Differential expression of MHC class II by donor-derived macrophages and host microglia late after transplant. (A) Schematic of transplantation regime for the identification of donor macrophages and host microglia. Lethally irradiated DBA2xF1.Csf1r-eGFP (H2^b/d) mice received 5 × 10⁶ TCD BM with no T cells or with 0.5 × 10⁶ CD3⁺ T cells from C57Bl/6.Csf1r-mApple (H2^b) donors to induce low-grade nonlethal cGVHD. (B) Representative flow cytometric dot plots for the identification of donor macrophages (mApple) and host microglia (MacGreen) within the CD45^dimCD11b⁺ population from digested coronal sections of the brains of cGVHD mice at days 14, 35, and 70 after transplant. Gated on forward and side scatter to identify live (Sytox Blue⁻) CD45⁺Ly6G⁻ cells. (C) Proportions of host microglia and donor BMDMs within the CD45^dimCD11b⁺ population in cGVHD brains (day 14: n = 3; day 35: n = 7; data pooled from 2 independent experiments; day 70: n = 13; data pooled from 4 independent experiments). (D) Representative histograms of MHC class II expression on TCD microglia compared with GVHD host microglia and donor BMDMs at days 35 and 70 after transplant. (E) Fold change of MHC class II mean fluorescence intensity (MFI) on GVHD host microglia and donor BMDMs relative to TCD host microglia at days 35 and 70 posttransplantation (day 35: n = 7; data pooled from 2 independent experiments; day 70: n = 9; data pooled from 2 independent experiments). (F) Representative ×100 original magnification confocal images demonstrating differential expression of MHC class II by donor macrophages (green arrows) and host microglia (white arrows) in situ in the hippocampus of GVHD and TCD mice 70 days after transplant. B6.Csf1r-eGFP donors used to facilitate identification of donor-derived macrophages (GFP⁺/Iba1⁺) compared with resident microglia (GFP⁻/Iba1⁺). Nuclei counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Original magnification ×100; scale bar, 10 μm. (G) Findings replicated in a second model of cGVHD (day 70 after transplant) where BALB/c donor BM plus CD3⁺ T cells were transplanted into B6.Csf1r-eGFP recipients. MHC class II expression restricted to donor Iba1⁺/GFP⁻ BMDMs (green arrows) and absent from host Iba1⁺/GFP⁺ microglia (white arrows). Original magnification ×100; scale bar, 10 μm. Data presented as mean ± standard error of the mean. Statistical significance calculated by 1-way analysis of variance with multiple comparisons (C) or unpaired Student t test (E). **P < .01, ***P < .001.