PTEN-Regulated AID Transcription in Germinal Center B Cells Is Essential for the Class-Switch Recombination and IgG Antibody Responses

Class-switch recombination (CSR) and somatic hypermutation (SHM) occur during the differentiation of germinal center B cells (GCBs). Activation-induced cytidine deaminase (AID) is responsible for both CSR and SHM in GCBs. Here, we show that ablation of PTEN through the Cγ1-Cre mediated recombination significantly influences the CSR and SHM responses. The GCs fail to produce the IgG1 B cells, the high affinity antibodies and nearly lost the dark zone (DZ) in Ptenfl/flCγ1Cre/+ mice after immunization, suggesting the impaired GC structure. Further mechanistic investigations show that LPS- and interleukin-4 stimulation induced the transcription of Cγ1 in IgM-BCR expressing B cells, which efficiently disrupts PTEN transcription, results in the hyperphosphorylated AKT and FoxO1 and in turn the suppression of AID transcription. Additionally, the reduced transcription of PTEN and AID is also validated by investigating the IgM-BCR expressing GCBs from Ptenfl/flCγ1Cre/+ mice upon immunization. In conclusion, PTEN regulated AID transcription in GCBs is essential for the CSR and IgG antibody responses.


PTEN-Regulated AID Transcription in GCBs
Frontiers in Immunology | www.frontiersin.org February 2018 | Volume 9 | Article 371 via nonhomologous end-joining (NHEJ) eventually completes the CSR by rejoining the two broken S regions (10,11). Previous studies suggested that the phosphatidylinositol-3-kinase (PI3K) and AKT signaling can both regulate the Ig gene rearrangement during B cell development and the CSR during GC responses (12)(13)(14)(15)(16)(17)(18). Phosphatase and tension homolog (PTEN) is known to negatively regulate PI3K-mediated growth, survival, proliferation and cellular metabolism of B cells (16,17,(19)(20)(21)(22). Thus PTEN deficiency alters B1, marginal zone B (MZB) and follicular B (FOB) cell subsets in Pten fl/fl CD19-Cre mice (16,17). Further study revealed that imbalanced PTEN and PI3K signaling impaired the μHC recombination in pro-B cells in Pten fl/fl mb1-Cre mice (12). Recently, emerging efforts have been placed to investigate the molecular mechanism of PTENand PI3K-tuned AKT signaling in regulating the strength of GC responses (14,15,23). B cell specific deficiency of PTEN in Pten fl/fl mb1 Cre/+ mice leads to the severe defects of B cell development at the bone marrow stage due to failed VJD recombination (12). The loss of the mature naïve B cell population in Pten fl/fl mb1 Cre/+ mice prevented the assessment of the function of PTEN in GCB-mediated CSR and antibody responses. As a solution, PTEN was recently knocked out in mature B cells in Pten fl/fl hCD20Tam Cre/+ mice, which demonstrated the importance of PTEN in regulating GC responses (23). Although mature B cell specific deficiency of PTEN in Pten fl/fl hCD20Tam Cre/+ mice excluded the B developmental defects as in the case of Pten fl/fl mb1 Cre/+ mice, the usage of Pten fl/fl hCD20Tam Cre/+ mice cannot explicitly separates the function of PTEN in mature B cell activation and proliferation upon antigen stimulation versus that in GC responses since GCBs were differentiated from activated mature naïve B cells after antigen stimulation. Here, to precisely assess the function of PTEN in GCB-mediated humoral responses in vivo, we used a mouse model with a PTEN deletion only in specific subsets of GCBs. Our results reveal that PTEN regulated AID transcription in GCBs is essential for the CSR and IgG antibody responses.

