Complementation between specific HLA-DR and HLA-DQ genes in transgenic mice determines susceptibility to experimental autoimmune encephalomyelitis

Complementation Between Specific HLA-DR and HLA-DQ Genes in Transgenic Mice Determines Susceptibility to Experimental Autoimmune Encephalomyelitis Pritam Das, Kristen M. Drescher, Annemieke Geluk, David S. Bradley, Moses Rodriguez, and Chella S. David ABSTRACT: To investigate the contribution of human leukocyte antigen (HLA) class II molecules in susceptibility to inflammatory demyelination, we induced experimental autoimmune encephalomyelitis (EAE) in transgenic (tg) mice expressing the HLA-DR3, HLA-DQ8 and HLA-DQ6 molecules in the absence of endogenous class II (Abo). Following immunization with mouse myelin, HLA-DR3 tg mice mounted strong T-cell proliferative responses, and developed inflammatory lesions and demyelination in the central nervous system with mild to moderate clinical symptoms of EAE. HLA-DQ8 and HLA-DQ6 tg mice elicited weak T-cell proliferative responses and did not develop clinical symptoms of EAE. HLA-DR3/DQ6 double tg mice immunized with mouse myelin experienced clinical disease similar to the single tg ABBREVIATIONS CNS central nervous system CFA complete Freund’s adjuvant CP chronic progressive EAE experimental autoimmune encephalomyelitis ELISA enzyme-linked immunosorbent assay HLA human leukocyte antigen IFN interferon IL interleukin

From the Departments of Immunology (P.D, K.M.D., D.S.B, M.R., C.S.D) and Neurology (K.M.D, M.R.), Mayo Clinic and Foundation, Rochester, Minnesota, USA and Department of Immunohematology and Blood Bank (A.G.), University Hospital, Leiden, The Netherlands. Address reprint requests to: Dr. Chella S. David, Department of Immunology; Mayo Clinic, 200 First St., SW, Rochester, MN 55905, USA; Tel: 507-284-8182; Fax: 507-266-0981; E-Mail: [email protected]. Received August 18, 1999; accepted August 19, 1999. The production of HLA tg mice was funded by National Institutes of Health (NIH) Grants AI14764 and CA24473 (C.S.D.). Morphology experiments were supported by NIH grants NS 24180 and NS 32129 (M.D.). P.D. is supported by a NIH training grant CA09127. K.M.D. is a Fellow of the National Multiple Sclerosis Society. We also thank the generous support of Ms. Kathryn Petersen for financing part of the animal costs of these experiments. Human Immunology 61, 279 –289 (2000) © American Society for Histocompatibility and Immunogenetics, 2000 Published by Elsevier Science Inc.

HLA-DR3 tg mice, indicating that expression of DQ6 in this line had no effect on disease. In contrast, HLA-DR3/ DQ8 double tg mice developed severe inflammatory lesions and clinical disease in response to immunization with mouse myelin. Our data suggest that in the presence of two susceptible class II alleles, namely HLA-DR3 and DQ8, there is additional selection and expansion of potential autoreactive T cells, resulting in enhanced severity of disease. Human Immunology 61, 279 –289 (2000). © American Society for Histocompatibility and Immunogenetics, 2000. Published by Elsevier Science Inc. KEYWORDS: transgenic mice; multiple sclerosis; MHC; TCR; EAE

LNC mAbs MHC MS PBL RR TCR tg

lymph node cells monoclonal antibodies major histocompatibility complex multiple sclerosis peripheral blood lymphocyte relapsing-remitting T-cell receptor transgenic mice

INTRODUCTION Genetic studies have documented associations between the presence or absence of certain human leukocyte antigen (HLA) class II alleles to susceptibility of particular autoimmune disorders [1– 8]. Several potential mechanisms have been proposed to explain these HLA associations with disease susceptibility. Polymorphic differences in major histocompatibility complex (MHC) class II molecules may influence susceptibility by selecting self-reactive T cells during thymic education [9]. Alternatively, nonsusceptible MHC class II alleles may either delete or fail to select disease-related self-reactive T cells. In the periphery, HLA-DR or -DQ molecules may pref0198-8859/00/$–see front matter PII S0198-8859(99)00135-4

