Blk haploinsufficiency impairs the development, but enhances the functional responses, of MZ B cells

Abstract

Blk was identified two decades ago as a B-cell-specific member of the Src family of tyrosine kinases. Recent studies, however, have discovered that Blk is expressed in many cell types outside of the B lineage, including early thymic precursors, interleukin-17-producing γδ T cells and pancreatic β-cells. In light of these recent discoveries, we performed a more comprehensive analysis of Blk expression patterns in hematopoietic cells and found that Blk is differentially expressed in mature B-cell subsets, with marginal zone (MZ) B cells expressing high levels, B1 B cells expressing intermediate-to-high levels and follicular (FO) B cells expressing low levels of Blk. To determine whether these differences in Blk expression levels reflected differential requirements for Blk in MZ, B1 and FO B-cell development, we analyzed the effects of reducing and eliminating Blk expression on B-cell development. We report that both Blk haploinsufficiency and Blk deficiency impaired the generation of MZ B cells. Moreover, although there were fewer MZ B cells in Blk(+/-) and Blk(-/-) mice as compared with Blk(+/+) mice, Blk-mutant MZ B cells were hyper-responsive to B-cell receptor stimulation, both in vitro and in vivo. Thus, this study has revealed a previously unappreciated role for Blk in the development and activation of MZ B cells.

Figure 1

Blk is differentially expressed in immature and mature B cell subsets. (a) Histograms show representative staining of the i.c. levels of Blk in gated populations of immature and mature B cell subsets in the BM, spleen and PerC of C57BL/6 mice. In the BM, these subsets include pre-pro-B (IgM⁻ B220⁺ CD43⁺ c-Kit^lo), pro-B (IgM⁻ B220⁺ CD43⁺ c-Kit⁻), pre-B (IgM⁻ B220⁺ CD43⁻), and immature (IgM⁺ B220^lo) B cells. In the spleen, the subsets include T1 (B220⁺ CD93⁺ IgM^hi CD23⁻), T2 (B220⁺ CD93⁺ IgM^hi CD23⁺), T3 (B220⁺ CD93⁺ IgM^lo CD23⁺), FO-I (CD19⁺ IgD^hi IgM^lo CD21⁺), FO-II (CD19⁺ IgD^hi IgM^hi CD21⁺), MZP (CD19⁺ IgD^hi IgM^hi CD21^hi), and MZ (CD19⁺ IgD^lo IgM^hi CD21^hi) B cells. In the PerC, the subsets include B1a (IgM⁺ CD5^lo CD11b⁺), B1b (IgM⁺ CD5⁻ CD11b⁺) and B2 (IgM⁺ CD5⁻ CD11b⁻) B cells. Cells from all tissues were stained and acquired on the same day. Data are representative of two independent experiments, with 3 mice per experiment. Solid gray histograms represent Blk staining levels in mature CD4⁺ T cells, which express negligible levels of Blk by real-time RT-PCR analysis. (b) Summary of the data presented in a. Comparison of i.c. Blk levels, presented as mean fluorescence intensity values, in gated immature and mature B cell subsets.

Figure 2

Comparison of Blk Expression Levels in B cells from Blk^+/+, Blk^+/− and Blk^−/− mice. Western blot analysis of B cell lysates from Blk^+/+, Blk^+/− and Blk^−/− mice (50 µg of protein/lane). Blots were probed with two different anti-Blk antibodies. Densitometric analysis of the bands shows a ~45% decrease in Blk levels in Blk^+/− B cells compared to Blk^+/+ B cells. Data are representative of three independent experiments.

Figure 3

Effects of Blk-haploinsufficiency and -deficiency on B cell development in the spleen and peritoneal cavity. (a) Phenotypic analysis of splenocytes from age-matched Blk^+/+, Blk^+/− and Blk^−/− mice. Left panel, dot plots show representative CD93 versus B220 staining profiles on total splenocytes. The percentages of immature transitional B cells (CD93⁺ B220⁺) and mature B cells (CD93⁻ B220⁺) are shown. Middle panels, dot plots show representative IgM versus CD23 staining profiles on gated immature transitional and mature B cell subsets. The percentages of T1, T2, T3 and FO-I B cells are shown. Right panel, dot plots show representative CD21 versus CD1d staining profiles on gated CD23⁻ IgM^hi B cells. The percentage of MZ B cells (CD21^hi CD1d⁺) is shown. (b) Dot plots show representative IgD versus IgM staining profiles on total splenocytes. The percentage of IgD^hi IgM^hi B cells is shown. Adjacent histograms show CD21 surface levels on gated IgD^hi IgM^hi B cells. The percentages of FO-II B cells (CD21⁺ IgD^hi IgM^hi) and MZP cells (CD21^hi IgD^hi IgM^hi) are shown. Data are representative of 4 to 6 mice per genotype. (c) Dot plots show representative CD11b versus CD5 staining on IgM⁺ PerC cells within the lymphoid gate. The percentages of B1a (CD11b⁺ CD5⁺ IgM⁺) and B1b (CD11b⁺ CD5⁻ IgM⁺) cells are shown. Data are representative of 3 to 5 mice per genotype.

