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. 2014 Sep 12;9(9):e107505.
doi: 10.1371/journal.pone.0107505. eCollection 2014.

PP4 Is Essential for Germinal Center Formation and Class Switch Recombination in Mice

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Free PMC article

PP4 Is Essential for Germinal Center Formation and Class Switch Recombination in Mice

Ming-Yu Chen et al. PLoS One. .
Free PMC article

Abstract

PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig) VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23(cre)PP4(F/F) mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab) or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23(cre)PP4(F/F) mice, and their B cells showed a reduced efficiency of class switch recombination (CSR) in vitro upon stimulation by LPS or LPS plus IL-4. When CD23(cre)PP4(F/F) mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs) in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Efficiency of pp4c deletion.
(A) Top panel: PCR analysis of genomic DNA from splenic B cells of WT (n = 2) and CD23crePP4F/F (n = 4) mice showing products representing the WT pp4c allele (+/+), the pp4c floxp allele (undeleted F/F), and the deleted pp4c allele (deleted F/F). Numbers above the top panel are individual mouse labels. Bottom panel: PCR analysis of CD14 as a loading control. (B) Top panel: RT-PCR analysis of mRNA from splenic B cells from the WT and CD23crePP4F/F mice in (A). Bottom panel: HPRT, loading control. (C) Top panel: Western blot to detect PP4 protein in the spenic B cells in (B). Numbers below this panel (% residual) are the relative protein levels quantified by Image J and normalized to the WT value (set to 100%). Bottom panel: β-tubulin, loading control. (D) Quantitation of the PP4 protein levels in the B cells in (C) after normalization to β-tubulin values. Data are the mean ± SD of samples assayed per group. For (A–D), results are representative of two independent experiments. (E) Quantitation of peripheral B cell counts in the spleen (SP) and lymph nodes (LN) of WT and CD23crePP4F/F mice (n = 40–50 mice/group). Data are the mean ± SD. (F) FACS profiles of B220 vs IgM and CD21 vs CD23 expression by WT and CD23crePP4F/F splenocytes (SP), and of B220 vs CD3 and IgM vs IgD expression by WT and CD23crePP4F/F total mesenteric LN cells. Numbers in quadrants represent the percentage of the indicated population relative to the total. Results are representative of 3 mice/group and of three independent experiments.
Figure 2
Figure 2. Characterization of B cells in CD23crePP4F/F mice.
(A) FACS assay of apoptosis of splenic B cells that were freshly isolated from WT and CD23crePP4F/F mice (n = 3/group) and subjected to AnnexinV vs 7AAD staining. Viabilities are shown for the immature (IM), follicular (FO) and marginal zone (MZ) B cell populations. Numbers in quadrants represent the percentage of the indicated population relative to the total. (B) Quantitation of the percentage of viable (AnnexinV7AAD) cells in the IM, FO and MZ B cell populations in (A). Data are the mean ± SD of triplicate samples from each group. For (A–B), results are representative of two independent experiments. (C) In vivo BrdU incorporation assay. Left panel: WT and CD23crePP4F/F mice (n = 3/group) were i.p. injected with BrdU twice daily (8 h apart) on 3 consecutive days. IM, FO and MZ splenocytes were freshly sorted, subjected to intracellular staining with anti-BrdU plus 7AAD, and analyzed by FACS. Right panel: Diagram indicating relative position of specific mitotic phase cells in the FACS profiles in the left panel. (D) Quantitation of the results in (C) showing the percentages of cells in the G0/G1, S and G2/M phases. Data are the mean ± SD of triplicate samples from each group. For (C–D), results are representative of two independent experiments. (E) In vitro BrdU incorporation assay. Splenic B cells were freshly isolated from WT (n = 3) or CD23crePP4F/F (n = 4) mice and cultured in BrdU-containing maintenance medium alone (Medium), or in maintenance medium containing 1 µg/ml anti-IgM (IgM low), 10 µg/ml anti-IgM (IgM high), 5 µg/ml anti-CD40 Ab (CD40), 1 µg/ml anti-IgM plus anti-CD40 Abs (IgM/CD40), 1 µg/ml LPS (LPS low), or 10 µg/ml LPS (LPS high). After 48 h, cells were analyzed by FACS. Data shown are the quantitation of the mean percentage ± SD of B cells in S phase relative to total B cells after 48 h stimulation. (F) Quantitation of the mean percentage ± SD of B cells in G2/M phase from the data in (E) after 48 h stimulation. (G) Quantitation of the mean percentage ± SD of activated (CD80+CD86+) B cells from the data in (E) after 24 h (d1) or 48 h (d2) of stimulation. (H) Quantitation of the mean percentage ± SD of CD40+CD25+ B cells from the data in (G) after 48 h (d2) stimulation. For (E–H), results are representative of two independent experiments.
