Tu81 - Multiplexed Single-cell Analysis Reveals Dysfunctional CD8+ CD103+ T Cells and Negatively Prognostic CD38+ Regulatory T Cells in Human Colorectal Cancer

Although T-cell infiltrates correlate with clinical outcomes in CRC, different T cell types, their functions and their influence on clinical outcomes remain unclear. Using a droplet-based single cell sequencing, we profiled 37,931 single T cells from tumors and adjacent normal colon of 16 treatment-naïve CRC patients with respect to transcriptome, TCR sequence, and 23 cell surface markers. Gene set enrichment analysis enabled us to link gene signature of each identified T cell type within tumors to the TCGA database.

 Among CD8⁺ T-cell infiltrates, we found two distinct cytotoxic T cell types differentiated into clonally-expanded exhausted T cells. GZMK⁺ KLRG1⁺ cytotoxic T cells with a less dysfunctional phenotype were enriched in CRC patients with good outcomes. Strikingly, GNLY⁺ CD103⁺ cytotoxic T cells, including intraepithelial lymphocytes (IELs), were not associated with good clinical outcomes, despite high co-expression of CD39 and CD103, markers which denote tumor-reactivity, possibly due to its dysfunctional phenotype.

 Among CD4⁺ T cell-infiltrates, we found two distinct regulatory T cells (Treg) subtypes associated with opposite clinical outcomes. While total Tregs, predominantly Helios⁺ cells, were associated with good outcomes, Helios^- CD38⁺ peripherally-induced Treg cells (pTregs) were strongly associated with bad outcomes independent of stage. CD38⁺ pTregs, which shared gene signatures with Th17 cells, possessed a highly suppressive phenotype, suggesting they are the elusive Treg population that inhibits anti-tumor immunity in CRC.  In summary, these observations support the potential for novel CRC therapies directed at CD38⁺ pTregs or CD8⁺ CD103⁺ T cells to augment existing T cell-targeted immunotherapies.