, M. E., R. Flierman, L. M. van Duivenvoorde, H. J. Witteveen, W. van Ewijk, J. M. van Laar, regel 33 R. R. de Vries, and R. E. Toes. 2005. Effective treatment of collagen-induced arthritis by regel 34 adoptive transfer of CD25+ regulatory T cells. Arthritis Rheum 52:2212-2221. regel 35 regel 36 regel 1 29. de Kleer, I., B. Vastert, M. Klein, G. Teklenburg, G. Arkesteijn, G. P. Yung, S. Albani, W. Kuis, regel 2 N. Wulffraat, and B. Prakken. 2006. Autologous stem cell transplantation for autoimmunity regel 3 induces immunologic self-tolerance by reprogramming autoreactive T cells and restoring the regel 4 CD4+CD25+ immune regulatory network. Blood 107:1696-1702. regel 5 30. Gavin, M. A., S. R. Clarke, E. Negrou, A. Gallegos, and A. Rudensky. 2002. Homeostasis and regel 6 anergy of CD4(+)CD25(+) suppressor T cells in vivo. Nat Immunol 3:33-41. regel 7 31. Komatsu, N., and S. Hori. 2007. Full restoration of peripheral Foxp3+ regulatory T cell pool by regel 8 radioresistant host cells in scurfy bone marrow chimeras. Proc Natl Acad Sci U S A 104:8959- regel 9 8964. regel 10 32. Condomines, M., P. Quittet, Z. Y. Lu, L. Nadal, P. Latry, E. Lopez, M. Baudard, G. Requirand, C. regel 11 Duperray, J. F. Schved, J. F. Rossi, K. Tarte, and B. Klein. 2006. Functional regulatory T cells regel 12 are collected in stem cell autografts by mobilization with high-dose cyclophosphamide and regel 13 granulocyte colony-stimulating factor. J Immunol 176:6631-6639. regel 14 33. Curotto de Lafaille, M. A., A. C. Lino, N. Kutchukhidze, and J. J. Lafaille. 2004. CD25- T cells regel 15 generate CD25+Foxp3+ regulatory T cells by peripheral expansion. J Immunol 173:7259- regel 16 7268. regel 17 34. Liang, S., P. Alard, Y. Zhao, S. Parnell, S. L. Clark, and M. M. Kosiewicz. 2005. Conversion of CD4+ regel 18 CD25- cells into CD4+ CD25+ regulatory T cells in vivo requires B7 costimulation, but not the regel 19 thymus. J Exp Med 201:127-137. regel 20 35. Wan, Y. Y., and R. A. Flavell. 2005. Identifying Foxp3-expressing suppressor T cells with a regel 21 bicistronic reporter. Proc Natl Acad Sci U S A 102:5126-5131. regel 22 36. Allan, S. E., S. Q. Crome, N. K. Crellin, L. Passerini, T. S. Steiner, R. Bacchetta, M. G. Roncarolo, and regel 23 M. K. Levings. 2007. Activation-induced FOXP3 in human T effector cells does not suppress regel 24 proliferation or cytokine production. Int Immunol 19:345-354. regel 25 7 Chapter 37. Wang, J., A. Ioan-Facsinay, E. I. van der Voort, T. W. Huizinga, and R. E. Toes. 2007. Transient regel 26 144 expression of FOXP3 in human activated nonregulatory CD4+ T cells. Eur J Immunol 37:129- regel 27 138. regel 28 38. Haas, J., B. Fritzsching, P. Trubswetter, M. Korporal, L. Milkova, B. Fritz, D. Vobis, P. H. Krammer, regel 29 E. Suri-Payer, and B. Wildemann. 2007. Prevalence of newly generated naive regulatory T cells regel 30 (Treg) is critical for Treg suppressive function and determines Treg dysfunction in multiple regel 31 sclerosis. J Immunol 179:1322-1330. regel 32 39. Small, T. N., E. B. Papadopoulos, F. Boulad, P. Black, H. Castro-Malaspina, B. H. Childs, N. Collins, regel 33 A. Gillio, D. George, A. Jakubowski, G. Heller, M. Fazzari, N. Kernan, S. MacKinnon, P. Szabolcs, regel 34 J. W. Young, and R. J. O’Reilly. 1999. Comparison of immune reconstitution after unrelated and regel 35 related T-cell-depleted bone marrow transplantation: effect of patient age and donor leukocyte regel 36 infusions. Blood 93:467-480. 40. van Amelsfort, J. M., K. M. Jacobs, J. W. Bijlsma, F. P. Lafeber, and L. S. Taams. 2004. CD4(+) regel 1 CD25(+) regulatory T cells in rheumatoid arthritis: differences in the presence, phenotype, regel 2 and function between peripheral blood and synovial fluid. Arthritis Rheum 50:2775-2785. regel 3 41. Ruprecht, C. R., M. Gattorno, F. Ferlito, A. Gregorio, A. Martini, A. Lanzavecchia, and F. Sallusto. regel 4 2005. Coexpression of CD25 and CD27 identifies FoxP3+ regulatory T cells in inflamed regel 5 synovia. J Exp Med 201:1793-1803. regel 6 42. Feger, U., C. Luther, S. Poeschel, A. Melms, E. Tolosa, and H. Wiendl. 2007. Increased frequency regel 7 of CD4+ CD25+ regulatory T cells in the cerebrospinal fluid but not in the blood of multiple regel 8 sclerosis patients. Clin Exp Immunol 147:412-418. regel 9 43. Bettelli, E., Y. Carrier, W. Gao, T. Korn, T. B. Strom, M. Oukka, H. L. Weiner, and V. K. Kuchroo. 2006. regel 10 Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regel 11 regulatory T cells. Nature 441:235-238. regel 12 44. Pasare, C., and R. Medzhitov. 2003. Toll pathway-dependent blockade of CD4+CD25+ T cell- regel 13 mediated suppression by dendritic cells. Science 299:1033-1036. regel 14 45. Valencia, X., G. Stephens, R. Goldbach-Mansky, M. Wilson, E. M. Shevach, and P. E. Lipsky. 2006. regel 15 TNF downmodulates the function of human CD4+CD25hi T-regulatory cells. Blood 108:253- regel 16 261. regel 17 46. Korn, T., J. Reddy, W. Gao, E. Bettelli, A. Awasthi, T. R. Petersen, B. T. Backstrom, R. A. Sobel, regel 18 K. W. Wucherpfennig, T. B. Strom, M. Oukka, and V. K. Kuchroo. 2007. Myelin-specific regulatory regel 19 T cells accumulate in the CNS but fail to control autoimmune inflammation. Nat Med 13:423- regel 20 431. regel 21 47. Szer, I. K., Y; Malleson, P; Southwood T., 2006. Arthritis in children & adolescents, first edition, regel 22 Oxford University Press, New York. regel 23 48. Hoffmann, P., R. Eder, L. A. Kunz-Schughart, R. Andreesen, and M. Edinger. 2004. Large-scale in regel 24 vitro expansion of polyclonal human CD4(+)CD25high regulatory T cells. Blood 104:895- transplantation cell in stem T cells Regulatory regel 25 903. 145 regel 26 49. Kreijveld, E., H. J. Koenen, L. B. Hilbrands, and I. Joosten. 2006. Ex vivo expansion of human regel 27 CD4+ CD25high regulatory T cells from transplant recipients permits functional analysis of regel 28 small blood samples. J Immunol Methods 314:103-113. regel 29 50. Bluestone, J. A. 2005. Regulatory T-cell therapy: is it ready for the clinic? Nat Rev Immunol regel 30 5:343-349. regel 31 51. Samsom, J. N. 2004. Regulation of antigen-specific regulatory T-cell induction via nasal and regel 32 oral mucosa. Crit Rev Immunol 24:157-177. regel 33 52. Albani, S., and B. Prakken. 2006. T cell epitope-specific immune therapy for rheumatic regel 34 diseases. Arthritis Rheum 54:19-25. regel 35 regel 36 regel 1 53. Kamphuis, S., W. Kuis, W. de Jager, G. Teklenburg, M. Massa, G. Gordon, M. Boerhof, G. T. Rijkers, regel 2 C. S. Uiterwaal, H. G. Otten, A. Sette, S. Albani, and B. J. Prakken. 2005. Tolerogenic immune regel 3 responses to novel T-cell epitopes from heat-shock protein 60 in juvenile idiopathic arthritis. regel 4 Lancet 366:50-56. regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 7 Chapter regel 26 146 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Summary8 and general discussion regel 1 SUMMARY regel 2 regel 3 The current treatment of JIA and RA consists of a generalized suppression of the immune regel 4 system and has become increasingly aggressive over the past decades with the earlier use regel 5 of potent corticosteroids and DMARDs. It has led to an improvement of the outcome of those regel 6 patients, however the drugs need to be administered continuously in order to remain effective, regel 7 and do not lead to a cure. The generalized immunosuppression also comes with a prize: regel 8 damaging side effects. Furthermore, some patients remain unresponsive to the currently regel 9 available treatment methods and for those severely ill children, autologous Bone Marrow regel 10 Transplantation (aBMT) has been shown to be a promising last resort. It leads to drug free regel 11 disease remission in a majority of patients for a long period of time. However, some patients regel 12 have been shown to get a disease relapse in due time after all. regel 13 Therefore in this thesis we had two main goals: regel 14 1) The search for a therapy aimed at suppressing processes that are at the basis of regel 15 disease pathogenesis. It overcomes unnecessary side effects and might lead to a regel 16 permanent cure of disease. We searched for strategies aimed at specific tolerance regel 17 induction to antigens present at the site of disease, and methods to optimalize this regel 18 antigen specific tolerance. regel 19 2) Unraveling the underlying working mechanism of aBMT, particularly focused on the regel 20 role of regulatory T cells (Tregs), which are cells that are known to keep autoimmunity regel 21 and inflammation under control. In exploring the working mechanism, we eventually regel 22 aim to further improve the clinical effect and prevent the relapses. regel 23 regel 24 In the first part of the thesis, we show in chapter 3 that nasal administration of the disease- regel 25 8 Chapter triggering antigen HSP60 peptide 180-188 was able to moderately suppress disease in regel 26 148 experimental Adjuvant Arthritis (AA). We were able to improve this effect by administration of regel 27 an altered peptide ligand of 180-188, alanine 183, both in the prevention of disease and in the regel 28 treatment of established disease. The treatment induced regulatory T cells that produced IL-4, regel 29 TGFβ and IL-10, but was dependent on IL-10 production. regel 30 In chapter 4 we show an alternative method to improve the arthritis suppressive effect of 180- regel 31 188 treatment by first shutting down the predominant pro-inflammatory environment in sick regel 32 animals. This could be achieved by preceding nasal tolerance induction by neutralization of regel 33 TNFα, a pro-inflammatory cytokine that is known to be at the basis of arthritis. This led to regel 34 the induction of CD4+ regulatory T cells, producing IL-10 and IL-4, and expressing CD25 and regel 35 Foxp3. regel 36 In the second part of the thesis we focused on unraveling the working mechanism of aBMT in regel 1 the Proteoglycan Induced Arthritis (PGIA) model. We show that irradiation followed by aBMT regel 2 induced a rapid decrease of arthritis followed by stabilization of disease at a level below its regel 3 severity before aBMT (chapter 5). The effect was not attributable to the conditioning regimen regel 4 alone, but in the first phase after aBMT suppression of the pro-inflammatory Th17 auto-antigen regel 5 specific response occurred, which may have created the ideal window for the next phase of regel 6 stabilization of disease. In this second phase, we found a crucial role for CD4+CD25+Foxp3+ regel 7 Tregs in inducing long-term restoration of the immune balance (chapter 6). regel 8 regel 9 Altogether, in this thesis, we employed two experimental arthritis models in order to move regel 10 forward towards a more specific treatment of arthritis. We were able to improve the arthritis regel 11 suppressive effect of antigen specific mucosal tolerance induction by use of an altered peptide regel 12 ligand and by combination therapy with an anti-inflammatory agent, and unravelled the role of regel 13 regulatory T cells in the immune deviation induced. Secondly, we showed that aBMT leads to a regel 14 restoration of the immune balance by regulatory T cells. regel 15 The insight provided in both treatment methods, can open the way to further improvement of regel 16 the treatment of arthritis in the future and in the prevention of disease relapses post aBMT. regel 17 Regulatory T cells may play an important role in this respect, by either inducing them in vivo, regel 18 eg by mucosal tolerance induction post aBMT, or by the administration of ex vivo generated regel 19 and/or expanded Tregs. Furthermore, by unravelling the underlying processes induced by regel 20 aBMT, future therapies may be devised in which a similar immunological mechanism can be regel 21 induced, without the unwanted side effects of severe conditioning that precedes aBMT. regel 22 regel 23 regel 24

