B cell receptor signal transduction in the GC is short-circuited by high phosphatase activity

Abstract

Germinal centers (GCs) generate memory B and plasma cells, which are essential for long-lived humoral immunity. GC B cells with high-affinity B cell receptors (BCRs) are selectively expanded. To enable this selection, BCRs of such cells are thought to signal differently from those with lower affinity. We show that, surprisingly, most proliferating GC B cells did not demonstrate active BCR signaling. Rather, spontaneous and induced signaling was limited by increased phosphatase activity. Accordingly, both SH2 domain-containing phosphatase-1 (SHP-1) and SH2 domain-containing inositol 5 phosphatase were hyperphosphorylated in GC cells and remained colocalized with BCRs after ligation. Furthermore, SHP-1 was required for GC maintenance. Intriguingly, GC B cells in the cell-cycle G(2) period regained responsiveness to BCR stimulation. These data have implications for how higher-affinity B cells are selected in the GC.

Fig. 1

Spontaneous and ligand-induced BCR signaling in GC, non-GC and resting B cells. (A) BCR-linked basal signaling in gated populations of GC, non-GC and naïve B cells from instantly-fixed total splenocytes harvested from d13 NP-CGG immunized mice (fig. S1). Fixed cells were treated with or without calf intestinal phosphatase (CIP), then labeled with antibodies specific for phosphorylated proteins. (A) Histograms show representative results of tyrosine phosphorylation from CIP-treated (red line) and untreated (blue line) cells, overlaid for direct comparison. y-axes show relative cell number. Bar charts are net median fluorescence intensity (MFI) indicating basal phosphorylation calculated by subtracting MFI of CIP treated from CIP untreated cells. Error bars are SEM from at least 5 independent experiments each using cells pooled from spleens of at least two mice for each group. * p < 0.05 and ** p < 0.01 MFI of GC or non-GC compared to naïve. (B) Response of GC, non-GC and resting B cells to BCR ligation. Total splenocytes from d13 NP-CGG immunized mice were stimulated ex-vivo with anti-μ (15μg/ml) for 5 minutes. Levels of Syk, Blnk, Tyr, p38 and Erk phosphorylation in gated GC and non-GC were measured. Profiles of GC unstimulated (red filled area), GC anti-IgM stimulated (red open line), non-GC unstimulated (blue filled area) and non-GC anti-IgM stimulated (blue open line) are overlaid for direct comparison.

Fig. 2

Analysis of phosphatase-dependent regulation of BCR signaling in GC cells. (A) Total splenocytes from d13 NP-CGG immunized mice were stimulated with 5mM (green line) or 10mM (blue line) H₂O₂ followed by detection of p-Syk and p-Blnk by flow cytometry in gated GC and non-GC Ag-specific cells. Figure is representative of 3 independent trials each of 3 mice. (B) Assessment of interaction between BCR ligation and phosphatase inhibition. Total splenocytes from immunized mice as in (A) were stimulated with 15μg/ml anti-IgM (red), 5mM H₂O₂ (blue) or both (green), and generation of p-Syk was assessed by flow cytometry. Representative of 3 or more experiments. (C) Indo1AM-loaded total splenocytes from immunized mice were stimulated with 15μg/ml anti-IgM (red), 5 mM H₂O₂ (blue) or both (green). Stimuli were added after acquiring events for 5 minutes to establish a basal level. Profiles of stimulants in gated GC (top) and non-GC (bottom) populations are overlaid. y-axis is indo1 violet to blue fluorescence ratio, an indicator of intracellular Ca²⁺ levels. (D) Compiled responses of GC and non-GC cells treated as in (C). Background-subtracted MFI was calculated from gates drawn before (180s time, background) and after (200s time, beginning at approximately the initial peak of response to anti–μ) stimulation. Bars show mean + SEM of net MFI from 4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (E) Ca²⁺ flux to ionomycin, used as positive control indicating equal responsiveness of GC and non-GC cells.

Fig. 2

Analysis of phosphatase-dependent regulation of BCR signaling in GC cells. (A) Total splenocytes from d13 NP-CGG immunized mice were stimulated with 5mM (green line) or 10mM (blue line) H₂O₂ followed by detection of p-Syk and p-Blnk by flow cytometry in gated GC and non-GC Ag-specific cells. Figure is representative of 3 independent trials each of 3 mice. (B) Assessment of interaction between BCR ligation and phosphatase inhibition. Total splenocytes from immunized mice as in (A) were stimulated with 15μg/ml anti-IgM (red), 5mM H₂O₂ (blue) or both (green), and generation of p-Syk was assessed by flow cytometry. Representative of 3 or more experiments. (C) Indo1AM-loaded total splenocytes from immunized mice were stimulated with 15μg/ml anti-IgM (red), 5 mM H₂O₂ (blue) or both (green). Stimuli were added after acquiring events for 5 minutes to establish a basal level. Profiles of stimulants in gated GC (top) and non-GC (bottom) populations are overlaid. y-axis is indo1 violet to blue fluorescence ratio, an indicator of intracellular Ca²⁺ levels. (D) Compiled responses of GC and non-GC cells treated as in (C). Background-subtracted MFI was calculated from gates drawn before (180s time, background) and after (200s time, beginning at approximately the initial peak of response to anti–μ) stimulation. Bars show mean + SEM of net MFI from 4 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. (E) Ca²⁺ flux to ionomycin, used as positive control indicating equal responsiveness of GC and non-GC cells.

