Brown Matthew E, Thirawatananond Puchong, Peters Leeana D, Kern Elizabeth J, Vijay Sonali, Sachs Lindsey K, Posgai Amanda L, Brusko Maigan A, Shapiro Melanie R, Mathews Clayton E, Bacher Rhonda, Brusko Todd M
Diabetes Institute, College of Medicine, University of Florida, Gainesville, FL, USA.
Department of Pathology, Immunology, and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA.
Diabetologia. 2025 Feb;68(2):397-418. doi: 10.1007/s00125-024-06329-8. Epub 2024 Dec 5.
AIMS/HYPOTHESIS: Immunotherapeutics targeting T cells are crucial for inhibiting autoimmune disease progression proximal to disease onset in type 1 diabetes. There is an outstanding need to augment the durability and effectiveness of T cell targeting therapies by directly restraining proinflammatory T cell subsets, while simultaneously augmenting regulatory T cell (Treg) activity. Here, we present a novel strategy for preventing 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.
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.
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. Cr-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.
目的/假设：靶向T细胞的免疫疗法对于抑制1型糖尿病发病初期自身免疫性疾病的进展至关重要。迫切需要通过直接抑制促炎T细胞亚群，同时增强调节性T细胞（Treg）活性，来提高T细胞靶向疗法的持久性和有效性。在此，我们提出一种新策略，使用靶向与1型糖尿病风险相关的T细胞共刺激受体CD226的阻断单克隆抗体，预防非肥胖糖尿病（NOD）小鼠模型中的糖尿病发生。
雌性NOD小鼠在7 - 8周龄时用抗CD226进行治疗，然后监测糖尿病发病率及治疗作用机制。
与同型对照治疗组相比，抗CD226治疗的NOD小鼠在12周时胰岛炎严重程度降低（0.84倍，p = 0.0002），在30周时疾病发病率降低（风险比0.41，p = 0.015）。治疗后5周进行的流式细胞术分析显示，抗CD226治疗小鼠脾脏中常规CD4 T细胞（0.87倍，p = 0.030）和CD8效应记忆T细胞（0.78倍，p = 0.0018）的增殖减少。胰腺Treg的表型分析显示，抗CD226治疗后CD25表达增加（2.05倍，p = 0.0073），信号转导和转录激活因子5（STAT5）磷酸化增加（1.39倍，p = 0.0007），脾脏Treg在体外对CD4应答T细胞（Tresp）的抑制增强（1.49倍，p = 0.0008，Treg:Tresp比例为1:2）。使用体外四聚体染色（0.50倍，p = 0.0317）和单细胞T细胞受体测序（0.61倍，p = 0.022）方法，抗CD226治疗的小鼠胰腺中胰岛特异性葡萄糖-6-磷酸酶催化亚基相关蛋白（IGRP）反应性CD8 T细胞的频率降低。铬释放试验表明，抗CD226治疗的自身反应性细胞毒性T淋巴细胞对β细胞的细胞介导裂解减少（0.61倍，p < 0.0001，效应细胞:靶细胞比例为1:1）。
结论/解读：CD226阻断可降低T细胞细胞毒性并改善Treg功能，是恢复1型糖尿病免疫调节的一种有针对性的合理方法。
