Conclusions/interpretation
CD226 blockade reduces T cell cytotoxicity and improves Treg function, representing a targeted and rational approach for restoring immune regulation in type 1 diabetes.
Methods
Female NOD mice were treated with anti-CD226 at 7-8 weeks of age and then monitored for diabetes incidence and therapeutic mechanism of action.
Results
Compared with isotype-treated controls, anti-CD226-treated NOD mice showed reduced insulitis severity (0.84-fold, p=0.0002) at 12 weeks and decreased disease incidence (HR 0.41, p=0.015) at 30 weeks. Flow cytometric analysis performed 5 weeks post treatment demonstrated reduced proliferation of conventional CD4+ T cells (0.87-fold, p=0.030) and CD8+ (0.78-fold, p=0.0018) effector memory T cells in spleens of anti-CD226-treated mice. Phenotyping of pancreatic Tregs revealed increased CD25 expression (2.05-fold, p=0.0073) and signal transducer and activator of transcription 5 (STAT5) phosphorylation (1.39-fold, p=0.0007) following anti-CD226, with splenic Tregs displaying augmented suppression of CD4+ responder T cells (Tresps) (1.49-fold, p=0.0008, 1:2 Treg:Tresp) in 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 both ex vivo tetramer staining (0.50-fold, p=0.0317) and single-cell T cell receptor sequencing (0.61-fold, p=0.022) approaches. 51Cr-release assays demonstrated reduced cell-mediated lysis of beta cells (0.61-fold, p<0.0001, 1:1 effector:target) by anti-CD226-treated autoreactive cytotoxic T lymphocytes. Conclusions/interpretation: CD226 blockade reduces T cell cytotoxicity and improves Treg function, representing a targeted and rational approach for restoring immune regulation in type 1 diabetes.