General discussion General regel 25 149 regel 26 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 GENERAL DISCUSSION regel 2 regel 3 PART I: Antigen specific immunomodulation and the beneficial effect of an regel 4 altered peptide ligand and combination therapy with anti-inflammatory regel 5 therapy regel 6 regel 7 Conventional treatment of JIA and RA consists of non specific suppression of disease. Due regel 8 to this approach, treatment needs to be administered continuously to remain effective and regel 9 does not lead to a cure. Furthermore, the generalized and prolonged immunosuppression may regel 10 lead to severe side effects. Therefore the new challenge in this field is how to further improve regel 11 disease outcome while also decreasing side effects. regel 12 regel 13 Antigen specific immunomodulation regel 14 Antigen specific immunomodulation would fit both criteria: through the mucosal administration regel 15 of an antigen present at the site of inflammation, tolerance to the antigen can be achieved with regel 16 subsequent clinical improvement and without the unnecessary side effects due to general regel 17 immunosuppression. Furthermore, it may be able to induce a specific shift in auto reactive regel 18 and/or pro-inflammatory T cell repertoire, which could have a lasting effect on the immune regel 19 balance. Thus, in theory, antigen specific immune modulation could contribute to developing regel 20 therapeutic strategies that ultimately even may lead to a cure. regel 21 In Adjuvant Arthritis (AA), the nasal administration of the disease triggering antigen, Heat regel 22 Shock Protein (HSP) 60 peptide 180-188, leads to clinical improvement1. However, the disease regel 23 suppression achieved is not optimal, and leaves room for improvement. In the first part of this regel 24 thesis, we show in experimental arthritis that mucosal tolerance induced to HSP60 peptide regel 25 8 Chapter 180-188 can be enhanced in two ways: by administering an altered peptide ligand, alanine regel 26 150 183, and by preceding the tolerance induction by an anti-inflammatory agent, anti TNFα. regel 27 regel 28 1. Use of an altered peptide ligand (APL) regel 29 regel 30 Improvement of disease remission by use of an APL in experimental arthritis regel 31 By nasal administration of an altered peptide of HSP60 peptide 180-188, alanine 183, in which regel 32 one amino acid at position 183 has been changed (leucine>alanine), the disease suppression regel 33 is superior to when the wild type epitope, peptide 180-188, is administered nasally. The disease regel 34 suppression can be achieved when alanine 183 is administered prior to disease induction regel 35 and thus as a preventive means, but also when full blown disease has developed. This is of regel 36 relevance to the clinical situation, since in human arthritis, treatment can not be started in a preventive manner, but is initiated at a time that disease is already present and often at an advanced stage. In addition to the clinical improvement seen by the administration of an APL in experimental regel 1 arthritis, we booked progress in unraveling the underlying mechanism induced regel 2 immunologically. Several mechanisms have been proposed to play a role in mucosal tolerance regel 3 induction, depending on the dose and route of the administered antigen. Either clonal anergy regel 4 may be induced by encounter of the antigen in the mucosal milieu, or the encounter may lead regel 5 to clonal deletion of the antigen specific T cells. Lastly, a deviation of pro-inflammatory T cells regel 6 to a regulatory/suppressive phenotype may be induced2. Earlier studies already showed that regel 7 the improved clinical effect can not be attributed to the increased binding affinity of the APL regel 8 to MHC class II, since the native peptide 176-190 has even higher binding capacity but is less regel 9 effective in preventing AA3. Instead, we now show that administration of alanine 183 leads to an regel 10 immune deviation by the induction of Tregs producing IL-10, IL-4 and TGFβ locally at the site of regel 11 tolerance induction (the mandibular lymph nodes) and at the site of inflammation (the inguinal regel 12 lymph nodes). This provides a conceptual step forward as it shows the efficacy of antigen regel 13 specific mucosal immune modulation. A better understanding of the working mechanism of regel 14 antigen specific therapy may provide guidelines for the development of methods with similar regel 15 underlying immunological deviation and similar clinical effect but without the side effects regel 16 seen in clinical trials with APLs (see below). regel 17 regel 18 Disappointing results of the use of APL in clinical trials regel 19 Unfortunately, in clinical trials thus far, the use of APLs has not been as successful as was to regel 20 be expected based on animal studies. Two phase II placebo controlled trials using an APL in regel 21 Multiple Sclerosis (MS) were even terminated early4;5. MS is a chronic demyelinating disease regel 22 characterized by an inflammatory attack on the myelin sheath in the central nervous system. regel 23 Like RA and JIA, it is considered a Th1 type disease, and the dominant epitope of myelin basic regel 24 protein (amino acids 83-99 of MBP), represents a candidate antigen for disease triggering T discussion General regel 25 cells. Two APLs of MBP83-99 were designed and tested in phase II clinical trials; however, the 151 regel 26 subcutaneous (s.c.) injection of the APLs was tolerated poorly at the dose tested. In the trial regel 27 by Bielovika et al. no clear correlation to worsening of disease was found in the small group regel 28 that was tested, however 3 patients did show exacerbation of disease that could be linked regel 29 to encephalitogenic capacity of the APL in two patients4. The trial by Kappos et al. was also regel 30 suspended early due to the occurrence of hypersensitivity reactions in 9% of the patients, regel 31 found to correlate to the induction of a Th2 type response by the APL. No increase in clinical regel 32 relapses nor new enhancing lesions were found in this trial either5. regel 33 In type I diabetes, a phase I clinical trial with NBI-6204, an APL of the insulin B epitope, induced regel 34 immune deviation from a Th1 to a tolerant Th2 phenotype, and a phase II clinical trial is regel 35 currently under way to assess whether this immunological deviation corresponds with clinical regel 36 efficacy6. regel 1 Future perspectives for the use of APL regel 2 The disappointing results of the use APLs in MS warrant caution. However at the same time, regel 3 some interesting conclusions can be drawn from the results. Despite the fact that the APLs regel 4 were poorly tolerated when administered s.c., they were able to induce immune deviation regel 5 in the pathogenic T cells to a tolerant phenotype. But before further clinical use, firstly, regel 6 preclinical studies will need to provide better insight in the optimal dosing regimen, since regel 7 most complications were seen at higher doses. Secondly, the trials were promising in that regel 8 they showed immune deviation from a Th1 to a Th2 phenotype; however they came with regel 9 subsequent hypersensitivity reactions in a proportion of patients. Therefore the strength of regel 10 this immune deviation will need to be kept within limits. Thirdly, the route of administration regel 11 deserves consideration. The administration of an APL s.c. is likely to have a different effect than regel 12 when the APL is administered via the tolerant environment of the mucosa. The exacerbation regel 13 of disease that was seen in a few of the patients is of course worrisome, but deserves re- regel 14 evaluation once the above-mentioned parameters have been optimalized. Most importantly, regel 15 the use of APLs in a clinical setting encounters one major principal problem: every human has regel 16 a different MHC haplotype, which is essentially different from the experimental setting with regel 17 genetically identical animals. For this reason, in case of human APL therapy an individualized regel 18 APL would have to be constructed to be specifically fit for that individual’s HLA haplotypes. This regel 19 may prove to be a limiting factor in future clinical use. regel 20 regel 21 2. Improvement of disease remission by combination therapy with anti TNFα regel 22 prior to mucosal tolerance induction in experimental arthritis regel 23 Next, we moved on to a different approach to improve the clinical effect achieved by nasal regel 24 administration of the wild type epitope 180-188, namely by preceding the mucosal tolerance regel 25 8 Chapter induction by an anti inflammatory agent. This is based on the hypothesis that the reason for regel 26 152 the suboptimal clinical improvement of 180-188 may be, that at the time of administration regel 27 when disease is full blown, the local inflammatory environment may have reached a threshold regel 28 beyond which is it not possible anymore to induce antigen specific immunomodulation. regel 29 Thus we hypothesized that we could create a milieu suitable for tolerance induction by prior regel 30 administration of an anti-inflammatory agent. regel 31 Interestingly, by giving a single low dose of Etanercept, a soluble TNF receptor fusion protein, regel 32 followed by mucosal tolerance induction to HSP60 peptide 180-188, significant suppression regel 33 of arthritis was seen to the same extent as a full course of Etanercept therapy. This implies regel 34 that lower doses of anti TNFα can be given, resulting in lower cost and less long term side regel 35 effects. Interestingly, two distinctly different immunological mechanisms were at the basis regel 36 of equivalent clinical suppression of arthritis, when comparing full dose Etanercept therapy with the combination treatment of anti TNFα and 180-188 peptide. Where anti TNFα treatment regel 1 induced mainly immune suppression, combination treatment was able to induce active regel 2 modulation through induction of IL-10 production by effector T cells as well as the induction of regel 3 CD4+CD25+ regulatory T cells, again producing IL-10 and expressing Foxp3. regel 4 regel 5 Combination therapy in other experimental models regel 6 Similarly promising results were recently published by Bresson et al. in experimental diabetes7. regel 7 Type I diabetes mellitus is a T cell mediated autoimmune disease that leads to progressive regel 8 destruction of insulin producing β cells in the pancreas. Bresson et al. studied the effect of regel 9 combination therapy in the NOD (non-obese diabetic) mouse model by preceding proinsulin regel 10 peptide treatment with anti CD3 treatment. Recent onset diabetes could be reversed more regel 11 potently than when anti CD3 or the peptide was given alone. This combination treatment regel 12 induced Tregs; the level of CD4+CD25+Foxp3+ cells increased and insulin specific production regel 13 of IL-10, TGFβ and IL-4 by Tregs was enhanced. regel 14 regel 15 Working mechanism of combination therapy regel 16 The reason that the induction of tolerance is facilitated by the combination with anti TNFα regel 17 and anti CD3 probably lies in the fact that anti TNFα and anti CD3 lead to a suppression of regel 18 the dominant pro-inflammatory environment, thus decreasing the background ‘noise’ and regel 19 creating an environment in which the antigen specific immune response can be modulated. regel 20 In addition, the fact that both have been shown to create a tolerogenic environment has likely regel 21 been instrumental as well. Several studies have shown that anti TNFα treatment can improve regel 22 Treg function and numbers in RA patients8-10. Also, anti TNFα treatment was shown to induce regel 23 a shift to a more anti-inflammatory cytokine profile in PBMCs (Peripheral Blood Mononuclear regel 24 Cells) and T cells of RA patients11. In NOD mice, it was demonstrated that anti CD3 treatment led discussion General regel 25 to a decrease in the amount of auto aggressive T cells and an expansion of CD4+CD25+ cells in 153 regel 26 draining lymph nodes. Disease suppression was mediated through production of TGFβ12. regel 27 regel 28 Mucosal tolerance induction in clinical trials regel 29 In several autoimmune diseases (AID) attempts have been made at inducing mucosal tolerance regel 30 with varying success. Since the ‘one disease-triggering antigen’ is not known in most AID, regel 31 attempts mainly focus on structural antigens present at the site of inflammation. In RA patients, regel 32 tolerance induction to major constituents of articular cartilage, such as chicken and bovine regel 33 type II collagen and human cartilage glycoprotein 39 was able to induce clinical improvement regel 34 in some studies, but not all13-15. In early onset type I diabetes, the oral administration of insulin regel 35 was not found beneficial in several trials16-18. Recently a trial was set up, where nasal insulin regel 36 regel 1 was administered in a preventive fashion. Infants were selected based on the presence of regel 2 HLA-DQB1 susceptibility alleles for type I diabetes and short acting insulin was administered regel 3 as soon as the presence of two or more auto antibodies was detected. However, even this regel 4 early administration of nasal insulin soon after seroconversion to auto antibody positivity in regel 5 genetically susceptible individuals, could not delay or prevent the occurrence of type I diabetes regel 6 and therefore the trial was terminated early19. regel 7 A different approach was taken both in the field of arthritis and type I diabetes by administration regel 8 of a HSP peptide. HSPs are present in all cells and are up regulated during stress. During a regel 9 pro-inflammatory response, due to stress and tissue degeneration they become increasingly regel 10 available, as such again forming a new target for the immune system and thus keeping loops regel 11 of inflammation intact. In both RA/JIA and type I diabetes, HSPs have indeed been identified regel 12 as important self-antigens and promising results were obtained by mucosal administration of regel 13 an HSP peptide in clinical trials. To 15 patients with early, active RA dnaJP1, which is derived regel 14 from the heat shock protein dnaJ, was administered orally in a Phase I/IIa clinical trial20. It regel 15 induced immune deviation from pro-inflammatory to modulatory T cell responses, leading regel 16 to significant reduction in TNFα and IFNγ production and an increase in IL-10 and IL-4. Even regel 17 though the clinical effect was not a primary focus of this phase I/IIa trial, no worsening of regel 18 disease was seen. Disease improvement was recently confirmed in a subsequent double blind regel 19 placebo controlled pilot phase II clinical trial in which 160 patients received dnaJP1 or placebo regel 20 orally once per day for six months. The dnaJP1 peptide treatment showed encouraging clinical regel 21 and immunological effects, suggesting that induction of immune tolerance to an inflammatory regel 22 ubiquitous antigen may indeed translate into clinical improvement of the disease (results regel 23 submitted for publication). In several placebo controlled clinical trials adults and children with regel 24 type I diabetes, the administration of DiaPep277, an immunogenic peptide of HSP60, was safe regel 25 8 Chapter and well tolerated, but effects on preservation of insulin production differed. In most trials, regel 26 154 a beneficial effect was seen on preservation of C-peptide levels over time due to DiaPep277 regel 27 treatment, however only one group describes concomitant preservation of endogenous regel 28 insulin secretion up to 18 months of treatment21;22. In other studies, the beneficial effect on regel 29 C peptide levels was not supported by a decrease in HbA1c nor daily insulin requirement23-25. regel 30 Interestingly, immunological analysis showed that the induction of IL-10 production and a regel 31 decrease in auto antigen specific T cell proliferation due to DiaPep277 treatment correlated with regel 32 β cell preservation, and thus may serve as immunological biomarkers for clinical efficacy26. regel 33 regel 34 regel 35 regel 36 Future perspectives for mucosal tolerance induction and combination therapy regel 1 Mucosal tolerance induction regel 2 The reason for the varying success of mucosal tolerance induction deserves further study. regel 3 First, the choice of antigen administered should be re-evaluated. Possibly, we can move away regel 4 from aiming to find ‘the one disease triggering antigen’ and move towards antigens present regel 5 at the site of inflammation, such as HSPs. As mentioned before, HSPs are present in all cells regel 6 and are up regulated during stress, for example in an arthritic joint. Due to tissue damage, regel 7 they may become even more readily available and thus continue to provide antigenic targets regel 8 for the immune system. In addition to their role in arthritis and type I diabetes, HSP antigens regel 9 can be of relevance in other AIDs as well. Secondly, timing and dosage of the antigen are regel 10 likely to be of critical importance. Lastly, in some trials, the disease process might have been regel 11 at too advanced a stage to be able to achieve antigen specific modulation. In those cases a regel 12 combination therapy, in which the pro-inflammatory state is put to rest first, might prove to regel 13 be beneficial. regel 14 regel 15 Combination therapy (Figure 1) regel 16 In our experimental arthritis model, we found a beneficial effect of preceding mucosal tolerance regel 17 induction by anti TNFα therapy. The clinical improvement thus achieved may be based on regel 18 two pillars: 1) by shutting down the pro-inflammatory environment, making antigen specific regel 19 immunomodulation possible and 2) by creating a more tolerogenic milieu, as anti TNFα was regel 20 shown to be able to do8;9. In this respect, since anti CD3 has shown in type I diabetes to be regel 21 able to also induce a more tolerogenic environment, anti CD3 combination therapy could be regel 22 attempted in JIA and RA as well27. regel 23 Furthermore, epitope specific immunotherapy also has the potential to act in synergy with regel 24 more traditional DMARDs. In the recent phase II trial with dnaJP1 in RA, unexpectedly the first discussion General regel 25 results were obtained of combination treatment of a DMARD and mucosal tolerance induction 155 regel 26 (results submitted for publication). Interestingly, the clinical effect of dnaJP1 was clearly regel 27 enhanced in patients using hydroxychloroquine (HCQ). HCQ is traditionally an anti-malaria drug regel 28 that, due to its immunomodulatory effects, is also used in the treatment of RA. The explanation regel 29 of the enhanced effect of dnaJP1 tolerance induction due to HCQ, may in part lay in the fact regel 30 that HCQ is known to decrease TNFα and IL-6 production. HCQ’s main effect is exerted through regel 31 blockade of the processing of proteins by antigen presenting cells (APC)28-30. This creates an regel 32 environment of low self-presentation of proteins, whereby the dnaJP1 peptide might be more regel 33 easily presented, as it does not need to be processed in order to be presented by APC. In this regel 34 way, such peptides may have a greater impact on the regulatory immune system. regel 35 regel 36   regel 1 Lastly, it is possible that combination of methotrexate (MTX) with antigen specific tolerance regel 2 induction may also be beneficial. MTX administration may create a better milieu for antigen regel 3 specific immunomodulation, by creating an anti-inflammatory environment and maybe even  regel 4 a more tolerogenic   microenvironment  via its action  on rapidly  proliferating  effector  T cells  and    regel 5 a decrease in TNFα production31;32. In this respect, further study of the immune regulatory regel 6 mechanism of MTX would be of interest. regel 7  regel 8   regel 9  regel 10 regel 11    regel 12   regel 13  regel 14  regel 15    regel 16     regel 17  regel 18  regel 19   regel 20    regel 21  regel 22  regel 23  regel 24  regel 25 8 Chapter  regel 26 156 Figure 1. Combination therapy creates a beneficial milieu for antigen specific immuno-  regel 27 modulation regel 28            regel 29  regel 30 regel 31  regel 32  regel 33 regel 34 regel 35 regel 36