MaTErialS anD METHODS
Mice, Cell Culture C57BL/6J (B6) background Pten fl/fl mice (a kind gift from Dr. Wei Guo, Tsinghua University) were mated to Cγ1-Cre transgenic mice (a kind gift from Dr. Tomohiro Kurosaki, Osaka University and Dr. Klaus Rajewsky, Max Delbrück Center) in which expression of Cre is controlled by the endogenous promoter of the B cellspecific gene Cγ1. Offspring carrying Cγ1-Cre and two copies of the floxed Pten allele or Cγ1-Cre plus two copies of the WT Pten allele were used in the analyses as homozygous mutant (Pten fl/fl Cγ1 Cre/+ ) or WT (Pten +/+ Cγ1 Cre/+ ) mice, respectively. All mice were maintained under specific pathogen-free conditions and used in accordance of governmental and institutional guidelines for animal welfare. Primary B cells were negatively isolated from the spleen of Pten +/+ Cγ1 Cre/+ or Pten fl/fl Cγ1 Cre/+ mice as previously reported (24). Single cell suspensions were cultured in RPMI-1640 medium supplemented with 10% FBS, 50 µM β-mercaptoethanol (Sigma-Aldrich), penicillin/streptomycin antibiotics (Invitrogen) and Non-Essential Amino Acids (Invitrogen). B cells were stimulated for 4 days using 10 µg/mL LPS (Sigma) alone or LPS plus 50 ng/mL interleukin-4 (IL-4) (R&D) or 1 µg/mL anti-CD40 (eBioscience) alone or anti-CD40 plus 50 ng/mL IL-4 (R&D) in order to drive primary B cells class-switch in vitro.

immunization, EliSa assay, and immunohistochemistry
For mice GC Flow cytometry analysis, 6-week-old Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice were injected intraperitoneally with 1 × 10 9 sheep red blood cells (SRBCs, Bioren, China) or emulsified BSA in Alum adjuvant then analysis at day 7 after the immunization. Qβ virus-like particles (VLPs) were expressed in E. coli strain JM109 with exogenous expression plasmid pQ10 and then purified. The CpG contained VLPs were obtained by packaging VLPs with CpG ODN G10 in vitro as described (25). 6-week-old Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice were injected intraperitoneally with 10 µg VLP in 400 µL PBS for the immunization. Mice were analyzed at day 7 or day 14 after immunization. For NP-antigen specific T-cell-dependent immunization, 6-week-old Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice were injected on footpad with 10 µg NP33-KLH in 20 µL PBS and boost at day 35. Mice were bled at the indicated days after immunization.
Total RNA was extracted using TRIzol (GIBCO) according to the manufacturer's instructions and was subjected to the reverse transcription to make cDNA. For PCR of post-switch Cγ1, transcript was amplified using the following primers pair: (

Microscopy instruments
Confocal images were captured using Olympus FV-1000 microscope equipped with 10 × objective lens and a 405, a 473, a 549, and a 635 nm laser. The acquisition was controlled by FV10-ASW4.0 software.

image Process and Statistical analyses
Images were analyzed by Image Pro Plus (Media Cybernetics) software following our previous protocols (27). Statistical tests were performed with Prism 5.0 (GraphPad). Two-tailed t tests were used to compare end-point means of different groups. Statistical significant results (p) are indicated as: *p < 0.05; **p < 0.01, and ***p < 0.001.

rESUlTS
normal Development and Homeostasis of B Cells in Pten fl/fl Cγ1 Cre/+ Mice To detect the PTEN function in GCBs, we generated the Pten fl/fl Cγ1 Cre/+ mice by breeding Pten fl/fl mice to Cγ1-Cre mice. In Pten fl/fl Cγ1 Cre/+ mice, PTEN would only be knocked out in B cells with Cγ1 transcription, which are ideally the IgG1-BCR expressing B cells ( Figure 1A). We avoided to breed Pten fl/f mice with Aicda Cre/+ mice since the Pten fl/fl Aicda Cre/+ mice have been reported to develop severe submandibular hair loss, skin thickening and the manifestation of squamous papillomas (28).
We first examined the development and homeostasis of B cells in Pten fl/fl Cγ1 Cre/+ mice and confirmed that PTEN deletion in IgG1-BCR expressing B cells did not affect B cell development in the bone marrow and peripheral lymphoid organs ( Figure 1B). Further flow cytometry analysis of the splenic IgM and IgG1-BCR expressing B cells showed comparable amounts of surface IgM or IgG1 BCRs in Pten fl/fl Cγ1 Cre/+ versus Pten +/+ Cγ1 Cre/+ control mice ( Figure 1C). impaired antibody responses in Pten fl/fl Cγ1 Cre/+ Mice Pten fl/fl Cγ1 Cre/+ mice showed B cell normal development that allowed us to examine the humoral responses upon the immunization with either the T cell-dependent (TD) antigen NP33-KLH or the Qβ VLPs as reported (25). Age and gender matched Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice were undergone footpad injection with 10 µg NP33-KLH in 20 µL PBS and boost at day 35 for the TD antigen immunization (Figure 2A). For Qβ virus immunization, 6-week-old Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice were immunized intraperitoneally with 10 µg VLP in 400 µL PBS ( Figure 2B). ELISA analyses showed that the IgM antibody responses upon the induction by both NP-KLH and VLP were significantly higher in the Pten fl/fl Cγ1 Cre/+ mice compared to the control Pten +/+ Cγ1 Cre/+ mice (Figures 2A,B). However, the production of not only IgG1 but also IgG2b and IgG3 were significantly blunted in the Pten fl/fl Cγ1 Cre/+ mice (Figures 2A,B). We hypothesized that the decreased IgG antibody responses may be due to a damaged CSR reaction within the GC of Pten fl/fl Cγ1 Cre/+ mice.

