DPPIV Inhibition in a Murine Model of Mixed Chimerism and Transplantation Tolerance

DPPIV inhibition in a murine model of mixed chimerism and transplantation tolerance

Doctoral thesis at the Medical University of Vienna for obtaining the degree of

Doctor of philosophy submitted by Dr. med. univ. Elisabeth Schwaiger

Supervisor: Prof. Dr. Thomas Wekerle Department of Surgery Medical University of Vienna Waehringer Guertel 18-20 1090 Vienna, Austria

Vienna, 03/05/2017 Declaration

I, Dr.med.univ. Elisabeth Schwaiger, declare that the work presented in this thesis is the result from original research performed at the Department of Surgery, Medical University of Vienna, and has not been previously submitted to any other university. As part of the PhD Program (N094) this work was done in the thematic program “Immunology” of the Medical University of Vienna and funded by a grant from the Austrian Science Fund (FWF, SFB F2310 dedicated to Prof. Dr. Thomas Wekerle). There was no financial interest or any relationship with financial interest related to the work of this thesis.

All studies were designed and performed by me and my coauthors under the guidance and supervision of Prof. Dr. Thomas Wekerle (Department of Surgery, Medical University of Vienna). With the support of Veerle Matheeussen, a research assistant of the Fund for Scientific Research-Flanders, DPPIV enzymatic activity assays were conducted in Flanders, Belgium. My laboratory colleagues Christoph Klaus, Nina Pilat, Ulrike Baranyi and Haley Ramsey (Department of Surgery, Medical University of Vienna) assisted me with the animal experiments and data analysis. During the course of this thesis, I participated in the performance of experiments and data acquisition for the research articles additionally mentioned in this manuscript.

The interpretation and analyzation of the experiments was done by me and the manuscript was exclusively written by me under the supervision of Prof. Dr. Thomas Wekerle. The literature used in this manuscript is cited and can be found in the reference section. Figures and Tables presented herein were designed by me or otherwise cited appropriately.

ii Table of Contents

Declaration ...... ii

Table of contents ...... iii

List of Figures ...... vi

List of Tables ...... vii

Abstract ...... viii

Zusammenfassung ...... ix

Publications arising from this thesis ...... xi Original articles ...... xi Review articles ...... xii

Abbreviations ...... xiii

Acknowledgements ...... xvi

CHAPTER ONE: INTRODUCTION

1.1 Organ transplantation ...... 1

1.2 Immunological tolerance ...... 2 1.2.1 Tolerance by deletional and regulatory mechanisms ...... 3 1.2.2 Tolerance induction ...... 8 1.2.3 Costimulatory signals in tolerance induction ...... 8

1.3 Chimerism ...... 14 1.3.1 Mixed hematopoietic chimerism ...... 15 1.3.2 Costimulatory blockade and new developments for the induction of mixed chimerism ...... 17

1.4 Clinical translation ...... 20

1.5 Hurdles to the induction of tolerance ...... 25

1.6 Hematopoietic stem cells & engraftment ...... 27 1.6.1 The role of CD26/DPPIV in bone marrow transplantation...... 28 1.6.2 CD26/DPPIV and its pleiotropic immunologic effects ...... 29 1.6.3 CD26/DPPIV inhibition and its widespread use in the clinic ...... 30

1.7 Aims of this thesis ...... 32

iii

CHAPTER TWO: RESULTS

2.1 Prologue ...... 33

2.1 First paper ...... 34 Dipeptidyl peptidase IV (DPPIV/CD26) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning ...... 34 2.1.2 Interlude ...... 45

2.2 Second paper ...... 46 Anti-LFA-1 or rapamycin overcome costimulation blockade-resistant rejection in sensitized bone marrow recipients ...... 46 2.2.1 Interlude ...... 60

2.3 Third paper ...... 61 Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade ...... 61 2.3.1 Interlude ...... 74

2.4 Fourth paper...... 75 Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade...... 75 2.4.1 Interlude ...... 85

2.5 Review article ...... 86

Hurdles to the induction of Tolerogenic Mixed Chimerism ...... 86 2.5.1 Interlude ...... 93

CHAPTER THREE: DISCUSSION

3.1 General discussion ...... 94

3.2 Conclusion & future perspectives ...... 96

CHAPTER FOUR: MATERIAL & METHODS

4.1 Animals ...... 98

4.2 Bone marrow transplantation protocol ...... 98

iv 4.3 DPPIV Inhibition ...... 99

4.4 Assay for DPPIV inhibition ...... 100

4.5 Flow cytometric analysis ...... 100

4.6 Skin grafting ...... 101

4.7 Statistical methods ...... 101

References: ...... 102

Curriculum Vitae ...... 117

Publications Elisabeth Schwaiger ...... 121

v List of Figures

Figure 1: Human T cell development in the thymus Figure 2: Different functions of Aire in medullary thymic epithelial cells Figure 3: T-cell activation through different signals Figure 4: CD28 driven expression of various costimulatory receptors Figure 5: Conditioning regimens for tolerance induction (Massachusetts General Hospital) Figure 6: Different substances facilitating toxicity reduction of conditioning regimens Figure 7: Schematic representation of the hematopoietic and stromal stem cell development Figure 8: Functions of human CD26+ T-cells in vitro Figure 9: Schematic representation of sitagliptin on the glucose metabolism

vi List of Tables

Table 1: Profiles and clinical results of patients conditioned for tolerance induction (Massachusetts General Hospital) Table 2: Demographics and clinical results of patients conditioned for tolerance induction (Northwestern Memorial hospital) Table 3: Different protocols used for DPPIV inhibition

vii Abstract

In organ transplantation the development of new pharmaceuticals has improved short-term allograft survival but long-term survival is still limited as the host’s alloreactive immune system cannot be fully controlled and as side effects of immunosuppressive therapies often affects patient life’s. Thereby, the development of minimally toxic bone marrow transplantation (BMT) protocols for the induction of donor specific tolerance via mixed hematopoietic chimerism remains a promising strategy. An enhanced engraftment of transplanted hematopoietic stem cells (HSCs) would allow a reduction of conditioning toxicities and thereby may facilitate a more widespread clinical application. Herein the stromal derived factor-1 (SDF-1, CXCL12) and its receptor CXCR4 play a crucial role as they are responsible for trafficking HSCs to the bone marrow niche. Their chemotactic effectiveness is downregulated by the enzymatic activity of CD26, dipeptidyl peptidase IV (DPPIV). Inhibition of DPPIV in the donor BM or additionally in the recipient, has been reported to enhance HSC engraftment in specific experimental settings typically using myeloablative conditioning or nonmyeloablative conditioning of immunodeficient recipients. We therefore investigated whether DPPIV inhibition might improve BM engraftment in a minimally toxic murine bone marrow transplantation (BMT) protocol using limited numbers of unsorted allogeneic (i.e. Balb/c to B6) or syngeneic (i.e. CD45-congenic) BMCs after nonmyeloablative conditioning. By applying sitagliptin, used in the treatment of type 2 diabetes, or Diprotin A, a trip-peptide (Ile-Pro-Ile), we were able to nearly completely block DPPIV enzymatic activity at time of transplantation. Especially by using sitagliptin, recipients reached more than 98% DPPIV inhibition at peak exposure. Successful HSC engraftment was demonstrated and donor skin graft tolerance was induced in all groups. Nevertheless, at time of follow up (>20 weeks), levels of multilineage chimerism were comparable with and without DPPIV inhibition and no difference in skin graft tolerance was detectable. It seemed that in this model of BMT with minimal conditioning, the beneficial effects of DPPIV inhibition did not suffice to have a potent effect on BM engraftment. Further research has to be done to identify mechanisms allowing engraftment of allogeneic bone marrow after minimal conditioning.

viii Zusammenfassung

Dank neuer pharmakologischer Entwicklung im Bereich der Organtransplantation konnte das kurzfristige Transplantatüberleben verbessert werden, dementgegen zeigt sich jedoch das Langzeitüberleben weiterhin begrenzt. Dies ist einerseits durch die spenderspezifische Alloreaktivität, aber auch durch zahlreich auftretende medikamentöse Nebenwirkungen bedingt, welche das Patientenleben maßgeblich beeinflussen. Die Entwicklung eines minimal- toxischen Modelles der Knochenmarktransplantation, zur Implementierung einer spenderspezifischen Toleranz mithilfe eines gemischten hematopoetischen Chimärismus, präsentiert sich hierbei vielversprechend. Eine verbesserte Einwanderung transplantierter Stammzellen ins Knochenmark würde eine Reduktion der Toxizität bisheriger Konditionierungsprotokolle ermöglichen und eine breitere klinische Durchführbarkeit bedeuten. Der stromal derived factor-1 (SDF-1, CXCL12) und sein Rezeptor CXCR4 spielen eine entscheidende Rolle, da sie für die Einwanderung von Stammzellen in die Knochenmarknische verantwortlich sind. Ihre chemotaktische Wirkung wird durch die enzymatische Aktivität von CD26, der Dipeptidylpeptidase IV (DPPIV), reduziert. In experimentellen Modellen, die typischerweise auf myeloablativer Konditionierung oder nicht- myeloablativer Konditionierung von immun-defizienten Empfängern beruhten, konnte durch eine Inhibierung der DPPIV Aktivität von Knochenmarkszellen sowie zusätzlich der Empfänger, das Anwachsen hematopoetischer Stammzellen positiv beeinflusst werden. Wir untersuchten in einem minimal toxischen Mausmodell der Knochenmarkstransplantation, ob eine DPPIV Hemmung das Anwachsen einer begrenzten Anzahl von nicht-gesorteten allogenen (d.h. Balb/c auf B6) oder syngenen (d.h. CD45-congenen) Knochenmarkszellen nach nicht-myeloablativer Konditionierung verbessern kann. Unter Verwendung von Sitagliptin, welches für die Therapie des Typ II Diabetes mellitus eingesetzt wird, sowie von Diprotin A, einem Tripeptid (Ile-Pro-Ile), wurde eine beinah komplette Blockade der DPPIV enzymatischen Aktivität zum Zeitpunkt der Transplantation erreicht. Speziell durch eine Behandlung der Empfänger mit Sitagliptin, gelang eine über 98% Inhibierung in der Spitzenexposition. Das Anwachsen von Knochenmarkszellen und die nachfolgende spenderspezifische Toleranz der Hauttransplantate war in allen Gruppen erfolgreich. Dennoch wurde über den Beobachtungszeitraum (>20 Wochen) kein Unterschied hinsichtlich des multilineären Chimärismus oder des Hauttransplantat-Überlebens, im Vergleich zur Kontrollgruppe ohne DPPIV Inhibierung, festgestellt.

ix Es scheint, als ob in diesem Model der Knochenmarkstransplantation mit minimaler Konditionierung, die positive Wirkung der DPPIV Inhibierung unzureichend für einen deutlichen Effekt hinsichtlich eines verbesserten Anwachsens von Knochenmarkszellen ist. Weitere wissenschaftliche Erkenntnisse sind hierbei notwendig um den Mechanismus des Anwachsens von syngenen oder allogenen Stammzellen nach nicht-myeloablativer Konditionierung entschlüsseln zu können.

x Publications arising from this thesis

Original articles

1. Dipeptidyl peptidase IV (DPPIV) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning. Schwaiger E, Klaus C, Matheeussen V, Baranyi U, Pilat N, Ramsey H, Korom S, De Meester I, Wekerle T. Exp. Hematology, 2012 Feb; 40(2):97-106

2. Anti-LFA-1 or rapamycin overcome costimulation blockade-resistant rejection in sensitized bone marrow recipients. Ramsey H, Pilat N, Hock K, Klaus C, Unger L, Schwarz C, Baranyi U, Gattringer M, Schwaiger E, Wrba F, Wekerle T. Transpl. Int. 2013 Feb;26(2):206-18.

3. Rapamycin and CTLA4Ig synergize to induce stable mixed chimerism without the need for CD40 blockade. Pilat N, Klaus C, Schwarz C, Hock K, Oberhuber R, Schwaiger E, Gattringer M, Ramsey H, Baranyi U, Zelger B, Brandacher G, Wrba F, Wekerle T. Am J Transplant. 2015 Jun;15(6):1568-79. doi: 10.1111/ajt.13154. Epub 2015 Mar 17

4. Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade Bigenzahn S, Pree I, Klaus C, Pilat N, Mahr B, Schwaiger E, Nierlich P, Wrba F, Wekerle T. J Immunol Res. 2016;2016:8635721. Epub 2016 Oct 31.

xi Review articles

1. Hurdles to the induction of tolerogenic mixed chimerism. Pilat N, Klaus C, Schwaiger E, Wekerle T. Transplantation. 2009 May 15;87(9Suppl): S79-84.

xii Abbreviations

Aire autoimmune regulator AMR antibody mediated rejection APCs antigen presenting cells Bcl-xL B-cell lymphoma-extra large BM bone marrow BMC bone marrow cell BMT bone marrow transplantation CB costimulation blockade CCL3 chemokine C-C motive ligand 3 CD cluster of differentiation CML chronic myeloid leukemia CSF colony stimulating factor CTLA4 cytotoxic T-lymphocyte-associated protein 4 CTLA4-Ig cytotoxic T-lymphocyte-associated protein 4 immunoglobulin CXCL12 C-X-C motive chemokine 12 CXCR4 C-X-C motive chemokine receptor 4 DBMT donor bone marrow transfusion DC dendritic cell DN double negative DP double positive DPPIV dipeptidyl peptidase IV DST donor specific transfusion eTACs extrathymic Aire expressing cells FC facilitating cell Flt3LG FMS-related tyrosine kinase 3 ligand FOXP3 forkhead box protein P3 G-CSF granulocyte colony-stimulating factor GIP glucose-dependent insulinotropic polypeptide GLP-1 glucagon like peptide - 1 GLP-1 glucagon-like peptide-1 GVHD graft versus host disease GVHD graft versus host disease

xiii HSC hematopoietic stem cell HSCs hematopoietic stem cells HSCT hematopoietic stem cell transplantation HSCT hematopoietic stem cell transplantation ICAM-1 intercellular adhesion molecule 1 ICOS inducible T-cell co-stimulator IDO indoleamine 2,3-dioxygenase Ig Immunoglobulin IL interleukin iTregs induced regulatory T-cells KIR killer cell immunoglobulin-receptor KLRG1 killer cell lectin-like receptor G1 LFA-1 lymphocyte function-associated antigen-1 mAb monoclonal antibody MHC major histocompatibility complex MHC major histocompatibility complex mTECs medullary thymic epithelial cells mTOR mammalian target of rapamycin NHP nonhuman primate model NK cells natural killer cells NKT-cells natural killer T-cells NOs nitric oxide synthase nTregs natural regulatory T-cell PBSC peripheral blood stem cells PD-1 programmed death 1 PI-3K phosphoinositide-3-kinase pMHC peptide-MHC complex SCF stem cell factor SDF-1 stromal derived factor 1 SP single positive TBI total body irradiation TCR T-cell receptor TF tissue factor Th1 T helper 1

xiv Th2 T helper 2 TIM T-cell immunoglobulin domain and mucin domain Tmem memory T-cells TNF tumor necrosis factor TNFRS TNF receptor superfamily TPO thrombopoietin Treg regulatory T-cell VCAM-1 vascular cell adhesion molecule 1

Acknowledgements

First of all, I would like to thank Prof. Dr. Thomas Wekerle for offering me the opportunity to work as a PhD student in his transplant-lab and for providing excellent mentoring and encouraging scientific support throughout all the years. I would also like to give a special thanks to Prof. Dr. Ferdinand Muehlbacher who always supported generously research in the field of transplantation at the Medical University of Vienna.

A very special thanks to all my colleagues at the transplant-lab who assisted me patiently with their knowledge in practical scientific work and for helping me to perform my research. Thank you for all your input and constructive discussions during my time as a PhD student. I would also like to thank Veerle Matheeussen for her exceptional help with the enzymatic assays.

Finally, I would like to express my deepest gratitude to my family and friends who were always there for me, encouraged and supported me all over the years.

xvi CHAPTER ONE: INTRODUCTION

1.1 Organ transplantation

Organ transplantation has become a state-of-the-art treatment for end stage organ failure but the number of patients on the waiting list is constantly increasing and organ shortage still remains a major problem. In 2013 more than 50.000 patients were waitlisted for a kidney transplant at the European union (data from the European commission) and 19.426 transplantations were performed according to the European Renal Association - European Dialysis and Transplant Association (ERA-EDTA).1 To counteract this ongoing divide between available organs and waitlisted patients, tremendous efforts have been made to extend the donor pool and improve long-term allograft survival. The limitation of long-term allograft survival lies on the one hand within the restriction of immunosuppressive therapy to fully control hosts immune response and on the other hand, because patients suffer from various drug side effects that can contribute to the problem of death with functioning graft. These drug side effects might not only reduce allograft and patient survival but can also harm recipient’s psychological constitution enormously. Out of an immunological perspective, chronic antibody-mediated rejection (AMR) represents the leading cause of early and late allograft injury followed by allograft loss. 2-4 Herein, plasmapheresis, immunoadsorption, modulation of B cell immunity, intravenous immunoglobulin (IVIG) therapy, anti-cluster of differentiation (CD)20 antibody rituximab, the proteasome inhibitor bortezomib or the anti-C5 antibody eculizumab have been used as therapeutic strategies.5,6 While several of these strategies have shown to effectively reverse episodes of acute AMR, treatment of chronic AMR still represents a major challenge. Hence, the establishment of immunological tolerance would not only erase the problem of immunological allograft damage due to rejection episodes but would also prevent multiple side effects due to chronic immunosuppressive therapy.

1 1.2 Immunological tolerance

Immunological tolerance is defined as a state in which the immune system does not mount a destructive response to a set of antigens or to tissues expressing those antigens, but remains capable of responding to other antigens.7 Therefore, immunological tolerance is of great interest as it may prevent chronic rejection, obviates the need for life long immunosuppressive therapy and can thereby limit their toxicity and metabolic side effects. The phenomenon of achieving immunological tolerance through hematopoietic chimerism dates back into 1945 when Ray Owen studied the inheritance of red blood cell antigens in the cattle and discovered the neonatal-transplant tolerance.8 At the same time, Frank Macfarlane Burnet disclosed the nature of self-recognition by the immune system. The development of immunocompetence was described as a slow process in adolescent animals and further, during the embryonic development, as a process of self-recognition through which ‘tolerance is acquired by fetal exposure to ‘non-self’ constituents’.9 The creation of the clonal selection theory was followed thereafter, and this early studies provided the basis for using hematopoietic stem cell transplantation (HSCT) for the induction of tolerance. 60 years ago, these findings were followed by a novel approach to treat bone marrow (BM) injury after lethal irradiation, used in the treatment for hematological malignancies, by transplantation of hematopoietic stem cells (HSCs).10 Henceforward, stem cell transplantation has become a state of the art treatment for hematological malignancies and for other severe immunodeficiency syndromes. In this protocols graft versus host diseases (GVHD) and the toxicity of the preparative treatment therapies including life-threatening immunosuppression through myeloablation, constitutes a formidable severe hazard for the recipient, which would not be acceptable in solid organ transplantation.11 Nowadays morbidity and mortality in the clinical translation decreased due to the development of reduced-intensity conditioning protocols, better prophylaxis and therapy against GVHD and novel antimicrobial drugs. Further development facilitated the successful accomplishment of an organ transplant following bone marrow transplantation (BMT) for hematological indication, even when major histocompatibility complex (MHC) loci were mismatched. 12-14 The beneficial effect of this protocols was, that many of these patients did not require any chronic immunosuppression after organ transplantation.

2 1.2.1 Tolerance by deletional and regulatory mechanisms Deletional tolerance. Central tolerance by clonal deletion relies primarily on an intra-thymic positive and negative selection of T-cells.15 Initially, T-cell progenitors migrate from the capillaries in the thymus near the corticomedullary junction. At this stage, T-cells are double- negative (DN). While moving to the sub-capsular zone, T-cells undergo a process of T-cell receptor (TCR)-β rearrangement additionally to the expression of a pre-TCRα complex. Thymocytes receiving signals through their TCR-β/pre-TCRα complex survive and DN T- cells refine into double-positive (DP). 16 17 18 These DP cells migrate through the cortex and encounter cortical-thymic epithelial cells. At this stage, a commitment to the thymus-derived regulatory T-cell lineage (i.e DP FOXP3 thymocytes) can already occur19. Following the affinity model, cells expressing a no or too low affinity TCR for the presented self-peptide major histocompatibility complex (MHC), die by neglect, which accounts for 80- 90% of lost thymocytes15. DP-thymocytes displaying an intermediate affinity for the self MHC survive and develop into CD4 or CD8 single positive (SP) thymocytes 15, a process known as positive selection. Following positive selection, SP thymocytes migrate into the thymic medulla for their 4-5day residency before they become part of the peripheral T-cell repertoire. 15,20,21 By encountering the peptide-MHC complexes (pMHC) in the thymic medulla, SP thymocytes that display a high affinity TCR undergo apoptosis (negative selection) or can develop into regulatory T-cells (agonist selection).22 It seems that the number of MHCII+ epitopes presented by thymic dendritic or medullary epithelial cells and the affinity between TCR and the presented peptide MHCII+ determines T- cell selection as it was shown in a model of polyclonal CD4+ T-cell tolerance.23 Thymocytes showing a high affinity TCR for the pMHC are very sensitive to deletion. An adequate signal for a high affinity TCR from only one encounter might be sufficient enough for a thymic deletion and crucial in the context of partial clonal deletion where tissue-restricted epitopes are expressed by only few MHC+ APCs in the thymus.23 Thymocytes with TCR-specificity for epitopes that are largely expressed by pMHC thymic dendritic or medullary cells can receive multiple signals and hence deletion may occur with even lower affinity.24 23

3 Figure 1: Human T-cell development in the thymus Regulatory T-cell ,#&'$%* %* +$%* /$+)79><=A($# &*#1Front Immunol38 The autoimmune regulator (Aire) plays a key role in thymic tolerance development and is primarily expressed in the thymic medulla. Interestingly, Aire was not only shown to promote expression of various tissue-specific genes but also to promote expression of disease-associated autoantigens and cancer germline genes by medullary thymic epithelial cells (mTECs).25,26 A transgenic mouse system demonstrated that in the absence of Aire, autoreactive cells can escape into the periphery, wheras, if Aire was present, these autoreactive cells would be deleted. 27,28 Deletion was even thought to be primarily mediated by Aire expression in mTECs and data suggest, that even thymic B cells may express low levels of Aire and thereby participate in T- cell selection.29 Concerning regulatory T-cells (Tregs), patients with Aire mutations showed decreased Treg numbers and FOXP3 expression and further, in vitro assays revealed a lower suppression capacity suggesting a potent role for Aire for Treg development. 30,31 Additionally, in a transgenic murine model it was demonstrated that Tregs can be selected by specific Aire- expressed antigens on mTECs32 and in a model using an endogenous antigen that was Aire dependent, antigen-specific Tregs failed to develop in the absence of Aire, illustrating the role of Aire in the selection of specific Tregs. 33

4 Figure 2: Different functions of Aire in medullary thymic epithelial cells

(.'%)2%-(&#) % $$+%*&#(%%/&%79><=B("3%()&%%+(%3+1*, $$+% Aire also plays a role in peripheral tolerance. In humans, Aire has been found in lymph nodes at both the transcript and the protein level. 34,35 Aire expression is also present in secondary lymphoid organs, such as lymph nodes, tonsils and gut associated lymphoid tissue.34 These characteristics suggest a potentially new cell population that has been termed extrathymic Aire expressing cells (eTACs). 36 ETACs may play a role in maintaining peripheral T-cell tolerance, when T-cells escape thymic negative selection. Nevertheless, next to positive and negative selection of evolving thymocytes, also B cells can be centrally deleted in the BM for the purpose of establishing tolerance. One mechanism behind this phenomenon was demonstrated to be receptor editing when developing B cells encounter a membrane-bound self Ag in the bone marrow. Thereby these cells withdraw their B cell receptor, display low levels of CD19 and produce a secondary diverse immunoglobulin (Ig) repertoire. 37 Split tolerance. Split tolerance can be described as a state where despite persistence of mixed hematopoietic chimerism, allogeneic tissue-specific antigens that are not expressed by hematopoietic cells, cause rejection while other alloantigens from the same donor are

5 tolerated.38,39 Thereby, minor histocompatibility antigen distributions seem to trigger this alloreactive tissue-specific rejection. In a murine model of bone marrow transplantation after nonmyeloablative conditioning, split tolerance was induced when CLTA4 Ig and rapamycin was applied after depletion of recipients CD4 T-cells.40 In this experimental model, long term MHC mismatched hematopoietic chimerism occurred and tolerance to donor heart allografts was maintained while donor skin grafts were rejected. Chimeras presented donor specific tolerance and deletion of donor reactive T-cells in vitro but minor histocompatibility mismatched skin grafts were rejected while minor histocompatibility matched skin grafts were accepted.40 Thereby, it seems that CD8 T-cells caused split tolerance through the direct pathway in presence of the CD40 costimulatory signal.

Nevertheless, another form of split tolerance was described by Fudaba et al where hematopoietic and kidney allografts may share minor histocompatibility antigens that are able to sensitize hosts hematopoietic cells.41 This phenomenon was described in a human HLA- identical combined kidney-bone marrow transplantation protocol with nonmyleoablative conditioning, where donor bone marrow was rejected while renal allograft tolerance sustained viable despite a in vitro alloresponse.41 Regulatory mechanisms. Next to central clonal deletion, regulatory mechanisms have been identified to be crucial for the induction and maintenance of tolerance. Concerning transplantation, peripheral regulation of alloreactivity seems to be based on a cell-contact or cytokine dependent mechanism 42,43,44 and can occur in secondary lymphoid tissues and in the allograft. 45,46 Various cell types have been identified to show regulatory potency. Regulatory activity, so far, has been attributed to regulatory macrophages47, dendritic cells48, myeloid- derived suppressor cells49, mesenchymal stromal cells 50,51, CD4+ regulatory cells 45, CD8+ 52 regulatory cells and to TFH cells , regulatory B cells andγδ6##s with the need for further investigations.53 Especially in tolerance inducing protocols using hematopoietic chimerism after nonmyeloablative condtitioning, regulatory mecahnisms can complete central deletion and support donor specific tolerance.54 Herein, a special interest lies within the Treg population. Adoptive Treg cell therapy has shown in murine models of BMT that regulation is present during the follow up despite less or incomplete clonal deletion. Moreover, the degree of tolerance is more complete due to Treg cell induced tolerance toward minor histocompatibility mismatched antigens. 55,54,44 This is of special interest as in nonhuman primate models and in

6 clinical trials, mixed hematopoietic chimerism for tolerance induction often occurs to be only transient on the long-term. 56,57

Accommodation, as a regulatory mechanism, diverges from tolerance as it describes the appearance of humoral anti-donor reactivity that is accompanied by an upregulation of cytoprotective molecules and does not injure the graft. 7,58,59,60 This phenomenon was first characterized in ABO-incompatible kidney transplantation when surprisingly, kidney allografts survived after an only transient elimination of anti-donor reactive blood group (A or B) antibodies from recipients. 61,62,58 Different mechanisms have been postulated to be responsible for accommodation but it seems that allografts become somehow resistant to anti-donor reactivity as C4d deposition, as a sign for complement fixation, stands for intact antibody binding but is lacking cellular lysis which implicates regulatory activity.58 Thereby, an upregulation of potent protective gens in the graft seems to control the immune system from rejection.59,60 Numerous molecules have been demonstrated to be involved in this process, as an upregulation of cytotoxic-cell genes like Granzyme A and B, Killer cell lectin-like receptor G1, Cathepsin, Perforin, and Chymase 1, were observed to be more present in tolerated grafts than in rejected grafts.59 Additionally, cytoprotective molecules like nitric oxide synthase (NOs), indoleamine 2,3 dioxygenase, adrenomedullin receptor, apelin, interleukin (IL)-27 p28 sub-unit, Tissue factor pathway inhibitor-2, B-cell lymphoma-extra large (Bcl-xL) and endothelial cell protein C receptor seem to be upregulated in these tolerated grafts.59 Hence, the upregulation of the cytotoxic or pro-inflammatory cell genes cannot be seen as a predictive marker for graft failure but do more refer to a balanced active immune process involving both effector and regulatory cells. 59,63

7 1.2.2 Tolerance induction Since Ray Owen discovered that dizygotic bovine twins develop hematopoietic chimerism by sharing a common placental circulation and became tolerant to MHC mismatched skin from each other, a step was set towards manipulating one’s immune system for achieving “Acitvely acquired tolerance”.8,64 Early experimental attempts for the induction of chimerism and tolerance focused on a complete myeloablation by a lethal dose of TBI for creating space in the bone marrow and suppressing recipient’s immune system by depleting alloreactive T-cells. Protocols followed where the elimination of the existing T-cell pool relied on depleting antibodies and non-myeloablative TBI.65 These findings were refined by targeting radiation to the thymus, which allowed a further reduction in the dose of TBI for successful BM engraftment. Taken together, these studies relied on a global non-selective reduction of recipient’s T-cell pool.66

1.2.3 Costimulatory signals in tolerance induction Within the discovery that the activation of the immune system depends on signaling through multiple pathways, costimulatory blockade was found to be helpful in tolerance inducing protocols. For a full activation, survival, clonal expansion and differentiation of a T-cell, costimulatory signals are indispensable, when once the primary signal of the T-cell receptor encounters a certain antigen-peptide exposed by the MHC. Therefore, antibodies and receptor fusion proteins were discovered to block this costimulatory signals at the time of antigen exposure67,68 and by modulating this specific pathway, classical T-cell anergy was in induced in vitro.69 Following, a more selectively targeting of the T-cell alloresponse was enabled. Concerning their structure, costimulatory molecules providing costimulatory signals, can be classified by the immunoglobulin family [CD28, cytotoxic lymphocyte-associated-antigen 4 (CTLA4), programmed death 1 (PD-1), inducible T-cell co-stimulator (ICOS)], the tumor necrosis factor (TNF) superfamily (CD40, OX 40, CD137), the adhesion molecule family [lymphocyte function-associated antigen-1 (LFA-1), CD2] and the discovered T-cell immunoglobulin domain and mucin domain (TIM) molecules, cell surface proteins mainly on T helper 1 (Th1) and T helper 2 (Th2) cells, antigen presenting cells (APCs), dendritic cells (DCs), macrophages and kidney epithelial cells. 70,71,72

8 One of the best analyzed costimulatory molecules is CD28. CD28 is expressed on the majority of human T-cells. In mice it is constitutively expressed on all T-cell subsets, whereas in humans only 95% of CD4+ and 50% of CD8+ T-cells express CD28.73,74 When a TCR encounters a MHC bound peptide, the T-cell enters the cell cycle. Hence, a second signal is required to fulfill the entire activation process and induce cell division.75 The costimulatory molecule CD28 on T-cells interacts with CD80 and CD86 (B7.1 and B7.2) ligands on APCs. Thereby, the CD28 signal reduces the number of ligand engaged TCRs needed for an initial T-cell activation.CB Together with TCR activation, the CD28/B7 binding increases the expression of the IL2 receptor α chain (CD25), the costimulatory molecule CD40L (CD154), increases the cytokine production such as IL2 and interferon gamma (IFN6γ) and enhances the expression of anti-apoptotic molecules (i.e. Bcl-xl) on the surface of T-cells. Following, by binding of IL2 and IL15 to their receptors, which share a common gamma chain, the phosphoinositide-3-kinase (PI-3K) and the mammalian target of rapamycin (mTOR) pathways are activated, growth signals are delivered and the T-cell enters the cell cycle.CC

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9 CD28 is also critical for the maintenance of an immune response as an initial activation and proliferation can be lost within 48-72 hours in the absence of CD28.75 T-cells lacking signals through that costimulatory molecule become functionally inactivated with reduced proliferation, differentiation and cytokine production and finally become anergic.78 The T-cell enters then a state of non-responsiveness to a TCR engagement and a following CD28 signal can no longer revers the established anergy. 75,68 CTLA-4 (CD152) acts a negative regulator upon T-cell activation by competitively and preferentially binding B7.1 but also B7.2, as it shares structural similarities to CD28, outcompeting its signal.79,80 After the inhibitory mechanism of CTLA-4 has been postulated to be an intrinsic signal81, new data emerged showing CTLA-4 as a cell-extrinsic inhibitor.82 CTLA-4 can bind its ligands followed by a process of trans-endocytosis and degradation inside the CTLA-4 expressing cells.82 CD28 can therefore no longer bind to CD80/CD86 and deliver costimulatory signals. Hence, CTLA4 gives a negative feedback loop that downregulates the T-cell response. 83,84 An indirect induced blockade of CD28 via the fusion protein CTLA4-Ig was found to inhibit the effects on T-cell activation due to costimulatory signal transduction and thereby prevents potential alloreaction. CTLA4-Ig, combines the extracellular domain of CTLA-4 with the FC portion of human IgG1 that binds CD80/86 (B7.1/B7.2) with high avidity. 85 Interestingly, in a murine model of heart transplantation, the usage of CTLA-4-Ig on day 2 instead at time of engraftment, prolonged allograft survival.86 It was also demonstrated, that the induction of CD4+ T-cell anergy requires costimulation transmitted by the B7 signal and an interaction between the CTLA-4 molecule and its ligand B.7, as the absence of the B7 signal leads to a loss of the CTLA-4-Ig tolerogenic capability.87 Moreover, blocking the CTLA-4 signal during the initial response with an anti-CTLA4- monoclonal antibody (mAb), resulted experimentally in an abrogation of tolerance and therefor shows its importance in the induction and maintenance of tolerance.87 This effect is probably due to the necessity of CTLA-4 upregulation upon T-cell activation and engagement with its ligands for activating its negative costimulatory function. Following, a delayed administration of CTLA4-Ig was applied in many experimental protocols for tolerance induction. 86,88-90

In the meantime, CTLA-4 Ig developed clinically into a by the FDA approved effective immunosuppressant (LEA 29Y, belatacept) which its registered under the trade name

10 Nulojix®. Vincenti et al demonstrated superiority of belatacept in patient and graft survival and a significantly increased mean eGFR compared to calcineurin inhibitors 7 years after kidney transplantation.91 Hence, a more selectively targeting of the T-cell alloresponse was enabled.