  PART II: Role of Tregs and source of Bone Marrow graft in aBMT regel 1 Despite progress in the treatment of patients with JIA and RA, with earlier use of potent regel 2 immunosuppressants and the addition of biologicals, some patients still remain unresponsive. regel 3 For those severely ill patients, autologous BMT is the last resort. It induces a drug free disease regel 4 remission in a majority of patients. Elegant work in JIA patients undergoing aBMT provided regel 5 insight in the underlying mechanism behind disease remission. It showed that disease regel 6 remission is mediated on one hand by (preferential) homeostatic proliferation of Tregs regel 7 during the lymphopenic phase of the reconstitution of the immune system and on the other regel 8 hand renewed thymopoiesis of naive Treg. Furthermore, a deviation of auto reactive T cells regel 9 from a pro-inflammatory phenotype before aBMT, to a tolerant phenotype after aBMT was regel 10 seen33. Further insight was obtained in Systemic Lupus Erythematosus (SLE) patients. SLE regel 11 is a systemic autoimmune disease, characterized by the generation of pathogenic antibodies regel 12 directed against a variety of auto antigens, including nuclear and cytoplasmic antigens. In regel 13 refractory SLE patients, conditioning followed by aSCT (Stem Cell Transplantation) has shown regel 14 the potential to induce long-term drug-free disease remission. In a recent paper, Alexander et regel 15 al. provide immunological follow up data for 7 transplanted SLE patients and show that immuno regel 16 ablation followed by aSCT leads to the depletion of auto reactive T and B cells and the generation regel 17 of a ‘juvenile’ tolerant adaptive immune system by thymus derived Foxp3+ Tregs34. regel 18 However, important questions regarding the working mechanism and optimal conditions for regel 19 transplantation are still unanswered. Therefore, in the second part of this thesis, we turned to regel 20 an experimental arthritis model: Proteoglycan Induced Arthritis. regel 21 regel 22 The need for a clinically relevant arthritis model: Proteoglycan Induced Arthritis regel 23 (PGIA) regel 24 The PGIA model has been set up and characterized extensively by Tibor Glant and co-workers discussion General regel 25 from Rush University in Chicago. PGIA is induced in susceptible female BALB/c mice by two 157 regel 26 injections of human PG in DDA. It is initiated by Th1 cells, but also dependent on B cells, and regel 27 leads to a progressive poly-arthritis. It has a relapsing-remitting course, equivalent to arthritis regel 28 in humans35;36. regel 29 We set up an autologous BMT model in PGIA. Conditioning followed by transplantation of regel 30 pseudo-autologous BM from syngeneic donor animals that were at the same stage of disease, regel 31 led to rapid improvement of disease. Fairly rapidly relapses occurred as well, but the arthritis regel 32 never returned to the level before aBMT, which is an important step forward in the treatment regel 33 of these severely ill patients and indicates that a restoration of the immune balance has taken regel 34 place. The protective effect of aBMT was also projected in the decrease of histological damage regel 35 in the joints post aBMT. regel 36 regel 1 Our data convincingly show, that the clinical improvement post aBMT was not merely regel 2 attributable to the conditioning regimen itself, as mice undergoing conditioning alone, though regel 3 they showed initial improvement, in time regained the most severe and sustained arthritis. regel 4 regel 5 Effect of composition of the autologous BM graft on the outcome of aBMT regel 6 We found that BM from sick animals, that were at the same stage of disease and of which regel 7 T cells were not depleted, had the best clinical effect, compared to BM from sick animals of regel 8 which T cells were depleted and compared to BM from healthy animals. These observations regel 9 are of particular interest due to the link with the clinical situation, since in JIA and RA patients, regel 10 autologous BM is harvested during a phase of active disease. BM is part of the lymphocyte regel 11 recirculation network and serves as a reservoir for (memory) T lymphocytes and even regel 12 preferentially for regulatory T cells (Tregs)37;38. Thus it can be hypothesized that a memory -PG regel 13 specific- T cell pool resides in the BM of sick animals. After transplantation of the BM into an regel 14 irradiated recipient, possibly these auto reactive T cells are ‘re-educated’ to become tolerant regel 15 Tregs in the lymphopenic environment33. Secondly, we found a role for Foxp3+ CD4+CD25+ regel 16 Tregs in the second, stabilization phase of PGIA after aBMT. This suggests that both ‘naturally regel 17 occurring’ Foxp3+ Tregs and re-educated auto antigen specific Tregs originating from the graft regel 18 may play a role. regel 19 Also, the results make the rationale for T cell depletion of the graft a point of discussion. The regel 20 rationale for depleting T cells from the graft stems from the idea of eliminating potentially regel 21 auto reactive T cells. However, in a pilot multi-centre randomised trial in 33 RA patients, when regel 22 CD34+ selection was performed as a way of depleting T cells, no additional effect on time to regel 23 relapse nor disease remission was achieved compared to unmanipulated grafts39. On the other regel 24 hand, in JIA patients the depletion of T cells from the graft even led to a major complication, regel 25 8 Chapter Macrophage Activation Syndrome (MAS). Due to the lack of control by T cells, macrophages regel 26 158 could become overly activated and extremely destructive40. Larger studies are needed to regel 27 better decide on the usefulness of T cell depletion from the BM graft, while also preventing regel 28 damaging complications of MAS. regel 29 regel 30 ABMT leads to long-term restoration of immune balance mediated by Tregs regel 31 We found that the disease remission post aBMT can be divided up into two phases (Figure 2): regel 32 regel 33 1) An initial phase of rapid, maximal clinical improvement regel 34 This phase coincides with a relative increase in CD4+CD25+ cells, however those cells regel 35 are not expressing high levels of Foxp3 yet, nor are they potently suppressive. There is a regel 36 predominance of memory T cells and memory Tregs over their naïve counterparts. Probably at this stage, the CD4+CD25+ cells are in a state of homeostatic proliferation during lymphopenia regel 1 and the absence of T effector cells and inflammation due to the conditioning explains the lack regel 2 of disease. In this initial phase the most important observation is a rapid decrease in the pro- regel 3 inflammatory auto-antigenic insult. This is reflected in a decrease of auto antibody formation regel 4 and a strongly reduced capacity to mount a Th17 response. It can be hypothesized that this regel 5 strong reduction of the pro-inflammatory response is essential for the success of the next regel 6 phase: the achievement of lasting immune tolerance. regel 7 regel 8 2) A second phase of stabilization of disease regel 9 During this phase some degree of relapse occurs, but disease stabilizes at a level below the regel 10 severity before aBMT. This process is regulated by CD4+CD25+ Tregs that now express high regel 11 levels of Foxp3, corresponding to a potently suppressive and naïve phenotype, thus probably regel 12 emerging from the thymus. The critical importance of the Tregs is further underscored by the regel 13 fact that rapid amelioration of disease occurs after administration of a CD25 depleting antibody. regel 14 The stabilization of disease cannot be broken by re-boost with the disease-triggering antigen, regel 15 indicating that a long-term restoration of immune homeostasis has taken place. regel 16 Interestingly, we thus found a correlation between the level of expression of Foxp3 on Tregs regel 17 and their functionality. Chauhan et al. recently obtained similar results in an experimental regel 18 allograft transplantation model. They also describe the correlation between the level of regel 19 regel 20 Foxp3 expression and the functionality of the Tregs, both with regards to regulatory cytokine 41 regel 21 production as well suppression of T cell activation . regel 22

