Sclerosis Model HLA-DR15 and TCR Transgenic Multiple High Incidence of Spontaneous Disease in An

High Incidence of Spontaneous Disease in an HLA-DR15 and TCR Transgenic Multiple Sclerosis Model

This information is current as Stephan Ellmerich, Marcin Mycko, Katalin Takacs, of September 28, 2021. Hanspeter Waldner, Faisal N. Wahid, Rosemary J. Boyton, Rosalind H. M. King, Paul A. Smith, Sandra Amor, Amy H. Herlihy, Rachel E. Hewitt, Mark Jutton, David A. Price, David A. Hafler, Vijay K. Kuchroo and Daniel M. Altmann

J Immunol 2005; 174:1938-1946; ; Downloaded from doi: 10.4049/jimmunol.174.4.1938 http://www.jimmunol.org/content/174/4/1938

References This article cites 36 articles, 11 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/174/4/1938.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

High Incidence of Spontaneous Disease in an HLA-DR15 and TCR Transgenic Multiple Sclerosis Model1

Stephan Ellmerich,* Marcin Mycko,§ Katalin Takacs,* Hanspeter Waldner,§ Faisal N. Wahid,* Rosemary J. Boyton,* Rosalind H. M. King,¶ Paul A. Smith,† Sandra Amor,† Amy H. Herlihy,‡ Rachel E. Hewitt,ʈ Mark Jutton,* David A. Price,# David A. Haﬂer,§ Vijay K. Kuchroo,§ and Daniel M. Altmann2*

Multiple sclerosis (MS) is thought to involve CD4 T cell recognition of self myelin, many studies focusing on a pathogenic role for anti-myelin, HLA-DR15-restricted T cells. In experimental allergic encephalomyelitis, it is known which epitopes trigger disease and that disease is associated with determinant spread of T cell reactivity. Characterization of these events in human MS is critical

for the development of peptide immunotherapies, but it has been difficult to define the role of determinant spread or define which Downloaded from epitopes might be involved. In this study, we report humanized transgenic mice, strongly expressing HLA-DR15 with an MS- derived TCR; even on a RAG-2 wild-type background, mice spontaneously develop paralysis. Disease, involving demyelination and axonal degeneration, correlates with inter- and intramolecular spread of the T cell response to HLA-DR15-restricted epitopes of myelin basic protein, myelin oligodendrocyte glycoprotein, and ␣B-crystallin. Spread is reproducible and progressive, with two of the epitopes commonly described in responses of HLA-DR15 patients. The fact that this pattern is reiterated as a consequence of CNS tissue damage

in mice demonstrates the value of the transgenic model in supplying an in vivo disease context for the human responses. This http://www.jimmunol.org/ model, encompassing pathologically relevant, spontaneous disease with the presentation of myelin epitopes in the context of HLA-DR15, should offer new insights and predictions about T cell responses during MS as well as a more stringent test bed for immunotherapies. The Journal of Immunology, 2005, 174: 1938–1946.