Another costimulatory molecule is CD40, a member of the TNF superfamily that is constitutively expressed on dendritic cells, macrophages, B-cells and platelets92,93 but also on epithelial cells, endothelial cells, fibroblasts and keratinocytes.94 CD40 binds to CD154 (CD40L), primarily expressed on activated T-cells, but also mast cells, eosinophils, dendritic and endothelial cells express CD154.94,95 After engagement of CD40 on dendritic cells, cytokine production is increased, costimulatory molecules are induced and further, crosspresentation of antigens can take place.96 Moreover, signal downstreaming leads to an upregulation of MHC- and costimulatory molecules of the B7 family,augments DC survival and enhances cytokine production like TNFαand IL12. 92 This process effectively triggers T-cell differentiation and activation. 96,97 By blocking the CD40:CD154 pathway, autoimmune diseases and allograft rejection has been targeted. 98,99,92 It has shown to be highly effective in experimental transplantation models for preventing acute rejection and prolonging allograft survival.89,97 Moreover an additional benefit was seen when the CD40:CD154 pathway was blocked together with CTLA4-Ig 100, donor specific transfusion (DST) or rapamycin101, but it was not efficient in preventing chronic rejection on its own. 102,103 While mAb blocking CD154 (CD40L) showed promising effects in rodents and NHP models, the use of the anti-human CD154 antibody was accompanied with severe thromboembolic complications in humans in a clinical phase I trial. 104-108 It was found out, that this thromboembolic effect was due to expression of CD154 on platelets where it participates in the stabilization of thrombi. 109 Moreover, CD154 also binds to its ligand CD40 on the vascular endothelium und upregulates adhesion molecules such as E-selectin, the vascular cell adhesion molecule 1 (VCAM-1), the intercellular adhesion molecule 1 (ICAM-1) and the tissue factor (TF) expressed in endothelial cells.110 Due to the thromboembolic side effects, occurring with the blockade of the CD40:CD154 pathway by this anti-human CD154 antibody, its further development was suspended. A new designed anti-CD40 mAb seemed to prolong islet allograft survival in rhesus macaques by acting synergistically with belatacept 111 and an anti-human CD40 mAb, developed for entering the phase I of a clinical trial as ASKP1240, proved to be save and well tolerated in healthy subjects.112 Recently a new anti-human CD154 domain antibody (dAb, BMS-986004)

11 that lacks the crystallizable fragment (Fc) was also developed for effectively blocking the CD40:CD154 pathway without presenting the devastating side effect of platelet activation3 ) antibody proved efficacious and safe in a non-human primate model with a significant prolongation in allograft survival. 113

Figure 4: CD28 driven expression of various costimulatory receptors

CD28-mediated co-stimulation: a quantitative support for TCR signaling (© 2003 Oreste Acuto and Frédérique Michel, Nat Rev Immunol) Other costimulatory molecules are the inducible costimulatory molecule (ICOS, CD278) on T lymphocytes belonging to the CD28 superfamily, CD27 and CD30, membrane proteins on a subpopulation of T and B lymphocytes belonging to the TNF receptor superfamily (TNFRS7 and TNRSF8), OX40 (CD134) on activated T-cells belonging to the TNFRSF4, 4-1BB on activated T lymphocytes belonging to the TNFRSF9, and LFA-1. LFA-1 is a member of the integrin family, that represents a unique alpha chain (CD11a) and a beta chain (CD18), for which they are recognized and interacts with ICAM-1 on APCs. It plays a role in T-cell trafficking, activation, costimulation and in the stabilization of the immunological synapse, ensuring optimum activation of T-cells. 114,115

12 Moreover, LFA-1 is also expressed on B cells as blockade of LFA-1 in combination with other immunosuppressive agents enabled the inhibition of anti-donor antibody production 116,117,118 and LFA-1 is expressed on memory T-cells (Tmem), suggesting a potential role for its blockade for preexisting anti-donor T-cell memory. 119,92 Efalizumab, an anti-LFA-1 mAb, has been approved by the FDA for treatment of severe plaque psoriasis but was removed from the market a few years later in June 2009 due to an increased risk of progressive multifocal leukenzephalopathy.120 Efalizumab acts by binding to CD11a and thereby bocks reversibly the binding of LFA-1 to its ligand ICAM-1. It attenuates T-cell activation and inhibits cell migration during an active immune response.121 In kidney transplantation, efalizumab has been evaluated in a phase I/II trial in a multi drug regimen, combined with cyclosporine A. It seemed to reduce rejection episodes, but unfortunately it also showed a higher incidence of posttransplant lymphoproliferative disease in the higher dose group.122 TIMs are members of the type I transmembrane glycoprotein family and were described as cell- surface proteins for differentiation between Th1 and Th2 cells. Therefore, promising results in immune regulation in autoimmune and allergic diseases were suggested. 123,124. Further they seem to have a broad immunological function like T-cell activation, induction of T-cell apoptosis, T-cell tolerance and they may be involved in the sweep of APCs to clear apoptotic cells.125 Concerning experimental models for tolerance induction, low affinity anti-TIM 1 mAb synergizes with rapamycin and prolong cardiac allograft survival by inhibiting Th1 alloresponses126, whereas blocking TIM 3 costimulation seemed to accelerate the development of autoimmune disease and abrogates tolerance in islet allograft models. Therefore, it presents to be a negative regulator for the T(H)1 immune response.127

13 1.3 Chimerism

In 1959, after treatment with a high dose of total body irradiation (TBI, 850R), patients with an acute lymphoblastic leukemia were successfully transplanted with syngeneic bone marrow. These patients showed a rapid clinical and hematological recovery after lethal irradiation, but leukemia recurred after a few months. 128,129 Nowadays, HSC transplantation has developed to a standard of care treatment procedure for various malignant hematological- and autoimmune diseases. Not only for various forms of leukemia, myelodysplastic syndromes, lymphoproliferative disorders, multiple myeloma and immunodeficiency syndromes, but also promising results for autologous HSCT for diseases like multiple sclerosis, systemic sclerosis, crohn’s disease and systemic lupus have been shown.130,131,132 Herein, a complete or near complete elimination of all blood elements followed by an allogeneic or autologous stem cell transplantation for reconstitution is necessary – a state designated to as “full chimerism”. Within the last decades, it has been published, that patients who received a bone marrow transplantation for the treatment of hematological diseases and needed a solid organ transplantation for end stage organ disease, accepted an allograft without immunosuppression when given from the same donor. 14,133,134,135 Nevertheless, following these findings of solid organ allograft tolerance after the establishment of full chimerism for hematological diseases, steps were taken to the induction of tolerance for end stage organ diseases solely, based on mixed hematopoietic chimerism, a state in which donor and recipient hematopoietic cells coexist (>1%<100%).

14 1.3.1 Mixed hematopoietic chimerism For the induction of donor specific tolerance for solid organ transplantation only, mixed hematopoietic chimerism shows various advantages over full hematopoietic chimerism, as it represents a reduced risk for graft versus host disease (GVHD), a better preserved immunocompetence and the need for less toxic conditioning regimens. 136 The mechanism of mixed hematopoietic chimerism relies on the engraftment of donor derived HSC following bone marrow transplantation in conditioned recipients. Adjacent to recipient stem- and precursor cells in the bone marrow, donor stem- and precursor cells develop into cells of all hematopoietic cell lineages in the same individual, generating systemic-tolerance to their MHC- and, depending on the protocol, to their non-MHC formations.137 These cells migrate and populate the recipient thymus, where a specialized microenvironment support their homing, survival, proliferation, differentiation, migration and selection.138,139 The contribution of this so-called ‘central tolerance’ achieved by clonal deletion of self-reactive and donor-reactive lymphocytes, distinguishes the chimerism concept from any other approaches. As already described above, mainly thymic epithelial and APCs are responsible for tolerizing donor reactive lymphocytes which seem to correlate with the extent of clonal deletion of donor reactive cells in mixed chimeras. 21,15 Further, concerning the maintenance of tolerance it may be possible that activated peripheral T-cells reenter the tymic medulla for providing a source of donor specific antigens leading to central clonal deletion of self-reactive lymphocytes.140 It was also found out that regulatory T- cells can recirculate back to the thymus and represent an activated and differentiated phenotype with which they perform a regulatory function through an inhibition of the development of interleukin-2 (IL-2) dependent de novo regulatory T-cells.141 It also seemed that B-cells have a role in central tolerance as only thymic B cells express AIRE, accompanied by an MHC II and CD80 upregulation and immunoglobulin class switching. Therefore, thymic B cells require a CD40 signal upon interaction with autoreactive CD4+ T- cells and with a represented endogenous self-antigen, they contribute to negative selection.29 Next to peripheral T-cells including regulatory T-cells and B cells, also dendritic cells seem to migrate back to the thymus and participating in the process of central clonal deletion. This phenomenon has been reported to occur in a three step adhesion cascade, necessary for successful central clonal deletion of reactive thymocytes encountering this immigrated peripheral antigen presenting dendritic cells. 142

15 Nevertheless, for the implementation of mixed hematopoietic chimerism, pre-existing mature T-cells represent a major barrier. A reduction of pre-existing mature donor reactive T-cells is necessary for the prevention of a premature rejection of transplanted bone marrow cells over the MHC-barrier. 143,144 While in previous protocols, recipients T-cells needed to be completely degraded by myeloablation or T-cell depleting antibodies, recent protocols are capable of tolerizing T-cells more precisely and leaving the T-cell pool generously intact. 136

A major contribution to the development for the induction of donor specific tolerance via mixed chimerism was the implementation of costimulation blockade (CB) following bone marrow transplantation. Progressive peripheral clonal deletion, i.e. apoptosis of mature T-cells, are achieved by entering a state of anergy, generated by costimulation blockade in the presence of an antigen.100,145 Wekerle et al uncovered this specific mechanism of deletion in tolerance induction in 1998. Thereby, the deletion of donor reactive T-cells, presenting Vβ5 and Vβ11 containing TCRs that bind superantigens, encoded in the background genome from B6/B10 mice, expressed by I-E (MHC II) positive B10.A cells, was analyzed.100 It was demonstrated, that only a combined application of CTLA-4 Ig and MR1 (anti CD154 mAb, anti CD40L) can induce a blockade of the CD28 and the CD40 costimulatory pathway after a 3 Gy TBI conditioning regimen and transfusion of donor bone marrow cells (BMCs). Thereby an ongoing deletion and over the time stable chimerism and donor specific tolerance was achieved.100 Herein, the induction of mixed hematopoietic chimerism was feasible without a complete destruction of donor’s T-cell pool. Concerning the establishment of tolerance among mature T-cells, donor-specific tolerance is already detectable bevor a complete clonal deletion takes place, indicating a considerable contribution of non-deletional mechanisms like regulation.146,147,148,149,136 Nevertheless, costimulation blockade alone has not proven to be efficient enough to control rejection of the transplanted bone marrow by premature donor reactive cells. 136 Today, strategies have evolved to overcome this alloreactive barrier and induce mixed chimerism in the experimental and clinical setting. All sharing one general goal - the discovery of less toxic conditioning for a widespread clinical application for the induction of donor specific tolerance in organ transplantation.

16 1.3.2 Costimulatory blockade and new developments for the induction of mixed chimerism After including costimulatory blockade into BMT protocols, minimally toxic conditioning regimens for the induction of hematopoietic mixed chimerism and donor specific tolerance have evolved. A major step was taken in the year 2000 when Wekerle et al published the first experimental protocol that completely spared any cytoreductive treatment when very high doses of fully MHC mismatched BMCs (approximately 2x108 unseparated) were transplanted by using costimulation blockade. 150 As these high numbers of BMCs would not be feasible in the clinical setting, further research was set on the discovery of new strategies to reduce toxicity and enhance engraftment of HSCs in these protocols. One of the strategies to reduce the toxicity was to add a one-month short-course immunosuppressive treatment, starting with the day of BMT as it would be required in the clinical translation.151 Blaha et al investigated in 2003 the impact of methylprednisolone, mycophenolate mofetil, rapamycin, FTY720, tacrolimus and cyclosporine A for the induction of mixed hematopoietic chimerism and donor specific tolerance in a fully MHC mismatched murine model of BMT.151 It was demonstrated that an additional treatment with tacrolimus or cyclosporine A after successful initiation of hematopoietic chimersim by a nonmyeloablative conditioning regimen, negatively affected chimerism in the second month after transplantation and donor specific tolerance was predominately impeded.151 An inhibitory effect of calcineurin inhibitors on apoptosis of alloreactive T-cells was held responsible for this phenomenon, as the early deletion of donor reactive T-cells seemed to be blocked and occurred to a significantly lesser degree at later time points.151 The other immunosuppressive drugs investigated in this study seemed to act more synergistically to the standard-protocol. Moreover, it was shown that the additional usage of methylprednisolone plus mycophenolate mofetil plus rapamycin allowed the reduction of the conditioning regimen to one gray when clinically feasible numbers of BMCs (15-20x106) were transplanted.151 In 2008 the effect of granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBCS), transplanted instead of BMCs for the induction of mixed chimerism under the use of costimulation blockade, was investigated. Thereby, a beneficial effect was lacking and the population of T-cells transplanted within the PBCS seemed to reject donors HSCs, as only recipient T-cell depletion combined with costimulation blockade led to a successful engraftment of PBCS without GVHD.152 This phenomenon was investigated more precisely when Hock et al uncovered that due to allorecognition, additionally transplanted donor CD4+ T-cells can trigger an IL-6 dependent recipient T-cell bystander activation that abrogates the

17 effect of anti-CD154 mAb (MR1) and leads to donor BM rejection in a murine model of BMT using nonmyeloablative conditioning and costimulation blockade.153

Natural killer (NK) cells and natural killer T (NKT) cells also attracted some interest for the induction of mixed chimerism. Nierlich et al published in 2010 the effect of NK and NKT-cells on the induction of mixed chimerism using CB. At that time NKT-cells were known to have various effects including augmenting or downmodulating the immune response. In this study, NKT-cells seem to trigger NK cells towards rejection of BMC and fail to abrogate chimerism in the absence of NK and T-cells. Hence, NKT-cells stimulated in CB based BMT protocols, prevented induction of chimerism and tolerance.154 Further, it was shown that mTOR inhibition and a singular blockade of the CD28 costimulation was able to induce mixed chimerism and donor-specific tolerance to cardiac allografts.40 After recipient conditioning of 2 Gy TBI with or without anti-CD4 and/or anti CD8 depleting Ab, mice were transplanted with fully MHC mismatched or minor antigen matched BMCs. After the establishment of chimerism, MHC-mismatched/minor antigen matched skin grafts survived, whereas MHC-mismatched/minor antigen mismatched skin grafts were rejected besides the survival of cardiac allografts, describing a potent role for minor antigen disparities in allograft rejection and split tolerance. Moreover, it was found out, that a CD28 costimulatory blockade solely was only able to tolerize CD8+ cells but not CD4+ cells. 40 Cippà et al focused on targeting memory cells, known to be resistant to costimulation blockade, by treatment with ABT-737 for Bcl-2/Bcl-XL inhibition.155 In a murine model of BMT using fully MHC mismatched BMCs, costimulation blockade (CD154) and donor-specific transfusion prior transplantation, blocking the anti-apoptotic Bcl-2/Bcl-XL pathway was sufficient for a reduction and re-sensitization of alloreactive Tmem.155 Also an induction therapy with LFA-1 or rapamycin showed effectiveness in preventing rejection of transplanted donor bone marrow cells into sensitized mice using costimulation blockade and low dose TBI.156

In 2010 Pilat et al published that Tregs led to long-term multilineage chimerism and donor specific skin graft tolerance without cytoreductive conditioning.55 Hence, by using costimulation blockade, moderate doses of allogeneic BMCs and a short-course of rapamycin, mixed chimersim and donor-specific tolerance was induced. The effectiveness of this setting was tested for several populations of polyclonal Tregs [forkhead box protein P3 (FOXP3)- transduced, natural and TGF-β induced Tregs]. In this study, again, it was found out, that

18 regulatory and deletional mechanisms were both evident for the maintenance of tolerance. Nevertheless, besides an already induced tolerance, the deletion process needed more time to be fully established in chimeras compared to prior protocols using cytoreductive conditioning.55 Further, Pilat et al demonstrated in a murine model of BMT for the induction of heart and skin graft tolerance, that protocols involving Tregs are more likely to induce and maintain tolerance to tissue specific antigens, i.e. antigens that are not restricted to hematopoietic cells, after nonmyeloablative conditioning.54 Thereby, for the induction of mixed hematopoietic chimerism and tolerance, in vitro activated recipient’s natural Tregs showed superiority to TGFβ induced Tregs in a dose dependent manner when only limited numbers (0.5x106) of Tregs were available.157 In this protocols, two mechanisms seem to be responsible for the beneficial effect of Tregs: Tregs suppress rejection of transplanted BM and Tregs establish a state where suppression-linked mechanisms defend the allograft from ongoing rejection of tissue specific antigens.137 Interestingly, by using myeloablative conditioning (9Gy TBI) long-term tolerance to tissue specific antigens was established even in the absence of additional Treg treatment, indicating for a persistent T-cell population responsible for tissue specific antigen rejection after nonmyeloablative conditioning. 137

Transplantation of a kidney first followed by a donor bone marrow transfusion (DBMT) several months later has been described as delayed tolerance and seem to be a step towards clinical translation. In 2007 Koyama et al published that CD8+ memory T-cell depletion was necessary to induce mixed chimerism and delayed renal allograft tolerance when DBMT was followed four months after kidney transplantation in a nonhuman primate model.158 This points to one of the key distinctions between rodents housed in pathogen free conditions and higher animal models or humans, where the exposure to environmental pathogens and the accumulation of memory cells complicate existing protocols and make it harder to induce tolerance using clinically acceptable conditioning protocols. Hence, in 2016 Hotta et al presented a role for induced Tregs (iTregs) converted from non-Tregs in a nonhuman primates (NHP) model.159 After simultaneous BMT or delayed BMT following heart, lung or kidney allograft transplantation,nonmyeloablative conditioned tolerant recipients showed a donor-specific loss of CD8+ T-cells and a proliferation of CD4+ T-cells. A main proportion of the expanded CD4+ T-cells were detected to be FOXP3+ in tolerant animals but not in animals that rejected their grafts. Herein the reason for the development of a delayed tolerance after transient hematopoietic chimerism seemed to lie in the induction of iTregs from non-Tregs by donor antigens. 159

19 1.4 Clinical translation

One of the first case-reports for clinical tolerance induction has been described in 1991 when two patients suffering from acute leukemia received a conventional HLA identical BMT and subsequently a kidney transplant from the same donor after developing end stage kidney disease. These grafts were accepted without immunosuppressive therapy.12 Many other reports followed and the induction of tolerance after solid organ transplantation was achieved.160,161,162

At time, the clinical protocols for the induction of chimerism are based on myleosuppressive treatment, immunosuppressive treatment and a source of allogeneic HSC. The first human trial was published in 1999 by the Boston group where 6 patients suffering from chronic renal insufficiency due to multiple myeloma received bone marrow transplantation and a kidney graft from the same HLA identical donor. 163 7 years after transplantation, 3 patients were off any immunosuppression with preserved kidney function, indicating a state of “operational tolerance”.41 Following thereafter in a subsequent trial, published from the Massachusetts General Hospital in Boston, 10 end-stage renal disease patients received a combined bone marrow and kidney transplant from a HLA mismatched living related donor after a nonmyeloablative conditioning.164,165 Herein the conditioning protocol, that consisted initially of cyclophosphamide, anti CD-2 mAb and thymic irradiation was extended with rituximab and prednisolone after the 3rd patient developed an irreversible humoral rejection. A further modification was performed due to development of donor specific antibodies. Consequently, two additional doses of rituximab were added to the protocol and cyclosporine A was switched to tacrolimus.166 Cyclosporin A/tacrolimus was administered postoperatively and discontinued during several months. All patients developed transient chimerism, 7 patients discontinued their immunosuppression successfully for more than 4 years, 4 patients withdraw their immunosuppression successfully for 4.5-11.4 years, 3 patients had to retake their immunosuppression due to recurrence of disease or antibody mediated rejection and 3 patients lost their graft due to thrombotic microangiopathy or rejection.165 Besides the persuading results of long term immunosuppression free graft survival, except one recipient all the other recipients suffered from acute kidney injury, the so called “engraftment syndrome”. This “engraftment syndrome” occurred about 10 days after transplantation and seemed to be associated with the hematopoietic recovery of the host and probably homeostatic proliferation of memory T-cells additional to a rapid loss of chimerism.165 Interestingly, allograft biopsies from the first

20 immunosuppressive free recipients showed six times higher FOXP3 levels and similar levels of Granzym B mRNA compared to patients receiving standard immunosuppression, implicating a considerable role for regulatory mechanisms.164

Figure 5: Conditioning regimens for tolerance induction ()) +)**)%(# &)' *#8

21 Table 1: (& #)%# % #()+#*)&'* %*)&% * &%&(*&#(% %+* &% (Massachusetts General Hospital)

Long-Term Results in Recipients of Combined HLA-Mismatched Kidney and Bone Marrow Transplantation Without Maintenance Immunosuppression (© 2014 Kawai T et al, Am J Transplant) Another trial has been published by Scandling et al in 2008 from the Stanford University School of Medicine, where patients received a HSCT from a HLA matched donor after a conditioning regimen with low intensity total lymphoid irradiation and anti-thymocyte globulin 10 days following kidney transplantation.167 This patients developed stable mixed chimerism despite withdrawal of immunosuppression 6 months after transplantation. 167 The same group published further data where 38 HLA matched and mismatched living donor kidney transplant recipients were subsequently transplanted with a CD34+ enriched hematopoietic cell transfusion containing a defined number of CD3+ T-cells after conditioning including total a lymphoid irradiation (10 doses with 80 to 120 cGy) and anti-thymocyte globulin.57 Immunosuppressive drugs were withdrawn in 16 of 22 HLA matched patients, with a median follow up of 29 months. Concerning the HLA mismatched cohort, 2 patients developed transient chimerism and were withdrawn from immunosuppression for 3.5 and 5.5 months until mild rejection episodes (Banff I) were observed. After the treatment of this rejection episodes, patients maintained immunosuppression. The “engraftment syndrome“,

22 reported by the Boston group, occurred in only one patient. No case of GVHD and no graft loss among living patients was reported and only one patient loss due to an accident occurred. 57

Another interesting approach was published by Leventhal et al from the Northwestern Memorial hospital in Chicago, when 19 out of 25 HLA mismatched kidney graft recipients who meanwhile finished a follow up period of more than 18 months, were transplanted in a clinical phase II trial (FDA-IDE 139479).168 Hence, after a conditioning regimen that included fludarabine, cyclophosphamide and 200 cGy TBI, a living donor kidney graft was transplanted together with facilitating cells enriched HSC. Facilitating cells are described to be a cell population consisting of tolerogenic CD8+/TCR- cells, assimilable to precursor plasmacytoid dendritic cells, that are attributed to be adjuvant in stem cell engraftment without inducing GVHD. 168-173 After one year, 12 of these initially 25 transplanted patients developed a durable chimeric state and were successfully weaned of immunosuppression (i.e. consisting of tacrolimus and in one case after the development of thrombotic microangiopathy, sirolimus, and mycophenolate mofetil). Infections were reported next to successful vaccination of recipients without loss of chimerism or rejection.168 Interestingly, no case of GVHD was published for a long time but changed recently when Leventhal et al published in the 8 year follow up data two cases of GVHD. One patient with acute gastrointestinal GVHD was successfully treated with steroids but developed mild chronic GVHD, and another patient presented a fatal course of gastrointestinal GVHD with a combined CMV infection eleven months after transplantation. 174

Table 2: Demographics and clinical results of patients conditioned for tolerance induction (Northwestern Memorial hospital)

Immune reconstitution/immunocompetence in recipients of kidney plus hematopoietic stem/facilitating cell transplants (© 2015 Leventhal et al, Transplantation) For the clinical translation of tolerance inducing protocols, murine and nonhuman primate models are indispensable. Nevertheless, the complex human immune system and the persistent existing safety concerns, including the risk of infections and GVHD, still presents a major challenge. In the 3 described clinical studies above, about 50% from the HLA mismatched cohort of the Northwestern Memorial Hospital and about 40% from the HLA matched cohort from the Massachusetts General Hospital in Boston were able to withdraw their immunosuppression. Herein, patients were conditioned prior transplantation allowing the application of this protocol only to living donor kidney recipients. In the cohort from the Stanford University School of Medicine only 50% of the HLA matched kidney transplant recipients were able to discontinue immunosuppression on the long term but no patient of the HLA mismatched cohort. Just recently, there were the first cases of GVHD by Leventhal et al 174 published, making further improvements in safety and efficacy of these protocols necessary. The interesting approach described by the Stanford University School of Medicine is that tolerance was induced following kidney transplantation and therefore brings the induction of tolerance closer to a broad clinical implementation as it may involve deceased donor kidney recipients one day.

1.5 Hurdles to the induction of tolerance

One of the main barriers for the clinical translation of transplantation tolerance is the heterologous immunity and the presence of donor-reactive memory T-cells in the recipient. Heterologous immunity describes the response of the immune system to an infectious agent, where cross-reactive specificities appear.175 Hence, even without encountering a specific antigen, memory or/and memory-like T-cells can arise, leading to a barrier for tolerance induction. The mechanism behind this phenomenon can be explained by viral infection models. 176,177 Due to an immune response to viral epitopes, a cross-reaction to allo-MHC molecules can occur.175 On the one hand, following a molecular mimicry a viral/host MHC may show similarities to an allo-MHC molecule, and on the other hand, an incomplete allelic exclusion at the TCRα locus can lead to a second allo-specific TCR.175 Additionally, following a lymphopenic state T-cells can proliferation and generate memory- like cells that represent a lower threshold for activation.175 This so called homeostatic proliferation, that often follows profound T-cell depletion, induces the generation of lymphocytes that are largely resistant to CB and tolerance induction.178 Hence, clinical trials involving a profound T-cell depletion as induction therapy for preventing rejection are at risk for the emergence of homeostatic proliferation. 179-181,182,183 In addition to heterologous immunity, donor reactive memory T-cells may arise from antigen- specific sensitization due to prior transplantation, blood transfusion or pregnancy.184,185 Tmem are phenotypically and functionally different from their naive counterparts as they possess a lower activation threshold, respond rapidly upon re-stimulation186 and show reduced requirements for the TCR signal and an independence for costimulatory signals.92,186,187 They seem to express high levels of adhesion molecules (LFA-1, CD4), cytokine receptors (CD122) and anti-apoptotic molecules of the bcl-2 family.188,189,190 These memory T-cells can cycle without the need for TCR triggering signal in the absence of an antigen 191 and they are highly refractory to a variety of therapeutic interventions. This is probably due to their reduced requirements for activation and their expression of anti-apoptotic molecules. It seems that their so-called self-renewal is dependent on certain cytokines like IL15, which has been demonstrated to be critical for the maintenance of memory CD8 T-cells. 192,193 Furthermore, CB is probably not effective in tolerizing donor specific memory T-cells that developed from prior exposure to donor antigens or viral pathogens.177,194-196 It was published, that even CB induced tolerance can be broken by an active transfer of CD4+ donor specific

25 memory cells in a murine system. Hence, CD4+ cells can respond via the indirect pathway and thereby induce a CD8+ triggered allograft response that overcomes tolerance.197 Another critical component in the activation mechanism of memory cells is the impact of the precursor frequency. Studies demonstrated that memory T-cells as well as naive alloreactive T- cells presented at high frequency, obviate the need for costimulation and therefore play a role in CB resistant rejection of the allograft.177,198,199 As already described above, Nierlich et al found a potent role for NKT triggered rejection of allograft HSCs by NK cells as a barrier to stem cell engraftment.154 The mechanism of NK cells has been described as the ability to kill cells if they lose one or more MHC class I molecules, independently of a prior exposure to an antigen.200 Several models have been described on how their inhibitory killer cell immunoglobulin-receptor (KIR) recognizes MHCI and thereby prevents autoreactivity. 200-202 Special conditioning regimenshave been set up to tolerize NK cells and avoid rejection of bone marrow cells in animal models.203,204,205,206 Taken together, these findings have an important meaning to the clinical implication of CB and tolerance induction. Homeostatic proliferation, heterologous immunity and pre-sensitization create a complexity in the immune system where precise information about the MHC disparities in non-human primate models and in humans is still lacking and where the clinical translation of such tolerance inducing protocols is furthermore impeded.

Figure 6: Different substances facilitating toxicity reduction of conditioning regimens

26 1.6 Hematopoietic stem cells & engraftment

Nowadays HSCT have a widespread use in the clinic for treating patients with cancer, in which a graft versus tumor effect is utilized, and for hematological and immune disorders, like leukemia, lymphoma and different kinds of immunodeficiency syndromes.130,207,131 Moreover, HSCs may have the potency to form and regenerate all kinds of cells like skeletal, cardiac muscle, blood vessels and hepatocytes. 208-211,212 For the induction of hematopoietic chimerism and donor specific tolerance, as already described above, HSCs were also used for transplantation. Now it is known, that the distribution of HSCs is about 1 out of 10,000 to 15,000 BMCs and in the periphery the proportion falls down to 1 out of 100,000 blood cells.213 The challenge lies within the efficient isolation of these HSCs for getting an appropriate amount necessary for a successful engraftment of cells in the BM. Thy-1+CD34+Lin-/low activity has been primarily attributed to human HSCs, and this HSCs can be isolated from fetal and adult BM, umbilical cord blood and cytokine mobilized peripheral blood. 214,215 Additional, CD49f seem to be a specific HSC marker on cells being very efficient in generating multilineage grafts, and CD49f is lost within the development of multipotent progenitors.216 In mice especially the Rh123lo subset of Thy1.1+Sca1+Lin-/lowcKit+ have proven to mark long-term self-renewing HSCs.217,218 CD34+ expressed by human and mouse blood cells have also proven to mark stem- and progenitor cells that are sufficient in self-renewal.219 Several approaches have been under investigation to enhance the amount of collected stem cells for an appropriate and successful transplantation. Cytokines have been used for mobilization and the attempt to expand HSCs in vitro is under development. Recombinant colony stimulating factors (CSF) can stimulate CD34+ cells, that leads to a multiple increase of their amount in the peripheral blood.220 Following, those CD34+ cells can then be collected by leukocyte apheresis and used for transplantation.219 The bone marrow niche, consisting of non-hematopoietic and hematopoietic components, creates a unique environment for HSCs, where homing and maintenance as well as the protection from differentiation and from apoptotic stimuli is controlled with highest accuracy.221 Mesenchymal stem cells, navigated by sympathetic nerve fibers, are capable of producing several factors like stem cell factor (SCF), FMS-related tyrosine kinase 3 ligand (Flt3LG), VCAM-1, thrombopoietin (TPO), granulocyte macrophage-, macrophage- and granulocyte colony stimulating factor (G-CSF), C-X-C motive chemokine 12 (CXCL12),

27 CXCL8 and the chemokine C-C motive ligand 3 (CCL3). Hence, proliferation, differentiation, mobilization and homing of HSC can take place. 221-224,225 The stromal derived factor 1 (SDF-1), a chemokine also known as CXCL12, is expressed in the bone marrow on cells lining the stem cell niche and has been identified to play a major role for directing HSCs to different sides of the bone marrow niche. 221,226,227 SDF-1 provides signals by binding exclusively to the C-X-C motive chemokine receptor 4 (CXCR4), which is expressed on mouse HCSs and on CD34+ cells, and for that take part in the process of HSC mobilization and transplantation.228,229,230,231 Nevertheless, getting an appropriate amount of HSCs still remains still a challenge for translating the experimental approaches into the clinical setting.

Figure 7: Schematic representation of the hematopoietic and stromal stem cell development

1.6.1 The role of CD26/DPPIV in bone marrow transplantation As HSC numbers are limited, an efficient engraftment of these cells is very essential. We mentioned it above, that one of the main players in mobilization and homing of HSCs is the SDF-1 (CXCL12) that binds to its receptor CXCR4, expressed on Sca-1+ lin- mouse and CD34+ HSCs. 229,232 DPPIV, also known as the enzymatic activity of CD26, is an ectopeptidase that cleaves the N-terminus of polypeptid-chains., Hence, by a premature cleavage of SDF-1 via DPPIV, the chemotactic function of SDF-1 is reduced and less HSCs home to the bone marrow and engraft. 229,233

28 In several, very specific murine models of BMT, inhibition of the enzymatic activity of CD26 seemed to have engraftment augmenting beneficial effects.234-238 Christopherson et al demonstrated a positive effect on the engraftment when BMCs were incubated prior to transplantation with Diprotin A, an enzymatic inhibitor that consists of three amino acids (Ile- Pro-Ile).229 Herein, a myeloablative conditioning protocol was used. Three years later Kawai et al published in a nonmyeloablative murine model of BMT using immunodeficient recipients, that a sole pre-incubation of CD34+ HSCs with Diprotin A is not as effective as an additional intravenous treatment at time of transplantation.237 According to that point, it remained unclear whether an inhibition of DPPIV enzymatic activity in a murine model of mixed chimerism using nonmyeloablative conditioning and limited numbers of BMCs has a beneficial effect on the engraftment.

1.6.2 CD26/DPPIV and its pleiotropic immunologic effects An inhibition of the enzymatic activity of CD26, DPPIV, seemed to have an immunosuppressive effect too as an irreversible inhibition of DPPIV was able to abrogate acute rejection in lung-239 and heart transplantation models240 and reduced ischemia/reperfusion injury. 241 An experimental murine model of pancreas islet transplantation had shown, that DPPIV inhibition via sitagliptin increased the levels of active glucagon-like peptide-1 (GLP-1) in the plasma and the beta cell mass through cell proliferation and additionally inhibits beta cell 29 apoptosis. 242 These pleiotropic effects can be attributed to the DPPIV mediated truncation of chemokines, outlining as functional mediators. A costimulatory function of CD26 and its enzymatic activity DPPIV in T-cell activation upon stimulation was also revealed. Via crosslinking of CD26 and CD3, IL-2 can be prominently increased in vitro. 243,244 Liu et al revealed in another in vitro model a chemotactic function for CD26 by decreasing thrombospondin-1 expression on T-cells, influencing their migration and motility, and therefore may play a role in autoimmunity.245 In patients with chronic myeloid leukemia (CML), CD26 and its enzymatic activity seem to have a potent role in the extramedullary spread. BCR/ABL1+ cells were shown to express high levels of CD26/DPPIV and thereby cleave SDF-1.246 This leads to an impaired homing of leukemic cells to the bone marrow niche and an abnormal extramedullary spread. In this study by Herrmann et al a xenotransplantation assay revealed that leukemic cells, which were incubated with vildagliptin for DPPIV enzymatic activity inhibition prior transplantation into irradiated immunodeficient mice, reduced their engraftment. 246

1.6.3 CD26/DPPIV inhibition and its widespread use in the clinic One of the best known clinical applications for DPPIV inhibition is the popular use of gliptins in the oral therapy of type 2 diabetes. Due to an inhibition of the enzymatic activity DPPIV, the cleavage of incretin hormons, mainly glucagon-like peptide-1 (GLP-1) and the glucose- dependent insulinotropic polypeptide (GIP), can be effectively downsized. Incretin hormones are secreted from endocrine cells (L-cells) in the gastrointestinal tract and insulin secretion is increased in response to orally uptaken glucose during ingestion of food.247 It was demonstrated that GLP-1 has a main effect on pancreatic beta-cell function and promotes insulin synthesis and inhibits glucagon secretion. 248,249 Hence, inhibiting the cleavage of this gastrointestinal hormons has proven to be beneficial in the treatment of typ 2 diabetes. 250,251 Since the first DPPIV inhibitor, sitagliptin, has been approved years ago for the treatment of type 2 diabetes, many other gliptins followed.252,253 Thereby the main gliptins nowadays used are saxaplitin, sitagliptin, vildagliptin and linagliptin, with a pharmacological profile of linagliptin that doesn’t require a dose adjustment in patients with renal impairment.254 One of their main advantages is the low risk of hypoglycemia and that it is itself not accompanied by a gain in body weight.247 Some meta-analyses of DPPIV inhibitors have demonstrated questionable risks for infections including nasopharynigitis, urinary tract infections and headache compared to controls. 255,256 Furthermore, it seemed that there is an increased risk in the usage of DPPIV inhibitors and

30 GLP-1 analoga for pancreatitis, pancreatic and thyreoid cancer and other neoplasms.257 It was postulated, that these substances can increase the ductal cell turnover in patients with obesity and type 2 diabetes. Many other neoplasms also showed changes in DPPIV expression and a loss of DPPIV activity may thereby be connected to a more aggressive tumor behavior and metastatic potential.257 Enhanced risks for cardiac and vascular disorders for DPPIV inhibitors have been uncovered too, but the statistical significance was marginal and further studies and meta-analysis will be needed to prove this theses. 247

Figure 9: Schematic representation of sitagliptin on the glucose metabolism

31 1.7 Aims of this thesis

The induction of tolerance has proven very effective in various experimental settings by the development of mixed chimerism. Though attempts have been made to successfully translate the mixed chimerism approach to the clinical setting, there are still hurdles that impede its clinical implementation.258 One of the main problems is due to toxic conditioning protocols, which are necessary to enable successful engraftment of clinically available numbers of HSCs. Therefore, new ways have to be discovered for enhancing stem cell engraftment under nontoxic conditioning for allowing a widespread clinical translation of such tolerance inducing strategies. As BMT protocols that used myeloablative conditioning or non-myeloablative conditioning with sorted CD34+ or lin- stem cells or unsorted BMC in an in utero transplantation model, have proven beneficial for enhancing HSC engraftment by DPPIV inhibition,234-238 we hypothesized that DPPIV inhibition would promote engraftment of allogeneic unsorted BMC under non-myeloablative conditioning in our murine model of BMT.

