Figure 7.
CXCL12 mislocalization limits B-cell development in TIMP deficiency. (A) CXCL12 protein concentration in media obtained from in vitro BMSC cultures, measured by sandwich ELISA. BMSCs were cultured for 2 weeks (WT, n = 3; QT3+/−, n = 3). Statistics by the unpaired Student t test; ***P < .001. (B) Soluble CXCL12 protein concentration in BM from WT and QT mice at 4 weeks of age, assayed by sandwich ELISA (WT, n = 3; QT, n = 3). Data represent mean plus or minus SEM; groups were compared by the unpaired Student t test; **P < .01. (C) Transmigration of pro-B, pre-B mature, and total B cells toward recombinant CXCL12. Data presented as percentage of input cell number migrated to the bottom chamber from both WT (n = 5) and QT3+/− (n = 4) BM. Statistics by 1-way ANOVA; *P < .05,**P < .01, ***P < .001. (D) Transmigration of pro-B, pre-B mature, and total B cells toward WT and QT3+/− BMSC culture media as well as positive and negative controls. Data presented as percentage of cell migrated to the bottom chamber for both WT (n = 2) and QT3+/− (n = 2) BM. (E) Representative images of CXCL12 (red), B220 (magenta), and LepR (green) staining of BM (diaphysis) from WT, QT3+/−, and QT tibia. Image showing B220+ B cells localizing around LepR+CXCL12+ (yellow; arrow) mesenchymal cells in WT, whereas in TIMP-deficient mice, morphology of mesenchymal cells is altered and with few B cells (arrowhead) around them. Insets, Magnified images of selected areas with arrows pointing to LepR+ cell processes. Scale bars, 150 µm (top panels), 40 µm (bottom panels), and 3× (insets). (F) Flow cytometric quantification of LepR+ cells in BM of 4-month-old WT and QT3+/− mice (gating strategy in supplemental Figure 7D). Statistics by the Student t test with *P < .05. (G) Summary schematic showing HSCs generating B-cell subsets. TIMP-deficient HSCs show cell-intrinsic defects, at least in part, due to the altered expression of transcription factors (TFs) critical for B-cell development. Within the BM niche, LepR+ mesenchymal cells provide IL7 for pro-B-cell proliferation and survival. CXCL12 production by LepR+ cells facilitate pro-B-cell retention to the vicinity of LepR+ cells for optimal IL7 dose. Differentiation into pre-B-cell requires CXCL12-mediated mobilization of precursors away from mesenchymal cells. LepR+ cells are fewer and have smaller processes in TIMP-deficient BM. Additionally, excessive soluble CXCL12 (both active and inactive forms) disrupts association of the developing B-cell population within this suboptimal stromal niche consequently reducing their numbers. DAPI, 4′,6-diamidino-2-phenylindole.

CXCL12 mislocalization limits B-cell development in TIMP deficiency. (A) CXCL12 protein concentration in media obtained from in vitro BMSC cultures, measured by sandwich ELISA. BMSCs were cultured for 2 weeks (WT, n = 3; QT3+/−, n = 3). Statistics by the unpaired Student t test; ***P < .001. (B) Soluble CXCL12 protein concentration in BM from WT and QT mice at 4 weeks of age, assayed by sandwich ELISA (WT, n = 3; QT, n = 3). Data represent mean plus or minus SEM; groups were compared by the unpaired Student t test; **P < .01. (C) Transmigration of pro-B, pre-B mature, and total B cells toward recombinant CXCL12. Data presented as percentage of input cell number migrated to the bottom chamber from both WT (n = 5) and QT3+/− (n = 4) BM. Statistics by 1-way ANOVA; *P < .05,**P < .01, ***P < .001. (D) Transmigration of pro-B, pre-B mature, and total B cells toward WT and QT3+/− BMSC culture media as well as positive and negative controls. Data presented as percentage of cell migrated to the bottom chamber for both WT (n = 2) and QT3+/− (n = 2) BM. (E) Representative images of CXCL12 (red), B220 (magenta), and LepR (green) staining of BM (diaphysis) from WT, QT3+/−, and QT tibia. Image showing B220+ B cells localizing around LepR+CXCL12+ (yellow; arrow) mesenchymal cells in WT, whereas in TIMP-deficient mice, morphology of mesenchymal cells is altered and with few B cells (arrowhead) around them. Insets, Magnified images of selected areas with arrows pointing to LepR+ cell processes. Scale bars, 150 µm (top panels), 40 µm (bottom panels), and 3× (insets). (F) Flow cytometric quantification of LepR+ cells in BM of 4-month-old WT and QT3+/− mice (gating strategy in supplemental Figure 7D). Statistics by the Student t test with *P < .05. (G) Summary schematic showing HSCs generating B-cell subsets. TIMP-deficient HSCs show cell-intrinsic defects, at least in part, due to the altered expression of transcription factors (TFs) critical for B-cell development. Within the BM niche, LepR+ mesenchymal cells provide IL7 for pro-B-cell proliferation and survival. CXCL12 production by LepR+ cells facilitate pro-B-cell retention to the vicinity of LepR+ cells for optimal IL7 dose. Differentiation into pre-B-cell requires CXCL12-mediated mobilization of precursors away from mesenchymal cells. LepR+ cells are fewer and have smaller processes in TIMP-deficient BM. Additionally, excessive soluble CXCL12 (both active and inactive forms) disrupts association of the developing B-cell population within this suboptimal stromal niche consequently reducing their numbers. DAPI, 4′,6-diamidino-2-phenylindole.

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