Identification of immunodominant T cell epitopes of human glutamic acid decarboxylase 65 by using HLA-DR(alpha1*0101,beta1*0401) transgenic mice

Abstract

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 HLA-DRB1*0405 alleles lend susceptibility, while HLA-DRB1*0403 confers protection. We have utilized HLA-DR(alpha1*0101,beta1*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.

Figure 1

The repertoire of mouse T cells in DR(α*0101,β*0401) transgenic mice. FACS analyses of peripheral blood lymphocytes from HLA-transgenic mice of different genetic background. Cells were stained using mouse CD4, hCD4, and human DR-specific reagents in standard protocols. FITC, fluorescein isothiocyanate; PE, phycoerythrin. (Upper Left) CD4⁺ T cell levels of progeny of DBA/1J mice backcrossed once with I-Aβ^0/0 mice (27% CD4⁺ T cells). (Upper Right) Analysis for a DR0401 mouse without hCD4 on an I-Aβ^0/0 background (3.8% CD4⁺ T cells). (Lower Left and Right) Heterozygous (13.3% CD4⁺ T cells) and homozygous (31.1% CD4⁺ T cells) HLA-DR, hCD4 mice, respectively. The DR4 and hCD4 are part of a single transgene in the C-line mice.

Figure 2

Representative plot to demonstrate definition of epitope sequence. The analysis for hybridoma 216 is shown. IL-2 production was measured as fluorescence units. Analysis was carried out using DR0401-positive EBV-transformed cells as APCs. (A) Hybridoma 216 responds specifically to a pool containing peptides in the region p230–300. (B) Hybridoma 216 recognizes only p271–285 within that pool. (C) The T cell response is DR0401 restricted as shown by specific blocking of response in the presence anti-HLA-DR antibodies.

Figure 3

Representative plots to demonstrate minimization analysis of epitope sequence. The specific epitope regions were mapped utilizing a set of variant peptides. These were designed to truncate the N and C termini of the putative core epitope as shown. The protocol used was as described for Fig. 2. (A) Hybridoma 91 recognizes LIAFTSEHS within p271–285. (B) Hybridoma 63 recognizes FTSEHSHFS within p271–285. (C) Hybridoma 55 recognizes (M)NILLQYVV within p116–130.

Figure 4

Summary of identified immunodominant epitopes of GAD65. Boxed sequences represent mapped immunogenic sequences. Thick-lined boxes correspond to immunodominant regions; thin-lined boxes correspond to single-hybridoma-recognized regions. The PEVKEK sequence refers to the area of homology between GAD65 and Coxsackie viral sequence. The black underlining shows DR0401 peptide binding motif sequences (thin line, permissive motifs; thick line, strong motifs) and gray arrowed underlining indicates DR0405 sequences (thin line, permissive motifs; thick line, strong motifs) as predicted using pmotif software and a motif from ref. .