The first aim of my thesis was to investigate if DPPIV enzymatic activity can be efficiently reduced by using two different DPPIV inhibitors (sitagliptin and Diprotin A) in our murine model of BMT.

Further, I wanted to assess if an DPPIV inhibition can enhance the engraftment of limited numbers of unsorted BMCs in an allogeneic murine model including CB after minimal- toxic conditioning (1Gy TBI).

The next aim was, to test if DPPIV inhibition showed different effects on the engraftment of a limited number of unsorted BMCs in a congenic murine model compared to an allogeneic murine model including CB.

32 CHAPTER TWO: Results 2.1 Prologue

Improvement of current BMT protocols for tolerance induction in solid organ transplantation is necessary to allow a safe clinical implementation in a wider range of indications. Therefore, efforts have been made to reduce the toxicity of existing conditioning protocols and to support the engraftment of clinically feasible numbers of BMCs. For the homing of HSCs to the bone marrow niche, the SDF-1 and its ligand CXCR4 are well known to have a prominent role but their chemotactic activity can be reduced by the enzymatic capability of CD26 (DPPIV). We therefore investigated in a congenic and allogeneic murine model of BMT whether an enzymatic blockade of DPPIV would allow an enhanced engraftment of limited numbers of BMCs after minimal toxic conditioning. Further, we developed different minimal toxic murine BMT protocols and aimed to overcome some of the existing hurdles that impedes a successful clinical implementation of tolerance induction.

33 2.1 First paper Dipeptidyl peptidase IV (DPPIV/CD26) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning

34 Experimental Hematology 2012;40:97–106 Dipeptidyl peptidase IV (DPPIV/CD26) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning Elisabeth Schwaigera, Christoph Klausa, Veerle Matheeussenb, Ulrike Baranyia, Nina Pilata, Haley Ramseya, Stephan Koromc, Ingrid De Meesterb, and Thomas Wekerlea aDivision of Transplantation, Department of Surgery, Vienna General Hospital, Medical University of Vienna, Austria; bDepartment of Pharmaceutical Sciences, Laboratory of Medical Biochemistry, University of Antwerp, Belgium; cDivision of Thoracic Surgery, University Hospital Zurich, Switzerland

(Received 10 August 2011; revised 26 September 2011; accepted 31 October 2011)

In order to develop minimally toxic bone marrow transplantation (BMT) protocols suitable for use in a wider range of indications, it is important to identify ways to enhance BM engraftment at a given level of recipient conditioning. CXCL12/stromal cell-derived factor-1a plays a crucial physiological role in homing of hematopoietic stem cells to BM. It is regulated by the ectopeptidase dipeptidyl peptidase IV (DPPIV; DPP4) known as CD26, which cleaves dipep- tides from the N-terminus of polypeptide chains. Blocking DPPIV enzymatic activity had a beneﬁcial effect on hematopoietic stem cell engraftment in various but very speciﬁc experimental settings. Here we investigated whether inhibition of DPPIV enzymatic activity through Diprotin A or sitagliptin (Januvia) improves BM engraftment in nonmyeloablative murine models of syngeneic (i.e., CD45-congenic) and allogeneic (i.e., Balb/c to B6) BMT (1 Gy total body irradiation, 10–15 3 106 unseparated BM cells/mouse). Neither Diprotin A administered in vivo at the time of BMT and/or used for in vitro pretreatment of BM nor sitagliptin administered in vivo had a detectable effect on the level of multilineage chimerism (follow-up O20 weeks). Similarly, sitagliptin did not enhance chimerism after allogeneic BMT, even though DPPIV enzymatic activity measured in serum was profoundly inhibited (O98% inhibition at peak exposure). Our results provide evidence that DPPIV inhibition via Diprotin A or sitagliptin does not improve engraftment of unseparated BM in a nonmyeloablative BMT setting. Ó 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Allogeneic bone marrow transplantation (BMT) has thera- end-stage renal disease simultaneously received a kidney peutic potential for a wide range of indications. Its clinical and BM graft from a human leukocyte antigen–mismatched application remains limited mainly to the treatment of living related donor [2,3]. Most recipients in this small life-threatening diseases because of substantial toxicities study became operationally tolerant. However, the non- associated with currently available BMT regimens. myeloablative conditioning regimen was associated with Transplantation of donor BM to induce mixed hema- substantial side effects, such as profound leukopenia, topoietic chimerism is an attractive experimental rendering this regimen virtually unacceptable in the approach to induce robust and lasting donor-speciﬁc routine organ transplantation setting. Therefore, less toxic tolerance in organ transplantation [1]. The clinical rele- BMT regimens achieving sufﬁcient engraftment with vance of this tolerance strategy has recently been und- reduced myelosuppressive conditioning still need to be erscored by a pilot trial in which patients suffering from developed to allow a more widespread application of this strategy [4]. In the experimental setting, less toxic mixed chimerism Offprint requests to: Thomas Wekerle, M.D., Division of Transplanta- protocols have been generated gradually during the last tion, Department of Surgery, Vienna General Hospital, Waehringer Guertel several decades. The use of costimulatory blockersdsome 18, 1090 Vienna, Austria; E-mail: [email protected] of which are already under clinical development as

0301-472X/$ - see front matter. Copyright Ó 2012 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. doi: 10.1016/j.exphem.2011.10.010

35 98 E. Schwaiger et al./ Experimental Hematology 2012;40:97–106

Table 1. Experimental protocols

Group TBI HSCT (cells/mouse) CB Additional treatment Mouse strain

A115 106 BMC - Congenic B115 106 BMC 5 mM Diprotin A in vitro (15 min) Congenic C110 106 BMC - Congenic D110 106 BMC 5 mM Diprotin A in vitro (15 min) & in vivo (72h) Congenic (4 mM Diprotin A with BMiv &5mM Diprotin A sc 2/day) E110 106 BMC Sitagliptin in vivo (4 mg/mouse/2/day) (72h) Congenic F115 106 BMC þ - Allogenic G115 106 BMC þ Sitagliptin in vivo (4 mg/mouse/2/day) (48h) Allogenic immunosuppressive drugs [5,6]das part of BMT protocols a congenic murine model using 1 Gy total body irradiation has allowed us to further reduce conditioning substantially (TBI) and conventional doses of BM. Moreover, we provide [7–11]. Short-course rapamycin [12,13] and therapeutic evidence that the clinically approved DPPIV inhibitor sita- administration of regulatory T cells [1,14] have led to the gliptin [35] completely blocked DPPIV/CD26 enzymatic most advanced murine minimum conditioning protocols. activity in vivo, but nevertheless did not increase BM However, translation to nonhuman primate models has re- engraftment in either allogeneic or congenic models of non- vealed that only transient chimerism is achieved with proto- myeloablative BMT. cols that establish permanent chimerism in mice [15,16]. Development of adjunctive treatments capable of promoting engraftment of a given dose of BM at a certain level Materials and methods of recipient myelosuppression is a critical goal toward clin- Animals ical translation of the mixed chimerism approach. Female C57BL/6NCrl (H-2b, CD45.2, denoted B6 herein), Balb/c The chemoattractant stromal cell–derived factor-1 (H-2d), and C3H/N (H-2k) were purchased from Charles River (CXCL12) binding to CXCR4 on hematopoietic stem cells Laboratories (Sulzfeld, Germany), female B6.SJL-Ptprca Pep3b/ (HSCs) plays an important role in regulating trafficking of BoyJ mice (H-2b, CD45.1, denoted CD45.1 B6 herein) were HSCs to BM [17]. Dipeptidylpeptidase IV (DPPIV/CD26) purchased from the Jackson Laboratory (Bar Harbour, ME, is an ectopeptidase that cleaves stromal cell–derived USA). All mice were housed under specific pathogen-free condi- factor-1 and thereby abrogates its chemotactic function tions and were used between 6 and 12 weeks of age. All experi- [18] with the consequence of reduced homing of HSCs to ments were approved by the local review board of the Medical their BM niches [19,20]. Specific inhibition of DPPIV/ University of Vienna, and were performed in accordance with national and international guidelines of laboratory animal care. CD26 via Diprotin A, an enzymatic inhibitor consisting of three amino acids (Ile-Pro-Ile), enhanced BM engraft- BMT protocol ment in certain murine BMT models [21–25]. Notably, Treatment protocols per group are listed in Table 1. For the con- Christopherson et al. showed a benefit on engraftment genic setting (groups A through E) CD45.1 B6 were used as recip- when BM was incubated with Diprotin A before trans- ients and B6 (i.e., CD45.2) as donors. For the allogeneic setting plantation into myeloablated congenic recipients [20]. (groups F and G) B6 were used as recipients, Balb/c as donors Combining in vivo with in vitro treatment with Diprotin A and C3H as third party. Recipients received 1 Gy TBI (day –1) 6 6 was found to further enhance its efficacy [24]. However, it and either 10 10 or 15 10 unseparated congenic or allogeneic BM cells (day 0), as indicated. Allogeneic recipients received remains undetermined whether DPPIV/CD26 inhibition costimulation blockade consisting of anti-CD154 monoclonal promotes engraftment of unseparated BM in the nonmye- antibody (MR1, 1 mg, day 0), and hCTLA4Ig (abatacept, 0.5 mg, loablative mixed chimerism setting. day 2) [12]. Indicated groups of mice received the following DPPIV An immunosuppressive role of DPPIV inhibition has also inhibition regimens. BM in vitro pretreated with Diprotin A been suggested in organ transplantation models (not involving (group B); BM in vitro pretreated with Diprotin A plus in vivo BMT), as an irreversible inhibitor of DPPIV abrogated acute recipient treatment with Diprotin A (group D); in vivo recipient rejection in rat lung and heart transplantation models treatment with sitagliptin (group E and G). Groups A, C, and F [26,27] and reduced ischemia/reperfusion injury [28]. This served as controls without DPPIV inhibition. Anti-CD154 mono- effect may be due to DPPIV/CD26-mediated truncation of clonal antibody was purchased from BioXCell (West Lebanon, mediators (such as cytokines and chemokines) [29,30],to NH, USA), hCTLA4Ig (abatacept) was generously provided by a potential costimulatory function of CD26, or to so far Bristol-Myers, Squibb Pharmaceuticals (Princeton, NJ, USA). unknown off-target effects of the inhibitor used [31–34]. DPPIV inhibition We report that neither in vitro DPPIV/CD26 enzymatic Donor BM was harvested, washed, and resuspended in BM inhibition of donor BM using Diprotin A nor additional medium containing M199 (Sigma M 4530), DNAse (Sigma systemic inhibition led to enhanced BM engraftment in D-4527), gentamycin, and HEPES (1M) as described [12,36]. The

36 E. Schwaiger et al./ Experimental Hematology 2012;40:97–106 99 control groups A, C, and F received BM in medium alone. In vitro Skin grafting pretreatment of BM with Diprotin A (groups B, D) was performed Full-thickness tail skin from Balb/c mice and fully mismatched by incubating BM with a concentration of 5 mM Diprotin A for 15 C3H (third party) was grafted 2 to 8 weeks after allogeneic min at room temperature [22] (group B) or for 15 min at 37C [21] BMT and visually inspected thereafter at short intervals. Grafts (group D). Subsequently, bone marrow cells (BMC) were washed were considered rejected when !10% remained viable. in BM medium, counted, and resuspended. For in vivo recipient treatment with Diprotin A (group D), BMC were resuspended in Statistics 1 mL BM medium containing 4 mmol Diprotin A, which was in- A two-sided Student’s t test was used to compare chimerism levels jected intravenously and 100 mL phosphate-buffered saline con- between the groups. Skin graft survival was calculated according taining 5 mmol Diprotin A injected subcutaneously every 12 to the Kaplan–Meier product limit method and compared between hours for 3 days (days 0, 1, and 2). This daily dose of Diprotin groups using the log-rank test. A was chosen as it showed therapeutic effects in other murine models [37,38]. For in vivo recipient treatment with sitagliptin (groups E and G), sitagliptin tablets (100 mg) were suspended Results in 2 mL cold phosphate-buffered saline under aseptic conditions, m resulting in 4 mg/80 L. Four milligrams sitagliptin was adminis- Inhibition of DPPIV with Diprotin A does not improve tered by oral gavage every 12 hours on days 0 and 1 (group G) or engraftment of unseparated congenic BM after on days 0, 1, and 2 (group E) post-BMT. Diprotin A was purchased nonmyeloablative conditioning from Sigma Aldrich and sitagliptin (Januvia) was kindly provided by Merck (Vienna, Austria). To investigate the effect of DPPIV inhibition on the engraftment of unseparated BM in the absence of alloreactivity, we Assay for DPPIV activity first used a CD45.2 / CD45.1 congenic donor-recipient DPPIV enzymatic activity was assayed by using glycyl-prolyl-4- combination [36,41]. CD45.1 B6 mice conditioned with 1 methoxy-b-naphthylamide (Gly-Pro-4-Me-b-NA) as fluorogenic Gy TBI received 15 106 unseparated CD45.2 BMCs substrate as described previously [39,40]. In a 96-well plate, that were or were not pretreated in vitro with the DPPIV 5 mL serum samples were mixed with 0.5 mM Gly-Pro-4-Me-b- inhibitor Diprotin A (n 5 9/group) [22]. Multilineage NA in 50 mM Tris buffer (pH 8.3) in a final volume of 110 mL. chimerism was followed in blood by flow cytometry. DPPIV activity was determined kinetically during 5 min at 37 C Lasting chimerism developed in all mice in both groups. b l 5 by measuring the velocities of 4-Me- -NA release ( ex 340 nm, During a period of 21 weeks post-BMT, chimerism levels l 5 430 nm) from the substrate using an Infinite 200 (Tecan em were comparable between Diprotin A–pretreated (group Group Ltd., Switzerland) (all reagents were purchased from Sigma-Aldrich). Fluorescence intensity was related to a 4-Me-b- B) and nontreated (group A) groups in all tested lineages NA standard curve. The reversibility of the inhibitors in the serum at all analyzed time points (note: treated and nontreated samples and the dilution of these samples in the assay make it groups were done in parallel within one experiment to necessary to create a calibration curve with known concentrations allow optimal comparability) (Fig. 1A). At the end of of the inhibitors in murine serum to estimate the percentage follow-up, mean percentages (6standard deviation) of in vivo inhibition of DPPIV enzymatic activity in the serum. chimerism were 35.7% (67.0%) vs 39.9% (67.7%) among Percentage inhibition was calculated by comparing DPPIV enzy- CD4 cells, 23.0% (64.7%) vs 29.3% (69.1%) among CD8 matic activity of treated mice to control mice, which were not cells, 40.6% (610.7%) vs 51.3% (613.4%) among B cells, enzymatically inhibited (defined as 100% activity). In order to and 26.7% (610.2%) vs 39.5% (614.9%) among myeloid limit the number of blood draws per mouse, groups were split in cells (Diprotin A pretreatment vs control group; p 5 NS for two and blood was taken only once a day for each mouse (either all lineages). Similarly, there were no differences in chime- at 2 hours or 12 hours post–DPPIV inhibitor administration). rism levels between groups in BM and spleen (p 5 NS, Flow cytometric analysis Fig. 1B). Two-color flow cytometric analysis was used to distinguish donor Because a beneficial effect has been described when and recipient cells of particular lineages by staining with fluores- in vitro Diprotin A pretreatment of donor BM was cein isothiocyanate–conjugated antibodies against CD4, CD8, combined with in vivo recipient treatment with Diprotin A B220, MAC-1, and biotinylated CD45.2 or 34-2-12 (H-2Dd, de- [24], we tested whether such a combination regimen would tected with phycoerythrin-streptavidin) and irrelevant isotype impact engraftment in the nonmyeloablative congenic controls [12,36]. Propidium iodide staining was used to exclude þ þ setting (group D). In addition to in vitro pretreatment of dead cells. The net percentage of CD45.2 or 34-2-12 live cells the BM, Diprotin A was injected in vivo together with the among different cell lineages was calculated. Mice were consid- BM and every 12 hours thereafter for 3 days post-BMT ered chimeric if they demonstrated at least 2% of donor cells (n 5 5). Again, chimerism levels were similar with (group within the myeloid lineage plus at least one lymphoid lineage. Surface staining was performed according to standard procedures D) and without (group C) DPPIV inhibition at all tested 5 and flow cytometric analysis was done on a Coulter Cytomics time points (follow-up 22 weeks; p NS for all time FC500. CXP software (Coulter, Vienna, Austria) was used for points) (Fig. 2A). At the end of follow-up, mean percent- acquisition and analysis. Antibodies were purchased from Becton ages of chimerism were 50.5% (65.8%) vs 43.7% (66.4%) Dickinson (San Diego, CA, USA). among CD4 cells, 36.4% (64.7%) vs 29.3% (66.5%)

37 100 E. Schwaiger et al./ Experimental Hematology 2012;40:97–106

Figure 1. Chimerism following transplantation of congenic BMCs pretreated in vitro with Diprotin A. Recipient mice were conditioned with 1 Gy TBI and received 15 106 congenic CD45.2 BMCs (n 5 9/control group A, n 5 9/Diprotin A–treated group B). BMCs of group B were treated in vitro with 5 mM Diprotin A before transplantation. The levels of chimerism in blood over time (A), was determined by ﬂow cytometry and is presented as means for Diprotin A–treated (squares) and untreated (dotted line with diamonds) groups. In (B) chimerism in BM and spleen at the end of follow-up is depicted in box and whisker plots. No signiﬁcant differences were noted between both groups.

38 E. Schwaiger et al./ Experimental Hematology 2012;40:97–106 101

Figure 2. Chimerism after transplantation of congenic BMCs after combined treatment with Diprotin A in vitro and in vivo and after in vivo treatment with sitagliptin. Recipient mice were conditioned with 1 Gy TBI and received 10 106 congenic CD45.2 BMCs (n 5 6/control group C, n 5 5/Diprotin A–treated group D, n 5 7/sitagliptin-treated group E). In group D, BMC were treated in vitro with 5 mM Diprotin A before transplantation and, in addition, recipients were treated in vivo with Diprotin A (4 mmol IV day 0 and 5 mmol Diprotin A subcutaneously every 12 hours for 3 days). Recipients of group E were treated with sitagliptin orally. Levels of chimerism in blood over time (A) were determined by ﬂow cytometry and are presented as means for Diprotin A–treated (squares), sitagliptin-treated (dotted line with triangle) and untreated (dotted line with diamonds) groups. In (B) chimerism in BM and spleen at the end of follow-up is depicted in box and whisker plots. No signiﬁcant differences were noted between both groups. among CD8 cells, 56.2% (66.7%) vs 54.9% (69.5%) Collectively, these experiments demonstrate that DPPIV among B cells, and 54.2% (610.0%) vs 41.6% (611.5%) inhibition with Diprotin A does not lead to a detectable among myeloid cells. Chimerism levels in BM and spleen improvement of the engraftment of unseparated congenic were also comparable among groups. BM in nonmyeloablatively conditioned recipients.

39 102 E. Schwaiger et al./ Experimental Hematology 2012;40:97–106

Sitagliptin inhibits DPPIV enzymatic activity more Sitagliptin does not improve engraftment in a mixed effectively than Diprotin A chimerism model of allogeneic BMT with To assess whether the failure to detect an engraftment effect nonmyeloablative conditioning and costimulation is due to insufficient DPPIV inhibition achieved with in vivo blockade Diprotin A treatment, serum DPPIV activity was measured. DPPIV inhibition was shown to affect alloreactivity in At peak exposure (2 hours after administration of Dipro- models of heart and lung transplantation [26,27]. As little tin A, group D), DPPIV serum activity decreased to 78.1% is known, however, about the effect of DPPIV inhibitors and 55.1% in two (randomly selected) mice, whereas in allogeneic BMT, we tested whether sitagliptin may DPPIV activity remained essentially unchanged in one improve engraftment in an allogeneic mixed chimerism mouse (Fig. 3A). At the time of trough exposure (12 hours model of limiting conditioning. B6 mice conditioned with postadministration), enzymatic activity declined to 51.7%, 1 Gy TBI (day –1), were transplanted with 15 106 fully 47.1%, and 16.1% in three remaining mice of the group mismatched Balb/c BMCs (day 0), and treated with anti- (Fig. 3A). Thus, although Diprotin A inhibits DPPIV CD154 monoclonal antibody (day 0) and CTLA4Ig (day activity, inhibition is only moderate. þ2), as described previously [12]. This BMT regimen is Sitagliptin (Januvia) is a specific DPPIV inhibitor that insufficient to induce reliable chimerism (and tolerance) has recently been approved for the treatment of type 2 dia- by itself, but chimerism and tolerance can be achieved betes [42]. We hypothesized that a more complete (and through adjunctive treatments, such as rapamycin [12]. clinically relevant) inhibition of DPPIV may be achieved One group was treated with sitagliptin orally (group G, with this drug. Sitagliptin has a half-life of about 12 to n 5 6) and was compared to one left untreated (group F, 14 hours [43] and is administered once daily at a dose of n 5 6). 100 mg (i.e., roughly 1.3 mg/kg) in the clinical setting of Again, sitagliptin nearly completely inhibited DPPIV type 2 diabetes. We measured DPPIV activity in serum of enzymatic activity after 2 hours (0.9%, 1.0%, 1.0%, mice treated with 4 mg sitagliptin orally every 12 hours 1.8%) and activity remained substantially decreased in 2 (160 mg/kg twice daily) (group E). Complete inhibition of 4 mice (32.2%, 32.5%, 81.4%, O85%) after 12 hours of DPPIV enzymatic activity at peak exposure (2 hours (Fig. 3C). As expected, without sitagliptin, mean chimerism postadministration) was observed in all four tested mice levels were low with varying individual levels and several (0.7%, 0.4%, 0.5%, and 0.5% enzymatic activity) mice had no detectable T-cell chimerism at the end of (Fig. 3B). At trough level 12 hours after the last dose, enzy- follow-up (Fig. 4A). Sitagliptin treatment, however, did matic DPPIV activity decreased to a mean of 31.9% (i.e., not improve chimerism levels. Chimeric mice (six of seven 68% inhibition) (16.2%, 22.1%, 57.4% in the remaining chimeras in the sitagliptin-treated group vs five of six in the three mice of the group) (Fig. 3B). Thus, in vivo treatment control group) showed chimerism levels of 1.6% (61.1%) with sitagliptin leads to more effective inhibition of DPPIV vs 1.8% (62.5%) among CD4 cells, 0.4% (60.6%) vs activity at peak exposure than Diprotin A. However, with 3.5% (63.1%) among CD8 cells, 7.5% (63.8%) vs 8.8% neither compound, a complete DPPIV inhibition for the (64.1%) among B cells, and 16.6% (612.5%) vs 21.8% entire treatment period was achieved, as moderate enzy- (610.3%) among myeloid cells for each lineage (p 5 matic activity at trough levels could still be observed. NS). Similarly, chimerism levels and rates were also comparable between groups in spleen (Fig. 4B). Despite superior DPPIV inhibition, sitagliptin does not Donor and third-party skin was transplanted to assess improve engraftment of unseparated congenic BMT after donor-specific tolerance. Third-party grafts were promptly nonmyeloablative conditioning rejected in both groups. Donor skin graft survival was To assess whether sitagliptin affects engraftment, we followed significantly prolonged in both groups but tolerance was hematopoietic chimerism in sitagliptin-treated recipients of not achieved, consistent with the poor T-cell chimerism unseparated congenic BM (n 5 7, group E). Again, no enhanced (p 5 0.5 sitagliptin-treated vs untreated recipients) [8,44]. engraftment was detected on DPPIV inhibition (p 5 NS at all Taken together, these results indicate that sitagliptin time points). Twenty-two weeks post-BMT mean chimerism does not improve engraftment of allogeneic BM under levels were 48.8% (65.7%) vs 43.7% (66.4%) among CD4 limiting recipient conditioning and consequently does not cells, 34.5% (63.0%) vs 29.3% (66.5%) among CD8 cells, improve tolerance induction in this setting. 60.1% (69.8%) vs 54.9% (69.5%) among B cells, and 48.6% (611.1%) vs 41.6% (611.5%) among myeloid cells (treated group E vs control group C) (Fig. 2A). Chimerism levels in Discussion BMandspleen(Fig. 2B) were also comparable (p 5 NS). Induction of donor-specific transplantation tolerance Thus, with the more specific, clinically approved DPPIV through mixed chimerism would be a potential new indica- inhibitor sitagliptin, no beneficial effect was detectable on the tion for allogeneic BMT. During the last 2 decades, the engraftment of congenic BM in nonmyeloablated recipient toxicity of the recipient conditioning was gradually reduced mice. in murine chimerism models [1]. Nevertheless cytotoxic

40 E. Schwaiger et al./ Experimental Hematology 2012;40:97–106 103

Figure 3. DPPIVenzymatic activity in serum after in vivo inhibition with Diprotin A or sitagliptin. DPPIVenzymatic activity in serum was measured 2 (peak) and 12 hours (trough) after in vivo treatment with Diprotin A (group D) or sitagliptin (group E). DPPIVenzymatic activity at peak and trough exposure is depicted for Diprotin A– (A) and sitagliptin-treated [(B) congenic, (C) allogeneic model] groups (n 5 3–4 randomly selected mice per group, congenic BMT). conditioning used to achieve engraftment of clinically Inhibition or genetic ablation of DPPIV/CD26 showed feasible numbers of BMCs keeps impeding clinical transla- promising engraftment-promoting effects in several murine tion of such protocols. Minimally toxic BMT regimens models especially when limited numbers of donor cells would also be of interest to allow other potential indications were used [25]. DPPIV enzymatic activity inhibition was of BMT [9]. effective in congenic (CD45-congenic) [20,38], xenogeneic

41 104 E. Schwaiger et al./ Experimental Hematology 2012;40:97–106

Figure 4. Chimerism and skin graft survival following transplantation of allogeneic BMCs after in vivo treatment with sitagliptin. Recipient mice were transplanted with 15 106 allogeneic BMCs after 1 Gy TBI (day –1) and costimulation blockade consisting of anti CD-154 monoclonal antibody (day 0) and CTLA4Ig (day 2). Four milligrams sitagliptin per mouse were administered twice a day (day 0–2). (A) Mean percent of blood chimerism among different cell lineages over time are depicted for sitagliptin-treated (bold lines with squares) and untreated (dotted lines with triangles) groups. No signiﬁcant differences were noted between both groups. (B) Chimerism in spleen was similar in both groups at the end of follow-up. (C) Approximately 8 weeks post-BMT, mice were grafted with donor and third-party skin. Skin graft survival was comparable in both groups (p 5 0.5).

(human cells into nonobese diabetic severe combined tation) [22] systems. Engraftment-enhancing effects of immune-deﬁcient recipients) [23,25], and allogeneic (major DPPIV inhibition were not only observed with separated þ þ histocompatibility complex–mismatched in utero transplan- (Sca1 lin murine BMCs or CD34 human mobilized

42 E. Schwaiger et al./ Experimental Hematology 2012;40:97–106 105

peripheral blood/cord blood cells, respectively) [20,24,25], Models in which an engraftment-enhancing of DPPIVinhibi- but also with unseparated populations [20,22,38].We tion was found used either myeloablative conditioning or therefore chose nonmyeloablative protocols using unsepa- nonmyeloablative conditioning of immunodeficient recipi- rated BMCs for evaluating DPPIV enzymatic inhibition, ents [20,21,23–25,38]. To the best of our knowledge, our as such systems are commonly used in the clinical setting studies are the first to investigate the effect of DPPIV inhibi- (but to the best of our knowledge have not been studied tion on BM engraftment in nonmyeloablatively conditioned in the DPPIV inhibition context before). In addition, we tar- wild-type recipients. In the environment of this different geted DPPIV in a fully allogeneic model, which would be conditioning regimen, DPPIV inhibition might be unable to similar to the clinical situation of tolerance induction in affect engraftment. Furthermore, an engraftment-enhancing organ transplantation. In these settings, we did not detect effect of DPPIV inhibition on allogeneic HSC has been a significant effect of DPPIV enzymatic activity inhibition shown so far only after myeloablative conditioning or in utero on BM engraftment (despite using Diprotin A treatment transplantation [21,22]. The potential effect on allogeneic schedules proven effective in other settings). BM engraftment after nonmyeloablative conditioningd In order to dissect whether a potential effect of DPPIV which is of relevance for clinical BMTdhas not been ascer- inhibition is due to modulating alloreactivity or HSC tained previously. Our results suggest that DPPIV inhibition engraftment, we used two established models of nonmye- is of limited therapeutic value in this setting. Besides, the loablative BMT. The CD45.2 / CD45.1 congenic model current study is the first to investigate the use of costimulation is almost free of relevant immunological barriers [36,41]. blockade together with DPPIV inhibition. Although an inter- In the 1-Gy allogeneic protocol, immunosuppressiond action cannot be ruled out, such interference would be unable through rapamycin [12] or administration of T regulatory to explain the observed lack of an effect in the congenic cells [45]dleads to engraftment and lasting chimerism in system, in which no costimulation blockade was used. this otherwise unsuccessful BMT protocol. In summary, although DPPIV enzymatic inhibition had Several DPPIV inhibitors have recently been approved for been demonstrated to have engraftment-promoting effects treatment of type 2 diabetes. We therefore tested whether si- in several specific models of HSC transplantation, our tagliptin enhances the engraftment of congenic or allogeneic studies provide evidence that DPPIV inhibition with Dipro- BM after nonmyeloablative TBI. As compared to Diprotin A, tin A or with sitagliptin does not lead to improved engraft- sitagliptin is both a more potent and specific DPPIV inhibitor ment of unseparated BM after nonmyeloablative recipient [46] that would be of importance for translating experimental conditioning. results to the clinical setting. Despite inhibition of DPPIV enzymatic serum activity, no beneficial effect of sitagliptin Acknowledgment was detectable in our studies. This work was supported by a grant from the Austrian Science Maximum inhibition of DPPIV serum activity (measured Fund (FWF, SFB F2310 to T.W.). Veerle Matheeussen is a research 2 hours after in vivo administration) was more profound with assistant of the Fund for Scientific Research Flanders (Belgium, sitagliptindachieving almost 100% inhibitiondthan with FWO-Vlaanderen). Diprotin A (Fig. 3). A rapid recovery of DPPIV systemic activity after Diprotin A treatment was also noted by Christo- Conflict of interest disclosure pherson and colleagues [20], but this transient inhibition was No financial interest/relationships with financial interest relating nevertheless sufficient to positively affect engraftment in the to the topic of this article have been declared. myeloablative setting. Kim and colleagues reported prolongation of islet graft survival with sitagliptin [47]. Sitagliptin was given in chow with a sitagliptin uptake of roughly 10 References mgd48 mg per day resulting in 78% to 88% DPPIV inhibi- 1. Pilat N, Wekerle T. Transplantation tolerance through mixed chimerism. Nat Rev Nephrol. 2010;6:594–605. tion. Notably, almost the full inhibitory effect was observed 2. Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal trans- even with the lowest consumed dose of 10 mg (approximately plantation without maintenance immunosuppression. N Engl J Med. 78% inhibition). In the clinical setting of type 2 diabetes, si- 2008;358:353–361. tagliptin is administered once daily, as it has a half-life of 3. Fehr T, Sykes M. Clinical experience with mixed chimerism to induce about 12 to 14 hours [43]. We therefore reasoned that oral transplantation tolerance. Transpl Int. 2008;21:1118–1135. 4. Pilat N, Klaus C, Schwaiger E, Wekerle T. Hurdles to the induction of tol- administration every 12 hours (at a dose of 160 mg/kg twice erogenic mixed chimerism. Transplantation. 2009;87(Suppl):S79–S84. daily compared to the clinical dose of approximately 1.3 mg/ 5. Vincenti F, Larsen C, Durrbach A, et al. Costimulation blockade with kg/day) would result in sufficient drug exposure during the belatacept in renal transplantation. N Engl J Med. 2005;353:770–781. critical period of HSC engraftment. It seems unlikely that 6. Pree I, Wekerle T. New approaches to prevent transplant rejection: the degree or duration of DPPIV inhibition is responsible costimulation blockers anti-CD40L and CTLA4Ig. Drug Discov Today Ther Strateg. 2006;3:41–47. for the lack of a detectable engraftment effect in our models. 7. Wekerle T, Sayegh MH, Hill J, et al. Extrathymic T cell deletion and allo- We think that several factors might be responsible for the geneic stem cell engraftment induced with costimulatory blockade is fol- lack of an effect of DPPIV inhibition in our present study. lowed by central T cell tolerance. J Exp Med. 1998;187:2037–2044.

106 E. Schwaiger et al./ Experimental Hematology 2012;40:97–106

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44 2.1.2 Interlude In order to improve existing bone marrow transplantation protocols we aimed for a new strategy that would enhance engraftment of BMCs after minimal toxic conditioning (1Gy). By inhibiting the enzymatic activity of CD26 (DPPIV), we searched for a beneficial interference of the SDF-1/CXCR4 axis to enhance the homing of limited numbers of BMCs to the bone marrow. After a DPPIV inhibition of the transplanted bone marrow or an additionally inhibition of recipient’s DPPIV activity with Diprotin A, or after a profound inhibition of recipients DPPIV activity with sitagliptin, only similar levels of chimerism and no prolonged skin graft survival was detectable at time of follow up (> 20 weeks).

