Microbiota depletion does not affect circulating and splenic T cell populations, as well as costimulatory molecule expression on microglia in aGVHD. (a–l) Flow cytometry analysis of the spleen and PBMCs isolated from the blood on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). Quantification of (a) CD69 on CD3⁺ CD4⁺ cells, (b) CD69 on CD3⁺ CD8⁺ cells, (c) percentage of CD3⁺ CD45⁺ cells, (d) percentage of CD3⁺ CD4⁺ cells, (e) percentage of CD3⁺ CD8⁺ cells, (f) IFN-γ in CD3⁺ CD8⁺ cells, and (g) IFN-γ in CD3⁺ CD4⁺ cells in PBMCs isolated from the blood. Quantification of the (h) percentage of CD3⁺ CD45⁺ cells, (i) percentage of CD3⁺ CD4⁺ cells, (j) percentage of CD3⁺ CD8⁺ cells, (k) IFN-γ in CD3⁺ CD8⁺, and (l) IFN-γ in CD3⁺ CD4⁺ cells in the spleen. The experiment was performed once (a–e and h–j) or two times (f, g, k, and l). n = 4–10 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. *P < 0.05, ***P < 0.001. (m) Quantification of the brain weight on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). The experiment was performed once. n = 4 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. (n–p) Flow cytometry analysis of CD45^int CD11b⁺ cells in the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). Quantification of percentage of (n) P2RY12 and (o) H2-k^d+ cells, along with (p) representative histograms of each group. The experiment was performed once (o) or two times (n) and results (mean ± SEM) pooled. n = 4–7 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. (q) Flow cytometry analysis of CD11b⁺ CD45^int cells in the brain on D+14 after allo-HCT or syn-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics. Quantification of the percentage of CD45^int CD11b⁺ cells. Experiments were performed two times and results (mean ± SEM) pooled. n = 8–10 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. (r and s) Histology of brain samples stained for Iba-1⁺ cells and analyzed on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). Quantification of the absolute number (per mm²) of Iba-1⁺ cells in the (r) hippocampus and (s) cerebellum. The experiment was performed once (r) or three times (s) and results (mean ± SEM) pooled. n = 3–10 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. (t–w) Flow cytometry analysis of CD11b⁺ CD45^hi cells and CD11b⁺ CD45^int cells in the brain on D+14 after allo-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics or vehicle (water). Quantification of (t) TLR4 on CD11b⁺CD45^hi cells, (u) MHC II on CD11b⁺ CD45^int cells, (v) CD40 on CD11b⁺ CD45^int cells, and (w) CD80 on CD11b⁺ CD45^int cells. The experiment was performed once (w), three times (t and v), or four times (u) and results (mean ± SEM) pooled. n = 4–17 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test or the Mann–Whitney U test. **P < 0.01. (x–z) Flow cytometry analysis of CD11b⁺ CD45^int cells in the brain on D+14 after allo-HCT or syn-HCT from SPF BALB/c (H2-k^d+) recipients treated with antibiotics. Quantification of (x) P2RY12, (y) CD11c, and (z) TLR4. The experiment was performed once. n = 3 or 5 per group. Each data point represents a biological replicate. P values were calculated using two-sided Student’s unpaired t test. **P < 0.01.