Orthogonal Gene Engineering Enables Novel Synthetic States of Powerful Tumor-rejecting CD8+ T Cells Escaping Canonical Exhaustion

To date, no immunotherapy approaches have managed to fully overcome T-cell exhaustion, which remains a mandatory fate for chronically activated effector cells and a major therapeutic challenge. Understanding how to reprogram CD8+ TILs away from exhausted effector states remains an elusive goal. Our work provides for the first-time evidence that orthogonal gene-engineering of T cells to secrete an IL-2-variant binding the IL-2R?? and the alarmin IL-33 reprogrammed adoptively transferred T cells to acquire a novel, synthetic state, which deviated from canonical exhaustion and displayed superior effector functions. These cells successfully overcame homeostatic barriers in the host, and led – in the absence of lymphodepletion or exogenous cytokine support – to high levels of engraftment and tumor regression. Our work unlocks the novel opportunity of rationally engineering synthetic CD8+ T-cell states endowed with the ability to avoid exhaustion and control advanced solid tumors. Overall design: CD45+ bulk TILs were independently sorted at at days 5 (T1), 12 (T2) and 26 (T3) post cell transfer Groups: Non-Treated tumor-bearing mice (G5); Mice received ACT using Untransduced OT1 T cells (G1);Mice received ACT using PD1d/IL2V-secreting OT1 (G2); Mice received ACT using PD1d/33-secreting OT1 (G3); Mice received ACT using PD1d/2V/33-secreting OT1 (G4)

迄今为止，尚无任何免疫治疗策略能够完全克服T细胞耗竭（T-cell exhaustion）——这仍是慢性活化效应T细胞的必然命运，也是临床治疗的重大难题。探索如何将CD8+肿瘤浸润淋巴细胞（tumor-infiltrating lymphocytes, TILs）重编程以脱离耗竭效应状态，仍是一个难以实现的目标。

本研究首次提供证据表明：对T细胞进行正交基因工程改造，使其分泌可结合白细胞介素2受体（IL-2R）与警报素IL-33的IL-2变体，可将过继转移的T细胞重编程为一种全新的合成状态——该状态脱离了经典的耗竭表型，并展现出更优异的效应功能。此类细胞可成功克服宿主体内的稳态屏障，且无需淋巴细胞清除或外源性细胞因子支持，即可实现高比例植入并促使肿瘤消退。

本研究为理性设计合成型CD8+ T细胞状态开辟了全新机遇——此类细胞可规避耗竭并控制晚期实体瘤。

实验整体设计：于细胞移植后第5天（T1）、第12天（T2）及第26天（T3）独立分选CD45+总肿瘤浸润淋巴细胞。实验分组如下：未处理的荷瘤小鼠（G5）；接受未转导OT1 T细胞过继细胞治疗（adoptive cell therapy, ACT）的小鼠（G1）；接受分泌PD1d/IL2V的OT1 T细胞过继细胞治疗的小鼠（G2）；接受分泌PD1d/33的OT1 T细胞过继细胞治疗的小鼠（G3）；接受分泌PD1d/2V/33的OT1 T细胞过继细胞治疗的小鼠（G4）。