G6PC2, also known as islet-specific glucose 6-phosphatase catalytic subunit-related protein (IGRP), is a major target of autoreactive CD8(+) T cells in both diabetic human subjects and the non-obese diabetic (NOD) mouse. However, in contrast to the abundant literature regarding the CD8(+) response to this antigen, much less is known about the potential involvement of IGRP-reactive CD4(+) T cells in diabetogenesis. The single previous study that examined this question in NOD mice was based upon a candidate epitope approach and identified three I-A(g7)-restricted epitopes that each elicited spontaneous responses in these animals. However, given the known inaccuracies of MHC class II epitope prediction algorithms, we hypothesized that additional specificities might also be targeted. To address this issue, we immunized NOD mice with membranes from insect cells overexpressing full-length recombinant mouse IGRP and measured recall responses of purified CD4(+) T cells using a library of overlapping peptides encompassing the entire 355-aa primary sequence. Nine peptides representing 8 epitopes gave recall responses, only 1 of which corresponded to any of the previously reported sequences. In each case proliferation was blocked by a monoclonal antibody to I-A(g7), but not the appropriate isotype control. Consistent with a role in diabetogenesis, proliferative responses to 4 of the 9 peptides (3 epitopes) were also detected in CD4(+) T cells purified from the pancreatic draining lymph nodes of pre-diabetic female animals, but not from peripheral lymph nodes or spleens of the same animals. Intriguingly, one of the newly identified spontaneously reactive epitopes (P8 [IGRP(55-72)]) is highly conserved between mice and man, suggesting that it might also be a target of HLA-DQ8-restricted T cells in diabetic human subjects, an hypothesis that we are currently testing.