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  

    discussion General

 regel 25

      159 regel 26

    regel 27    regel 28        regel 29  regel 30  regel 31  regel 32  regel 33  regel 34  regel 35  regel 36 Figure 2. Immune reconstitution post aBMT can be divided up into two phases                                                                       

  regel 1 Future perspectives for aBMT regel 2 regel 3 In an experimental setting regel 4 We showed in the second part of this thesis that aBMT leads to a long-term restoration of the regel 5 immune balance in PGIA and that this process is orchestrated by Tregs. The improvement of regel 6 disease can be divided up into two phases: the initial, rapid clinical improvement, due to a regel 7 decrease in auto antibody production and IL-17 production, leading to a lack of inflammation regel 8 and a lack of effector T cells. The second phase is a phase of stabilization of disease, in which regel 9 Tregs play an active role, but the exact origin of those Tregs, and whether they are of host regel 10 or donor origin has not been determined yet. They may be naïve Tregs originating from the regel 11 thymus combined with Tregs from the graft and/or host Tregs that have preferentially survived regel 12 irradiation. Whether the Tregs are from donor or host origin and tracing of Tregs in vivo would be regel 13 an interesting future focus of research in an experimental setting. regel 14 1) Chimerism regel 15 It deserves further study to determine the origin of the Tregs that orchestrate disease regel 16 stabilization post aBMT. When Tregs from the donor can be distinguished from Tregs from the regel 17 host, e.g. by performing a male to female transplantation or a Thy1.1 to Thy1.2 transplantation, regel 18 post aBMT the origin of the Tregs can be visualized. regel 19 2) In vivo tracing of Tregs regel 20 Secondly, it will be interesting to expand the knowledge on the role of the Tregs post aBMT by regel 21 assessing the in vivo kinetics and homing of the Tregs. For this purpose, Tregs of the graft could regel 22 be traced in vivo by labeling them prior to addition to the graft with e.g. CFSE or luciferase. regel 23 3) Mesenchymal Stem Cell Transplantation regel 24 In recent years, interest in the role of mesenchymal stem cell (MSC) transplantation as a regel 25 8 Chapter therapy for arthritis has grown. MSCs are multipotent mesenchymal stromal cells that can regel 26 160 easily be expanded in vitro, while retaining the potential to differentiate along various lines of regel 27 mesenchymal origin, including chondrocytes, osteoblasts and adipocytes42;43. Large quantities regel 28 can thus easily be obtained for therapeutical purposes and as such they can serve as building regel 29 blocks for replacement of tissues and reparation of damaged structures in diseases such as regel 30 JIA and RA. Interestingly, the regenerative effects of MSCs do not merely rely on their ability regel 31 to structurally contribute to tissue repair, but they have also been found to possess potent regel 32 immunomodulatory and anti-inflammatory effects. After in vivo administration, they migrate to regel 33 injured tissues where they can inhibit the release of pro-inflammatory cytokines and promote regel 34 survival of damaged cells. They are thus interesting candidates for future therapy and their regel 35 use is currently being tested in several experimental models of autoimmune disease. regel 36 In Experimental Autoimmune Encephalomyelitis (EAE), a model for MS, the systemic injection regel 1 of MSCs at disease onset was able to ameliorate disease and decreased infiltration of regel 2 the central nervous system by T cells, B cells and macrophages44. The application of MSCs regel 3 in experimental arthritis models has thus far had conflicting results. In one study, a single regel 4 administration of MSCs in CIA prevented the occurrence of severe irreversible damage to bone regel 5 and cartilage. The MSCs exerted their immunomodulatory effects by educating antigen specific regel 6 CD4+CD25+CD27+ Tregs45. In another CIA study, MSCs, derived from a murine MSC cell line, regel 7 were unable to confer clinical benefit and the presence of TNFα was found responsible in in regel 8 vitro experiments for the reversal of the immunosuppressive effect46. The as of yet unpublished regel 9 results of the same group using freshly cultured MSCs instead of MSCs from a cell line were regel 10 more promising, in that a preventive systemic injection was able to suppress arthritis47. Thus, regel 11 MSCs are promising candidates for therapy of several autoimmune diseases, however due to regel 12 the conflicting data, further pre-clinical studies are needed before translation to the clinics can regel 13 be made. In this respect, combination therapy with anti TNFα could be of benefit. regel 14 regel 15 For translational therapy regel 16 We found a key role for Tregs in the long-term restoration of the immune balance post aBMT. regel 17 Unfortunately, the disease is not in complete remission post aBMT, but some degree of relapse regel 18 occurs, even though disease is at a lower level than before aBMT. Therefore, future therapies regel 19 should be devised in which those relapses too can be prevented. Two approaches can be taken, regel 20 both by profiting further from the beneficial role of Tregs; either by in vivo induction of Tregs regel 21 post aBMT or by the use of in vitro induced or expanded Tregs. regel 22 1) In vivo induced Tregs regel 23 In the first part of this thesis, we describe how nasal tolerance induction leads to in vivo regel 24 induction of Tregs with subsequent clinical improvement. This disease improvement could be discussion General regel 25 enhanced by prior administration of anti TNFα, which creates a window for antigen specific 161 regel 26 immunomodulation by shutting down the pro-inflammatory environment. By conditioning regel 27 and aBMT a similar window is created, which can also be instrumental in the induction of regel 28 antigen specific tolerance to PG, the disease trigger in this model. Broere et al. have recently regel 29 shown that oral administration of PG can lead to disease improvement48. Timing and dosage regel 30 of mucosal administration of PG post aBMT first require exact testing in this model, however regel 31 provide a promising approach to the prevention of relapses post aBMT. regel 32 2) In vitro expanded Tregs regel 33 Treg therapy has received considerable attention in recent years and has been the focus of regel 34 extensive study. Because of the important role Tregs have been found to play in protection regel 35 from and remission of autoimmunity, attempts have been made to design a way to use them regel 36 regel 1 in a therapeutic fashion. Due their low frequency in peripheral blood of patients (up to 5-10% of regel 2 CD4+ T cells), they need to be expanded in order to gain numbers large enough, without losing regel 3 their suppressive function. Experience has been gained in this respect by several groups49-51 regel 4 and the first clinical trials in which Tregs will be administered are about to start. Caution will regel 5 have to be taken with respect to the expansion of Tregs though. First of all, while expanding regel 6 Tregs, a thin line between Tregs and effector T cells exists. Foxp3 was considered to be an regel 7 exclusive marker for Tregs, however has recently been found to be transiently up regulated regel 8 in human effector T cells as well52. Therefore careful phenotypical as well as functional regel 9 characterization will have to be performed after they have been expanded and prior to injecting regel 10 them in patients. Secondly, expanded Tregs thus far are not antigen specific. Ideally, devising a regel 11 method in which Tregs for a specific antigen can be expanded would be preferable. regel 12 regel 13 regel 14 CONCLUSION regel 15 regel 16 In this thesis, we describe and characterize several ways to improve disease outcome of regel 17 experimental arthritis with subsequent implications for translational therapy. Our principal aim regel 18 was to identify methods to more specifically obtain long lasting disease remission with fewer regel 19 side effects. We particularly focused on the underlying immunological mechanisms induced. regel 20 The first part of the thesis focuses on characterizing and improving antigen specific therapy. regel 21 Nasal administration of an antigen present at the site of inflammation has previously been regel 22 shown to induce antigen specific immune tolerance. We were able to improve this mucosal regel 23 tolerance induction in two ways: 1) by nasal administration of an altered peptide ligand, in regel 24 which one amino acid has been replaced at a critical binding site, leading to the induction of regel 25 8 Chapter regulatory T cells that produce IL-10 and TGFβ at the site of tolerance induction as well as the regel 26 162 site of inflammation; 2) by preceding mucosal tolerance induction by the administration of regel 27 anti TNFα, which shuts down the dominant pro-inflammatory milieu and is able to induce a regel 28 more tolerogenic environment. This resulted in active immune regulation through the induction regel 29 of CD4+CD25+Foxp3+ regulatory T cells producing IL-10. These studies have provided a regel 30 significant step forward in the process of antigen specific immunomodulation by improving regel 31 the clinical outcome and unraveling the immunological mechanisms involved. They are now regel 32 ready to be translated to the human situation. regel 33 In the second part of the thesis we focused on the underlying working mechanism of aBMT in regel 34 experimental arthritis, aiming to improve disease outcome in severely ill treatment resistant regel 35 patients and aiming for a permanent restoration of the immune balance. We designed a BMT regel 36 protocol in PGIA and studied conditions of optimal composition of the BM graft. We found that the graft from a sick donor of which T cells are not depleted led to the best clinical improvement, regel 1 indicating either the presence of regulatory T cells in this graft, or the fact that auto reactive T regel 2 cells, if present, did not prevent clinical improvement, probably due to the tolerogenic milieu regel 3 they repopulated in post aBMT. Secondly, we found that aBMT rapidly suppressed the pro- regel 4 inflammatory Th17 auto-antigen specific response, which may have created the ideal window regel 5 for the next phase, in which stabilization of the disease took place. In this second phase, we regel 6 found an important role for CD4+CD25+ Foxp3+ Tregs, indicating that indeed a long-term regel 7 restoration of the immune balance took place post aBMT. regel 8 By unravelling the underlying processes induced by aBMT, future therapies may be devised in regel 9 which a similar immunological mechanism can be induced, without the unwanted side effects regel 10 of severe conditioning that precedes aBMT. regel 11 regel 12 Proposal for ideal future treatment regimen regel 13 Based on the results presented in this thesis, we would like to propose our view on an regel 14 optimal, future treatment regimen as depicted in Figure 2. Depending on the stage of disease regel 15 at presentation, 3 paths can be followed on the road to the final goal of antigen specific regel 16 immunomodulation, which holds promise of a long-term remission of disease with few side regel 17 effects. In recent onset arthritis, antigen specific immunomodulation may succeed without regel 18 the need for prior intervention. However, when the arthritis is more advanced, the patient may regel 19 benefit from prior suppression of the pro-inflammatory environment by a combination strategy. regel 20 Anti TNFα may achieve this effect while also creating a more tolerogenic environment. Thirdly, regel 21 in patients with treatment resistant disease, immunoablation followed by aBMT may provide regel 22 a last resort. Ideally, post aBMT antigen specific immunomodulation is induced in order to regel 23 prevent relapses from happening and keep the restored immune balance post aBMT in tact regel 24 for a long period of time. discussion General regel 25 163 regel 26 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36                                                                      regel 1  regel 2 regel 3   regel 4 regel 5 regel 6 regel 7 regel 8   regel 9  regel 10  regel 11 regel 12 regel 13 regel 14 regel 15   regel 16    regel 17 regel 18 regel 19 regel 20 regel 21    regel 22  regel 23 Figure 3. Proposed future treatment paradigm for arthritic diseases  regel 24 regel 25 8 Chapter  regel 26 164 Reference List regel 27 regel 28 1. Prakken BJ, Roord S, van Kooten PJ et al. Inhibition of adjuvant-induced arthritis by interleukin-   regel 29 10-driven regulatory cells induced via nasal administration of a peptide analog of an arthritis- regel 30 related heat-shock protein 60 T cell epitope. Arthritis Rheum. 2002;46:1937-1946. regel 31 2. Weiner HL. Oral tolerance for the treatment of autoimmune diseases. Annu.Rev.Med. regel 32 1997;48:341-351. regel 33 3. Prakken BJ, Van Der ZR, Anderton SM et al. Peptide-induced nasal tolerance for a mycobacterial regel 34 heat shock protein 60 T cell epitope in rats suppresses both adjuvant arthritis and regel 35 nonmicrobially induced experimental arthritis. Proc.Natl.Acad.Sci.U.S.A 1997;94:3284-3289. regel 36                                                                     