xperimental allergic encephalomyelitis (EAE)3 is a pro- are abundant data on the specific T cell responses that must be totypic model of autoimmunity in which many basic te- targeted to alleviate disease, while in human studies, in which a E nets of autoimmune disease originate (1). Efforts to im- multitude of T cell epitopes is implicated, this is not the case. An plement peptide-based immunotherapies for multiple sclerosis additional problem is that, in many EAE models, disease progres- (MS) aim to build on the large dataset of EAE tolerogenic and sion is associated with spread of T cell responses, although de- by guest on September 28, 2021 antagonist peptide therapies (2, 3). However, translation from a pendence of disease on spread differs somewhat between EAE peptide-induced murine model to the complex pattern of myelin models, raising questions about the generality of this effect in CNS responses found in humans with a spontaneous and variable dis- disease (4–7). The role of epitope spread in relapsing and remitting ease such as MS offers a daunting challenge. In mouse EAE, there MS is unclear. Heterogeneity in HLA genotype and disease phenotype and the long timescale of disease progression make this *Human Disease Immunogenetics Group, Department of Infectious Diseases and difficult to study (8, 9). An appreciation of the nature and extent of Transplantation Biology Group, Medical Research Council Clinical Sciences Centre, epitope spread in MS will be critical to the successful development † Imperial College, Hammersmith Hospital, London, United Kingdom; Department of of peptide therapies, which may need to be different in patients Neuroinflammation, Division of Neuroscience, Imperial College and Department of Immunobiology, Biomedical Primate Research Centre, Rijswijk, The Netherlands; with long-term compared with recent onset disease (10). ‡Biological Imaging Centre, Medical Research Council Clinical Sciences Centre, Im- § The response against myelin basic protein (MBP) 83–99 is an perial College, London, United Kingdom; Center for Neurologic Diseases, Brigham ϩ and Women’s Hospital, Harvard Medical School, Boston, MA 02115; ¶Department of immunodominant one in DR15 MS patients (11), while compar- Clinical Neurosciences, Royal Free and University College Medical School, London, ʈ ison of T cell responses to myelin from normal and MS brain United Kingdom; Peter Medawar Building for Pathogen Research, University of ␣ Oxford, Oxford, United Kingdom; and #Vaccine Research Center, National Institute emphasizes the importance of responses to B-crystallin in MS- of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD derived tissue (12). Furthermore, ␣B-crystallin is one of the most 20892 abundantly induced transcripts in MS brain (13). Other compari- Received for publication September 14, 2004. Accepted for publication November sons of T cell responses from patients and controls identify 11, 2004. epitopes from myelin oligodendrocyte glycoprotein (MOG) and The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance proteolipoprotein (14, 15). with 18 U.S.C. Section 1734 solely to indicate this fact. An optimal MS model for addressing these issues would be 1 This work was funded by grants from the Multiple Sclerosis Society of Great Britain spontaneous, introducing no inherent bias through being rooted in and Northern Ireland and the Biotechnology and Biological Sciences Research particular modes of EAE induction, would be pathologically se- Council. vere and faithful to the human disease, and would be immunoge- 2 Address correspondence and reprint requests to Dr. Daniel M. Altmann, Human Disease Immunogenetics Group, Department of Infectious Diseases, Imperial Col- netically humanized. In this way, HLA-restricted epitopes impli- lege, Hammersmith Hospital, Du Cane Road, London W12 ONN, U.K. E-mail ad- cated in disease progression or amelioration in the reductionist dress: [email protected] context of the mouse model could be readily investigated in the 3 Abbreviations used in this paper: EAE, experimental allergic encephalomyelitis; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; MS, mul- human setting. One of the first reported TCR transgenic disease tiple sclerosis; PNS, peripheral nervous system; SP, single positive. models involving expression of a murine EAE-derived TCR

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 1939 showed a high incidence of spontaneous disease that was depen- 5 ϫ 103 per well with CD4ϩ or CD8ϩ effector cells. At 4 h, supernatants dent to some extent on the environment in which the mice were were removed from each well for counting in an automated gamma housed (16). The immunogenetics of human MS have previously counter. Specific lysis was calculated according to the formula: (mean sam- ple cpm Ϫ mean spontaneous release)/(mean total cpm Ϫ mean sponta- been modeled by expressing HLA class II genes, or HLA-DR with neous release) ϫ 100. a matched TCR, in transgenic mice (17–20). Disease in HLA-DR/ TCR transgenics has been achieved by injection of myelin peptides Paralysis scores under EAE-inducing conditions or by crossing onto RAG-2 knock- Mice were scored for spontaneous disease, as follows: 0, normal; 1, limp outs, eliminating T regulatory cells, although a high incidence of tail; 2, impaired righting reflex or waddling gait; 3, partial hind limb pa- spontaneous disease in humanized models has rarely been ob- ralysis; 4, total hind limb paralysis; 5, total limb paralysis; 6, moribund. served. In this study, we describe a unique model in which there is Histopathology a very high incidence of spontaneous disease, allowing the analysis of CNS pathology that is faithful to clinical MS and accompanied Mice were perfused with 1% paraformaldehyde/1% gluteraldehyde/1% dextran/PIPES buffer. Tissues were postfixed in osmium tetroxide and em- by epitope spread on a RAG-sufficient background and in the con- bedded in Durcupan resin. Semithin sections were stained with thionin and text of human HLA molecules. The model thus offers clear advan- acridine orange, and ultrathin sections with uranyl acetate and lead citrate. tages over existing ones for testing immunotherapies. Alternatively, 5-␮m wax sections were prepared and stained with H&E or luxol fast blue/cresyl fast violet. Materials and Methods Line 7 mice Results

