Genetic ablation of donor BMDM CSF1R signaling. (A) Illustration of transplantation of 5 × 10⁶ TCD BM cells and 0.5 × 10⁶ CD3⁺ T cells from Scl-Cre^ERT2 × ZsGreen^fl/fl donors to lethally irradiated B6D2F1 recipients. Timeline shows the experimental design for tamoxifen treatment and harvest of Scl-Cre^ERT2 × ZsGreen^fl/fl BM recipients. Mice were treated with tamoxifen for 5 days beginning at day 35 or day 70 after transplant to induce Cre-recombinase activity and euthanized at day 70 or day 90, respectively. (B) Representative flow cytometry dot plots and quantification for ZsGreen^neg and ZsGreen^pos cells within the CD45^dimCD11b⁺ population at day 70 post-transplant; n = 6 mice; data representative of 1 independent experiment. (C) Representative flow cytometry dot plots and quantification for ZsGreen^neg and ZsGreen^pos cells within the CD45^dimCD11b⁺ population at day 90 after transplant; n = 6 mice; data representative of 1 independent experiment. (D) Transplant design for the transfer of either CX3CR1cre^ERT2 × CSF1R^wt/wt Ai9 BM or CX3CR1cre^ERT2 × CSF1R^fl/fl Ai9 BM plus enriched WT splenic CD3⁺ T cells into lethally irradiated B6.Csf1r-eGFP × DBA2 F1 recipients. Experimental timeline illustrates the delivery of tamoxifen at day 35 for 5 consecutive days to induce Cre-recombinase activity. Two recipients per group were euthanized at day 39 to confirm induction of TdTomato (Ai9) expression and loss of CSF1R-dependent monocyte populations. Remaining mice were sacrificed at day 70 after transplant. (E) Clinical scores of recipients. Arrow indicates the initiation of tamoxifen treatments at day 35; n = 10 to 12 mice per group. (F) Representative flow cytometry dot plots illustrating the CD45^dimCD11b⁺ cell population within live Ly6G^negCD45^pos cells. Donor BMDM are gated as CD45^dimCD11b^posTdTomato^posMacGreen GFP^neg and host microglia are CD45^dimCD11b^posMacGreen GFP^posTdTomato^neg. (G) Absolute number of CD45^dimCD11b⁺TdTomato⁺ donor BMDM in the brain; n = 4 mice per group. (H) MHC class II expression on CD45^dimCD11b⁺TdTomato⁺ donor BMDM in the brain (mean fluorescence intensity [MFI]); n = 4 mice per group. (I) Absolute number of CD8⁺ T cells in the live CD3⁺ population in the brain; n = 4 mice per group. (J) Absolute number of CD4⁺ T cells in the live CD3⁺ population in the brain; n = 4 mice per group. (K) mRNA expression of Ifng and Ccl2 as detected by qRT-PCR; n = 5 to 6 mice per group. (L) Time recipients spent mobile in the FST, out of a total time of 180 seconds, at day 70 after transplant; n = 7 mice per group. Statistics calculated with Student unpaired t test. Data presented as mean ± SEM. ∗∗P < .01. mRNA, messenger RNA; qRT-PCR, quantitative reverse transcription polymerase chain reaction; SEM, standard error of the mean.