Exhausted T cells in the tumor microenvironment are major targets of immunotherapies. However, the exhaustion status of CD8⁺ tumor-infiltrating lymphocytes (TILs) in bladder cancer has not been comprehensively evaluated. Herein, we examined distinct exhaustion status of CD8⁺ TILs based on the level of programmed cell death-1 (PD-1) and thymocyte selection-associated high mobility group box protein (TOX) expression in urothelial bladder cancer. We also evaluated the reinvigoration of exhausted CD8⁺ TILs upon ex vivo treatment with inhibitory checkpoint blockers. TOX-expressing PD-1^highCD8⁺ TILs had the highest expression of immune checkpoint receptors (ICRs), the most terminally exhausted features, and the highest tumor antigen reactivity among PD-1⁺CD8⁺ TILs. Bladder cancer patients with a high percentage of PD-1^highTOX⁺CD8⁺ TILs had more progressed T-cell exhaustion features and higher programmed death-ligand 1 expression in tumor tissues. TIGIT was the most frequent co-expressed ICR on PD-1⁺CD8⁺ TILs, and TIGIT blockade enhanced the PD-1 blockade-mediated cytokine production by CD8⁺ TILs from bladder cancer patients. Our findings provide an improved understanding of the heterogeneous exhaustion status of CD8⁺ TILs and additional immunotherapy strategies to improve outcomes of bladder cancer patients.

肿瘤微环境中耗尽的T细胞是免疫疗法的主要目标。但是，尚未全面评估膀胱癌中CD8 ⁺肿瘤浸润淋巴细胞（TILs）的衰竭状态。在本文中，我们根据尿路上皮膀胱癌中程序性细胞死亡1（PD-1）和与胸腺细胞选择相关的高迁移率族框蛋白（TOX）表达的水平，检查了CD8 ⁺ TILs的衰竭状态。我们还评估了用抑制性检查点阻断剂进行离体治疗后用尽的CD8 ⁺ TIL的恢复活力。表达TOX的PD-1^高CD8 ⁺TILs在PD-1 ⁺ CD8 ⁺ TILs中具有最高的免疫检查点受体（ICR）表达，最末端耗尽的特征以及最高的肿瘤抗原反应性。PD-1^高TOX ⁺ CD8 ⁺ TILs百分比高的膀胱癌患者在肿瘤组织中具有更多的进展性T细胞衰竭功能和更高的程序性死亡配体1表达。TIGIT是PD-1 ⁺ CD8 ⁺ TILs上最常见的共表达ICR ，TIGIT阻断增强了膀胱癌患者CD8 ⁺ TILs的PD-1阻断介导的细胞因子产生。我们的发现提供了对CD8异质性耗竭状态的更好理解⁺ TIL和其他免疫疗法可改善膀胱癌患者的预后。