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erentially bind and present autoantigens to disease related T cells, which are then activated and initiate an immune response [10, 11]. Similarly, immune reactivity of T cells directed against a bacterial or viral antigen may cross-react with a self-antigen (molecular mimicry), resulting in the activation and expansion of autoreactive T cells [12]. However, to our knowledge experimental evidence to support any of these mechanisms in humans has not been demonstrated. Multiple sclerosis (MS) is a chronic immune-mediated demyelinating disease of the central nervous system (CNS), characterized by focal infiltration of T cells and macrophages into the white matter, resulting in neurological dysfunction [13]. Initial reports documented a weak association of the HLA class I antigens HLA-A3 and -B7 with MS [14, 15], but a stronger association with the HLA-DR2/DQ6 extended haplotype in Caucasian MS patients [16 –20]. Correlations to other MHC class II molecules, such as HLA-DR1, -DR3, and -DR4, were demonstrated in other ethnic populations [21–25]. While it is favored that MS-associated HLA-DR molecules present autoantigenic peptides to initiate the disease process [26], some studies suggest that susceptibility to MS may be associated with the HLA-DQ locus [27, 28]. Depending on the disease course, these HLA associations in MS can be further differentiated. The two major forms of clinical disease in MS are chronic progressive (CP) and relapsing-remitting (RR). These subtypes manifest at different ages, and differ with respect to sex distribution and clinical findings [29]. Both CP and RR MS have been associated with the DR2 (DRB1*1501)/DQ6 (DQB1*0602) extended haplotype [29, 30]. An additional risk for CP MS was conferred by a DQB1 RFLP seen in HLA-DR4/DQ8 and HLA-DR7/ DQ9 haplotypes, whereas individuals with the HLADR3/DQ2 haplotype were at higher risk for RR MS [30]. These observations raise the question of whether a single allele, a combination of two alleles, or a complex haplotype confers an increased risk of susceptibility to demyelination and clinical disease. Linkage disequilibrium between certain DR and DQ genes adds to the difficulty in analyzing their individual effects. In summary, linkage analysis provides strong evidence for the involvement of HLA alleles in MS, although these association appear to be complex. To study the role of MHC class II alleles in the pathogenesis of inflammatory demyelinating disease, we generated transgenic (tg) mice expressing HLA-DR2, HLA-DR4, HLA-DR3, HLA-DQ8, and HLA-DQ6 molecules. These tg lines were then mated to class II deficient mice (Abo) to eliminate the effects of endogenous class II molecules. To simulate a heterozygous human haplotype, we also generated double tg mice expressing both the HLA-DR3 and DQ8 or DQ6 mol-

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ecules. We induced experimental autoimmune encephalomyelitis (EAE), a well-characterized murine model of MS, in these tg mice to explore the contribution of these genes to pathology and clinical illness. MATERIALS AND METHODS tg Mice The HLA-DQ8 (DQA1*0301, DQB1*0302), HLADR3 (DRA1*0101, DRB1*0301), and HLA-DQ6 (DQB1*0601, DQA1*0103) tg mice were produced, as previously described [31–33]. Each strain was bred onto the mouse class II knockout (Abo) background [34]. The Abo.DQ8 and Abo.DQ6 mice were mated with the Abo.DR3 mice to obtain the Abo.DR3/DQ8 and Abo.DR3/DQ6 double tg lines. Transgene negative littermates were used as controls. All mice used in this study were bred and maintained in the pathogen free Immunogenetics Mouse Colony of Mayo Clinic according to National Institutes of Health and institutional guidelines. Flow Cytometry Expression of HLA-DR and HLA-DQ molecules on peripheral blood lymphocytes (PBLs) was analyzed by flow cytometry using monoclonal antibodies (mAbs) L227 and IVD12, specific for HLA-DR [35] and HLA-DQ [36], respectively, as described previously [31]. The Tcell receptor (TCR) V␤ usage of CD4⫹ T cells was determined on PBLs with mAbs specific for: V␤2 (B20.6.5) [37], V␤4 (KT4-10) [38)], V␤5.1,2 (MR9.4) [39], V␤ 5.1 (MR9.8) [39], V␤6 (44.22.1) [40], V␤7 (TR.310) [41], V␤8.1,2 (KJ16 –133) [42], V␤8.2 (F23.2) [43], V␤9 (MR10 –2), V␤11 (RR–153) [44], V␤14 (14.2) [45], and V␤17 (KJ23a) [46], as described previously [31]. Myelin Antigen and MBP Peptides Mouse myelin extract was prepared from brains and spinal cords of normal mice over sucrose gradients by the method of Norton and Poduslo, as previously described [47]. Overlapping peptides (20 mer, overlap by 10 amino acids) to the entire sequence of human MBP were synthesized by the Peptide Core Facility at the Mayo Clinic using an automated 430A peptide synthesizer (Applied Biosystems). T-Cell Proliferation Assay Mice were immunized at the base of the tail and in each hindlimb footpad with 200 ␮g of the mouse myelin extract emulsified in complete Freund’s adjuvant (CFA) (Difco Laboratories, Detroit, MI, USA). Ten days later, mice were sacrificed and draining lymph node cells (inguinal, popliteal, and para-aortic) were harvested. Cells

