American Society of Hematology

Figure 6.

$TIMP-deficient microenvironment is suboptimal for B-cell development. (A) Schematic showing the experimental procedure for WT BM transplants into WT or QT3+/−. Recipients were irradiated twice for a total of 11 Gy and 2 million BM cells from donor mice were injected 24 hours later. After 16 weeks, recipient BM were analyzed by flow cytometry. (B) Total BM cells (left panel) and donor-derived (CD45.2−) total BM cell count (right panel), 16 weeks posttransplantation, in recipient mice (WT→WT, n = 3; WT→QT3+/−, n = 3). Data represent mean plus or minus SEM; groups were compared by the unpaired Student t test; *P < .05. Experiment was repeated twice, and similar results were seen. (C) Percentage and absolute count of donor’s (CD45.2−) B cells, pre-B-cell and pro-B-cell experiment in panel B (WT→WT, n = 4; WT→QT3+/−, n = 4). Data represent mean plus or minus SEM; groups were compared by the unpaired Student t test; *P < .05. (D) Percentage (left) and absolute number (right) of B-cell developmental fractions MLP, CLP, Fr-A, Fr-B and C, Fr-C′ of transplanted cells (CD45.2−) in recipient mice from the experiment in panel B. Data represent mean plus or minus SEM; groups were compared by 2-way ANOVA; ***P < .001. (E) Schematic outlining the protocol to analyze WT and QT3+/− B-cell development in vitro. Pro-B cells were FACS-sorted from WT or QT3+/− mice, plated in 96-well plates, and cultured with conditioned media from WT or QT3+/− BMSC cultures. (F) Number of pro-B (left) and pre-B (right) cells after 6 days’ culture of FACS-sorted pro-B cells, with or without conditioned media of WT-BMSCs or QT3+/− BMSCs, expressed as fraction of total cells plated. **P < 0.01.$

TIMP-deficient microenvironment is suboptimal for B-cell development. (A) Schematic showing the experimental procedure for WT BM transplants into WT or QT3^+/−. Recipients were irradiated twice for a total of 11 Gy and 2 million BM cells from donor mice were injected 24 hours later. After 16 weeks, recipient BM were analyzed by flow cytometry. (B) Total BM cells (left panel) and donor-derived (CD45.2⁻) total BM cell count (right panel), 16 weeks posttransplantation, in recipient mice (WT→WT, n = 3; WT→QT3^+/−, n = 3). Data represent mean plus or minus SEM; groups were compared by the unpaired Student t test; *P < .05. Experiment was repeated twice, and similar results were seen. (C) Percentage and absolute count of donor’s (CD45.2⁻) B cells, pre-B-cell and pro-B-cell experiment in panel B (WT→WT, n = 4; WT→QT3^+/−, n = 4). Data represent mean plus or minus SEM; groups were compared by the unpaired Student t test; *P < .05. (D) Percentage (left) and absolute number (right) of B-cell developmental fractions MLP, CLP, Fr-A, Fr-B and C, Fr-C′ of transplanted cells (CD45.2⁻) in recipient mice from the experiment in panel B. Data represent mean plus or minus SEM; groups were compared by 2-way ANOVA; ***P < .001. (E) Schematic outlining the protocol to analyze WT and QT3^+/− B-cell development in vitro. Pro-B cells were FACS-sorted from WT or QT3^+/− mice, plated in 96-well plates, and cultured with conditioned media from WT or QT3^+/− BMSC cultures. (F) Number of pro-B (left) and pre-B (right) cells after 6 days’ culture of FACS-sorted pro-B cells, with or without conditioned media of WT-BMSCs or QT3^+/− BMSCs, expressed as fraction of total cells plated. **P < 0.01.

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