CSF1R blockade during established cGVHD. Lethally irradiated B6D2F1 mice received 5 × 10⁶ TCD BM cells from a B6.Csf1r-eGFP donor with or without 0.5 × 10⁶ CD3⁺ RFP⁺ T cells on day 0. M279 or isotype control antibody was administered from days 51 to 70 after transplant. CNS cGVHD was assessed on day 70 after transplant. (A) Frequency of CD45^dimCD11b⁺ cells in the brain at day 70 after transplant; n = 3 to 4 mice per group, representative of 2 independent experiments. (B) Absolute number of CD45^dimCD11b⁺ cells in the brain at day 70 after transplant; n = 3 to 4 mice per group, representative of 2 independent experiments. (C) Representative confocal images of GFP⁺/IBA1⁺ donor BMDM and GFP^–/IBA1⁺ host microglia, and MHC class II expression in the hippocampus. Nuclei counterstained with DAPI. (D) Quantification of the total number of IBA1⁺ cells in the hippocampus at day 70 after transplant; n = 4 to 5 mice per group; data representative of 2 independent experiments. (E) Violin plot showing the quantification of the size of IBA1⁺ cells in the hippocampus from 4 to 5 mice per group, expressed as pixels squared. Bold line represents the median. (F) Representative confocal images of GFP⁺/IBA1⁺ donor BMDM and GFP^-/IBA1⁺ host microglia, and MHC class II expression in the cortex. Nuclei counterstained with DAPI. (G) Quantification of the total number of IBA1⁺ cells in the cortex at day 70 after transplant; n = 4 to 5 mice per group; data representative of 2 independent experiments. (H) Violin plot showing the quantification of the size of IBA1⁺ cells in the cortex from 4 to 5 mice per group, expressed as pixels squared. Bold line represents the median. (I) Absolute number of donor graft-derived RFP⁺CD3⁺CD8⁺ T cells in the brain at day 70 after transplant; n = 3 to 4 mice per group; data from 1 experiment. (J) Absolute number of donor graft-derived RFP⁺CD3⁺CD4⁺ T cells in the brain at day 70 after transplant; n = 3 to 4 mice per group; data from 1 experiment. (K) mRNA expression of Ifng and Ccl2 in brains of transplant recipients as detected by qRT-PCR; n = 5 to 11 mice per group; data pooled from 2 independent experiments. (L) Time recipients spent mobile in the FST out of a total time of 180 seconds at day 70 after transplant; n = 5 to 15 mice per group; data pooled from 3 independent experiments. (C,F) Original magnification, ×20; scale bar, 50 μm. Insets: original magnification, ×100; scale bar, 10 μm. White arrows indicate IBA1⁺/GFP^–/MHC class II^- host microglia. Green arrows indicate IBA1⁺/GFP⁺/MHC class II⁺ donor BMDM. Statistics calculated by (A-B,I-J,L) ordinary 1-way ANOVA with Tukey multiple comparisons test, (D-E,G-H) Kruskall-Wallis nonparametric 1-way ANOVA with Dunn multiple comparisons test, (K) Mann-Whitney nonparametric t test (GVHD + iso mAb vs TCD + iso mAb or GVHD + M279 vs TCD + iso mAb). Data presented as mean ± SEM, except (E,H) presented as median. ∗P < .05; ∗∗P < .01; ∗∗∗∗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.