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.

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.

Graphical Abstract

中文翻译：

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抑制 CD226 共刺激通过增强调节性 T 细胞和减少效应 T 细胞功能来抑制 NOD 小鼠的糖尿病发展

 目标/假设

靶向 T 细胞的免疫疗法对于抑制 1 型糖尿病发病近端的自身免疫性疾病进展至关重要。迫切需要通过直接抑制促炎 T 细胞亚群来增强 T 细胞靶向疗法的持久性和有效性，同时增强调节性 T 细胞 （Treg） 活性。在这里，我们提出了一种在 NOD 小鼠模型中预防糖尿病发病率的新策略，使用靶向 1 型糖尿病风险相关 T 细胞共刺激受体 CD226 的阻断单克隆抗体。

 方法

雌性 NOD 小鼠在 7-8 周龄时接受抗 CD226 治疗，然后监测糖尿病发病率和治疗作用机制。

 结果

与同种型处理的对照相比，抗 CD226 处理的 NOD 小鼠在 12 周时显示胰岛素严重程度降低 （0.84 倍，p = 0.0002），在 30 周时疾病发生率降低 （HR 0.41，p = 0.015）。 治疗后 5 周进行的流式细胞术分析显示，抗 CD226 处理小鼠脾脏中常规 CD4⁺ T 细胞 （0.87 倍，p=0.030） 和 CD8⁺ （0.78 倍，p=0.0018） 效应记忆 T 细胞的增殖减少。胰腺 Tregs 的表型分析显示，抗 CD226 后 CD25 表达增加（2.05 倍，p=0.0073）和信号转导和转录激活因子 5 （STAT5） 磷酸化（1.39 倍，p=0.0007），脾脏 Tregs 显示对 CD4⁺ 反应性 T 细胞 （Tresps） 的抑制增强（1.49 倍，p=0.0008,1：2 Treg：Tresp）体外抗 CD226 处理的小鼠表现出胰岛特异性葡萄糖-6-磷酸酶催化频率降低胰腺中亚基相关蛋白 （IGRP） 反应性 CD8 ⁺ T 细胞，使用离体四聚体染色 （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。⁵¹Cr 释放试验显示抗 CD226 处理的自身反应性细胞毒性 T 淋巴细胞对 β 细胞介导的裂解减少 （0.61 倍，p<0.0001,1：1 效应子：靶标）。

结论/解释

CD226 阻断可降低 T 细胞细胞毒性并改善 Treg 功能，代表了恢复 1 型糖尿病免疫调节的靶向和合理方法。

 图形摘要