Dysfunctional tumor-infiltrating Vδ1 + T lymphocytes in microsatellite-stable colorectal cancer

Abstract

Although γδ T cells are known to participate in immune dysregulation in solid tumors, their relevance to human microsatellite-stable (MSS) colorectal cancer (CRC) is still undefined. Here, using integrated gene expression analysis and T cell receptor sequencing, we characterized γδ T cells in MSS CRC, with a focus on Vδ1 + T cells. We identified Vδ1⁺ T cells with shared motifs in the third complementarity-determining region of the δ-chain, reflective of antigen recognition. Changes in gene and protein expression levels suggested a dysfunctional effector state of Vδ1⁺ T cells in MSS CRC, distinct from Vδ1⁺ T cells in microsatellite-instable (MSI). Interaction analysis highlighted an immunosuppressive role of fibroblasts in the dysregulation of Vδ1⁺ T cells in MSS CRC via the TIGIT-NECTIN2 axis. Blocking this pathway with a TIGIT antibody partially restored cytotoxicity of the dysfunctional Vδ1 phenotype. These results define an operative pathway in γδ T cells in MSS CRC.

Fig. 1. Single-cell RNA- and TCR sequencing reveal favored Vδ1+ usage with expanded CDR3 identity patterns in γδ T cells of MSS CRC.

CD45 + /CD45– cells were sorted from human MSS CRC and distant HC of resection tissue and processed for 10X genomics RNA and αβ-, γδ-TCR sequencing. a Graphical abstract of the work flow. CRC tissue and healthy distant colon was obtained from 17 and 16 patients, respectively. Single-cell RNA-sequencing and αβ-/γδ-TCR sequencing were performed from seven patients. b UMAP of cells in HC and CRC displayed according to the similarity of their transcriptome. c Marker genes of depicted cell subsets. d Circos plots displaying chain pairing of TRDV and TRGV in HC compared to CRC. e STARTRAC expansion index of γδ T cells (left) and αβ T cells (right). αβ T cells but not γδ T cells showed an increasing index indicative of antigen-specific clonal expansion. f Percentage of top 10 expanded patterns of identity in the CDR3 region of γδ T cells in CRC and corresponding HC using GLIPH2. g Visual comparison of tree map graphs showing the identity pattern in the CDR3 region of γδ T cells in HC and CRC. In tree maps, each rectangle reflects a unique pattern, as indicated, where the size of a spot equals the percentage. Violin plots display median and quartiles (e); two-tailed paired t-test (e, f). EGC, enteric glial cells. ILC, innate lymphoid cells. p values are shown on the graphs.

Fig. 2. TRDV1+ cells demonstrate profound dysregulation but not exhaustion in MSS CRC.

γδ T cells subsets were analyzed according to the expression of marker genes TRDV1, TRDV2 and TRDV3. (a) UMAP based on the expression of TRDV1, TRDV2 and TRDV3 in HC and CRC. On the right side, donut charts illustrating the quantities of cells, indicating the counts of TRDV1, TRDV2 and TRDV3 expressing cells in both HC and CRC. b Top 10 differentially overexpressed genes for each subset TRDV1, TRDV2 and TRDV3. c Expression of individual genes displayed as percentage of positive cells for TRDV1 and TRDV2 in HC and CRC reveal distinct effector states of both subsets (n = 16 for HC, n = 17 for CRC). d Expression of TRDV1 in MSI and MSS of TCGA-COAD (n = 217 for MSS, n = 121 for MSI). e TRDV1+ cells per sample in HC, MSS CRC and MSI CRC in the Pelka et al. data set (n = 36 for HC, n = 34 for MSI, n = 28 for MSS). f Expression of individual genes displayed as percentage of positive cells for TRDV1 in HC, MSS CRC and MSI CRC using Pelka et al. (n = 36 for HC, n = 28 for MSS, n = 34 for MSI). g, h Depiction of exhaustion scores using genes sets by Zhang et al.. and Lui et al.. as previously published of TRDV1+ cells in HC and CRC (left: n = 16 for HC, n = 17 for CRC, right: n = 36 for HC, n = 28 for MSS, n = 34 for MSI). Data points and error bars represent the mean ± SD (c, e, f), Violin plots display median, quartiles ±SD (d, g, h); one-way ANOVA with Fisher’s LSD (c, f), two-tailed unpaired t-test (d, e, g, h). p values are shown on the graphs.

Fig. 3. γδ T cells are reduced in MSS CRC compared to HC and upregulate co-expression of inhibitory receptors.

