Antibiotics increase immune cell infiltration in the brain during aGVHD. (a–c) Analysis of the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). (a and b) Quantification of the percentage of CD3⁺ CD45⁺ cells using flow cytometry (a) along with (b) representative flow cytometry plots of each group. (c) Quantification of absolute numbers of CD3⁺ CD45⁺ cells. Experiments were performed four (a and b) or five times (c) and results (mean ± SEM) pooled. n = 14–22 per group. Each data point represents a biological replicate. P values were calculated using the Mann–Whitney U test. *P < 0.05, **P < 0.01. (d–f) Histology of brain samples stained for CD3⁺ cells on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). The scatter plot shows absolute number (per mm²) of CD3⁺ cells in the (d) cortex and (e) cortical meninges, along with (f) representative images of each group (scale bars, 20 μm; inset, 10 μm). Experiments were performed three times and results (mean ± SEM) pooled. n = 11–14 per group. Each data point represents a biological replicate. P values were calculated using either two-sided Student’s unpaired t test or the Mann–Whitney U test. *P < 0.05, **P < 0.01. (g and h) Analysis of the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics and transplanted on D0 with different T cell numbers. (g) Quantification of the percentage of CD3⁺ CD45⁺ cells using flow cytometry along with (h) representative flow cytometry plots of each group. Experiments were performed two times and results (mean ± SEM) pooled. n = 11–14 per group. Each data point represents a biological replicate (n = 1). P values were calculated using the Kruskal–Wallis test. *P < 0.05, **P < 0.01. (i–y) Flow cytometry analysis of the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). (i) Quantification of the percentage of CD44⁺ CD62L⁺ cells along with (j) representative flow cytometry plots of indicated groups. Quantification of the (k) ratio of CD3⁺ CD8⁺ cells to CD3⁺ CD4⁺ cells, (l) IFN-γ in CD3⁺ CD8⁺ cells, (n) IFN-γ in CD3⁺ CD4⁺ cells, (p) CD69 on CD3⁺ CD45⁺ cells, (r) CD25 on CD3⁺ CD45⁺ cells, (t) percentage of Ly6G⁺ cells, and (v) CD206⁺ cells, and (x) E-cadherin on CD31⁺ CD45⁻ cells. Representative histograms of (m) IFN-γ in CD3⁺ CD8⁺ cells, (o) IFN-γ in CD3⁺ CD4⁺ cells, (q) CD69 on CD3⁺ CD45⁺ cells, and (s) CD25 on CD3⁺ CD45⁺ cells and representative flow cytometry plots of (u) Ly6G⁺ and (w) CD206⁺ cells in each group. The experiment was performed once (i and j), two times (r, s, v, w, x, and y), three times (p, q, t, and u), or four times (k–o) and results (mean ± SEM) pooled. n = 5–20 per group. Each data point represents a biological replicate. P values were calculated using either two-sided Student’s unpaired t test or the Mann–Whitney U test. *P < 0.05, **P < 0.01.