Frequency and staining characteristics of MR1–5-OP-RU and MR1–6-FP tetramer+populations identified with species-matched and species-mismatched tetramer reagents. Bar charts and heatmap summaries, displaying the following: A, frequencies of species-matched (red histograms) and species-mismatched (black histograms) MR1–5-OP-RU (RU, black dots) and MR1–6-FP (FP, white dots) tetramer+ cells in human CD3+ PBMC (n = 8), cattle CD3+ PBMC (n = 8), pig-tailed macaque CD8+ PBMC (n = 5), MAIT cell boosted mouse spleen (n = 7)–derived αβTCR+ lymphocytes, and sheep CD8+ PBMC (n = 6). Histograms depict mean frequency ± SEM with each individual datapoint shown. B, species-matched (red histograms) and species-mismatched (black histograms) MR1–5-OP-RU tetramer (RU) geometric mean fluorescence intensity (gMFI) fold change over the MR1–5-OP-RU tetramer- population (background) and (C) SD of the MR1–5-OP-RU tetramer+ (RU) gMFI as described in (A). Differences between the frequencies obtained from species-matched and species-mismatched MR1–5-OP-RU staining were evaluated using a one-way ANOVA with Giesser-Greenhouse correction or, where there are missing values, a mixed effect model with repeated measures, followed by Dunnett’s multiple comparison test, comparing staining with species-matched MR1–5-OP-RU tetramer and all other species-mismatched MR1–5-OP-RU tetramers, with individual variance computed for each comparison. Only statistically significant differences (p value < 0.05) are indicated. Data are combined from either one (pig-tailed macaque), two (human, cattle, and sheep), or three (mouse) separate experiments. 5-OP-RU, 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil; 6-FP, 6-formylpterin; PBMC, peripheral blood mononuclear cell; MAIT, mucosal-associated invariant T; MR1, MHC-I related protein 1; TCR, T cell receptor.