Flow cytometry analysis of γδ T cell abundance and subsets in human MSS CRC compared to HC. a Gating strategy for γδ T cells from human mononuclear cells isolated from CRC. b Significant increase in CD3+ cells depicted as mean percentage of viable CD45+ cells in CRC compared to HC (n = 29 per group). c Significant decrease of TCRγδ + cells in CRC depicted as mean percentage of viable CD3+ cells compared to HC (n = 29 per group). d Representative image of multicolor fluorescent immunohistochemistry staining of TCRγδ + cells (TCRγδ PE/ CD3 A647) in HC and CRC. e Significantly more Vδ1 + populate HC and CRC compared to Vδ2 + , assessed by flow cytometry and depicted as percentage of positive cells of viable TCRγδ + CD3 + cells (n = 29 per group). Vδ1 + (f) and Vδ2 + (g) display different T cell effector states according to their CD27 and CD45RA expression (n = 16 per group). Temra: CD45RA + CD27–, Tem: CD45RA–CD27–, Tcm: CD45RA–CD27 + , Tnaive: CD45RA + CD27 + . h Expression of inhibitory checkpoint receptors on Vδ1 + (n = 13), Vδ2 + (n = 11) and TCRγδ negative T cells (n = 13) in HC (white) and CRC (gray). i Co-expression of depicted receptors on Vδ1 + , Vδ2 + and TCRγδ negative T cells in HC (white) and CRC (gray; n = 11 per group). Co-expression was assessed by first gating on PD-1+ cells and then calculating the percentage of double-positive cells. Data points and error bars represent the mean ± SD (b, c, e−i); two-tailed paired t-test (b, c, e, i), one-way ANOVA with Fisher’s LSD (f−h); Temra, effector memory RA; Tem, effector memory; Tcm, central memory. p values are shown on the graphs.

Fig. 4. Dysfunctional cytotoxic potential of Vδ1 + T cells from MSS CRC can be restored in vitro.

Flow cytometry analysis of intracellular cytokine staining of Vδ1 + and Vδ2 + T cells in HC or CRC. a Overlay of representative histograms and corresponding bar plots of tested cytokines by flow cytometric analysis in Vδ1 + and Vδ2 + T cells in HC (light cyan) or CRC (light pink) and stimulated (PMA/iono) HC (cyan) or CRC (pink, n = 16 per group). In CRC, Vδ1 + display reduced expression of TRAIL, TNF-α and IFN-γ which is reversible upon stimulation. b Percentage of Ki-67 + Vδ1 + , Vδ2 + and TCRγδ negative T cells in HC (white) compared to CRC (gray; n = 8 per group). Increased expression of Ki-67 in Vδ1 + and TCRγδ negative T cells in CRC indicate in tissue proliferation in contrast to the Ki-67 negative Vδ2 + T cells which potentially influx from the periphery. Overlay of representative histograms of Ki-67 expression in HC (cyan) and CRC (pink). c Increased Ki-67+ expression of Vδ1 + T cells isolated of CRC was mirrored in elevated proliferation (gray) during IL-2 alone or CD3/CD28 stimulation as compared to HC (white; n = 3 per group). d Pairwise comparison of killing assay with Vδ1 + and Vδ2 + T cells with two different CRC lines (HT29 in black, SW480 in blue; n = 14 per group). e Percentage of dead HT29 after exclusion of baseline HT29 cell death in a killing assay with Vδ1 + and Vδ2 + T cells isolated from HC (cyan) and CRC (pink). Two conditions were tested: Vδ1 + and Vδ2 + T cells with (cyan, pink) and without PMA/iono stimulation (light cyan, light pink; n = 5 per group). f CD107a expression of Vδ1 + and Vδ2 + T cells from HC (cyan) and CRC (pink) after killing of HT29 as in (e). g Representative FACS plot of dead HT29 in a killing assay with Vδ1 + T cells with no stimulation and stimulated with PMA/ionomycin. Data points and error bars represent the mean ± SD (a−f); one-way ANOVA with Fisher’s LSD (a−f); Ctrl; Control; p values are shown on the graphs.

Fig. 5. Impaired killing capacity by Vδ1 + T cells can be reinstated after TIGIT blockade.

a Number of predicted overall interactions of depicted cell clusters in HC and CRC using CellChat. b UMAP of CD45+ and CD45– cells of HC and CRC displayed according to the similarity of their transcriptome. Fibroblasts clusters are highlighted. c Significant receptor-ligand interactions of fibroblasts cell clusters to γδ T cells as predicted by CellChat in HC and CRC. d Marker gene expression of fibroblast clusters. e Co-expression analysis of differentially overexpressed genes of each fibroblast cluster in CRC. f Gating strategy of fibroblast isolation for experiment as performed in (g). g Viable Vδ1 + T cells and fibroblasts were isolated from HC and CRC, fibroblasts were seeded, with or without antibody-blocking of TIGIT, Vδ1 + T cells were added and killing assay with HT29 was performed. h Percentage of dead HT29 after exclusion of baseline HT29 cell death in a killing assay as stated in (g); n = 8 per group. i TRDV1 expression of patients with disease stages I, II, III, IV in the MSS CRC cohort of the TCGA-COAD (n = 29 for I, n = 64 for II, n = 77 for III, n = 38 for IV). j Correlation between COL1A2, IL6 with TRDV1, TIGIT expressing cells in the MSS cohort of the TCGA-COAD. Data points and error bars represent the mean ± SD (h), Violin plots display median, quartiles ±SD (i); Wilcoxon Rank Sum test (c). one-way ANOVA with Fisher’s LSD (h), two-tailed paired t-test (i), Pearson Correlation (j). p values are shown on the graphs.