45 2.2 Second paper

Anti-LFA-1 or rapamycin overcome costimulation blockade-resistant rejection in sensitized bone marrow recipients

46 Transplant International ISSN 0934-0874

ORIGINAL ARTICLE Anti-LFA-1 or rapamycin overcome costimulation blockade- resistant rejection in sensitized bone marrow recipients

Haley Ramsey,1* Nina Pilat,1* Karin Hock,1 Christoph Klaus,1 Lukas Unger,1 Christoph Schwarz,1 Ulrike Baranyi,1 Martina Gattringer,1 Elisabeth Schwaiger,1 Fritz Wrba2 and Thomas Wekerle1

1 Division of Transplantation, Department of Surgery, Medical University of Vienna, Vienna, Austria 2 Institute of Clinical Pathology, Medical University of Vienna, Vienna, Austria

Keywords Summary anti-LFA-1, costimulation blockade, mixed chimerism, rapamycin, T memory cells, While costimulation blockade-based mixed chimerism protocols work well for tolerance. inducing tolerance in rodents, translation to preclinical large animal/nonhuman primate models has been less successful. One recognized cause for these difficul- Correspondence ties is the high frequency of alloreactive memory T cells (Tmem) found in the Thomas Wekerle MD, Division of (pre)clinical setting as opposed to laboratory mice. In the present study, we there- Transplantation, Department of Surgery, fore developed a murine bone marrow transplantation (BMT) model employing Vienna General Hospital, Waehringer Guertel 18, Vienna 1090, Austria. recipients harboring polyclonal donor-reactive Tmem without concomitant Tel.: 43 1 40400 5621; humoral sensitization. This model was then used to identify strategies to over- fax: 43 1 40400 6872; come this additional immune barrier. We found that B6 recipients that were e-mail: [email protected] enriched with 3 9 107 T cells isolated from B6 mice that had been previously grafted with Balb/c skin, rejected Balb/c BM despite costimulation blockade with Conflicts of Interest anti-CD40L and CTLA4Ig (while recipients not enriched developed chimerism). The authors of this manuscript have no Adjunctive short-term treatment of sensitized BMT recipients with rapamycin or conflicts of interest to disclose. anti-LFA-1 mAb was demonstrated to be effective in controlling Tmem in this *Haley Ramsey and Nina Pilat are co-first model, leading to long-term mixed chimerism and donor-specific tolerance. authors of this manuscript. Thus, rapamycin and anti-LFA-1 mAb are effective in overcoming the potent barrier that donor-reactive Tmem pose to the induction of mixed chimerism and tol- Received: 7 December 2011 erance despite costimulation blockade. Revision requested: 15 January 2012 Accepted: 25 October 2012 Published online: 13 December 2012 doi:10.1111/tri.12021 recipient conditioning regimens that are substantially more Introduction intense (i.e., cytotoxic and myelotoxic) than those used in Numerous treatment protocols inducing mixed hemato- rodents [11]. Notably, costimulation blockade, which is poietic chimerism lead to robust transplantation tolerance sufficient for inducing mixed chimerism in nonmyeloablat- in rodents [1]. Recently, the mixed chimerism strategy also ed mice [12–14], fails to establish chimerism in MHC-mis- led to operational tolerance in most participants of clinical matched nonhuman primates [15,16]. Moreover, while proof-of-principle trials [2,3]. Translation of less toxic and mixed chimerism induces B cell tolerance in rodents [17], thus clinically more acceptable, experimental chimerism evidence for B cell immunity – albeit of unknown clinical protocols, however, has been associated with substantial consequence – was observed in the clinical setting [2,18]. setbacks [4]. While permanent mixed chimerism can be The different frequencies of memory T cells (Tmem) in induced in rodents with minimal conditioning [5–9], long- patients and nonhuman primates on one side and (usually term chimerism in large animals is much more difficult to young) laboratory mice kept under protected conditions achieve [10]. In nonhuman primates, in particular, macro- on the other side has emerged as a critical factor accounting chimerism is usually detectable only transiently despite for the difficulty in translating tolerance protocols from

47 Ramsey et al. Overcoming memory barrier in tolerance induction bench to bedside [19,20]. Tmem respond more rapidly to least 6 weeks post-BMT) for either means of Tmem genera- antigen recognition, are less dependent on ‘conventional’ tion or tolerance assessment, respectively. Grafts were visu- costimulation pathways (i.e., CD28 and CD40) and are ally inspected daily and considered to be rejected when less more resistant to regulation than na€ıve T cells [21]. Even in than 10% remained viable. the absence of previous exposure to alloantigen, alloreactive Tmem are generated through heterologous immunity and Detection of donor-specific antibodies (DSA) homeostatic proliferation [22,23]. Humoral sensitization has been investigated in murine mixed chimerism models Serum was collected from skin recipients and heat deacti- and has been recognized as sizable barrier [24,25]. While vated before incubation with thymocytes from B6, Balb/c, humoral sensitization can be generally avoided in renal and C3H/N mice. After a 30 min incubation period, cells transplantation through pretransplant cross-match assays were washed and labeled with FITC-anti-mouse IgG1/2 detecting donor-specific antibodies (DSA), no assays for (BD Pharmingen, San Diego, CA, USA) to detect cell- evaluating sensitization at the T cell level are yet available bound anti-donor antibodies (DSA) via flow cytometric for routine use in the clinical setting [26]. Such T cell sensi- analysis. tization causes costimulation blockade-based tolerance protocols developed in na€ıve mice to fail when applied to Generation and isolation of Tmem cells recipients enriched with T cells sensitized to the donor [27]. Likewise, concomitant or previous exposure to infec- Three weeks after skin grafting, mice were tested by flow tions leads to the failure of otherwise successful costimula- cytometry for DSA. T cells were isolated from spleen and tion blockade-based murine mixed chimerism protocols lymph nodes of DSA-positive mice 3 weeks post skin graft- owing to heterologous immunity [22,28]. In allosensitized ing through MACS separation (Pan T cell Exclusion Kit; recipients, Tmem were shown to persist even after lethal Miltenyi Biotec, Bergisch Gladbach, Germany). Purity of irradiation (10 Gy) mediating rejection of BM [25]. It separated cells was >96%. remains to be determined, however, if and how an isolated Tmem barrier without concomitant humoral sensitization Mixed lymphocyte reaction (MLR) can be overcome in recipients of a costimulation blockade- based mixed chimerism regimen. Mixed lymphocyte reactions were performed as described To closer model the clinical setting, we therefore devel- previously [7]. Briefly, 4 9 105 responder splenocytes oped a nonmyeloablative murine protocol of mixed chime- were plated in triplicates and incubated with 4 9 105 rism in which recipients contain functionally relevant irradiated (30 Gy) stimulator cells of either Balb/c numbers of polyclonal alloreactive Tmem. Moreover, we (donor), C3H (third party), B6 (host) mice, or with demonstrate that anti-LFA-1 and rapamycin are effective in medium only. After 72 h, cells were pulsed with 3H-thy- controlling Tmem reactivity in this new BMT model. midine (Amersham Biosciences, Sunnyvale, CA, USA) and incubated for approximately 18 h. Incorporated radioactivity was measured using scintillation in a Materials and methods b-counter. Stimulation indices (SI) were calculated in Animals relation to medium controls. Female C57Bl/6 (B6, H-2b, CD45.2), Balb/c (H-2d), and C3H/N (H-2k) mice were purchased from Charles River BMT protocol Laboratories (Sulzfeld, Germany). Congeneic CD45.1 recipients (B6.SJL-Ptprc Pepc/BoyJ) were purchased from Two or 3 Gy TBI was administered to B6 recipients Charles River Italy (Calco, Italy). All mice were housed 1 day before receiving 20 9 106 unseparated Balb/c bone under specific pathogen-free conditions and were used marrow cells (BMC) and costimulation blockade (1 mg between 6 and 10 weeks of age. All experiments were of MR1 hamster anti-mouse-CD40L (CD154) mAb on approved by the local review board of the Medical Univer- day 0 and 0.5 mg of human CTLA4Ig (abatacept) on day sity of Vienna and were performed in accordance to 2) [7]. Where indicated groups received in addition national and international guidelines of laboratory animal either rapamycin (0.1 mg d-1, d0, and d2) (Alexis Bio- care. chemicals, San Diego, CA, USA) or blocking anti-LFA-1 mAb (M17/4) (0.5 mg, d-1 and d2). Anti-CD40L and anti-LFA-1 mAbs were purchased from BioXCell (West Skin grafting Lebanon, NH, USA) and hCTLA4Ig was generously pro- Donor Balb/c or third party C3H/N full thickness tail skin vided by Bristol-Myers, Squibb Pharmaceuticals (Prince- was grafted onto na€ıve B6 mice or B6 BMT recipients (at ton, NJ, USA).

48 Overcoming memory barrier in tolerance induction Ramsey et al.

splenocytes or lymph nodes were prepared from experi- Flow cytometric analysis of chimerism, deletion, mental and naive animals, responders were resuspended in and intracellular IFNc expression cell culture media and plated in triplicates in 96-well PVDF Two-color flow cytometric analysis was used to distinguish Membrane ELISPOT plates (Millipore, Billerica, MA, USA) donor and host cells of particular lineages by staining with at a 2:1 ratio (8 9 105 responder cells per well) with fluorescin isothiocyanite-conjugated antibodies against 4 9 105 stimulators (host, donor, or third party) or med- CD4, CD8, B220, MAC1, and biotinylated antibody against ium alone. The mouse IFN-c ELISPOT kit (eBioscience, H-2Dd (34-2-12, developed with phycoerythrin Frankfurt, Germany) was used according to the manufac- streptavidin) and isotype controls. To analyze the expres- turer’s instructions, freshly prepared AEC substrate (Sigma, sion of Vb subunits among splenocytes, staining was per- St. Louis, MO, USA) was added for 10 min at room formed with fluorescin isothiocyanite (Fitc) antibodies temperature for spot development. Analysis was performed against Vb8.1/2, Vb11, and Vb5.1/2 (or isotype control) on a Bioreader 5000 (BIOSYS, Pasadena, CA, USA) with and phycoerythrin-conjugated (PE) antibodies against CD4 Bioreader 10.8 software. Spot development was calculated and CD8 (antibodies from Becton Dickinson, San Diego, in relation to medium controls. CA, USA and Biolegend, San Diego, CA, USA). To analyze Vb subunits in the thymus, Vb8.1/2, Vb11, and Vb5.1/2 Statistical analysis (or isotype control) expression was measured on gated single positive thymocytes (CD4+ [PE] CD8 [Cy5] and For comparing the rates of chimerism between groups Fish- CD4 [PE] CD8+ [Cy5]). Propidium iodide staining was er’s exact test was used. A two-tailed Student’s t-test was used to exclude dead cells. The net percentage of donor chi- used for comparing Vb deletion and levels of chimerism merism was calculated by subtracting control staining from between groups. A value of P < 0.05 was considered to be quadrants containing donor and host cells expressing a par- statistically significant. Survival was calculated according to ticular lineage marker, and by dividing the net percentage the Kaplan–Meier product limit method and compared of donor cells by total net percentage of donor plus host between groups using the log-rank test. Graph Pad Prism cells of that lineage. Mice were considered chimeric if they software was used for creating Kaplan–Meier survival showed at least 2% donor cells within the myeloid lineage curves. and within at least one lymphoid lineage [7,29,30]. Intra- cellular staining for IFN-c was performed as per the manu- Results facturer’s instructions (Cytofix/Cytoperm kit; Biolegend). Cells from spleen or lymph nodes were resuspended in cell Enriching BMT recipients with donor-reactive Tmem culture media containing brefeldin (GolgiPlug; BD Pharm- abrogates chimerism and tolerance despite costimulation ingen) and plated in 96-well plates at a 1:1 ratio with irradi- blockade ated donor splenocytes (1 9 106 cells per well) and First, a model mimicking the clinical situation of T cell sen- restimulated for 3–4 h. Cells were washed and analyzed by sitization without accompanying humoral sensitization was staining with fluorochrome-conjugated antibodies (Bioleg- established (Fig. 1a). Na€ıve B6 mice were grafted with Balb/c end) to CD4 and CD8, followed by intracellular staining skin and upon confirmation of the development of donor- ® with Alexa Fluor 647 anti-mouse IFN-c (Biolegend). Flow specific antibodies 3 weeks postgrafting (indicative for T cytometric analysis was done on a Coulter Cytomics cell sensitization) (Fig. 1b), T cells were isolated for transfer FC500. CXP software (Coulter, Brea, CA, USA) was used into na€ıve B6 mice (which went on to serve as BMT recipi- for acquisition and analysis. ents). These cells showed significantly increased reactivity to Balb/c (P < 0.01), but not third party C3H (P = 0.085) stimulators in in vitro MLR assays (Fig. 1c) in comparison Histological analysis to na€ıve BL6 cells. (Note: for reasons of simplicity the trans- Sections of 4 lm were cut from paraffin-embedded tis- ferred cell population is referred to as ‘Tmem’ hereafter.) sue fixed in 4.5% formalin (with a buffered pH of 7.5), Similarly, freshly isolated Tmem cells reacted with substan- stained with hematoxylin–eosin and Giemsa according to tially higher levels of IFN-c secretion in response to donor standard protocols, and analyzed by an experienced (but not third party antigen) in ELISPOT assays as com- pathologist. pared to na€ıve T cells (Fig. 1e). In addition, intracellular IFN-c production was increased among Tmem upon polyclonal and donor-specific stimulation (Fig. 1f). Phenotypic ELISPOT analysis of Tmem cells by flow cytometry revealed an IFN-c secretion was induced in response to 18–20 h of increased percentage of CD4 and CD8 effector and central ex vivo stimulation with allogeneic stimulators. In brief, memory cells in comparison to na€ıve T cells (Fig. 1d).

49 Ramsey et al. Overcoming memory barrier in tolerance induction

Figure 1 Generation of a BMT model using Tmem-enriched recipients. (a) Na€ıve B6 mice were grafted with Balb/c tail skin to sensitize them toward Balb/c. Three weeks later, T cells were isolated through magnetic bead separation and transferred to another set of na€ıve B6 mice that served as recipients of Balb/c BM 1 week later. (b) To ensure that T cells are only transferred after successful sensitization, serum from each skin grafted mouse was tested by flow cytometry for the presence of high levels of donor-specific antibodies (IgG1/2) against Balb/c (gray filled curve), B6 (black curve), and C3H (dotted curve) thymocytes (shown in comparison to serum from a na€ıve B6 control – upper panel). Results from one representative mouse are shown. (c) In MLR assays, T cells isolated from sensitized mice showed increased proliferation in response to Balb/c stimulators, but not to C3H third party stimulators (compared with na€ıve B6). Mean stimulation indices (SI) of recipient responder cells against B6 (n = 3) (black), Balb/c (n = 3) (white), and C3H/N (n = 2) (gray) stimulator are shown. Error bars indicate standard deviation. (d) Phenotypic analysis of pooled isolated Tmem cells (n = 3) showed an increase in Tmem specific markers in both CD4/8 effector and central memory cells. (e) IFN-c specific ELISPOT analysis shows enhanced donor-specific memory responses with MACS separated pan T cells isolated from sensitized mice in comparison to pan T cells from na€ıve mice and unsorted splenocytes from na€ıve mice (n = 2 per group). Responses against B6 (white), Balb/c (black), and C3H (gray) stimulators are shown. (f) Fre- quency of intracellular IFNc responses in CD8+ and CD4+ splenocytes from pooled sensitized (dark gray, n = 2) and pooled na€ıve mice (light gray, n = 2) in response to donor antigen are shown.

50 Overcoming memory barrier in tolerance induction Ramsey et al.

Collectively, these data provide evidence that the ‘Tmem’ age chimerism in the majority of recipients (6/32 chimeras population generated for use in this model show pheno- in recipients enriched with Tmem compared with 29/37 typic and functional properties characteristic of memory/ chimeras in control BMT recipients without T cell trans- effector T cells. fer, pooled data from seven experiments, P < 0.01) Increasing numbers of sensitized T cells were adminis- (Fig. 2b and d). Moreover, BMT recipients enriched with tered to B6 mice which 1 week later underwent BMT (15– 3 9 107 Tmem rejected donor skin whereas most recipi- 20 9 106 Balb/c BMC, 2 or 3 Gy TBI, and costimulation ents without cell transfer accepted donor skin for a long blockade). Mice receiving 1 9 105 Tmem cells (4/5 chime- term (while rejecting third party grafts, not shown) ric, not shown), 1 9 106 (3/4 chimeric, not shown), or (Fig. 2c). A total of 3 9 107 transplanted Tmem consist of 1 9 107 (3/5 and 4/5 chimeric) after 2 Gy TBI, and mice 52.6% CD4 (15 2 9 106 cells) and 45.7% CD8 receiving 2 9 107 Tmem cells after 3 Gy TBI showed no (13 1 9 106 cells), close to a 1:1 ratio. When either significant abrogation of chimerism in comparison to the 1.5 9 107 CD4+ or 1.5 9 107 CD8+ cells isolated from respective BM control groups (3/5 and 5/8 chimeric) sensitized mice were transferred into BMT recipients (Fig. 2a). Transfer of 2 9 107 Tmem cells after 2 Gy (0/5 (which is comparable to the amount of CD4 and CD8 chimeric) and 3 9 107 Tmem after 3 Gy abrogated chi- cells contained in bead-separated 3 9 107 T cells as merism (0/4 chimeric) (Fig. 2a). Results from multiple assessed by flow cytometry), chimerism was not abrogated repeat experiments confirmed that the transfer of 3 9 107 (4/6 and 3/4 chimeric; 3 Gy TBI; data not shown), indi- Tmem after 3 Gy TBI reproducibly abrogated multi-line- cating that it is neither solely the CD4 subset nor solely

Figure 2 Enriching BMT recipients with donor-reactive Tmem abrogates chimerism and tolerance despite costimulation blockade. (a) Increasing numbers of sensitized T cells were administered to B6 mice which 1 week later underwent BMT (15 9 106 Balb/c BMC with 2 Gy or 20 9 106 BMC with 3 Gy TBI and costimulation blockade). Chimerism was abrogated when 2 9 107 Tmem (2 Gy) and 3 9 107 Tmem cells (3 Gy) were transferred. Mean percentage chimerism among various leukocyte lineages in blood is shown over time as determined by ﬂow cytometry. Levels of mean donor B220 (■), Mac1 (▲), and CD4 (♦) chimerism are shown over time. Numbers of chimeric mice are denoted at the end of follow-up. (b) Two-color ﬂow cytometric analysis of multi-lineage chimerism is shown for a representative BMT recipient without cell transfer (top) and a BMT recipient enriched with 3 9 107 Tmem (bottom) (8 weeks post-BMT). Numbers indicate net percentage of donor chimerism in each lineage. (c) Tmem and control mice were grafted with Balb/c donor skin. While most control BMT recipients without cell transfer (broken line, ▲, n = 15) accepted donor grafts long- term, Tmem-enriched BMT recipients rapidly rejected donor skin (solid line, ●, n = 10 P = 0.0074). (d) T cells isolated from mice sensitized to C3H, from mice sensitized to Balb/c (‘Tmem’), T cells from na€ıve mice or no T cells were transferred to BMT recipients. The percentage of mice that became chimeric with each of these protocols is shown. Data are shown from a total of nine separate experiments with each experimental group being performed at least two times.

51 Ramsey et al. Overcoming memory barrier in tolerance induction the CD8 subset contained in 3 9 107 Tmem that causes not signiﬁcantly reduce the rate of chimerism (6/8 vs. 29/ rejection. To investigate whether transferred Tmem need 37 without cell transfer, P = 0.34), neither did the transfer to be donor-speciﬁc to abrogate chimerism, T cells from of T cells isolated from na€ıve B6 mice [8/15 chimeras vs. B6 mice grafted with C3H skin were used for adoptive 29/37 (BMT control), P = 0.055; vs. 6/32 (Tmem), transfer. Transfer of Tmem from C3H sensitized mice did P = 0.037] (Fig. 2d). Consequently, the transfer of

Figure 3 Stable long-term multi-lineage hematopoietic chimerism in presensitized BMT recipients after administration of additional anti-LFA-1 or rapamycin. Mean percent chimerism among CD4 cells (a), CD8 cells (b), B cells (c) and myeloid cells (d) is shown over time in peripheral blood for Tmem- enriched BMT recipients treated in addition with anti-LFA-1 (Δ, n = 14), rapamycin (○, n = 14) or no additional treatment (Tmem control, ■, n = 31) (compared with BMT recipients without cell transfer (BMT control, ●, n = 37) All groups received costimulation blockade. (e) Mean levels of B cell (black bars) and myeloid (gray bars) chimerism within bone marrow of anti-LFA-1 (n = 10) and rapamycin-treated Tmem-enriched recipients (n = 6) were not signiﬁcantly different in comparison to those of BMT recipients without cell transfer (n = 9; 20 weeks post-BMT) (Tmem control n = 6).

52 Overcoming memory barrier in tolerance induction Ramsey et al.

3 9 107 Tmem sensitized to Balb/c together with 3 Gy TBI was chosen for subsequent investigations. Thus, we have developed a model in which BMT recipients enriched in donor-reactive Tmem cells reject donor BM despite costimulation blockade, preventing the development of chimerism and tolerance.

Stable long-term multi-lineage hematopoietic chimerism in presensitized BMT recipients after administration of additional anti-LFA-1 or rapamycin Next, we aimed to identify drugs that would control Tmem cells in BMT recipients overcoming T cell sensitization. When LFA-1 – which is upregulated on Tmem and whose blockade acts synergistically with costimulation blockade [36,37] – was blocked with an anti-LFA-1 mAb at the time of BMT (0.5 mg, d-1 and d2), long-term (>3 months), multi-lineage chimerism was induced in the majority of Tmem-enriched BMT recipients (10/14, P < 0.001) (Fig. 3a –d). Likewise, when rapamycin – which has multiple mechanisms of action with regard to na€ıve, regulatory, and memory T cells [38] – was used as adjunctive treatment (0.1 mg Figure 4 Tolerance in presensitized BMT recipients after administration d-1, d0, and d2), chimerism was successfully established in of additional anti-LFA-1 or rapamycin. (a) Anti-LFA-1 (□, dashed line, Tmem-enriched BMT recipients (12/14, P < 0.001) (Fig. 3a n = 14) and rapamycin- (D, dotted line, n = 13) treated groups showed –d). Chimerism rates achieved in Tmem-enriched recipients donor skin graft survival comparable to BMT recipients without cell transfer (●, solid line, n = 15) whereas Tmem-enriched BMT recipients treated with anti-LFA-1 or rapamycin were comparable without additional treatment rapidly rejected donor grafts (♦, dash-dot with those observed in BMT recipients without cell transfer line, n = 15). (b) Tmem-enriched BMT recipients treated with rapamycin (29/37, P = 0.2 and 0.3), indicating that the additional or anti-LFA-1 showed donor-specific hyporesponsiveness in MLR assays engraftment-inhibiting barrier of Tmem was successfully performed 8 weeks post-BMT (P=0.0045 for Rapa, P=0.0019 for overcome. Successful chimerism induction was also evident anti-LFA-1; SI anti-donor compared with na€ıve B6 mice; P = 0.1593 for in BM in rapamycin- and anti-LFA-treated mice at the end Rapa, P = 0.1709 for anti-LFA-1 compared with Tmem control; n = 3 of follow-up (Fig. 3e). for each group). Mean SI indices of recipient responder cells against B6 (black), Balb/c (white), and C3H/N (gray) stimulator are shown. Error bars indicate standard deviation. Tolerance in presensitized BMT recipients after administration of additional anti-LFA-1 or rapamycin Transferred memory T cells do not proliferate in BMT recipients To assess whether anti-LFA-1 or rapamycin-treated chimeras developed donor-specific tolerance, donor Balb/c and To follow adoptively transferred cell populations post- C3H tail skin was grafted ca 6 weeks post-BMT. Balb/c skin BMT, CD45.2 B6 Tmem or na€ıve T cells were transferred survived long-term (Fig. 4a) while third party skin was rap- into CD45.1 B6 recipients. Nine days post-BMT (i.e., idly rejected in all groups (MST = 12 days, data not shown). 16 days post T cell transfer) no evidence for proliferation Histological analysis at the end of follow-up revealed that of transferred T cells in response to donor BMT was found donor grafts of Tmem-enriched recipients were comparable in either treated or untreated groups as levels of CD45.2 to BMT recipients without cell transfer in that they showed cells in blood were not increased over baseline (2 days negligible eosinophil and neutrophil infiltration and only before BMT, i.e., 5 days post-transfer) (Fig. 5a). Overall, moderate intraepithelial infiltrates (data not shown). levels of CD45.2 T cells were not statistically different In vitro MLR assays were performed at the end of the fol- between groups. However, a trend toward higher numbers low-up (8–20 weeks post-BMT). As shown in Fig. 4b, both of persisting Tmem (in particular CD4+) was observed with rapamycin and anti-LFA-1-treated BMT recipients demon- rapamycin and anti-LFA-1 (compared with untreated strated donor-specific hyporesponsiveness. Taken together, Tmem recipients) (Fig. 5b). Analysis of spleen and lymph these data reveal that sensitized BMT recipients nodes revealed similar results with no statistical significance additionally treated with anti-LFA or rapamycin develop between Tmem-enriched groups with or without additional donor-specific tolerance. anti-LFA-1 or rapamycin. Thus, we think that it is unlikely

53 Ramsey et al. Overcoming memory barrier in tolerance induction

Figure 5 Anti-LFA-1 and rapamycin treatment has no detectable influence on proliferation and survival of transferred memory cells after BMT and leads to abrogation of effector function of transferred Tmem. There was no statistically significant difference regarding proliferation and survival of transferred cells between Tmem recipients treated with either anti-LFA-1 or rapamycin or without additional treatment (pretreatment d5 post-transfer/d-2 post-BMT: n = 7na€ıve T cells, n = 19 Tmem-enriched; 16 days post-transfer/9 days post-BMT: n = 4na€ıve T cells, n = 2 Tmem-enriched, n = 4 anti-LFA-1, n = 3 rapamycin) in total (a) CD45.2 cells and (b) T cell subpopulations. (c) IFNc specific ELISPOT analysis shows enhanced donor- specific memory responses in Tmem-enriched recipients which was abrogated in BMT recipients treated with anti-LFA-1 or rapamycin (and in BMT controls; 8 weeks post-BMT, n = 2 in all groups). Responses against B6 (white), Balb/c (black), and C3H/N (gray) stimulators are shown. (d) Represen- tative histograms showing donor-specific IFNc responses in CD8+ (upper panel) and CD4+ (lower panel) lymphocytes (8 weeks post-BMT). that either relevant proliferation of transferred Tmem cells post-transplant, IFN-c production was analyzed in T cells or rapid elimination occurred in rapamycin or anti-LFA-1 taken from BMT recipients upon ex vivo stimulation with treated Tmem-enriched BMT recipients. More extensive donor antigen (8 weeks post-BMT). In ELISPOT assays, analyses are required, however, to definitively define the Tmem-enriched BMT recipients showed high levels of kinetics of transferred CD4 and CD8 cells in detail. IFN-c secretion in response to donor, but not third-party antigen. In contrast, IFN-c secretion in Tmem-enriched recipients treated with rapamycin or anti-LFA-1 was signif- Anti-donor memory T cell response is eliminated through icantly lower and comparable to naive control animals rapamycin or anti-LFA-1 (Fig. 5c). As ELISPOT assays mainly assess CD8 Tmem To evaluate whether memory T cell responses are in fact reactivity, we also evaluated IFN-c production by intracel- attenuated through rapamycin and or anti-LFA-1 lular FACS staining. Here, both CD4+ and CD8+ T cells of

54 Overcoming memory barrier in tolerance induction Ramsey et al.

Tmem-enriched BMT recipients produced substantially nonhuman primates or transplant patients. The murine more IFN-c than BMT controls. Both rapamycin and anti- model developed in the current study was designed to allow LFA-1 drastically reduced production of IFN-c in both the evaluation of treatments for their efficacy to control the CD4 and CD8 cells (Fig. 5d). Together, these results indi- Tmem response in recipients of mixed chimerism cate that the enhanced donor-specific CD4 and CD8 T regimens. memory cell response seen in Tmem-enriched BMT recipi- While TCR-transgenic systems facilitate detailed mecha- ents becomes virtually eliminated through rapamycin or nistic studies [16], the described Tmem-enriched model anti-LFA-1 by 8 weeks post-BMT. offers the advantage that recipients contain a polyclonal repertoire of Tmem which presumably encompasses a broad spectrum of affinities and specificities, as is usually Tolerant chimeras treated with anti-LFA-1 and rapamycin the case in the clinical setting. Moreover, both CD4 and show peripheral and central deletion of donor-reactive CD8 Tmem contribute to the elicited rejection in the cur- T cells rent model. Interestingly, another study identified virus- As clonal deletion is a major mechanism of most chimerism induced alloreactive CD8+ central memory T cells to be pri- protocols, we determined if donor-reactive T cells are marily responsible for memory-mediated rejection [22], deleted in Tmem-enriched recipients. The frequency of cer- whereas in our model, transfer of CD8+ cells alone did not tain superantigen-reactive T cell populations corresponds to abrogate chimerism (at least not at the cell doses tested). the deletion of ‘truly reactive’ donor-specific T cells (as Titrating the number of Tmem that is transferred revealed assessed by T cells with a donor-reactive transgenic TCR that certain frequencies of Tmem can be sufficiently con- [39]) and was thus used as a surrogate marker. Developing trolled through costimulation blockade allowing chimerism thymocytes whose TCRs contain Vb11 and Vb5 bind to su- to be induced (Fig. 2a). Once a threshold is crossed, how- perantigens presented by I-E, and are deleted in Balb/c ever – in our model 2–3 9 107 transferred T cells from sen- which are I-E positive mice, but not in B6 mice, which do sitized mice – donor BM is rejected despite costimulation not express I-E [40–42]. Deletion of CD4 cells was noted in blockade. This chimerism-abrogating effect is antigen-spe- peripheral blood as early as 2 weeks post-BMT in Tmem- cific as the transfer of equal numbers of T cells from mice enriched mice receiving anti-LFA-1 (data not shown). Six sensitized to an unrelated third party skin donor did not weeks post-BMT, substantial deletion of CD4+ Vb11 and prevent chimerism induction (Fig. 1d). These results are Vb5 (but not control Vb8) cells in peripheral blood was evi- consistent with the empirical observation that costimula- dent in anti-LFA-1 and rapamycin-treated recipients in tion blockade-based immunosuppressive therapy is par- comparison to na€ıve B6 mice (and to Tmem control mice) tially, but not completely, effective in nonhuman primates (Fig. 6a). At such early time points, clonal deletion of CD4 and in renal transplant patients (that can be expected to (but not CD8) T cells was also observed in the spleen of mice harbor alloreactive Tmem) [43–46]. treated with anti-LFA-1 or rapamycin (8 weeks post-BMT) A number of selected drugs that we screened in this (Fig. 6b). Deletion of Vb5/Vb11 CD4+ single positive thy- model failed to overcome the sensitization barrier. Neither mocytes was also evident in anti-LFA-1 and rapamycin-trea- bortezomib, anti-TNFa (infliximab) nor anti-IL7 – all with ted animals at this time point (but not in Tmem controls), reported beneficial effects on Tmem in other settings [31– demonstrating that central clonal deletion occurs in these 35] – were effective as adjunctive treatments in our model mice (Fig. 6c). Moreover, deletion of CD8+ splenocytes (data not shown). However, two drugs – anti-LFA-1 and became detectable late after BMT (20 weeks post-BMT, data rapamycin, both approved for clinical application were not shown). As CD8 T cells – in contrast with CD4 cells – are identified to control Tmem-triggered rejection allowing not deleted extrathymically (as they do not efficiently bind chimerism and tolerance induction in Tmem-enriched to the superantigen-presenting MHC II), but only intrathy- recipients. Indeed, anti-LFA-1 and rapamycin were revealed mically at the double positive stage of development, this to abrogate the enhanced responses in both CD4 and CD8 deletion observed among CD8 splenocytes provides addi- T memory cell subsets. LFA-1, a b2 integrin composed of a tional evidence for central clonal deletion in these chimeras. unique a-chain (CD11a) noncovalently linked to a b-chain Taken together, these data suggest peripheral and central (CD18), has an important role in T cell adhesion and acti- clonal deletion of donor-reactive T cells in Tmem-enriched vation and is upregulated on Tmem [47]. Anti-LFA-1 on BMT recipients treated with anti-LFA-1 or rapamycin. its own prolongs heart and islet, but not skin allografts and acts synergistically with anti-CD40L, CTLA4Ig, and mTOR inhibitors in various experimental transplant models Discussion [48,49]. Notably, CD8 Tmem responses not inhibited by Overcoming the barrier of Tmem activation remains a con- costimulation blockade are susceptible to anti-LFA-1 siderable challenge when tolerance protocols are applied to treatment [50,51]. Recently, an anti-LFA mAb – used in

55 Ramsey et al. Overcoming memory barrier in tolerance induction

Figure 6 Tolerant chimeras treated with anti-LFA-1 and rapamycin show peripheral and central deletion of donor-reactive T cells. (a) The frequencies of Vb11 and Vb5 CD4+ cells (isolated from peripheral blood) were signiﬁcantly reduced in anti-LFA-1 and rapamycin-treated Tmem-enriched BMT recipients (compared with na€ıve B6) at 6 weeks post-BMT, suggesting peripheral clonal deletion of donor-reactive CD4 T cells (anti-LFA-1 n = 7, rapamycin n = 12, Tmem control = 3, BMT control n = 6, na€ıve Balb/C n = 3, and naive B6 n = 3). (b) The frequencies of Vb11 and Vb5 among CD4+ and among CD8+ splenocytes were measured at 8 weeks post-BMT (anti-LFA-1 n = 3, rapamycin n = 3, Tmem control = 3, BMT control n = 3, na€ıve Balb/C n = 3, and naive B6 n = 3). Signiﬁcantly lower levels of CD4+ Vb11 cells (and numerically lower levels of CD4+ Vb5 cells) were evident in anti- LFA-1 and rapamycin-treated groups (compared with na€ıve B6). No such deletion was observed at this early time point among CD8+ cells, suggesting that the deletion of CD4 T cells early post-BMT took place extrathymically (8 weeks post-BMT; anti-LFA-1 n = 3, rapamycin n = 3, Tmem control = 3, BMT control n = 3, na€ıve Balb/C n = 3, and naive B6 n = 3). (c) Flow cytometric analysis of thymocytes revealed reduced frequencies of Vb11 and Vb5 among CD4+ single positive cells in anti-LFA-1 (n = 3) and rapamycin-treated recipients (n = 3) in comparison to na€ıve B6 control mice (n = 3), indicating central clonal deletion of donor-reactive thymocytes (Tmem control n = 3, BMT control n = 3 and na€ıve Balb/C n = 3). (Panel a–c) Na€ıve B6 are shown in black, na€ıve Balb/c in white, BMT control in light-gray, control Tmem-enriched recipients in dark-gray, Tmem-enriched recipients treated with anti-LFA-1 in white with black vertical stripes, Tmem-enriched recipients treated with rapamycin in white with black horizontal stripes. combination with belatacept, a second generation CTLA4Ig response [52]. A humanized anti-CD11a mAb (efalizumab) – was demonstrated to prolong islet allograft survival in a was approved for treatment of severe psoriasis [53] and has nonhuman primate model by controlling the Tmem been evaluated in renal [54] and in islet transplant recipi-

56 Overcoming memory barrier in tolerance induction Ramsey et al. ents [55]. While efalizumab was recently withdrawn by the The model presented herein is expected to enhance the manufacturer because of the occurrence of progressive predictive value of murine mixed chimerism studies and multi-focal leukoencephalopathy in rare cases, this devastat- should be helpful in the development of clinically viable ing complication occurred with long-term use. Thus, short- tolerance protocols. Anti-LFA-1 and rapamycin have been term therapy with efalizumab for appropriate indications identified as drugs with efficacy in controlling Tmem in might still be considered acceptably safe. recipients of nonmyeloablative BMT and are attractive can- Although underlying mechanisms still need to be clarified, didates for evaluation in preclinical and clinical mixed chi- the engraftment enhancing effect seen with anti-LFA-1 might merism studies. be due altered Tmem trafficking impairing rejection. Besides, anti-LFA-1 might affect Treg function. Recent data suggest Authorship that treatment with anti-LFA-1 and CTLA4-Ig leads to a selective enrichment of Tregs (CD4+CD25+FoxP3+)in HR, NP: designed and performed research, analyzed data, peripheral lymph nodes in a fully allogeneic murine skin wrote paper. CK, LU, CS, UB, MG, KH, ES and FW: per- transplantation model [56]. Simultaneously, within the same formed research. TW: designed research, analyzed data, model, activated effector cells were found to undergo wrote paper. increased apoptosis within lymph nodes. As well, in another allogeneic murine transplant model, anti-LFA-1 was found Funding to promote retention of Tregs in lymph nodes, as well as inhibition of cytokine production in Tmem cells [57]. The authors have declared no funding. Inhibition of mTOR (with rapamycin or its derivatives) leads to a series of effects that is considerably more complex Acknowledgements than has been initially recognized [58]. In particular, mTOR inhibition enhances the Tmem responses to viral This work was supported by the Austrian Science Fund infections [59], but not the Tmem response to transplant (FWF) (TRP151-B19 and Doctoral Programme W1212). antigens [60]. mTOR inhibition has also been noted to pro- We thank Elizabeth Hablit for her secretarial assistance. mote regulatory T cell generation and function [61]. In the context of chimerism induction in na€ıve recipients, mTOR inhibitors promote engraftment of allogeneic BM by them- References selves [7,49,62] and in synergy with regulatory T cell ther- 1. Pilat N, Wekerle T. Transplantation tolerance through apy [63]. While rapamycin failed to promote tolerance to mixed chimerism. Nat Rev Nephrol 2010; 6: 594. anti-donor memory cells in a previous study [22], the cur- 2. Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched rent results extend the beneficial effects of mTOR inhibi- renal transplantation without maintenance immunosup- tion in BMT recipients to those that contain a substantial pression. N Engl J Med 2008; 358: 353. frequency of donor-reactive Tmem. This discrepancy may 3. Scandling JD, Busque S, Dejbakhsh-Jones S, et al. Tolerance be because of the different dosing regimens of rapamycin. and chimerism after renal and hematopoietic-cell transplan- The therapeutic effect of rapamycin on alloreactive mem- tation. N Engl J Med 2008; 358: 362. ory T cell responses observed in the current experiments is 4. Kean LS, Gangappa S, Pearson TC, Larsen CP. Trans- in line with recent reports on the distinct effects of mTOR plant tolerance in non-human primates: progress, current 6 inhibition on this Tmem subset [59,60]. Another possible challenges and unmet needs. Am J Transplant 2006; : mechanism of rapamycin in this model might be its effect 884. 5. Wekerle T, Kurtz J, Ito H, et al. 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57 Ramsey et al. Overcoming memory barrier in tolerance induction

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59 2.2.1 Interlude As costimulation-based protocols for the induction of mixed chimerism and transplant tolerance work well in the experimental setting of small animal models, the translation into nonhuman primate models and into the clinical setting is still impeded. The complex immune system from nonhuman primates and from transplant recipients including a memory T-cell pool, represents a major hurdle for the successful induction of tolerance.

