PhD Candidate University of Colorado - Anschutz Medical Campus - Aurora, CO Denver, Colorado, United States
Disclosure(s):
Utibeabasi Ettah, RN, B.Pharm, MS, PhD in view: No financial relationships to disclose
Introduction/Rationale: Loss of immune tolerance to β-cell antigens, including insulin and GAD65, drives type 1 diabetes (T1D). MHC class II alleles shape T cell repertoires, with DR4/DQ8 conferring susceptibility and DR15/DQ6 protection in T1D. The mechanism by which protective alleles bias regulatory T cells (Treg) selection and function is unclear. We hypothesize that DR15/DQ6 promote tolerance by selecting Tregs with a distinct, regulation-favoring TCR recognition mode
Methods: To test this, we combined structural, biophysical, and repertoire analyses of human DR15-GAD65 and DQ6-insulin–specific TCRs. Mutagenesis mapped critical peptide residues mediating binding and signaling. In vivo, antigen-specific CD4⁺ Tregs were enumerated from NOD.FoxP3-eGFP mice by flow cytometry.
Results: Structural analysis of DR15–GAD65 Treg TCRs revealed a CDR3α-dominated, peptide-centric docking topology with minimal MHCβ engagement. A van der Waals clamp at GAD65 P5Y/P7W stabilized TCR–pMHC interactions and prolonged complex lifetime despite modest equilibrium affinity, establishing a kinetic signature of tolerogenic recognition. Alanine-scanning confirmed a flexible, low-constraint peptide footprint. For DQ6–InsB10-21 TCRs, mutagenesis and binding assays indicate a C-terminal-shifted docking mode, suggesting a recognition topology favoring regulatory engagement. To test whether these signatures are characteristic of peripherally induced Tregs (pTregs) under autoimmune pressure, insulin-specific CD4⁺ Tregs were enumerated from pancreatic lymph nodes and islets of NOD.FoxP3-eGFP mice via flow cytometry with Nrp-1. Single-cell RNA-seq will be used to define clonotypes and transcriptional programs, assessing whether this unconventional recognition are features of pTregs.
Conclusion: These findings establish a structural and kinetic blueprint for protective HLA–Treg interactions, highlighting unconventional TCR footprints as mechanistic features that may enforce peripheral tolerance in T1D.
