Different treatments have distinct effects on microbiota and immune cells. (A–C) Experimental plan of vedolizumab treatment (A), antibiotic regimen (B), and BMT (C) in Rag1w/w (W/W) mice. (D) Colitis score based on immunohistochemical analysis of 12-wk-old wild-type (+/+, n = 15), and W/W mice untreated (UNT, n = 20), or treated with VDZ (n = 8), antibiotics (ANMV, n = 15), or BMT (n = 15). Bars show the mean with SEM. (E) Bar plots showing absolute counts of total TCRβ+ T cells isolated from the LP of 12-wk-old +/+ mice (n = 15), and W/W mice left untreated (UNT, n = 20), or treated with VDZ (n = 8), antibiotics (ANMV, n = 15), or BMT (n = 15). Bars show the mean with SEM. (F) RNAscope analysis of colon biopsy isolated from untreated W/W mice, or mice treated with VDZ, ANMV, or BMT showing Ifng (40×, scale bar: 20 μm) and Cxcl9 transcript expression (10×, scale bar: 100 μm). (G) Subset distribution of cytokine production by Th cells after restimulation isolated from the LP of 12-wk-old +/+ mice (n= 15), and W/W mice left untreated (UNT, n = 20), or treated with VDZ (n = 8), antibiotics (ANMV, n = 15), or BMT (n = 15). (H) UMAP plot (left panel) showing the expression profiles of CD4+ T cells isolated from ANMV-treated W/W mice. Colors represent cells clustered together on the basis of similarity of global gene expression. The stacked bar plot (right panel) shows cluster distribution in each condition. Single-cell samples were obtained from four to five pooled mice/group. (I and J) Serum levels of IFN-γ (I) and CXCL10 (J) of 12-wk-old +/+ mice (n= 7), and W/W mice left untreated (UNT, n = 17), or treated with VDZ (n = 5), antibiotics (ANMV, n = 19), or BMT (n = 5). Error bars show the mean with SEM. (K) Alpha diversity of fecal microbiota collected from +/+ (n = 4) or W/W mice before (UNT, n = 10) and after BMT (n = 10). Significance was determined by a Wilcoxon rank test. (L) Relative abundance of Firmicutes in the fecal microbiota from +/+ (n = 4) or W/W mice before (UNT, n = 10) and after BMT (n = 10). Significance was computed from the linear mixed-effects model ∼Group + (1|SubjectID) using the MaASLin2 R package. (M) Parallel univariate analysis comparing the fecal metabolomes of W/W mice after BMT to before BMT (n = 10/group) with notable metabolite families highlighted by color. FDR levels as calculated via a Benjamini–Hochberg correction are indicated. +/+ mice were internally bred (C57BL/6J [CD45.2] or B6.SJL-Ptprca Pepcb/BoyJ [CD45.1]). Data are from more than three independent experiments. Groups were compared using a Kruskal–Wallis test with Dunn’s multiple comparisons correction (B, E, and G). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. VDZ, vedolizumab.