For investigating the role of memory T-cells in the murine system, a memory T-cell enriched model of BMT was established. Therefore, memory T-cells from sensitized B6 mice were isolated and transferred into B6 mice receiving BMT. A dose dependent effect of the transferred memory T-cells was observed leading an antigen specific rejection of donor BM despite costimulation blockade. Among different tested substances, anti-LFA-1 and rapamycin seemed to work synergistically with costimulation blockade and were able to abrogate the alloreactive effect of memory T-cells on BM engraftment and skin graft tolerance. Hence, anti-LFA-1 and rapamycin showed substantial potency to overcome the barrier caused by donor-reactive memory T-cells for the induction of transplantation tolerance.

60 2.3 Third paper

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

61 American Journal of Transplantation 2015; 15: 1568–1579 C Copyright 2015 The American Society of Transplantation Wiley Periodicals Inc. and the American Society of Transplant Surgeons doi: 10.1111/ajt.13154

Rapamycin and CTLA4Ig Synergize to Induce Stable Mixed Chimerism Without the Need for CD40 Blockade

y y N. Pilat1, , C. Klaus1, , C. Schwarz1, K. Hock1, whereas fully mismatched grafts are rejected, suggest- R. Oberhuber2, E. Schwaiger1, M. Gattringer1, ing that non-MHC antigens cause graft rejection and split tolerance. H. Ramsey1, U. Baranyi1, B. Zelger3, G. Brandacher5, F. Wrba4 1, Abbreviations: BM, bone marrow; BMT, bone marrow and T. Wekerle * transplantation; FACS, fluorescence-activated cell sorter; H&E, hematoxylin and eosin; mAbs, monoclo- 1Section of Transplantation Immunology, Department of nal antibodies; MLR, mixed lymphocyte reaction; MST, Surgery, Medical University of Vienna, Austria median survival time; TBI, total body irradiation 2Department of Visceral, Transplant, and Thoracic Surgery, Center of Operative Medicine, Innsbruck Received 24 September 2014, revised 12 November Medical University, Austria 2014 and accepted for publication 30 November 2014 3Institute of Pathology, Medical University of Innsbruck, Austria 4Institute of Clinical Pathology, Medical University of Vienna, Austria Introduction 5Department of Plastic and Reconstructive Surgery, Vascularized Composite Allotransplantation (VCA) Current immunosuppressive regimens are remarkably Laboratory, Johns Hopkins University School of successful at controlling acute rejection, but the rate of Medicine, Baltimore, MD annual graft attrition after the first year posttransplant has Corresponding author: Thomas Wekerle, changed little (1), and immunological factors remain a major [email protected] y cause of graft loss (2). Besides, morbidity caused by Both authors contributed equally. immunosuppressants is substantial. Therefore, tolerance strategies replacing non-specific immunosuppression are of considerable interest.

The mixed chimerism approach achieves donor-specif- Most tolerance approaches developed over the last decade ic tolerance in organ transplantation, but clinical use is rely on costimulation blockade (3), with the CD28 and CD40 inhibited by the toxicities of current bone marrow (BM) pathways being studied most extensively. The CD40: transplantation (BMT) protocols. Blocking the CD40: CD154 costimulatory pathway is crucial for both B and T cell CD154 pathway with anti-CD154 monoclonal anti- immunity (4). CD40 signaling is critical for APC activation bodies (mAbs) is exceptionally potent in inducing required both for full T cell activation (5) and for mixed chimerism, but these mAbs are clinically not immunoglobulin class switch. CD40 expression was shown available. Defining the roles of donor and recipient CD40 in a murine allogeneic BMT model, we show that to be important for CD8 T cell differentiation and the CD4 or CD8 activation through an intact direct or CD4 T formation of CD8 memory subsets (6,7). Recently, CD40 cell activation through the indirect pathway is suffi- expression on CD8 T cells has been recognized to cient to trigger BM rejection despite CTLA4Ig treat- contribute to alloreactivity (8). CD40 ligand (CD154, ment. In the absence of CD4 T cells, CD8 T cell CD40L, gp39) is expressed on activated CD4 T cells, CD8 activation via the direct pathway, in contrast, leads T cell subsets, monocytes, platelets and is present in to a state of split tolerance. Interruption of the CD40 soluble form in serum (9). Moreover, CD40L expression on signals in both the direct and indirect pathway of DCs is important in CD4-independent priming of CD8 T cell allorecognition or lack of recipient CD154 is required responses (10). It remains controversial whether CD154 for the induction of chimerism and tolerance. We transmits a cell-intrinsic signal to T cells (11–13), which developed a novel BMT protocol that induces mixed chimerism and donor-specific tolerance to fully mis- would be of relevance for the development of antibodies matched cardiac allografts relying on CD28 costimula- interfering with the CD40:CD154 pathway. Recently, it was tion blockade and mTOR inhibition without targeting shown that the absence of CD40 signaling leads to the CD40 pathway. Notably, MHC-mismatched/minor protection of tubular epithelium from apoptosis and thereby antigen-matched skin grafts survive indefinitely preserved kidney allograft function (14).

62 CD40 in Chimerism-Based Tolerance

Antibodies directed against CD154 are exceptionally potent in Antibodies and fluorescence-activated cell sorter the prevention of humoral and cellular inflammatory re- (FACS) analysis sponse (15). Anti-CD154 mAb prolongs graft survival when Multi-color flow cytometric analysis of multi-lineage macrochimerism and given alone (16), and leads to indefinite survival in combination Vb-subunit expression was performed as described previously (20,37). Chimerism was calculated as the net percentage of donor MHC class Iþ with donor-specific transfusion (17), CTLA4Ig (16) or rapamy- d cin (18) in rodent models. Moreover, anti-CD154 is the (H-2D , 34-2-12) cells among specific leukocyte lineages (20,37). Mice were considered chimeric if donor cells were detectable by flow cytometry within backbone of numerous mixed chimerism protocols in both the myeloid lineage and at least one lymphoid lineage. both rodents and non-human primates (19). Anti - CD154 mAbs used alone (13,20–22) or in combination with CTLA4Ig (20,23,24) – allowed the induction of mixed Anti-donor antibodies Recipient serum harvested >3 months post-BMT was heat-inactivated and hematopoietic chimerism through allogeneic BMT without incubated with recipient-type, donor-type and third-party-type thymocytes the need for global destruction of the recipient T cell repertoire. (which are low in Fc-receptors, reducing background staining). Binding of Several attempts at replacing anti-CD154 mAb in such serum IgG Abs to thymocytes was analyzed by flow cytometry using FITC- regimens have failed so far (19,20). As anti-CD154 mAbs conjugated rat anti-mouse IgG1 and IgG2a/2b (BD Biosciences, San Jose, CA). were associated with severe thromboembolic complications (25,26), their clinical development was temporarily Mixed lymphocyte reaction (MLR) suspended. Antibodies directly targeting CD40 (27–31), and MLRs were performed as described in detail previously (23). Stimulation more recently so-called domain antibodies targeting indices (SI) were calculated in relation to medium controls. Results represent CD40L (32) are under development as potential alternative averaged data of triplets from pooled animals, which were normalized but it is unknown, if and when they will become clinically (divided through recipient) so that reaction against self (recipient/recipient) available. was set as baseline.

The mixed chimerism approach has successfully establis- Skin grafting hed tolerance in some renal transplant patients in pilot Full thickness tail skin from Balb/c, B10.D2 or C3H was grafted 4–6 weeks trials (33–35). Routine clinical application, however, has so after BMT and visually inspected thereafter at short intervals. Grafts were far been prevented by the toxicities and risks involved considered to be rejected when less than 10% remained viable. in current BMT protocols. Translation of costimulation blockade-based protocols with a more acceptable toxicity Heart transplantation profile has been stalled by the lack of clinically available Primarily vascularized cervical heterotopic heart transplantation was compounds. With abatacept/belatacept (36) the first com- performed 6–8 weeks after BMT with a modified cuff-technique for pounds targeting the CD28 pathway are now available but revascularization as described previously (38). Cardiac allograft survival was drugs interfering with the CD40:CD154 pathway are still determined by daily palpation. wanting. Histologic analysis In the present study, we define the roles of donor and 4 mm sections were cut from paraffin-embedded tissue fixed in 4.5% recipient CD40 in the establishment of mixed chimerism and formalin (with a buffered pH of 7.5), stained with hematoxilin and eosin tolerance through allogeneic BMT and present a chimerism/ (H&E) and Giemsa according to standard protocols, and analyzed by an tolerance protocol devoid of anti-CD154 treatment. experienced pathologist in blinded fashion. Cardiac allografts were scored according to the ISHLT guidelines (39).

Materials and Methods Statistics A two-sided Student’s t-test was used to compare percentages of Vb-family Animals positive cells, chimerism levels between groups and SI values, Fisher’s Female C57BL/6 (B6, recipient, H-2b), Balb/c (donor, H-2d), B10.D2 (H-2d, exact test was used to compare chimerism rates between groups. Skin and minor antigen matched to B6), C3H/HeNCrl (C3H, third party, H-2k) and SJL heart allograft survival were calculated according to the Kaplan–Meier (third party, H-2s) mice were purchased from Charles River Laboratories product limit method and compared between groups using the log-rank test. (Sulzfeld, Germany). CD40-/- (B6 and Balb/c background) and CD154-/- (B6 A p value less than 0.05 was considered to be statistically significant. background) mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were housed under specific pathogen-free conditions and were used at 6–12 weeks of age. All experiments were approved by the local Results review board of the Medical University of Vienna. Ablating recipient CD154 but not donor or recipient BMT protocol CD40 is sufficient for the induction of chimerism and Groups of age-matched B6 recipients received 2 Gy total body irradiation (d- donor-specific tolerance 1) and costimulation blockade with CTLA4Ig (0.5 mg, d2 and d4) with or To define the role of the CD40 pathway donor and/or without anti-CD154 mAb (anti-CD40L, MR1, 1 mg, d0) and rapamycin (0.1 mg/mouse, d-1, d0 and d2) as indicated. Groups of mice received a recipient mice deficient in either CD40 or CD154 were used depleting dose of anti-CD4 (GK1.5, 1.8–mg/mouse, d-5/d-1) and/or anti CD8 in an established mixed chimerism model entailing non- (2.43, 1.4 mg, d-5/d-1). 15–20 106 unseparated BM cells from Balb/c or myeloablative total body irradiation, a moderate dose of B10.D2 donors were injected intravenously on d0. allogeneic BM cells and costimulation blockade which

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63 Pilat et al induces lasting chimerism and tolerance (40). Omission of of split tolerance as they promptly rejected donor skin while anti-CD154 from this protocol results in uniform abrogation maintaining donor chimerism (median survival time [MST] of chimerism and tolerance induction (WT ! WT), whereas ¼ 8 days; p ¼ n.s. compared to na€ıve controls; data not CD154-/- recipients grafted with WT BM developed shown). Importantly, chimeras also show donor-specific chimerism and donor-specific skin graft tolerance (Table 1, tolerance in vitro and deletion of donor-reactive T cells (data Figure 1A,B). Additionally, chimeric mice exhibited donor- not shown), suggesting that tissue specific minor antigens specific tolerance in in vitro MLR assays (data not shown). trigger donor skin rejection. Thus, CTLA4Ig alone is unable Thus neither target cell depletion nor a CD154 signal are to prevent CD4 cells from rejecting allogeneic BM and is required mechanisms of anti-CD154 mAbs but rather insufficient for tolerizing CD8 cells in the absence of CD4 blocking recipient CD154 is sufficient. cells to a degree necessary for the acceptance of donor skin. Transplantation of CD40-/- BM into WT recipients and WT BM into CD40-/- recipients prevented chimerism induction To define which T cell subset(s) are not tolerized in CD40 (Figure 1A) and tolerance in vitro (MLR, data not shown), deficient donor or recipient, CD4 or CD8 cells were again indicating that eliminating CD40 signalling in either the depleted at the time of BMT in respective combinations. direct or the indirect pathway of allorecognition alone is Depletion of CD8 cells again led to a failure of chimerism insufficient (despite treatment with CTLA4Ig). Only when induction when CD40-/- BM was transplanted, suggesting both donor and recipient were lacking CD40, induction of that lack of CD40 on donor APCs is insufficient for CD4 T chimerism and tolerance was successful (Figure 1A,B). cell tolerization. Thus, CD4 activation through the intact Thus, interruption of the CD40 signals in both the direct indirect pathway is sufficient to trigger BM rejection and indirect pathway of allorecognition, but not either (Figure 1A,B). In contrast to WT BM, recipients of pathway alone, allows engraftment of allogeneic BM under CD40-/- BM depleted of CD4 cells developed chimerism CTLA4Ig treatment (summarized in Table 1 and illustrated in and skin graft tolerance (Table 1). Thus the lack of CD8 Figure 1C). tolerization on the skin graft level is not due to CD4 depletion including the depletion of CD4þ Tregs, but rather results from CD40 signalling in direct antigen presentation. Donor cells lacking CD40 fail to tolerize recipient CD4 T cells Notably, the lack of recipient CD40 invariably led to rejection The presence of either CD4 or CD8 T cells alone is sufficient of WT BM irrespective of CD4 or CD8 depletion, suggesting to reject MHC-mismatched BM in recipients receiving non- that activation of either CD4 or CD8 through the direct myeloablative TBI only (21). In the WT ! WT combination, pathway is sufficient to trigger BM rejection. Alternatively, the elimination of CD4 cells or CD4 plus CD8 cells, but not this might be due to the fact that CD40 is critically required CD8 cells alone allowed chimerism to be established for thymic development of Tregs, resulting in substantially without anti-CD154 (using CTLA4Ig alone) (Table 1). reduced Treg numbers in these mice (41). Collectively, However, after CD4 depletion chimeras showed a state these results also indicate that CTLA4Ig is effectively

Table 1: The role of CD40:CD154 pathway in chimerism and tolerance induction. Induction of chimerism and tolerance using various WT and CD40:CD154 pathway knockout models. Tolerance among chimeras was assessed by skin grafts (Y ¼ yes, N ¼ no; aCD4 ¼ CD4 T cell depleting Ab, aCD8 ¼ CD8 T cell depleting Ab; n.d. ¼ not done) Donor Recipient Anti-CD154 mAb CTLA4Ig Depleting antibody Chimerism Skin tolerance

WT WT Y Y N 17/22 13/17 WT WT N Y N 0/5 n.d. WT WT Y Y aCD4 4/5 2/3 WT WT Y Y aCD8 5/5 3/5 WT WT N Y aCD4 4/5 0/4 WT WT N Y aCD8 0/5 n.d. WT WT N Y aCD8aCD4 5/5 4/5

WT CD154-/- N Y N 7/8 5/7 CD40-/- CD40-/- N Y N 8/8 8/8

WT CD40-/- N Y N 0/5 n.d. WT CD40-/- NYaCD4 0/5 n.d. WT CD40-/- NYaCD8 0/5 n.d.

CD40-/- WT N Y N 0/8 n.d. CD40-/- WT N Y aCD4 3/5 3/3 CD40-/- WT N Y aCD8 0/5 n.d.

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64 CD40 in Chimerism-Based Tolerance

Figure 1: Tolerization of CD4 T cells requires blockade of indirect pathway of allorecognition. Groups of wildtype (WT) and knockout (CD40-/-, CD154-/-) B6 mice received 2 Gy TBI and ~15–20 x 106 Balb/c BMCs (WT or CD40-/-), CTLA4Ig and T cell depleting antibodies where indicated. (A) Long-term donor (H-2Dd) chimerism among leukocytes of the myeloid (Mac1þ) lineage in various groups (WT! CD154-/-, 7/8; CD40-/-! CD40-/-, 8/8; CD40-/-!WT, 0/8; CD40-/-!WT CD4 depleted, 3/5; CD40-/-!WT CD8 depleted, 0/5; WT! CD40-/-, 0/5 chimeras; WT! CD40-/- CD4 depleted, 0/5; WT! CD40-/- CD8 depleted, 0/5) was assessed by FCM analysis of peripheral blood at multiple time points (mean SD; representative data from four independent experiments). (B) Donor-specific tolerance in various WT and KO combinations was assessed by grafting donor 4–6 weeks post-BMT. Donor-specific tolerance could be demonstrated in CD40-/- BMT recipients grafted with CD40-/- BM (n ¼ 8, filled triangle, ~) and in CD154-/- recipients grafted with WT BM (n ¼ 8, filled square, &) and CD4- T cell depleted WT recipients grafted with CD40-/- BM (n ¼ 5, dotted line, open circle, ). All other combinations rapidly rejected donor- specific skin grafts (WT!WT, n ¼ 5, filled circle, *; CD40-/-!WT, n ¼ 8, dotted line, open triangle, 4; CD8 depleted CD40-/-! CD8 depleted WT, n ¼ 5, dotted line, open square, &). Representative data from 4 independent experiments are shown. (C) Schematic illustration showing the presence of CD40:CD154 costimulatory signal in direct (left panel) and indirect (right panel) pathways of allorecognition in various donor-recipient combinations. preventing rejection of allogeneic skin under conditions Rapamycin promotes engraftment of allogeneic BM in which CD40-mediated T cell activation is eliminated in in the presence of an intact CD40:CD154 both the direct and indirect pathway (WT ! CD154-/- and costimulation pathway CD40-/- ! CD40-/-) or in which CD8 cells can only be Based on the above findings, interventions that efficiently effectively activated through the indirect pathway of control directly alloreactive CD4 and CD8 responses and allorecognition (CD40-/- ! WT þ CD4 depletion). Taken indirectly alloreactive CD4 responses would be required to together, these results provide evidence that anti-CD154 allow chimerism and tolerance induction without anti- mAb is required to control directly alloreactive CD4 and CD154mAb. Mammalian target of rapamycin (mTOR) has CD8 cells and indirectly alloreactive CD4 cells. CTLA4Ig been demonstrated to play a critical role in T cell activation alone is only sufficient to tolerize indirectly alloreactive CD8 upon antigen encounter by the TCR. Inhibition of mTOR T cells. pathway leads to anergy in vivo and in vitro (even in the

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65 Pilat et al presence of costimulatory signalling) (42). The mTOR CTLA4Ig/rapa treatment fails to tolerize skin-specific inhibitor rapamycin (sirolimus) has been shown to promote minor antigens tolerance by its inhibitory effect on conventional T cells and To define the mechanism why donor hearts but not donor its positive effect on Tregs (43). Notably, rapamycin exerts an skin is accepted with the CTLA4Ig/rapa regimen, we next immunosuppressive effect on CD8 T cells in the context of assessed whether tolerance to MHC and/or minor antigens an allograft (44). The mTOR pathway also plays an important is achieved. The Balb/c ! B6 combination used in the role in the maturation and function of DCs, thereby current study crosses both MHC and multiple minor antigen preventing intact allograft rejection. As mTOR inhibition barriers. Minor antigens, in particular skin specific antigens, using rapamycin has been proposed to broadly modulate failed to be tolerized in some previously reported several parts of the alloresponse, we tested whether short- chimerism studies (45,46). Therefore, we transplanted term treatment with rapamycin overcomes the need for B10.D2 BM (H-2d) which is MHC-mismatched to B6 but CD40:CD154 blockade. Rapamycin together with CTLA4Ig shares the same minor antigen background into B6 (termed hereafter ‘CTLA4Ig/rapa’ regimen) led to the recipients (H-2b). Again, persistent mixed chimerism was engraftment of 15 106 Balb/c BMC in B6 recipients achieved with CTLA4Ig/rapa, but notably this time irradiated with 2 Gy TBI in the majority of recipients (9/10 donor skin (B10.D2) was accepted for the length of follow chimeras; p ¼ 1.000 vs CTLA4Ig/anti-CD154; p ¼ 0.002 vs. up (> 100 days) (Figure 2C). Thus, the CTLA4Ig/rapa CTLA4Ig only, Figure 2A). Rapamycin alone without protocol leads to skin tolerance towards MHC antigens. To CTLA4Ig, in contrast, did not lead to BM engraftment (0/5 directly test whether it is the lack of tolerance towards chimeras, P ¼ 0.002 vs. CTLA4Ig/rapa, Figure 2A). Chime- minor antigens that causes donor skin graft rejection in rism in BMT recipients treated with CTLA4Ig/rapa regimen Balb/c chimeras, we grafted B10.D2 skin and hearts unto was of multi-lineage nature, with substantial donor popula- recipients of Balb/c BM treated with CTLA4Ig/rapa. Both tions present in all tested leukocyte lineages (27.1 þ/ 8.9% B10.D2 skin and heart grafts were accepted indefinitely CD4 chimerism, 10.9 þ/ 4.3% CD8 chimerism, 36.7 þ/ (>100 days) (Figure 3A–C). Moreover, Balb/c chimeras that 5.0% B cell chimerism, 71.6 þ/ 19.7% myeloid chimerism; had rejected Balb/c skin were challenged with a B10.D2 n ¼ 8, 6 months post-BMT, representative data from one of skin graft (n ¼ 5), which survived long-term (>100d in all seven independent experiments) (Figure 2B). Chimerism mice, one graft was lost due to a technical error; Figure 3D) levels in peripheral blood correlated with chimerism in without signs of rejection, whereas second Balb/c grafts lymphoid organs (BM and spleen, data not shown). Pooled (n ¼ 5) were again promptly rejected. These results provide data from multiple repeat experiments reveal that chimerism evidence that tissue-specific antigens expressed by Balb/c induction using CTLA4Ig/rapa tended to be more reliable skin, but not Balb/c hearts, are responsible for the rejection than using CTLA4Ig/anti-CD154 mAb (43/47 chimeras vs. 24/ of donor skin in CTLA4Ig/rapa-induced Balb/c chimeras. 31 chimeras which is consistent with historic data from our Besides, linked suppression is not a mechanism in the lab (37,40)), although the difference did not reach statistical CTLA4Ig/rapa chimeras as minor antigens are not tolerized significance (p ¼ 0.1027). despite robust tolerance towards MHC antigens (47).

CTLA4Ig plus rapamycin induces donor-specific The CTLA4Ig/rapa regimen leads to in vitro and tolerance to donor heart allografts humoral tolerance. To assess tolerance, skin, and heart transplants were To further evaluate tolerance induced by the CTLA4Ig/ performed 4–8 weeks post-BMT. All BMT recipients rapa protocol, in vitro MLR assays were performed. treated with CTLA4Ig/rapa accepted donor hearts for the Responsiveness towards donor was reduced almost to length of follow-up (> 100 days, Figure 2C) and histological the level of self-reactivity in both Balb/c and B10.D2 analysis revealed only mild pathological changes (interstitial chimeras while third party reactivity was preserved and/or perivascular infiltrates without myocyte damage or (20 weeks post-BMT) (Figure 4A). Furthermore, no anti- vasculitis, Figure 2D). donor Abs were detectable in chimeras, whereas substantial levels of Abs towards third-party grafts were We also grafted BMT recipients with donor and third party demonstrated (Figure 4B). Interestingly, although Balb/c skin which is commonly regarded as a stringent test for skin grafts were rejected, these mice lacked detectable assessing transplantation tolerance. Chimeras induced levels of anti-donor Abs, suggesting that cellular but not with CTLA4Ig/rapa treatment showed significant prolonga- humoral immunity towards minor antigens is responsible tion of donor skin graft survival, while promptly rejecting for skin graft rejection in these chimeras. third party skin grafts (MST ¼ 40 days for Balb/c skin; MST ¼ 12 for third-party C3H skin; p < 0.0001) (Figure 2C). Importantly, CTLA4Ig combined with rapamycin led to a Central and peripheral deletion contribute to significant prolongation of skin graft survival, whereas tolerance in CTLA4Ig/rapa chimeras combined with CD4 depletion it did not (CTLA4Ig/Rapa vs. We further evaluated whether clonal deletion of donor- CTLA4Ig/anti-CD4 p ¼ 0.0001). However, contrary to previ- reactive T cells contributes to tolerance as it is one of the ous protocols with CTLA4Ig plus anti-CD154 mAb, donor main mechanisms observed in previous mixed chimerism skin was not permanently accepted. models (20). In chimeras, the frequency of certain

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66 CD40 in Chimerism-Based Tolerance

Figure 2: Costimulation blockade with CTLA4Ig synergizes with rapamycin to induce mixed chimerism and donor-specific tolerance. (A) Groups of B6 mice were grafted with Balb/c BM (15 x 106) after 2 Gy total body irradiation (TBI). Additional treatment consisted of combined costimulation blockade with CTLA4Ig and MR1 (8/8 chimeras), CTLA4Ig alone (0/5 chimeras), the combination of CTLA4Ig and rapamycin (9/10 chimeras) or rapamycin (0/5 chimeras) alone. Long-term donor (H-2Dd) chimerism among leukocytes of the myeloid (Mac1þ) lineage was assessed by FCM analysis of peripheral blood at multiple time points and is shown as mean percent (mean SD; representative data from six independent experiments) (B) The combination of CTLA4Ig and rapamycin led to multi lineage chimerism as shown by substantial levels of donor cells in all tested lineages (T cells, B cells and myeloid lineage). FCM plots from a representative BMT recipient treated with CTLA4Ig and rapamycin are shown (25 weeks post-BMT). Multi lineage chimerism persisted and remained stable for the length of follow up (up to nine months post-BMT), suggesting successful engraftment and survival of donor hematopoietic stem cells. (C) Survival of donor-specific skin grafts was significantly prolonged (n ¼ 13, solid circle, *; log rank, p < 0.0001) whereas 3rd party grafts were rapidly rejected (C3H, n ¼ 5, dotted line, open square, &). Donor-specific heart allografts survived indefinitely (n ¼ 6, open triangle, 4). Groups of mice grafted with B10.D2 BM showed indefinite survival of donor-specific skin grafts (B10.D2, n ¼ 9, solid square, &). Data are representative for three independent experiments. (D) Representative heart allografts are shown (left, Balb/c - full mismatch; right, B6 - syngeneic graft; 100d posttransplantation, H&E staining, original magnification, 200).

superantigen-reactive T cell populations (Vb11 and Vb5 but Discussion not Vb8 in the strain combination used) correlates with the deletion of truly alloreactive, donor-speciﬁc T cells (48,49) The present study reveals that CD40 signals contribute to and hence is a useful surrogate marker to measure clonal alloreactivity in allogeneic BMT through both the direct and deletion in WT recipients. We observed a substantial the indirect pathway of allorecognition, which both have to deletion of CD4 cells in peripheral blood of chimeras early be controlled to achieve engraftment and tolerance. The after BMT (data not shown), which progressed over time need for CD40 blockade can be obviated through treatment and was signiﬁcant for both markers 14 weeks post-BMT with CTLA4Ig together with rapamycin that allows engraft- (Figure 4C) in blood cells and splenocytes (Figure 4D). ment of conventional doses of allogeneic BM. Central tolerance was directly assessed among CD4 single- positive thymocytes of long-term chimeras (20 weeks post- More than a decade ago, the introduction of the costimu- BMT) (Figure 4E). lation blockers CTLA4Ig and anti-CD154 mAb as part of

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67 Pilat et al

Figure 3: Minor antigen mismatch is responsible for skin allograft rejection in CD40:CD154 costimulation independent tolerance. (A) Groups of B6 mice were grafted with fully mismatched (Balb/c) BM (15 x 106) after 2 Gy TBI and treatment with CTLA4Ig and rapamycin. Tolerance was assessed by grafting MHC mismatched (B10.D2) skin and heart allografts. Donor-MHC type heart allografts (n ¼ 5, solid circle, *) and skin allografts (n ¼ 8, dotted line, triangle, 4) survived indefinitely. (B) Heart grafts were harvested at day 100 after transplantation. A representative B10.D2 (MHC mismatch) graft stained with H&E is shown (original magnification, 200). Histopathologic analysis of the heart grafts showed results comparable to those achieved in the fully mismatched setting, with only mild signs of rejection (C) Representative histology of B10.D2 skin grafts (upper panel; syngeneic control, lower panel) from CTLA4Ig/Rapa treated chimeras is shown. (H&E staining magnification x20; 105d post skin grafting). Skin grafts remained macroscopically intact for the length of follow up - with histopathological analysis revealing no signs of rejection - but were comparable to syngeneic controls. (D) Balb/c BM chimeras that rejected Balb/c skingrafts were re-challenged with either minor matched (B10.D2, n ¼ 5, solid circle, *) or fully mismatched (Balb/c, n ¼ 5, dotted line, open circle, ) skingrafts.

BMT regimens allowed engraftment of fully allogeneic BM omitted in several settings (13,21), CD40 blockade for the ﬁrst time without global destruction of the recipient remained a critical component of all reported regi- T cell repertoire (20,24). Subsequently, numerous related mens (23,24,50–52). To the best of our knowledge the protocols have been developed. While CTLA4Ig could be protocol of the present study is the ﬁrst to avoid global T cell

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68 CD40 in Chimerism-Based Tolerance

Figure 4: Chimeras induced by CTLA4Ig and rapamycin show T cell and humoral tolerance in vitro and demonstrate central and peripheral deletion of donor-reactive T cells. (A) Chimeras treated with CTLA4Ig/rapamycin BMT protocol showed specific hyporesponsiveness in vitro (20 weeks post-BMT). SIs were calculated by dividing the mean cpm from responses against recipient (white columns; B6), donor (light grey columns, Balb/c; dark grey columns, B10.D2), or third-party (black columns; SJL) cells by mean background cpm (normalized by self-reactivity). (B) We tested BMT recipients for the presence of anti-donor antibodies late after BMT (>3 months) by flow cytometric assessment of recipient serum binding to donor cells. Chimeras uniformly failed to develop detectable levels of anti-donor antibodies in serum, while antibodies against third-party antigens were evident. Sera reactivity with syngeneic (B6; dotted thin line), donor (Balb/c; black thick line) or third-party graft-specific (C3H; grey thick line) thymocytes is shown in representative mice. (C–D) Deletion of donor-reactive T cells in chimeras in comparison to na€ıve B6 mice is shown for full (Balb/c BM recipients, dark grey columns) and MHC mismatched donor/recipient combinations (B10.D2 BM recipients, light grey columns). Donor specific deletion of CD4þ T cells (assessed by multi-colour flow cytometry in selected mice) is shown for (C) peripheral blood (14 weeks post-BMT) (D) and splenocytes (SPL, 20 weeks BMT). (E) Intrathymic deletion indicating central tolerance was directly assessed in CD4 single-positive (SP) thymocytes (THY, 20 weeks post-BMT). White bars denote na€ıve B6 controls, black bars denote na€ıve Balb/c controls. (5 mice per group, data representative for multiple independent experiments; mean SD, * p < 0.05, ** p < 0.01, *** p < 0.001). Deletion was evident, but less pronounced, suggesting that both central and peripheral deletional and non-deletional tolerance mechanisms contribute to donor-specific tolerance in CTLA4Ig/rapa treated chimeras.