Damaged CSr in Pten fl/fl Cγ1 Cre/+ Mice
To test whether or not PTEN deletion in IgG1 + B cells will damage the CSR within the GC reaction, GCs were induced by the immunization of SRBC in both Pten fl/fl Cγ1 Cre/+ and Pten +/+ Cγ1 Cre/+ control mice. Flow cytometry analyses of the splenic B cells at day 7 after SRBC injection unexpectedly demonstrated an increased but not decreased levels of GCBs in Pten fl/fl Cγ1 Cre/+ mice than the control Pten +/+ Cγ1 Cre/+ mice even though the size of the spleen of both types of mice was comparable (Figures 3A,B). Remarkably, further analyses showed that the IgM-BCR expressing GCBs were obviously increased while the class-switched IgG1-BCR abnormal gC Structure and SHM in Pten fl/fl Cγ1 Cre/+ Mice It is known that GC contains the light zone (LZ) and the dark zone (DZ). GCBs undergo consecutive and cyclic phases of proliferation and SHM in the DZ, followed by the migration to the LZ, where they capture and internalize antigen for the acquisition of survival signals from follicular helper T cells (3,29,30). We thus quantified the formation of LZ and DZ within the GC and found that DZ and LZ compartmentalization was severely disturbed in the Pten fl/fl Cγ1 Cre/+ mice upon the immunization by both SRBC and VLP (Figures 4A-C). Moreover, we observed that the number of mice upon immunization with 10 µg NP33-KLH at day 0. NP-specific antibody titers in IgM, IgG1, IgG2b, and IgG3 isotypes were examined by ELISA using NP8-BSA (5 µg/ml) as the coating Ag. The data represent the median ± interquartile range from three independent experiments with six mice per group at the indicated time point and were analyzed with Kruskal-Wallis test. (B) The production of virus-like particle (VLP)-specific antibodies in Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice upon the immunization (i.p.) with 10 µg VLP at day 0, day 7, and day 14. VLP specific antibody titers in IgM, IgG and IgG1, IgG2b, IgG2c, or IgG3 isotype were examined by ELISA experiment by using VLP (2 µg/ml) as the coating Ag. The data represent the median ± interquartile range from three independent experiments with six mice per group at the indicated time point and were analyzed with Kruskal-Wallis test. ***p < 0.001.  (14,15). Since an essential step in the selection of high affinity GCBs is the recruitment of LZ GCBs into the GC DZ (31, 32), it is not a surprise that the SHM in GCBs was significantly damaged in Pten fl/fl Cγ1 Cre/+ mice upon the immunization by TD antigen NP33-KLH ( Figure 4D). Lastly, it should be noted that even though the GC DZ formation was impaired in the Pten fl/fl Cγ1 Cre/+ mice, the size of the GCs was normal in the spleen section as detected by immunofluorescence and the number of GCs per spleen section was even higher in these PTEN KO mice (Figures 5A,B), which are consistent with the results that Pten fl/fl Cγ1 Cre/+ mice exhibited an increased levels of GCBs than the control Pten +/+ Cγ1 Cre/+ mice upon immunization (Figure 3A; Figure S1B in Supplementary Material).
repression of aiD induction in gCBs from Pten fl/fl Cγ1 Cre/+ Mice  It is well known that AID, which is specifically induced in the GCBs, is a crucial enzyme responsible for both CSR and SHM (6). Thus, we hypothesized that the PTEN expression level in these Pten fl/fl Cγ1 Cre/+ mice shall be influenced before the CSR reaction to drive the switch of IgM-BCR to IgG-BCR expressing B cells, which might subsequently impair the function of AID. To test this hypothesis, splenic B cells from Pten fl/fl Cγ1 Cre/+ and Pten +/+ Cγ1 Cre/+ mice were stimulated with LPS and IL-4 to induce CSR in vitro. To specifically examine the B cells without effective CSR, we sorted the IgM-BCR expressing B cells from the LPS-and IL-4 stimulated splenic B cells (Figure 6A). WB of these stimulated IgM-BCR expressing B cells detected the reduced PTEN and AID protein expression in the cells derived from Pten fl/fl Cγ1 Cre/+ mice compared to the Pten +/+ Cγ1 Cre/+ control mice ( Figure 6B). Meanwhile, these IgM-BCR expressing B cells  also showed hyper-phosphorylated AKT and FoxO1 ( Figure 6B). These results suggested that the AID transcription was affected by the hyper-phosphorylation of AKT and FoxO1 since AKT are known to inhibit the expression and function of AID (16,23). Indeed, RT-PCR assay demonstrated that the level of PTEN and AID mRNA was markedly reduced in the stimulated IgM-BCR expressing B cells from Pten fl/fl Cγ1 Cre/+ mice than those B cells from the Pten +/+ Cγ1 Cre/+ control mice (Figure 6C, top and Figure 6D). The transcription of Cγ1 was also detected in both the murine control and KO IgM-BCR expressing B cells, which readily explained the Cγ1-mediated PTEN deletion in the IgM-BCR expressing B cells in Pten fl/fl Cγ1 Cre/+ mice (Figure 6C, top and Figure 6D). We future validated these conclusions by utilizing purified GBCs in vivo from SRBC immunized Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice. We sorted the IgM-BCR expressing GCBs at day 7 after SRBC immunization ( Figure S3 in Supplementary Material). RT-PCR of IgM-BCR expressing GCBs from Pten fl/fl Cγ1 Cre/+ mice also detected the significantly reduced transcription of PTEN and markedly reduced AID (Figure 6C, bottom and Figure 6D). The transcription of Cγ1 was also detected in IgM-BCR expressing GCBs from both Pten +/+ Cγ1 Cre/+ and Pten fl/fl Cγ1 Cre/+ mice ( Figure 6C, bottom and Figure 6D), which readily explained the Cγ1-mediated PTEN deletion in the IgM-BCR expressing GCBs. All these results demonstrated that the PTEN expression level in Pten fl/fl Cγ1 Cre/+ mice was significantly impaired in GCBs. Thus, PTEN regulated AID transcription through PI3K-AKT signaling pathway in GCBs controls the CSR, IgG antibody response, and SHM.

