Intratumoral CD4+ T cells mediate anti-tumor cytotoxicity in human bladder cancer. Intratumoral CD4+ T cells mediate anti-tumor cytotoxicity in human bladder cancer

Responses to anti-PD-1 immunotherapy occur but are infrequent in bladder cancer. The specific T cells that mediate tumor rejection are unknown. T cells from human bladder tumors and non-malignant tissue were assessed with single-cell RNA and paired T cell receptor (TCR) sequencing of 30,604 T cells from 7 patients. We find that the states and repertoire of CD8+ T cells are not altered in tumors compared with non-malignant tissues. In contrast, single-cell analysis of CD4+ T cells demonstrates several tumor-specific states, including multiple distinct states of regulatory T cells. Surprisingly, we also find multiple cytotoxic CD4+ T cell states that are clonally expanded. These CD4+ T cells can kill autologous tumor in an MHC class II-dependent fashion and are suppressed by regulatory T cells. Further, a gene signature of cytotoxic CD4+ T cells in tumors predicts a clinical response in 244 metastatic bladder cancer patients treated with anti-PD-L1. Overall design: Fresh bladder tumors and adjacent normal bladder tissue were obtained from surgical resection (cystectomy). These were treated before surgery with anti-PD-L1 therapy, chemotherapy, or no systemic therapy. Fresh tissues were digested to single cells and subjected to droplet-based single-cell RNA sequencing (scRNA-seq, 10X Genomics). Libraries for T cell receptor (TCR sequencing) were generated using consensus TCR primers from the full-length cDNA generated during 10X Genomics RNAseq library preparation.

抗PD-1免疫治疗（anti-PD-1 immunotherapy）在膀胱癌中虽可起效，但响应率较低。介导肿瘤排斥反应的特异性T细胞仍未明确。研究人员对7名患者的30604个T细胞开展单细胞RNA测序与配对T细胞受体（T cell receptor, TCR）测序，以此分析源自人类膀胱肿瘤及非恶性组织的T细胞。
我们发现，与非恶性组织相比，肿瘤组织中CD8+ T细胞（CD8+ T cells）的状态与受体库并未发生显著改变。与之相反，对CD4+ T细胞（CD4+ T cells）的单细胞分析揭示了多种肿瘤特异性状态，包括多种不同亚型的调节性T细胞（regulatory T cells）。令人意外的是，我们还发现了多个克隆扩增的细胞毒性CD4+ T细胞（cytotoxic CD4+ T cells）状态。这类CD4+ T细胞可通过依赖MHC II类分子（MHC class II）的方式杀伤自体肿瘤，且会被调节性T细胞抑制。
此外，肿瘤内细胞毒性CD4+ T细胞的基因特征可预测244名接受抗PD-L1（anti-PD-L1）治疗的转移性膀胱癌患者的临床响应情况。
实验设计：研究人员通过外科膀胱切除术（cystectomy）获取新鲜膀胱肿瘤组织及邻近正常膀胱组织。部分患者在术前接受了抗PD-L1治疗、化疗，或未接受任何全身治疗。将新鲜组织消化为单细胞悬液后，采用基于液滴的单细胞RNA测序（droplet-based single-cell RNA sequencing, scRNA-seq, 10X Genomics）进行检测。T细胞受体测序文库则利用10X Genomics RNA测序文库制备过程中生成的全长cDNA，通过共识TCR引物构建而成。