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. 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.