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69 Pilat et al depletion in allogeneic BMT without requiring anti-CD154/ Due to the current lack of available drugs blocking the CD40 anti-CD40 treatment. pathway, our aim was to develop an anti-CD154 mAb-free BMT protocol. mTOR inhibition through rapamycin has Despite the critical role of its blockade in experimental pleiotropic effects on effector or conventional T cells and BMT-based mixed chimerism protocols, the detailed Treg broadly modulating several arms of alloreactivity (58). function of CD40 in BM rejection and tolerization remains In the context of BMT rapamycin promotes BM engraft- incompletely understood. Previous studies reported con- ment (31,37,40,51,59). It also synergizes with belatacept in flicting results as to the mechanism of anti-CD154 mAb the clinical setting of renal transplantation (60). Combining with some evidence pointing to an effector function of the rapamycin and CTLA4Ig treatment, permanent BM engraft- mAb being required (11,13,53) and some not (12). Employ- ment was accomplished with a clinically feasible dose of ing CD154 deficient recipients our results demonstrate that fully mismatched BMC transplanted under non-myeloabla- the lack of CD154 on recipient cells is sufficient for allowing tive TBI. Long-term multilineage chimerism indicated stem BM engraftment if CTLA4Ig is administered. Thus, neither a cell engraftment. Donor-specific tolerance was evident to cell intrinsic CD154 signal nor target cell destruction but heart grafts and in in vitro assays (including lack of donor solely the blockade of CD40 signals is a critical mechanism specific Abs) and was associated with intra- and extra- of anti-CD154 mAb treatment. Results obtained with donor/ thymic deletion of donor-reactive T cells. Survival of donor recipient combinations lacking the CD40 receptor suggest skin grafts, however, was only prolonged, but not perma- that a CD40 costimulatory signal via either the direct or nent. Thus, distinct mechanisms seem to prevent donor indirect pathway alone is sufficient to trigger rejection of marrow rejection in this new protocol that do not donor BM. Consequently CD40 needs to be controlled in necessarily involve interruption of the CD40 pathway. both pathways to achieve BM engraftment. Rapa likely acts – at least in part – by promoting selective survival of regulatory CD4 T cells (43), which in turn Moreover, using a strain combination in which donor mice suppress CD8 cells. Moreover, Rapa was shown to lack CD40 we could demonstrate that donor BM lacking potently inhibt MHC class I-restricted CD8þ (and class II- CD40 causes rejection despite CTLA4Ig, indicating that the restricted CD4 þ) T cell-mediated graft rejection (61). indirect pathway of T cell activation is sufficient to prevent Importantly, it has recently been revealed that Rapa chimerism induction in this model. Importantly, CD28 has the potency to inhibit survival of allo-specific CD8 costimulatory blockade alone was not able to tolerize CD4þ T cells by DC modulation and subsequently altered T cells in this setting (CD40-/- ! WT þ anti-CD8), but could costimulation (44,62). tolerize CD8þ T cells leading to indefinite skin graft survival (CD40-/- ! WT þ anti-CD4). Thus, only CD8 cells faced with Skin-specific minor antigens were identified as responsible an impaired direct but intact indirect pathway of allorecog- for this state of ‘split tolerance’ by the use of B10.D2 skin nition can be controlled sufficiently by CTLA4Ig to allow grafts (that are MHC-matched to Balb/c but minor antigen engraftment and indefinite skin graft acceptance. CTLA4Ig matched to B6) that were accepted indefinitely in Balb/c together with CD4 T cell depletion leads to a state of split chimeras even when grafted after the rejection of Balb/c tolerance, as chimerism is successfully induced but skin skin. Split tolerance was also observed in other models grafts are rapidly rejected. The failure to induce donor- devoid of anti-CD154 mAb treatment (46,63). Moreover specific tolerance in this setting was not due to the CD8 cells have been shown to be responsible for split depletion of CD4 cells and the subsequent lack of tolerance in models that are based on regulatory tolerance CD4þCD25þ Tregs, as CD4 T cell depletion with donor mechanisms, leading to skin but not cardiac allograft BM lacking CD40 was sufficient to induce donor specific rejection (64). This is in line with data from our knock-out tolerance. experiments, suggesting that CD8 cells are responsible for this split tolerant state which leads to skin graft rejection in The use of CD40 deficient recipient mice led to abrogation the presence of CD40 costimulation via the direct pathway of chimerism despite CD4 or CD8 cell depletion, indicating of antigen presentation. Of note, as many chimerism that directly alloreactive CD4 or CD8 cells are sufficient to models reported include no minor mismatches (20,65–68) trigger BM rejection and cannot be controlled by CTLA4Ig. they would not be able to find this type of ‘split tolerance’ The response of the direct pathway of antigen recognition and need to be interpreted accordingly. Noteworthy, we might also be augmented in this setting as Tregs are mainly recently could show that BMT protocols employing anti- activated through the indirect pathway and might lose CD154 mAb (plus CTLA4Ig) are not able to reliably induce some of their regulatory potency (54,55). CD4þCD25þ complete immunological tolerance either. Cardiac allografts Tregs have been shown to be more potent in the prevention revealed signs of chronic rejection including chronic of allograft rejection if they are specific for both direct and allograft vasculopathy, which was not seen when Tregs indirect pathway of allorecognition (56), likewise CD8þ were additionally transferred at the time of BMT (69). Tregs are suggested to display alloantigen recognition via both pathways (57). Deficiency of properly activated donor- Recipients grafted with either fully mismatched or MHC specific Tregs might also be an important cause for the mismatched BM demonstrated indefinite survival of MHC failure of chimerism and tolerance induction in this setting. mismatched skin (and heart) allografts. Interestingly, fully

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70 CD40 in Chimerism-Based Tolerance mismatched skin grafts do not cause humoral responses 6. Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell can upon rejection, which is of particular interest as the be a temporal bridge between a CD4þ T-helper and a T-killer cell. humoral response towards minor antigens is clinically is Nature 1998; 393: 474–478. relevant (70,71). 7. Bourgeois C, Rocha B, Tanchot C. A role for CD40 expression on CD8þ T cells in the generation of CD8þ T cell memory. Science New York, NY 2002; 297: 2060–2063. In summary, our results identify a novel BMT protocol that 8. Liu D, Ferrer IR, Konomos M, Ford ML. Inhibition of CD8þ T cell- induces mixed chimerism and donor-specific tolerance to derived CD40 signals is necessary but not sufficient for Foxp3þ fully mismatched cardiac allografts using CTLA4Ig and induced regulatory T cell generation in vivo. J Immunol 2013; 191: mTOR inhibition, without T cell depleting agents or mAbs 1957–1964. targeting the CD40:CD154 pathway. This regimen might be 9. Larsen CP, Pearson TC. The CD40 pathway in allograft rejection, a safe and clinically testable approach for deliberate acceptance, and tolerance. Curr Opin Immunol 1997; 9: 641–647. induction of tolerance as well as for the treatment of 10. Johnson S, Zhan Y, Sutherland RM, et al. Selected Toll-like non-malignant hematopoietic disorders such as hemoglo- receptor ligands and viruses promote helper-independent cytotox- binopathies (72–74). ic T cell priming by upregulating CD40L on dendritic cells. Immunity 2009; 30: 218–227. 11. Blair PJ, Riley JL, Harlan DM, et al. CD40 ligand (CD154) triggers a Acknowledgments short-term CD4(þ) T cell activation response that results in secretion of immunomodulatory cytokines and apoptosis. J Exp Med 2000; 191: 651–660. This work was supported by the Austrian Science Fund (FWF, F2310, 12. 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Christoph Klaus designed and performed research, ana- Prevention of collagen-induced arthritis with an antibody to gp39, lyzed data, wrote manuscript, Nina Pilat designed and the ligand for CD40. Science New York, NY 1993; 261: 1328–1330. performed research, analyzed data, wrote manuscript, 16. Larsen CP, Elwood ET, Alexander DZ, et al. Long-term acceptance Christoph Schwarz performed research, Karin Hock per- of skin and cardiac allografts after blocking CD40 and CD28 formed research, Rupert Oberhuber performed research, pathways. Nature 1996; 381: 434–438. 17. Hancock WW, Sayegh MH, Zheng XG, Peach R, Linsley PS, Turka Elisabeth Schwaiger performed research, Martina Gat- LA. Costimulatory function and expression of CD40 ligand, CD80, tringer performed research, Haley Ramsey performed and CD86 in vascularized murine cardiac allograft rejection. 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60. Ferguson R, Grinyo J, Vincenti F, et al. Immunosuppression with 67. Fehr T, Haspot F, Mollov J, Chittenden M, Hogan T, Sykes M. belatacept-based, corticosteroid-avoiding regimens in de novo Alloreactive CD8 T cell tolerance requires recipient B cells, kidney transplant recipients. Am J Transplant 2011; 11: 66–76. dendritic cells, and MHC class II. J Immunol 2008; 181: 165–173. 61. Blazar BR, Taylor PA, Sehgal SN, Vallera DA. Rapamycin, a potent 68. Lucas CL, Workman CJ, Beyaz S, et al. LAG-3, TGF-beta, and cell- inhibitor of T-cell function, prevents graft rejection in murine intrinsic PD-1 inhibitory pathways contribute to CD8 but not CD4 T- recipients of allogeneic T-cell-depleted donor marrow. Blood 1994; cell tolerance induced by allogeneic BMT with anti-CD40L. Blood 83: 600–609. 2011; 117: 5532–5540. 62. Fischer RT, Turnquist HR, Wang Z, Beer-Stolz D, Thomson AW. 69. Pilat N, Farkas AM, Mahr B, et al. T-regulatory cell treatment Rapamycin-conditioned, alloantigen-pulsed myeloid dendritic cells prevents chronic rejection of heart allografts in a murine mixed present donor MHC class I/peptide via the semi-direct pathway chimerism model. J Heart Lung Transplant 2014; 33: 429–437. and inhibit survival of antigen-speciﬁc CD8(þ) T cells in vitro and in 70. Zhang Q, Reed EF. Non-MHC antigenic targets of the humoral vivo. Transpl Immunol 2011; 25: 20–26. immune response in transplantation. Curr Opin Immunol 2010; 22: 63. Luo B, Nanji SA, Schur CD, Pawlick RL, Anderson CC, Shapiro AM. 682–688. Robust tolerance to fully allogeneic islet transplants achieved by 71. Zou Y, Stastny P, Susal C, Dohler B, Opelz G. Antibodies against chimerism with minimal conditioning. Transplantation 2005; 80: MICA antigens and kidney-transplant rejection. N Engl J Med 370–377. 2007; 357: 1293–1300. 64. Tsang JY, Tanriver Y, Jiang S, et al. Conferring indirect 72. Kean LS, Durham MM, Adams AB, et al. A cure for murine sickle allospeciﬁcity on CD4 þ CD25þ Tregs by TCR gene transfer cell disease through stable mixed chimerism and tolerance favors transplantation tolerance in mice. J Clin Invest 2008; 118: induction after nonmyeloablative conditioning and major histo- 3619–3628. compatibility complex-mismatched bone marrow transplantation. 65. Sykes M. Mechanisms of transplantation tolerance in animals and Blood 2002; 99: 1840–1849. humans. Transplantation 2009; 87: (9 Suppl) S67–S69. 73. Kean LS, Manci EA, Perry J, et al. Chimerism and cure: 66. Fehr T, Wang S, Haspot F, et al. Rapid deletional peripheral CD8 hematologic and pathologic correction of murine sickle cell T cell tolerance induced by allogeneic bone marrow: Role of disease. Blood 2003; 102: 4582–4593. donor class II MHC and B cells. J Immunol 2008; 181: 4371– 74. Adams AB, Durham MM, Kean L, Shirasugi N. Ha J. Williams MA 4380. et al. 2001; 167: 1103–1111.

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73 2.3.1 Interlude Treatment with anti-CD154 mAb was very effective in murine models of BMT using minimal toxic conditioning, but is clinically unavailable. Therefore, a BMT protocol without blocking the CD40:CD154 pathway and without a global T-cell depletion was aimed. For understanding the mechanisms of the CD40:CD154 pathway in allorecognition, various murine combinations lacking the CD40 or CD154 receptor were employed. CD40 costimulatory signal triggered BM rejection via the direct or the indirect pathway and moreover, even in CD40 deficient recipients. BM was rejected directly by CD4+ and CD8+ T- cells despite treatment with CTLA4-Ig. Hence, a blockade of the CD40:CD154 signal for direct and indirect allorecognition or the lack of recipients CD154 signal is required for inducing chimerism and tolerance.

After combining a mTOR inhibitor (rapamycin), known for its various effects on different T- cell subsets, with CTLA4-Ig, permanent BM engraftment of a clinically feasible number of BMC after nonmyeloablative conditioning was facilitated. Nevertheless, this protocol led to a state of split tolerance as heart allografts were accepted additionally to donor BM but skin graft survival was only prolonged. This finding was confirmed by the long-term acceptance of MHC mismatched/minor antigen matched skin grafts leading to the suggestion that tissue specific antigens expressed by Balb/c skin grafts were able to trigger rejection in this protocol.

74 2.4 Fourth paper Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade

75 Hindawi Publishing Corporation Journal of Immunology Research Volume 2016, Article ID 8635721, 9 pages http://dx.doi.org/10.1155/2016/8635721

Research Article Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade

Sinda Bigenzahn,1 Ines Pree,1 Christoph Klaus,1 Nina Pilat,1 Benedikt Mahr,1 Elisabeth Schwaiger,1 Patrick Nierlich,1 Friedrich Wrba,2 and Thomas Wekerle1

1 Section of Transplantation Immunology, Department of Surgery, Medical University of Vienna, Waehringer Guertel 18, 1090 Vienna, Austria 2Institute of Clinical Pathology, Medical University of Vienna, Waehringer Guertel 18, 1090 Vienna, Austria

Correspondence should be addressed to Thomas Wekerle; [email protected]

Received 20 July 2016; Revised 27 September 2016; Accepted 10 October 2016

Academic Editor: Stuart Berzins

Copyright © 2016 Sinda Bigenzahn et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Mixed chimerism and tolerance can be successfully induced in rodents through allogeneic bone marrow transplantation (BMT) with costimulation blockade (CB), but varying success rates have been reported with distinct models and protocols. We therefore b investigated the impact of minor antigen disparities on the induction of mixed chimerism and tolerance. C57BL/6 (H2 )mice 7 received nonmyeloablative total body irradiation (3 Gy), costimulation blockade (anti-CD40L mAb and CTLA4Ig), and 2×10 d bone marrow cells (BMC) from either of three donor strains: Balb/c (H2 ) (MHC plus multiple minor histocompatibility antigen d a (mHAg) mismatched), B10.D2 (H2 )orB10.A(H2 ) (both MHC mismatched, but mHAg matched). Macrochimerism was followed over time by flow cytometry and tolerance was tested by skin grafting. 20 of 21 recipients of B10.D2 BMC but only 13 of 18 ofBalb/c BMC and 13 of 20 of B10.A BMC developed stable long-term multilineage chimerism (𝑝 < 0.05 for each donor strain versus B10.D2). Significantly superior donor skin graft survival was observed in successfully established long-term chimeras after mHAg matched BMT compared to mHAg mismatched BMT (𝑝 < 0.05). Both minor and major antigen disparities pose a substantial barrier for the induction of chimerism while the maintenance of tolerance after nonmyeloablative BMT and costimulation blockade is negatively influenced by minor antigen disparities.

1. Introduction [4–7]. The potential impact of mHAg has not been formally tested, however. Donor specific tolerance through mixed chimerism can be mHAg are polymorphic non-MHC proteins that are able achieved in various animal models by nonmyeloablative BMT to induce a T cell response due to allelic variations between and CB [1]. Success rates of chimerism and tolerance induc- donor and recipient [8]. mHAg play a prominent role in tion have typically been high when donor-recipient strain hematopoietic stem cell transplantation (HSCT) in many combinations were used which cross only MHC barriers regards [9]. Striking differences in engraftment of purified but share the same mHAg background [2, 3]. However, this hematopoietic stem cells and/or development and severity setting does not reflect the clinical situation where mHAg of GVHD depending on mHAg disparities were found in disparities exist universally. When switching to a different murine MHC-matched donor host strain combinations [10]. donor-recipient combination, which crosses MHC plus mul- In the human setting, disparities in mHAg increase not only tiple mHAg barriers (Balb/c → BL6), retrospective review of the rates of rejection and graft-versus-host-disease (GVHD) pooled results revealed that overall only approximately 75% of [11] but also the efficacy of graft-versus-leukemia-effects mice developed lasting chimerism and of those that became (GVL) [12]. Recently, mHAg have also been reported to play chimeric approximately 15% rejected donor type skin grafts an essential role in the persistence of donor chimerism [13].

76 2 Journal of Immunology Research

d mHAg also play a role in solid organ transplantation [14]. and a biotinylated antibody against H-2D (34-2-12, devel- In mice, skin allografts differing only in minor antigens are oped with phycoerythrin streptavidin) and irrelevant isotype rejected with the same pace as MHC disparate allografts [15]. controls. To analyze the expression of V𝛽-subunits staining Moreover, a single mHAg disparity was sufficient to induce was performed with FITC-conjugated antibodies against chronic rejection of cardiac allografts in a congenic mouse V𝛽8.1/2 and V𝛽11 and PE-conjugated antibodies against model [16]. In clinical studies of kidney transplantation only CD4. Propidium iodide (PI) staining was used to exclude limited and controversial data exist regarding the impact of dead cells. Mice were considered chimeric if they showed mHAg disparities on graft survival [17]. Humoral immunity detectable donor cells within the myeloid lineage plus at least to specific mHAg, such as antibodies to angiotensin type one lymphoid lineage. An Epics XL-MCL flow cytometer 1 receptor (AT1R) and endothelin type A receptor (ETAR), (Beckman Coulter, IL Alliance, Vienna, Austria) was used for have been shown to correlate with an increased incidence of acquisition and EXPO32 ADC Software, Applied Cytometry acute rejection and inferior long-term graft survival in kidney Systems, was used for analysis of flow cytometric data. and heart allografts [18]. However, so far the role of mHAg disparities in the induc- 2.5. Histological Staining. Four micrometer sections were cut tion and maintenance of tolerance through mixed chimerism from paraffin-embedded tissue fixed in 4.5% formalin (pH of has not been clearly defined. Therefore, we investigated 7.5), stained with hematoxylin-eosin and Giemsa according to different donor host strain combinations displaying MHC standard protocols, and analyzed by an experienced patholo- a d mismatches only (either H2 or H2 )orMHCplusmultiple gist in blinded fashion according to The Banff 2007 Working d mHAg mismatches (H2 ). Classification of Skin-Containing Composite Tissue Allograft Pathology [19].

2. Materials and Methods 2.6. Statistics. Atwo-tailedStudent’st-test was used for com- b d paring percentages of V𝛽-positive populations and levels of 2.1. Animals. Female C57BL/6 (BL6: H2 ), Balb/c (H2 ), and k chimerism within several cell lineages. The chi-square test C3H/N (H2 ) mice were obtained from Charles River Lab- d was used for comparing rates of chimeras between groups. oratories (Sulzfeld, Germany) and B10.D2 (H2 ) and B10.A a Skin graft survival was calculated according to the Kaplan- (H2 ) from The Jackson Laboratory (Bar Harbor, ME). All Meier product limit method and compared between groups mice were housed under specific pathogen free conditions by using the log-rank test. The Fisher Exact Test was used to and used between 8 and 10 weeks of age. All experiments were compare histologically categorized skin grafts [19] of different approved by the local review board of the Medical University donor groups. A 𝑝 value less than 0.05 was considered to be of Vienna and were performed in accordance with national statistically significant. and international guidelines of laboratory animal care. 3. Results 2.2. Conditioning Protocol and BMT. Age-matched female BL6 recipients underwent nonmyeloablative total body irra- 3.1. Induction of Stable Multilineage Chimerism through BMT diation (TBI, 3 Gy, d −1) prior to the intravenous injection of 7 Plus CB Is Impeded by mHAg Disparities. Three groups of approximately 2×10 unseparated bone marrow cells (BMC) mice were treated with a previously published nonmyeloab- b from Balb/c, B10.D2, or B10.A donors as previously described lative BMT protocol [4–6]. BL6 recipients (H2 ) received 7 [4–6]. Additionally mice were injected intraperitoneally with 2×10 BMC from different donor strains with mismatches of an anti-CD154 mAb (MR1; 1 mg d0) and hCTLA4Ig (0.5 mg MHC with/without additional mHAg mismatches after 3 Gy d+2). Anti-CD154 mAb was purchased from Bioexpress Inc. TBI (d −1) together with CB consisting of a single dose each (New Hampshire, USA) and hCTLA4Ig was generously proof anti-CD154mAb (1 mg MR1, d0) and CTLA4Ig (0.5 mg, d d vided by Bristol-Myers Squibb Pharmaceuticals (Princeton, d2). Balb/c (H2 ), B10.D2 (H2 ,samebackgroundasBL6), a New Jersey). and B10.A (H2 ,samebackgroundasBL6)micewereused as donors (Figure 1(a)) to investigate the potential influence 2.3. Skin Grafting. Full thickness tail skin from sacrificed of mHAg on top of the burden of MHC mismatch and the Balb/c, B10.D2, or B10.A mice, respectively (donor specific), influence of the specific MHC haplotype on the induction k a d and C3H/N (H2 ; 3rd party) was grafted 7 or 15 weeks after of long-term multilineage chimerism (H2 versus H2 ). In BMT. Recipient mice were anesthetized through intraperi- BL6 recipients of Balb/c BMC high levels of multilineage toneal injection of a mixture of ketamine (100 mg/kg) and chimerism (tested in CD4, CD8, B cells, and myeloid cells, xylazine (5 mg/kg) before attachment of skin grafts at the Figure 1(b)) were initially induced in 17 of 18 mice. At 16 weeks lateral thoracic wall. Skin grafts were visually inspected after BMT only 13 of 18 mice stayed chimeric (pooled data of thereafter at short intervals. Rejection was defined as less than two independent experiments). This result is consistent with 10% viable tissue. numerous previous experiments using this protocol showing that chimerism is lost in approximately 25% of recipients 2.4. Flow Cytometric (FCM) Analysis. Two-color FCM was over time. In contrast, 21 of 21 BL6 mice receiving BMC of used to distinguish donor and recipient cells of particular B10.D2 developed multilineage chimerism which remained lineages, by staining with fluorescein isothiocyanate- (FITC-) stableinallbutoneanimalovertime(𝑝 < 0.05 versus conjugated antibodies against CD4, CD8, B220, and MAC1 Balb/c after week 16 after BMT, Figure 1(c)). Interestingly

77 Journal of Immunology Research 3

(i) 3 Gy TBI (d−1) (ii) Anti-CD154mAb (d0, 1 mg) b (iii) CTLA4Ig (d2, 0.5 mg) C57BL/6 (H2 ) 7 b null (iv) BMC (d0, 2×10 ) Class I K and D - , L Class II A -b, E null

Donor strain Balb/c B10.D2 B10.A a d H2 Haplotype H2

Alleles Class I K, D, and L -d Class I K -k, D, and L -d Class II A and E -d Class II A and E -k

Mismatch versus C57BL/6 MHC plus MHC only MHC only

Multiple minor mm No minor mm No minor mm

(maj+min+) (maj+min−) (maj+min−)

(a) -2-12 -2-12 PI SSC 34 34

FSC FSC CD4 CD8 -2-12 -2-12 34 34

B220 Mac-1 (b) Rates of chimeras Donor strain Time point Balb/c B10.D2 B10.A p value versus B10.D2 w2 17/18 21/21 17/20 w6 15/18 20/21 16/20 ∗ ∗ w1613/18 20/21 13/20 ∗ ∗ ∗ w1913/18 20/21 13/20 p < 0.05

(c)

Figure 1: Continued.

78 4 Journal of Immunology Research

∗ ∗∗ 100 100

80 80 ∗ 60 60 ∗ 40 40

20 20 % donor CD4 cells % donor % donor CD8 cells % donor 0 0 w2 w6 w12 w19 w2 w6 w12 w19 ∗∗ ∗ ∗ ∗∗ ∗ ∗ ∗ ∗ 100 ∗∗ ∗ 100

80 ∗∗ 80

60 60

40 40 ∗∗ 20 20 % donor B220 cells % donor % donor MAC1 cells MAC1 % donor 0 0 w2 w6 w12 w19 w2 w6 w12 w19

Balb/c donors B10.A donors Balb/c donors B10.A donors B10.D2 donors B10.D2 donors (d)

100 CD4 100 CD8

80 80

60 60

40 40

20 20 % of CD8 donor cells CD8 donor % of % of CD4 donor cells CD4 donor % of 0 0 Balb/c B10.D2 B10.A Balb/c B10.D2 B10.A

B220 MAC1 100 100

80 80

60 60

40 40

20 20 % of Mac1 donor cells donor Mac1 % of % of B220 donor cells B220 donor % of 0 0 Balb/c B10.D2 B10.A Balb/c B10.D2 B10.A (e) Figure 1: mHAg disparities impede rates of chimerism achieved through BMT with CB. (a) depicts the BMTprotocolused with distinct donor-recipient combinations. (b) FACS plots show total donor (34-2-12 mAb recognizes H-Dd)and recipient type cells in CD4, CD8, B cells, and myeloid cells of one representative recipient of Balb/c BMC at the end of observation period.Dead cells were excluded through propidium iodide (PI) staining. (c) Rates of chimeras were determined at different time points afterM B T. Early multilineage chimerism was induced in almost all mice. Maintenance of chimerism was observed in all but one recipient mouse of MHC mismatched, but mHAg matched d B10.D2 (H2 ) bone marrow throughout the observation period. In contrast, chimerism rates dropped in mice, which were transplanted with d BMCofMHC and mHAg mismatched Balb/c (H2 ) mice over time similarly to mice, which receivedMHC mismatched but mHAg matched a B10.A (H2 )BMC(𝑝 < 0.05 for both versus B10.D2 BMC recipients after week 16).d ( ) Chimerism levels among CD4 cells, CD8 cells, B cells, and myeloid cells were measured by FCM at different time points afterM B T. All groups of recipients showed relatively similar chimerism levels among T cell lineages throughout the observation period. Significantly higher chimerism was observed in B cells among recipients of d MHC mismatched, but mHAg matched B10.D2 (H2 ) bone marrow compared to mice transplanted with MHC and mHAg mismatched Balb/c d ∗∗ (H2 )BMC( 𝑝 <0.01at all measured time points). Myeloid cell chimerism was significantly lower in recipients of MHC mismatched,but a d ∗ ∗∗ mHAg matched B10.A (H2 ) after week 6 after BMTcompared to recipients of Balb/c (H2 )BMC( 𝑝 <0.05and 𝑝 <0.01at indicated time points). Mean percent of chimerism, interquartile range (box), and SD (whiskers) of long-term chimeras are shown as box-and-whisker blots (representative data from 1 of 2 independent experiments). (e) Chimerism levels of long-term chimeras after transplantation of Balb/c BMC, B10.D2 BMC, or B10.A BMC, respectively, at the end of observation period.Individual percent, mean percent of chimerism, and SD (error bars) are shown as scatter plot (pooleddata of two independent experiments).

79 Journal of Immunology Research 5 the rate of chimeras also dropped in BL6 recipientsof B10.A B10.D2 and B10.A donor grafts in contrast to Balb/c grafts BMC from initially 17 of 20 immediately after BMTto13 (Figure 2(b)). Histological analysis of those donor grafts of 20 at week 16 (𝑝 < 0.05 versus B10.D2, Figure 1(c)). that were retained until the end of follow-up revealed that These data indicate that mHAg mismatches pose a barrier Balb/c donor grafts exhibited signs of chronic rejection, like to establishing long lasting multilineage chimerism through sparse infiltration with lymphocytes and mast cells together d BMTwithCB.Additionally the MHC haplotype (H2 versus with focally dense lymphocytic-mononuclear cell infiltration. a H2 ) also seems to influence bone marrow (BM) engraftment In comparison no incidence of dense focal lymphocytic with this CB-based nonmyeloablative protocol. infiltration was seen, in B10.D2 donor grafts (Figure 2(c)). Analyzing lineage-specific blood chimerism levels in suc- Skin grafts were scored by a blinded pathologist according cessful long-term chimeras it was noted that B cell chimerism to The Banff 2007 Working Classification of Skin-Containing was significantly higher in recipients of B10.D2 BMCcom- Composite Tissue Allograft Pathology [19]. Moderate (grade pared to recipients of Balb/c BMC at each investigated time 2; 2/3) and mild signs of inflammation (grade 1; 1/3) were point (e.g., B10.D2 versus Balb/c: 81.9% ±7.2versus 61.3% ± found in Balb/c grafts, whereas no signs of inflammation ∗∗ ∗ 9.4 at week 19, Figure 1(d), 𝑝 <0.01, 𝑝 <0.05), whereas (grade 0) were seen in 4/4 B10.D2 grafts (𝑝 < 0.05 versus CD8 and myeloid cell chimerism levels were comparable Balb/c donors) and 2/4 B10.A grafts (2/4 grade1,𝑝=n.s. between these two groups. CD4 chimerism was significantly versus Balb/c, Figure 2(d)). higher in recipients of Balb/c BMC at week 19 after BMT Taken together, these data suggest that mHAg disparities (B10.D2 versus Balb/c: 36.08% ± 14.53 versus 58.75% ± 12.13, increase the rate of chronic skin graft rejection in recipients ∗ Figure 1(d), 𝑝 <0.01). Significantly lower chimerism levels with persistent levels of mixed chimerism. were observed in myeloid lineages in B10.A BMC recipients compared to recipients of Balb/c BMC from week 6 on. No 3.3. Deletion of Donor-Reactive T Cells Differs among Donor consistent differences in T cell chimerism (CD4- and CD8 Strains. Peripheral and central clonal deletion are important cells) and B cell chimerism levels were observed between mechanisms for the induction and maintenance of tolerance long-term chimeras after BMT from B10.A or Balb/c donors. in models of mixed chimerism induced by BMTplusCB Nonetheless, CD4 and CD8 T cell chimerism levels were 25.56 ± 10.43 [20]. Notably not all mice become chimeric with such a significantly higher (B10.A versus Balb/c: CD4: protocol and not all chimeric mice accept donor skin grafts versus 13.51 ± 4.82 and CD8: 24.55 ± 7.83 versus 15.48 ± 𝛽 8.86 indefinitely. We followed V 11+ CD4+ T cells, which in ) in recipients of B10.A BMC 6 weeks after BMTbut this model recognize endogenous superantigens presented by declined below that of Balb/c BMC recipients by week 19 37.71±25.44 58.75±12.13 donor MHC II (I-E) but not recipient MHC class II and thus (B10.A versus Balb/c: CD4: versus , serve as surrogate markers for donor-alloreactive T cells. Like 𝑝 < 0.05 ). Chimerism levels of individual BMT recipients in previous experiments Balb/c long-term chimeras displayed are shown in Figure 1(e). The total level of donor chimerism markeddeletion of V𝛽11+ CD4+ T cells by week 4 after BMT among leukocytes was 62.41% versus 62.26% versus 53.41% in (2.04% ±0.80[𝑛=8]versus5.20% ±0.17in na¨ıve BL6 mice recipients of Balb/c versus B10.D2 versus B10.A bone marrow, [𝑛=2]). Compared to Balb/c chimeras, a similar degree of respectively (at 19 weeks after BMT). deletion was observed in B10.D2 chimeras (1.99% ±0.69[𝑛= a These results suggest that bothd in ividual MHC haplo- 11], 𝑝=n.s.). Interestingly, transplantation of B10.A (H2 , type and minor antigen disparities influence the degree of mHAg matched)bonemarrowled to a significantly more chimerism in distinct lineages. pronounced early deletion compared to Balb/c and B10.D2 (0.67% ± 1.62 [𝑛=9], 𝑝 < 0.05 versus Balb/c and B10.D2 3.2. mHAg Disparities Are a Hurdle for Induction of Donor donors, both lineages). In contrast irrelevant V𝛽8.1/2+ CD4+ Specific Tolerance. Specific skin graft acceptance is consid- T cells were not deleted in either group after BMT, indicating ered as a stringent test to indicate transplantation tolerance. the specificity of the deletion for superantigens presented by To investigate a possible influence of mHAg mismatches on the donor (Figure 3(a)). Ten weeks afterM B T the degree of skin graft acceptance, donor and 3rd party tail skin was deletion was significantly enhanced in long time chimeras transplanted 2-3 months after BMT. Among successfully after Balb/c and B10.D2 BMT(0.93% ±0.22[𝑛=8]and established chimeras long-term donor skin graft survival 1.26% ±0.27[𝑛=11], 𝑝 < 0.01 versus week 4 for both (>130 days) was observed in 10 of 13 recipients of Balb/c lineages) but still was significantly less pronounced than in BMC. This rate is similar to our previous experience with recipients of B10.A BMC(0.23% ±0.16[𝑛=9], 𝑝 < 0.01 this protocol [4, 6, 7]. In contrast, recipients of B10.D2 or versus Balb/c and B10.D2, both lineages; Figure 3(b)). Con- B10.A BMCshowed a significantly better long-term donor d k skin graft survival (B10.D2: 16/17, B10.A: 12/12, Figure 2(a), cluding, the type of MHC (i.e. I-E versus I-E ) may influence ∗ 𝑝 <0.05for Balb/c versus B10.D2 and B10.A donors; pooled the extent and kinetic of deletion of donor-reactive T cells. data of two independent experiments). However, in recipients of Balb/c BMC no significant difference in chimerism levels 4. Discussion of long-term chimeras, which rejecteddonor skin grafts in comparison to tolerant animals was observed (Figure 2(b)). In this studyweprovideevidence that mHAg disparities Regarding the macroscopic appearance of donor skin play a decisive role in the induction and maintenance of grafts of long-term chimeras, no shrinking, thickening, or tolerance in recipients conditioned with nonmyeloablative loss of surface aspect was observed in mHAg matched BMTand CB, in which mHAg both impede the engraftment

80 6 Journal of Immunology Research

∗ 100

40 % graft survival % graft 20

0 0 50 100 150 Days post skin grafting

Balb/c donors 3rd party B10.D2 + B10.A donors (a)

Week 2 Week 6 Week 19 25 100 100 20 80 80 15 60 60 10 40 40 % of donor cells donor % of % of donor cells donor % of 5 cells donor % of 20 20 0 0 0 CD4 CD8 B220 Mac1 CD4 CD8 B220 Mac1 CD4 CD8 B220 Mac1

Rejected Rejected Rejected Tolerant Tolerant Tolerant (b) mHAg matched mHAg mHAg mismatched mHAg

Balb/c donor B10.D2 donor B10.A donor (c)

Balb/c donor B10.D2 donor B10.A donor (d)

Figure 2: Continued.