Effect of HLA-DR and HLA-DQ Transgenes on EAE Susceptibility

were resuspended in complete RPMI containing 5% horse serum, with serum supplements at a concentration of 1 ⫻ 107 cells/ml. Cells were then plated at 1 ⫻ 106 cells/well in a 96-well tissue culture plate in a total volume of 200 ␮l, in the presence of titrating amounts of myelin, Concavalin A (positive control) and medium (background control). The cells were incubated for 48 h at 37°C, and then pulsed with [3H]-thymidine for an additional 18 h. [3H]-thymidine uptake was measured using a scintillation counter (Beckman Instruments, Palo Alto, California) and mean counts per minute (cpm) of triplicate samples were calculated. For in vitro inhibition experiments, mAbs specific for CD4 (GK1.5), CD8 (TIB 105), HLA-DQ (IVD12), HLA-DR (L227), H2-E␤b (Y-17), and H-2E␣ (14.4.4) was added (20 ␮ of culture supernatants/well) to LNCs challenged in vitro with mouse myelin (20 ␮g/ml). For analysis of hMBP peptides, mice were immunized at the base of the tail and in each hind limb footpad with 100 ␮g of each peptide emulsified in CFA. Draining lymph node cells were harvested, resuspended in medium, and plated in the presence of 20 ␮g of the recall peptide/well. [3H]-thymidine uptake was then measured as before. Induction of EAE At age 8 –12 weeks, EAE was induced by subcutaneous immunization with the mouse myelin extract (400 ␮g) emulsified 1:1 with CFA on both sides of the flanks. Pertussis toxin (200 ng) (Sigma Chemicals, St. Louis, MO, USA) was administered intravenously on day 0 and 48 h later. Mice were assessed for clinical disease using standard EAE scoring criteria: (0) no disease, (1) tail atony, (2) hind-limb weakness, (3) hind-limb paralysis, (4) hind limb paralysis and forelimb paralysis or weakness, and (5) moribund [48]. Mice of both sexes were used in this study. Histopathology For histological analysis, mice were sacrificed by an overdose of pentobarbital, and brain and spinal cord tissues were frozen in OCT embedding compound. Sections were stained with hematoxylin and eosin. For determination of demyelination, mice were perfused with Trump’s fixative by intracardiac puncture following the development of EAE. Spinal cord blocks (1 mm) were embedded in glycol methacrylate plastic and stained with a modified erichrome stain with cresyl violet counterstain, as previously described [49]. Measurement of Cytokines Culture supernatants of lymph node cells after in vitro stimulation for 72 h in the presence of mouse myelin (20 ␮g) were assessed for cytokines, including interferon (IFN)-␥, interleukin (IL)-6, IL-12, IL-4, and IL-10, by