ϫ Characterization of TCR/HLA expression and function Downloaded from (C57BL/6 CBA)F2 oocytes were microinjected with an HLA- DRA1*0101 cosmid, as previously described (21), and clone 11, a full- A number of TCR and HLA-DR15/A␤0 transgenic lines was gen- length DRB1*1501 cosmid encompassing extensive, flanking 5Ј and 3Ј erated, of which one, termed line 7, is described in this work. Flow regulatory sequence. Founder 24 was mated for more than six generations ␤0 ␤0 cytometric analysis of thymocytes showed that 97% of CD4 sin- with C57BL/6 A mice to yield DR15 A mice. To generate mice ex- ␤ pressing an anti-MBP 85–99/DR15 TCR, rearranged V␣3.1J␣40 and gle-positive (SP) thymocytes carry the human V 2 TCR (Fig. 1), V␤2.1J␤2.1 segments amplified from genomic DNA of the Ob1A12 T cell and the receptor is also present on 79% of CD8 SP cells. In the clone were subcloned into pT␣Cass and pT␤Cass (12, 22). A number of spleen, 97% of CD4 SP cells express the transgenic TCR (Fig. 2a). http://www.jimmunol.org/ TCR-positive founders was generated, of which one, termed line 7, is de- Analysis of MHC class II expression in the mice shows strong scribed in this work. A subline was generated by two generations of back- crossing to C57BL/6 RAG-2Ϫ/Ϫ mice. HLA-DR15 expression, with no expression of mouse/human class II mixed pairs (Fig. 2b). Flow cytometry Naive splenocytes from TCR transgenics show a strong prolif- The phenotype of lymphocyte populations was assessed by flow cytometry erative (Fig. 3a) and IFN-␥ response (Fig. 3b) to low doses of on a BD Biosciences FACSCalibur running CellQuest software and using MBP 85–99 peptide. CD4 and CD8 cells can recognize MBP 85– fluorochrome conjugated Abs against CD4, CD8, B220 (BD Biosciences), HLA-DR␣, HLA-DR␤, and human V␤2 (Serotec). All staining reactions ϫ 5

