MAIT cells develop in response to bloom of riboflavin synthesizers during weaning. (A) 16S rRNA gene sequencing of fecal microbiota sampled from untreated WT mice at the indicated ages. Data represent the mean relative abundance (n = 3–4 mice/group). (B) qPCR of the ribD gene relative to the 16S V3–V4 region for the samples described in A (n = 3–4/group). (C) WT mice received ampicillin (ABX) by daily oral gavage during the indicated 2-wk periods followed by a fecal microbiota transplant (FMT) from untreated age-matched mice 24 h following cessation of ampicillin. Animals were then analyzed at 8 wk of age. (D) Representative flow cytometry of αβ T cells from murine ear pinnae, with identification of MAIT cells using dual MR1 tetramers. (E and F) The number (E) and frequency (F) of MAIT cells in the ear pinnae of mice treated with ampicillin during the indicated ages (n = 5/group). (G and H) The number (G) and frequency (H) of MAIT cells magnetically enriched from the thymus of mice treated with ampicillin from 2 to 4 wk old as described in C (n = 5/group, representative [rep.] of 2 experiments [expts.]). (I and J) The number (I) and frequency (J) of immature (CD24⁺CD44⁻) and mature (CD24⁻CD44⁺) MAIT cells magnetically enriched from the thymus of 4-wk-old mice at the conclusion of ampicillin treatment (n = 5/group, rep. of 2 expts.). Data represent the mean ± SEM, with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as calculated by ordinary one-way ANOVA with Šidák’s multiple comparison correction (E and F), two-tailed, unpaired t test (G and H), or multiple unpaired t tests with Holm–Šidák’s multiple comparison correction (I and J).