CSF1R blockade during aGVHD induces region-specific microglia depletion. Lethally irradiated C57BL/6 recipients received 10 × 10⁶ TCD BM with or without 5 × 10⁶ enriched splenic T cells from BALB/c donors on day 0. M279 or isotype control antibody was administered from days 0 to 14. Behavior and CNS inflammation parameters were assessed on day 14 after transplant. (A) Representative confocal images of IBA1⁺ microglia and MHC class II expression in the hippocampus of GVHD and TCD mice at day 14 after transplant after treatment with isotype control antibody or M279; original magnification, ×20; scale bar, 50 μm. Insets demonstrate microglia morphology; original magnification, ×60; scale bar, 20 μm. Nuclei counterstained with DAPI. (B) Quantification of the total number of IBA1⁺ cells in the hippocampus at day 14 after transplant; n = 3 to 5 mice per group; data pooled from 2 independent experiments. (C) Violin plot showing the quantification of the size of IBA1⁺ cells in the hippocampus from 3 to 5 mice per group, expressed as pixels squared. Bold line represents the median. (D) Representative confocal images of IBA1⁺ microglia and MHC II expression in the cortex of GVHD and TCD mice at day 14 after transplant after treatment with isotype control antibody or M279; original magnification, ×20; scale bar, 50 μm. White arrows indicate IBA1⁺/MHC class II⁺ microglia. Nuclei counterstained with DAPI. (E) Representative confocal images demonstrating cortical microglia morphology; original magnification, ×60; scale bar, 20 μm. Nuclei counterstained with DAPI. (F) Quantification of the total number of IBA1⁺ cells in the cortex at day 14 after transplant; n = 3 to 5 mice per group; data pooled from 2 independent experiments. (G) Violin plot showing the quantification of the size of IBA1⁺ cells in the cortex from 3 to 5 mice per group, expressed as pixels squared. Bold line represents the median. (H) Representative images of IBA1⁺ cells in the cortex of GVHD and TCD mice at day 14 after transplant maintained on either a control diet or a diet containing the CSF1R small molecule inhibitor, PLX5622; original magnification, ×20; scale bar, 50 μm. Nuclei counterstained with DAPI. (I) Representative images demonstrating cortical microglia morphology; original magnification, ×60; scale bar, 20 μm. Nuclei counterstained with DAPI. (J) Quantification of the total number of IBA1⁺ cells in the cortex at day 14 after transplant; n = 3 to 5 mice per group; data from 1 independent experiment. (K) mRNA expression of Tnf and Ccl12 in the brain at day 14 after transplant as detected by qRT-PCR. Statistics calculated by ordinary 1-way ANOVA with Tukey multiple comparisons test (B,F,J,K [Ccl2]), Kruskal-Wallis nonparametric 1-way ANOVA with Dunn multiple comparisons test (C,G), and Mann-Whitney nonparametric unpaired t test (K [Tnf]). Data presented as mean ± SEM, except (C,G) presented as median. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean.