Experimental autoimmune encephalomyelitis induced in B6.C-H-2bm12 mice by myelin oligodendrocyte glycoprotein: effect of MHC class II mutation on immunodominant epitope selection and fine epitope specificity of encephalitogenic T cells

Journal of Neuroimmunology 96 Ž1999. 9–20

Experimental autoimmune encephalomyelitis induced in B6.C-H-2 bm12 mice by myelin oligodendrocyte glycoprotein: effect of MHC class II mutation on immunodominant epitope selection and fine epitope specificity of encephalitogenic T cells Itzhack Mendel, Hanan Gur, Nicole Kerlero de Rosbo ) , Avraham Ben-Nun Department of Immunology, The Weizmann Institute of Science, RehoÕot 76100, Israel Received 20 August 1998; accepted 2 December 1998

Abstract The effect of the bm12 mutation on susceptibility to MOG-induced EAE, TCR repertoire and fine epitope specificity of the encephalitogenic T-cells, was assessed. prMOG35-55 was encephalitogenic for H-2 bm12 and H-2 b mice. Despite only minor differences in TCRVb expression and fine epitope specificity, H-2 bm12r and H-2 brprMOG35-55-specific T-cells failed to recognize AbrprMOG35-55 and Abm12rprMOG35-55, respectively. rhMOG-induced EAE was milder in H-2 bm12 mice, possibly as a result of co-dominant responses to prMOG35-55 and to the non-encephalitogenic pMOG94-116, rather than a single dominant response to prMOG35-55 in H-2 b mice. q 1999 Elsevier Science B.V. All rights reserved. Keywords: MHC class II; Antigen presenting cells; bm12 mutation; Experimental autoimmune encephalomyelitis; Multiple sclerosis

1. Introduction Experimental autoimmune encephalomyelitis ŽEAE., the purported model for multiple sclerosis ŽMS., is a CD4q T cell mediated inflammatory disease of the central nervous system, induced in susceptible laboratory animals by active immunization with myelin proteins and peptides thereof, or by passive transfer of T cells specific for the encephalitogen into naive syngeneic recipients ŽRaine, 1984.. Myelin basic protein ŽMBP. and proteolipid protein ŽPLP., the two quantitatively major proteins, were long thought to be the only encephalitogenic components ŽSobel et al., 1994; Tuohy, 1994.. Recently, however, we and others ŽAmor et al., 1994; Linington et al., 1993; Mendel et al., 1995, 1996; Johns et al., 1995; Kerlero de Rosbo et al., 1995; Ben-Nun et al., 1996. demonstrated that a quantitatively Abbreviations: EAE, Experimental autoimmune encephalomyelitis; MS, Multiple sclerosis; MOG, Myelin oligodendrocyte glycoprotein; bm12, B6.C-H-2 bm 12 ; MBP, Myelin basic protein; PLP, Proteolipid protein; rhMOG, Recombinant human MOG; prMOG, Synthetic rat MOG peptide; phMOG, Synthetic human MOG peptide; TCR, T cell receptor; SI, Stimulation index ) Corresponding author. Tel.: q972-8-9342568; Fax: q972-89344141; E-mail: [email protected]

minor myelin protein, myelin oligodendrocyte glycoprotein ŽMOG. is also strongly encephalitogenic, an observation highly relevant to MS where, for 50% of patients, the reactivity of peripheral blood lymphocytes is directed predominantly to MOG, rather than to MBP or PLP ŽKerlero de Rosbo et al., 1993, 1997.. In murine EAE induced with MBP or PLP, the region of the molecule encompassing the encephalitogenic epitope generally varies depending on the MHC class II background of the susceptible mouse strain. Thus, in H-2 u ŽPLrJ, B10.PL. and H-2 s ŽSJLrJ. mice, the most susceptible to EAE induction with MBP ŽZamvil and Steinmann, 1990., the epitopes recognized by the encephalitogenic T cells are located within amino acids 1–11 and 84–96, respectively ŽZamvil and Steinmann, 1990.. In PLP-induced EAE, the epitopes recognized by the various susceptible strains vary considerably; thus, PLrJ ŽH-2 u . mice respond to amino acid residues 43–64 ŽWhitam et al., 1991.; C3H ŽH-2 k . mice to amino acid residues 215–232 ŽEndoh et al., 1990.; Biozzi ABrH ŽH-2 dq1 . respond to residues 56–70 ŽAmor et al., 1993.; two encephalitogenic epitopes were delineated to amino acids residues 105–115 and 106–116 in SWR ŽH-2 q . mice ŽTuohy et al., 1989.; and encephalitogenic codominant epitopes corresponding

0165-5728r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 5 7 2 8 Ž 9 9 . 0 0 0 0 9 - 0