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capture enzyme-linked immunosorbent assay (ELISA), according to manufacturer protocols (Genzyme, Cambridge, MA, USA). Control samples were cultured in the absence of stimulation. Briefly, plates were coated with capture antibody overnight at 4°C, washed, and blocked for 1 h. Samples and diluted standards were then incubated for 1 h at 37°C, followed by incubation with biotinylated secondary antibody. Plates were washed and incubated with horseradish peroxidase conjugated streptavidin. The plates were developed using 3,3, 5,5⬘tetramethylbenzidine as the substrate, and read at 405 nm using a Model 3550 BIORAD microplate reader. A standard curve was plotted from the mean absorbance for each standard, and cytokine levels were calculated from the standard curve. Statistical differences in the severity of EAE among groups was determined using the MannWhitney rank sum test. RESULTS tg Expression and Selection of T-Cell Repertoire Representative surface expression of each transgene on PBLs was analyzed by flow cytometry. Approximately 14 –18% of PBLs derived from Abo.DR3 tg mice expressed the DR molecule. In the Abo.DQ8 and Abo.DQ6 tg mice, 25–35% of PBLs expressed DQ molecules. No endogenous class II molecules were detected in any of the tg strains. In the Abo.DR3/DQ8 and Abo.DR3/DQ6 double tg mice, the expression levels of both transgenes were complemented in the PBL population. Analysis of V␤ TCR expression within the CD4⫹ population of PBLs demonstrated that all the HLA tg lines expressed a variety of V␤ TCRs, with the exception of mice expressing the HLA-DR3 molecule, wherein V␤5 and V␤11 were deleted. T-Cell Proliferative Responses Against Myelin Antigen To examine the immune response of HLA-tg mice against mouse myelin, in vitro LNC proliferative responses were assessed. Mice were immunized with 200 ␮g of myelin in CFA. Ten days later, the mice were sacrificed, and the draining lymph nodes were pooled from 3 mice for analysis. As shown in Fig. 1A, lymph node cells from the Abo.DR3 mice generated strong proliferative responses against mouse myelin. This response was inhibited by both anti-CD4 mab and antiHLA-DR mab (Fig. 2A), indicating the response was mediated by CD4⫹ T cells and restricted by the tg HLA-DR molecule. T-cell lymph node cells from Abo.DQ8 mice (Fig. 1A) elicited a weak proliferative response against mouse myelin. As before, both antiCD4 mab and anti-HLA-DQ mab inhibited the T-cell response (Fig. 2B). Interestingly, LNCs from Abo.DR3/

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FIGURE 1 T-cell response to mouse myelin in Abo, Abo.DR3, Abo.DQ8, and Abo.DR3/DQ8 tg mice (A), and in Abo, Abo.DR3, Abo.DQ6, and Abo.DR3/DQ6 tg mice (B). Ten days after in vivo priming with mouse myelin, purified LNCs from tg mice primed with mouse myelin pooled from 3 mice/group, were challenged in vitro with increasing doses of mouse myelin. Control samples were cultured in medium alone. Proliferation was determined by [3H]-thymidine incorporation, as described in Materials and Methods. Data represent the mean of three mice (each sample assayed in triplicate).

DQ8 double tg mice immunized with mouse myelin mounted proliferative responses that were stronger than those seen in either parental tg line (Fig. 1A). The response against mouse myelin was also assessed in mice expressing the HLA-DQ6 gene, and mice expressing

FIGURE 2 Inhibition of myelin-specific proliferative response in (A) Abo.DR3 and (B) Abo.DQ8 tg mice. Purified LNCs from mouse myelin primed tg mice (pooled, 3/group) were co-cultured with mouse myelin in vitro and mAbs specific for the HLA-DQ, HLA-DR, CD4, CD8 H-2Ab, or H-2Eb, as described in Materials and Methods. The effect of blocking is shown as the percent of inhibition: 1 ⫺ (␦ cpm of cultures ⫹ experimental mAb) ⫼ (␦ cpm of cultures ⫹ irrelevant control mAb) ⫻ 100.

both the HLA-DR3 and DQ6 genes. In humans, the HLA-DQ6 (DQB1*0601) gene is in linkage disequilibrium with HLA-DR2 (DRB1*1502). Although this haplotype is not known to be linked to MS, DRB1*1502 restricted T cells specific for myelin antigens have been identified in MS patients [50], while the role of DQ6 in

TABLE 1 Development of EAE in transgenic micea Number of mice with maximum severity score

