Cancer-specific tissue-resident memory T-cells express ZNF683 in colorectal cancer

Abstract

Background: Tissue-resident memory T (Trm) cells are associated with cytotoxicity not only in viral infection and autoimmune disease pathologies but also in many cancers. Tumour-infiltrating CD103⁺ Trm cells predominantly comprise CD8 T cells that express cytotoxic activation and immune checkpoint molecules called exhausted markers. This study aimed to investigate the role of Trm in colorectal cancer (CRC) and characterise the cancer-specific Trm.

Methods: Immunochemical staining with anti-CD8 and anti-CD103 antibodies for resected CRC tissues was used to identify the tumour-infiltrating Trm cells. The Kaplan-Meier estimator was used to evaluate the prognostic significance. Cells immune to CRC were targeted for single-cell RNA-seq analysis to characterise cancer-specific Trm cells in CRC.

Results: The number of CD103⁺/CD8⁺ tumour-infiltrating lymphocytes (TILs) was a favourable prognostic and predictive factor of the overall survival and recurrence-free survival in patients with CRC. Single-cell RNA-seq analysis of 17,257 CRC-infiltrating immune cells revealed a more increased zinc finger protein 683 (ZNF683) expression in cancer Trm cells than in noncancer Trm cells and in high-infiltrating Trm cells than low-infiltrating Trm in cancer, with an upregulated T-cell receptor (TCR)- and interferon-γ (IFN-γ) signalling-related gene expression in ZNF683⁺ Trm cells.

Conclusions: The number of CD103⁺/CD8⁺ TILs is a prognostic predictive factor in CRC. In addition, we identified the ZNF683 expression as one of the candidate markers of cancer-specific Trm cells. IFN-γ and TCR signalling and ZNF683 expression are involved in Trm cell activation in tumours and are promising targets for cancer immunity regulation.

Fig. 1. CD103, which drives Trm cell generation, correlates with lymphatic activation in CRC.

a ITGAE-correlated genes encoding CD103 in TCGA-COAD and TCGA-READ. b Bar graph showing enriched terms across the gene sets correlated with ITGAE (correlation coefficient >0.4), coloured by P values. c Bean plots of cytotoxic cytokines, immune checkpoint molecules, and Trm cells are considered core genes of CRC in TCGA. The genes were categorised according to the median mRNA expression of CD8A and ITGAE as cut-off values into high CD8A and high ITGAE (H–H), high CD8A and low ITGAE (H–L), low CD8A and high ITGAE (L–H), and low CD8A and low ITGAE (L–L) groups. d Kaplan–Meier curve of OS for the four groups. e Immunohistochemical staining for CD103 at invasive margins of resected CRC tissue (left: high invasion; right: low invasion). f Kaplan–Meier curves of OS and RFS by CD103⁺ and CD8⁺ cell count. Trm cells tissue-resident memory T cells, CRC colorectal cancer, ITGAE integrin subunit alpha E, TCGA The Cancer Genome Atlas, COAD colon adenocarcinoma, READ rectum adenocarcinoma, mRNA messenger RNA, OS overall survival, RFS recurrence-free survival.

Fig. 2. Trm cells represent the most activated subset of CD8⁺ T cells in CRC.

a t-SNE plots of ~12,000 live and singlet-gated CD45⁺ single-cell transcriptomes obtained from resected CRC tissue showing gene expression encoding Trm cell markers, cytotoxic cytokines, and immune checkpoint molecules. b Trm (red) and non-Trm (green) clusters are defined according to ITGAE (encoding CD103) and CD8A expression. c Correlation between Trm and non-Trm cell clusters: mean (log₁₀ [molecules/cell/gene]). d Volcano plot showing DEGs between Trm and non-Trm cell clusters. Dot lines represent an FDR of <0.05 and a fold change of >2. e Violin plot of the expression of cytotoxic cytokines and immune checkpoint molecule genes in the two clusters in (b). f Top 3 NES of hallmark gene sets in GSEA between Trm and non-Trm cell clusters. g Venn diagrams overlapping transcripts differentially expressed with a fold change of >2 between Trm and non-Trm cell clusters in lung cancer (left) and CRC (right). Trm cells tissue-resident memory T cells, CRC colorectal cancer, ITGAE integrin subunit alpha E, t-SNE t-distributed stochastic neighbour embedding, DEG differentially expressed gene, FDR false discovery rate, NES normalised-enrichment scores, GSEA gene set enrichment analysis.

Fig. 3. Characteristics of cancer-specific Trm cells in CRC.

a t-SNE plots of singlet-gated CD45⁺ single-cell transcriptomes obtained from resected CRC and adjacent normal colon tissue and gene expression of ITGAE and CD8A. Tumour Trm (orange) and non-Trm (blue) cell clusters are defined according to ITGAE and CD8A expression. b Volcano plot showing the DEGs between Trm and non-Trm cell clusters. c Immunohistochemical staining of CRC tissues at the invasive margins; CD103 staining of Case 1 (high invasion; upper left), CD8 staining of Case 1 (high invasion; upper right), CD103 staining of Case 2 (low invasion; lower left) and CD8 staining of Case 2 (low invasion; lower right). d t-SNE plots of singlet-gated CD45⁺ single-cell transcriptomes obtained from Trm high- and low invasion cases of resected CRC. High (orange) and low (dark yellow) Trm invasion are defined according to ITGAE and CD8A expression. e Volcano plot showing DEGs between Case 1 and Case-2 Trm cell clusters. f Venn diagram of upregulated genes in Fig. 2e, tumour Trm cells, and high invasion Trm cells. Trm cells tissue-resident memory T cells, CRC colorectal cancer, ITGAE integrin subunit alpha E, t-SNE t-distributed stochastic neighbour embedding, DEG differentially expressed gene.

Fig. 4. ZNF683⁺ Trm cells enhance tumour suppression through positive feedback by autocrine IFN-γ.

a Distribution of CD8A +, ITGAE + and ZNF683⁺ cells in the t-SNE plot. b GSEA comparing ZNF683⁺ and ZNF683^– Trm cell clusters. c Violin plot showing the expression of cytotoxic cytokines and immune checkpoint molecules in association with ZNF683 expression. d The hypothesis of signals surrounding ZNF683. e TBX21–ZNF683 expression ratio in Trm cells. f t-SNE plot of cells expressing ZNF683 and TBX21 (red, ZNF683; blue, TBX21). g High ITGAE and high-CD8A cases with high expression of ZNF683 in the TCGA database; TOP4 signalling was elevated in GSEA was IFN-γ and TCR signalling. ZNF683 zinc finger protein 683, Trm cells tissue-resident memory T cells, IFN-γ interferon-gamma, t-SNE t-distributed stochastic neighbour embedding, GSEA gene set enrichment analysis, TCGA The Cancer Genome Atlas, ITGAE integrin subunit alpha E, TCR T-cell receptor.