γδ T Cells Control Gut Pathology in a Chronic Inflammatory Model of Colorectal Cancer.

Gut γδ T cells are intraepithelial lymphocytes with reported roles in maintaining the gut barrier against infection and epithelial stress., In addition, absence of γδ T cells leads to faster polyp formation in an inflammation unassociated model of colorectal cancer (CRC) and γδ T cells reportedly showed antitumor activity in the context of human CRC., Patients with inflammatory bowel disease with early CRC have heightened risk for surgery with a different evolutionary history and genomic characteristics compared with sporadic CRC.5, 6, 7 To investigate the unknown role of γδ T cells in inflammatory bowel disease–associated CRC, we used a new murine model (chronic AOM-DSS).

We observed diminishing γδ T cells in colonic intraepithelial lymphocytes of AOM-DSS mice, associated with reduced trends in epithelial-anchorage-molecules and TCR γδ genes (Supplementary Figure 1). To validate the pathologic impact of γδ T cells’ loss, we investigated polyp formation in the presence or not of γδ T cells (Figure 1A and B and Supplementary Figure 2A-F). Interestingly, we observed a body weight difference in this model only after the 2nd cycle of DSS (Figure 1A), in contrast to acute DSS colitis. At the termination (Day 58), gross pathologic parameters of colon were significantly increased in TCRδ-/- mice (Supplementary Figure 2B). TCRδ-/- mice with AOM-DSS showed increased histologic inflammation and increased polyp formation (Figure 1B and Supplementary Figure 2C). Polyps in the distal colon were limited to small regions in WT mice but were multifocal to widespread in TCRδ-/- mice (Supplementary Figure 2D and E). Quantification with Ki67 and Bcl2 revealed that polyps in TCRδ-/- mice were more prevalent in the distal colon compared with the proximal colon with increased proliferation and antiapoptosis (Supplementary Figure 2F). Thus, absence of γδ T cells chronically induced early polyp formation qualitatively and quantitatively.

Figure 1

Lack of γδ T cells enhances gut pathology and tumorigenesis in a chronic AOM-DSS model. (A) Representative comparative body weight loss curve from 2 independent experiments (n = 30/strain). (B) Day 58 histologic scores (1-cm colon) from animals as shown in Supplementary Figure 2B. (C, D) Volcano plots showing the DEGs comparing KO versus WT in each model. Cutoffs: fold change ≥2; adjusted P ≤ .05. KO, knock-out.

To identify the early molecular events underlying gut pathology mediated by loss of γδ T cells, we focused on an early time point after the 2nd cycle of DSS. Polyps were not grossly observed in the colon of WT or TCRδ-/- mice but were observed microscopically in the distal colon of TCRδ-/- mice (Supplementary Figure 2G). Because polyp initiates in the distal region, this location provided opportunity to observe differential gene expression (RNAseq) without the bias of number and shape of polyps. Principal component analysis revealed well-separated clusters of AOM-DSS treatment from respective naive groups and WT cluster was separated from TCR δ-/- cluster within AOM-DSS treatment (Supplementary Figure 2H).

We identified a massive transcriptional reprogramming with 445 upregulated differentially expressed genes (DEGs) and 1297 downregulated DEGs in AOM-DSS treatment groups comparing TCR δ-/- mice versus WT counterpart (Figure 1C and D). The number of DEGs in untreated naive mice was much lower emphasizing the requirement of tissue insult to reveal γδ T cells’ functions. Pathway enrichment analysis on DEGs (CPDB, Supplementary Figure 3A and B) has revealed upregulation of several signaling pathways including destructive structural pathways and while some beneficial structural pathways were downregulated. Similar results were obtained using DAVID pathway analysis (data not shown). In detailed analysis, inflammation and inflammation-associated oncogenes were upregulated, whereas some anti-inflammatory, antiangiogenesis, gut homeostasis, structural integrity, and tumor suppression genes were significantly downregulated (Supplementary Figure 3C-M). Overall, we identified a profound γδ T cell–dependent transcriptional reprograming of gut tissues that emerges early and affects broad cellular functions.

We wondered whether γδ T cells’ presence could delay the appearance of an immunoregulatory environment, a hallmark of protumor condition. We evaluated several cells and observed augmentation of Helios+Foxp3+ natural Tregs (nTregs) and 2 myeloid cell types: mast cells and conventional dendritic cell 1 (cDC1), known to induce/maintain/associate with CD4+Tregs.8, 9, 10 nTregs were augmented in the intraepithelial lymphocytes in contrast to the LP fraction of TCR δ-/- mice on Day 58 following AOM-DSS treatment (Supplementary Figure 4A and B and Figure 2A and B).

Figure 2

γδ T cells control immunoregulatory phenotype in IEL and correlate negatively in COAD. (A) Cell numbers of Tregs in IEL. (B) Cell numbers for Mast cells (left panel, live CD45+Epcam-CD3-B220-ckit+) and cDC1s (right panel, live CD45+Epcam-CD3-CD19-MHCII+F4/80-CD11c+CD11b-CD103+). (C) Expression of TRDC at different stages of COAD/READ tumors and related normal. (D) Median expression of top 100 upregulated genes (KO vs WT in AOM+DSS) in the same dataset. IEL, intraepithelial lymphocytes; KO, knock-out; ns, not significant.

Analyzing transcriptomic data from CRC tumor patients, we found that TCR delta chain constant region (TRDC) gene expression was consistently lost in early to late stages of tumors (along with TRGC and known epithelial-anchorage-molecules but reverse trend in FOXP3) (Figure 2C and Supplementary Figure 4C-G). Interestingly, the median expression of the top 100 upregulated genes in the absence of γδ T cells (TCRδ-/- vs WT, AOM-DSS treatment) was robustly upregulated at different stages of colon (but not rectal) adenocarcinoma (Figure 2D). Therefore, loss of γδ T cells and the associated upregulation of the gene signature observed in early tumorigenesis in our mouse model are also found in cancer patient samples.

Our data reveal a profound protective feature of gut γδ T cells in a relevant chronic gut pathology model that was confirmed to be present in CRC patient samples. These results shed light on the physiologic relevance of these cells in intestinal epithelial barrier integrity, and as critical sentinels against early tumorigenesis. Further investigations involving targeted approaches for differentially expressed candidate genes identified in our datasets, in the context of the murine model, would be helpful. Manipulation of these cells may confer therapeutic benefits in CRC, and their broader spectrum of applications warrant further investigation.