Transfer of donor Ifngr^–/– BM alleviates CNS cGVHD. (A) Absolute number of CD45^dimCD11b⁺GFP^– donor BMDM in the CD45^posSytox^neg (live) population; n = 9 mice per group; data pooled from 2 independent experiments. (B) MHC class II expression on CD45^dimCD11b⁺GFP^– donor BMDM (mean fluorescence intensity [MFI]); n = 9 mice per group; data pooled from 2 independent experiments. (C) Absolute number of CD45^dimCD11b⁺GFP⁺ host microglia in the CD45^posSytox^neg (live) population; n = 9 mice per group; data pooled from 2 independent experiments. (D) MHC class II expression on CD45^dimCD11b⁺GFP⁺ host microglia; n = 9 mice per group, data pooled from 2 independent experiments. (E) Representative confocal images of IBA1⁺ cells and MHC II expression in the hippocampus at day 70 after transplant. Green arrows indicate host microglia (GFP⁺IBA1⁺MHCII^–) and white arrows indicate donor BMDM (GFP^–IBA1⁺MHCII⁺); original magnification, ×20; scale bar, 50 μm. Nuclei counterstained with DAPI. (F) Absolute number of CD8⁺ T cells in the live CD3⁺ population in the brain; n = 4 to 5 mice per group; data representative of 2 independent experiments. (G) Absolute number of CD4⁺ T cells in the live CD3⁺ population in the brain; n = 4 to 5 mice per group; data representative of 2 independent experiments. (H) mRNA expression of Ifng and Ccl2 in the brain as detected by qRT-PCR; n = 6 mice per group, data representative of 2 independent experiments. (I) Time recipients spent mobile in the forced swim test, out of a total time of 180 seconds, at day 70 after transplant; n = 11 mice per group; data pooled from 2 independent experiments. (J) UMAP plot of the 54 789 nuclei isolated from the brains of recipients of WT BM + T, Ifngr^–/– BM + WT T, and WT TCD BM, showing cell populations identified via unsupervised clustering. (K) UMAP plot demonstrating subclustering of the microglia/PVM/BMDM cluster into 2 distinct populations. (L) Depiction of myeloid cell proportions comprised of population 0 (microglia/PVM) and population 1 (BMDM) across samples. (M-P) Volcano plots show -log₁₀ of the adjusted P value and average log₂ fold change (FC) for all genes in the listed comparison, with highlighting of those that are significantly upregulated (avg_log2FC > 0.6 + P < .05, red) or downregulated (avg_log₂FC < 0.6 + P > .5, blue). Gray dots indicate genes that are not differentially expressed. (M) Volcano plot for microglia in recipients of WT BM + T vs WT TCD BM. (N) Volcano plot for microglia in recipients of Ifngr^-/- BM + T vs WT BM + T. (O) Volcano plot for BMDM in recipients of WT BM + T vs WT TCD BM. (P) Volcano plot for BMDM in recipients of Ifngr^–/– BM + T vs WT BM + T. Statistics calculated by (A-D) Student unpaired t test; (F-G,I) ordinary 1-way ANOVA with Tukey multiple comparisons test; (K) Mann-Whitney nonparametric t test (WT BM + T vs Ifngr^–/– BM + WT T or WT BM + T vs WT BM TCD). Data presented as mean ± SEM. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; DAPI, 4′,6-diamidino-2-phenylindole; mRNA, messenger RNA; PVM, perivascular macrophages; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean.