Type 1 Diabetes (T1D) is an autoimmune disease marked by direct elimination of insulin-producing β cells by autoreactive T effectors. Recent T1D clinical trials utilizing autologous Tregs transfers to restore immune balance and improve disease has prompted us to design a novel Tregs-based antigen-specific T1D immunotherapy. We engineered a Chimeric Antigen Receptor (CAR) expressing a single-chain Fv recognizing the human pancreatic endocrine marker, HPi2. Human T cells, transduced with the resultant HPi2-CAR, proliferated and amplified Granzyme B accumulation when co-cultured with human, but not mouse β cells. Furthermore, following exposure of HPi2-CAR transduced cells to islets, CD8⁺ lymphocytes demonstrated enhanced CD107a (LAMP-1) expression, while CD4⁺ cells produced increased levels of IL-2. HPi2-CAR Tregs failed to maintain expansion due to a persistent tonic signaling from the CAR engagement to unexpectantly HPi2 antigen present on Tregs. Overall, we show lack of functionality of HPi2-CAR and highlight the importance of careful selection of CAR recognition driver for the sustainable activity and expandability of engineered T cells.

1 型糖尿病 (T1D) 是一种自身免疫性疾病，其特征是自身反应性 T 效应器直接消除产生胰岛素的 β 细胞。最近利用自体 Tregs 转移来恢复免疫平衡和改善疾病的 T1D 临床试验促使我们设计一种新型的基于 Tregs 的抗原特异性 T1D 免疫疗法。我们设计了一种嵌合抗原受体 (CAR)，它表达了一种识别人类胰腺内分泌标记物 HPi2 的单链 Fv。用所得 HPi2-CAR 转导的人类 T 细胞在与人类（而非小鼠 β 细胞）共培养时增殖并放大颗粒酶 B 的积累。此外，将 HPi2-CAR 转导细胞暴露于胰岛后，CD8 ⁺淋巴细胞表现出增强的 CD107a (LAMP-1) 表达，而 CD4 ⁺细胞产生更高水平的 IL-2。HPi2-CAR Tregs 未能维持扩张，因为从 CAR 接合到 Treg 上意外存在的 HPi2 抗原的持续强直信号。总的来说，我们展示了 HPi2-CAR 缺乏功能，并强调了仔细选择 CAR 识别驱动程序对工程 T 细胞的可持续活动和可扩展性的重要性。