Inhibition of CD226 Co-Stimulation Suppresses Diabetes Development in the NOD Mouse by Augmenting Tregs and Diminishing Effector T Cell Function

Abstract

AbstractAims/hypothesisImmunotherapeutics targeting T cells are crucial for inhibiting autoimmune disease progression proximal to disease onset in type 1 diabetes. A growing number of T cell-directed therapeutics have demonstrated partial therapeutic efficacy, with anti-CD3 (α-CD3) representing the only regulatory agency-approved drug capable of slowing disease progression through a mechanism involving the induction of partial T cell exhaustion. There is an outstanding need to augment the durability and effectiveness of T cell targeting by directly restraining proinflammatory T helper type 1 (Th1) and type 1 cytotoxic CD8+T cell (Tc1) subsets, while simultaneously augmenting regulatory T cell (Treg) activity. Here, we present a novel strategy for reducing diabetes incidence in the NOD mouse model using a blocking monoclonal antibody targeting the type 1 diabetes-risk associated T cell co-stimulatory receptor, CD226.MethodsFemale NOD mice were treated with anti-CD226 between 7-8 weeks of age and then monitored for diabetes incidence and therapeutic mechanism of action.ResultsCompared to isotype-treated controls, anti-CD226 treated NOD mice showed reduced insulitis severity at 12 weeks and decreased disease incidence at 30 weeks. Flow cytometric analysis performed five weeks post-treatment demonstrated reduced proliferation of CD4+and CD8+effector memory T cells in spleens of anti-CD226 treated mice. Phenotyping of pancreatic Tregs revealed increased CD25 expression and STAT5 phosphorylation following anti-CD226, with splenic Tregs displaying augmented suppression of CD4+T cell respondersin vitro.Anti-CD226 treated mice exhibited reduced frequencies of islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP)-reactive CD8+T cells in the pancreas, using bothex vivotetramer staining and single-cell T cell receptor sequencing (scTCR-seq) approaches.51Cr-release assays demonstrated reduced cell-mediated lysis of beta-cells by anti-CD226-treated autoreactive cytotoxic T lymphocytes.Conclusions/interpretationCD226 blockade reduces T cell cytotoxicity and improves Treg function, representing a targeted and rational approach for restoring immune regulation in type 1 diabetes.Research in ContextWhat is already known about this subject?The co-stimulatory receptor CD226 is upregulated upon activation and is highly expressed on NK cell subsets, myeloid cells, and effector T cells.A single nucleotide polymorphism in CD226 (rs763361; C>T) results in a Gly307Ser missense mutation linked to genetic susceptibility for type 1 diabetes.Global knockout ofCd226and conditionalCd226knockout in FoxP3+Tregs reduced insulitis severity and diabetes incidence in NOD mice, indicating a crucial role for CD226 in disease pathogenesis.What is the key question?Can CD226 blockade reduce T cell cytotoxicity and improve Treg function to diminish diabetes incidence in NOD mice?What are the new findings?Anti-CD226 treatment reduced insulitis, decreased disease incidence, and inhibited splenic CD4+and CD8+effector memory T cell proliferation.Pancreatic Tregs from anti-CD226 treated mice exhibited increased CD25 expression; splenic Tregs displayed augmented STAT5 phosphorylation and suppressive capacityin vitro.Anti-CD226 treatment reduced IGRP-specific pancreatic CD8+T cell frequencies, and reduced autoreactive CD8+T cell-mediated lysis of beta-cellsin vitro.How might this impact on clinical practice in the foreseeable future?CD226 blockade could reduce autoreactive T cell cytotoxicity, enhance Treg function, and slow disease progression in high-risk or recent-onset type 1 diabetes cases.