<b>Treg-specific CD226 Deletion Reduces Diabetes Incidence in NOD Mice by Improving Regulatory T Cell Stability</b>

<p>Co-stimulation serves as a critical checkpoint for T cell activation, and several genetic variants affecting co-stimulatory pathways confer risk for autoimmune diseases. A single nucleotide polymorphism (<i>rs763361</i>) in the <i>CD226</i> gene encoding a co-stimulatory receptor increases susceptibility to multiple autoimmune diseases, including type 1 diabetes. We previously found that <i>Cd226</i> knockout protected non-obese diabetic (NOD) mice from disease, but the impact of CD226 on individual immune subsets remained unclear. Our prior reports implicate regulatory T cells (Treg), as human CD226⁺ Tregs exhibit reduced suppressive function. Hence, we hypothesized that genomic <i>Cd226</i> gene deletion would increase Treg stability, and Treg-specific <i>Cd226</i> deletion would inhibit diabetes in NOD mice. Indeed, crossing NOD.<i>Cd226</i>^<em>-/-</em> and a NOD Treg-lineage tracing strain resulted in decreased pancreatic Foxp3-deficient “ex-Tregs.” We generated a novel Treg-conditional knockout (Treg^∆^{<em>Cd226</em>}) strain that displayed decreased insulitis and diabetes incidence. CD226-deficient pancreatic Tregs had increased expression of the co-inhibitory counter-receptor TIGIT. Moreover, NOD splenocytes treated with a TIGIT-Fc fusion protein exhibited reduced T cell proliferation and IFN-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 co-stimulatory pathway to halt autoimmunity.</p><p><br></p><p><b>Keywords: </b>CD226, NOD mouse, regulatory T cell, type 1 diabetes, conditional knockout, Treg stability</p><p><b><u>Highlights</u></b></p><p>· We previously found that <i>Cd226</i> genomic knockout in NOD mice reduced insulitis severity and diabetes incidence, but the impact on individual immune subsets remained unclear.</p><p>· Human CD226⁺Tregs exhibit reduced suppressive function suggesting <i>Cd226</i> gKO would increase Treg stability<u>,</u> and Treg-specific <i>Cd226</i> deletion would inhibit diabetes in NOD.</p><p>· Treg-conditional CD226 KO reduced insulitis and delayed diabetes onset in female NOD mice while CD226 gKO NODs displayed reduced ex-Tregs in pancreas and increased TIGIT expression on Tregs.</p><p>CD226/TIGIT imbalance contributes to Treg instability in NOD mice and highlights the potential for therapeutic targeting this co-stimulatory pathway in type 1 diabetes.</p>