Treg-Specific CD226 Deletion Reduces Diabetes Incidence in NOD Mice by Improving Regulatory T-Cell Stability

Costimulation serves as a critical checkpoint for T-cell activation, and several genetic variants affecting costimulatory pathways confer risk for autoimmune diseases. A single nucleotide polymorphism (rs763361) in the CD226 gene encoding a costimulatory receptor increases susceptibility to multiple autoimmune diseases, including type 1 diabetes. We previously found that Cd226 knockout protected NOD mice from disease, but the impact of CD226 on individual immune subsets remained unclear. Our prior reports implicate regulatory T cells (Tregs), as human CD226(+) Tregs exhibit reduced suppressive function. Hence, we hypothesized that genomic Cd226 gene deletion would increase Treg stability and that Treg-specific Cd226 deletion would inhibit diabetes in NOD mice. Indeed, crossing NOD.Cd226(−/−) and a NOD Treg-lineage tracing strain resulted in decreased pancreatic Foxp3-deficient “ex-Tregs.” We generated a novel Treg-conditional knockout (Treg(Δ)(Cd226)) strain that displayed decreased insulitis and diabetes incidence. CD226-deficient pancreatic Tregs had increased expression of the coinhibitory counter-receptor T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT). Moreover, NOD splenocytes treated with TIGIT-Fc fusion protein exhibited reduced T-cell proliferation and interferon-γ production following anti-CD3/CD28 stimulation. This study demonstrates that a CD226/TIGIT imbalance contributes to Treg instability in NOD mice and highlights the potential for therapeutic targeting this costimulatory pathway to halt autoimmunity. ARTICLE HIGHLIGHTS: We previously found that Cd226 genomic knockout (gKO) in NOD mice reduced insulitis severity and diabetes incidence, but the impact on individual immune subsets remained unclear. Human CD226(+) regulatory T cells (Tregs) exhibit reduced suppressive function, suggesting Cd226 gKO would increase Treg stability, and Treg-specific Cd226 deletion would inhibit diabetes in NOD mice. Treg-conditional Cd226 KO reduced insulitis and delayed diabetes onset in female NOD mice, while Cd226 gKO NOD mice displayed reduced Foxp3-deficient Tregs in pancreas and increased T-cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT) expression on Tregs. CD226/TIGIT imbalance contributes to Treg instability in NOD mice and highlights the potential for therapeutic targeting this costimulatory pathway in type 1 diabetes.