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to amino acid sequences 139–151 and 178–191 were demonstrated for SJLrJ ŽH-2 s . mice ŽTuohy et al., 1989, 1992; Greer et al., 1992.. In contrast, in MOG-induced EAE, the MOG sequence of amino acids 35–55 contains the encephalitogenic epitopes for several mouse strains of the same or different MHC class II backgrounds ŽAmor et al., 1994; Kerlero de Rosbo et al., 1995; Mendel et al., 1995, 1996; Ben-Nun et al., 1996., as well as for Lewis rats ŽLinington et al., 1993; Johns et al., 1995.. Thus, a synthetic peptide encompassing amino acids 43-57 could induce EAE in Biozzi ABrH mice ŽAmor et al., 1994., and we demonstrated that a synthetic peptide encompassing amino acids 35-55 ŽprMOG35-55. of the rat MOG sequence is highly encephalitogenic in several H-2 u and H-2 b mouse strains ŽKerlero de Rosbo et al., 1995; Mendel et al., 1995, 1996; Ben-Nun et al., 1996.. In SJLrJ ŽH-2 s . mice, however, prMOG35-55, although immunogenic, could not induce disease ŽKerlero de Rosbo et al., 1995., and the encephalitogenic epitope was found to be located within a different region of the MOG molecule, at amino acids 92–106 ŽAmor et al., 1994.. These studies emphasize the influence of MHC genes on encephalitogenic epitope selection associated with susceptibility to EAE induced with different encephalitogens. Our studies on EAE induced by prMOG35-55 in H-2 u and H-2 b mice also show the association of specific clinical expression with the MHC class II background of the mice ŽKerlero de Rosbo et al., 1995; Mendel et al., 1995, 1996; Ben-Nun et al., 1996.. Thus, regardless of the strain, mice of H-2 b background immunized with prMOG35-55 develop the ‘classical’ form of EAE characterized by caudo-rostral ascending paralysis typical of EAE induced by MBP and PLP ŽMendel et al., 1995, 1996; Ben-Nun et al., 1996.. In contrast, the disease induced by the same peptide in mice of H-2 u background, PLrJ and B10.PL, is markedly different, with delayed onset and unpredictable clinical signs more reminiscent of MS ŽKerlero de Rosbo et al., 1995; Ben-Nun et al., 1996.. Passive transfer of prMOG35-55-reactive T cells from H-2 b and H-2 u mice also results in different clinical expression in their respective naive syngeneic recipients ŽMendel et al., 1995, 1996; Ben-Nun et al., 1996.. The encephalitogenic prMOG35-55-reactive T cells from H-2 b and H-2 u mice recognize different core epitopes delineated to prMOG40-48 and prMOG36-45, respectively ŽKerlero de Rosbo et al., 1995; Mendel et al., 1996.. For both H-2 b and H-2 u mice, the respective minimal T cell epitope for prMOG35-55-reactive T cells was also the minimal encephalitogenic epitope ŽMendel et al., 1996, unpublished data.. It therefore appears that in prMOG35-55-induced EAE, differences in fine specificity of the encephalitogenic epitope recognized in the context of different MHC class II backgrounds can result in disease with differential clinical expression. As a result of a gene conversion event, the B6.C-H-2 bm12 Žbm12. mice differ from their congenic C57BLr6J mice by only three amino acid residues at positions 67, 70 and

71 of their Ab subunit ŽMcKenzie et al., 1979; McIntyre and Seidman, 1984; Mengle-Gaw et al., 1984.. Such alteration, although somewhat subtle, is nevertheless located on one of the a helices forming the peptide binding groove ŽBrown et al., 1988. and may alter the shape of the groove to change its specificity and its mode of interaction with the T cell receptor ŽKobori et al., 1992.. The susceptibility of H-2 b mice to MOG-induced EAE makes it possible to study the effect of minor alterations of MHC class II on disease development and reactivity to immunodominant epitopes on the MOG molecule, using the congenic strains bm12 and C57BLr6J mice.

2. Materials and methods 2.1. Mice Female C57BLr6J ŽH-2 b ., C3H.SW ŽH-2 b . and B6.CH-2 bm12 ŽH-2 bm12 . were purchased from Jackson Laboratories ŽBar Harbor, ME.. All mice were 2–3 months old when used in the experiments. 2.2. MOG peptides prMOG35-55 and truncated peptides thereof ŽKerlero de Rosbo et al., 1995; Mendel et al., 1995, 1996., and overlapping synthetic peptides ŽphMOG. spanning the Iglike extracellular domain of human MOG ŽMendel et al., 1996; Kerlero de Rosbo et al., 1997., were as described previously. Recombinant Ig-like domain of human MOG ŽrhMOG. was prepared as previously described ŽMendel et al., 1996.. 2.3. T cell lines to MOG peptides T cell lines specific for prMOG35-55 and for phMOG94-116 were selected in vitro as described previously ŽBen-Nun and Lando, 1983. from LN cells of mice which had been primed 9 days before with the relevant peptide Ž150 mg. emulsified in complete Freund’s adjuvant ŽCFA. containing 250 mg Mycobacterium tuberculosis ŽMt. H37Ra ŽCat. No: 3114-25, Difco Laboratories, Detroit, MI.. All T cell lines were maintained in vitro in medium containing IL-2 with alternate stimulation with the relevant antigen, every 10-14 days as previously described ŽBen-Nun and Lando, 1983., except that the source of IL-2 was culture supernatant from IL-2-secreting C63 fibroblast line kindly provided by Dr. Strominger’s laboratory, Department of Biochemistry, Harvard University. 2.4. T cell proliferatiÕe responses Line T cells Ž1.5 = 10 4rwell. were cultured in microtiter wells in the presence of irradiated Ž2500 rad. syngeneic spleen cells Ž5 = 10 5rwell. as previously de-

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scribed ŽBen-Nun and Lando, 1983.. To analyse the primary response, popliteal lymph node cells Ž5 = 10 5 . isolated from mice injected s.c. in the hind foot pads with MOG peptides, prMOG35-55 or phMOG94-116 Ž150 mg peptide in CFA containing 250 mg Mt., or with rhMOG Žapproximately 80 mg in CFA containing 500 mg Mt. were cultured in microtiter wells as previously described ŽBen-Nun and Lando, 1983.. The antigen was added Ž0.5 and 2 mgrwell. in triplicate cultures. The cultures were incubated for 48 h at 378C in humidified air containing 7.5% CO 2 . w3 Hx-thymidine Ž1 mCirwell. was added for the last 16 h of the incubation and the cultures were then harvested and counted using a Matrix 96 Direct beta counter ŽPackard Instr., Meriden, CT.. The proliferative response is measured as the w3 Hx-thymidine incorporation expressed as mean counts per minute Žcpm. of triplicate cultures. 2.5. Induction of EAE with MOG peptides, prMOG35-55 and phMOG94-116, and with rhMOG Mice were injected s.c. at one site in the flank with 200 ml of emulsion composed of 300 mg MOG peptide in CFA containing 500 mg Mt. An identical booster immunization was given at one site on the other flank one week later, and mice received 400 ng pertussis toxin ŽPT. in 500 ml PBS in the tail vein, immediately and 48 h after the first immunization. The same protocol was followed for induction of EAE with rhMOG, except that the initial and booster immunizations were composed of approximately 80 mg of rhMOG in CFA containing 500 mg Mt. Following the encephalitogenic challenge, mice were observed daily and clinical manifestations of EAE were scored on a scale of 0–6 as previously described ŽBen-Nun and Lando, 1983..

