IL-7–responsive bone marrow cells are pro-B cells and early pre-B cells that express IL-7R, IL-3R, and c-kit. (A) Flow cytometric analysis of IL-7R, IL-3R, B220, and c-kit expression of IL-7–responsive normal bone marrow cells. The cells are IL-7R⁺, predominantly CD19⁺ and c-kit⁺, and B220⁺, CD43⁺. They also express IL-3R at low density, and after gating, these cells were found to coexpress IL-7R and B220 (lower histograms). Bone marrow cells from a 7-week-old B6J129SVF₂ mouse were cultured for 10 days on NIH-3T3 cells that secrete mIL-7 (T220-29 cells) and then stained, fixed, and analyzed by flow cytometry. Fifty thousand cells were analyzed without gating for size. The percentage of positive cells is shown in the quadrant. Histograms: dotted line, isotype-matched control antibody; heavy line, antibody. (B) IL-3 and SCF induce the proliferation of IL-7–responsive cells. IL-7–responsive cells (wild-type bone marrow cells cultured for 9 days on T220-29 cells) were washed and cultured without feeder cells in the presence of rmIL-7 (5 ng/mL), rmIL-3 (10 ng/mL), or rmSCF (100 ng/mL). The cells were pulse-labeled with [³H]-thymidine and the incorporation of [³H]-thymidine was measured as counts per minute (cpm). Error bars show the standard deviation of 12 replicates. (C) IL-3 and IL-7 both activate STAT5 in IL-7R⁺ B-lymphoid progenitors from normal bone marrow. A gel mobility shift assay was performed after 10 hours of IL-7 starvation by using +/+ bone marrow cells that had been cultured for 14 days on T220-29 cells. The addition of anti-Stat5A antiserum50 reduced STAT5-DNA complex formation, whereas control serum did not. The arrowhead indicates the position of the Stat5-DNA complex.