Figure 3
Figure 3. B cell-specific PP4 deficiency reduces Ig production in vivo.
(A) Quantitation of serum levels of the indicated classes of Igs in non-immunized WT and CD23crePP4F/F mice (n = 8–10/group). Data are values for individual mice, and horizontal bars are geometric means. Results shown are from one experiment. (B, C) Quantitation of serum levels of TNP-specific IgG1 (B) and IgG3 (C) in WT and CD23crePP4F/F mice (n = 5–6/group) before immunization (d0), or at days 7, 14, 21, 35 and 49 post-immunization with TNP-KLH. Data are presented as in (A) and are from one experiment. (D) FACS profiles of CD38 vs IgG1 and CD38 vs IgG3 expression by B220+PNAIgMIgD gated splenocytes isolated from WT or CD23crePP4F/F mice (n = 3–6/group) at day 7 post-injection of PBS (control; left panels) or TNP-KLH (right panels). (E) Quantitation of the percentages of IgG1 + (left) and IgG3 + (right) switched B cells among total B cells from the mice in (D). Data are values for individual mice, and horizontal bars are geometric means. For (D–E), results are representative of two independent experiments. (F–H) Quantitation of serum levels of TNP-specific IgM (F), IgG1 (G) and IgG3 (H) in WT and CD23crePP4F/F mice (n = 8/group) before immunization (d0), or at days 7, 14, 21 and 28 post-immunization with TNP-Ficoll. Data are presented as in (A) and are from one experiment.
Figure 4
Figure 4. Impaired GC formation in CD23crePP4F/F mice immunized with TNP-KLH.
(A) Immunohistochemical (IHC) images of PNA (dark blue) vs B220 (brown) expression in spleen sections of WT and CD23crePP4F/F mice (n = 3–6/group) at day 7 post-injection of PBS (control) or TNP-KLH. Data in the right panels are higher magnification images of the red rectangular areas in the center panels. (B) FACS profiles of B220 vs PNA expression by splenocytes from the mice in (A) at day 7 post-injection of PBS or TNP-KLH. (C) FACS profiles of CD95 vs GL7 expression by B220+-gated splenocytes from the mice in (A) at day 7 post-injection of PBS or TNP-KLH. (D) Quantitation of the percentage of GL7+CD95+ GC B cells among total B cells from the data in (C). Data are values for individual mice, and horizontal bars are geometric means. For (A–C), results are representative of two independent experiments.
Figure 5
Figure 5. Impaired GC formation in CD23crePP4F/F mice immunized with TNP-Ficoll or infected with H1N1 virus.
(A) IHC images of PNA (dark blue) vs B220 (brown) expression in spleen sections of WT and CD23crePP4F/F mice (n = 3–4/group) at day 14 post-injection of PBS or TNP-Ficoll. Data in the right panels are higher magnification images of the red rectangular areas in the center panels. Red arrows indicate GCs. (B) FACS profiles of B220 vs PNA expression in total splenocytes of the mice in (A). TNP-Ficoll-immunized WT and CD23crePP4F/F mice (n = 3–4/group) from those in (A) that were given a second injection of TNP-Ficoll at day 100 (Re-boost). (C) Quantitation of the percentage of PNA+B220+ GC B cells among total splenocytes of the mice in (B). Data are values for individual mice, and horizontal bars are geometric means. (D) FACS profiles of CD95 vs GL7 expression by B220+ splenic B cells isolated from WT and CD23crePP4F/F mice (n = 6/group) at day 15 post-injection of PBS or H1N1 virus. (E) Quantitation of the percentage of GL7+CD95+ GC B cells among total B cells from the data in (D). (F) FACS profiles of CXCR4 vs GL7 expression by B220+ splenic B cells from the mice in (D). (G) Quantitation of the percentage of GL7+CXCR4+ centroblasts among total B cells from the data in (F). For (A–G), results are representative of two independent experiments.
Figure 6
Figure 6. Impaired Ig class switching in PP4-deficient B cells.
(A) FACS profiles of IgG1 vs IgG3 expression by B220+IgMIgD gated B cells that were isolated from WT and CD23crePP4F/F mice (n = 3/group) and stimulated in vitro with LPS for 4 days. (B) Quantitation of the percentage of IgG3 +-switched B cells among total B cells from the data in (A). Data are the mean ± SD of triplicates. For (A–B), results are representative of two independent experiments. (C) FACS profiles of IgG1 vs IgG3 expression by B220+IgMIgD-gated B cells that were isolated from WT and CD23crePP4F/F mice (n = 3/group) and stimulated in vitro with LPS plus IL-4 for 4 days. Data were analyzed as for (A). (D) Quantitation of the percentage of IgG1 +-switched B cells among total B cells from the data in (C). For (C–D), results are representative of