Fig. 5

Effects of PD-L1 blockade on migration of immune cells to the brain of TBI mice. After 7 days of TBI (6 days of PD-L1 blockade), whole brain was harvested from each mouse after removal of blood cells by perfusion with heparinized-PBS, followed by preparation of brain single-cell suspension. Cells were stained with the Zombie Violet™ FVD to exclude FVD-positive dead cells, followed by incubation with CD16/CD32 Abs for blocking FcR binding, and fluorochrome-conjugated Abs against mouse CD45, CD3, CD4, CD8, CD11b, Ly-6G, Ly-6C, F4/80, B220, and PD-1. Brain-infiltrating leukocytes (LYM) were defined as CD45^High cells that could be distinguished from CD45^Low glia cells. Within the CD45^High population, polymorphonuclear neutrophils (PMNs) were identified by Ly-6G expression, T cells as CD45^HighCD3⁺ cells, B cells as CD45⁺B220⁺, and M/Mɸ as CD45⁺CD11b⁺Ly-6G⁻. M/Mɸ were further divided into Ly-6C^High and Ly-6C^LowF4/80^High subsets. A Gating strategy for flow cytometric analysis of immune cells in the brain of TBI mice. B Representative flow plots of brain cells from the gated M/Mϕ (M) showing % of Ly-6C^High versus Ly-6C^LowF4/80^High M/Mϕ in the brain of TBI mice treated with PD-L1 Ab versus IgG. C Pooled data of Ly-6C^High versus Ly-6C^LowF4/80^High M/Mϕ in the brain of TBI mice treated with PD-L1 Ab versus IgG (n = 5). *p < 0.05. D Representative flow plots showing PD-1 expressing T cells. E Representative IHC of CD45⁺ leukocytes (red) in injured cortex from mice treated with PD-L1 Ab versus IgG (n = 5). F ELISA results of MCP-1 in the brain lysates (n = 3), MCP-1 in CSF and serum samples (n = 5). G The levels of PD-L1 and MCP-1 in the brain of TBI mice were present at opposite patterns of regulation. ANOVA test with Dunn’s corrections was used for comparing sham versus TBI. ns, not significant; *p < 0.05; ***p < 0.001