were performed on 5 10 cells using standard procedures. Briefly, the by guest on September 28, 2021 cells were incubated with the Abs at 4°C for 30 min, washed three times, and resuspended in FACS buffer before assessment. T cell peptide responses T cell responses were analyzed in HL-1 medium (Cambrex) to MBP, MOG, or ␣B-crystallin peptides, added to triplicate wells at a final concentration of 50 ␮g/ml, unless indicated otherwise. In some cases, T cells were prepared by centrifugation of disrupted CNS cells over a 30–70% Percoll density gradient. For CNS cell assays, 2 ϫ 104 infiltrate cells/well were used with 3 ϫ 105 irradiated (2000 R) splenocytes as APC and peptide added at a final concentration of 50 ␮g/ml. Peptide panels representing the sequences of human MBP, MOG, and ␣B-crystallin were made by Biosynth International. Tissue culture supernatant was removed from each well after 48 h for ELISA measurement of IFN-␥ and IL-4 responses (R&D Systems). In some cases, long-term T cell lines were generated against myelin peptides by four or more cycles of restimulation at 14-day intervals using irradiated HLA-DR15 transgenic spleen cells as APC to present peptide added at 50 ␮g/ml and cultured between restimulations in medium containing 20 U/ml rIL-2. To investigate adoptive transfer of disease, T cell lines were propagated by repeated restimulation with spread epitopes. Cells were given by i.v. injection of 5 ϫ 106 cells to HLA-DR15 transgenic recipients, given 24 h after irradiation of mice with 400 R. At the time of cell transfer and again 24 h later, mice received an i.v. injection of 200 ng of pertussis toxin (Valeant Pharmaceuticals). To analyze responses of purified populations, CD4ϩ and CD8ϩ cells were negatively purified from line 7 spleen using rat anti-mouse anti-CD4/ CD8 Abs and anti-rat IgG Dynal beads (Dynal Biotech). Anti-DR Abs were also used to eliminate any DRϩ cells. CD4ϩ or CD8ϩ cells were added at 3 ϫ 105 cells/well in 96-well plates supplemented with 3 ϫ 105 ϩ ␮ DR15 APC and MBP 85–99 at concentrations from 0 to 25 g/ml. IL-2 FIGURE 1. Flow cytometric analysis of thymus. Plots show represen- at 1 U/ml was added to the wells containing the CD8ϩ cells. To test for left killing by selected populations of line 7 cells, splenocytes were stimulated tative staining of lymphoid populations from a wild-type control ( ) and ␮ ϩ ϩ line 7 transgenics (right). The V␤2-positive population within each of the with 1 g/ml MBP 85–99 for 48 h, and CD4 and CD8 cells were Ϫ Ϫ negatively selected, as described above. PGF HLA-DR15ϩ human B lym- four demarcated regions is plotted in CD4 8 double-negative, ϩ ϩ ϩ ϩ phoblastoid cells were labeled with 100 ␮Ci of chromium51 and pulsed CD4 CD8 double-positive, CD8 , and CD4 , respectively. Controls are with 1 ␮g/ml MBP 85–99 for2hat37°C. PGF target cells were added at DR15 negative, human TCR negative, and H2-A positive. 1940 SPONTANEOUS DISEASE IN HLA-DR15/TCR TRANSGENIC MS MODEL

a very low incidence of disease, unless injected with MBP peptide or crossed onto RAG-2 knockouts, while a line reported by us showed poverty of movement, but only rare paralysis (19, 20). Approximately 60% of line 7 mice develop paralysis by 6 mo. This phenotype is greatly exacerbated by crossing onto a RAG-2-null background, suggesting a possible role of regulatory cells.

Histopathological changes in line 7 mice Paralysis in the RAG-sufficient line was associated with profound histopathological changes. Several mice of various ages were analyzed, with representative findings shown in Fig. 5. CNS and peripheral nerves of wild-type control, age-matched mice showed no histological abnormalities (data not shown). In transgenics, in- flammation is associated with myelin loss in the spinal cord and cerebellum. Analysis of mice aged under 4 mo shows demyelination, for example in the lumbar cord, with extensive infiltrates and perivascular cuffing (Fig. 5, a and b). Mice with chronic, overt paralysis show widespread axonal degeneration (Fig. 5, c, e, and f), Downloaded from associated with dense areas of lymphocytic infiltration, predominantly in the spinal cord, most marked distally and in the brain (Fig. 5d). This involves extensive infiltration of the pia arachnoid and perivascular cuffing of blood vessels in cord and brain (Fig. 5d). Lesions are widespread in the spinal cord, but particularly evident in the dorsal and lateral columns. In the brain, lesions are found in the cerebellum, brain stem, and origin of the fifth nerve. http://www.jimmunol.org/ In younger animals, many of the lesions associated with these infiltrates are demyelinating. In older animals, axonal degeneration is more frequently found. Collections of axonal organelles are found in some demyelinated and remyelinated axons in spinal cord lesions (Fig. 5f), indicating cessation of axonal flow. Infiltrating lymphocytes are often closely associated with large, unmyelinated axons (Fig. 5e), suggesting active demyelination. Lesions in the

peroneal nerves and dorsal root ganglia are more focal, but follow by guest on September 28, 2021 the same pattern. In the peripheral nervous system (PNS), both sensory (dorsal root ganglion) and motor nerves contained lesions.

