The CD4^-CD8^- MAIT cell subpopulation is a functionally distinct subset developmentally related to the main CD8⁺ MAIT...

Mucosa-associated invariant T (MAIT) cells are unconventional innate-like T cells that recognize microbial riboflavin metabolites presented by the MHC class I-like protein MR1. Human MAIT cells predominantly express the CD8α coreceptor (CD8⁺), with a smaller subset lacking both CD4 and CD8 (double-negative, DN). However, it is unclear if these two MAIT cell subpopulations distinguished by CD8α represent functionally distinct subsets. Here, we show that the two MAIT cell subsets express divergent transcriptional programs and distinct patterns of classic T cell transcription factors. Furthermore, CD8⁺ MAIT cells have higher levels of receptors for IL-12 and IL-18, as well as of the activating receptors CD2, CD9, and NKG2D, and display superior functionality following stimulation with riboflavin-autotrophic as well as riboflavin-auxotrophic bacterial strains. DN MAIT cells display higher RORγt/T-bet ratio, and express less IFN-γ and more IL-17. Furthermore, the DN subset displays enrichment of an apoptosis gene signature and higher propensity for activation-induced apoptosis. During development in human fetal tissues, DN MAIT cells are more mature and accumulate over gestational time with reciprocal contraction of the CD8⁺ subset. Analysis of the T cell receptor repertoire reveals higher diversity in CD8⁺ MAIT cells than in DN MAIT cells. Finally, chronic T cell receptor stimulation of CD8⁺ MAIT cells in an in vitro culture system supports the accumulation and maintenance of the DN subpopulation. These findings define human CD8⁺ and DN MAIT cells as functionally distinct subsets and indicate a derivative developmental relationship.