Human leukocyte antigen (HLA)-DQ8 transdimer (HLA-DQA1*0501/DQB1*0302) confers exceptionally high risk in autoimmune diabetes. However, little is known about HLA-DQ8 transdimer-restricted CD4 T cell recognition, an event crucial for triggering HLA-DQ8 transdimer-specific anti-islet immunity. Here, we report a high degree of epitope overlap and T cell promiscuity between susceptible HLA-DQ8 and HLA-DQ8 transdimer. Despite preservation of putative residues for T cell receptor (TCR) contact, stronger disease-associated responses to cross-reactive, immunodominant islet epitopes are elicited by HLA-DQ8 transdimer. Mutagenesis at the α chain of HLA-DQ8 transdimer in complex with the disease-relevant GAD65250-266 peptide and in silico analysis reveal the DQ α52 residue located within the N-terminal edge of the peptide-binding cleft for the enhanced T cell reactivity, altering avidity and biophysical affinity between TCR and HLA-peptide complexes. Accordingly, a structurally promiscuous but nondegenerate TCR-HLA-peptide interface is pivotal for HLA-DQ8 transdimer-mediated autoimmune diabetes.