Inter- and intramolecular determinant spread during spontaneous disease As a preliminary to analysis of the relationship between disease progression and epitope spread, we quantified ingress of T cells into the CNS during the preclinical phase of disease (Fig. 6). That is, we needed some estimate of the immunopathological events in the CNS that may lead to epitope spread before paralysis is detectable. Indeed, we found that T cells progressively accumulate in FIGURE 2. Flow cytometric analysis of spleen. a, Human TCR expres- the CNS of line 7 mice throughout adult life. Eighteen-week mice sion in CD4 and CD8 splenic populations, and b, HLA-DR15 mice do not were then separated by spontaneous disease score into groups with express class II DR␣E␤ mixed pairs: spleen cells were stained for B220 vs score 0, 1–2, or Ͼ3, and each group was analyzed for epitope the DR␤ Ab, Tu39, or 17.33s, an Ab that we have previously demonstrated to be sensitive for the detection of DR␣E␤ mixed pairs. Line 7 lympho- spread; a representative experiment is shown in Fig. 7a. Three cytes stain strongly for the human pair, but not for the mixed pair. percent of CD4 SP cells do not express the transgenic TCR, and dual TCR␣ cells can further widen the range of Ags recognized. Mice showing no overt signs of clinical disease showed epitope 99, both with respect to proliferation and cytotoxicity, and thus spread, both within MBP, to MBP 38–59, but also to an ␣B-crys- may contribute to the disease phenotype (Fig. 3, e–h). However, tallin epitope, 161–175. These mice responded to three of the CD8 cells respond poorly and at a higher peptide concentration, epitopes tested. The development of overt disease (score 1–2) was ϩ presumably because, unlike murine CD4 cells, which can make associated with more extensive epitope spread though the C ter- a functional interaction with HLA class II (23), CD8 cells lack a minus of MBP, again also including MBP 38–59 and ␣B-crystal- functional coreceptor interaction. In line with this, responses can lin. These mice responded to eight of the epitopes tested. Among be blocked using Abs to murine CD4, but not CD8 (Fig. 3g). the MBP epitopes specifically recognized in the context of disease were MBP 101–120 and MBP 142–161, echoing findings in terms Spontaneous paralysis in TCR transgenics of spread responses following actively induced disease (20). Mice Line 7 TCR transgenics spontaneously develop paralysis from 4 to with a disease score Ͼ3 were in generally poor health with small, 5 mo (Fig. 4). This phenotype thus differs from two other TCR lymphopenic spleens. They showed a refocused T cell response, transgenic lines expressing the Ob1A12 receptor: one line showed recognizing four of the tested epitopes. At this stage in the disease, The Journal of Immunology 1941

FIGURE 3. Response of naive transgenic spleen cells to MBP peptide. Responses of three transgenics (closed symbols) and three wild-type controls (open symbols) were analyzed with respect to proliferation

