CD4+ mucosal-associated invariant T cells express highly diverse T cell receptors

Mucosal-associated invariant T (MAIT) 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 in humans express a conserved CD8αβ+ TRAV1-2 T cell receptor (TCR) recognizing microbially derived vitamin B2 intermediates presented by the evolutionarily conserved major histocompatibility complex class I-related molecule, MR1, there is an emerging appreciation for diverse MAIT cell subsets that possess distinct functions including CD4+ MAIT cells that remain underexplored. In this study, we adopted an unbiased single-cell TCR-sequencing approach in MR1-5-OP-RU-tetramer-reactive T cells. We discovered that CD4+ MAIT cells are enriched with highly diverse TRAV1-2- TCRs. To specifically characterize this TCR repertoire, we analyzed VDJ sequences across 2 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 segment, CDR3α, and TRBV sequences. TRAV1-2- MAIT cell TCRs were also enriched after in vitro culture with interleukin-2 and Mycobacterium 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 for them.