Mice

% Incidence (number/total number)

Mean onset day ⫾ SE

1

2

3

4

5

Ab .DR3 Abo.DR3/DQ8 Abo.DR3/DQ6 Abo.DQ6 Abo.DQ8 Abo

60 (12/20) 80 (16/20) 60 (6/10) 0 (0/10) 0 (0/20) 0 (0/20)

14.4 ⫾ 6 9.5 ⫾ 3 13.2 ⫾ 3 — — —

3 — 2 — — —

2 — — — — —

7 3 4 — — —

— 1 — — — —

— 12b — — — —

o

Mice were immunized with 400 ␮g mouse myelin/CFA and scored for disease, as described in Materials and Methods. Results represent the summary of two experiments using 5–10 mice/group. b p ⬍ 0.01 Mann-Whitney rank sum test, Abo.DR3 vs. Abo.DR3/DQ8 mice. EAE, experimental autoimmune encephalomyelites a

FIGURE 3 Representative photomicrographs of inflammatory lesions in the brain and spinal cords of tg mice. H & E. Magnification ⫻80. (A) Presence of perivascular inflammatory infiltrates in the spinal cord white matter of an Abo.DR3 tg mouse on day 14 postimmunization. (B) In an Abo.DR3 /DQ8 double tg mouse on day 9 post-immunization, the inflammatory infiltrate extends diffusely into the white matter parenchyma. (C) An acute perivascular meningeal inflammatory lesion is visible in the cortex of an Abo.DR3 tg mouse. (D) A large inflammatory lesion in the brain of an Abo.DR3/DQ8 double tg mouse shows diffuse infiltration.

FIGURE 4 Representative photomicrographs of demyelination in the spinal cords of tg mice following induction of EAE. Spinal cord sections were embedded in glycol methacrylate plastic, and stained with a modified erichrome/cresyl violet stain. Magnification ⫻200. (A) At 21 days post-immunization with mouse myelin, no inflammation or demyelination was observed in the Abo.DQ6 tg mice. (B) At 14 days post-immunization with mouse myelin (EAE score of 3), the Abo.DR3 tg mice experienced meningeal inflammation and demyelination. (C) At 21 days post-immunization with mouse myelin, no inflammation or demyelination was observed in the Abo.DQ8 tg mice. (D) At day 12 post-immunization with mouse myelin, the Abo.DR3/DQ8 double tg mice displayed severe meningeal inflammation and widespread demyelination, which extended into the white matter parenchyma (EAE score of 5).

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this haplotype remains unclear. Upon immunization with mouse myelin, the Abo.DQ6 mice elicited weak proliferative responses (Fig. 1B) against myelin, whereas the Abo.DR3/DQ6 double tg mice mounted moderate T-cell proliferative responses (Fig. 1B). These results indicate that the tg HLA molecules can present myelin epitopes, albeit at different intensities, effectively to CD4⫹ T cells in the tg lines. Induction of EAE in HLA tg Mice We then examined the development of EAE in these mice following immunization with the mouse myelin extract. As shown in Table 1, 60% (12/20) of the Abo.DR3 tg mice developed clinical symptoms of EAE, with a mean day of onset of 14.4, and maximum severity score ranging from 1–3. As expected, no clinical symptoms of disease were seen in tg negative (Abo) littermate controls, indicating that disease was restricted by the expression of the DR3 molecule. Following the initial acute phase of disease, the majority of Abo.DR3 mice (EAE score of 3) went into remission (clinical score decreased from 3 to 1), and did not relapse for the remainder of the test period (10 weeks). Immunization of the Abo.DQ8 and Abo.DQ6 tg mice with mouse myelin resulted in no clinical symptoms or histological characteristics of EAE (Fig. 4A, C; Table 1). In contrast, severe disease developed in the Abo.DR3/DQ8 tg mice using the same immunization protocol (Table 1). The evolution of disease in these mice was dramatic, in that until 7–9 days post-immunization, the mice displayed no clinical symptoms of EAE. However, within the next 24 hours, these mice developed severe disease, with the majority becoming moribund or dead. In contrast, Abo.DR3/DQ6 tg mice developed clinical disease similar to the parental Abo.DR3 mice (Table 1), suggesting that the presence of DQ6 molecule had no effect on incidence and severity of disease in this tg line. Histologic analysis revealed moderate degrees of inflammation in the white matter parenchyma of the spinal cords (Fig. 3A) and brain (Fig. 3C) of the Abo.DR3 tg mice. However, the inflammatory infiltrates were more extensive in the spinal cords (Fig. 3B) and brain (Fig. 3D) of Abo.DR3/DQ8 mice. In the Abo.DR3 mice, the infiltrates tended to localize in perivascular regions, whereas in the Abo.DR3/DQ8, inflammatory cells infiltrated widely throughout the white matter. Demyelination was analyzed in spinal cord sections embedded in plastic. No inflammation or demyelination was observed in the spinal cords of Abo.DQ6 (Fig. 4A) of Abo.DQ8 (Fig. 4C) mice. In contrast, moderate demyelination was observed in perivascular lesions in the Abo.DR3 (Fig. 4B) mice. More severe inflammation and demyelination was observed in the Abo.DR3/DQ8 mice (Fig. 4D), with