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Blood 2004;104:895-903. regel 16 50. Kreijveld E, Koenen HJ, Hilbrands LB, Joosten I. Ex vivo expansion of human CD4+ CD25high regel 17 regulatory T cells from transplant recipients permits functional analysis of small blood regel 18 samples. J.Immunol.Methods 2006;314:103-113. regel 19 51. Bluestone JA. Regulatory T-cell therapy: is it ready for the clinic? Nat.Rev.Immunol. regel 20 2005;5:343-349. regel 21 52. Allan SE, Crome SQ, Crellin NK et al. Activation-induced FOXP3 in human T effector cells does regel 22 not suppress proliferation or cytokine production. Int.Immunol. 2007;19:345-354. regel 23 regel 24 regel 25 8 Chapter regel 26 168 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Nederlandse samenvatting regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 samenvatting Nederlandse regel 26 170 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 DEEL 1: Tolerantie inductie in arthritis door antigeen specifieke immuun regel 1 modulatie regel 2 regel 3 Huidige behandeling van reumatische aandoeningen: aspecieke onderdrukking van de regel 4 ziekte regel 5 Juveniele Idiopathische Arthritis (JIA) en Reumatoide Arthritis (RA) behoren tot de meest regel 6 voorkomende autoimmuun ziekten. Ze hebben een golvend beloop met remissies en regel 7 exacerbaties, en tijdens de systemische inflammatoire episodes worden de gewrichten regel 8 aangetast. Uitgebreid onderzoek in de afgelopen jaren is gericht geweest op het ontravelen van regel 9 het onderliggende mechanisme van het autoimmuun proces. Er blijkt sprake te zijn van een regel 10 initiele T cel gemedieerde fase, gevolgd door zich zelf instand houdende cycli van niet specieke regel 11 inflammatie, uiteindelijk leidend tot gewrichtsschade. Waarschijnlijk spelen lokale antigenen regel 12 in het synovium en gewricht een rol in het proces, maar het ‘ene ziekte makende’ antigeen is regel 13 niet bekend, en bestaat waarschijnlijk ook niet. De behandeling van JIA en RA kan een ware regel 14 uitdaging zijn en de ziektes kunnen leiden tot aanzienlijke morbiditeit en zelfs mortaliteit. regel 15 De huidige behandeling van (jeugd)reuma bestaat voornamelijk uit onderdrukking van de regel 16 tweede, inflammatoire fase van de ziekte. Hiermee kan ziekte remissie bereikt worden, maar regel 17 het leidt niet tot genezing, echter slechts tot tijdelijke verbetering van de ziekte. Daarbij regel 18 treden soms onnodige bijwerkingen op, zoals infecties en kanker. Ook is een aantal patiënten regel 19 ongevoelig voor deze methoden. regel 20 regel 21 Een elegantere benadering: antigeen specifieke immuunmodulatie regel 22 Het induceren van tolerantie ten opzichte van een antigeen dat aanwezig is ter plaatse van regel 23 de inflammatie heet antigeen specifieke tolerantie inductie. Het bewerkstelligen van antigeen regel 24 specifieke immuun tolerantie zou een patient gegeneraliseerde immuun suppressie kunnen samenvatting Nederlandse regel 25 besparen en zou mogelijk kunnen leiden tot een blijvende verbetering, wellicht genezing, zonder 171 regel 26 onnodige bijwerkingen. Antigeen specifieke T cel tolerantie kan bereikt worden door het oraal of regel 27 nasaal toedienen van een relevant antigeen; dit wordt respectievelijk orale of nasale tolerantie regel 28 inductie genoemd. In meerdere experimentele autoimmuun modellen is aangetoond dat orale regel 29 toediening van een ziektemakend autoantigeen kan leiden tot significante onderdrukking van regel 30 de ziekte. Het klinische effect kan vaak zelfs nog overtroffen worden door nasale toediening regel 31 van het antigeen. regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 Kandidaten voor mucosale tolerantie inductie: Heat shock eiwitten regel 2 Voor de inductie van antigeen specifieke tolerantie moeten antigenen geidentificeerd worden regel 3 die een rol spelen in de pathogenese van de ziekte. Er is waarschijnlijk geen sprake van slechts regel 4 1 ziektemakend antigeen, dus het is nuttiger om op zoek te gaan naar stoffen die aanwezig regel 5 zijn op de plek van de inflammatie en een rol spelen in het in stand houden van het ziekte regel 6 proces. Heat shock proteinen (HSP) zijn zulke antigenen. Ze zijn aanwezig in alle cellen, en regel 7 worden opgereguleerd tijdens stress. Doordat ze wijdverspreid voorkomen en afkomstig zijn regel 8 van bacterieen worden HSP-peptiden gezien als een ‘gevaar’ en daardoor wekken ze een pro- regel 9 inflammatoire respons op. Door middel van deze repons worden pathogenen geklaard uit het regel 10 lichaam, maar door cellulaire stress komen ook weer nieuwe lichaamseigen HSP peptiden vrij. regel 11 Deze peptiden vormen opnieuw een target voor het immuun systeem, en hierdoor worden regel 12 cycli van inflammatie in stand gehouden. Deze HSP peptiden worden vervolgens herkend door regel 13 regulatoire T cellen die verdere weefsel schade kunnen voorkomen. Als deze regulatoire functie regel 14 defect is, zullen de loops van inflammatie aanhouden en zal de autoimmuniteit voort duren. regel 15 regel 16 De inductie van regulatoire T cellen: een belangrijk doel in de zoektocht naar genezing van regel 17 arthritis regel 18 Doordat JIA en RA pro-inflammatoire aandoeningen zijn, is deviatie van de pathogene T cellen regel 19 naar een tolerant, regulatoir fenotype een van de doelen in de zoektocht naar genezing van regel 20 de ziekte. Onderzoek in meerdere diermodellen van autoimmuun ziekten heeft inderdaad regel 21 aangetoond dat regulatoire T cellen (Tregs) een belangrijke rol spelen in het beperken van regel 22 inflammatie en het onderdrukken van de ziekte. regel 23 Regulatoire T cellen kunnen onderverdeeld worden in 2 subtypes: naturally occuring Tregs regel 24 (nTregs) en Tregs die geinduceerd worden in de periferie (induced Tregs = iTregs). regel 25 samenvatting Nederlandse 1) NTregs ontstaan tijdens de ontwikkeling in de thymus en behelzen 5-10% van de regel 26 172 perifere CD4+ T cellen. Ze brengen CD4, de α keten van de IL-2 receptor (CD25), regel 27 CTL associated antigen 4 (CTLA-4) en glucocorticoid induced tumor necrosis regel 28 receptor (GITR) tot expressie aan hun oppervlakte. Maar hun meest onderscheidende regel 29 marker is transcriptie factor Foxp3, welke ook een belangrijke rol speelt in hun regel 30 ontwikkeling. NTregs oefenen hun suppressieve functie uit door het ondrukken van regel 31 de proliferatie van effector T cellen. Dit gebeurt door middel van een direct contact- regel 32 afhankelijk mechanisme en/of door de productie van suppressieve cytokines, zoals regel 33 IL-10 en TFGβ. regel 34 2) De tweede groep, de iTregs, worden in de periferie geinduceerd, en kunnen in regel 35 verschillende subtypes onderverdeeld worden, waarvan Tr1, Th3 en adaptieve Tregs regel 36 de best beschreven vormen zijn. Ze worden geinduceerd door contact met een antigeen in een anti-inflammatoire/suppressieve omgeving, bijv. door mucosale regel 1 tolerantie inductie. Zij oefenen hun suppressieve functie uit door de productie van regel 2 cytokines, bijv IL-10 (Tr1) en TGFβ (Th3). regel 3 regel 4 Antigeen specifieke tolerantie inductie door middel van HSP peptides in experimentele arthritis regel 5 (Adjuvant Arthritis) regel 6 Diermodellen worden regelmatig gebruikt om meer informatie te krijgen over de pathogenese regel 7 van ziekteprocessen, en tevens voor het ontwikkelen van nieuwe therapeutische strategieen. regel 8 De rol van HSPs in antigeen specifieke immunomodulatie is duidelijk aangetoond in het regel 9 Adjuvant Arthritis (AA) model. Het is een veelgebruikt model, met nauwe histopathologische regel 10 gelijkenis met humane RA en JIA. Het kan geinduceerd worden in Lewis ratten door middel van regel 11 2 immunisaties met hitte geinactiveerd Mycobacterium Tuberculosis (Mt) op dag 0 en dag 21 regel 12 (Figuur 1). Het is een T cel afhankelijke aandoening die vanzelf overgaat, en een T cel kloon regel 13 specifiek voor de 180-188 sequentie van mycobacterieel HSP60 kan de ziekte overbrengen regel 14 naar naieve dieren. regel 15 In AA leidt de nasale toediening van ditzelfde 180-188 peptide tot zowel preventie van arthritis regel 16 als onderdrukking van reeds bestaande ziekte. De suppressie van de ziekte is echter nog niet regel 17 optimaal, en het verbeteren van dit effect was één van de doelen van dit proefschrift. regel 18  regel 19 regel 20    regel 21  regel 22  regel 23  regel 24 Nederlandse samenvatting Nederlandse regel 25  173 regel 26      regel 27  regel 28  regel 29 Figuur 1. Het Adjuvant Arthritis model (AA) regel 30  In Lewis ratten leiden twee injecties van Mt in het adjuvant CFA tot een vorm van arthritis, die uiteindelijk regel 31  spontaan herstelt. regel 32  regel 33 regel 34 regel 35 regel 36 regel 1 DEEL 2: Tolerantie inductie door middel van autologe Beenmerg Transplantatie regel 2 (aBMT) regel 3 regel 4 Autologe BMT voor ernstige, onbehandelbare ziekte regel 5 Ondanks de vooruitgang in de behandelstrategieen van JIA en RA zijn sommige patienten nog regel 6 steeds ongevoelig voor deze behandelmethoden. Voor deze ernstig zieke patienten is autologe regel 7 BMT een laatste uitweg gebleken, aangezien aBMT bijv in JIA leidt tot medicatie-vrije ziekte regel 8 remissie in een merendeel van de patienten tijdens een follow up van 12-60 maanden na regel 9 transplantatie. Echter, aangezien de genetische aanleg van patienten niet verandert in een regel 10 aBMT setting, is het te verwachten dat de ziekte uiteindelijk toch weer terug keert. Inderdaad regel 11 is bij een aantal BMTpatienten na jaren van medicatie vrije ziekte remissie de ziekte toch weer regel 12 terug gekeerd. Daarnaast is het werkingsmechanisme van aBMT nog onvoldoende bekend, regel 13 en daarom was er behoefte aan een klinisch relevant dier model waarin dit mechanisme regel 14 onderzocht kon worden. regel 15 regel 16 Proteoglycaan geinduceerde arthritis (PGIA) regel 17 PGIA kan geinduceerd worden in BALB/c muizen door 2 injecties van humaan PG in het regel 18 adjuvant DDA. Het leidt tot een progressieve poly arthritis die geinitieerd wordt door PG regel 19 specifieke T cellen, maar ook B cellen zijn van cruciaal belang voor de ziekte (Figuur 2). PGIA is regel 20 uitgebreid bestudeerd door de groep van Tibor Glant in Chicago en de ziekte heeft klinische en regel 21 histopathologische gelijkenis met humane arthritis. Daarnaast heeft PGIA een golvend beloop, regel 22 met remissies en exacerbaties, vergelijkbaar met de klinische situatie. Daarom is PGIA een regel 23 waardevol model om het onderliggende werkings mechanisme van aBMT te bestuderen, en regel 24 het kan bovendien gebruikt worden om strategieen te ontwikkelen om de resultaten van aBMT regel 25 samenvatting Nederlandse te verbeteren, dan wel vervangende behandelmethoden te vinden met vergelijkbaar resultaat. regel 26 174 Dit waren belangrijk doelen van het tweede deel van dit proefschrift. regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36   regel 1 regel 2 regel 3 regel 4 regel 5 regel 6    regel 7 regel 8  regel 9 Figuur 2. Het Proteoglycan Arthritis model (PGIA) regel 10 regel 11 In BALB/c muizen leiden twee injecties van humaan PG in het adjuvant DDA tot een progressieve polyarthritis met een golvend beloop van remissies en exacerbaties, waarbij nooit spontaan herstel regel 12 optreedt. regel 13 regel 14 Doelen van dit proefschrift regel 15 In dit proefschrift hadden we twee doelen: regel 16 1. Een zoektocht naar het onderdrukken van processen die aan de basis staan van regel 17 het ziekteproces. Hiermee kunnen onnodige bijwerkingen voorkomen worden en het regel 18 zou kunnen leiden tot een permanente verbetering van de ziekte en dus tot genezing. regel 19 Hierbij zochten wij naar methoden die leiden tot specifieke tolerantie inductie tegen regel 20 antigenen die zich ter plaatse van de inflammatie bevinden, en naar methoden om regel 21 deze tolerantie inductie te optimaliseren. regel 22 2. Het ontravelen van het onderliggende werkingsmechanisme van autologe BMT, regel 23 waarbij we vooral geinteresseerd waren in de rol van regulatoire T cellen (Tregs). Tregs regel 24 zijn in staat om autoimmuniteit en inflammatie te onderdrukken. Door het in samenvatting Nederlandse regel 25 kaart brengen van werkingsmechanisme van aBMT hopen we het klinische effect 175 regel 26 verder te verbeteren en relapses van de ziekte in de toekomst te voorkomen. regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 Resultaten regel 2 regel 3 DEEL 1: Verbetering van het effect van nasale tolerantie inductie met HSP60 regel 4 peptide 180-188 met behulp van een altered peptide ligand en anti TNFα. regel 5 In het eerste deel van dit proefschrift beschrijven we in hoofdstuk 3 dat nasale toediening regel 6 van het ziekteverwekkende antigeen, HSP60 peptide 180-188, leidt tot een suboptimale regel 7 onderdrukking van Adjuvant Arthritis (AA). Door een altered peptide ligand van 180-188 regel 8 toe te dienen, genaamd alanine 183, werden zowel de preventie van de ziekte alswel de regel 9 onderdrukking van reeds bestaande ziekte versterkt. Door de behandeling met alanine 183 regel 10 werden regulatoire T cellen geinduceerd, die IL-4, TGFβ en IL-10 produceerden, maar waarbij het regel 11 effect alleen afhankelijk was van IL-10. regel 12 In hoofdstuk 4 laten we een alternatieve methode zien om het suboptimale effect van nasale regel 13 toediening van 180-188 te verbeteren. Dit werd bewerkstelligd door eerst het pro-inflammatoire regel 14 milieu, dat overheerst ten tijde van actieve ziekte, te onderdrukken met behulp van anti TNFα. regel 15 TNFα is een cytokine dat aan de basis staat van arthritis. Door voorafgaand aan de nasale regel 16 tolerantie inductie anti-TNFα toe te dienen, werd de onderdrukking van de ziekte verstrekt. regel 17 Het leidde tot de inductie van CD4+ regulatoire T cellen, die IL-10 en IL-4 produceerden en CD25 regel 18 en Foxp3 tot expressie brachten. regel 19 regel 20 DEEL 2: Autologe BMT (aBMT) leidt tot restoratie van de immuun homeostase regel 21 in arthritis met behulp van Tregs. regel 22 De focus van het tweede deel van dit proefschrift lag op het ontravelen van het onderliggende regel 23 werkingsmechanisme van aBMT in het PGIA model. We tonen aan dat bestraling gevolgd regel 24 door aBMT leidde tot een snelle onderdrukking van de ziekte, gevolgd door een stabilisatie regel 25 samenvatting Nederlandse van de ziekte op een niveau onder de ernst van de arthritis voor aBMT (hoofdstuk 5). Het regel 26 176 klinische effect was niet het gevolg van alleen de bestraling, maar in de eerste fase na BMT regel 27 werd suppressie van autoantigeen specifieke pro-inflammatoire Th-17 cellen gevonden. Door regel 28 de onderdrukking van IL-17 werd mogelijk een optimaal milieu gecreerd, waarin de volgende regel 29 fase van stabilisatie van de ziekte bewerkstelligd kon worden. In deze tweede fase vonden we regel 30 een belangrijke rol voor CD4+CD25+Foxp+ Tregs in het herstellen van de immuun balans op de regel 31 lange termijn (hoofdstuk 6). regel 32 regel 33 regel 34 regel 35 regel 36 Conclusie en implicaties voor de toekomst regel 1 regel 2 In dit proefschrift werden twee experimentele dier modellen toegepast om te werken aan een regel 3 meer specifieke behandeling van arthritis. Het effect van mucosale tolerantie inductie kon regel 4 versterkt worden door een altered peptide ligand te gebruiken of door combinatie therapie regel 5 met een anti-inflammatoir middel, en de rol van regulatoire T cellen bij deze processen werd regel 6 beschreven. Tevens werd beschreven hoe aBMT leidt tot restoratie van de immuun balans met regel 7 behulp van regulatoire T cellen. regel 8 De hiermee verkregen inzichten in beide behandel methoden kan in de toekomst helpen bij regel 9 het verbeteren van de behandeling van arthritis en tot het voorkomen van relapses na aBMT. regel 10 Regulatoire T cellen spelen een belangrijke rol hierin en door ze actief te induceren, bijv. in vivo regel 11 door mucosale tolerantie inductie na aBMT, of door Tregs toe te dienen, die in vitro geinduceerd regel 12 en geexpandeerd zijn. Tot slot kunnen door het ontdekken van het werkingsmechanisme van regel 13 aBMT in de toekomst methoden ontwikkeld worden met hetzelfde werkingsmechanisme, maar regel 14 zonder de bijwerkingen van de zware conditionering die vooraf gaat aan aBMT. regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24