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2.8. PCR analysis of TCR Vb gene expression by MOGreactiÕe T cell lines To obtain data on the potential expression of Vb proteins for which antibodies were not available to us, analysis of TCR Vb gene usage by encephalitogenic prMOG3555-reactive line T cells was also carried out by PCR amplification of expressed TCR genes and Southern blotting analysis of the amplified products, as described previously ŽMatsuoka et al., 1993..

3. Results 3.1. A b r prMOG35-55-specific T cells do not proliferate in response to prMOG35-55 presented by APC expressing the A b m 12 mutation To determine whether or not the three amino acids difference between the Abm12 and the Ab molecules affects the presentation of relevant peptides to prMOG35-55specific line T cells raised on an Ab background, irradiated spleen cells from bm12 mice as a source of APC were tested for their ability to present prMOG35-55, prMOG3752 and prMOG40-55 to AbrprMOG35-55-specific line T cells. As shown in Fig. 1, line T cells raised from C3H.SW mice proliferated strongly to all three peptides in the presence of syngeneic Ab APC, with stimulation indices ŽSI. of 161, 126 and 94 for prMOG35-55, prMOG37-52 and prMOG40-55, respectively. In contrast, in the presence of bm12 APC, the same AbrprMOG35-55-specific T cells did not proliferate to any significant extent in response to any of the three peptides. Thus, SI values were very low, reaching values of 3.1, 3.4 and 4.1 for prMOG35-55, prMOG37-52 and prMOG40-55, respectively ŽFig. 1.. The failure of bm12 APC to present prMOG35-55 to

2.6. AdoptiÕe transfer of EAE with MOG-specific T cells Cell transfer experiments were conducted as described previously ŽBen-Nun and Lando, 1983. with prMOG35-55or phMOG94-116-reactive line T cells. Briefly, reactive line T cells were stimulated in vitro with the relevant peptide as described above. Three days later, 1 = 10 7 cells in 0.5 ml PBS were injected in the tail vein of irradiated Ž400 rads. naive syngeneic recipients. Mice were followed and scored daily as above. 2.7. Cytofluorometric analysis of TCR Vb gene expression by MOG-reactiÕe T cell lines TCR Vb gene expression of prMOG35-55-reactive line T cells was analyzed by flow cytometry using available monoclonal anti-Vb TCR antibodies exactly as described previously ŽMendel et al., 1995..

Fig. 1. Ab rprMOG35-55-specific line T cells do not proliferate to antigen presented by APC expressing the Abm 12 mutation. Ab rprMOG35-55specific line T cells Ž1.5=10 4 rwell. raised in C3H.SW mice ŽMendel et al., 1996. were tested in microtiter wells for their proliferation to prMOG35-55, prMOG37-52 and prMOG40-55 Ž1 mgrwell. in the presence of irradiated spleen cells Ž5=10 5 APCrwell. from H-2 b ŽC3H.SW. mice or from bm12 mice.

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AbrprMOG35-55-specific T cells, may be due to inefficient binding of prMOG35-55 to the Abm12 molecule which would also affect its immunogenicity. 3.2. prMOG35-55 is immunogenic and encephalitogenic for bm12 mice As can be seen in Fig. 2A, prMOG35-55 is highly immunogenic for bm12 mice, inducing T cells which proliferated to prMOG35-55 with SI ) 10 when tested for primary and secondary responses, indicating that the inability of bm12 APC to present prMOG35-55 to Abr prMOG35-55-specific line T cells is not due to an inability of the bm12 APC to bind the peptide per se. Furthermore, prMOG35-55 was as highly encephalitogenic for bm12

mice as for C57BLr6J mice ŽFig. 2B., inducing EAE with the same clinical expression and progression in the two congenic strains, i.e., typical caudo-rostral ascending paralysis with early disease onset Ž10–12 days after the first inoculation., similar to the disease induced with MBP or PLP. 3.3. prMOG35-55-specific T-cells from bm12 mice are encephalitogenic To ascertain that in bm12 mice, as in C57BLr6J mice, prMOG35-55-induced EAE is also mediated by prMOG35-55-specific T cells, T cell lines were raised from bm12 and C57BLr6J mice injected with prMOG3555, as described in Section 2, and expanded in vitro. After five rounds of in vitro stimulation with prMOG35-55, the expanded line T cells were highly reactive and specific to prMOG35-55 ŽSI s 18 and SI s 45 for AbrprMOG35-55and Abm12rprMOG35-55-specific T cells, respectively. ŽFig. 3A. and were transferred into their respective naive irradiated syngeneic recipients Ž10 7 line T cells per mouse.. As can be seen in Fig. 3B, Abm12rprMOG35-55-specific T cells are highly encephalitogenic, inducing in bm12 mice a chronic EAE identical to that induced by AbrprMOG3555-specific T cells in C57BLr6J mice. 3.4. A b m 12 r prMOG35-55-specific T cells proliferate poorly to prMOG35-55 presented in the context of A b

Fig. 2. prMOG35-55 is highly immunogenic for bm12 mice and induces EAE of similar incidence and severity in bm12 mice as EAE induced by the same peptide in H-2 b mice. ŽA. Immunogenicity of prMOG35-55 in bm12 mice. LN cells from bm12 mice immunized 9 days previously with prMOG35-55 in CFA were tested for their proliferative response to prMOG35-55, before ŽPrimary response. and after ŽSecondary response. one round of stimulation in vitro with prMOG35-55. The response to 2.0 mg of PPD was also tested as a positive control of cell proliferation Žprimary response. and as a control of specificity Žsecondary response. for the prMOG35-55-reactive T cells. ŽB. prMOG35-55 is as encephalitogenic for bm12 mice as for C57BLr6J mice. C57BLr6J and bm12 mice were injected for EAE induction with prMOG35-55 as described ŽSection 4.. The mean daily clinical score"S.E.M. is shown for each group of five mice. Disease incidence was 100% in both groups.

