Exploring the colorectal cancer metabolome as a source of host-derived Mucosal-Associated Invariant T cell activating ligands

Abstract

Mucosal-associated invariant T (MAIT) cells, a subset of innate-like T cells, are strategically located in barrier tissues where they respond to microbial and metabolic cues via the monomorphic antigen-presenting molecule MR1 - major histocompatibility complex class I-related protein. While their role as watchers on the wall, or microbial metabolite sensors has been well established, their contributions to various pathologies remain poorly defined. MAIT cells have been discovered in several tumor types, including colorectal cancer (CRC), where frequencies and functional states have been variable but skew toward either pro- or anti- tumor effects. The environment within tumors is characterised by metabolic reprogramming, raising the possibility that tumors may generate endogenous MR1-binding ligands. However, to date there is no direct evidence of cancer cells producing ligands capable of engaging MAIT cells in a TCR dependent manner. To address this unresolved question, we aimed to determine whether soluble factors derived from CRC cells are sufficient to activate MAIT cells and if these responses were MR1 dependent. Primary human MAIT cells were isolated, expanded in vitro, and co-cultured with MR1-expressing THP-1 antigen-presenting cells in the presence of supernatants from four human CRC cell lines (SW480, HCT116, HT29, and Caco-2). MAIT cell activation was assessed by measuring surface activation markers, cytokine production, and degranulation, with MR1-blocking antibodies used to evaluate dependence on MR1-mediated antigen presentation. We further examined whether tumor conditioning alters MAIT cell effector capacity and whether hypoxic culture conditions modulate the generation of MAIT-activating signals. Finally, we assessed the reciprocal axis by stimulating CRC cells with IL-17A and IL-17F and performing transcriptional profiling. CRC-derived supernatants did not induce robust or consistent MAIT cell activation. Observed responses were modest, variable across donors, and not consistently reduced by MR1 blockade, indicating limited MR1-dependent signaling. Tumor conditioning did not enhance MAIT cell responsiveness, and hypoxia did not substantially alter activation. In contrast, IL-17A stimulation of CRC cells induced transcriptional programs enriched for inflammatory and tumor-associated pathways, including NF-κB and TNF signaling, whereas IL-17F elicited weaker responses. These findings do not support a model in which CRC cells broadly generate potent MR1 ligands, and instead suggest that MAIT cell activity in tumors may be driven by MR1-independent signals or require additional microenvironmental cues.