Mucosal-associated invariant T (MAIT) cells are unconventional T cells recognizing microbial metabolites, presented by the invariant MR1 protein. Upon activation, MAIT cells rapidly secrete cytokines and exert cytotoxic functions, and may thus be highly relevant also in tumor immunity. MAIT cells accumulate in colon tumors, but in contrast to other cytotoxic T cell subsets, their presence in tumors has been associated with worse patient outcome. Here we investigated if exhaustion may contribute to reduced anti-tumor immunity by MAIT cells. Freshly isolated lymphocytes from colon tumors, unaffected tissue and blood from the same patients were analyzed by flow cytometry to detect MAIT cells with effector functions that are relevant for tumor immunity, and their expression of inhibitory receptors and other exhaustion markers. Our studies show that MAIT cells with a PD-1highTim-3+CD39+ terminally exhausted phenotype and an increased proliferation accumulate in colon tumors. The exhausted MAIT cells have reduced polyfunctionality with regard to production of important anti-tumor effector molecules, and blocking antibodies to PD-1 partly improved activation of tumor-infiltrating MAIT cells in vitro. We conclude that the tumor microenvironment leads to exhaustion not only of conventional T cells, but also MAIT cells, and that checkpoint blockade therapy may be useful also to reinvigorate tumor-infiltrating MAIT cells.

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In colon cancer, tumor progression and patient outcome is affected by the cytokine balance in the tumors. IFNγ, TNFα and Granzyme B expression are associated with favorable patient outcome, while high IL-17 expression is associated with accelerated tumor progression. However, knowledge of the regulation and activation of unconventional T cell subsets in colon tumors is limited. The aim of this study was to characterize unconventional T cells in colon tumors and unaffected tissue, determine their capacity to produce cytokines affecting tumor progression as well as the In vitro cytotoxic capabilities of MAIT and γδ T cells. Using flow cytometry, we show that MAIT cells accumulate in colon tumors and that the frequencies of γδ T cells are reduced in the tumor epithelium. Using polyclonal stimulation, we show that IFNγ production by tumour infiltrating MAIT cells is impaired whilst tumour infiltrating γδ T have an increased expression of IFNγ, TNFα and Granzyme B. IL-17 expression was also elevated in tumour infiltrating γδ T cells, but at lower levels than the TH1 - associated cytokines. Tumor infiltrating MAIT cells had an exhausted (PD-1highTim-3+) phenotype compared to MAIT cells from unaffected tissue. Analyzing cytokine expression, we show that while no single molecule is lost the polyfunctional capacity of tumour infiltrating MAIT cells is decreased compared to MAIT cells from unaffected tissue. Altogether, this study shows that γδ T cells and MAIT cells contribute to the cytokine balance in colon tumors with a TH1 – dominated profile and that they have potent cytotoxic capacity, which may reduce tumor progression and improve patient outcome.

The presence of activated T cells in colorectal cancer (CRC) tissues is a strong predictor of patient survival. Our previous research have shown that regulatory T cells (Treg) are able to reduce T-cell transendothelial migration in vitro and accumulation of effector T cells in intestinal tumors in vivo in a murine model for intestinal adenomas (APCMin/+). In this study we investigated the effect of Treg depletion on the cytotoxic potential of conventional αβ T cells and γδ T cells in intestinal adenomas. We used the APCMin/+ \DEREG mouse model, which harbours a high affinity diphtheria toxin receptor under the control of the FOXP3 promoter, to deplete Treg in tumor bearing mice. We found that the number of CD8αβ T cells per mg tissue in the lamina propria was significantly increased in the Treg depleted intestinal adenomas in comparison to the non-depleted tumors. We could confirm this finding with IHC staining in tissue sections. The number of CD8αα T cells and γδ T cells remained unchanged. Furthermore, ex vivo frequencies in the lamina propria of Granzyme B+ CD8αβ T cells were increased in the Treg depleted intestinal adenomas. Following in vitro stimulation of lymphocytes from lamina propria with PMA/ionomycin, there was a trend towards a higher frequency of IFNγ+ CD8αβ T cells in Treg depleted adenomas, but frequencies of CD107a+ CD8αβ T cells were decreased. The frequency of TNF+CD8αβ T cells was elevated in the tumor in comparison to the unaffected tissue regardless of Treg depletion. We are investigating the combined effect of Treg depletion and PD-1 immunotherapy on the cytotoxic phenotype of T cells in the intestinal adenomas. The results indicate that the modulation of Treg in colorectal cancer could have apositive effect on T cell cytotoxicity.