Both H-2 b and H-2 bm12 mice can mount a strong T cell response to prMOG35-55, indicating that APC from both strains can present the peptide adequately to their syngeneic T cells. However, as described above, bm12 APC do not present prMOG35-55 to AbrprMOG35-55-specific T cells. To analyze if Abm12rprMOG35-55-specific T cells would recognize prMOG35-55 as presented by APC from H-2 b mice, the encephalitogenic prMOG35-55-specific line T cells raised from bm12 mice were tested for their proliferation to prMOG35-55, prMOG37-52 and prMOG40-55, in the presence of H-2 bm12 or H-2 b APC. As can be seen in Fig. 4, Abm12rprMOG35-55-specific T cells responded strongly to all three peptides presented in the context of Abm12 , with SI of 12.2, 10.4 and 13.3 for proliferation to prMOG35-55, prMOG37-52 and prMOG40-55, respectively. In contrast, the response by Abm12rprMOG35-55-specific T cells to the same peptides presented in the context of Ab was poor ŽSI s 5.0, 3.9 and 5.0 for prMOG35-55, prMOG37-52 and prMOG40-55, respectively. ŽFig. 4., albeit not as drastically reduced as that observed when Abm12 APC presented the same peptides to AbrprMOG35-55-specific T cells ŽFig. 1.. Failure of Ab and Abm12 APC to efficiently cross-present prMOG35-55 non-syngeneically to H-2 bm12 and H-2 b prMOG35-55-specific T cells suggested that, despite the ability of the respective APC to bind prMOG35-55, the

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Fig. 3. Abm 12 rprMOG35-55-specific line T cells transfer EAE to naive syngeneic recipients. The proliferative response to prMOG35-55 Ž1 mgrwell. of prMOG35-55-specific line T cells from C57BLr6J and bm12 mice was tested after five rounds of stimulation in vitro with prMOG35-55 as described ŽSection 4. ŽA., and 1=10 7 prMOG35-55specific line T cells were injected four days later in the tail vein of naive irradiated syngeneic recipients ŽB.. The mean daily clinical score"S.E.M. is shown for four mice injected. Disease incidence was 100% in both groups.

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specific T cells, the proliferative response of Abm12r prMOG35-55 line T cells to peptides representing prMOG35-55 truncated at the N- orrand C-terminal ends was analyzed and compared with the proliferative response of AbrprMOG35-55-specific T cells. Results shown in Table 1 indicate that prMOG39-48 and prMOG40-50 are the shortest MOG peptides tested which could induce stimulation, albeit partial ŽSI s 3.6 and SI s 7.2 for prMOG39-48 and prMOG40-50, respectively., of the bm12 cells, as compared to the stimulation by prMOG35-55 ŽSI s 22.6. ŽTable 1.. These two peptides appear to represent two nested epitopes for encephalitogenic Abm12rprMOG35-55-specific T cells, as truncation of the amino acid residue 48 to yield prMOG39-47 abrogated the ability of the peptide to induce proliferation, whilst amino acid residue 39 was not required for stimulation by all the cells as prMOG40-55 and prMOG40-50 could both induce proliferation of the line T cells ŽSI s 21.5 and 7.2, respectively; Table 1.. The recognition of two nested epitopes within prMOG35-55 by Abm12rprMOG35-55-specific line T cells contrasts with the recognition of a single core epitope delineated to prMOG40-48 in AbrprMOG35-55specific line T cells ŽMendel et al., 1996.. In addition, it would appear that minimal epitopes of at least ten amino acids in length, i.e., amino acid residues 39–48 and 40– 49r40–50, are required for stimulation of encephalitogenic Abm12rprMOG35-55-specific line T cells, rather than a nonameric sequence, i.e., amino acid residues 40–48, which is sufficient to stimulate AbrprMOG35-55-specific line T cells ŽMendel et al., 1996.. In contrast to H-2 b mice, the minimal stimulatory epitopes prMOG39-48 and prMOG40-49r40-50 were not encephalitogenic, and the smallest encephalitogenic peptide which included the prMOG39-48 stimulatory epitope was prMOG37-48 ŽTable 1..

mode of binding may be different, inducing encephalitogenic T cells with different fine epitope specificity. Accordingly, Abm12rprMOG35-55-specific T cells were analyzed for their fine epitope specificity, and studies were carried out to determine whether the minimal encephalitogenic epitope within prMOG35-55, which we delineated to prMOG40-48 in H-2 b mice is the same or different in bm12 mice. 3.5. Fine epitope specificity of encephalitogenic A b m 12 r prMOG35-55-specific T-cells and analysis of the minimal encephalitogenic sequence within prMOG35-55, for bm12 mice To determine whether prMOG35-55 presented in the context of Abm12 resulted in a shift in the minimal epitope recognised by encephalitogenic Abm12 rprMOG35-55-

Fig. 4. Abm 12 rprMOG35-55-specific T cells proliferate poorly to prMOG35-55 presented in the context of Ab. Abm 12 rprMOG35-55specific line T cells were tested in microtiter wells for their proliferation to prMOG35-55, prMOG37-52 and prMOG40-55 Ž1 mgrwell. in the presence of irradiated spleen cells Ž5=10 5 APCrwell. from bm12 mice or from H-2 b ŽC3H.SW. mice.