(a), IFN-␥ production (b), IL-4 (c), and TNF-␣ (d). Downloaded from CD4ϩ (e) and CD8ϩ (f) puriﬁed cells respond to MBP 85–99. The proliferative response of line 7 T cells is inhibited by anti-HLA-DR and anti-CD4, but not by anti-CD8 (g): anti-CD8 (squares), anti-HLA-DR (tri- angles), anti-CD4 (circles), and anti-CD4 plus anti- CD8 (diamonds). h, Puriﬁed CD4 and CD8 line 7 cells both have the capacity to lyse HLA-DR15 peptide- http://www.jimmunol.org/ pulsed targets. L7 splenocytes were stimulated with 1 ␮g/ml MBP 85–99 for 48 h, and CD4ϩ (diamonds) and CD8ϩ (squares) cells were added to MBP 85–99 PGF DR15ϩ B cells. by guest on September 28, 2021

histopathology shows very advanced degeneration, and CNS pre- tially spreads to MBP 38–59, and then, correlating with the period sentation of myelin peptides may be limited. In addition to MBP during which more T cells infiltrate the CNS and mice progress to 38–59 and ␣B-crystallin 161–175, identified in the studies above full-blown paralysis, to MOG 82–96, and to ␣B-crystallin 161– as HLA-DR15-presented disease-associated epitopes, preliminary 175. This model thus echoes the type of hierarchy of spread seen studies identified an additional spread epitope, MOG 82–96. These in EAE, except that it is in this study demonstrated in spontaneous three epitopes were used for more detailed analysis in groups of disease for HLA-DR-restricted self epitopes with direct relevance mice at various stages of disease. A clear and reproducible pattern to MS. Although the data shown in Fig. 7b clearly demonstrate a of progressive spread was identified (Fig. 7b). The response ini- hierarchy of spread for these epitopes correlating with age, there is

FIGURE 4. Disease in line 7 and line 7-RAG-deﬁ- cient mice. The mean disease score (left) is shown for a cohort of 10 mice using either the line 7 (Œ) or line 7 RAGϪ/Ϫ mice (f); percentage of affected mice is shown on the right. 1942 SPONTANEOUS DISEASE IN HLA-DR15/TCR TRANSGENIC MS MODEL

FIGURE 5. Histopathological analysis of disease. CNS and PNS tissue from representative mice was analyzed: a, resin section from dorsal column, cervical spinal cord of a 4-mo mouse, disease score 2; arrows indicate demyelination adjacent to the pia, while scat- tered degenerated axons are indicated with open arrow- heads, bar ϭ 50 ␮m, stain is thionin and acridine orange; b,asa, but taken from a mouse with score 4, showing extensive demyelination, with perivascular cuffing around some blood vessels (arrows); c, thoracic spinal cord lateral column from disease score 4, line 7 mouse, showing axonal degeneration (arrows), bar ϭ Downloaded from 20 ␮m; d, 9 mo old, disease score 4 line 7 mouse showing extensive perivascular cuffing in the structures un- derlying the cerebellum, bar ϭ 100 ␮m; e, electron micrograph taken from the lateral column of the lumbar spinal cord of the same mouse, as shown in d, showing demyelinated axons (arrows) and lymphocytic infiltrate http://www.jimmunol.org/ (asterisk), bar ϭ 1 ␮m; f, electron micrograph, as in c, showing early axonal degeneration (arrows) and demyelination (asterisk). by guest on September 28, 2021

no clear distinction by disease score. However, clinical hind-limb cells, as well as a population of CD4ϩ8ϩTCRϩ cells, not detected in paralysis is an extreme measure of CNS damage, and it should not the peripheral population (Fig. 9a). An extrathymic CD4ϩ8ϩ double- surprise us that, with greater than 105 myelin-reactive Th1 cells in positive population has been described as having a highly activated the brain, it is possible to get local tissue breakdown and priming phenotype in other models (25). CNS-derived T cells proliferate in of responses to spread epitopes before the appearance of full- response to MBP 80–99, MBP 38–59, and ␣B-crystallin 161–175 blown paralysis. Responses to these spread epitopes are inhibited by Ab to HLA-DR and not class I (Fig. 8). T cell responses to epitopes in the region of MBP 38–59 and MOG 82–96 have previously been described in the responses of HLA-DR15 MS patients (15, 24). It is likely that the responses to spread epitopes are due to peptide recognition by dual TCR␣ T cells, as T cell lines selected against the spread epitopes show altered TCR V␣ usage compared with the starting population (data not shown). Long- term T cell lines cultured from diseased line 7 mice against MOG 82–96 transfer disease to naive HLA-DR15 recipients, indicating that the responses to spread epitopes can contribute to pathogenesis (Table I). When passive disease was induced in HLA-DR15 recipients by transfer of anti-MBP-85–99 T cell lines, the pattern of spread observed for spontaneous disease was reiterated, with the development of responses to the MOG and ␣B-crystallin spread epitopes (data not shown).

