Figure 2.
B-cell defect detectable in fraction B and C in TIMPless mice. (A) Percentage (top) and total number (bottom) of live B220+ B cells and CD11b+Gr-1+ myeloid cells in the BM (2 femurs and 2 tibiae) WT, QT3+/−, and QT mice of different ages. Mature B cell as a percentage of total BM cells is shown for WT (1 month, n = 21; 2 months, n = 17; 6 months, n = 2; 24 months, n = 7), QT3+/− (1 month, n = 18; 2 months, n = 13; 6 months, n = 2; 24 months, n = 10), and QT (1 month, n = 13; 2 months, n = 8) BM. The absolute number (bottom) of live B220+ B cells in WT (1 month, n = 15; 2 months, n = 12; 6 months, n = 2; 24 months, n = 7), QT3+/− (1 month, n = 13; 2 months, n = 9; 6 months, n = 2; 24 months, n = 10), and QT (1 month, n = 7; 2 months, n = 3) BM. The B-cell percentage data were compared for using the ANOVA Bonferroni multiple comparisons test (1-month WT vs QT3+/−, QT3+/− vs QT) and the 2-tailed unpaired Student t test (2-month WT vs QT3+/−, QT3+/− vs QT). Absolute B-cell numbers were compared using Sidak multiple comparison (1-month WT vs QT, 2-month WT vs QT) and the 2-tailed unpaired Student t test (1-month WT vs QT3+/−, 24-month WT vs QT3+/−). For both, data represent mean plus or minus SEM with *P < .05, **P < .01, and ***P < .001, and ****P < .0001. (B) Schematic of B-cell developmental stages from HSCs in BM. (C) Percentage (left) and absolute number (right) of B-cell developmental fractions (Fr.) in WT, QT3+/−, and QT BM. These values are for 1 month (WT, n = 8; QT3+/−, n = 9; QT, n = 4), 6 month (WT, n = 2; QT3+/−, n = 2), and 24 months (WT, n = 4; QT3+/−, n = 7) old mice. Data represent mean plus or minus SEM and statistical comparison was by 2-way ANOVA using the Sidak multiple comparisons test between the age groups; *P < .05 and **P < .01. (D) Schematic showing the experimental method for assessing in vitro response of B cells to cell death cues. Splenic B cells were plated with a variety of death-inducing cues and incubated overnight (O/N) at 37°C before viability analysis by flow cytometry. (E) Schematic showing the experimental method for assessing proliferation of splenic B cells in response to LPS, as well as class switch recombination in response to IL4. Splenic B cells were dyed with CFSE before plating with LPS and IL4. After 4 days of culture at 37°C, CFSE levels and the number of IgG1+ cells were determined by flow cytometry. (F) B-cell response to death-inducing cues, including no treatment (NT), irradiation at 2 Gy (Irr), heat shock for 10 minutes (HS), and dexamethasone at a concentration of 0.1 nM (Dex 0.1 nM). This graph represents WT n = 3 and QT n = 1, but this experiment was repeated with slightly different conditions each time for a total of WT n = 9 and QT n = 3. (G) Proliferation index (average number of divisions undergone by a cell) of WT and QT splenocytes after 4 days in culture with LPS and IL4 (schematic shown in panel E). WT (n = 6), QT (n = 2); statistics by the unpaired Student t test. (H) Class switch recombination of splenocytes to IgG1+ cells throughout 7 generations, plotted as the frequency of cells that expressed IgG1 per generation. WT (n = 6), QT (n = 2), each in technical triplicate. Statistics by 2-way ANOVA. (I) Serum levels of IgG and IgM of 4-week-old mice as measured by ELISA. WT (n = 5), QT (n = 5); statistics by the unpaired Student t test. LMPP, lymphoid-primed multipotent progenitor; LT-HSC, long-term HSC; MPP, multipotent progenitor; ST-HSC, short-term HSC.

B-cell defect detectable in fraction B and C in TIMPless mice. (A) Percentage (top) and total number (bottom) of live B220+ B cells and CD11b+Gr-1+ myeloid cells in the BM (2 femurs and 2 tibiae) WT, QT3+/−, and QT mice of different ages. Mature B cell as a percentage of total BM cells is shown for WT (1 month, n = 21; 2 months, n = 17; 6 months, n = 2; 24 months, n = 7), QT3+/− (1 month, n = 18; 2 months, n = 13; 6 months, n = 2; 24 months, n = 10), and QT (1 month, n = 13; 2 months, n = 8) BM. The absolute number (bottom) of live B220+ B cells in WT (1 month, n = 15; 2 months, n = 12; 6 months, n = 2; 24 months, n = 7), QT3+/− (1 month, n = 13; 2 months, n = 9; 6 months, n = 2; 24 months, n = 10), and QT (1 month, n = 7; 2 months, n = 3) BM. The B-cell percentage data were compared for using the ANOVA Bonferroni multiple comparisons test (1-month WT vs QT3+/−, QT3+/− vs QT) and the 2-tailed unpaired Student t test (2-month WT vs QT3+/−, QT3+/− vs QT). Absolute B-cell numbers were compared using Sidak multiple comparison (1-month WT vs QT, 2-month WT vs QT) and the 2-tailed unpaired Student t test (1-month WT vs QT3+/−, 24-month WT vs QT3+/−). For both, data represent mean plus or minus SEM with *P < .05, **P < .01, and ***P < .001, and ****P < .0001. (B) Schematic of B-cell developmental stages from HSCs in BM. (C) Percentage (left) and absolute number (right) of B-cell developmental fractions (Fr.) in WT, QT3+/−, and QT BM. These values are for 1 month (WT, n = 8; QT3+/−, n = 9; QT, n = 4), 6 month (WT, n = 2; QT3+/−, n = 2), and 24 months (WT, n = 4; QT3+/−, n = 7) old mice. Data represent mean plus or minus SEM and statistical comparison was by 2-way ANOVA using the Sidak multiple comparisons test between the age groups; *P < .05 and **P < .01. (D) Schematic showing the experimental method for assessing in vitro response of B cells to cell death cues. Splenic B cells were plated with a variety of death-inducing cues and incubated overnight (O/N) at 37°C before viability analysis by flow cytometry. (E) Schematic showing the experimental method for assessing proliferation of splenic B cells in response to LPS, as well as class switch recombination in response to IL4. Splenic B cells were dyed with CFSE before plating with LPS and IL4. After 4 days of culture at 37°C, CFSE levels and the number of IgG1+ cells were determined by flow cytometry. (F) B-cell response to death-inducing cues, including no treatment (NT), irradiation at 2 Gy (Irr), heat shock for 10 minutes (HS), and dexamethasone at a concentration of 0.1 nM (Dex 0.1 nM). This graph represents WT n = 3 and QT n = 1, but this experiment was repeated with slightly different conditions each time for a total of WT n = 9 and QT n = 3. (G) Proliferation index (average number of divisions undergone by a cell) of WT and QT splenocytes after 4 days in culture with LPS and IL4 (schematic shown in panel E). WT (n = 6), QT (n = 2); statistics by the unpaired Student t test. (H) Class switch recombination of splenocytes to IgG1+ cells throughout 7 generations, plotted as the frequency of cells that expressed IgG1 per generation. WT (n = 6), QT (n = 2), each in technical triplicate. Statistics by 2-way ANOVA. (I) Serum levels of IgG and IgM of 4-week-old mice as measured by ELISA. WT (n = 5), QT (n = 5); statistics by the unpaired Student t test. LMPP, lymphoid-primed multipotent progenitor; LT-HSC, long-term HSC; MPP, multipotent progenitor; ST-HSC, short-term HSC.

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