DiSCUSSiOn
We  The observation of the pre-transcription of Cγ1 in IgM-BCR expressing B cells is consistent with the examination of the Cγ1 reporter mice, which reported the Cγ1 reporter gene expression in 85-95% of the GCB fraction 10-14 days after immunization with SRBC (33). Not a surprise, hyper-phosphorylated AKT and FoxO1 are observed as a result of the drastically reduced The hyper-phosphorylated AKT and FoxO1 in turn influence the AID expression in IgM-BCR expressing GCBs and reduce the differentiation of DZ GCBs partially through downregulation of the chemokine receptor CXCR4 (14,15). However, whether or not the reduced expression of AID can directly contribute to the decrease in the DZ GCBs deserves further investigation. In the literature, the important functions of PI3K-AKT pathway on the regulation of cell growth, survival, proliferation, cell cycle and cellular metabolism were also reported (16, 19-22, 34, 35). Thus, it is also of interest to investigate how these events can influence the formation of GC structures.
In conclusion, our research provides an alternative mechanistic explanation for the significantly impaired CSR in PTEN deficient GCBs in addition to the recent published studies showing that constitutive PI3K activation or ablation of FOXO1 impairs AID targeting to particular switch regions. which leads to the partly lost CSR (14,15). Our results demonstrate that PTEN regulated AID transcription in GCBs is essential for the CSR and IgG antibody response, and SHM.

ETHiCS STaTEMEnT
All animal protocols used in this study are approved by the IACUC (Institutional Animal Care and Use Committee) of Tsinghua University and performed in accordance with guidelines of the IACUC. The laboratory animal facility has been accredited by AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International). The assurance identification number is 15-LWL2 and was issued by Dr. Zai Chang, the vice chair of IACUC of Tsinghua University, Beijing, China.
aUTHOr COnTriBUTiOnS WL and JW conceived, designed, and drafted the article; JW and SL performed experiments and laboratorial analysis; BH and MY supported the materials; JW and WL wrote the manuscript; WL, HQ, ZD, BH, MY, and JW reviewed and approved the manuscript final version.