Single cell analysis of anti-insulin CD4 T cells in pre-diabetic NOD mice and humans reveals molecular mechanisms of disease.

Background: The single residue polymorphism at b57 in HLA-DQ8 and I-A^g7 is the dominant genetic feature of T1D. The molecular mechanisms supporting this association are unknown. We had shown that a “P9 switch model” of T cell recognition was controlled by b57 for the recognition of HEL antigen; here we expand the observation to the anti-insulin response in mice in which the switch recognition of insulin B12-20 peptide drives the early autoreactivity in the islet. HLA-DQ8 insulin tetramers have been developed to study anti-islet reactivity in human blood samples.

 Methods: Utilizing I-A^g7 and HLA-DQ8 tetramers, specific for insulin peptides, the anti-insulin response in mice and T1D patients was monitored. Antigen-specific CD4 T cells were sorted from blood, spleen, pancreatic lymph nodes, and islets of NOD mice, and peripheral blood of humans for single cell gene expression analysis and sequencing.

 Results: In mice, early islet infiltration is dominated by CD4 T cells specific for the 12-20 peptide of insulin which retains no charge at the P9 position. This population is the first to present the hallmarks of activation and effector functions in islets. As predicted, the majority of the TCR CDR3b of these cells display an acidic residue in their N-terminal portion and recognition is totally dependent on this residue. Gene expression profiling revealed a cytokine signature and absence of TCR activation for lymph node resident cells, whereas islet infiltrating cells presented a typical TCR activation profile with upregulation of co-receptors, signaling molecules and cytokines defining T_H1 cells. Associated to this differential expression profile, paired ab TCR sequencing revealed polyclonality of the anti-insulin response in the lymph node, and marked oligoclonality in the islets, indicating a local in situ expansion. Importantly, in both mouse and man, activated anti-insulin CD4 T cells could be isolated from peripheral blood.