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multiple demyelinated axons distributed away from perivascular infitrates. Cytokine Profiles of LNC in Response to Mouse Myelin Expression of particular cytokines by autoreactive T cells has been implicated in the development and progression of immune-mediated diseases, such as EAE and MS [26]. To examine whether the presence of particular HLA class II transgenes altered the cytokine profiles and, thus, disease phenotype, cytokines produced in vitro by LNCs cultured with mouse myelin were analyzed. Transgenic mice (3/group) were immunized with mouse myelin and 10 days later, draining LNCs were removed and stimulated with mouse myelin. Pooled culture supernatants were analyzed for IFN-␥, IL-12, IL-6, IL-4, and IL-10 by capture ELISA. As shown in Table 2, moderate levels of IFN-␥, IL-12, and IL-6 were produced by LNCs from Abo.DR3 mice, with undetectable amounts of IL-4 and negligible amounts of IL-10. LNCs of Abo.DQ8 mice produced lower amounts of each cytokine. In contrast, LNCs of Abo.DR3/DQ8 produced the highest levels of all cytokines examined, particularly, IFN-␥ levels. A simple explanation for the increased severity of disease seen in the Abo.DR3/DQ8 compared to Abo.DR3 may be attributed to the higher levels of Th1 cytokines, such as IFN-␥. Alternatively, the altered cytokine levels produced by T cells in these mice could be a direct consequence of their increased proliferation in response to mouse myelin. While the severity of EAE has been reported to correlate with the increase in TH1 cytokines, it cannot be discounted that perhaps the increase in IL-10 and IL-4 may influence disease development and/or progression. T-Cell Response Against Human MBP Peptides To examine the contribution of each HLA class II molecule in the T-cell selection of MBP epitopes, T-cell proliferative responses against overlapping peptides encompassing the entire human MBP sequence were analyzed. Although in these experiments mouse myelin was used for immunizations, because of the implications in these experiments for human MS, we examined the Tcell responses against human MBP peptides. All strains of mice were immunized with individual hMBP peptides to test T-cell reactivity in vivo. Ten-days post-immunization, the mice were sacrificed, and lymph node cells were stimulated in vitro with the same hMBP peptides. T cells from Abo.DR3 mice mounted strong proliferative responses against two hMBP peptides (hMBP 31–50 and hMBP 121–140, Fig. 5A), whereas T cells from the Abo.DQ8 mice generated moderate responses against two regions of the hMBP sequence (hMBP 71–90, 81– 100 and 121–140, 131–150, respectively, Fig. 5B). In-

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TABLE 2 Cytokine expression by LNCs of transgenic mice in response to mouse myelina Mice Abo.DR3 Abo.DQ8 Abo.DR3/DQ8

In vitro antigen

IFN-␥

IL-12

IL-6 (pg/ml)

