CSF1R blockade exacerbates aGVHD. Lethally irradiated C57BL/6 recipients underwent transplantation with 10×10⁶ TCD BM cells with or without 5 × 10⁶ CD3⁺ T cells from BALB/c donors. M279 or isotype control antibody was administered from days 0 to 14. Mice were euthanized on day 14 after transplant for assessment of the brain. (A) Absolute number of host microglia (CD45^dimCD11b⁺H2Dd^–) in brains of recipients at day 14 after transplant within the live (SytoxBlue^neg) Ly6G^neg population; n = 7 to 8 mice per group; data pooled from 2 independent experiments. (B) MHC class II expression (mean fluorescence intensity [MFI]) on host microglia at day 14 after transplant; n = 7 to 8 mice per group; data pooled from 2 independent experiments. (C) Absolute number of Ly6C^hiMHC II⁺ intermediate inflammatory monocytes within the live CD45^hiCD11b⁺ population in the brains of transplant recipients at day 14 after transplant; n = 3 to 4 mice per group, representative of 2 independent experiment. (D) Absolute number of CD8⁺ T cells in the brain at day 14 after transplant, within the live CD45⁺CD11b^–CD3⁺ population; n = 7 to 8 mice per group; data pooled from 2 independent experiments. (E) Absolute number of CD4⁺ T cells in the brain at day 14 after transplant, within the live CD45⁺CD11b^–CD3⁺ population; n = 7 to 8 mice per group; data pooled from 2 independent experiments. (F) mRNA expression of Tnf, Ccl2, and Ifng in the brain at day 14 after transplant as detected by qRT-PCR; n = 4 to 12 mice per group; data pooled from 2 independent experiments. (G) Time recipients spent mobile in the FST, out of a total time of 180 seconds, at day 14 after transplant; n = 5 to 12 mice per group; data pooled from 3 independent experiments. (H) Representative confocal images of IBA1⁺ microglia in the hippocampus ([left panel] original magnification, ×20; scale bar, 50 μm) of naïve CX3CR1cre × CSF1R^flfl mice. Higher magnification illustrates altered morphology ([right panel] original magnification, ×100; scale bar, 10 μm). Nuclei counterstained with DAPI. (I) Quantification of IBA1⁺ cells in the hippocampus. (J) Violin plot showing the quantification of the size of IBA1⁺ cells in the hippocampus from 3 mice per group, expressed as pixels squared. Bold line represents the median. (K) Representative confocal images of IBA1⁺ microglia in the cortex ([left panel] original magnification, ×20; scale bar, 50 μm) of naïve CX3CR1cre × CSF1R^flfl mice. Higher magnification illustrates altered morphology ([right panel] original magnification, ×100; scale bar, 10 μm). Nuclei counterstained with DAPI. (L) Quantification of IBA1⁺ cells in the cortex. (M) Violin plot showing the quantification of the size of IBA1⁺ cells in the cortex from 3 mice per group, expressed as pixels squared. Bold line represents the median. (N) mRNA expression of Sall1, Tnf, and Ccl12 in the brain as detected by qRT-PCR. (O) Time naïve CX3CR1cre × CSF1R^wt/wt and CX3CR1cre × CSF1R^flfl mice spent mobile in the FST out of a total time of 180 seconds. Statistics calculated by: (A-E) ordinary 1-way ANOVA with Tukey multiple comparisons test; (F) Kruskal-Wallis nonparametric 1-way ANOVA with Dunn multiple comparisons test on ΔCT values; (G) ordinary 1-way ANOVA with Tukey multiple comparisons test (GVHD + iso mAb vs GVHD + M279 vs TCD + iso mAb) and Student unpaired t test (TCD + iso mAb vs TCD + M279); (M-N) Mann-Whitney nonparametric t test (comparisons performed on ΔCT values for qRT-PCR data); (O) Student unpaired t test. Data presented as mean ± SEM, except for (J,M) 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.