CD4 ⁺ Mucosal-associated Invariant T (MAIT) cells express highly diverse T cell receptors

Mucosal-associated invariant T cells are highly conserved innate-like T cells in mammals recognized for their high baseline frequency in human blood and cytotoxic effector functions during infectious diseases, autoimmunity, and cancer. While the majority of these cells express a conserved CD8αβ+ TRAV1-2 T cell receptor recognizing microbially-derived Vitamin B2 intermediates presented by the evolutionarily conserved major histocompatibility complex I-related molecule, MR1, there is an emerging appreciation for diverse subsets that may be selected for in humans with distinct functions, including subpopulations that co-express CD4. Prior work has not examined T cell receptor (TCR) heterogeneity in CD4 ⁺ MAIT cells, largely due to bias of identifying human MAIT cells as CD8 ⁺ TRAV1-2 ⁺ cells. In this study, we adopted an unbiased single-cell TCR-sequencing approach of total MR1-5-OP-RU-tetramer-reactive T cells and discovered that CD4 ⁺ MAIT cells express highly diverse TRAV1-2 negative TCRs. To specifically characterize this TCR repertoire, we analyzed VDJ sequences of single MR1-5-OP-RU tetramer ⁺ MAIT cells across two datasets and identified distinct TCR usage among CD4 ⁺ MAIT cells including TRAV21, TRAV8 (TRAV8-1, TRAV8-2, TRAV8-3), and TRAV12 families (TRAV12-2, TRAV12-3), as well as more variable J chain and CDR3 sequences. Non-TRAV1-2 MAIT cell TCRs were also enriched after in vitro expansion, including with Mycobacterial tuberculosis . These results indicate that mature human CD4 ⁺ MAIT cells adopt distinct TCR usage from the canonical TRAV1-2 ⁺ CD8 ⁺ subset and suggest that alternative MR1 ligands in addition to riboflavin intermediates may select them.