Multiplexed single-cell analysis reveals prognostic and nonprognostic T cell types in human colorectal cancer

Abstract

Clinical outcomes in colorectal cancer (CRC) have been correlated with T cell infiltrates, but the specific populations of T cells, their functions, and how they influence clinical outcomes remains unclear. To comprehensively investigate the diverse phenotype and function of T cells in CRC, 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. Our single-cell analysis identified phenotypically and functionally distinguishable effector CD4 + and CD8 + T celltypes within human tumors. We employed single-cell gene signatures from these T cell subsets to query the TCGA database to assess the prognostic significance of these subsets. Among CD8 + Tcell 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) with a more dysfunctional phenotype, were not associated with good clinical outcomes, despite high co-expression of CD39 and CD103, markers which denote tumor-reactivity. Together, this suggests that tumor-reactive cytotoxic T cells are effectively targeted to the tumor, yet their presence alone does not contribute to anti-tumor activity due to their impaired function, as reflected in clinical outcomes. 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. These findings highlight the potential utility of these 4 subpopulations in predicting clinical outcomes independent of stage. Furthermore, 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.

Automated protein-protein structure prediction of the T cell receptor-peptide major histocompatibility complex

“…To design TCRs to target the CEA 571-579 peptide restricted to the HLA-A2 (MHC), TCR clonotypes were identified utilizing the single cell RNA V(D)J sequenced T cells resected from colorectal tumors of 16 CRC patients (21). This technique identifies TCR clonotypes by matching the amino acids from the CDR3 regions of the and β chain of the TCR (18)(19)(20)(21). First, T cells were identified by the expression of CD3D and CD8A (and the absence of CD4 expression), as only CD8+ T cells can bind to the HLA-A2 (MHC).…”

“…T lymphocyte single cell RNA-Seq data was made available to us from the Han group and has been previously published (20,46). Raw data was first put through a quality control process to exclude cells with less than 200 unique genes, more than 7,500 unique genes, and/or more than 10% mitochondrial gene expression.…”

“…Addressing the second challenge has been difficult as the identification of patientspecific TCR repertoires has involved methods that are low-throughput or limited to a single chain (15)(16)(17). However, recent breakthroughs in single-cell sequencing allow determination of the CDR3 regions of the and β chain of the TCR in a highthroughput manner (18)(19)(20)(21). This technological breakthrough facilitates an unprecedented exploration of the vast TCR information space and allows the scientific community to refocus attention on fundamental questions related to recombination, maturation, and intersecting diversity of patient-specific TCR repertoires.…”

Intestinal cellular heterogeneity and disease development revealed by single-cell technology

“…Treatment of a CD40activating antibody could expand Th1-like and CD8 + memory T cells in colon cancer (Zhang et al, 2020a). Furthermore, the scRNA-seq analysis of 37,931 T cells from 16 CRC patients suggests the phenotypically and functionally distinguishable CD4 + and CD8 + effector T cell types are associated with clinical outcomes (Masuda et al, 2022). The GZMK + /KLRG1 + cytotoxic CD8 + T cells with a less dysfunctional phenotype were enriched in CRC patients with good outcomes.…”

Prioritizing prognostic-associated subpopulations and individualized recurrence risk signatures from single-cell transcriptomes of colorectal cancer

“…tumor cytotoxicity 39 . Besides, the results of other cell subpopulations were consistent with previous studies (Figure 3C).…”