The Human T Cell Response to Myelin Oligodendrocyte Glycoprotein: A Multiple Sclerosis Family-Based Study 1 Niklas K. U. Koehler,* Claude P. Genain,* Barbara Giesser, † and Stephen L. Hauser* Myelin oligodendrocyte glycoprotein (MOG) is an encephalitogenic myelin protein and a likely autoantigen in human multiple sclerosis (MS). In this work, we describe the fine specificity and cytokine profile of T cell clones (TCC) directed against MOG in three nuclear families, comprised of four individuals affected with MS and their HLA-identical siblings. TCC were generated from PBMC by limiting dilution against a mixture of eleven 20-mer overlapping peptides corresponding to the encephalitogenic ex- tracellular domain of human MOG (aa 1–120). The frequency of MOG peptide-reactive T cells was surprisingly high (range, 1:400 to 1:3,000) and, unexpectedly, cloning efficiencies were highest at low seeding densities of 10 2 or 10 3 PBMC per well. A total of 235 MOG peptide-reactive TCC were produced, all of which were CD4  CD8  TCR  TCR  . All 11 MOG peptides were rec- ognized by the TCC, and different epitopes of MOG appeared to be immunodominant in the HLA-identical siblings. The patterns of cytokine secretion by TCC from single individuals were generally similar. The healthy individuals exhibited Th2-, Th0-, and T regulatory cell 1-like cytokine profiles, whereas TCC from one sibling with MS had a striking Th1-like phenotype, producing high levels of IFN- and TNF-, and low IL-4 levels. Thus, MOG-reactive T cells appear to constitute an important part of the natural T cell repertoire, a finding that could contribute to the development of autoimmunity to this protein. The Journal of Immunology, 2002, 168: 5920 –5927. M ultiple sclerosis (MS) 3 is an autoimmune disease thought to be caused by a concerted attack of T cells, B cells, and macrophages against myelin components of the CNS (1, 2). The immune responses against the quantitatively major myelin proteins, specifically myelin basic protein (MBP) and proteolipid protein, have been well characterized, both in an- imal models (experimental allergic encephalomyelitis) and in hu- man MS. MBP appears to be a critical T cell autoantigen in MS (2); however, because MBP is expressed in central and peripheral nervous system myelin, additional factors must be present to ex- plain the specificity of MS for CNS white matter. Myelin oligo- dendrocyte glycoprotein (MOG) is a minor myelin protein that is exclusively expressed in the CNS and is located on the extracel- lular membrane of oligodendrocytes, their processes, and the out- ermost myelin lamellae (3–7). In several animal species, most no- tably non-human primates, MOG is highly encephalitogenic and induces a primary demyelinating disease that closely mimics hu- man MS (8 –14). Large concentric areas of macrophage infiltra- tion, autoantibody deposition, and vesicular demyelination are characteristic of MOG-induced experimental allergic encephalo- myelitis (15, 16). In humans, autoimmunity against MOG also appears to play a causative role in the pathogenesis of MS. MOG- specific autoantibodies have been detected in situ in actively de- myelinating MS lesions (15). High levels of reactivity of PBMC against MOG (17–19), high precursor frequencies of MOG-reac- tive T cells in serum and cerebrospinal fluid (CSF), and CSF au- toantibodies (20 –22) have also been associated with MS. In this study, a limiting dilution technique was used to generate MOG- reactive T cell clones (TCC) from haploidentical siblings belong- ing to MS-prone families. The fine specificity and cytokine pat- terns of the TCC were also analyzed. Our data indicate that MOG peptide-reactive T cells occur with surprisingly high frequency in the normal T cell repertoire and raise the possibility that individual differences exist in the response to MOG that may be relevant to the pathogenesis of MS. Materials and Methods Subjects Individuals from three MS families with multiple affected members were studied (Table I). Family I consisted of two unaffected parents (I-Uf and I-Um), two daughters (I-Ms1 and I-Us1) discordant for MS, and two sons (I-Ms2 and I-Us2) also discordant for MS. The affected daughter (I-Ms1) was treated with IFN--1b, while the affected son (I-Ms2) was untreated. Families II and III consisted of a sibling pair discordant for MS (II-Ms1 and II-Us1; III-Ms1 and III-Us2); in each, the affected individual was untreated (Table I). All affected individuals had relapsing-remitting, clinically definite MS (23). HLA typing was performed by a PCR method as previously described (23). All siblings within a given family were HLA-haploidentical. Antigens For T cell cloning and epitope mapping studies, a series of eleven 20-mer peptides overlapping by 10 aa and corresponding to the entire sequence of the extracellular domain of human MOG (aa 1–120) were synthesized us- ing standard F-moc chemistry and purified 95% as verified by HPLC and mass spectrometry (Research Genetics, Huntsville, AL). In addition to the peptides, 82 positively reacting TCC from family I were tested for reac- tivity against recombinant proteins corresponding to the extracellular do- mains of recombinant human (rh)MOG and recombinant rat (rr)MOG, pro- duced in Escherichia coli and purified as described previously (24). The purity of these recombinant proteins was confirmed by SDS-PAGE and silver staining. For 26 MOG-reactive TCC from family I, fine mapping of *Department of Neurology, University of California, San Francisco, CA 94143; and † Department of Neurology, University of Arizona, Tucson, AZ 85724 Received for publication April 16, 2001. Accepted for publication March 25, 2002. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 1 N.K.U.K. was a postdoctoral fellow of the Deutsche Forschungsgemeinschaft (KO 1719/1-1). C.P.G. was a Harry Weaver Neuroscience Scholar of the National Multiple Sclerosis Society. 2 Address correspondence and reprint requests to Dr. Stephen L. Hauser, Department of Neurology, University of California, San Francisco, CA 94143. E-mail address: hauser@itsa.ucsf.edu 3 Abbreviations used in this paper: MS, multiple sclerosis; TCC, T cell clone; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; rh, recombinant human; rr, recombinant rat; Tr1, T regulatory cell 1; CSF, cerebrospinal fluid. The Journal of Immunology Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00