Osr2 functions as a mechanical checkpoint to augment CD8+ T cell exhaustion [TIL_scRNA_CD8]

The alteration in extracellular matrix (ECM) architecture and stiffness becomes one of the hallmarks of cancer. Whether the mechanical property of ECM contributes to the functionality of CD8+ T cells, a key component of anti-tumor response, remains largely unknown. Here we report that mechanical stress drives T cell exhaustion program via Osr2, a transcription factor that had been poorly investigated in T cells. Osr2 is highly and selectively induced in CD8+ T cells when encountering stiff matrix or mechanical force signaling (MFS). Integrated genetic and multi-omics analyses reveals that Osr2 is enriched in the terminal exhausted tumor infiltrating lymphocytes and its expression is modulated by the combined TCR and MFS mediated by Piezo1/Calcium/CREB axis. Importantly, depletion of Piezo1 or Osr2 alleviates T cell exhaustion, which is associated with enhanced anti-tumor immunity mediated by antigen specific T cells or CAR-T cells in solid tumor models. On the contrary, forced Osr2 expression in CD8+ T cells augment their exhaustion and dampens tumor control. In agreement with these observations, high Osr2 expression is correlated with poor prognosis in multiple human malignancies. Mechanistically, Osr2 recruits HDAC3, which in turn establishes epigenetic reprogramming to suppress cytotoxic genes expression and to promote CD8+T cell exhaustion. Thus, our results unravel a mechanical signaling cascade centered by Osr2 that serves as a key driver for CD8+ T cell exhaustion and suggest that mechanical checkpoints could be promising targets for cancer immunotherapy. Overall design: Single cell transcriptional features of WT OT-I T cells following adoptive co-transfer into MC38-OVA bearing hosts for 14 days.

细胞外基质（extracellular matrix, ECM）的结构与刚度改变是癌症的标志性特征之一。ECM的机械特性是否会影响CD8+ T细胞（CD8+ T cells）——抗肿瘤免疫应答的关键组成部分——的功能，目前仍未明确。本研究发现，机械应激通过Osr2驱动T细胞耗竭程序，Osr2是一种此前在T细胞中研究较少的转录因子（transcription factor）。当CD8+ T细胞遭遇刚性基质或机械力信号（mechanical force signaling, MFS）时，Osr2会被高度且选择性地诱导表达。整合遗传与多组学分析（multi-omics analyses）显示，Osr2在终末耗竭肿瘤浸润淋巴细胞（terminal exhausted tumor infiltrating lymphocytes）中富集，其表达受Piezo1/钙离子/CREB信号轴（Piezo1/Calcium/CREB axis）介导的T细胞受体（T cell receptor, TCR）与机械力信号共同调控。值得注意的是，在实体瘤模型中，敲除Piezo1或Osr2可缓解T细胞耗竭，这与抗原特异性T细胞或嵌合抗原受体T细胞（chimeric antigen receptor T cells, CAR-T）介导的增强型抗肿瘤免疫密切相关。反之，在CD8+ T细胞中强制过表达Osr2会加剧其耗竭，并削弱肿瘤控制效果。与上述观察一致，在多种人类恶性肿瘤中，高表达Osr2与不良预后相关。从机制层面来看，Osr2会招募组蛋白去乙酰化酶3（histone deacetylase 3, HDAC3），进而建立表观遗传重编程（epigenetic reprogramming）以抑制细胞毒性基因（cytotoxic genes）的表达，并促进CD8+ T细胞耗竭。因此，本研究揭示了以Osr2为核心的机械信号级联反应，其作为CD8+ T细胞耗竭的关键驱动因素，同时提示机械检查点（mechanical checkpoints）有望成为肿瘤免疫治疗（cancer immunotherapy）的潜在靶点。整体实验设计：将野生型OT-I T细胞（WT OT-I T cells）过继共转输至携带MC38-OVA的宿主小鼠，14天后采集样本进行单细胞转录特征（single cell transcriptional features）分析。