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Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) α-chain, TRAV1-2–TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)–related class I–like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-d-ribityllumazine (rRL-6-CH₂OH), specifically detect all human MAIT cells. Tetramer⁺ MAIT subsets were predominantly CD8⁺ or CD4⁻CD8⁻, although a small subset of CD4⁺ MAIT cells was also detected. Notably, most human CD8⁺ MAIT cells were CD8α⁺CD8β^−/lo, implying predominant expression of CD8αα homodimers …