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Table 1 In vitro stimulatory effect of truncated peptides overlapping prMOG 35-55 on prMOG 35-55-specific line T cells raised in C3H.SW ŽH-2 b . and bm12 ŽH-2 bm 12 . mice and their encephalitogenicity in vivo

a b

Incidence of disease. ND: not done.

3.6. The TCR Vb gene usage of encephalitogenic A b m 12 r prMOG35-55 line T cells is as diÕerse as that of encephalitogenic A b r prMOG35-55 line T cells, but shows minor differences in the Vb genes expressed The inability of the encephalitogenic Abm12rprMOG3555-specific line T cells to recognize antigen presented in the context of Ab , together with their difference in fine epitope specificity as compared to AbrprMOG35-55-

specific line T cells, may be related to changes at the level of their TCR expression. TCR Vb gene usage was analysed by FACS and PCR, after four rounds of in vitro stimulation with prMOG35-55 ŽTable 2.. The Abm12r prMOG35-55-specific line T cells expressed diverse TCR Vbs ŽTable 2., as observed for AbrprMOG35-55-specific line T cells ŽMendel et al., 1995, 1996.. FACS analysis using available MAb specific to various TCR Vbs showed a high expression of TCR Vb8 Ž30%., as well as expres-

Table 2 Distribution of Vbs expressed by encephalitogenic prMOG35-55-reactive T cells TCR

TCR expression by prMOG35-55-specific T cells from H-2 b and H-2 bm12 mice Detected by FACS Ž% of TCR ab T cells.

Detected by PCR Vb1 Vb2 Vb3 Vb4 Vb5 Vb6 Vb7 Vb8 Vb9 Vb10 Vb11 Vb12 Vb13 Vb14 Vb15 Vb16 a b

C57BLr6J qqa y y y y q y qqq y y y y y q qq y

bm12 qqq y y y y q y qqq y y y y q y y q

q , qq, qqq indicate relative intensity of the signal detected on the autoradiogram. Mab not available.

C57BLr6J )b 0 0 0 ND 7 0 43.1 ND 0 ND ND ND 2.5 ) )

bm12 ) 0 0 0 0 10 0 30 0 0 0 0 4 ND ) )

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sion of TCR Vb6 Ž10%., and TCR Vb13 Ž4%. ŽTable 2.. PCR analysis also revealed a high level of Vb1 expression, and a relatively low expression of Vb16 ŽTable 2.. Comparison of the TCR Vb expression by encephalitogenic Abm12rprMOG35-55-specific line T cells with that of encephalitogenic AbrprMOG35-55-specific line T cells ŽTable 2. indicates that the TCR Vb expression of these cells is as diverse in the two congenic strains, with a total of 5 TCR Vbs expressed by each T cell line. In both lines, the expression of TCR Vb8 predominates, albeit at a somewhat reduced level in the H-2 bm12 line Ž30% as compared to 43% in the H-2 b line.; Vb1 appears to be highly expressed in both lines, and the level of Vb6 expression does not differ significantly Ž10% in the H-2 bm12 line as compared to 7% in the H-2 b line.. The encephalitogenic Abm12rprMOG35-55-specific line T cells, however, do not express TCR Vb15 or TCR Vb14, both of which were detected in the H-2 b line, with TCR Vb15 apparently at a high level ŽTable 2.. Instead, the encephalitogenic Abm12rprMOG35-55-specific line T cells express TCR Vb13 and Vb16. Whether or not the differences in TCR Vb expressed by the Abm12rprMOG35-55-specific line T cells are related to the occurrence of the two nested epitopes recognized within prMOG35-55, rather than the single core sequence required by AbrprMOG35-55-specific line T cells ŽMendel et al., 1996., will need to be determined at the level of T cell clones. 3.7. bm12 mice are less susceptible than H-2 b mice to EAE induced by the Ig-like extracellular domain of MOG In C3H.SW ŽH-2 b . mice, EAE induced with a recombinant preparation representing the Ig-like extracellular domain of human MOG ŽrhMOG. is as severe as EAE induced with prMOG35-55, which is the single immunodominant region of MOG for these mice ŽMendel et al., 1996.. To analyze if the change in minimal encephalitogenic epitope within the encephalitogenic prMOG35-55 may influence EAE induced with rhMOG, H-2 bm12 and H-2 b mice were injected for EAE with rhMOG as described in Section 2, and scored daily for disease development. As can be seen in Fig. 5, C57BLr6J Žfour out of five. and C3H.SW Žthree out of five. mice developed severe signs of EAE 10–12 days after the first inoculation, reaching a maximal clinical score of 3, without recovery. In contrast, although bm12 mice were susceptible to rhMOG-induced EAE, the resulting disease was weaker; only two of the five mice injected developed disease with maximal clinical scores of 1 and 3, respectively ŽFig. 5.. Moreover, the mouse with the mildest disease Žclinical score of 1. subsequently recovered fully. As EAE induced by prMOG35-55 in bm12 mice does not differ from that induced in H-2 b mice by the same peptide, these results were somewhat unexpected and may reflect alterations in epitope immunodominance in response to rhMOG by bm12

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Fig. 5. The clinical severity and incidence of EAE induced by rhMOG are greatly reduced in bm12 mice as compared to rhMOG-induced EAE in H-2 b mice. C3H.SW, C57BLr6J and bm12 mice were injected for disease development with rhMOG as described ŽSection 4., and were followed daily for clinical signs of EAE. The mean daily clinical score" S.E.M. is shown for each group of five mice. Disease incidence was 4r5, 3r5 and 2r5 for C57BLr6J, C3H.SW and bm12 mice, respectively. Similar data were obtained in at least two other experiments with the same mouse strains.

