Type 1 diabetes (T1D) is caused by the T-cell mediated destruction of the insulin-producing beta cells. The HLA genes, HLA DQ8 and HLA DQ2, confer the highest risk of developing T1D implicating CD4+ T cell responses in autoimmune beta-cell destruction. Efforts to identify the antigens and epitopes recognized by CD4+ T cells in T1D have been hampered by the technical challenges associated with detecting these rare cells in patient blood. To address this we have developed a protocol to isolate T cells from the residual pancreatic islets of deceased organ donors who suffered from T1D. Analysis of CD4+ T-cell clones from one donor, who has the high T1D risk HLA type: HLA DR3-DQ2; DR4-DQ8. We tested 53 clones from this donor for responses to 13 overlapping peptides that cover the entire sequence of human proinsulin. Fifteen of 53 clones (28%) recognized epitopes derived from the C-peptide of human proinsulin. Six distinct clones recognized the same core C-peptide epitope (PI40-50), while two clones recognized PI50-60. Analysis of the clone’s HLA restriction revealed that all, but one pair of clones with identical TCRs, were restricted by HLA DQ8 (A1*0301, B1*0302). The remaining clones were restricted by an HLA DQ8 transdimer, formed from the DQ2 alpha chain and the DQ8 beta chain (DQA1*0501, DQB1*0302). To our knowledge, this is the first analysis of the antigen specificity of human islet-infiltrating CD4+ T cells. Our work confirms that central role that CD4+ T-cell responses play in the pathogenesis of human T1D and greatly extends our knowledge of HLA DQ8 restricted T-cell responses. We anticipate that epitopes recognized by human islet-infiltrating CD4+ T cells will be strong candidates for developing effective antigen-specific therapies.