Antigenic stimulation in conjunction with cytokine is required for mediating IL-17A production in human MAIT cells

Abstract Mucosal-associated invariant T (MAIT) cells are donor unrestricted T cells capable of both antigen-specific adaptive responses and cytokine driven innate-like functions. Although human MAIT cells uniformly express RORγt and IL23R, they generally produce IFN-γ, and only a small fraction produces IL-17. Recent studies show that combined TCR and cytokine stimulation can elicit functional heterogeneity in blood-derived MAIT cells. Here, we investigate the role of IL-23/IL-23R signaling in mediating the function and transcriptional profiles of lung MAIT cell clones. We demonstrate that BAL-derived lung MAIT cell clones exhibit distinct cytokine profiles and variable IL23R expression. Short-term IL-23 stimulation triggers clone-specific transcriptional programs and IL23R-dependent upregulation of type 17-associated genes. Prolonged conditioning of lung MAIT cell clones with TCR (5-OP-RU) and cytokine (IL-23) stimulation induces stable IL-17A production along with unique transcriptional changes. TCR + IL-23 conditioning alone upregulates clone-specific and shared cytoskeletal/structural gene programs, whereas subsequent PMA/Ionomycin stimulation further induces IL-12 family signaling and metabolic genes. Together, these findings demonstrate that IL23R expression and TCR signaling are required for IL-17A production, highlighting that these conditions may be met in tissue environments where MR1-specific antigens and proinflammatory cytokines coexist. Competing Interest Statement The authors have declared no competing interest. Data availability statement All R studio code used in the generation of this analysis is freely available and uploaded to github (https://github.com/kaindylan/IL23R-MR1T-Paper/tree/main). All raw data was deposited to dbgap (accessions number: phs003810.v1.p1). The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files. The source data underlying the graphs in the paper can be found in the Supplementary Data. The accession number for the atomic coordinates of CB964 A2 TCR-MR1-5-OP-RU, along with associated structure factors, have been deposited at the protein databank (www.rcsb.org) with accession code 9MS0. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.