Nederlandse samenvatting Nederlandse regel 25 177 regel 26 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 samenvatting Nederlandse regel 26 178 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Dankwoord regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 Dankwoord regel 26 180 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Ik kijk terug op een geweldig promotie traject, waarvan het eerste gedeelte zich afspeelde in regel 1 San Diego in het lab van Salvo Albani, gevolgd door een periode in Utrecht in het lab van Berent regel 2 Prakken. In deze periodes ben ik begeleid en geholpen door een groot aantal mensen die ik regel 3 hierbij graag zou willen bedanken. regel 4 regel 5 Allereerst mijn begeleiders regel 6 Berent, wat ben ik je ongelofelijk dankbaar voor de mooie promotie tijd die ik heb gehad. regel 7 Ondanks het feit dat je me volgens eigen zeggen hebt geprobeerd te ontmoedigen onderzoek regel 8 bij je te komen doen in San Diego, heb ik dat niet opgepikt en besloot ik toch voor een jaar regel 9 onderzoek bij je te gaan doen. Tijdens die periode in 1998 wist je al snel mijn enthousiasme regel 10 voor dit onderzoek te wekken en werd de basis van dit proefschrift gelegd. Ik besef me goed regel 11 in wat voor een luxe positie ik verkeerde in die tijd: minstens 1x per dag spraken we elkaar, regel 12 je leerde me de basis beginselen van de immunologie, en zelfs het pipetteren en hanteren regel 13 van de dieren bracht je me zelf bij. Je hebt over de jaren heel wat belangrijke gebeurtenissen regel 14 in mijn leven meegemaakt, en stond altijd voor me klaar. Bedankt voor je altijd aanwezige regel 15 interesse in alles wat ik doe, zowel in als buiten het lab. Ik vond en vind het een hele bijzondere regel 16 samenwerking, en hoop dat die hier niet ophoudt. regel 17 Salvo, thank you very much for your inspiring guidance during the times I worked in your lab in regel 18 San Diego. The fact that I extended both my stays in your lab by 6 months says enough I would regel 19 say! I vividly remember meeting you, the professor of the lab, for the first time. You picked regel 20 me up in your jeep, put me on the back seat with my suitcase on my lap, and started laughing regel 21 once the rain started pouring down through the not so solid soft roof! Next thing I knew, I was regel 22 drinking marguerita’s with you in the bar next to the lab... I enjoyed the work, the people in the regel 23 lab, the city and its surroundings, not so much the discussions about Macintosh vs Microsoft, regel 24 but overall, I wouldn’t have wanted to miss it for the world. Thank you very much for taking me Dankwoord regel 25 to the whales in Mexico, it is still one of my most special trips ever. Thanks also for still staying 181 regel 26 in touch and caring about my future. regel 27 Wietse, dank voor de altijd inspirerende en gezellige promovendi lunch besprekingen. Onder regel 28 het genot van een broodje kroket (ik wel hoor!) of een gezonder broodje kaas, wist je altijd weer regel 29 de spijker op zijn kop te slaan en kwam je met innovatieve ideeen voor vervolg experimenten. regel 30 Femke, pas laat in mijn onderzoekstraject kwam je bij de groep, maar je hebt een enorme regel 31 invloed gehad op mijn project. Ik vond het altijd erg prettig om met je van gedachten te wisselen regel 32 en daar zijn veel goede ideeen uit voort gekomen. Bovendien hebben we ook naast het werk al regel 33 vele leuke avonden en congressen samen meegemaakt. Leuk dat je binnenkort weer in Utrecht regel 34 terug bent, maar voor nu moet je vooral lekker genieten daar in San Diego! regel 35 regel 36 regel 1 Nico, dank voor je begeleiding bij de BMT projecten; jouw klinische ervaring en kennis waren regel 2 hierbij onmisbaar. En de potjes met geld, die je altijd weer ergens vandaan weet te halen voor regel 3 congressen of drankjes tijdens congressen, kwamen steeds goed van pas! regel 4 regel 5 The Albani lab and other friends in San Diego regel 6 The ‘basis’ of this thesis lies in San Diego. In 1998 and 2001 I spent a year and a year and a regel 7 half respectively in the lab of Salvo Albani, and I am not quite sure whether it was the exciting regel 8 lab work or the beautiful city and surroundings that made me extend my stay each time I was regel 9 there... Certainly the people I met there and am still in touch with played a big part in it. regel 10 Erika, thank you so much for your friendship over the years. Despite the fact that we live regel 11 worlds apart, you always manage to call at least once a month, and in this way we stay well regel 12 informed about each other’s lives. We attended each other’s weddings and I am very happy regel 13 you are going to be present at the defense of this thesis as well. I hope there will be many more regel 14 life events to share in the future. The next one is coming up around August! regel 15 Gisella, thanks a lot for all your help and guidance in the lab in San Diego. You taught me a lot, regel 16 and took care of ‘the boy’ while I was away! I am very happy we manage to stay in touch, mainly regel 17 thanks to the fact that you check in on Skype every once in a while. Thank you very much for regel 18 coming to our wedding, we hope to be able to visit you in Switzerland some time soon. regel 19 Ray, you always managed to find me (or my friends) a place to stay every time I decided to regel 20 come back to San Diego, and I was even privileged to live at your ‘near-beach-front' house regel 21 in clairemont (in case of an earth quake that would destroy everything in between). You regel 22 regularly organized trips and parties, especially around dutch holidays. And without you I regel 23 would not have been able to take a diving class, since I depended on you dragging me through regel 24 the waves! Ilkay, I am very happy you met Ray, so we became good friends as well! Gijs and regel 25 Dankwoord I really enjoyed your wedding in Istanbul and it will be great to have the two of you and Aydin regel 26 182 living nearby in Holland for a few months this year. Hopefully you may even be there when I regel 27 defend this thesis. Thanks a lot for your friendship throughout the years, and I hope to stay in regel 28 touch for many more years to come! regel 29 Tho, thanks a lot for all your help on the rat experiments in San Diego. The days would often regel 30 start early around 7 am and would often not end until 10 pm, but you were always there next regel 31 to me, without even complaining once. I have wonderful memories of our time together in the regel 32 lab, and even better memories of our (although not so private) date to the Lion King musical in regel 33 LA. Hope you can come visit us some time in the future! regel 34 Arash and family, thank you very much for being a family away from home in San Diego regel 35 during the time I was there. I wish all of you the best and hope to see you again some time. regel 36 Nicole, thank you very much for your secretarial assistance during my stay in San Diego. regel 1 Thanks for always being able to extend my visa every time I decided to stay longer. But most regel 2 of all thanks for all the fun times we had together. It was great that you came to visit Holland regel 3 once before, and we hope you will come again some time this year! regel 4 regel 5 Het Prakken Lab regel 6 Beste Wilco, Mark en Mariska, onze analisten en steunpilaren, veel dank voor al jullie hulp. regel 7 Wilco, je was altijd bereikbaar en behulpzaam als ik je nodig had. Dank voor je hulp met onder regel 8 andere de muizen, ratten, luminex en het maken van figuren. Met name heel veel dank voor je regel 9 hulp in de laatste fase; zelfs tijdens een avondwandeling met de hond wist ik je nog te vinden regel 10 en loosde je me door de laatste figuren heen.Mark , naast de altijd gezellige praatjes en je hulp regel 11 in het lab, heb ik nog het meest genoten van de heerlijke en gezellige dinertjes met zijn vieren. regel 12 Ik hoop dat we dat in toekomst regelmatig blijven doen! En Mariska, dank voor je hulp bij de regel 13 eindspurt! We hadden meteen prachtige data met de ELISA en ook al vroeg je je bij de expansie regel 14 volgens mij regelmatig af waar je eigenlijk mee bezig was als je weer een paar cellen per keer regel 15 doorzette, je gaf niet op! regel 16 regel 17 Berber, door de jaren heen zijn we goede vriendinnen geworden, en sta je altijd voor me klaar. Je regel 18 bent daarbij ook nooit te beroerd om Teun en Hidde te komen knuffelen! Dank voor de eindeloze regel 19 moeite die je hebt gedaan om uit Kaapstad over te vliegen om er op 9 april bij te kunnen zijn, regel 20 en ik vind het super als het alsnog lukt, maar het is eigenlijk natuurlijk veel te veel gevraagd. Ik regel 21 hoop op nog heel veel gezellige avondjes samen na je terugkomst uit Kaapstad! regel 22 Lianne en Huib, kamergenoten van het eerste deel van mijn onderzoeksperiode in Utrecht. regel 23 Lianne, fijn toch dat we zulke duidelijke afspraken kunnen maken over onze levensplanning regel 24 en ons er allebei ook nog aan houden! Ik hoop dat we regelmatig blijven afspreken en elkaar’s Dankwoord regel 25 kinderen ook nog groot zullen zien worden. Dank voor het feit dat je altijd geinteresseerd 183 regel 26 en betrokken bent in wat ik doe! Huib, dank voor je gezelligheid en geduld tijdens de soms regel 27 drukke tijden op de kamer. Ooit gaat het me nog wel eens lukken om je mobiele nummer in regel 28 mijn telefoon te krijgen! Succes met de laatste loodjes van jouw boekje, fijn dat je nu ook bijna regel 29 klaar bent. regel 30 Eva, heel veel dank voor je hulp als student in San Diego. Naast het feit dat je veel nuttig regel 31 werk hebt gedaan, vond ik het ook erg gezellig om samen met je in het buitenland gezeten te regel 32 hebben. De dutch dinners waren een groot succes, hoe kan het ook anders, en de ‘world dance’ regel 33 les komt nog regelmatig van pas op de dansvloer (hmm, of misschien toch niet meer zo vaak regel 34 tegenwoordig..)! Succes nu met je eigen promotie en opleiding! regel 35 regel 36 regel 1 Nathalie, mijn student op het muizen project, dank voor al je hulp. Je kon al meteen heel handig regel 2 met de muizen omgaan, en daarbij kwam het erg goed uit dat je totaal niet bang voor ze was! regel 3 Veel succes in de toekomst, hoewel ik niet denk dat we je in een lab terug zullen zien? regel 4 Naomi, ik weet niet eens meer hoeveel tijd we samen hebben doorgebracht in San Diego, maar regel 5 ik heb er goede herinneringen aan! Er is een mooie vriendschap uit voortgekomen, en hoewel regel 6 we elkaar nu wat minder vaak zien, hoop ik dat te kunnen veranderen als onze levens weer in regel 7 wat rustiger vaarwater zijn gekomen. regel 8 Ismé, je loopt altijd net een paar stappen voor op mij. Je ging me voor in San Diego, voor in het regel 9 lab van Berent, voor in het AGIKO traject en de opleiding in het WKZ en promoveerde al een paar regel 10 jaar geleden. Daarnaast waren we tegelijkertijd zwanger; jij van je tweede zoon en ik van onze regel 11 tweeling. Daarom kon ik door de jaren heen altijd bij je terecht voor vragen en advies, zowel regel 12 op werk vlak als privé. Steeds weer is dat heel waardevol gebleken en ik ben heel blij dat je er regel 13 telkens weer de tijd voor nam. Ik hoop dat we de komende jaren net zulk leuk contact blijven regel 14 houden! regel 15 Sylvia, helaas ben je alweer een tijdje weg uit het WKZ, maar toch lukt het je goed om contact regel 16 te houden. Ik kijk terug op een aantal hele gezellige congressen samen, en vond het super dat regel 17 je zelfs op kraamvisite bent geweest bij Teun en Hidde. Het etentje met zijn vieren gaat er vast regel 18 nu snel een keer van komen! regel 19 Yvonne, ook al zijn we maar kort buren geweest, het heeft me nuttige ‘inside’ informatie regel 20 opgeleverd over het wel en wee van Gijs en zijn vrienden tijdens de avond voor ons huwelijk! regel 21 Ik vind het heel knap hoe je al zeer snel volledig zelfstandig was als student in het lab, en regel 22 daarmee iedereen in een sneltrein vaart voorbij streefde. Succes met alles! regel 23 Ellen, heel veel dank voor het bijspringen bij het muizen werk tijdens mijn zwangerschap, regel 24 daarmee nam je heel wat stress uit handen. Ik ben blij dat je de overstap hebt gemaakt naar regel 25 Dankwoord onze groep! regel 26 184 Joost S., ik vond het altijd erg waardevol om met je van gedachten te wisselen over het PGIA regel 27 model. Succes met je experimenten! regel 28 Sytze, Annick, Lise, Alvin, Joost, Ruud, Annemarie, Selma, Lieke, Marloes, Jorg en Jeffrey, regel 29 dank voor de gezellige tijden in en buiten het lab! regel 30 regel 31 Erica en Heleen, onze secretaresses. Erica, al jaren sta je altijd klaar als er op het laatste regel 32 moment nog van alles geregeld moet worden en ook nu bij het afronden van het boekje regel 33 kwamen er regelmatig geruststellende mails mijn kant op. Gelukkig zien we elkaar nu sowieso regel 34 regelmatig op de creche! regel 35 Heleen, ondanks het feit dat jij zelf net een hele moeilijke periode had doorgemaakt, ben je regel 36 altijd bewonderenswaardig geinteresseerd geweest in ‘onze’ Hidde. Heel erg knap en lief van je, het geluk gaat nu ook vast jouw kant op komen. Veel dank voor alles! De Flex kamer regel 1 Coralie, Marieke vd Z, Marieke E, Sanne H en Jopje, met jullie bracht ik de laatste 1.5 jaar van regel 2 mijn onderzoek in het WKZ door. We refereerden er in die tijd al regelmatig aan, dat we soms regel 3 meer op de hoogte waren van elkaars wel en wee dan onze beste vrienden, omdat we zoveel regel 4 tijd samen doorbrachten. Zo kregen jullie wekelijks een update over de groei en het welzijn van regel 5 Teun en Hidde, en jullie waren steeds weer even geïnteresseerd in de echo plaatjes en mijn regel 6 verhalen! Erg lief en fijn dat jullie zo meeleefden en ik hoop dat we, hoewel we nu niet meer regel 7 bij elkaar op de kamer zitten, net zulk leuk contact zullen blijven houden. Nu op naar jullie regel 8 promoties! regel 9 Sanne N, Stefan, Gerwin, Lieke en Judith, veel plezier nu op de flexkamer en bedankt voor het regel 10 feit dat ik nog regelmatig aan mag komen waaien en dan altijd een plekje kan vinden achter regel 11 een van de bureau’s. regel 12 Martijn en Marije, gezellig dat jullie regelmatig kwamen bijkletsen op de flexkamer. Martijn, regel 13 ik vond het erg leuk om samen met jou paranimf te zijn bij Berber, veel succes met de regel 14 voorbereidingen van je huwelijk! regel 15 regel 16 Het lab van diergeneeskunde regel 17 Corlinda, Suzanne, Femke en Teun, heel veel dank voor jullie hulp en tips bij het opstarten regel 18 van het PGIA model. Ik kon altijd voor vragen en praktische tips bij jullie terecht, waar ik erg regel 19 dankbaar voor ben! Suzanne, heel veel sterkte, jij herinnert me er regelmatig aan, waarom we regel 20 dit onderzoek doen. Willem, dank voor je interesse en nuttige adviezen. regel 21 regel 22 Het hematologie lab regel 23 Henk, heel erg bedankt voor je hulp bij het opzetten van het beenmerg transplantatie model in regel 24 de muis. Je hebt me de technieken geleerd en ook bij het opzetten van de experimenten had je Dankwoord regel 25 altijd nuttige op- en aanmerkingen. Anton, bedankt voor het meedenken bij het opzetten van 185 regel 26 de experimenten. regel 27 regel 28 Paranimfen regel 29 Bas, ik vind het ontzettend knap, hoe attent en geïnteresseerd je altijd bent in mij en anderen. regel 30 Heel veel dank voor alle kaartjes, telefoontjes en vriendschap de afgelopen jaren en natuurlijk regel 31 in het bijzonder de laatste tijd. Hopelijk worden we ooit nog eens buren, hoewel ik zeker weet regel 32 dat dat geen vereiste zal zijn om ons leuke contact voort te zetten in de toekomst. Super dat regel 33 je naast me staat! regel 34 regel 35 regel 36 regel 1 Eef, we hebben ontzettend veel uren samen doorgebracht in het lab, en zijn in de jaren steeds regel 2 meer naar elkaar toe gegroeid. Ik heb veel bewondering voor je preciezie en enthousiasme regel 3 voor het onderzoek. Ons verblijf samen in San Diego was geweldig: de avondjes film kijken bij regel 4 jou ‘thuis’, de schoen van Gijs die het ‘geheim’ weg gaf, alle uurtjes bij de ratten, de heerlijke regel 5 wijntjes, mijn 3 koffers en jouw ene rugzak; het heeft een mooie basis gelegd voor ons regel 6 onderzoeks traject samen. Dank voor al je hulp en kritisch meedenken, leuk dat je naast me regel 7 staat! regel 8 regel 9 Vrienden regel 10 Lieve vrienden en vriendinnen, heel veel dank voor jullie vriendschap en interesse over de regel 11 jaren. Hopelijk breekt er nu een wat rustiger tijd aan, zodat ik jullie allemaal weer wat vaker op regel 12 kan komen zoeken! regel 13 regel 14 Mijn familie regel 15 Lieve schoonfamilie, Noud, Adriette, Saskia en Fleur, dank voor jullie interesse en in het regel 16 bijzonder voor de hulp bij het opvangen van de kinderen tijdens het afronden van dit regel 17 proefschrift. Het was een drukke tijd, en jullie stonden altijd voor ons klaar. Fijn dat je bijna regel 18 weer terug bent Saskia! regel 19 Lieve Simone en Karlijn, ik kan me geen betere schoonzussen wensen en Teun en Hidde geen regel 20 betere tantes! Jullie zijn altijd attent en geïnteresseerd, erg lief! regel 21 Lieve broers, Jasper en Floris, tijdens mijn promotie tijd hebben jullie met name genoten van regel 22 de tijd die ik in San Diego woonde, omdat jullie toen een mooi excuus hadden om op bezoek te regel 23 komen, wat jullie dan ook allebei gedaan hebben. Ik wacht rustig af tot jullie aankondigen dat regel 24 er wat te vieren valt... maar eerst maar eens dit feestje! regel 25 Dankwoord Lieve pap en mam, ik weet niet waar ik moet beginnen jullie te bedanken. Jullie hebben me regel 26 186 een mooie jeugd gegeven en dragen nog dagelijks bij aan het welzijn in ons leven. Heel veel regel 27 dank voor jullie liefde en onvoorwaardelijke steun over de jaren, en de goede zorgen voor Teun regel 28 en Hidde. Zonder jullie was dit boekje er nooit gekomen en ik draag het dan ook met veel plezier regel 29 aan jullie op. regel 30 regel 31 Lieve Teun en Hidde, jullie zijn de allerliefste, mooiste en grappigste kinderen op de hele wereld. regel 32 Jullie zijn veel belangrijker dan dit boekje, ik ben ontzettend trots op jullie! regel 33 Lieve Gijs, dit promotie traject was alleen al waardevol vanwege het feit dat ik er jou door heb regel 34 leren kennen in het lab van Berent! Ik ben intens gelukkig met jou en ons leventje samen, regel 35 bedankt voor het feit dat je er altijd voor me bent en zoveel moois toevoegt aan mijn leven. regel 36 Curriculum Vitae regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 Vitae Curriculum regel 26 188 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Sarah Roord werd geboren op 23 juni 1974 te Utrecht. Ze groeide samen met haar ouders en regel 1 haar twee broers op in Bunnik. In 1990 vertok het gezin naar Charottesville (Virginia, USA), waar regel 2 zij een jaar naar de Charlottesville Highschool ging. In 1993 slaagde zij voor haar gymnasium regel 3 examen aan het Stedelijk Gymnasium in Utrecht. In datzelfde jaar startte ze met de studie regel 4 geneeskunde aan de Rijksuniversiteit Leiden. Tijdens haar studie werkte ze enkele jaren met regel 5 veel plezier voor Eurotransplant International Foundation als allocatie coördinator. Voor haar regel 6 wetenschappelijke stage verbleef zij een jaar in San Diego (Californië, USA), waar ze onder regel 7 begeleiding van Prof. Dr. Salvo Albani de eerste experimenten voor dit proefschrift verrichtte. regel 8 In 2001 slaagde zij cum laude voor haar artsexamen en bracht zij opnieuw twee jaar door in regel 9 San Diego voor onderzoek, gefinancierd door een stipendium van het Ter Meulen Fonds van de regel 10 KNAW. In 2002 startte zij als assistent-geneeskundige in opleiding tot klinisch onderzoeker regel 11 (NWO-AGIKO) aan de opleiding kindergeneeskunde in het UMC Utrecht (opleider: Prof. Dr. Jan regel 12 Kimpen, thans Dr. Joost Frenkel) in combinatie met promotieonderzoek onder supervisie van regel 13 Prof. Dr. Wietse Kuis en Prof. Dr. Berent Prakken. Het perifere gedeelte van de opleiding heeft regel 14 zij van 2005 tot 2006 in de Isala Klinieken te Zwolle (opleider: Dr. Paul Brand) voltooid. Sarah regel 15 is getrouwd met Gijs Teklenburg; samen zijn zij de trotse ouders van Teun en Hidde (geboren regel 16 in 2007). regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24