mice. Accordingly, the response to rhMOG was analyzed both in bm12 and in C57Blr6J mice. 3.8. bm12 mice immunized with rhMOG mount a T cell response to an additional epitope located within amino acids 101-116 T cells from H-2 b mice immunized for EAE with rhMOG, recognize a single immunodominant encephalitogenic region, encompassed within prMOG35-55 ŽMendel et al., 1996.. To determine if the mutation in the Ab molecule in bm12 mice may result in differences in the recognition of rhMOG by bm12 T cells, possibly accounting for differences in disease expression with rhMOG, the primary response to overlapping synthetic human MOG peptides ŽphMOG. spanning rhMOG, by LN cells from bm12 mice and C57BLr6J mice immunized with rhMOG was assessed. As can be seen in Fig. 6A, the primary response by rhMOG-primed LN cells from C57BLr6J mice was directed against a single immunodominant region, encompassed within phMOG34-56, similarly to what we observed with rhMOG-primed LN cells from C3H.SW mice ŽMendel et al., 1996.. In contrast, rhMOG-primed LN cells from bm12 mice recognized two fully distinct immunodominant regions ŽFig. 6B.. As expected, the region encompassed by phMOG34-56, corresponding to the encephalitogenic prMOG35-55, was one of the immunodominant regions recognized on rhMOG by bm12 LN cells, indicating that, as in H-2 b mice, this amino acid sequence is recognized as a bona fide, and not a cryptic, epitope in bm12 mice. The second region recognized as immunodominant by rhMOG LN cells from bm12 mice resides within the MOG sequence encompassing amino

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Fig. 6. Primary and secondary T cell responses to overlapping phMOG in C57BLr6J and bm12 mice injected with rhMOG. LN cells from C57BLr6J ŽA. and bm12 ŽB. mice immunized 9 days previously with rhMOG were tested for their proliferative response to rhMOG and overlapping phMOG thereof, before Žprimary response. and after Žsecondary response. one round of in vitro stimulation with rhMOG. Each histogram represents the mean" S.D. of triplicate cultures. The stimulation index is given in parentheses.

acids 94–121, as indicated by the proliferative response of the rhMOG-primed LN cells to phMOG94-116 and phMOG101-121. As phMOG94-116 and phMOG101-121 stimulated the rhMOG-primed LN cells to the same extent ŽSI s 7.0 and 6.5, respectively., it is reasonable to assume that the epitope recognized within this region is located at the overlap of the two peptides, i.e., at amino acids 101–116. After one round of stimulation in vitro with rhMOG, the reactivity to phMOG94-116 and phMOG101-

121 by T cells derived from bm12 mice was no longer observed, and reactivity to phMOG34-56 Žand to some degree also to the adjacent overlapping peptide phMOG2446. only could be detected ŽFig. 6B.. Such an observation is surprising as the primary response of the bm12 LN cells to phMOG94-116rphMOG101-121 was as strong as that to phMOG34-56. It is relevant to point out in this context that T cell expansion with IL-2 following antigenic stimulation of the LN cells, as well as in subsequent cycles, is

I. Mendel et al.r Journal of Neuroimmunology 96 (1999) 9–20

17

Table 3 phMOG94-116-specific line T cells from bm12 mice, but not C57BLr6J mice, proliferate to rhMOG Proliferative responsea by phMOG94-116-specific line T cells from

None phMOG94-116 phMOG101-121 rhMOG a

Antigen

C57BLr6J

bm12

5.0 mgrml 10.0 mgrml 5.0 mgrml 10.0 mgrml 5.0 mgrml 12.5 mgrml

2925 " 170 19468 " 2135 Ž6.6. 24143 " 1036 Ž8.2. 10554 " 1210 Ž3.6. 14177 " 372 Ž4.8. 3007 " 171 Ž1.0. 3782 " 190 Ž1.3.

434 " 12 6006 " 671 Ž13.8. 8545 " 282 Ž19.6. 4867 " 280 Ž11.2. 6830 " 777 Ž15.7. 1303 " 166 Ž3.0. 1758 " 206 Ž4.0.

Expressed as mean c.p.m." S.D. ŽSI..

carried out in our laboratory with supernatant from IL-2 secreting cells, rather than with ConA supernatant. It is therefore possible that phMOG94-116rphMOG101-121reactive T cells induced by immunization with rhMOG in bm12 mice require factorŽs. other than IL-2 for growth, i.e., IL-4.

transfer of AbrphMOG94-116- or Abm12rphMOG94-116specific line T cells into their naive syngeneic recipients Ž1 = 10 7 cells per mouse. did not induce EAE Ždata not shown.. Thus, although phMOG94-116 is immunogenic for both bm12 and C57BLr6J mice, it does not represent an encephalitogenic epitope for these mice.

3.9. phMOG94-116, is a bona fide T cell epitope in bm12 mice, but cryptic in C57BLr 6J mice. phMOG94-116 is not encephalitogenic in bm12 or C57BLr 6J mice

4. Discussion

Recognition of phMOG94-116rphMOG101-121 by rhMOG-primed LN cells from bm12 mice, but not from C57BLr6J mice, raised the question as to whether this reactivity is specific for LNC bearing the Abm12 mutation or can this epitope also bind and be recognized in the context of Ab. Accordingly, bm12 and C57BLr6J mice were immunized with phMOG94-116 in CFA as described in Section 4, and phMOG94-116-reactive T cells were selected from draining LN cells. Proliferation assays indicated that phMOG94-116 is as immunogenic for C57BLr6J mice as for bm12 mice, and highly specific line T cells could be raised from mice of both strains ŽTable 3.. AbrphMOG94-116- and Abm12rphMOG94116-specific line T cells reacted also to phMOG101-121 ŽTable 3., indicating that the epitope recognized is shared by both peptides and therefore likely, as suggested above, to be located within the sequence which overlaps the peptides, i.e., amino acids 101–116. However, only the phMOG94-116-specific T cells from bm12 mice responded to rhMOG, albeit to a lesser extent ŽSI s 19.6 and 4.0 in response to phMOG94-116 and rhMOG, respectively; Table 3., indicating that for C57BLr6J mice, phMOG94-116, or more specifically the sequence spanning amino acids 101–116, is only recognized as a cryptic epitope. In view of the recognition of phMOG94116rphMOG101-121 as a co-immunodominant epitope by Abm12rrhMOG-reactive T cells, phMOG94-116 was tested for its potential encephalitogenic activity in both bm12 and C57BLr6J mice. Encephalitogenic challenge of bm12 or C57BLr6J mice with phMOG94-116 in CFA never resulted in clinical EAE Ždata not shown.. Similarly, passive