Fig. 3

Differences in phosphorylation and localization of SHIP-1 and SHP-1 in GC and naïve B cells after and without BCR ligation. (A) Top: SHIP-1 phosphorylation (Y1020) was determined by Western blot of lysates from unstimulated and anti-IgM stimulated (5 min) FACS-sorted GC, non-GC and naïve B cells. β-actin was used as a loading control. Bottom: Quantitation of blot shown in top is median fluorescence intensity (MFI) of gated p-SHIP-1 bands relative to total SHIP-1. Blots shown are representative of 3 similar experiments. (B) Top: Tyrosine phosphorylation of SHP-1 was determined by immunoprecipitation of total SHP-1 in FACS-sorted GC and naïve B cells both unstimulated and after 2 minutes of anti-IgM stimulation, followed by blotting with anti-pTyr (4G10). Bottom: Quantitation of blots shown in top is MFI of gated p-SHP-1 relative to β-actin, which was determined by western blot on parallel samples of the same lysates. Data representative of 4 similar experiments: (C, D) High throughput imaging cytometric analysis of SHP-1/BCR association. Total splenocytes were either left unstimulated or stimulated with anti-IgM (b.7–6) for 2, 5, 10, 15 and 30 minutes; see fig. S7A for 15 and 30 min summary data. (C) Representative images of GC (top row) and Non-GC (bottom row) B cells captured by the Amnis Imagestream X. (D) Co-localization of SHP-1 and BCR was measured in gated non-GC (λ₁⁺PNA^lo) and GC (λ₁⁺PNA^hi) as similarity scores in GC (shaded) and non-GC (open) B cells at multiple time points with respect to BCR ligation (all time points summarized in fig. S7A). The box depicts a gate drawn at <1.2 similarity, which was used to calculate the % of GC and non-GC cells demonstrating substantial SHP-1/BCR dissociation at various times after BCR ligation (fig. S7B). (E, F) Effect of deletion of SHP-1 in B cells on the ongoing GC response. The strategy for tamoxifen-induced deletion using a new B cell-specific inducible Cre (hCD20-TamCre) and SHP-1^fl/fl mice is detailed in fig. S8. SHP-1^fl/fl mice with or without (control) the Cre Tg were immunized with NP-CGG, treated from d 9–12 with tamoxifen and then analyzed at d 14. (E) Representative flow cytometric analysis of splenocytes from experimental (left) and control (right) mice, detecting Ag-specific GC cells as PNA⁺/NIP⁺ among gated B220⁺ B cells. Numbers are percentages of B cells in the gate. (F) Data from 3 independent experiments showing loss of GC B cells (B220+PNA+NIP+) upon SHP-1 deletion in B cells.

Fig. 4. BCR signal transduction in GC B cells at the G2/M phase of cell cycle

Mice at d13 post-immunization with NP-CGG were injected i.v. once with 3 mg of 5-Bromo-2′-deoxyuridine (BrdU) and sacrificed 1 h later. Splenocytes were isolated, and either treated with anti-IgM or not for 5′ and then fixed and stained as described in Materials and Methods. (A) Flow cytometry gating to identify GC B cells (left panel) and then separate them into cell cycle compartments based on DAPI and BrdU staining (right panel). (B) Phosphorylation of Syk in response to BCR ligation was measured in gated populations based on (A) and as labeled: G1 (left), S (center) and G2/M (right). Histograms of p-Syk staining in unstimulated cultures are grey-filled and of stimulated cultures are open. Data are representative of four independent mice from two independent experiments, all with similar results. (C–F) GC cells were analyzed on the Imagestream X for SHP-1 intensity and SHP-1/BCR colocalization during different phases of the cell cycle. GC B cells were prepared and treated as in (A) but analyzed on the Imagestream after staining as in Fig. 3. (C) DAPI and BrdU identify G1, S and G2/M phases. G2 and M phases were separated based on nuclear area and aspect ratio. (D) Analysis of total SHP-1 expression as a function of cell cycle. (E, F) Analysis of SHP-1/BCR colocolazation as a function of cell cycle. during the G2 phase compared to G1 and S phases. However, SHP-1 level increased back at the M phase. Moreover, ex vivo stimulation resulted in time-dependent dissociation of SHP-1 from the BCR only during the G2 phase. Data are representative of 2 experiments.