CXCR4 signaling and function require the expression of the IgD-class B-cell antigen receptor

Abstract

Mature B cells coexpress both IgM and IgD B-cell antigen receptor (BCR) classes, which are organized on the cell surface in distinct protein islands. The specific role of the IgD-BCR is still enigmatic, but it is colocalized with several other receptors on the B-cell surface, including the coreceptor CD19. Here, we report that the chemokine receptor CXCR4 is also found in proximity to the IgD-BCR. Furthermore, B cells from IgD-deficient mice show defects in CXCL12-mediated CXCR4 signaling and B-cell migration, whereas B cells from IgM-deficient mice are normal in this respect. CXCR4 activation results in actin cytoskeleton remodeling and PI3K/Akt and Erk signaling in an IgD-BCR-dependent manner. The defects in CXCR4 signaling in IgD-deficient B cells can be overcome by anti-CD19 antibody stimulation that also increases CXCL12-mediated B-cell migration of normal B cells. These results show that the IgD-BCR, CD19, and CXCR4 are not only colocalized at nanometer distances but are also functionally connected, thus providing a unique paradigm of receptor signaling cross talk and function.

Keywords: B-cell antigen receptor; CXCR4; IgD; cytoskeleton; signaling.

Fig. 1.

Signaling through CXCR4 is coupled to the BCR via Syk and the actin cytoskeleton. (A, Top) Schematic representation of BCR ablation in floxed B1-8HCknockin × mb1-creERT2 mice by tamoxifen administration. (Bottom) Representative FACS analysis for IgM and IgD expression in CD21^pos, B220^pos splenic B cells after 8 d of tamoxifen administration. (B) Calcium flux measurement of BCR^pos (black) and BCR^neg (purple) splenic B cells in response to 100 ng/mL CXCL12 (Left) or 1 µM Lat-A (Right). (C) Calcium flux measurement of WT splenic B cells treated with 1 µM Syk inhibitor (gray) and untreated control cells (black) and stimulated with CXCL12 (Left) or Lat-A (Right). (D) Migration of BCR^pos and BCR^neg splenic B cells toward CXCL12 over a period of 4 h. (E) Spreading of WT splenic B cells treated with Syk inhibitor or untreated control cells on surfaces coated with either anti-κ antibody or CXCL12. Cells were allowed to spread for 5 min, and then fixed and stained with DAPI and phalloidin to visualize F-actin. (Scale bar: 5 μm.) Quantification shows percentage of spreading cells per image. (F) Quantification of cellular F-actin in WT splenic B cells via staining with phalloidin and flow-cytometric analysis after different time points of CXCL12 stimulation. (G) Fab-PLA analysis of IgM–IgM and IgD–IgD proximity in resting WT splenic B cells compared with cells stimulated with CXCL12 or Lat-A. (Scale bar: 5 μm.) Calcium flux analyses in B and C are representative of four independent experiments. Migration analyses in D represent median of four or more independent experiments. F-actin analyses in E and F represent mean ± SD of three independent experiments. PLA analyses in G represent mean ± SD of six independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001.

Fig. S1.

Signaling through CXCR4 is coupled to the BCR via Syk and the actin cytoskeleton. (A) Expression of CXCR4 in BCR^pos (black) and BCR^neg (purple) splenic B cells by FACS analysis (Left) and mean fluorescence intensity (MFI) quantification (Right). (B) Calcium flux measurement of splenic B cells in response to 5 µg/mL anti-κLC in absence (control, black) and presence of 1 µM Syk inhibitor (gray). (C) Representative Western blot analysis of F- and G-actin fractions of splenic B cells in response to 100 ng/mL CXCL12 stimulation in the absence and presence of 1 µM Syk inhibitor. (D) Quantification of F-actin fraction dynamics in response to 100 ng/mL CXCL12 stimulation. Analysis of CXCR4 expression in A and calcium flux analyses in B are representative of six and four independent experiments, respectively. Quantification of F- and G-actin fractions in D represents mean ± SEM of four or more independent experiments. **P < 0.01.

Fig. 2.

