B7-H4 inhibits growth of neutrophil progenitors from BM. (A) B7-H4–deficient neutrophil progenitors have increased cell division. BM cells (2 × 10⁶) from indicated mice were labeled with CFSE and cultured for 3 days. Cells were harvested and doubly stained with anti–Gr-1/CD11b mAb. The dilution of CFSE in gated Gr-1⁺CD11b⁺ neutrophils was analyzed by flow cytometry. Data represent at least 3 independently performed experiments. (B) BM was harvested by flushing both femoral bones of WT B6 mice. Lineage⁺ cells were removed by PE-labeled anti–Gr-1, anti-CD11c, anti-Ter119, anti-CD11c, anti-CD3, anti-B220, and anti-CD19 (BD PharMingen) followed by MACS anti-PE microbeads and LD-negative selection columns (Miltenyi Biotec). The Lin⁻ cells in the BM were quantified and normally with 75% to 85% purity. Gr-1⁺/CD11b⁺ cells were completely depleted and were plated with 100 ng/mL SCF, 10 ng/mL G-CSF, and 40 μg/mL B7-H4Ig. Cells were harvested on 1 to 3 days for cell counting. Results are presented as triplicates of mean numbers with standard deviation. Data are a representative of 4 independent experiments. *P < .05. Error bars indicate SD (n = 3 wells of cultured cells). (C) Lin⁻ BM cells were prepared and in vitro differentiated as described in panel B. Cells were harvested from day 1 to day 3 and analyzed by staining with anti–Gr-1 and anti-CD11b mAb. (Top panel) Treated by control Ig. (Middle panel) Treated by B7-H4Ig. Cell division was also monitored by flow cytometric analysis of CFSE dilution (bottom panel). Closed symbol indicates control Ig; open symbol, B7-H4Ig.