Tu77 - Selective CD226 Knockout on Regulatory T Cells Reduces Diabetes in Female Non-obese Diabetic Mice

T cell co-stimulation serves as a critical checkpoint for T cell development and activation, and several genetic variants affecting co-stimulatory pathways confer risk for autoimmune diseases. A single nucleotide polymorphism in CD226 (rs763361; G307S) has been shown to increase susceptibility to type 1 diabetes (T1D), multiple sclerosis, and rheumatoid arthritis. Our group previously found that knockout of Cd226 protected non-obese diabetic (NOD) mice from disease and that CD226 activity on CD4⁺ T cells was required to confer disease through adoptive transfers into immunodeficient NOD.scid recipients. However, the impact of CD226 signaling on individual immune subsets remained unclear. Our efforts here focus on CD4⁺ regulatory T cells (Tregs) based on our work showing that human CD226⁺ Tregs exhibited reduced FOXP3⁺Helios⁺ purity and suppression following expansion. Crossing the NOD.Cd226^-/- and Treg-fate tracking strains (NOD.Foxp3-GFP-Cre.R26-YFP) resulted in increased “early” Tregs (p< 0.0001) and decreased ex-Tregs (GFP^-YFP⁺; p< 0.0001) in the pancreatic lymph nodes. We next generated a Treg-conditional KO (cKO) strain by crossing NOD.Cd226^fl/fl and NOD.Foxp3-Cre strains and observed selective CD226 knockout on Tregs. Treg cKO mice had decreased insulitis (p< 0.0001) and diabetes incidence (p=0.044) compared to control mice (NOD.Cd226^+/+.Foxp3^Cre/+). cKO mice had increased naïve (CD44^-CD62L⁺) and decreased effector/memory (CD44⁺CD62L^-) subset frequencies of Tregs (p=0.0149 and 0.0362) and conventional CD4⁺ T cells (p=0.0016 and 0.0040) compared to controls. These studies highlight the role for CD226 in controlling Treg lineage stability in the NOD mouse model of T1D and the potential for therapeutic targeting of this pathway to restore immunoregulation in autoimmune disorders.