Recipient BM Mφs persist and expand posttransplantation. (A) Schematic of the experimental BM transplantation model. Lethally irradiated MacGreen recipient mice were transplanted with 1000 syngeneic B6.SJL LSK cells and harvested at indicated time points posttransplant. (B-C) Representative flow cytometry analysis of naive (B; no-Tx) and posttransplant (C; 5 weeks shown) recipient MacGreen BM (see supplemental Figure 2A-C for representative isotype control staining). F4/80⁺Ly6G^neg cells (gate i) were subsequently gated into VCAM-1⁺GFP⁺ cells (gate iii) that were predominantly CD169⁺ BM Mφ (dark green gate), VCAM-1^negGFP^hi cells (gate iv) consisted of CD169^neg recipient monocytes (light green gate x), VCAM-1⁺GFP^neg cells (gate v) consisted of donor BM CD169⁺ Mφs (orange gate), and VCAM-1^negGFP^neg cells (gate vi) contained CD169^neg donor monocytes (yellow gate viii). Granulocytes were gated as F4/80^negLy6G⁺ cells (gate ii), which in BM were uniformly GFP⁺ or GFP^neg in no-Tx and posttransplant samples, respectively (not depicted). (D) Number of recipient (blue bars) and donor (white bars) granulocytes (gate ii) per femur across the transplantation time course. (E) Total number of recipient (dark green bars) and donor (orange bars) Mφs and recipient (light green bars) and donor (yellow bars) monocytes per femur across the transplantation time course. In panels D and E, data are presented as mean ± SD. Statistical analysis of recipient Mφs was performed using 1-way ANOVA Tukey’s multiple comparison test. In panel E, asterisks represent significant differences in recipient Mφ number compared with 5 weeks posttransplant (*P < .05, **P < .01, and ****P < .0001). Flow cytometry plots and histograms as well as associated gated cell percent frequencies are from a representative animal, and the percentage of positive cells is based on the preceding parent populations. Data are representative of 5 to 13 individual animal samples per time point from either 1 (weeks 3 and 32), 2 (week 5), or 3 (all other time points) biological replicate experiments. 300 000 events were collected from each experiment sample by flow cytometry. MO, monocyte; Tx, transplant.