Relationship between IL-7R signaling and CXCR4-mediated proB cell motility. (A) Total number of developing B cell subsets from femur and tibia (left) and from spleen (right) of mice conditionally deficient in IL-7Ra in B-lineage cells: Mb1^Cre/+ Il7ra^Fl/+ (black) and Mb1^Cre/+ Il7ra^Fl/Fl (gray) mice. (B) Mobilization index: ratio of proB and preB cell number in the spleen to that in the BM of Mb1^Cre/+ Il7ra^Fl/+ (Het, black) and Mb1^Cre/+ Il7ra^Fl/Fl (KO, gray) mice. Circles depict individual mice. (C) CXCR4 geometric mean fluorescence intensity (GMFI) in proB cells (left) and preB cells (right) gated as in A in Mb1^Cre/+ Il7ra^Fl/+ (Het, black) and Mb1^Cre/+ Il7ra^Fl/Fl (KO, gray) mice. (D) In vitro transwell (5 µm) migration assay of Mb1^Cre/+ Il7ra^Fl/+ (Het, black) and Mb1^Cre/+ Il7ra^Fl/Fl (KO, gray) BM cells toward CXCL12 gradients. Y axis indicates frequency of migrated cells; x axis shows CXCL12 concentration (nanograms per milliliter). (A–D) Lines indicate mean; circles depict individual mice (n = 3). (E) FAK expression in proB cells isolated from C57BL/6 mice untreated (littermate controls, black) or treated with anti–IL-7Ra blocking antibodies (gray) for 36 h. (F) α4 integrin expression in proB cells isolated from C57BL/6 mice treated with isotype control (black) or anti–IL-7Ra blocking antibody (gray) for 36 h. (E and F) Numbers inside histograms indicate geometric mean fluorescence intensity ± SD (n = 3 mice in each group). (G) Mean motility coefficient of proB cells in mice untreated (black) or treated with IL-7Ra blocking antibodies (gray) for 36 h. GFP⁺ proB cells were visualized in calvaria of lethally irradiated Cxcl12^dsRed/+ mice reconstituted with BM cells from Rag1^GFP/GFP mice. ProB cell movement was tracked for 30 min in vivo by IVM. Lines represent the mean motility coefficient from three separate videos per mouse. (H) Median velocity. Circles, individual cells. (I) Measurement of cell axis ratio of GFP⁺ proB cells before (black) and after (gray) treatment with anti–IL-7Ra blocking antibody. Each point represents an individual cell analyzed. (J) Frequency of GFP⁺ proB cells found in contact with Cxcl12-dsRed cells in mice treated with IL-7Ra blocking antibody (gray) or untreated (black). Circles, average from individual fields of view. (K) Model of the proB/IL-7⁺ cell circuit. *, P < 0.05; **, P < 0.005; ***, P < 0.0005; ****, P < 0.00005 by unpaired Student’s t test. Data in all panels are representative of two to four independent experiments.