Identification of immunodominant T cell epitopes of human glutamic acid decarboxylase 65 by using HLA-DR(a1*0101,b1*0401) transgenic mice (insulin-dependent diabetes mellitusyautoantigensyepitope mappingyT cell hybridomas)

Glutamic acid decarboxylase isoform 2 (GAD65; EC 4.1.1.15) has been identified as a key target autoantigen of insulin-dependent diabetes mellitus (IDDM). IDDM is genetically associated with the major histocompatibility complex (MHC), and particular alleles from the HLA-DQ and HLA-DR loci contribute to disease. Among DR4 subtypes, HLA-DRB1*0401, HLA-DRB1*0402, and HLADRB1*0405 alleles lend susceptibility, while HLA-DRB1*0403 confers protection. We have utilized HLA-DR(a1*0101,b1*0401) (hereafter referred to as DR0401), human CD4, murine class II null triple transgenic mice and recombinant GAD65 to generate T cell hybridomas, and we have used overlapping sets of peptides to map the immunodominant epitopes of this autoantigen. We have identified 10 immunogenic regions for GAD65, of which 6 are recognized by multiple hybridomas. These epitopes are also generated by human antigen-presenting cells and their presentation is DR0401 restricted, as shown by the use of typed human lymphoblastoid cell lines and antibody blocking experiments. Immunodominant GAD65 epitopes defined in transgenic mice correspond to GAD65 regions previously shown to elicit T cell responses specifically in DR0401 IDDM patients, underscoring the validity of this approach. Interestingly, although the major epitopes contain DR0401 binding motifs, one of the epitopes contains a DR0405 motif. Susceptibility to insulin-dependent diabetes mellitus (IDDM), though multifactorial, is markedly influenced by the major histocompatibility complex (MHC) class II genotype (1). The strongest associations of IDDM susceptibility in Caucasians are with the HLA-DQ loci, specifically with alleles that carry a neutral residue (Val, Ala, or Ser) at position 57 of the DQ b chain, such as the HLA-DQB1*0302 allele, while other alleles encoding an Asp residue at the same position are negatively related to IDDM susceptibility (1). In addition, 90% of patients with IDDM express HLA-DR3 or HLA-DR4 (2), and three separate studies have recently shown an association between certain HLA-DR4 alleles and IDDM. In populations of Mexican Americans (3), Sardinians (4), and Belgians (5), it was shown that HLA-DRB1*0405 lends susceptibility to IDDM, while HLA-DRB1*0403 confers a protective effect. HLA-DRB1*0405 increases the effect of HLA-DQB1*0302 when found on the same haplotype, while HLA-DRB1*0403 acts in a dominant manner to confer protection (4, 5), even when linked to DQB*0302 with the DRB1*0301-DQB1*0201 susceptibility haplotype present on the other chromosome. In northern Europeans HLA-DRB1*0401 and DRB1*0402 are also associated with disease. In relative terms, the contribution of HLA-DRB1*04 alleles to disease is in the following order: HLA-DRB1*0405, *0402, *0401, *0404, *0403, and *0406 (6). Although these associations have been known for a number of years, the molecular or biochemical basis for such HLA associations remains unclear. MHC class II molecules function at the level of both positive and negative selection of the T cell repertoire in the thymus, but HLA-DQ and HLA-DR molecules also select and present peptides to CD41 T cells in the periphery, and they may play a role in IDDM through the presentation of islet cell specific peptides to pathogenic T cells. Due to the presence of autoantibodies and T cell reactivity in patients, several b-islet cell proteins have been identified as potential disease targets (7). Eighty percent of prediabetic individuals followed in families with a history of diabetes, and most recent onset diabetic patients, have serum autoantibodies directed against glutamic acid decarboxylase (GAD; EC 4.1.1.15) (8). Furthermore, half of new-onset IDDM patients have T cell proliferative responses to GAD (9, 10). Finally, GAD isoform 2 (GAD65) has been used in tolerogenic regimens to prevent disease in susceptible nonobese diabetic (NOD) mice (11–13). The identification of the immunodominant epitopes of target autoantigens presented by the different HLA molecules is a key step toward delineating the role of MHC molecules in disease, as well as providing reagents for diagnosis and possible therapies for IDDM. Such efforts are difficult in human subjects due to the paucity of circulating autoreactive T cells in peripheral blood of patients, and the difficulty in long term maintenance of human T cell clones. In addition, the presence of multiple HLA molecules in each individual makes the determination of MHC restriction elements difficult, and many patients need to be analyzed before a clear pattern emerges. To circumvent these problems, we have utilized HLADR(a1*0101,b1*0401) (hereafter referred to as DR0401) transgenic mice to isolate T cell hybridomas, and a rapid screening assay to map the immunodominant epitopes recognized by these hybridomas. We have defined the immunodominant epitopes for GAD65 and demonstrated that these epitopes are processed by human antigen-presenting cells (APCs) and presented in an HLA-DR4-restricted manner. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y948082-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: APCs, antigen-presenting cells; EBV, Epstein–Barr virus; FACS, fluorescence-activated cell sorter; GAD, glutamic acid decarboxylase; GAD65, GAD isoform 2; HAT, hypoxanthiney aminopterinythymidine; hCD4, human CD4; IL-2, interleukin 2; MHC, major histocompatibility complex; TCR, T cell antigen receptor; DR040n, HLA-DR(a1*0101,b1*040n). ‡Present address: Cell Genesys, 344 Lakeside Drive, Foster City, CA 94404. ¶To whom reprint requests should be addressed.