Bm12 mice and their congenic H-2 b mice have been used to study the effect of minor alterations in MHC class II on antigen presentation and on epitope selection associated with immune responses linked to Ab. Until recently, the only autoimmune disease to which H-2 b mice were found to be susceptible, was experimental autoimmune myasthenia gravis ŽEAMG., and the bm12 mutation was found to convert C57BLr6J mice from highly susceptible to EAMG, to a congenic strain resistant to this disease ŽChristadoss et al., 1985; Bellone et al., 1991; Infante et al., 1991; Shenoy et al., 1993; Karachunski et al., 1995; Oshima and Atassi, 1995; Yang et al., 1998.. However, the bm12 mutation could also have the opposite effect, as the introduction of the H-2 bm12 MHC genes into NZB mice was shown to enhance susceptibility to autoimmune lupus erythematosus-like syndromes, with NZB.H-2 bm12 mice spontaneously generating high titers of autoantibodies to dsDNA and developing proteinuria, whereas NZB.H-2 b mice do not develop such symptoms ŽChiang et al., 1990.. Systemic lupus erythematosus and EAMG are autoimmune diseases which are mediated mostly by antibody. As we have recently shown, H-2 b mice can also be susceptible to the T cell-mediated autoimmune disease EAE, when the encephalitogen is MOG ŽMendel et al., 1995, 1996; BenNun et al., 1996.. The MOG-induced EAE, which we have recently characterized with respect to immunodominance, epitope specificity and TCR expression associated with disease development in H-2 b mice, now provides a welldefined model to study the effect of minor alterations of the MHC class II on T cell-mediated autoimmune diseases. The results presented here show that the influence of the bm12 mutation on MOG-induced EAE differed markedly

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depending on whether disease was induced with prMOG35-55, the immunodominant encephalitogenic region of MOG for H-2 b mice, or with the 121 amino acids-long rhMOG representing the extracellular domain of the MOG molecule. While bm12 mice were as highly susceptible as C57BLr6J mice to EAE induced with prMOG35-55, the bm12 mutation markedly reduced the clinical incidence and severity of rhMOG-induced EAE. Although there was no difference in susceptibility to prMOG35-55-induced EAE between C57BLr6J and bm12 mice, APC expressing Ab or Abm12 failed to cross-present prMOG35-55 to non-syngeneic prMOG35-55-specific T cells from the other congenic mice. The strong immunogenicity and encephalitogenicity of prMOG35-55 in bm12 mice indicate that this failure in cross-presentation could not be related to differences in binding affinity of the prMOG35-55 to Ab or Abm12 molecules. The bm12 mutation is therefore more likely to affect the mode of binding of prMOG35-55 or the mode of interaction with TCR, resulting in selection in bm12 mice of T cells expressing TCRs with altered positioning on the Abm12rprMOG35-55 complex, as a result of different Va and Vb gene usage. However, fine epitope specificity analysis of the Abm12rprMOG35-55 and AbrprMOG35-55 encephalitogenic T cells revealed only minor differences in their core epitopes. While the core epitope stimulatory for AbrprMOG35-55-specific T cells was delineated to prMOG40-48, Abm12rprMOG35-55-specific T cells recognized two nested epitopes within the 35-55 encephalitogenic region of MOG, prMOG39-48 and prMOG4050r40-49 Žalthough prMOG40-49 was not tested, residue 49 is essential for stimulation and prMOG40-49, rather than prMOG40-50, may be the minimal sequence for this nested epitope.. Thus, the core epitope for AbrprMOG3555 T cells is nonameric ŽprMOG40-48., whereas the Abm12rprMOG35-55-specific T cells require core epitopes of at least ten amino acids, corresponding to prMOG40-48 with an additional residue at the N- ŽprMOG39-48. or the C-terminus ŽprMOG40-49r40-50.. Hence, the three amino acids difference between the Abm12 and Ab molecules resulted only in subtle differences in core epitope recognition by the prMOG35-55 T cells for these congenic mice. Rather interestingly, analysis of the TCR Vb gene usage by prMOG35-55-specific T cells from C57BLr6J and bm12 mice also did not reveal major differences which could account for the failure of cross-presentation by congenic APC. The TCR Vb gene expression by Abm12rprMOG35-55-specific T cells was also diverse, and there was no difference in the predominantly expressed Vb genes. As neurological impairment and progression of the ‘classical’ EAE induced by prMOG35-55 was identical in both congenic strains, it would appear that the slight differences in the mode of binding by prMOG35-55 to Ab and Abm12 molecules, as well as the mode of interaction of the peptiderMHC complex with the TCRs are not sufficient to influence disease susceptibility or expression.