Signaling through CXCR4 is coupled to IgD, but not IgM. (A) Calcium flux measurement of WT (black), IgM^−/− (blue), and IgD^−/− (red) splenic B cells in response to stimulation with 5 µg/mL anti-κ (first panel), 100 ng/mL CXCL12 (second panel), or 1 µM Lat-A (third panel). (B) Migration of WT (black), IgM^−/− (blue), and IgD^−/− (red) splenic B cells toward CXCL12 over a period of 4 h. (C) Quantification of cellular F-actin in IgM^−/− (first panel, blue) and IgD^−/− (second panel, red) splenic B cells via staining with phalloidin and flow-cytometric analysis after different time points of CXCL12 stimulation in untreated (solid line) and Syk-inhibited (dashed line) cells. (D) Spreading of IgM^−/− and IgD^−/− splenic B cells treated with Syk inhibitor or untreated control cells on surfaces coated with either anti-κ antibody or CXCL12. Cells were allowed to spread for 5 min, and then fixed and stained with DAPI and phalloidin to visualize F-actin. Quantification shows percentage of spreading cells per image. (E) 1-PLA analysis of CXCR4-Igκ proximity in resting WT, IgM^−/−, and IgD^−/− splenic B cells. (Scale bar in D and E: 5 μm.) Calcium flux analyses in A are representative of four independent experiments. Migration analyses in B represent median of six independent experiments. F-actin analyses in C and D represent mean ± SD of three independent experiments. PLA analyses in E represent mean ± SD of five or more independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. S2.

Signaling through CXCR4 is coupled to IgD, but not IgM. (A) Expression of BCR (κLC) in WT (black), IgM^−/− (blue), and IgD^−/− (red) splenic B cells by FACS analysis (Left) and MFI quantification (Right). (B) Expression of CXCR4 in WT (black), IgM^−/− (blue), and IgD^−/− (red) B cells by FACS analysis (Left) and MFI quantification in splenic (Middle) and circulating B cells (Right). (C) FACS analysis for binding of fluorescently labeled CXCL12 binding on WT (black), IgM^−/− (blue), and IgD^−/− (red) B cells. (D) Histogram of the normalized total calcium release response (area under curve) for 0.05, 0.5, 5.0, and 10.0 µg/mL of anti-κLC stimulations. The calcium signal at the given concentration was normalized to total BCR expression as determined by maximum anti-κLC staining with 10 µg/mL antibody. (E) Histogram of the normalized calcium release response at different concentrations of CXCL12 stimulation, normalized to total BCR expression as determined by anti-κLC staining with 10 µg/mL antibody. (F) Calcium flux measurement of WT (black), IgM^−/− (blue), and IgD^−/− (red) splenic B cells in response to 100 ng/mL CXCL12 in presence of 1 µM Syk inhibitor. (G) 1-PLA analysis of CXCR4–IgD proximity in resting WT, IgM^−/−, and IgD^−/− splenic B cells. (Scale bar: 5 μm.) FACS analyses of BCR and CXCR4 expression and CXCL12 binding in A–C are representative of six independent experiments. Calcium flux analyses in D and E represent mean ± SEM of four independent experiments. Calcium flux analyses in F is representative of four independent experiments. PLA analyses in G represent mean ± SD of four independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.

Fig. 3.

Signaling through CXCR4 induces the Erk and Akt pathway in an IgD-dependent manner. Quantification of Western blot analysis of WT (gray), IgM^−/− (blue), and IgD^−/− (red) splenic B cells after stimulation with either 100 ng/mL CXCL12 or 1 µM Lat-A for 5 min compared with resting cells. Data represent mean ± SD of four independent experiments.

Fig. S3.

Signaling through CXCR4 induces the Erk and Akt pathway in an IgD-dependent manner. (A) Western blot analysis of WT (gray), IgM^−/− (blue), and IgD^−/− (red) splenic B cells after stimulation with either 100 ng/mL CXCL12 or 1 µM Lat-A for 5 min compared with resting cells. (B) Western blot analysis of WT (gray), IgM^−/− (blue), and IgD^−/− (red) splenic B cells after stimulation with either 5 µg/mL anti-κ or 1 µM Lat-A for 1, 5, and 10 min compared with resting cells.

