<p>All relevant data values for figures.</p>

CD8+ T cells are capable of specifically targeting and eliminating malignant tumor cells, but tumor cells can develop resistance mechanisms to escape CD8+ T cell-mediated killing. Here, we performed a whole genome CRISPR-Cas9 knockout screen under CD8+ T cells pressure and identified the E3 ubiquitin ligase CUL5 as an essential factor required for escaping CD8+ T cells killing in bladder cancer cells. We found that CUL5 knockout promoted the sensitivity of bladder cancer cells to CD8+ T cell-mediated killing both in vivo and in vitro. Mechanistically, CUL5 loss reduced the ubiquitination of PTBP1, which regulated alternative splicing of RUBCN pre-mRNA and led to an increase in the levels of the RUBCN-S isoform, thereby preventing immune evasion of bladder cancer cells by inhibiting autophagy. Importantly, CUL5 knockout significantly enhanced the efficacy of anti-PD-1 immunotherapy in a xenograft model. Collectively, these findings reveal a novel mechanism of bladder cancer immune evasion, providing potential targets for cancer immunotherapy.

CD8+ T细胞能够特异性靶向并清除恶性肿瘤细胞，但肿瘤细胞可发展出耐药机制以逃逸CD8+ T细胞介导的杀伤。本研究在CD8+ T细胞筛选压力下开展全基因组CRISPR-Cas9基因敲除筛选，鉴定出E3泛素连接酶CUL5是膀胱癌细胞逃逸CD8+ T细胞杀伤所必需的关键因子。研究发现，敲除CUL5可在体内及体外增强膀胱癌细胞对CD8+ T细胞介导杀伤的敏感性。从机制层面而言，CUL5缺失降低了PTBP1的泛素化水平，后者可调控RUBCN前体mRNA的可变剪接，使RUBCN-S亚型的表达水平升高，进而通过抑制自噬阻碍膀胱癌细胞的免疫逃逸。尤为重要的是，在异种移植模型中，敲除CUL5可显著增强抗PD-1免疫治疗的疗效。综上，本研究揭示了膀胱癌细胞免疫逃逸的全新机制，为肿瘤免疫治疗提供了潜在靶点。