Antibiotic use in early life preferentially inhibits MAIT cell development. (A) WT mice received antibiotics (ABX) by daily oral gavage from 2 to 4 wk and were co-housed with untreated age-matched mice for 1 wk. Animals were then analyzed at 8 wk. (B and C) Fold change (FC) in the number of T cells in the skin (B) and lungs (C) of 8-wk-old mice following treatment described in A relative to untreated controls (n = 5–6/group, 2 pooled expts.). (D and E) Mice received the indicated antibiotics from 2 to 4 wk and the relative abundance of the ribD gene within their feces (D) and the DNA content per fecal pellet (E) were determined prior to cohousing (n = 3/group, rep. of 2 expts.). (F) IL-2 detected in coculture assay incubated with depicted concentrations of 5-OP-RU, 60 pM 5-OP-RU with anti-MR1 antibody (60 + αMR1), or no 5-OP-RU added (0). (G) IL-2 detected in coculture assay incubated with supernatants of feces from mice during antibiotic treatment as described in A (n = 3–4/group, rep. of 2 expts.). (H) 16S rRNA gene sequencing of the feces from untreated 8-wk-old mice and animals that received ampicillin (Amp), vancomycin (Vanc), or metronidazole (Met) from 2 to 4 wk following the cessation of antibiotic use or at the conclusion of cohousing (CH). Data represent the mean (n = 3 mice/group). (I–L) The number (I and K) and frequency (J and L) of MAIT cells in the ear pinnae (I and J) and lungs (K and L) of mice at 8 wk old following treatment as described in A (n = 10–12/group, 2 pooled expts.). Data represent the mean ± SEM, with *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 as calculated by one sample t test relative to a hypothetical value of 1 (B and C) or ordinary one-way ANOVA with Šidák’s multiple comparison correction (D, E, G, I, and L).