{"doi":"10.1101/2024.07.16.603756","title":"Inhibition of CD226 Co-Stimulation Suppresses Diabetes Development in the NOD Mouse by Augmenting Tregs and Diminishing Effector T Cell Function","abstract":"Abstract Aims/hypothesis Immunotherapeutics 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. Methods Female NOD mice were treated with anti-CD226 between 7-8 weeks of age and then monitored for diabetes incidence and therapeutic mechanism of action. Results Compared 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 responders 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 and single-cell T cell receptor sequencing (scTCR-seq) approaches. 51 Cr-release assays demonstrated reduced cell-mediated lysis of beta-cells 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. Research in Context What 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 of Cd226 and conditional Cd226 knockout 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 capacity in vitro . Anti-CD226 treatment reduced IGRP-specific pancreatic CD8 + T cell frequencies, and reduced autoreactive CD8 + T cell-mediated lysis of beta-cells in 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2024,"id":488747,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9627,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":678329,"name":"Puchong Thirawatananond","orcid":"0000-0003-4973-7380","position":1,"is_corresponding":false},{"id":356514,"name":"Leeana D. Peters","orcid":"0000-0002-7878-0014","position":2,"is_corresponding":false},{"id":1333698,"name":"Elizabeth J. Kern","orcid":null,"position":3,"is_corresponding":false},{"id":1333699,"name":"S. Vijay","orcid":null,"position":4,"is_corresponding":false},{"id":880165,"name":"Lindsey K. Sachs","orcid":null,"position":5,"is_corresponding":false},{"id":251267,"name":"Amanda L. Posgai","orcid":"0000-0002-9491-0958","position":6,"is_corresponding":false},{"id":356517,"name":"Maigan A. Brusko","orcid":"0000-0002-4331-2202","position":7,"is_corresponding":false},{"id":479037,"name":"Melanie R. Shapiro","orcid":"0000-0003-2090-0877","position":8,"is_corresponding":false},{"id":389036,"name":"Clayton E. Mathews","orcid":"0000-0002-8817-6355","position":9,"is_corresponding":false},{"id":86476,"name":"Rhonda Bacher","orcid":"0000-0001-5787-476X","position":10,"is_corresponding":false},{"id":356521,"name":"Todd M. Brusko","orcid":"0000-0003-2878-9296","position":11,"is_corresponding":false},{"id":729988,"name":"Matthew E. Brown","orcid":"0000-0001-8230-5243","position":0,"is_corresponding":true}],"reference_count":97,"raw_metadata":null,"created_at":"2026-07-19T02:08:19.655720Z","pmid":"39071293","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}