Monoclonal Antibody Blockade of CD226 Decreases Spontaneous Diabetes in the NDO Mouse by Augmenting Treg and Diminishing Effector Functionality. Monoclonal Antibody Blockade of CD226 Decreases Spontaneous Diabetes in the NDO Mouse by Augmenting Treg and Diminishing Effector Functionality

Immunotherapeutics that modulate T cell activation represent a crucial component for inhibiting the autoimmune pathogenesis of type 1 diabetes (T1D) and improving the efficacy of β-cell replacement therapy. Here, we present a novel strategy for reducing diabetes incidence in the NOD mouse using monoclonal antibodies (mAbs) to block the T cell costimulatory receptor, CD226. Notably, female NOD mice treated with 600 µg of ⍺-CD226 mAbs between 7-8 weeks of age showed decreased disease incidence at 30 weeks and reduced insulitis severity compared to mice treated with an isotype control. Ex vivo analysis performed five weeks post-treatment revealed ⍺-CD226 mAbs persist in vivo, reducing the availability of CD226 on CD8+ T cells and Tregs. Flow cytometric analysis demonstrates that ⍺-CD226 mAbs inhibit the proliferation of both CD4+ and CD8+ T cells in vitro. Similarly, ex vivo samples had reduced CD4+ and CD8+ effector memory T cell proliferation following ⍺-CD226 mAb treatment. Splenocytes treated with ⍺-CD226 mAbs exhibited a more immunoregulatory cytokine profile with decreased IFN-γ and increased IL-10 production. This phenotype was further corroborated by 51Cr-release assays demonstrating reduced cell-mediated lympholysis (CML) of murine β-cells by ⍺-CD226 mAb-treated autoreactive cytotoxic lymphocytes. Ex vivo phenotyping of FOXP3+Helios+ Tregs revealed increased CD25 expression following ⍺-CD226 mAb treatment, with Tregs displaying augmented suppressive capacity of CD4+ responders during in vitro suppression assays. These data suggest that ⍺-CD226 mAbs both reduce T cell cytotoxicity and improve Treg function, with important therapeutic implications for the prevention or suspension of T1D. Overall design: NOD mice received three doses of either ⍺-CD226 mAb or Isotype Control between 7-8 weeks of age. At 12 weeks of age, CD3+ T cells enriched from pancreatic-draining lymph nodes (pLN) and collagenase-digested pancreas were stained with ADT antibodies before generating gene expression, V(D)J and surface protein libraries. ADT: Barcode Sequence TotalSeq-C0001 anti-mouse CD4: AACAAGACCCTTGAG TotalSeq-C0002 anti-mouse CD8⍺: TACCCGTAATAGCGT TotalSeq-C0073 anti-mouse CD44: TGGCTTCAGGTCCTA TotalSeq-C0112 anti-mouse CD62L: TGGGCCTAAGTCATC TotalSeq-C0848 anti-mouse TIGIT: GAAAGTCGCCAACAG