Curriculum Vitae Curriculum regel 25 189 regel 26 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 Vitae Curriculum regel 26 190 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 List of publications regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 publications of List regel 26 192 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 F van Wijk, S T Roord, B Vastert, I de Kleer, N Wulffraat, and B J Prakken. Regulatory T cells in regel 1 autologous stem cell transplantation for autoimmune disease. Autoimmunity 2008. Oct 28:1 regel 2 regel 3 Sarah T.A. Roord, Wilco de Jager, Louis Boon, Nico Wulffraat, Anton Martens, Berent Prakken, regel 4 Femke van Wijk. Autologous Bone Marrow Transplantation in autoimmune arthritis restores regel 5 immune homeostasis through CD4+CD25+FOXP3+ Regulatory T cells. Blood 2008. May regel 6 15;111(10):5233-41. Epub 2008 Feb 6. regel 7 regel 8 Van Emmen E, Roord ST, Brouwer AF, Kuiters GR, Bekhof J. Pustular and vesicular skin eruptions regel 9 in newborns. Ned Tijdschr Geneeskd 2007. Feb 3;151(5):277-283. regel 10 regel 11 Sarah T.A. Roord, Berent J. Prakken, Salvatore Albani. Present and future of combination regel 12 therapy of autoimmune diseases. Editorial. Future Rheumatol 2006. 1(6): 649-655. regel 13 regel 14 Roord ST, Zonneveld-Huijssoon E, Le T, Yung GP, Koffeman E, Ronaghy A, Ghahramani N, Lanza regel 15 P, Billetta R, Prakken BJ, Albani S. Modulation of T cell function by combination of epitope regel 16 specific and low dose anticytokine therapy controls autoimmune arthritis. PLoS ONE 2006. regel 17 Dec 20:1:e87. regel 18 regel 19 Prakken BJ, Samodal R, Le TD, Giannoni F, Yung PG, Scavulli J, Amox D, Roord S, de Kleer I, Bonnin regel 20 B, Lanza P, Berry C, Massa M, Billetta R, Albani S. Epitope-specific immunotherapy induces regel 21 immune deviation of proinflammatory T cells in rheumatoid arthritis. Proc Natl Acad Sci USA regel 22 2004. Mar 23;101(12):4228-33. regel 23 regel 24 G.L. Puga Yung, T.D. Le, S. Roord, B. Prakken and S. Albani. Heat shock proteins (HSP) for publications of List regel 25 immunotherapy of rheumatoid arthritis (RA). Inflamm. Res. 2003. 52: 443-451. 193 regel 26 regel 27 Berent J. Prakken, Sarah Roord, Arash Ronaghy, Marca Wauben, Salvatore Albani, Willen van regel 28 Eden. Heat shock protein 60 and adjuvant arthritis: a model for T cell regulation in human regel 29 arthritis. Springer Semin Immunopathol 2003. 25: 47-63. regel 30 regel 31 Massa M, Costouros N, Mazzoli F, De Benedetti F, La Cava A, Le T, De Kleer I, Ravelli A, Liotta regel 32 M, Roord S, Berry C, Pachman LM, Martini A, Albani S. Self epitopes shared between human regel 33 skeletal myosin and Streptococcus pyogenes M5 protein are targets of immune responses in regel 34 active juvenile dermatomyositis. Arthritis Rheum 2002. Nov; 46(11): 3015-25. regel 35 regel 36 regel 1 Van Eden W, Van Der Zee R, Van Kooten P, Berlo SE, Cobelens PM, Kavelaars A, Heijnen CJ, regel 2 Prakken B, Roord S, Albani S. Balancing the immune system: Th1 & Th2. Ann Rheum Dis. 2002. regel 3 Nov; 61 Suppl 2:ii25-8. Review. regel 4 regel 5 Prakken BJ, Roord S, van Kooten PJ, Wagenaar JP, van Eden W, Albani S, Wauben MH. Inhibition regel 6 of adjuvant-induced arthritis by Interleukin-10 driven regulatory cells induced via nasal regel 7 administration of a peptide analog of an arthritis-related heat shock protein 60 T cell epitope. regel 8 Arthritis Rheum 2002. Jul; 46(7): 1937-46. regel 9 regel 10 Ronaghy A, Prakken BJ, Takabayashi K, Firestein GS, Boyle D, Zwaifler NJ, Roord ST, Albani regel 11 S, Carson DA, Raz E. Immunostimulatory DNA sequences Influence the Course of Adjuvant regel 12 Arthritis. J Immunol 2002. Jan 1; 168(1): 51-56. regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 publications of List regel 26 194 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 List of abbreviations regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 abbreviations of List regel 26 196 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Adjuvant (induced) Arthritis AA/AIA regel 1 Altered peptide ligand APL regel 2 Antigen presenting cell APC regel 3 Area under the curve AUC regel 4 Autoimmune disease AID regel 5 Autologous bone marrow transplantation aBMT regel 6 Collagen induced arthritis CIA regel 7 Complete Freund’s adjuvant CFA regel 8 Cytotoxic T lymphocyte antigen-4 CTLA-4 regel 9 Dimethyl Dioctadecyl Ammonium Bromide DDA regel 10 Disease-modifying antirheumatic drug DMARD regel 11 Enzyme linked immunosorbent assay ELISA regel 12 Experimental autoimmune encephalomyelitis EAE regel 13 Fluorescence activated cell sorter FACS regel 14 Glucocorticoid-induced tumor necrosis factor receptor GITR regel 15 Hyddroxychloroquine HCQ regel 16 Heat shock protein HSP regel 17 Immunoglobulin Ig regel 18 Incomplete Freund’s adjuvant IFA regel 19 Interferon IFN regel 20 Intradermal i.d. regel 21 Intraperitoneal i.p. regel 22 Inguinal lymph node ILN regel 23 Interleukin IL regel 24 Juvenile Idiopathic Arthritis JIA abbreviations of List regel 25 Macrophage activation syndrome MAS 197 regel 26 Major histocompatibility complex MHC regel 27 Mandibular lymph node MLN regel 28 Mean Fluorescence Index MFI regel 29 Mesenchymal stem cell MSC regel 30 Methotrexate MTX regel 31 Monoclonal antibody MAB/mAb regel 32 Mycobacterium Tuberculosis Mt regel 33 Myelin basic protein MBP regel 34 Multiple Sclerosis MS regel 35 Non-obese diabetic NOD regel 36 regel 1 Ovalbumin OVA regel 2 Peripheral blood lymphocyte PBL regel 3 Peripheral blood mononuclear cell PBMC regel 4 Phosphate buffered saline PBS regel 5 Proteoglycan Induced Arthritis PGIA regel 6 Stem cell transplantation SCT regel 7 T helper Th regel 8 Transforming growth factor TGF regel 9 Regulatory T cells Treg regel 10 Tumor necrosis factor TNF regel 11 Rheumatoid arthritis RA regel 12 Subcutaneous s.c. regel 13 Systemic Lupus Erythematosus SLE regel 14 T cell receptor TCR regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 abbreviations of List regel 26 198 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36 Colour Figures    regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10  regel 11  regel 12 Figure 2. Combination therapy as well as a full course of Etanercept treatment led to reduction regel 13 of histological damage in the ankle joints.   regel 14 Chapter 4, page 74. regel 15    regel 16    regel 17      regel 18   