Responses of CNS-inﬁltrating T cells

We then purified lymphocytic infiltrates from the brain and spinal FIGURE 6. Progressive spontaneous ingress of T cells into the CNS of cord of paralyzed mice for more detailed analysis (Fig. 9). The ma- line 7 mice. Horizontal bars indicating the mean number of infiltrating ϩ ϩ ϩ ϩ jority of cells were TCR CD4 , but there were also TCR CD8 lymphocytes recovered from three mice per timepoint. The Journal of Immunology 1943

FIGURE 7. Determinant spread in diseased mice. a, Three mice per group at 18 wk of age and with a disease score of either 0, 1–2, or Ͼ3 were sacriﬁced, and splenic proliferative responses were analyzed in response to MBP and ␣B-crystallin peptides. Downloaded from Histograms indicate the mean from three mice Ϯ SD. b, Mice at different ages and disease scores were analyzed for splenic T cell proliferation to the indicated epitopes. Each row shows a different mouse, and T cell proliferative responses were as follows: Ϫ,

stimulation index Ͻ2; ϩ, stimulation index http://www.jimmunol.org/ 2–5; ϩϩ, stimulation index 5–10; ϩϩϩ, stimulation index Ͼ10. by guest on September 28, 2021

(Fig. 9b). When these proliferative responses are compared with potent IFN-␥ response, particularly to MBP 80–99 and to a lesser matched cultures containing the same number of splenic T cells, the extent to MBP 38–59 and ␣B-crystallin 161–175. responses of CNS cells are reduced and the response to MOG 82–96, which can be detected in splenocytes, is not readily detectable in the Discussion inﬁltrating cells. However, when cytokine production of CNS-inﬁl- EAE studies have yielded substantial progress in understanding the trating cells and spleen cells was compared, CNS T cells made a more pathogenesis of MS; yet, of many treatments developed for EAE, few have had major impact on clinical practice. If therapies based on a single antagonist peptide are to be applied (10), it is vital to understand whether the response involves epitope spread, and, if so, whether the antagonist treatment inhibits the spread response (3). This new model encompasses pathologically relevant, spontaneous disease with the presentation of myelin epitopes in the context of an immune system, in which the only class II molecule

Table I. Long-term T cell lines against both MBP 85–99 and the spread epitope, MOG 82–96, can transfer disease to HLA-DR15 transgenic recipients

T Cell Line Cells Number Affected Disease Scores (mean) FIGURE 8. Inhibition of the T cell response to spread epitopes. This was assessed using Ab to HLA-DR (a) or to H-2D (b); histogram shading: MBP 85–99 3/4 5, 4, 4, 0 (3.25) f, no mAb added; o,1␮g/ml mAb; s,5␮g/ml mAb. MOG 82–96 3/4 1, 1, 1, 0 (0.75) 1944 SPONTANEOUS DISEASE IN HLA-DR15/TCR TRANSGENIC MS MODEL