Fig. 4.

CD19 acts as a dominant positive regulator of CXCR4 and cytoskeleton disruption induced signaling. (A) Calcium flux measurement of WT (black), IgM^−/− (blue), and IgD^−/− (red) splenic B cells in response to stimulation with 10 µg/mL anti-CD19 (first panel), anti-CD19 plus CXCL12 (second panel), and anti-CD19 plus Lat-A (third panel). (B) Calcium flux measurement of BCR^pos (black) and BCR^neg (purple) splenic B cells in response to stimulation with 10 µg/mL anti-CD19 (first panel), anti-CD19 + CXCL12 (second panel), and anti-CD19 + Lat-A (third panel). (C) Migration of WT (black), IgM^−/− (blue), and IgD^−/− (red) splenic B cells toward CXCL12, anti-CD19, or anti-CD19 plus CXCL12 over a period of 4 h. (D) Migration of BCR^pos and BCR^neg splenic B cells toward anti-CD19 or anti-CD19 plus CXCL12 over a period of 4 h. Calcium flux analyses in A and B are representative of three independent experiments. Migration analyses in C and D represent median of five or more independent experiments. *P < 0.05; **P < 0.01; ns, not significant.

Fig. 5.

CD19 is the linchpin between CXCR4, the BCR, and the actin cytoskeleton. (A) Calcium flux measurement of WT (black) and CD19^−/− (green) splenic B cells in response to 100 ng/mL CXCL12. (B) Migration of WT (black) and CD19^−/− (green) splenic B cells toward CXCL12 over a period of 4 h. Calcium flux analyses in A and migration analysis in B are representative of four and six independent experiments, respectively. (C) Schematic representation shows plasma membrane localization of IgM and IgD on mature B cells and chemokine receptor CXCR4 that resides in the IgD–CD19 island. Upon engagement of CXCL12, CXCR4 triggers a multitude of signals including actin remodeling. As a result of actin remodeling, a Syk-dependent BCR signal, specifically through the IgD–BCR, is triggered, and a feedforward loop is initiated that induces Ca²⁺ influx, Akt/Foxo1 and Erk pathway activation in addition to F-actin reorganization. The later part of actin remodeling could also be initiated by cytoskeleton disruption using Lat-A, thus enabling the study of the effect of the chemokine-driven actin remodeling on the BCR.

Fig. S4.

CD19 expression and boosting effect of anti-CD19 Fab fragment antibody. (A) Expression of CD19 in WT (black), IgM^−/− (blue), and IgD^−/− (red) splenic B cells by FACS analysis (B) MFI quantification of CD19 expression. (C) Expression of CD19 in BCR^pos (black) and BCR^neg (purple) splenic B cells by FACS analysis. (D) MFI quantification of CD19 expression in BCR^pos (black) and BCR^neg (purple) splenic B cells. (E) Calcium flux measurement in WT mature B cells in response to 1 µM Lat-A (black) and 20 µg/mL anti-CD19 Fab fragment (dashed gray) stimulation. (F) Ca²⁺ influx in WT mature B cells in response to increasing doses of anti-CD19 Fab fragment at the indicated time point (1.), followed by 1 µM Lat-A treatment at the time point (2.). The concentrations of anti-CD19 Fab fragment, represented by the colored lines, are indicated in the legend table. For comparison, the Ca²⁺ influx after 1 µM Lat-A or 20 µg/mL anti-CD19 Fab fragment treatment are shown as solid and dashed gray lines, respectively. FACS analyses of CD19 expression in A–D are representative of six independent experiments. Calcium flux analyses in E and F are representative of three independent experiments. **P < 0.01; ***P < 0.001; ns, not significant.

Fig. S5.

CD19 is a dominant-positive regulator of CXCR4 signaling and cytoskeleton disruption induced signaling. (A) Western blot analysis of WT (gray), IgM^−/− (blue), and IgD^−/− (red) splenic B cells after stimulation with 10 µg/mL anti-CD19 or the combination of anti-CD19 with either Lat-A or CXCL12, for 5 min compared with resting cells. (B) Quantification of Western blot analysis data represents mean ± SD of four independent experiments.