     regel 19        regel 20    regel 21  

  regel 22          regel 23    

 regel 24      regel 25 figures Colour   regel 26 200     regel 27   regel 28     regel 29           regel 30  regel 31    regel 32                    regel 33 Figure 3. The combination therapy of Etanercept and HSP60 180-188 led to an antigen specific  regel 34 increase of IL-10 and IL-4 production and up regulation of CTLA-4 expression in CD4+ T cells in regel 35 draining Mandibular Lymphnodes (MLN).  regel 36 Chapter 4, page 76.             

       

            

        regel 1 regel 2 regel 3 regel 4 regel 5 regel 6   regel 7     regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14  regel 15   regel 16       regel 17       regel 18

         regel 19   Figure 4. Less histological  damage after aBMT in hind limb joints        Chapter 5, page 99. regel 20        regel 21                      regel 22               regel 23                                 regel 24    

 figures Colour   regel 25     201 regel 26       regel 27  regel 28    regel 29           regel 30   regel 31         regel 32              regel 33  regel 34  Figure 6. Suppression of IL-17 production post aBMT regel 35  Chapter 5, page 102.  regel 36                  

       

            

  regel 1 regel 2 regel 3 regel 4 regel 5 regel 6 regel 7 regel 8 regel 9 regel 10 regel 11 regel 12 regel 13 regel 14 regel 15 regel 16 regel 17 regel 18 regel 19 regel 20 regel 21 regel 22 regel 23 regel 24 regel 25 regel 26 regel 27 regel 28 regel 29 regel 30 regel 31 regel 32 regel 33 regel 34 regel 35 regel 36