FIGURE 9. Responses by CNS- infiltrating T cells. a, Brain- and spinal cord-infiltrating cells from three mice, score 3–4, were purified and analyzed by flow cytometry. The lower FACS plot indicates TCRϩ cells within the marked gate of CNS- infiltrating CD4ϩCD8ϩ cells. b,T cell proliferation, IFN-␥ release, and IL-4 release in response to spread Downloaded from epitopes in pooled cells from spleen (f) or CNS (u) of mice (three per group) with a disease score of 2–4. http://www.jimmunol.org/

for Ag presentation is the MS-associated HLA-DR15 heterodimer. damage. Of these epitopes, the ␣B-crystallin epitope is conserved The localization of disease lesions may be to some extent dictated between human and mouse, as is the 9-aa core of MOG 82–96 and by guest on September 28, 2021 by specificity of the TCR transgene (26), and thus, the approach is the MBP epitope, with the exception of a substitution from glycine helpful for delineating the in vivo pathogenicity of human T cell to serine in the mouse at residue 46. The ␣B-crystallin 161–175 clones. However, severity of the phenotype may be variable: we epitope was not identified in a small study of T cell responses by described a sister line, expressing the same human TCR, albeit at HLA-DR15 MS patients, although we have preliminary data that a lower level and limited to the CD4 lineage, showing a mild patient T cell lines can indeed be cultured in response to this phenotype with rare paralysis, but general poverty of movement epitope (28) (D. Price and R. Hewitt, unpublished observation). (20). As might be expected, the pattern of epitope spread in the Responses to ␣B-crystallin are believed to reflect its appearance context of milder neuropathology was different from the pattern within perivascular macrophages in MS lesions (29). In rodent seen in the line 7 mice described in this work. Thus, with differ- models, tolerance is more robust and T cell responses are often ential expression of the same TCR, we described different disease poor (30). From a danger hypothesis perspective, circumvention of severity, albeit with similar CNS localization and different patterns tolerance, whether in patients or transgenics, will depend on ␣B- of associated epitope spread. The latter point is not surprising, crystallin expression in an inflammatory context. We have previ- because one would expect the pattern of epitope spread to depend ously shown that in the case of another stress protein, heat shock largely on the extent of tissue damage rather than the initiating protein 60, autoreactivity can develop in the face of strong thymic TCR sequence. expression that would normally be associated with central toler- Epitope spread in this model was very reproducibly found to ance (31). MBP 38–59, MOG 82–96, and ␣B-crystallin 161–175. Some of This model offers new and unique features in comparison with these responses correlated with age rather than paralysis score previously published human TCR transgenics for MS research (presumably though correlating with the accumulation of subclin- (19). In an earlier example, mice were described that expressed the ical tissue damage), while responses to some epitopes such as same TCR, had a low frequency of the transgenic TCR-positive MBP 101–120 and 142–161 were more specifically correlated with cells, and could be actively induced to develop EAE after an ag- paralysis score. The response is hierarchical and progressive: as gressive regime of high dose peptide with pertussis toxin. The new mice age and become sicker, responses to more epitopes are found. model has little overlap with the earlier work, describing a highly The MOG 82–96 and MBP 38–59 spread responses echo the find- penetrant, spontaneous disease phenotype in mice, even when not ing that these are key HLA-DR15-restricted epitopes in MS (14, crossed to RAG knockouts. This makes this a unique and valuable 15, 24, 27). However, from the studies with human PBL, it is humanized model for the analysis of mechanisms and the testing of uncertain how and whether the responses are associated with therapeutics in a more stringent and relevant model than EAE in- pathogenesis. What our mouse/human hybrid model adds to this is duction. For example, as commented upon on the subject of prom- the answer that responses to these epitopes are indeed generated as ising drug trials in EAE models, “none of these three mouse mod- part of epitope spread, activated through pathogenic CNS tissue els of EAE develops spontaneously; instead, they are induced by The Journal of Immunology 1945 aggressive immunization protocols. So, it is hard to use the results epitopes involved in patient responses and for investigation of of testing potential drugs in these models to predict what will hap- immunotherapies. pen in human multiple sclerosis.” (32). Our new model for the first time fills this niche, and in the context of a humanized class II; so, Acknowledgments observations with respect to T cell specificities are closely appli- We thank Drs. D. Douek and N. Karandikar for use of their myelin peptide cable to human patient studies. 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