two independent experiments. (E) RT-PCR analysis of mRNA from B cells that were isolated from WT and CD23crePP4F/F mice (n = 4/group), and either left unstimulated or stimulated in vitro with LPS or LPS plus IL-4 for 4 days. Samples were analyzed to detect AID and the indicated switched transcripts. HPRT, loading control. (F) RT-PCR analysis of the indicated germline transcripts in the B cells in (E). Loading control was the same as in (E). For (E–F), results are representative of two independent experiments. (G) DC-PCR analysis of genomic DNA prepared from resting B cells isolated from WT and CD23crePP4F/F mice (n = 4/group). Genomic DNA was digested with EcoRI, ligated and utilized as template for PCR to detect the recombined Sµ-Sγ3 sequence (see Methods). nAChRe, loading control. (H) DC-PCR analysis of genomic DNA prepared from WT and CD23crePP4F/F B cells that were stimulated in vitro with LPS plus IL-4 for 4 days. The recombined Sµ-Sγ1 sequence was detected as illustrated in Figure S1. For (G–H), results are representative of two independent experiments.
Figure 7
Figure 7. PP4-deficient B cells exhibit increased etoposide-induced caspase 3 cleavage.
(A) FACS profiles of AnnexinV vs 7AAD staining of WT and CD23crePP4F/F B cells that were cultured for 6 h (not shown) or 24 h in maintenance medium alone or in maintenance medium containing 1 µM etoposide. (B) Quantitation of the percentage of viable (AnnexinV7AAD) cells among total B cells from the data in (A). Data are the mean ± SD of three mice per group. For (A–B), results are representative of two independent experiments. (C) FACS profiles of B220 vs cleaved caspase 3 staining of WT and CD23crePP4F/F B cells in the resting state (Resting), cultured in maintenance medium alone (Medium) or in maintenance medium containing 1 µM etoposide for 10 h or 15 h. (D) Quantitation of the percentage of B cells exhibiting cleaved caspase 3 among total B cells from data in (C). Data are the mean ± SD of three mice per group. For (C–D), results are representative of two independent experiments.
Figure 8
Figure 8. PP4-deficient B cells exhibit reduced CD40-mediated ERK and JNK activation, abnormal BCR-mediated IκBα degradation and reduced BCR/CD40-mediated S phase entry.
(A) Western blot (WB) analysis of splenic B cells that were isolated from WT and CD23crePP4F/F mice (n = 6/group) and left unstimulated (0), or stimulated in vitro with anti-CD40 Ab for the indicated times. Lysates were subjected to WB to detect p-Erk1/2 (T202/Y204), total Erk, p-JNK (T183/Y185), total JNK, IκBα, p-Akt/PKB (S473), and total Akt. Results are representative of three independent experiments. (B) WB analysis of splenic B cells that were isolated from WT and CD23crePP4F/F mice (n = 6/group) and left unstimulated (0), or stimulated with anti-IgM Ab for the indicated times. Lysates were analyzed as in (A). Results are representative of two independent experiments. (C) FACS profile of in vitro BrdU incorporation assay. Splenic B cells were freshly isolated from BCRHEL/CD23crePP4+/+ (n = 3) and BCRHEL/CD23crePP4F/F (n = 3) mice and cultured in BrdU-containing maintenance medium alone (Medium), or in maintenance medium containing 1 µg/ml HEL, 1 µg/ml anti-IgM plus anti-CD40 Abs (IgM/CD40) and 0.5 µg/ml LPS (LPS low). After 72 h, cells were analyzed by FACS. Numbers in upper quadrants represent the percentage of B cells in S phase relative to the total B cells after 72 h stimulation, while numbers in left quadrants represent the percentage of apoptotic B cells. (D) Quantitation of the mean percentage ± SD of B cells in S phase from the data in (C). β-tubulin, loading control. Results are representative of two independent experiments. (E) FACS profile of in vivo BrdU incorporation assay. BCRHEL/CD23crePP4+/+ (n = 4) and BCRHEL/CD23crePP4F/F (n = 4) mice were immunized with HEL in alum at day 0 and injected with BrdU from day 3 in 4 consecutive days as illustrated in Figure S5A. At day 7 after immunization, splenic B cells were harvested and analyzed by FACS. Numbers in quadrants represent the percentage of B cells in S phase (BrdU+) relative to total B cells. Results are representative of two independent experiments. (F) Quantitation of the mean percentage ± SD of B cells in S phase from the data in (E).

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The study was funded by National Science Council 100-2320-B-400-003-MY3 (http://www.most.gov.tw/mp.aspx?mp=7), National Health Research Institute IM-100-IM-101-PP-04 and IM-102-PP-04 (http://english.nhri.org.tw/NHRI_WEB/nhriw001Action.do). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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