Multimodal single-cell analysis of human TILs across multiple tumor types reveals heterogeneity and potential opportunities for personalized immunotherapy

Abstract Immune checkpoint blockade (ICB) efficacy varies among tumor types likely due to differences in tumor infiltrating lymphocyte (TIL) composition and function within the tumor microenvironment (TME). To help understand these differences, we conducted multimodal single-cell analysis of TILs including single-cell RNA sequencing (scRNA-seq), CITE-seq (oligo-tagged antibodies), and scTCR-seq (10× Genomics) in (non-small cell lung cancer: NSCLC; head and neck squamous cell carcinoma: HNSCC; renal cell carcinoma: RCC; and breast cancer: BrCa; n=48). We found that regulatory T cell (Treg) frequency was higher in HNSCC, whereas exhausted T cells (Tex) were higher in NSCLC and RCC. In contrast to other tumor types, Tex in RCC lacked the expression of CD103, a hallmark of tissue-resident T cells. On the other hand, expression of PD-1, TIM-3, and LAG-3 were more prominent in Tex in RCC. Interestingly, Tex in HNSCC showed higher expression of TIGIT than other tumor types. Previous work has demonstrated an increased presence of CD4+CD8+ double-positive T cells (DPT) in RCC, which was associated with better overall survival. Therefore, we used CITE-seq to identify DPT and then compared the composition of DPT among different tumor types. DPT were CD39+, a marker for tumor-reactive T cells, and the vast majority were transcriptionally categorized as CD8+ T cells in RCC, whereas DPT in other tumor types are mixture of CD4+ or CD8+ T cell subsets. We also found overlap of TCR profiles between DPT and CD8+ T cell subsets (Tex, ZNF683-CD8, and GZMK-CD8) in RCC. Together, multimodal single-cell analysis of TILs highlighted heterogeneity among tumor types that may provide insight into novel strategies to treat cancer. Supported by a research grant from GlaxoSmithKline and the Providence Portland Medical Foundation. Supported by a research grant from GlaxoSmithKline and the Providence Portland Medical Foundation.