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Science 336 (6085): 1178-1181

Copyright © 2012 by the American Association for the Advancement of Science

B Cell Receptor Signal Transduction in the GC Is Short-Circuited by High Phosphatase Activity

Ashraf M. Khalil1, John C. Cambier3, and Mark J. Shlomchik1,2,*

1 Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06520, USA.
2 Department of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.
3 Integrated Department of Immunology, National Jewish Health and University of Colorado Health Sciences Center, Denver, CO 80206, USA.


Figure 1
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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 day-13 NP-CGG immunized mice (fig. S1). Fixed cells were treated with or without calf intestinal phosphatase (CIP) and then labeled with antibodies specific for phosphorylated proteins. (A) Histograms show representative results of tyrosine phosphorylation from CIP-treated (red) and untreated (blue) cells, overlaid for direct comparison. The y axes show relative cell numbers. Bar charts show net median fluorescence intensity (MFI) indicating basal phosphorylation calculated by subtracting MFI of CIP-treated from CIP-untreated cells. Error bars denote SEM from at least five 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 cells. (B) Response of GC, non-GC, and resting B cells to BCR ligation. Total splenocytes from day-13 NP-CGG immunized mice were stimulated ex vivo with anti-μ (15 μg/ml) for 5 min. Levels of Syk, Blnk, Tyr, p38, and Erk phosphorylation in gated GC and non-GC cells were measured. Profiles of GC-unstimulated (red filled area), GC anti-IgM–stimulated (red open trace), non-GC–unstimulated (blue filled area), and non-GC anti-IgM–stimulated (blue open trace) cells are overlaid for direct comparison.

 

Figure 2
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Fig. 2. Analysis of phosphatase-dependent regulation of BCR signaling in GC cells. (A) Total splenocytes from day-13 NP-CGG immunized mice were stimulated with 5 mM (green) or 10 mM (blue) H2O2, followed by detection of p-Syk and p-Blnk by flow cytometry in gated GC and non-GC Ag-specific cells. These panels are representative of three independent trials each of three mice. (B) Assessment of interaction between BCR ligation and phosphatase inhibition. Total splenocytes from immunized mice as in (A) were stimulated with 15 μg/ml anti-IgM (red), 5 mM H2O2 (blue), or both (green), and generation of p-Syk was assessed by flow cytometry. Panels are representative of three or more experiments. (C) Indo1AM-loaded total splenocytes from immunized mice were stimulated with 15 μg/ml anti-IgM (red), 5 mM H2O2 (blue), or both (green). Stimuli were added after acquiring events for 5 min to establish a basal level. Profiles of stimulants in gated GC (top) and non-GC (bottom) populations are overlaid. The y axis shows the 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 (180-s time, background) and after (200-s time, beginning at approximately the initial peak of response to anti-μ) stimulation. Error bars show mean + SEM of net MFI from four independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Ca2+ flux to ionomycin, used as a positive control indicating equal responsiveness of GC and non-GC cells.

 

Figure 3
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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 (Tyr1020) was determined by Western blot of lysates from unstimulated and anti-IgM stimulated (5 min) fluorescence-activated cell–sorted GC, non-GC, and naïve B cells. β-actin was used as a loading control. (Bottom) Quantitation of blot shown in top is MFI of gated p-SHIP-1 bands relative to total SHIP-1. Blots shown are representative of three similar experiments. (B) (Top) Tyrosine phosphorylation of SHP-1 was determined by immunoprecipitation of total SHP-1 in fluorescence-activated cell–sorted GC and naïve B cells both unstimulated and after 2 min 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 four similar experiments. (C and 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 min (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 (Amnis, Seattle, Washington). (D) Colocalization of SHP-1 and BCR was measured in gated non-GC ({lambda}1+PNAlo) and GC ({lambda}1+PNAhi) 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 percentage of GC and non-GC cells demonstrating substantial SHP-1/BCR dissociation at various times after BCR ligation (fig. S7B). (E and 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 enzyme (hCD20-TamCre) and SHP-1fl/fl mice is detailed in fig. S8. SHP-1fl/fl mice with or without (control) the Cre Tg were immunized with NP-CGG, treated from days 9 to 12 with tamoxifen, and then analyzed at day 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 three independent experiments showing loss of GC B cells (B220+PNA+NIP+) upon SHP-1 deletion in B cells.

 

Figure 4
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Fig. 4. BCR signal transduction in GC B cells at the G2/M phase of the cell cycle. Mice at day 13 postimmunization with NP-CGG were injected intravenously once with 3 mg of 5-bromo-2'-deoxyuridine (BrdU) and sacrificed 1 hour later. Splenocytes were isolated and either treated with anti-IgM or not for 5', then fixed and stained as described in the supplemental materials and methods. (A) Flow cytometry gating to identify GC B cells (left) and then separate them into cell-cycle compartments based on 4',6-diamidino-2-phenylindole (DAPI) and BrdU staining (right). (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 (gray-filled) and stimulated cultures (open) are shown. Data are representative of four independent mice from two independent experiments, all with similar results. (C to 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 (green), S (blue), and G2/M phases (red/black). 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 and F) Analysis of SHP-1/BCR colocalization as a function of cell cycle during the G2 phase compared with the G1 and S phases. However, the 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 two experiments.

 


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