Figure 4

Effects of Blk-haploinsufficiency and -deficiency on the humoral responses to a T-independent antigen. Age-matched Blk^+/+, Blk^+/− and Blk^−/− mice were immunized intravenously with NP-Ficoll, a type 2 T-independent antigen. 7 days later, sera were collected and analyzed. Relative amounts of NP-specific serum Ig, IgM and IgG1 in immunized Blk^+/+, Blk^+/− and Blk^−/− mice. **p ≤ 0.01. Data represent 3 to 4 mice per genotype.

Figure 5

Effects of Blk-haploinsufficiency and -deficiency on B cell activation in vitro. (a) Upregulation of CD86 expression following BCR stimulation. Splenocytes from Blk^+/+, Blk^+/− and Blk^−/− mice were plated and then stimulated with varying doses (1.25, 2.5, 5, and 10 µg/ml) of goat anti-mouse F(ab')₂ anti-IgM for 24 hours. Histograms show CD86 expression on gated MZ (CD21^hi CD1d⁺ IgD^lo) and FO (CD23⁺ IgD^hi) B cells from Blk^+/+ (shaded histogram), Blk^+/− (gray histogram) and Blk^−/− (dark gray histogram) mice. Data are representative of three independent experiments. (b) Summary of FO B cell data presented in a. Comparison of the percentages of CD86⁺ FO B cells among Blk^+/+ (light gray bars), Blk^+/− (gray bars) and Blk^−/− (dark gray bars) mice as a function of antigen dose. *p ≤ 0.05, **p ≤ 0.01. (c) Summary of MZ B cell data presented in a. Comparison of the mean fluorescence intensity of CD86 on MZ B cells from Blk^+/+ (light gray bars), Blk^+/− (gray bars) and Blk^−/− (dark gray bars) mice as a function of antigen dose. **p ≤ 0.01. (d) Comparison of proliferative responses following BCR stimulation. Purified splenic B cells were labeled with CFSE and then stimulated with soluble anti-IgM (Fab’)₂ (10 µg/ml) or with soluble anti-IgM (Fab’)₂ (10 µg/ml) and anti-CD40 monoclonal antibody (1 µg/ml). After 72 hours, cells were harvested, stained with monoclonal antibodies against various surface antigens to identify and gate FO and MZ B cells subsets, and then proliferation was assessed by flow cytometric analysis. The percentage of cells within each CFSE peak is given. The overlay histograms show representative CFSE levels in unstimulated B cells. Data are representative of two independent experiments, with 4 mice per genotype.

Figure 6

Effects of Blk-deficiency on BCR signal transduction. Splenocytes from Blk^+/+ and Blk^−/− mice were stimulated for 0, 2, 5 and 10 minutes with 10 µg/ml of goat anti-mouse F(ab')₂ anti-IgM, stained with monoclonal antibodies against various surface antigens in addition to anti-pERK, and then analyzed by flow cytometric analysis. (a) Sample histograms for intracellular pERK in unstimulated (shaded histogram) and stimulated total B cells (left panel) and MZ B cells (right panel) from Blk^+/+ (top panel) and Blk^−/− (bottom panel) mice. Comparison of the kinetics and magnitude of ERK activation in B cells (b) and MZ B cells (c) from Blk^+/+ and Blk^−/− mice. Data are presented as fold change, which was calculated by dividing the geometric mean fluorescence intensity (MFI) of the stimulated sample (MFI_stim) by the MFI of the unstimulated sample (MFI_unstim). **p ≤ 0.01, #p ≤ 0.001. Data are representative of three independent experiments.

Figure 7

Aged Blk^+/− and Blk^−/− mice display autoimmune phenotypes. (a) Comparison of the absolute numbers of splenocytes and PerC cells in 5 to 6-month old Blk^+/+, Blk^+/− and Blk^−/− mice. Data represent 3 to 6 mice per genotype. (b) Comparison of the surface levels of CD86 on B1, MZ and FO B cells from aged Blk^+/+, Blk^+/− and Blk^−/− mice. Data are representative of 3 to 6 mice per genotype. Comparison of the number of MZ (c) and B1 (d) B cells in young (2 months of age) and aged (5 to 6 months of age) Blk^+/+, Blk^+/− and Blk^−/− mice. *p ≤ 0.05, **p ≤ 0.01, #p ≤ 0.001. Data are representative of 3 to 6 mice per genotype. (e) Sera were collected from 6-month-old Blk^+/+, Blk^+/− and Blk^+/− mice and analyzed for the presence of ANA by ELISA. Data are presented as OD₄₅₀ readings for a 1:100 dilution of serum. Symbols represent individual mice. Gray horizontal bar marks the upper limit (Blk^+/+ mean plus 2 standard deviations) of the normal range. Numbers of Blk^+/− and Blk^−/− mice that exhibit serum ANA levels above the upper limit of the normal range are shown.