81 Journal of Immunology Research 7

∗ 100

% of mice % of 40

0 Balb/c B10.D2 B10.A

Grade 4 Grade 1 Grade 3 Grade 0 Grade 2 (e)

b Figure 2: mHAg are a barrier for induction of donor specific tolerance through BMT plus CB. (a)2 BL6(H )recipientsofbonemarrowof d d a fully MHC mismatched Balb/c (H2 ,mHAgmismatched), B10.D2 (H2 ), or B10.A (H2 , both mHAg matched) mice were tested for donor specific tolerance through transplantation of donor type and 3rd party skin 7 or 14 weeks after BMT. Treatment with 3 Gy TBId ( −1): 7 transplantation of 2×10 BMC together with CB (1 mg anti-CD154mAb, d0; 0.5 mg CTLA4Ig, d2) leads to long lasting (>130 days) donor d skin graft acceptance in long-term chimeras, which had received BMC from MHC and mHAg mismatched Balb/c (H2 ) donors in 10 of 13 mice. Significantly better graft survival was observed in long-term chimeric recipients of MHC mismatched but mHAg identical bone ∗ marrow (28/29, B10.D2 and B10.A, 𝑝 <0.05;pooleddata from 2 independent experiments). Skin graft survival was calculated according ∗ to the Kaplan-Meier product limit method and compared by using the log-rank test. 𝑝 <0.05versus Balb/c donors. (b) Chimerism levels of long-term chimeras, which received Balb/c BMC were not significantly different in tolerant mice (circles I)compared to mice, which d rejected (squares ◼) donor skin grafts. 𝑝 = n.s. for all time points. (c) Macroscopical aspects of Balb/c (H2 , mHAg mismatched) donor skin grafts changedduring the observation period with grafts showing shrinking, thickening, and loss of surface structure. In comparison, donor d a grafts of B10.D22 (H )and B10.A (H2 , both mHAg matched)micestayed macroscopically unchanged from 7 days after skin grafting (when protecting bandage was removed) until the end of observation period.(d) Representative histology of donor skin grafts of Balb/c (left), B10.D2 (middle), and B10.A (right) 44 weeks after skin grafting. HE staining, magnification 160x, and Giemsa staining 160x (not shown) analyzed. (e) Classification of donor skin grafts 44 weeks after skin grafting according to The Banff 2007 Working Classification of Skin-Containing ∗ Composite Tissue Allograft Pathology [19] by a blinded expert pathologist. 𝑝 <0.05versus Balb/c. Most of Balb/c donor skin grafts showed histologically moderate (grade 2, 2/3) signs of inflammation whereas all B10.D2 donor skin grafts were free of inflammatory infiltratesd (gra e ∗ 0, 𝑛=4, 𝑝 <0.05). B10.A partially showed no (grade0,2/4)ormild signs of inflammation (grade1,2/4,𝑝=n.s. versus Balb/c).

of BM and promote the rejection of donor skin in successfully seems to be dispensable in fully mismatched combinations established mixed chimeras. (Balb/c to BL6) [25]. Alternatively, NKG2D can function as a Tolerance induction through donor BMT dependson costimulatory receptor to augment the response of na¨ıve CD8 two, mechanistically separate, events: successful engraftment T cells [26]. This could provide an alternative costimulatory of donor BM anddurable tolerization of all relevant donor route for CD8 T cells when CD40 and CD28 pathways antigens in the context of donor chimerism. The dfin ings are blocked. The role of NKG2D in CD8 T cell mediated presented herein indicate that mHAg influence both of these BM rejection has not yet been evaluated although it is well events. The frequency of successful long-term chimeras was established that H60 triggers an abnormally high number of higher in the absence of mHAg disparities when the MHC responding CD8 T cells [22]. haplotype was the same (13/18 for Balb/c versus 20/21 for Unexpectedly, significantly fewer mice developed long B10.D2). These results are in line with findings that MHC- lasting multilineage chimerism after receiving BM from matched BM is rejected in sublethally irradiated mice [21]. B10.A compared to B10.D2 donors (both mHAg matched Only some of the minor antigens that drive alloreac- to recipient). Since the only difference between B10.Ad an tivity in this strain combination (Balb/c to BL6) have been B10.D2 is the H2 haplotype these results suggest that the identified, with H60 provoking a particularly strong reaction MHC haplotype per se influences chimerism induction. Dis- [22]. H60 is a ligand for the activating receptor NKG2D tinct H2 haplotypes are known to stimulate varying numbers which is expressed on NK cells and on activated CD8 T of alloreactive T cells and thus exhibit a varying degree of cells, which are both effective mediators of allogeneic BM immunogenicity [27], which apparently also influences BM rejection. Intriguingly, Balb/c donor mice feature a higher rejection versus engraftment. Interestingly, if engraftment is surface expression of H60 than recipient BL6 mice [23]. successful, B10.A chimeras accepteddonor skin grafts to a NKG2D has been reported to enhance NK cell mediated comparable extent as B10.D2 chimeras without macroscop- BM rejection in semiallogeneic pairs (Balb/c to F1) [24] but ical and histological signs of chronic inflammation. Thus,

82 8 Journal of Immunology Research

V훽8.1/2 V훽11 V훽8.1/2 V훽11 25 6 25 6

5 5 20 20 4 4 15 15 3 3 10 10 ∗∗ 2 2 ∗∗

Mean% CD4+ PBL Mean%

Mean% CD4+ PBL Mean% 5 5 CD4+ PBL Mean% 1 ∗ 1 ∗

0 0 0 0 B6 B6 B6 B6 Balb/c Balb/c Balb/c Balb/c B10.A donors B10.A donors Balb/c donors Balb/c donors B10.A donors B10.A donors Balb/c donors Balb/c donors B10.D2 donors B10.D2 donors B10.D2 donors B10.D2 donors 4 weeks after BMT 4 weeks after BMT 10 weeks after BMT 10 weeks after BMT

(a) (b)

Figure 3: Deletion of donor-reactive T cells differs among donor strains. (a) Deletion of donor-reactive T cells was investigated through + + + determination of the percentage of V𝛽11 and V𝛽8.1/2 CD4 PBL by 2-color flow cytometric analysis 4 weeks after BMT. Deletion of + + d d V𝛽11 CD4 PBL in Balb/c (H2 ,mHAgmismatched, 𝑛=8)and B10.D2 (H2 ,mHAgmatched, 𝑛=11)BMCrecipientsdeveloped to a similar dimension irrespective of differing mHAg disparities of donor to recipient. In mice which were transplanted with BMC of B10.A a d (H2 ,mHAgmatched, 𝑛=9) a significant increase of early deletion compared to BMT of Balb/c (H2 ,mHAgmismatched)and B10.D2 d ∗ + + (H2 ,mHAgmatched) donors was observed ( 𝑝 <0.05versus Balb/c and B10.D2 BMC recipients). The percentage of V𝛽8 CD4 cells was not significantly reduced in any group compared to na¨ıve BL6 mice indicating the specificity of the deletion for superantigens presented by the donor. (b) Ten weeks after BMT the degree of deletion was significantly enhanced in long time chimeras after Balb/c and B10.D2 BMT + + + but still was significantly less pronounced than in recipients of B10.A BMC. Mean percentages of V𝛽11 and V𝛽8.1/2 CD4 PBL, interquartile range (box), and SD (whiskers) of long-term chimeras are shown as box-and-whisker blots. Representative data from one of two independent ∗ ∗∗ experiments. Statistical significance determined by log-rank test. 𝑝 <0.05versus Balb/c donors; 𝑝 <0.01versus week 4 after BMT (each group).

distinct MHC haplotypes impede BM engraftment to varying 5. Conclusion degrees but do not affect the success of skin graft tolerance in established mixed chimeras. This study reveals that mHAg disparities have a negative With regard to the second event, mHAg disparities impact on BM engraftment and tolerance maintenance in increased the rate of skin graft rejection in successfully a nonmyeloablative, CB-based chimerism model. Preclinical established chimeras (10/13 for Balb/c versus 28/29 for B10.D2 tolerance protocols should encompass mHAg disparities to and B10.A). In addition, the surviving skin grafts of Balb/c reflect the clinical settingd an need to induce mechanisms but not B10.D2 donors exhibited histological signs of chronic capable of durable tolerization of donor mHAg. inflammation. Thus, mHAg disparities can drive chronic rejection in the presence of stable mixed chimerism. Several Competing Interests groups attributed tissue specific antigens, which are not present in the BM but the skin, for this state of so-called The authors declare that there is no conflict of interests “split tolerance” [28]. Obviously, for clinical translation this regarding the publication of this paper. hurdle of mHAg disparities with its associated risk of “split tolerance” needs to be successfully overcome, which will Acknowledgments require specifically designed protocols. Recently, we could This work was supported by the Austrian Science Fund (FWF, demonstrate that the combination of regulatory cell therapy TRP151, and W1212 to Thomas Wekerle). with donor BM transplantation leads to a state of tolerance that encompasses donor mHAg [7]. Critically, this regimen References relies on extensive regulatory mechanisms, including linked suppression, that appear superior and indeed indispensable [1] N. Pilat and T. Wekerle, “Transplantation tolerance through for tolerization of donor mHAg (Pilat et al. JCI Insight 2016, mixed chimerism,” Nature Reviews Nephrology,vol.6,no.10, in press) pp. 594–605, 2010.

83 Journal of Immunology Research 9

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84 2.4.1 Interlude As different success rates and the appearance of the so called “split tolerance” in tolerance inducing protocols occurred, the role of minor antigen disparities in a costimulation based protocol using nonymeloablative conditioning was investigated. By employing MHC mismatched/minor histocompatibility antigen mismatched or MHC mismatched (fully or haploident)/minor histocompatibility antigen matched donor-recipient combinations, we studied their effect on the establishment of chimerism and skin graft tolerance in our murine model of BMT. We found out, that major and minor antigen disparities can impede the induction of mixed chimerism, whereas if chimerism is already established, minor antigen disparities can negatively affect allograft acceptance in an ongoing manner.

85 2.5 Review article

Hurdles to the induction of Tolerogenic Mixed Chimerism

86 © 2009 Lippincott Williams & Wilkins Pilat et al. S79 fection resulted in detectable serum levels of IFN-␤ and systemic infections in the clinic, with respect to their capacity to pro- injection of recombinant IFN-␤ was sufficient to prevent anti- mote transplant rejection. Future prospective clinical trials CD154ϩDST-mediated allograft acceptance. These findings should help determine whether specific types of bacterial in- may be clinically important, as type I IFN treatment can be fections can precipitate acute or chronic rejection. used to treat recurrence of hepatitis C or lupus flares in some patients, and thus, such treatment may have the capacity to REFERENCES promote acute allograft rejection in the clinic. 1. Taylor DK, Neujahr D, Turka LA. Heterologous immunity and homeo- Therefore, at least 2 pathways, MyD88 and type I IFN static proliferation as barriers to tolerance. Curr Opin Immunol 2004; 16: can independently oppose transplantation tolerance. It is 558. clear that not all bacteria induce production of type I IFNs 2. Adams AB, Pearson TC, Larsen CP. Heterologous immunity: An over- (our own unpublished observations), and whether additional looked barrier to tolerance. Immunol Rev 2003; 196: 147. 3. Pantenburg B, Heinzel F, Das L, et al. T cells primed by Leishmania pathways may interfere with transplantation tolerance re- major infection cross-react with alloantigens and alter the course of mains to be established. Of great clinical importance is the allograft rejection. J Immunol 2002; 169: 3686. question of whether bacterial or viral infections occurring 4. Welsh RM, Markees TG, Woda BA, et al. Virus-induced abrogation of after establishment of transplantation tolerance can reverse transplantation tolerance induced by donor-specific transfusion and anti-CD154 antibody. J Virol 2000; 74: 2210. established tolerance. Clearly, bacterial infections do occur in 5. Chen L, Wang T, Zhou P, et al. TLR engagement prevents transplanta- long-term stable transplant recipients (8), but prospective tion tolerance. Am J Transplant 2006; 6: 2282. clinical trials are lacking to determine whether this type of 6. Ahmed EM, Alegre ML, Chong A. The role of bacterial infections in infections can precipitate acute or chronic rejection. Like- allograft rejection. Exp Rev Clin Immunol 2008; 4: 281. wise, experimental models testing whether infections of any 7. Rubin RH. Temporal aspects of transplant infectious disease. Transpl Infect Dis 2003; 5: 63. kind can break established tolerance are lacking. Our unpub- 8. Fishman JA. Infection in solid-organ transplant recipients. N Engl lished data indicate that administration of CpG after cardiac J Med 2007; 357: 2601. or skin allografts are stably accepted is not sufficient to elicit 9. Walker WE, Nasr IW, Camirand G, et al. Absence of innate MyD88 signaling rejection. However, bacterial infections can induce several promotes inducible allograft acceptance. J Immunol 2006; 177: 5307. 10. McKay D, Shigeoka A, Rubinstein M, et al. Simultaneous deletion of simultaneous proinflammatory pathways that could cooper- MyD88 and Trif delays major histocompatibility and minor antigen ate to reverse established tolerance. mismatch allograft rejection. Eur J Immunol 2006; 36: 1994. 11. Tesar BM, Zhang J, Li Q, et al. TH1 immune responses to fully MHC CONCLUSIONS mismatched allografts are diminished in the absence of MyD88, a toll- like receptor signal adaptor protein. Am J Transplant 2004; 4: 1429. Our data indicate that TLR signaling and type I IFNR sig- 12. Goldstein DR, Tesar BM, Akira S, et al. Critical role of the Toll-like naling can both prevent the induction of transplantation toler- receptor signal adaptor protein MyD88 in acute allograft rejection. ance by costimulation-targeting therapies. It may be of interest J Clin Invest 2003; 111: 1571. to consider these pathways as therapeutic targets to facilitate 13. Thornley TB, Brehm MA, Markees TG, et al. TLR agonists abrogate transplantation tolerance to organs colonized by commensal costimulation blockade-induced prolongation of skin allografts. J Im- munol 2006; 176: 1561. bacteria. However, inhibiting such pathways should proceed 14. Porrett PM, Yuan X, LaRosa DF, et al. Mechanisms underlying block- with caution as such interventions may result in serious infec- ade of allograft acceptance by TLR ligands. J Immunol 2008; 181: 1692. tious complications. For instance, mice lacking IFN signaling are 15. Thornley TB, Phillips NE, Beaudette-Zlatanova BC, et al. Type 1 IFN exquisitely sensitive to viral infections that become rapidly lethal mediates cross-talk between innate and adaptive immunity that abrogates transplantation tolerance. J Immunol 2007; 179: 6620. (17). In addition, TLR signals have been described in some cases 16. Wang T, Chen L, Ahmed EM, et al. Prevention of allograft tolerance by to potentiate Treg function (18), although they can also inhibit bacterial infection with Listeria monocytogenes. J Immunol 2008; 180: 5991. regulation (19), such that antagonism of this pathway may pre- 17. Durbin JE, Hackenmiller R, Simon MC, et al. Targeted disruption of vent the induction of regulation, a tolerance mechanism impor- the mouse Stat1 gene results in compromised innate immunity to viral tant in both the induction and maintenance of tolerance by disease. Cell 1996; 84: 443. 18. Zanin-Zhorov A, Cahalon L, Tal G, et al. Heat shock protein 60 en- anti-CD154 (20). Elimination of bacterial load from colonized hances CD4ϩ CD25ϩ regulatory T cell function via innate TLR2 sig- donor organs may be an alternative approach to facilitate trans- naling. J Clin Invest 2006; 116: 2022. plantation tolerance, although the efficacy of this intervention 19. Pasare C, Medzhitov R. Toll pathway-dependent blockade of remains to be demonstrated in animal models. CD4ϩCD25ϩ T cell-mediated suppression by dendritic cells. Science 2003; 299: 1033. We conclude that TLR signals and bacterial infections 20. Lee I, Wang L, Wells AD, et al. Recruitment of Foxp3ϩ T regulatory can interfere with the induction of transplantation tolerance cells mediating allograft tolerance depends on the CCR4 chemokine and that closer attention may need to be paid to perioperative receptor. J Exp Med 2005; 201: 1037. Hurdles to the Induction of Tolerogenic Mixed Chimerism

Nina Pilat, Christoph Klaus, Elisabeth Schwaiger, and Thomas Wekerle

To date, organ transplant patients have to deal with the numerous side effects of life-long dependence on immunosuppressive drugs, whereas at the same time these drugs fail to prevent chronic rejection in many cases. Finding ways to

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establish donor-specific immunological tolerance thus remains one of the major goals in transplantation medicine. Tolerance through mixed chimerism can be achieved in rodents and in humans by the transplantation of hematopoietic stem cells. Widespread clinical application of this tolerance approach is, however, prevented by the toxicities of current bone marrow transplantation protocols in humans. Cytotoxic recipient conditioning and the hazard of graft-versus- host disease are unacceptable risks for organ transplant recipients. However, considerable progress has been made toward nontoxic conditioning regimens in animal studies. Translation of these findings into large animal models and the clinical setting is expected to be an important step toward broad clinical application of the mixed chimerism approach in organ transplantation. Keywords: Tolerance, Mixed chimerism, Organ transplantation. (Transplantation 2009;87: S79–S84)

HURDLES TO BE OVERCOME Specific Hurdles for the Mixed Chimerism Approach General Hurdles for Inducing Immunological Establishment of mixed chimerism by transplantation Tolerance of donor hematopoietic stem cells is a promising strategy for The development of new immunosuppressants has sig- inducing transplantation tolerance. Hematopoietic stem-cell nificantly improved short-time graft survival, but long-term transplantation was introduced as a novel approach to cancer outcome has changed little, being limited by chronic rejection, treatment approximately 50 years ago (5). Since then, much substantial morbidity and significant mortality. The induction progress has been made establishing stem-cell transplanta- of permanent donor-specific tolerance is a pressing unmet need tion as successful treatment of hematological malignancies in spite of five decades of intensive research in the field. and other life-threatening immunohematological disorders. In recent years, numerous factors have been identified, Morbidity and mortality after allogeneic stem-cell trans- which impede the induction of immunological tolerance and plantation have since been substantially reduced, especially explain—at least in part—the failure so far to achieve it in the through the development of reduced-intensity conditioning clinical setting (Fig. 1) (1). Among them, the clone size of allo- protocols, better prophylaxis against graft-versus-host disease reactive T cells is one of the most formidable barriers, requiring (GVHD), and novel antimicrobial drugs. Acute and chronic a substantial reduction (through depletion or deletion) of the GVHD and the toxicity of the host preparative treatment re- number of donor-reactive T cells before the remaining quan- main substantial hurdles of allogeneic stem-cell transplanta- tity of T cells can be rendered tolerant (2). Homeostatic pro- tion that prevent its use in a much wider group of indications liferation also markedly impedes tolerance induction and (6). With regard to toxicities of the conditioning protocols, is a problem occurring, for instance, after commonly used cytoreductive treatment in particular (i.e., myelosuppression T-cell–depleting induction therapy. A history of infections through irradiation or cytotoxic drugs; T-cell or B-cell deple- (heterologous immunity) and concomitant (viral) infections tion) is associated with serious medical risks. are additional hurdles. Finally, in the clinical setting, tolerance needs to be established in the face of a high frequency of The Clinical Experience With the Mixed memory cells (not existing to the same degree in “clean” Chimerism Approach for the Induction of mouse models). Memory T cells are rather resistant to toler- Tolerance ance induction and to T-cell–depleting antibodies, making Tolerance induction using mixed chimerism has been them an important obstacle in human tolerance (3, 4). On top tested in two prospective clinical series. In the first trial, highly of these immunological issues, the lack of reliable tolerance selected patients suffering from both end-stage renal failure and assays remains a critical logistical and ethical dilemma. These multiple myeloma simultaneously received bone marrow and a hurdles are relevant to all strategies of tolerance induction, kidney from a human leukocyte antigen-identical sibling (7). including mixed chimerism. Recipient conditioning consisted of cyclophosphamide, antithy- mocyte globulin, and thymic irradiation (plus a limited course of cyclosporine), leading to chimerism in all six of the six (6/6) patients, which was transient in four patients but turned into full chimerism in the other two patients. Three patients remain This work was supported in part by the Austrian Science Fund (FWF, SFB operationally tolerant without any immunosuppression after a F2310). Thomas Wekerle has received consulting income, honoraria, and travel reported follow-up of up to 7 years. Only one patient experi- grants in the past 3 years from Wyeth. He has also conducted research enced a rejection episode but immunosuppression treatment supported by or involving technology owned by the business Bristol was later successfully withdrawn. Notably, two patients devel- Myers Squibb. oped GVHD, both of them showing full chimerism. All other authors declare no potential conflicts of interest. Recently, the same group of investigators reported the Division of Transplantation, Department of Surgery, Vienna General Hos- pital, Medical University of Vienna, Vienna, Austria. successful induction of transient chimerism and tolerance in Address correspondence to: Thomas Wekerle, M.D., Division of Transplan- human leukocyte antigen-mismatched (haploidentical) living- tation, Department of Surgery, Vienna General Hospital, Waehringer related donor–recipient pairs with stable renal allograft function Guertel 18, 1090 Vienna, Austria. for up to 5.3 years after complete withdrawal of immunosup- E-mail: [email protected] Copyright © 2009 by Lippincott Williams & Wilkins pressive drugs in four recipients (8). This group of patients ISSN 0041-1337/09/0-80 did not suffer from concomitant indications for bone marrow DOI: 10.1097/TP.0b013e3181a2b9cc transplantation (BMT; such as multiple myeloma). Notably,

FIGURE 1. General and specific hurdles need to be overcome to induce tolerance through mixed chimerism. transient chimerism was achieved without the development The availability of clinically approved costimulation of GVHD. However, the conditioning regimen included pro- blockers is of great importance for routine clinical use of the found T-, B-, and natural killer (NK)-cell depletion and sub- mixed chimerism strategy (15, 16). Blockade of the CD154: stantial myelosuppression, leading to severe leukopenia CD40 pathway was the most promising approach in rodents. (nadir of absolute neutrophil counts [meanϮSD] 36Ϯ20/mm3) Moreover, anti-CD154 was able to enhance allograft survival and to capillary leak syndrome. These trials are ground- in non-human primate models, although development of allo- breaking in that they show in a prospective fashion that tol- antibodies was not inhibited (17). Unexpectedly, however, erance can be intentionally induced in the clinical setting. At anti-humanCD154 antibodies were associated with severe the same time, they are a clear reminder that the risk of thromboembolic complications (18) and had questionable GVHD and the toxicities of the recipient conditioning remain efficacy in the clinical setting. Clinical trials were put on hold, prohibitive obstacles for routine application. Hence, the de- and it is unlikely that an anti-CD40L monoclonal antibody velopment of minimally toxic BMT protocols remains a re- will become available in the near future. search priority. Blocking CD40 would be an alternative approach for targeting the CD154:CD40 pathway, and would probably PROGRESS IN THE DEVELOPMENT OF avoid thromboembolic complications. Several anti-CD40 an- MINIMUM-CONDITIONING BMT tibodies have been developed with some of them showing PROTOCOLS promising results in non-human primate models. One of Costimulation Blockade them has been shown to synergize with beletacept in prolong- Substantial progress toward nontoxic BMT regimens ing allograft survival (19). Thus, targeting of CD40 may be an has been achieved in mouse models over the last decade. alternative strategy for the development of clinically available Many of these advances have been systematically translated to therapeutic agents interfering with the CD154:CD40 co- large animal and non-human primate systems (9–11). stimulatory pathway; further investigation is required. The introduction of costimulation blockers as part of The fusion protein CTLA4Ig (abatacept) was designed BMT protocols allowed a drastic reduction of recipient con- to block the critical costimulation signal CD28. However, ditioning, leading to irradiation-free protocols which, how- CTLA4Ig had limited efficacy in non-human primate trans- ever, require mega doses of bone marrow (12, 13). Numerous plantation experiments, which was partly attributed to its low variations of such protocols have since been developed, all of avidity to CD86 resulting in incomplete blockade in vivo. them using anti-CD40L (CD154) monoclonal antibody with This led to the development of belatacept (formerly known as or without the fusion protein CTLA4Ig (14). Nevertheless, LEA29Y), a modified CTLA4Ig with higher affinity for B7 myelosuppression has remained a prerequisite for the en- (CD80 and CD86) target molecules and an improved in vivo graftment of clinically feasible doses of bone marrow (Fig. 2). immunosuppressive potency (20). Belatacept is currently be-

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FIGURE 2. A problematic relationship ex- ists between the clinical practicability of the transplanted bone marrow dose and the extent of the required recipient conditioning. The use of clinically feasible doses of bone marrow for the induction of mixed chimerism has so far required considerable cytoreductive conditioning of the recipient. New additional interventions have been developed that favorably shift this balance allowing the use of lower bone marrow doses with less toxic host conditioning. ing evaluated in several clinical trials as a replacement for protocols using transient immunosuppression would be ac- calcineurin inhibitors as the primary immunosuppressant. ceptable and indeed desirable for safety reasons, with the goal The results of a completed phase II study in renal transplan- of establishing drug-free tolerance thereafter. Studies investi- tation suggest that belatacept is as effective as cyclosporine A gating the compatibility of immuosuppressants revealed a in preventing acute rejection episodes (21). Thus, it is likely potent beneficial effect for rapamycin-based therapies, whereas that belatacept will become the first costimulation blocker calcineurin inhibitors were shown to prevent tolerance in- available for clinical use in organ transplant recipients. duction in these protocols (25, 26). Short-term treatment with rapamycin promotes the engraftment of allogeneic bone Peripheral Blood Stem Cells as a Stem-Cell Source marrow and allows reduction of total body irradiation at a In mouse models, mixed chimerism and tolerance can given dose of transplanted bone marrow or a reduction of the be induced with irradiation-free protocols by transplanting required number of BMCs in an irradiation-free setting (27). high doses of bone marrow cells (BMCs) (13, 22). Because Nevertheless, immunosuppression could not overcome the such high numbers of BMCs are clinically not available, a need for cytoreductive recipient treatment when clinically potential solution for this problem could be the use of gran- feasible doses of BMCs were transplanted. ulocyte colony-stimulating factor-mobilized peripheral blood stem cells (PBSCs). With this approach, the highest number Natural Killer Cells of hematopoietic stem cells can be obtained, however, sub- NK cells represent a potent barrier to BMC engraftment stantial biological and immunological differences between under limited recipient conditioning. The depletion of NK PBSCs and BMCs have been described. The frequency of pro- cells allows induction of chimerism with reduced numbers of genitor cells is lower in PBSCs than in bone marrow, but on a BMCs (28, 29). Recently, it has been found that NK-cell de- per-cell basis, progenitor cells contained in PBSC have a sim- pletion and short-course rapamycin treatment synergistically ilar engraftment capacity as those contained in bone marrow, promote bone marrow engraftment, leading to the induction leading to stable long-term chimerism in a CD45-congenic of tolerance in an irradiation-free (albeit cytotoxic) setting mouse model (23). Unexpectedly, allogeneic PBSCs fail to using feasible amounts of BMCs (Klaus, Wekerle et al., un- engraft in protocols based on T-cell depletion or costimula- published data). NK-cell alloreactivity seems to be crucial in tion blockade, which are otherwise successful when BMCs are costimulation blockade-resistant rejection, suggesting that its used. PBSCs even trigger the rejection of BMCs when co- inhibition may represent an important target in the clinical transplanted simultaneously. This detrimental effect was translation of tolerance-induction protocols. found to be mediated by the high frequency of mature donor T cells contained in PBSCs, which paradoxically trigger the rejection of donor hematopoietic cells. Hence, PBSCs are Indoleamine-2,3-dioxygenase more immunogenic and less tolerogenic in murine mixed As noted earlier, CTLA4Ig was designed to block the CD28 chimerism models and do not provide a straightforward so- pathway. Recently, CTLA4Ig and CTLA4-expressing regulatory lution for obtaining adequate cell numbers (24). T cells (Tregs) were proposed to act through an additional mechanism, namely the induction of indoleamine-2,3-dioxygenase, a Immunosuppression in Costimulation tryptophan-catabolizing enzyme impairing T-cell immunity Blockade-Based BMT Protocols (30, 31). Robust in vivo data in organ transplant models on the Conventional immunosuppressive drugs allow the re- role of indoleamine-2,3-dioxygenase are, however, still lacking. duction of recipient conditioning. For clinical application, In a nonmyeloablative BMT model, which critically depends on

CTLA4Ig, the activity of indoleamine-2,3-dioxygenase does not REFERENCES play a critical role in the induction of tolerance, as shown by 1. Ashton-Chess J, Brouard S, Soulillou J-P. Is clinical tolerance realistic blocking enzymatic activity in vivo (32). in the next decade? Transpl Int 2006; 19: 539. 2. Wells AD, Li XC, Li Y, et al. Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nature Med 1999; 5: Regulatory T Cells 1303. Central and peripheral clonal deletion are funda- 3. Lakkis FG, Sayegh MH. Memory T cells: A hurdle to immunologic tolerance. J Am Soc Nephrol 2003; 14: 2402. mental mechanisms underlying donor-specific tolerance 4. Golshayan D, Buhler L, Lechler RI, et al. From current immunosuppres- in murine mixed chimerism models. Because clone size of sive strategies to clinical tolerance of allografts. Transpl Int 2007; 20: 12. alloreactive T cells is one of the major hurdles to tolerance, 5. Appelbaum, Frederick R. Hematopoietic-cell transplantation at 50. peripheral deletion of preexisting, mature alloreactive T N Engl J Med 2007; 357: 1472. cells in the induction phase is of particular importance, 6. Copelan EA. Hematopoietic stem-cell transplantation. N Engl J Med 2006; 345: 1813. obviating the need for global T-cell depletion. In addition 7. Fudaba Y, Spitzer TR, Shaffer J, et al. Myeloma responses and tolerance to clonal deletion, nondeletional mechanisms contribute following combined kidney and nonmyeloablative marrow transplan- to tolerance in mixed chimeras. tation: In vivo and in vitro analyses. Am J Transplant 2006; 6: 2121. In a model using costimulation blockers and nonmy- 8. Kawai T, Cosimi AB, Spitzer TR, et al. HLA-mismatched renal trans- ϩ plantation without maintenance immunosuppression. N Engl J Med eloablative total body irradiation, early depletion of CD25 2008; 358: 353. cells or the neutralization of interleukin-2 prevented the in- 9. Kean LS, Adams AB, Strobert E, et al. Induction of chimerism in rhesus duction of tolerance after BMT. In contrast, CD25 depletion macaques through stem cell transplant and costimulation blockade- late after BMT did not interfere with established skin graft based immunosuppression. Am J Transplant 2007; 7: 320. tolerance in chimeras. These and other experiments suggest a 10. Kingsley CI, Nadig SN, Wood KJ. Transplantation tolerance: Lessons from experimental rodent models. Transpl Int 2007; 20: 828. role for Tregs in the induction phase but not for the mainte- 11. Kean LS, Gangappa S, Pearson TC, et al. Transplant tolerance in non- nance of tolerance in this particular BMT model (33). The human primates: Progress, current challenges and unmet needs. Am J extent to which regulation contributes to tolerance differs Transplant 2006; 6: 884. from model to model, depending on the specific BMT regi- 12. Wekerle T, Sayegh MH, Hill J, et al. Extrathymic T cell deletion and men used. Regulatory mechanisms seem to play a more pro- allogeneic stem cell engraftment induced with costimulatory blockade is followed by central T cell tolerance. J Exp Med 1998; 187: 2037. nounced role in minimum conditioning regimens, displaying 13. Wekerle T, Kurtz J, Ito H, et al. Allogeneic bone marrow transplanta- lower levels of chimerism (34). tion with co-stimulatory blockade induces macrochimerism and toler- Given the role of Tregs in mixed chimerism models, ance without cytoreductive host treatment. Nature Med 2000; 6: 464. their therapeutic use in such models would be of interest. 14. Pree I, Pilat N, Wekerle T. Recent progress in tolerance induction Tregs have potent effects in allotransplantation models, but through mixed chimerism. Int Arch Allergy Immunol 2007; 144: 254. 15. Snanoudj R, de Preneuf H, Creput C, et al. Costimulation blockade and its do not induce tolerance across major histocompatibility possible future use in clinical transplantation. Transpl Int 2006; 19: 693. complex barriers on their own. In BMT models, Tregs have 16. Pree I, Wekerle T. New appproaches to prevent transplant rejection: been reported to reduce the risk of GVHD while preserving Co-stimulation blockers anti-CD40L and CTLA4Ig. Drug Discov Today beneficial graft-versus-tumor effects (35). Tregs also inhibit Ther Strateg 2006; 3: 41. T-cell–mediated rejection of allogeneic bone marrow in a 17. Kirk AD, Burkly LC, Batty DS, et al. Treatment with humanized monoclonal antibody against CD154 prevents acute renal allograft rejection myeloablative system (36) and promote engraftment of allo- in nonhuman primates. Nat Med 1999; 5: 686. geneic bone marrow (37). In the latter model, full chimerism 18. Sidiropoulos PI, Boumpas DT. Lessons learned from anti-CD40L treat- was induced by Treg therapy using 5 Gy total body irradia- ment in systemic lupus erythematosus patients. Lupus 2004; 13: 391. tion, a dose of irradiation considered unacceptable by most 19. Adams AB, Shirasugi N, Jones TR, et al. Development of a chimeric anti-CD40 monoclonal antibody that synergizes with LEA29Y to pro- clinicians for organ transplant recipients. long islet allograft survival. J Immunol 2005; 174: 542. 20. Larsen CP, Pearson TC, Adams AB, et al. Rational development of LEA29Y (belatacept), a high-affinity variant of CTLA4-Ig with potent CONCLUSIONS AND PERSPECTIVES immunosuppressive properties. Am J Transplant 2005; 5: 443. 21. Vincenti F, Larsen C, Durrbach A, et al. Costimulation blockade with The concept of mixed chimerism leading to long-lasting belatacept in renal transplantation. N Engl J Med 2005; 353: 770. tolerance is successful in mouse models and in humans. Co- 22. Durham MM, Bingaman AW, Adams AB, et al. Cutting edge: Administra- stimulation blockers have allowed the development of less tion of anti-CD40 ligand and donor bone marrow leads to hematopoietic toxic BMT protocols using feasible numbers of BMCs. Tox- chimerism and donor-specific tolerance without cytoreductive condi- icity of current clinically tested BMT protocols, however, is tioning. J Immunol 2000; 165: 1. 23. Koporc Z, Bigenzahn S, Blaha P, et al. Induction of mixed chimerism still unacceptable for widespread application in organ trans- through transplantation of CD45-congenic mobilized peripheral blood plant patients. Recent results from mouse experiments are stem cells after nonmyeloablative irradiation. Biol Blood Marrow encouraging in that they suggest that strategies devoid of cy- Transplant 2006; 12: 284. totoxic recipient treatment might become available. Transla- 24. Koporc Z, Pilat N, Nierlich P, et al. Murine mobilized peripheral blood tion of these protocols into the clinical setting could lead us stem cells have a lower capacity than bone marrow to induce mixed chimerism and tolerance. Am J Transplant 2008; 8: 2025. closer to the major goal in transplantation research, the in- 25. Blaha P, Bigenzahn S, Koporc Z, et al. The influence of immunosup- duction of “true” tolerance. pressive drugs on tolerance induction through bone marrow transplantation with costimulation blockade. Blood 2003; 101: 2886. 26. Taylor PA, Lees CJ, Wilson JM, et al. Combined effects of calcineurin ACKNOWLEDGMENTS inhibitors or sirolimus with anti-CD40L mAb on alloengraftment under nonmyeloablative conditions. Blood 2002; 100: 3400. The authors thank Ulrike Baranyi for critical reading of 27. Blaha P, Bigenzahn S, Koporc Z, et al. Short-term immunosuppression the manuscript, and Liz Hablit for secretarial assistance. facilitates induction of mixed chimerism and tolerance after bone mar-

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row transplantation without cytoreductive conditioning. Transplanta- erance mechanisms in a murine model of mixed chimerism with co- tion 2005; 80: 237. stimulation blockade. Am J Transplant 2005; 5: 1237. 28. Kean LS, Hamby K, Koehn B, et al. NK cells mediate costimulation 34. Domenig C, Sanchez-Fueyo A, Kurtz J, et al. Roles of deletion and blockade-resistant rejection of allogeneic stem cells during nonmyeloa- regulation in creating mixed chimerism and allograft tolerance us- blative transplantation. Am J Transplant 2006; 6: 292. ing a nonlymphoablative irradiation-free protocol. J Immunol 2005; 29. Westerhuis G, Maas WGE, Willemze R, et al. Long-term mixed chimerism 175: 51. after immunologic conditioning and MHC-mismatched stem-cell trans- 35. Edinger M HP, Hoffmann P, Ermann J, et al. CD4ϩCD25ϩ regula- plantation is dependent on NK-cell tolerance. Blood 2005; 106: 2215. tory T cells preserve graft-versus-tumor activity while inhibiting 30. Finger EB, Bluestone JA. When ligand becomes receptor—Tolerance graft-versus-host disease after bone marrow transplantation. Na- via B7 signaling on DCs. Nat Immunol 2002; 3: 1056. ture Med 2003; 9: 1144. 31. Hainz U, Ju¨rgens B, Heitger A. The role of indoleamine 2,3-dioxygenase in 36. Joffre O, Gorsse N, Romagnoli P, et al. Induction of antigen-specific transplantation. Transpl Int 2007; 20: 118. tolerance to bone marrow allografts with CD4ϩCD25ϩ T lymphocytes. 32. Pree I, Bigenzahn S, Fuchs D, et al. CTLA4Ig promotes the induction of Blood 2004; 103: 4216. hematopoietic chimerism and tolerance independently of indoleamine- 37. Joffre O, Santolaria T, Calise D, et al. Prevention of acute and chronic 2,3-dioxygenase (IDO). Transplantation 2007; 83: 663. allograft rejection with CD4ϩCD25ϩFoxp3ϩ regulatory T lympho- 33. Bigenzahn S, Blaha P, Koporc Z, et al. The role of non-deletional tol- cytes. Nat Med 2008; 14: 88.

