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Synergistic role of riboflavin-auxotrophic Enterococcus for MR1 expression and in-tra-tumoral mucosal associated invariant T (MAIT) cell activation

ABSTRACT: The role of mucosal invariant T (MAIT) cells in the lung tumor microenvironment re-mains poorly understood, especially in the setting of immune checkpoint inhibitors. We identified intratumoral MAIT cells from paired single cell RNA and TCR sequencing datasets of tumor infil-trating CD3 T cells isolated from non-small cell lung cancer tumors in patients receiving neoadju-vant PD-1 blockade therapy. MAIT cells were subclustered to identify conventional MAIT-associ-ated TCR clonotypes, which were then tested for the recognition of bacteria using a MAIT TCR capture functional assay. Strikingly, surveillance of intratumoral bacteria in lung cancer patients revealed Enterococcus spp. that are not directly recognized by MAIT cells but, nevertheless can synergize with exogenous riboflavin biosynthesis-derived metabolites to induce expression of MR1 by antigen presenting cells (APC, dendritic cells, B cells and mononuclear phagocytes). Enhanced MR1 cell surface expression resulted from enterococcal-mediated perturbation of an endo-lysosomal vacuolar pathway with recycling of early endosomal MR1 to the APC cytoplasmic membrane. Riboflavin auxotrophic Enterococcus spp may therefore exercise their beneficial im-munomodulatory functions upon immune checkpoint blockade treatment, at least in part, by pro-moting intratumoral MR1 expression and innate-like T cell activation. Our results indicate that composition of the intratumoral microbiome during immune checkpoint inhibitor treatment has the potential to impact the function of human intratumoral MAIT cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE320009 | GEO | 2026/02/23

REPOSITORIES: GEO

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Project description:MAIT cells are multifunctional innate-like effector cells recognizing bacterial-derived vitamin B metabolites presented by the non-polymorphic MHC class I related-1 molecule (MR1). Activated MAIT cells can exert regulatory, pro-inflammatory, or cytotoxic responses that enable the direct killing of infected cells. While tremendous progress regarding the multifaceted roles of MAIT cells has been made in the last decade, our understanding of the MR1-mediated responses of MAIT cells upon their interaction with other immune cells is still incomplete. Here, we performed the first translatome study of primary human MAIT cells interacting with THP-1 monocytes in a bicellular system. We analyzed the interaction between MAIT and THP-1 cells in the presence of the activating 5-OP-RU or the inhibitory Ac-6-FP MR1-ligand. Using bio-orthogonal non-canonical amino acid tagging (BONCAT) we were able to enrich selectively those proteins that were newly translated during MR1-dependent cellular interaction. Subsequently, newly translated proteins were measured cell-type-specifically by ultrasensitive proteomics to decipher the coinciding immune responses in both cell types. This strategy identified over 2,000 MAIT and 3,000 THP-1 active protein translations following MR-1 ligand stimulations. Translation of both cell types was more efficiently induced by 5-OP-RU in comparison to Ac-6-FP, which correlated with higher conjugation frequencies and CD3 polarization at immunological synapses. In addition to known effector responses, protein translations uncovered type I and type II Interferon-driven protein expression profiles in both 5-OP-RU-stimulated MAIT and THP-1 cells. Interestingly, the translatome of THP-1 macrophages suggested that activated MAIT cells can impact M1/M2 polarization in these cells. Indeed, gene and surface expression of CXCL10, IL-1, CD80, and CD206 confirmed an M1-like phenotype of macrophages being induced in the presence of 5-OP-RU-activated MAIT cells. Furthermore, we validated that the Interferon-driven translatome was accompanied by the induction of an antiviral phenotype in THP-1 cells, which were found able to suppress viral replication following conjugation with MR1-activated MAIT cells. In conclusion, BONCAT translatomics extended our knowledge of MAIT cell immune responses at the protein level and discovered that MR1-activated MAIT cells are sufficient to induce M1 polarization and an anti-viral program of macrophages.

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Synergistic role of riboflavin-auxotrophic Enterococcus for MR1 expression and in-tra-tumoral mucosal associated invariant T (MAIT) cell activation

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