SignificanceMucosa-associated invariant T (MAIT) cells are unconventional innate-like T cells recognizing microbial riboflavin metabolites presented by the monomorphic MR1 molecule. Here, we show that the CD8+CD4− and CD8−CD4− subpopulations of human MAIT cells represent transcriptionally and phenotypically discrete subsets with distinct functional profiles. Furthermore, T cell receptor repertoire analysis, as well as MAIT cell data based on human fetal tissues, umbilical cord blood, and culture systems indicate that the CD8−CD4− subset may derive from the main CD8+CD4− MAIT cell pool. Thus, MAIT cells, a major antimicrobial effector T cell population in humans, segregate into two functionally distinct but developmentally related subsets separated by the expression of CD8. This functional difference may have significant implications in infectious and inflammatory diseases.

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Progress in our understanding of MR1-restricted Mucosa-associated Invariant T (MAIT) cells has raised an interest in harnessing these cells for immunotherapy. The innate-like response characteristics, abundance in the blood, donor-unrestricted nature, and tropism for tissues make MAIT cells suitable candidates for adoptive cell transfer therapies. However, reliable methods and tools to utilize MAIT cells in such approaches are lacking. Here, we established methodology for efficient expansion of human MAIT cells in culture with high purity and yield, preserved functional response toward their natural ligand, and with increased cytotoxic potential. The cultured MAIT cells retained their effector memory characteristics without signs of terminal differentiation, and expressed a more diverse set of chemokine receptors potentially widening their already broad tissue tropism. To investigate the potential of MAIT cells in a context outside their main role in controlling bacterial infection, we engineered cultured MAIT cells with a new TCR specificity to mediate effective antiviral HLA class I-restricted effector function. In summary, we developed robust and effective methodology for the expansion of human MAIT cells with enhanced cytolytic capacity, and for their engineering with a new specificity. These findings form a basis for the development of MAIT cells as a platform for adoptive immunotherapy. Introduction Mucosa-associated invariant T cells (MAIT) are a non-conventional T cell population at the bridge between innate and adaptive immunity (1, 2). Human MAIT cells express a semiinvariant TCR using the Vα7.2 (TRAV1-2) TCR segment paired with a restricted TCRβ repertoire, which specifically recognizes vitamin B2-derived microbial metabolites loaded on the highly conserved MR1 molecule (3-5). This limited TCR repertoire allows for the identification of MAIT cells based on the co-expression of Vα7.2 and CD161 (6), or more recently, using MR1 tetramers loaded with the stimulatory MAIT cell ligand 5-(2oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU) (7, 8). Abundant in the blood, where they account for 1-10% of total T cells (9, 10), MAIT cells are enriched in mucosal tissues, as well as in peripheral organs including the liver where they represent up to 30-50% of T cells (11). The metabolites recognized by MAIT cells are byproducts of the riboflavin biosynthesis pathway expressed by diverse species of bacteria, mycobacteria and fungi (12), and therefore in association with their location at sites of pathogen entry, MAIT cells are viewed as early sentinels responding to microbial infections. In response to antigen, MAIT cells produce pro-inflammatory cytokines such as IFNγ, TNF and IL-17A, and can release cytolytic effector molecules including granzymes and perforin leading to the lysis of the infected cells, the inhibition of bacterial growth, and the shaping of the local immune response (9, 13-15). MAIT cells can also be activated in a TCR and MR1-independent way via the pro-inflammatory cytokines IL-12 and IL-18,...

Bats are reservoirs for a large number of viruses which have potential to cause major human disease outbreaks, including the current coronavirus disease 2019 (COVID-19) pandemic. Major efforts are underway to understand bat immune response to viruses, whereas much less is known about their immune responses to bacteria. In this study, MR1-restricted T (MR1T) cells were detected through the use of MR1 tetramers in circulation and tissues of Pteropus alecto (Pa) bats. Pa MR1T cells exhibited weak responses to MR1-presented microbial metabolites at resting state. However, following priming with MR1-presented agonist they proliferated, upregulated critical transcription factors and cytolytic proteins, and gained transient expression of Th1/17-related cytokines and antibacterial cytotoxicity. Collectively, these findings show that the Pa bat immune system encompasses an abundant and functionally conserved population of MR1T cells with mucosal-associated invariant T-like characteristics, suggesting that MR1 and MR1T cells also play a significant role in bat immune defense.