Myelin Medium Myelin Medium Myelin Medium

364.6 0.1 74.9 0.1 1032.2 0.2

264.4 112.6 154.7 95.0 432.1 158.8

340.6 94.9 138.2 34.5 419.7 62.4

IL-4

IL-10

Undetectable — — — 18.0 —

15.8 — — — 41.7 —

Three mice/group were immunized with 200 ␮g of mouse myelin in CFA in the base of tails and hind footpad. Ten days later, draining lymph node cells were harvested and cultured in the presence of 20 ␮g mouse myelin or medium alone for 72 h. Pooled culture supernatants were then analyzed for the presence of cytokines using capture ELISA, as described in Materials and Methods. IFN, interferon; IL, interleukin; CFA, complete Freund’s adjuvant; ELISA, enzyme-linked immunosorbent assay

a

terestingly, T cells from the Abo.DR3/DQ8 double tg mice mounted the strongest proliferative responses to the previously identified Abo.DR3 and Abo.DQ8 restricted epitopes (Fig. 5C). In addition, T-cell reactivity to new epitopes (hMBP 11–30, 41– 60, and 141–160) was seen in the Abo.DR3/DQ8 tg line (Fig. 5C). T cells from the Abo.DQ6 mice reacted to only one hMBP peptide (hMBP 81–100, Fig. 6B). However, the response to hMBP 81–100 was completely suppressed by the introduction of the DR3 molecule in the Abo.DR3/DQ6 tg line (Fig. 6C). In contrast, the expression of DQ6 in this line had no effect on reactivity to the DR3 restricted epitopes, hMBP 31–50 and hMBP 121–140 (Fig. 6C). These results suggest that combinations of MHC class II genes can differentially regulate peptide-specific T-cell responses. DISCUSSION Numerous population-based studies have documented the association and linkage of certain HLA class II specificities with MS [14 –20]. Similarly, the role of various class II genes in disease progression has been studied, but controversy remains in regard to the role of particular alleles in the prognosis of long-term disability and disease phenotype. Strong linkage disequilibrium between the class II molecules, or perhaps the requirement of more than one allele or a combination of DR-DQ genes have made it difficult to determine the contribution of specific class II alleles in disease susceptibility and progression. To address some of these issues, we induced EAE, a well-characterized experimental model of inflammatory demyelination, in tg mice expressing the HLADR3, HLA-DQ8, and HLA-DQ6 molecules. Studies have documented the association of HLA-DR3 with a benign relapsing form of MS [29, 51], although other investigators have found no association [52] or associations with severe progressive forms of MS [53]. The geographic variation, environmental trigger, and genetic

backgrounds in the different populations chosen for study may explain these apparent contradictions. Following immunization with mouse myelin, the HLA-DR3 tg mice mounted strong T-cell proliferative responses, and developed perivascular inflammatory lesions and demyelination in the CNS, with mild to moderate symptoms of EAE. The milder form of EAE seen in our HLA-DR3 mice would support population studies in which DR3⫹ MS patients display a mild or benign relapsing type of disease. These observations are further supported by recent experiments from our group [54], in which the expression of the HLA-DR3 molecule was shown to reduce the severity of demyelination in the TMEV-induced mouse model of MS. The HLA-DR4/ DQ8 haplotype is associated with more chronic progressive forms of MS [55]. In a previous report, HLA-DR4 tg mice were shown to develop severe progressive forms of EAE upon immunization with encephalitogenic peptides [56]. On the other hand, studies investigating the role of HLA-DQ8 in MS susceptibility are limited, possibly due to its strong linkage disequilibrium with HLA-DR4. Upon immunization with mouse myelin, the HLA-DQ8 tg mice elicited weak T-cell proliferative responses, and did not develop clinical symptoms or histologic features of EAE. Since the HLA-DQ8 tg mice were not susceptible to EAE, we would conclude that the DQ8 molecules do not present an encephalitogenic epitope, or these mice lack autoreactive T cells restricted by DQ8 necessary for disease induction. To recreate the more common human situation, in which the HLA-DR and -DQ alleles are linked together, we generated mice expressing both the HLA-DR3 and DQ8 genes. While the HLA-DR3/DQ8 genes are not in linkage disequilibrium, they are part of the DR3/DQ2, DR4/DQ8 extended HLA haplotypes frequently found in individuals prone to several autoimmune diseases [8]. Immunization of the HLA-DR3/DQ8 double tg mice with mouse myelin resulted in the strongest T-cell proliferative responses compared to those seen in the single

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FIGURE 5 T-cell responses against hMBP peptides in Abo.DR3 (A), Abo.DQ8 (B), and Abo.DR3/DQ8 (C) tg mice. Ten days after in vivo priming with individual hMBP peptides, purified LNCs from tg mice primed with peptide, pooled from 2 mice/peptide, were challenged in vitro with the same immunized peptide (20 ␮g/well). Control samples were cultured in medium alone. Proliferation was determined by [3H]-thymidine incorporation, as described in Materials and Methods, and represented as the stimulation index.