TIM-1: A New Player in Transplant Immunity

Christophe Mariat,1,2 Nicolas Degauque,1,3 and Terry B. Strom1,4

Data have recently pointed to a possible regulatory function of T-cell immunoglobulin mucin protein 1 (TIM-1) during the alloimmune response. Cross-linking TIM-1 onto CD4ϩ T cells intensifies proinflammatory cytopathic allogeneic response and inhibits the development of natural and inducible regulatory T cells. The TIM-1 pathway might thus be a potential hurdle to the induction of tolerance in transplantation. Not unexpectedly, blocking the TIM-1 pathway is now emerging as a successful strategy to promote experimental peripheral tolerance. Keywords: T cell immunoglobulin mucin protein 1 (TIM-1), Allo-immunity, Transplant tolerance. (Transplantation 2009;87: S84–S86)

THE T-CELL IMMUNOGLOBULIN MUCIN teins (1). TIM gene family members reside in syntenic PROTEINS FAMILY chromosomal regions, 5q32.2 in human and 11B1.1 in The T-cell immunoglobulin mucin (TIM) proteins mouse, which has been linked to both atopy and autoimmune diseases, thereby suggesting that these proteins represent a newly discovered family of molecules that act ϩ in concert with T-cell receptor and costimulatory mole- might modulate CD4 T-cell response. cules to regulate the expansion and effector functions of A role for TIM-3 and TIM-2 in gating the Th1 and Th2 T-helper cells. The TIM proteins are type I membrane gly- compartments has been described (2–5). TIM-3 is predomi- coproteins expressed on T cells and containing common nantly expressed by Th1 cells (TIM-1 is considered as a specific structural motives, namely IgV domain, glycosylated mu- surface marker of terminally differentiated Th1 cells) and deliv- cin domain, and cytoplasmic domain. The mouse gene ers a death signal and, therefore, leads to the inhibition of aggres- family includes eight members (encoding TIM-1, TIM-2, sive Th1-mediated autoimmune and alloimmune responses. TIM-3, and TIM-4 proteins and the putative TIM-5 to TIM-2 which is in contrast specifically up-regulated in Th2 cells, TIM-8 proteins), whereas the human gene family includes mirrors the effect of TIM-3 on Th1 cell by providing an inhibi- three members encoding TIM-1, TIM-3, and TIM-4 pro- tory, albeit not death, signal on Th-2 cell. TIM-4 occupies a special place within the TIM family. Unlike the other members, TIM-4 is expressed on antigen C. Mariat and N. Deagauque contributed equally to this work and are co-first authors. presenting cells, not T cells, and has been identified as a nat- Terry B. Strom has filed patent applications regarding TIM-1 in the past three ural ligand for TIM-1 (6). years. TIM-1 was initially believed to be the human ortho- Nicolas Degauque and Christophe Mariat declare no potential conflicts of logue of TIM-2 and as such, to predominantly govern the interest. Th-2 axis. However, its immune function does not exactly fit 1 The Transplant Institute, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA. into the Th-1/Th-2 dichotomy and has remained rather elu- 2 Service de Ne´phrologie, Dialyse et Transplantation Re´nale, CHU de Saint- sive until recently. Etienne, Universite´ Jean Monnet, Saint-Etienne, France. 3 INSERM U643, Hoˆtel-Dieu, Universite´ de Nantes, Nantes, France. 4 Address correspondence to: Terry B. Strom, MD, The Transplant Institute, THE EMERGING ROLE OF TIM-1 IN T Beth Israel Deaconess Medical Center, 330 Brookline Avenue-E/CLS CELL DEPENDENT IMMUNE RESPONSE Room 608, Boston, MA 02215. TIM-1 was first described as KIM-1 (kidney injury E-mail: [email protected] Copyright © 2009 by Lippincott Williams & Wilkins molecule 1), an epithelial cell adhesion glycoprotein, which is ISSN 0041-1337/09/8709S-84 up-regulated by proximal tubules in postischemic kidney (7). DOI: 10.1097/TP.0b013e3181a2ba83 Rat KIM-1 gene exhibits a high homology of sequence with a

92 2.5.1 Interlude As short-term survival of transplanted organs has significantly improved in the last decades, long-term survival is still limited due to chronic allograft rejection and various side effects of current immunosuppressive therapies. Thereby, the induction of donor specific tolerance remains promising, though its widespread clinical implementation is still impeded. This review gives an overview on the hurdles existing for a clinical translation of tolerance inducing protocols.

93 CHAPTER THREE: DISCUSSION

3.1 General discussion %&(%*(%)'#%** &%1* %+* &%&&%&()' $$+%&#& *&#(%-&+###&- * - * (-#& $$+%&)+''()) &%- * &+*%/) %)&(!* &%%&(* *1('()%*) '(&$ ) % )*(*/ &( '(&#&% *(%)'#%* % '* %* )+(, ,#3 * * ) '& %*1 * )*# ) $%* & &%&( )' *&#(% * (&+ $ . $*&'& * $() $ ) #(/%# % ##/*(%)#*+*%)'() )*%*()( -&("&($&(- )'( # % # $'#$%** &%3=BD1=B?1>AE1=B@1=BC1>B< ## %&-1 * *&. */ & +((%* &% * &% % '(&*&&#)1* # $ *%+$()& ), ##%* &$'#. +$% $$+%)/)*$ %#+ %$$&(/6##)1 &$&)** '(&# (* &%%&%&()' %* & )7 % %&%6 %* & )8($ %) ##% %&(* # % # $'#$%** &%3 * (&( $&()*(*/* *-&+###&-+(* ((+* &%&* &% * &% % *&. */&(. )* %'(&*&&#)&(( % %)+ '(&*&&#) %&(%*(%)'#%** &%#&)( *&* # % #'(* 3 % * &% ) '(&,% % # &%(% % % % % %(*$%* & ) % &%% 1.%&% &(##&% $&#)& - * * ($/#&#* ,&% * &% % >>E1>B=1>?@&(*('('( % $$+%& %*( ' %*)- * %&%6$/#&#* ,&% * &% % >?B1>?C1>?D&( %%##&% 7+##/ $ )$* 8 %+*(&*(%)'#%** &%$&#3>?A$&% * %+$(&+)*)* * ,%**( +**&>B% *)%0/$* * , */7 81 )$)*&'#/$!&('(* % &$ %%*( " %& )*&* &%$((&-% 3 ) % #, * &$ % *&( 6=1 .'()) % * &% $((&- $ (&%, (&%$%*1>>B* * %)*&@1.'())&% )1>?= *(+)*( " %&* )*$##)*&* &%$((&-3>>E &* )*&&+("%&-#1 * )%,(% %,)* *&(1- * (% %*((% & * 6=5@ . ) / % * &% -&+# ,%*&+) % % .'( $%*# )** % & *&#(% %+* &% , $ . $( )$ +) % $ % $# *&. &% * &% % % # % #) #%+$()&+%)'(*##&% &()/%% )3 / +) % '(&* % &( ) *# '* % 7''(&, / * % >< &+() * * %(#/

94 &$'#*#/#&" %0/$* * , */** $&*(%)'#%** &%3 (/1) *# '* % '(&,)+'( &(*& '(&* %+)&(* )$ % * &%1#* &+ '(&* % )#(/ % %* %&* ($+( %$&#)&3>>E1>?C (%*#/*&* '+# ) *&(1- (%% %%(*$%*&,%# $ * %+$()&?@F##)&(6=F# %6##)-)$&%)*(*/ % * %( ' %*) * , */1>?C1>>E--(%&*#*&)%/&* &)'(&$ ) %()+#*)3 ()&%&(* * $ *1* * - *#+)$+( %$&#- * 5 $ 1- (%/##&(* , */ -)#))%%$ -( * &%##/&% * &%- * % 6##'#* &%3*( &% * &% %1* &)$ -(*(%)'#%*- * )&(* +$%?@F##)7=.=B% %)&# % * &%&?@F##)- * '(&* % -)%&*)+ %*%&+ 3>?C * (&( )- ) $ #( #,#) & $ . $*&'& * $( )$1 ##) -( * ( %+*- * '(&* %'( &(*(%)'#%** &%&(( ' %*)-( * &%##/*(* - * '(&* %&(,% ( ' %*4) %0/$* * , */-) $ % ) /% *(*$%* - * ) *# '* %3 &(&,(1 % &+( )/%% $&#1 * )% & ##&(* , */>B>)$*&+% #'+#&(% % %%(*$%**( % * &%3 %1'&*%* # %*(* &%&* ''# &)* $+#* &%#&"- * % * &% ) +%# "#/*&()'&%) #&(* $ )) %% #*&%)*$##%(*$%* %&+( ##&% $+( %$&#&3 &%&$(/&, * # $&%)*(* &% * &% % ( ,% +'(+#* &% & 6= % * &% $((&-+'&%$ %%*)1# " (( * &%&( $&* ('/3>B? )#)*&* )+)* &%* *% (( * &%( ,%% %$%*&6=α%6=β>B@$/(+ /$ % $##/*&. &% * &% %'(&*&&#)+) %=/ * *'&)) #/$ % $ 0)* *& % * &% %&+($+( %$&#&3

95 3.2 Conclusion & future perspectives %$/-&("- ,$&%)*(** *& % * &% %$+( %$&#&$ . $( )$- %&%#/# $ *%+$(&)-(*(%)'#%**(%&%6$/#&#* , &% * &% % +) % = / 3 ,(* #))1 % %*((% & * 6=5@ . )1 / #&" %* %0/$* #,&6=1)$* (/%&**& ,'&*%*(&# % &$ % & )3 &##&- %1* &+)&(*&. */(+* &%&. )* %'(&*&&#)+) %# % ##/, ## %+$()&) %*&#(% %+* &%# )&%)*(* )* *($&('(&$ ) %3 %1 % '(* +#(* * ,* &%&( ' %*)4%*+(#()+*#)&*(*$%*- * 6β %+ () &( ? *(%)+ () ) &- .'* &%# ()+#*) % * .'( $%*# )** %1 # %*&'(&*&&#),& &%//*&(+* ,&% * &% %3AA1=AC&(&,(1'(&*&&#) %,&#, % ( * ('/ ) &- )+'( &( */ &%(% % * $ %*%% & *&#(% % )% & (&% (!* &% % %&%$/#&#* , &% * &% ( ' %*)1 )' ##/ - % $ %&( %* % )'( * ) -( %,&#,3A@1=?C ) ) & )' # %*()* &( * # % # *(%)#* &% ) +$%) '()%* #( ,( */ & * ))+ .'()) % %* %) % (&% (!* &%)* ##('()%*)&%&* # %+))&('($*+((*#&))3 %+'&$ %)*(*/ )* ,#&'$%*&#6>5#6 % *&(6C?C36C?C %+) '&'*&) ), * %*( %) '* -/ %##&(* ,6##)%* (&(##&- %$+( % $&# & +) % &)* $+#* &% #&" * ,& % & /*&*&. &% * &% %3 * &%##/1 %* )'(&*&&#* %* 6*&#(&% *&/#&)'&( %-)(,()% $ . $( )$%*&#(%-) %+(&))+## (( (3>BA&(&,(16C?C -),%)+))+# %* )*# ) $%*&$ . $( )$%&%&()' *&#(% $'#&/ % $$&(/ 6##) % $+( % $&# & +) % &)* $+#* &% #&" 7%* =A@ $8 % &%&( )' *(%)+) &%3=AA % * ) )** % )*(*/ -) &+% - '(* +#(#/ *(*) $$&(/ 6##)1 ## '&'+#* &% "%&-% *& (#/ *&#( 0 % * (&(('()%*)$!&((( (*&* - )'(# % #''# * &%&)+ '(&*&&#)3 & )+$ +'1 % * #)* /()1 *($%&+) &(*) , % $ *& ,#&' *&#(% %+ %'(&*&&#)&('(&#&% %'* %*%(*)+(, ,#3,(* #))1% %*((%& * 6=5@. ) %&*)$%&**& ,) % %** %&+($+( %$&#& 1&%+((%*%-'(&$ ) %* ('+* &'* &%),&#,3()1%-) %%* & )

96 %$ $* )&( (%*&$ %* &%)& $$+%&)+''())%*)- * &)* $+#* &%#&" , #/'&) * ,'()'* ,* *)'(&*&&#)&(*&#(% %+* &% %)&# &(% *(%)'#%** &%$ *)+ *#&(- (# % #''# * &%&%/3

97 CHAPTER FOUR: MATERIAL & METHODS

4.1 Animals

Female C57BL6/6NCrl (H-2b, CD45.2), C3H/N (H-2k) and Balb/c (H-2d) were purchased from Charles River Laboratories (Germany). Female B6SJL-Ptprca Pep3b / BoyJ mice (H-2b, CD45.1, mentioned as CD45.1 B6) were purchased from the Jackson Laboratory (Bar Harbour, USA). Mice were housed under pathogen-free conditions and used between 8 and 12 weeks of age. All experiments were approved by the local review board of the Medical University of Vienna and performed in accordance with national and international guidelines of laboratory animal care.

4.2 Bone marrow transplantation protocol

For the congenic murine model, CD45.1 B6 mice were used as recipients and CD45.2 B6 mice as donors. For the allogeneic murine model, B6 mice were used as recipients, Balb/c mice were used as donors and C3H mice were used for transplantation of third party skin. Recipients were conditioned with 1 Gy of TBI prior transplantation at day -1. Either 10x106 or 15x106 unseparated congenic or allogeneic BMCs were transplanted at day 0 (see Table 3 below). Costimulation blockade was administered in allogeneic recipients at day 0 (anti-CD154 monoclonal antibody, MR1 at a dose of 1mg) and at day 2 (hCTLA4-Ig, abatacept at a dose of 0.5mg).151 Anti-CD154 monoclonal antibody was purchased from BioXCell (West Lebanon, USA), hCTLA4-Ig (abatacept) was provided by Bristol-Myers Squibb Pharmaceuticals (Princeton, USA) Treatment protocols concerning DPPIV inhibition were performed as listed in the Table 3 and described in detail below. Groups of mice received either Diprotin A or sitagliptin or served as controls without DPPIV inhibition. BM was pretreated with Diprotin A prior transplantation in vitro (group B, Table 3); BM was pretreated prior transplantation with Diprotin A (5mM) in vitro and additionally recipients received in vivo treatment with Diprotin A (4µM) (group D, Table 3); recipients were treated with sitagliptin (4mg/mouse/2x/day) in vivo (group E and G, Table 3). Controls did not receive any DPPIV inhibition (group A, C and F, Table 3).

Table 1. Experimental protocols

Group TBI HSCT (cells/mouse) CB Additional treatment Mouse strain

Table 3: Different protocols used for DPPIV inhibition

Dipeptidyl peptidase IV (DPPIV/CD26) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning (© 2012 Schwaiger E. et al, Experimental Hematology)

4.3 DPPIV Inhibition

BM of donor mice was harvested, washed and resuspended in bone marrow medium consisting of M199 (Sigma M 4530), DNAse (Sigma D-4527), gentamycin and HEPES (1M) as described.151 Control groups (A, C and F, Table 3) received BM in medium without DPPIV inhibition. In vitro pretreatment of donor BM with Diprotin A was performed by an incubation with 5mM Diprotin A for 15 minutes at room temperature (group B, Table 3)235 or for 15 minutes at 37°C (group D, Table 3)234. Following, BMC were washed in BM medium, counted and as needed resuspended. For in vivo treatment with Diprotin A (group D, Table 3), BMC were resuspended in 1ml BM medium containing 4 µmol Diprotin A and injected intravenously. For the in vivo treatment of group D, 100 µL phosphate-buffered saline containing 5 µmol Diprotin A was injected subcutaneously every 12 hours at days 0,1 and 2.261 For recipients who received in vivo treatment with sitagliptin (groups E and G, Table 3) 100 mg sitagliptin tablets were suspended in 2 ml phosphate-buffered saline under aseptic conditions. The suspension resulted in 4 mg sitagliptin/80 µl, which was administered per oral gavage every 12 hours on days 0 and 1 (group G, Table 3) or on days 0, 1 and 2 (group E, Table 3). Diprotin A was purchased from Sigma Aldrich and sitagliptin (Januvia®) was provided by Merck (Austria).

99 4.4 Assay for DPPIV inhibition

DPPIV activity was assayed using glycyl-prolyl-4-methoxy-β-naphthylamide (Gly-Pro-4-Me- β−ΝΑ) as fluorogenic substrate as described.2661267 5µl serum samples were mixed with 0.5 mM Gly-Pro-4-Me-β−ΝΑ in Tris buffer (50mM, pH 8.3) in a volume of 110 µL. DPPIV activity was detected kinetically during 5min (37°C) by measuring the velocities of 4-Me-

β−ΝΑ release (λex = 340nm, λem = 430nm) from the substrate using Infinite® 200 (Tecan Group Ltd, Switzerland). Therefor needed reagents were purchased from Sigma Aldrich. The fluorescence intensity was related to a standard curve (4-Me−β−ΝΑ). The reversibility of the inhibitors and the dilution of the samples in the assay necessitates the creation of a calibration curve with known concentrations of the inhibitors in the murine serum to allow an estimation of the percentage of in vivo inhibition of the DPPIV enzymatic activity. Inhibition was calculated by comparing DPPIV enzymatic activity of treated mice to control mice. In order to keep the amount of blood draw for each mouse acceptable, groups of mice were split and blood from each mouse was only taken once daily (either at 2 or 12 hours after administration of the DPPIV inhibitor).

4.5 Flow cytometric analysis

Flow cytometric analysis was employed to distinguish donor and recipient cells of particular lineages by two colors. Staining was performed either with fluorescein isothiocyanate- conjugated antibodies against CD4, CD8, B220 or MAC-1 or with biotinylated antibodies against CD45.2 or 34-2-12 (H-2d). Biotinylated antibodies were then detected with phycoerythrin-streptavidin. Additionally, irrelevant isotype controls were used151 and dead cells were excluded by using propidium iodide staining. The percentage of CD45.2+ or 34-2- 12+ cells among the different cell lineages was calculated. Mice were defined to be chimeric if they had at least 2% donor cells within the myeloid lineage and within at least one lymphoid lineage. Surface staining was performed according to the standard procedure and analysis were done on a Coulter Cytomics FC500. CXP software (Coulter, Austria) was used for acquisition and analysis. All antibodies were purchased from Becton Dickinson (San Diego, USA).

100 4.6 Skin grafting

In the allogeneic model, tail skin from Balb/c mice (fuly MHC mismatched) and C3H mice (fully MHC mismatched, third party) was transplanted 2 to 8 weeks after bone marrow transplantation and visually inspected at short intervals. If grafts remained below 10% viable, they were considered rejected.

4.7 Statistical methods Chimerism levels between groups were compared by a two-sided Student’s t test. Skin graft survival of different groups was calculated according to the Kaplan-Meier method and compared to each other by using the log-rank test. A p-value < 0.5 was considered statistically significant.

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114 242 Kim, Y. S. et al. Improved outcome of islet transplantation in partially pancreatectomized diabetic mice by inhibition of dipeptidyl peptidase-4 with sitagliptin. Pancreas 40, 855-860, doi:10.1097/MPA.0b013e318214832d (2011). 243 Tanaka, T., Kameoka, J., Yaron, A., Schlossman, S. F. & Morimoto, C. The costimulatory activity of the CD26 antigen requires dipeptidyl peptidase IV enzymatic activity. Proc Natl Acad Sci U S A 90, 4586-4590 (1993). 244 Ohnuma, K., Dang, N. H. & Morimoto, C. Revisiting an old acquaintance: CD26 and its molecular mechanisms in T cell function. Trends Immunol 29, 295-301, doi:10.1016/j.it.2008.02.010 (2008). 245 Liu, Z., Christensson, M., Forslow, A., De Meester, I. & Sundqvist, K. G. A CD26- controlled cell surface cascade for regulation of T cell motility and chemokine signals. J Immunol 183, 3616-3624, doi:10.4049/jimmunol.0804336 (2009). 246 Herrmann, H. et al. Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia. Blood 123, 3951-3962, doi:10.1182/blood-2013-10- 536078 (2014). 247 Goossen, K. & Graber, S. Longer term safety of dipeptidyl peptidase-4 inhibitors in patients with type 2 diabetes mellitus: systematic review and meta-analysis. Diabetes Obes Metab 14, 1061-1072, doi:10.1111/j.1463-1326.2012.01610.x (2012). 248 Barnett, A. DPP-4 inhibitors and their potential role in the management of type 2 diabetes. Int J Clin Pract 60, 1454-1470, doi:10.1111/j.1742-1241.2006.01178.x (2006). 249 Drucker, D. J. & Nauck, M. A. The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes. Lancet 368, 1696- 1705, doi:10.1016/S0140-6736(06)69705-5 (2006). 250 Wiedeman, P. E. & Trevillyan, J. M. Dipeptidyl peptidase IV inhibitors for the treatment of impaired glucose tolerance and type 2 diabetes. Curr Opin Investig Drugs 4, 412-420 (2003). 251 Herman, G. A. et al. Pharmacokinetics and pharmacodynamics of sitagliptin, an inhibitor of dipeptidyl peptidase IV, in healthy subjects: results from two randomized, double-blind, placebo-controlled studies with single oral doses. Clin Pharmacol Ther 78, 675-688, doi:10.1016/j.clpt.2005.09.002 (2005). 252 Alexander, G. C., Sehgal, N. L., Moloney, R. M. & Stafford, R. S. National trends in treatment of type 2 diabetes mellitus, 1994-2007. Arch Intern Med 168, 2088-2094, doi:10.1001/archinte.168.19.2088 (2008). 253 Nauck, M. A., Vilsboll, T., Gallwitz, B., Garber, A. & Madsbad, S. Incretin-based therapies: viewpoints on the way to consensus. Diabetes Care 32 Suppl 2, S223-231, doi:10.2337/dc09-S315 (2009). 254 Forst, T. & Pfutzner, A. Linagliptin, a dipeptidyl peptidase-4 inhibitor with a unique pharmacological profile, and efficacy in a broad range of patients with type 2 diabetes. Expert Opin Pharmacother 13, 101-110, doi:10.1517/14656566.2012.642863 (2012). 255 Amori, R. E., Lau, J. & Pittas, A. G. Efficacy and safety of incretin therapy in type 2 diabetes: systematic review and meta-analysis. JAMA 298, 194-206, doi:10.1001/jama.298.2.194 (2007). 256 Richter, B., Bandeira-Echtler, E., Bergerhoff, K. & Lerch, C. Emerging role of dipeptidyl peptidase-4 inhibitors in the management of type 2 diabetes. Vasc Health Risk Manag 4, 753-768 (2008). 257 Labuzek, K. et al. Incretin-based therapies in the treatment of type 2 diabetes--more than meets the eye? Eur J Intern Med 24, 207-212, doi:10.1016/j.ejim.2013.01.009 (2013).

115 258 Pilat, N., Klaus, C., Schwaiger, E. & Wekerle, T. Hurdles to the induction of tolerogenic mixed chimerism. Transplantation 87, S79-84, doi:10.1097/TP.0b013e3181a2b9cc (2009). 259 Buhler, L. H. et al. Induction of kidney allograft tolerance after transient lymphohematopoietic chimerism in patients with multiple myeloma and end-stage renal disease. Transplantation 74, 1405-1409, doi:10.1097/01.TP.0000034627.37442.A4 (2002). 260 Leventhal, J. et al. Chimerism and tolerance without GVHD or engraftment syndrome in HLA-mismatched combined kidney and hematopoietic stem cell transplantation. Sci Transl Med 4, 124ra128, doi:10.1126/scitranslmed.3003509 (2012). 261 Broxmeyer, H. E. et al. AMD3100 and CD26 modulate mobilization, engraftment, and survival of hematopoietic stem and progenitor cells mediated by the SDF-1/CXCL12- CXCR4 axis. Ann N Y Acad Sci 1106, 1-19, doi:10.1196/annals.1392.013 (2007). 262 Koporc, Z. et al. Induction of mixed chimerism through transplantation of CD45- congenic mobilized peripheral blood stem cells after nonmyeloablative irradiation. Biol Blood Marrow Transplant 12, 284-292, doi:10.1016/j.bbmt.2005.11.011 (2006). 263 Ponomaryov, T. et al. Induction of the chemokine stromal-derived factor-1 following DNA damage improves human stem cell function. J Clin Invest 106, 1331-1339, doi:10.1172/JCI10329 (2000). 264 Herberg, S. et al. Total body irradiation is permissive for mesenchymal stem cell- mediated new bone formation following local transplantation. Tissue Eng Part A 20, 3212-3227, doi:10.1089/ten.TEA.2013.0663 (2014). 265 Cippa, P. E. et al. Targeting apoptosis to induce stable mixed hematopoietic chimerism and long-term allograft survival without myelosuppressive conditioning in mice. Blood 122, 1669-1677, doi:10.1182/blood-2012-09-453944 (2013). 266 Scharpe, S. et al. Assay of dipeptidyl peptidase IV in serum by fluorometry of 4- methoxy-2-naphthylamine. Clin Chem 34, 2299-2301 (1988). 267 Matheeussen, V. et al. Expression and spatial heterogeneity of dipeptidyl peptidases in endothelial cells of conduct vessels and capillaries. Biol Chem 392, 189-198, doi:10.1515/BC.2011.002 (2011).

116 Curriculum Vitae

Name: Dr. med. univ. Elisabeth Schwaiger Phone: 0043 40400 43910 Address: Department of Internal Medicine III, Division of Nephrology and Dialysis Medical University Vienna (MUV) Mail: [email protected] Date of birth: 15.07.1981 Place of birth: Judenburg, Austria Citizenship: Austria

Education: Okt.1999 – Apr.2006: Medical studies at the Medical University of Graz Since Nov. 2006: Doctoral programme of medical science (PhD, N094) at the Transplantation Laboratory of Prof. Dr. Wekerle, Department of Surgery, MUV

Clinical education: Nov. 2008 – Apr. 2009: Residency, University Clinic of Trauma Surgery, MUV Apr. 2009 – Aug.2010: Residency, University Clinic of Surgery, MUV Aug. 2010 – Nov. 2015: Residency, University Clinic for Internal Medicine III, Department of Nephrology and Dialysis, MUV Nov. 2015: Licence for Internal Medicine, Austria Since Nov 2015: Medical specialisation in Nephrology at the Department of Nephrology and Dialysis, MUV

117 Scientific education: Nov. 2006 – Nov. 2008: PhD student (funded through FWF, SFB-F23, Mechanisms of Establishment and Maintenance of Immunological Tolerance, Haematopoietic Chimerism for the Induction of Tolerance) at the Transplantation Laboratory (Prof. Dr. Wekerle), Department of Surgery, MUV Aug. 2010 – Aug. 2016: PhD student at the VIETAC lab, Department of Internal Medicine, Division of Nephrology and Dialysis, MUV

Clinical studies: Nov. 2006 – Nov 2008: Pharmacist within the Bristol-Meyers-Squibb Belatacept study IM103 027 (Belatacept Evaluation of Nephroprotection as First- line Immunosuppression Trial-Extended Criteria), MUV Nov. 2006 – Nov 2008: Pharmacist of the Bristol-Meyers-Squibb Belatacept study IM103 008 (Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression Trial), MUV Nov. 2006 – Nov 2008: Pharmacist of the Bristol-Meyers-Squibb Belatacept study IM103 045 (Belatacept as First-line immunosuppression in Phase II studies of liver transplantation), MUV Apr. 2008 – Nov. 2008: Pharmacist of the Amevive-Study 0485-CL-E201 Proof of Concept with Alefacept in Kidney Transplantation (Efficacy and safety of Alefacept in combination with Tacrolimus MMF and Steroids in de novo kidney transplantation), MUV Since Jan. 2016: Subinvestigator of the Chiesi-Study Prot.Nr. CCD-06235AA1-01 (Multicentre, Open Label, Randomized, Two-Arm, Parallel- Group Study To Assess Efficacy and Safety of Envarsus® compared with Tacrolimus Used As Per Current Clinical Practice In The Initial Maintenance Setting In De Novo Kidney Transplant Patients), MUV Since Jun. 2016: Subinvestigator in the Bristol-Meyers-Squibb Belatacept study IM103-116 (Evaluation of the Benefits and Risks in

118 Maintenance Renal Transplant Recipients Following Conversion to Nulojix® (Belatacept) - based Immunosuppression), MUV

Talks and poster presentations at scientific meetings: Austrotransplant 2007: CD26-Inhibition in a Congenic Murine Model of Mixed Chimerism, talk TTS 2008: CD26/DPPIV Enzymatic Inhibition in a Murine Model of Mixed Chimerism, poster ÖGAI 2008: DPPIV Inhibition in a Murine Model of Mixed Chimerism, poster Austrotransplant 2008: DPPIV-Inhibition as a strategy to enhance stem cell engraftment in a Murine Model of Mixed Chimerism, talk Wagrain 2008: DPPIV-Inhibition as a strategy to enhance stem cell engraftment in a Murine Model of Mixed Chimerism, talk Austrotransplant 2012: HLA-Antikörper vermittelte Komplementfixation, C1q- versus C4d Fixations Assays, talk Austrotransplant 2013: Klassische Komplementfaktoren als Trigger des Prozone- Effekts bei Luminex-basierter Detektion von HLA- Alloantikörpern, talk Peritransplant Immunadsorption bei Nierentransplantat- empfängern mit präformierten Spender-spezifischen Antikörpern, talk ABMR bei ABO- & HLA-AK-inkompatibler Nierentransplantation, talk WTC 2014: Peri-transplant immunoadsorption for donor-specific antibody- positive recipients of a deceased donor allograft with or without a positive current cytotoxic crossmatch, poster

119 Teaching: 2009: Seminar with internship (Themenspezifische Untersuchungstechniken, Chirurgische Wundversorgung) 2013 - 2016: Seminar with internship (Fall basiertes Lernen) 2015 - 2017: Seminar with internship (BL 9 – Krankheit, Manifestation und Wahrnehmung, allgemeine Arzneimitteltherapie) 2017: Seminar with internship (Themenspezifische Untersuchungstechniken – 8. Semester)

Professional society memberships: Since Nov. 2006: Austrian Society of Transplantation Feb. 2009-2010: Austrian Society of Surgery 2009 and 2015: European Society of Organ Transplantation (ESOT) Since 2011: Austrian Society of Nephrology Since 2015: Austrian Society of Intensive Care and Emergency Medicine

Dr. Elisabeth Schwaiger Vienna, 03.05.2017

120 Publications Elisabeth Schwaiger

Original papers

1. Deceased Donor Kidney Transplantation Across Donor-Specific Antibody Barriers - Predictors of Antibody–Mediated Rejection Schwaiger E1, Eskandary F1,2, Kozakowski N3, Bond G1, Kikic Z1, Yoo D2, Fischer G.F. 4, Oberbauer R1, Böhmig GA1. Nephrol Dial Transplant. 2016 Mar 24. pii: gfw027

2. Minor Antigen Disparities Impede Induction of Long Lasting Chimerism and Tolerance through Bone Marrow Transplantation with Costimulation Blockade Bigenzahn S, Pree I, Klaus C, Pilat N, Mahr B, Schwaiger E, Nierlich P, Wrba F, Wekerle T. J Immunol Res. 2016;2016:8635721. Epub 2016 Oct 31.

4. Bortezomib in late antibody-mediated kidney transplant rejection (BORTEJECT Study): study protocol for a randomized controlled trial. Eskandary F, Bond G, Schwaiger E, Kikic Z, Winzer C, Wahrmann M, Marinova L, Haslacher H, Regele H, Oberbauer R, Böhmig GA1. Trials. 2014 Apr 3;15:107. doi: 10.1186/1745-6215-15-107

5. Complement component C3 activation: the leading cause of the prozone phenomenonaffecting HLA antibody detection on single-antigen beads Schwaiger E, Wahrmann M, Bond G, Eskandary F, Böhmig GA. Transplantation. 2014 Jun 27;97(12):1279-85

121 6. ABO antibody and complement depletion by immunoadsorption combined with membrane filtration--a randomized, controlled, cross-over trial. Eskandary F, Wahrmann M, Biesenbach P, Sandurkov C, König F, Schwaiger E, Perkmann T, Künig S, Derfler K, Zlabinger GJ, Böhmig GA. Nephrol Dial Transplant. 2014 Mar;29(3):706-14

7. Anti-LFA-1 or rapamycin overcome costimulation blockade-resistant rejection in sensitized bone marrow recipients. Ramsey H, Pilat N, Hock K, Klaus C, Unger L, Schwarz C, Baranyi U, Gattringer M, Schwaiger E, Wrba F, Wekerle T Transpl. Int. 2013 Feb;26(2):206-18.

8. Solid phase detection of C4d-fixing HLA antibodies to predict rejection in high immunological risk kidney transplant recipients. Bartel G, Wahrmann M, Schwaiger E, Kikic Z, Winzer C, Hörl WH, Mühlbacher F, Hoke M, Zlabinger GJ, Regele H, Böhmig GA Transpl Int. 2013 Feb;26(2):121-30

9. Modified solid-phase alloantibody detection for improved crossmatch prediction Wahrmann M, Hlavin G, Fischer G, Marinova L, Schwaiger E, Hörl WH, Zlabinger GJ, Körmöczi GF, König F, Böhmig GA Hum Immunol. 2013 Jan;74(1):32-40.

10. Persistent molecular microchimerism induces long-term tolerance towards a clinically relevant respiratory allergen. Baranyi U, Pilat N, Gattringer M, Linhart B, Klaus C, Schwaiger E, Iacomini J, Valenta R, Wekerle T Clin. Exp. Allergy. 2012 Aug;42(8):1282-92

122 11. Dipeptidyl peptidase IV (DPPIV) inhibition does not improve engraftment of unfractionated syngeneic or allogeneic bone marrow after nonmyeloablative conditioning. Schwaiger E, Klaus C, Matheeussen V, Baranyi U, Pilat N, Ramsey H, Korom S, De Meester I, Wekerle T Exp. Hematology, 2012 Feb; 40(2):97-106

12. Bortezomib fort the treatment of chronic antibody-mediated kidney allograft rejection. Schwaiger E, Regele H, Wahrmann M, Werzowa J, Haidbauer B, Schmidt A, Böhmig GA. Clin. Transpl. 2010:391-6

123 Reviews

1. Antibody-mediated rejection: analyzing the risk, proposing solutions. Arias M1, Rush DN, Wiebe C, Gibson IW, Blydt-Hansen TD, Nickerson PW, Sellarés J, López-Hoyos M, San Segundo D, Crespo-Leiro MG, Marzoa-Rivas R, Barge-Caballero E, Paniagua-Martín MJ, Román A, Serón D, Böhmig G, Schwaiger E Transplantation. 2014 Aug 15;98 Suppl 3:S3-21.

2. Prevention and treatment of alloantibody-mediated kidney transplant rejection. Bartel G, Schwaiger E, Böhmig GA Transpl Int. 2011 Dec;24(12):1142-55

3. Hurdles to the induction of tolerogenic mixed chimerism. Pilat N, Klaus C, Schwaiger E, Wekerle T Transplantation. 2009 May 15;87(9Suppl):S79-84.

Weitere Veröffentlichungen:

1. FSGS Rekurrenz nach Nierentransplantation. Schwaiger E, Böhmig G. Nephroscript Nr 4 / 2012

2. DFS – Diabetisches Fußsyndrom. Schwaiger E, Thallinger Ch. Facharzt für Dermatologie, 2/2011

124