Pathogen-reactive MAIT TCRα chains are not confined to TRAV1-2/TRAJ33 rearrangements. Human CD8⁺ MAIT cells from four individuals were stimulated overnight using A549 cells infected with M. smegmatis, S. typhimurium, or C. albicans. Live, pathogen-reactive MAIT cells were then isolated by flow cytometry based on coexpression of TRAV1-2 and TNF. Unbiased molecular analysis of expressed TRA gene products was performed as described in Materials and methods. Aggregate TRAJ gene profiles from all four donors in response to each pathogen (n = 3) are shown at the top; data from individual donors are shown underneath as indicated. Each sequence was counted once regardless of frequency. Total sequence counts are indicated in each case.