FIGURE 6 T-cell response against hMBP peptides in Abo.DR3 (A), Abo.DQ6 (B), and Abo.DR3/DQ6 (C) tg mice. Ten days after in vivo priming with individual hMBP peptides, purified LNCs from tg mice primed with peptide, pooled from 2 mice/peptide, were challenged in vitro with the same immunized peptide (20 ␮g/well). Control samples were cultured in medium alone. Proliferation was determined by [3H]-thymidine incorporation, as described in Materials and Methods, and represented as the stimulation index.

tg mice. Inflammatory demyelinatimg lesions and severe clinical symptoms of EAE developed in these mice, with the majority of mice succumbing to death 1–2 weeks post-immunization. One possible mechanism to explain the severe disease in this double tg line is that, following presentation of an HLA-DR3 restricted encephalitogenic epitope, the DQ8 molecule may present additional CNS antigens (cryptic epitopes), exposed as a consequence of myelin injury. However, such a phenomenon would most likely develop with time and clinical symptoms occur relatively quickly, thus, reducing the likelihood of this possibility. To determine the cytokine profile in these mice, we examined the expression of both TH1 type (IFN-␥ and IL-12) and TH2 type (IL-4, IL-6, and IL-10) cytokines in

LNCs in response to mouse myelin. Increased levels of all cytokines tested were detected in the HLA-DR3/DQ8 double tg mice compared to the parental strains, which could potentially influence the disease outcome. Since LNCs from the double tg mice proliferated more vigorously than the parental strains, the increased levels of cytokines could be due to the presence of the increased number of T cells in culture. A more plausible explanation for the increased severity of disease seen in the HLA-DR3/DQ8 mice may involve both antigen presentation and TCR repertoire selection. Specifically, disease may be initiated via antigen presentation and activation of T cells in the periphery by a specific HLA class II-peptide, T-cell complex. The increased severity of disease may be due to an increase in the frequency of

Effect of HLA-DR and HLA-DQ Transgenes on EAE Susceptibility

autoreactive T cells that are present and/or selected in mice bearing the susceptible HLA-DR3/DQ8 combination. To test this possibility, we analyzed T-cell responses to peptides derived from human MBP, a major component of myelin. Indeed, strong T-cell responses were generated to several human MBP epitopes in the Abo.DR3/DQ8 double tg mice, including responses to epitopes previously not seen in either single tg Abo.DR3 or DQ8 mice. This would suggest that, in the presence of two predisposing class II alleles, there is additional selection and expansion of potential autoreactive T cells, an outcome of which may be reflected in enhanced severity of disease. Furthermore, coexpression of the DR3 molecule in the Abo.DR3/DQ6 tg line suppressed the recognition of peptide MBP 81–100, which was the only epitope recognized in the single Abo.DQ6 tg mice. In contrast, there were enhanced T-cell responses to this epitope in the Abo.DR3/DQ8 mice compared to the single DQ8 tg mice. This observation is particularly interesting, considering the importance of MBP 81–100 in HLA-DR-restricted T-cell responses in human MS [12]. In closing, these results indicate that epistatic interactions between MHC class II genes can differentially regulate antigen-specific T-cell responses, which ultimately may determine susceptibility or resistance to autoimmune disease.

7. 8. 9.

10.

11. 12.

13.

14. 15. 16.

ACKNOWLEDGMENTS

We thank Drs. Shen Cheng, Paul Zhou, Gunter Hammerling, and Jeanine Baisch for generating the tg mice, Julie Hanson for mice breeding, Michelle Smart for tissue typing, and Kevin Pavelko for photography.

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