The bm12 mutation was reported to affect reactivity or epitope specificity in response to insulin ŽHochman and Huber, 1984., poly ŽGlu60 Ala30 Tyr 10 . ŽLei et al., 1982., H–Y antigen ŽMichaelides et al., 1981., hen egg lysozyme ŽKobori et al., 1992., and acetylcholine receptor ŽAchR. ŽChristadoss et al., 1985; Bellone et al., 1991; Infante et al., 1991; Shenoy et al., 1993; Karachunski et al., 1995; Oshima and Atassi, 1995; Yang et al., 1998.. Abolition of susceptibility to EAMG in H-2 bm12 mice appears to be linked to a lack of recognition of immunodominant epitopes ŽBellone et al., 1991; Infante et al., 1991; Karachunski et al., 1995., or to recognition as cryptic in H-2 bm12 mice, of an epitope which is immunodominant in H-2 b mice ŽBellone et al., 1991.. Such epitope repertoire shifts do not appear to be due to lack of binding of the epitopes by the Abm12 molecule, as T cells could be raised to the peptides encompassing the epitopes ŽInfante et al., 1991; Kobori et al., 1992; Yang et al., 1998.. However, analyses of the ability of Ab and Abm12 molecules to cross-present immunodominant AchR epitopes to non-syngeneic specific T cells originating from the other congenic mice have yielded controversial data, ranging from total absence of cross-presentation by Ab and Abm12 APC non-syngeneically ŽOshima and Atassi, 1995; Yang et al., 1998., through partial cross-presentation ŽBellone et al., 1991; Infante et al., 1991; Yang et al., 1998., and on to fully effective cross-presentation ŽBellone et al., 1991.. These results indicate that the H-2 restriction of the H-2 b or H-2 bm12 is highly variable depending on the T cell clone, regardless of the ability of the relevant MHC to bind the epitope, suggesting that the distinction between the two class II MHC molecules may be at the level of the TCRs expressed by the specific T cells. Analysis of TCR V gene usage of T cells specific for the immunodominant epitopes recognized by Th cells in EAMG revealed restricted TCR Va Žmostly Va 8. and Vb Žmostly Vb6. gene expression by C57BLr6J T cells responding to the immunodominant epitopes, while TCR gene usage by bm12 T cells was much less restricted ŽInfante et al., 1992; Karachunski et al., 1995; Yang et al., 1998.. Analysis of the response to hen egg lysosyme ŽHEL. peptide p74-96 by specific T cells from bm12 and C57BLr6J mice revealed only minor differences in epitope specificity, comparable to our observations in epitope specificity of prMOG35-55-specific T cells from bm12 and C57BLr6J mice. Nevertheless, and as we observed in the present study, HEL p74-96-specific T cells recognized p74-96 only when presented by self Ab or Abm12 molecules ŽKobori et al., 1992.. However, reactivity to HEL p74-96 both by cloned Abr and Abm12rHEL p74-96-specific T cells was associated predominantly with TCR Va 11 and Vb3, and the failure in cross-presentation was attributed to different joining regions, in both a and b TCR chains ŽKobori et al., 1992.. In contrast, both Abr and Abm12rprMOG35-55 encephalitogenic T cells exhibited diverse TCR Vb gene usage, the pattern of which did not vary significantly between the congenic strains. Under-

I. Mendel et al.r Journal of Neuroimmunology 96 (1999) 9–20

standing the lack of cross-presentation of prMOG35-55 by Ab and Abm12 expressing APC to specific T cells non-congenically requires clonal analysis and sequencing the CDR3 regions of the a and b TCR genes expressed by the clones derived from both congenic strains. It should be noted, however, that the TCR usage by AbrprMOG35-55 T cell clones is highly heterogeneous and no CDR3 consensus sequence associated with the recognition of prMOG35-55 could be demonstrated ŽMendel et al., manuscript in preparation.. In contrast with the subtle differences in the autoimmune response to prMOG35-55 which did not affect prMOG35-55-induced EAE in bm12 mice, the effect of the bm12 mutation on EAE induced with rhMOG was much more pronounced. The incidence and clinical severity of the disease induced with rhMOG in bm12 mice were greatly reduced, as compared to rhMOG-induced EAE in H-2 b mice. Analysis of the epitopes recognized upon primary response to rhMOG by LN cells from C57BLr6J and bm12 mice, revealed a major alteration in epitope repertoire of the bm12 rhMOG-reactive T cells, whereby an additional epitope, probably located within amino acids 101-116 of the MOG molecule, is also recognized as co-dominant with the encephalitogenic region encompassing amino acids 35-55. Upon further selection of the Abm12rrhMOG-reactive T cells with medium containing IL-2, reactivity to amino acids 101-116 was no longer observed. Attempts to induce EAE either actively with phMOG94-116, or passively with phMOG94-116-specific T cells were unsuccessful in bm12 mice, as well as in C57BLr6J mice which recognize phMOG94-116r101– 121 only as a cryptic epitope. Accordingly, we have postulated that reactivity to amino acids 101–116 upon immunization of bm12 mice with rhMOG is associated with down-regulation of the disease induced by the region encompassing amino acids 35–55. The possibility that the phMOG94-116r101-121-specific T cells, induced in bm12 mice upon immunization with rhMOG, may have a regulatory role in the rhMOG-induced EAE is now being further investigated by analyzing their cytokine profile and determining if passive transfer of these cells into syngeneic recipients immunized for EAE with prMOG35-55 will result in decreased incidence and severity of the disease. Thus, minor alterations in the MHC class II molecule can have a different effect on disease susceptibility depending on the encephalitogen used to induce EAE. In bm12 mice, EAE induced with prMOG35-55 representing the major encephalitogenic epitope, differed markedly from EAE induced with rhMOG, even though rhMOG-induced disease is associated with a predominant response against amino acids 35–55 of the MOG molecule ŽMendel et al., 1996., which is also observed in bm12 mice. The minor alterations in fine epitope specificity induced by the bm12 mutation do not affect prMOG35-55-induced EAE indicating that upon immunization of bm12 mice with rhMOG, the prMOG35-55-reactive T cells should have been as

19

encephalitogenic as the prMOG35-55-reactive T cells induced in C57BLr6J mice immunized with rhMOG. The reduction in disease incidence and clinical severity of rhMOG-induced EAE in bm12 mice therefore appears to be linked to reactivity to another co-dominant epitope, down-regulating disease expression.

Acknowledgements This work was supported by the National Multiple Sclerosis Society of New York, USA; the Minerva Foundation, Munich, Germany; and the European Union, BIOMED 2. A. Ben-Nun is the incumbent of the Eugene and Marcia Appelbaum Professorial Chair. H. Gur is a recipient of the Harris Award, The Sackler Faculty of Medicine, Tel Aviv University, Israel.

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