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 cell-specific 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/flCγ1^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/flCγ1^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.

For mice GC Flow cytometry analysis, 6-week-old Pten^+/+Cγ1^Cre/+ and Pten^fl/flCγ1^Cre/+ mice were injected intraperitoneally with 1 × 10⁹ 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/flCγ1^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/flCγ1^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.

To detect VLP or NP-specific IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 in immunized mice (6 Pten^+/+Cγ1^Cre/+ and 6 Pten^fl/flCγ1^Cre/+), 2 µg/mL VLP, 5 µg/mL NP₈-BSA or 5 µg/mL NP₃₀-BSA were coated on maxisorb plates (Nunc) and incubated overnight at 4°C. All these plates were blocked with 0.3% gelatin in PBS buffer (2 h at 37°C), followed by addition of pre-diluted mice serum into each well and incubated at 37°C for 1 h. 1:10,000 diluted HRP conjugated goat anti-mouse IgM, IgG, IgG1, IgG2b, IgG2c, and IgG3 were used to detect VLP or NP-specific antibodies also the innate immune antibodies. Then followed the protocol as published before for the final readout results (26).

For immunohistochemistry 6-week-old Pten^+/+Cγ1^Cre/+ and Pten^fl/flCγ1^Cre/+ mice were injected intraperitoneally with 1 × 10⁹ SRBC in PBS. Spleen sections were frozen from day 7 after immunization in OCT compound (Sakura Finetek). After that spleen sections were stained with Alexa Fluor 488 anti-mouse/human CD45R/B220 (#103225, BioLegend), PE anti-mouse FAS (#152608, BioLegend), and APC anti-mouse CD3ε (#100312, BioLegend) by following the protocol described by Sandrine Sander (14).

Primary splenic B cells or cultured cells were lysed by 2× Lysis buffer. Proteins were extracted by 10% Bis-Tris PAGE (Life technologies) with the cocktail of proteinase inhibitors (Life technologies) in it, transferred to polyvinylidene fluoride (PVDF). The electroblotted membranes were blocked by TBST containing 5% non-fat milk (BD) and were probed with anti-PTEN, anti-β-actin, anti-p-AKT 473, or anti-p-FoxO1/3a primary antibodies overnight at 4°C then immunoblotted with HRP-conjugated secondary antibodies (Dako). PTEN (138G6) Rabbit antibody (#9559), Phospho-AKT (Ser473) rabbit antibody (#4060), phospho-FoxO1 (Thr24)/FoxO3a (Thr32) rabbit antibody (#9464), and β-actin (13E5) rabbit antibody (#4970) were purchased from Cell Signaling Technology. AID rabbit antibody was a kind gift from Dr. Feilong Meng (Shanghai Institute of Biochemistry and Cell Biology, China).

Pure Pten^+/+Cγ1^Cre/+ and Pten^fl/flCγ1^Cre/+ splenic B cells were stimulated for 4 days with 10 µg/ml LPS and 50 ng/mL IL-4. Pre-incubated IgM-BCR expressing B cells were sorted by using FACSAria III Cell Sorter (BD). SRBC immunized Pten^+/+Cγ1^Cre/+ and Pten^fl/flCγ1^Cre/+ mice IgM-BCR expressing GCBs were first enriched by CL-7 marker (GL-7 monoclonal antibody, Biotin, eBioscience; streptavidin microbeads, Miltenyi Biotec) by using the LS columns (Miltenyi Biotec). On the basis of enrich, cells were sorted by using FACSAria III Cell Sorter (BD) after labeling with IgM-BCR expressing GCBs dyes (anti-CD19-APC, anti-FAS-PE, Alexa Fluor 488 goat Fab anti mouse IgM μ chain and ef450-streptavidin for GL-7).

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: (Cγ1, 429 bp) 5′-GGC CCT TCC AGA TCT TTG AG-3′ (Cγ1 forward), 5′-GGA TCC AGA GTT CCA GGT CAC T-3′ (Cγ1 reverse). For amplification of the PTEN (387 bp) transcript, the primer pair of 5′-TTG AAG ACC ATA ACC CAC CAC AG-3′ (PTEN forward) and 5′-GGC AGA CCA CAA ACT GAG GAT TG-3′ (PTEN reverse) was used. Forward primer for AID (349 bp): 5′-GAG GGA GTC AAG AAA GTC ACG CTG GA-3′, reverse primer for AID: 5′-GGC TGA GGT TAG GGT TCC ATC TCA G-3′. Forward primer for control GAPDH (566 bp): 5′-TGT GTC CGT CGT GGA TCT GA-3′ and reverse primer for GAPDH: 5′-TTG CTG TTG AAG TCG CAG GAG-3′. PCR conditions were 94°C for 3 min, 94°C for 1 min, 60°C for 45 s, 72°C for 45 s, 30 cycles (from step 2 to step 4), and 72°C for 10 min for final extension.

Cells were preincubated with FcBlock (anti-CD16/32, eBiosence) to minimize nonspecific staining. Cells were then stained with cocktails of various mAbs (anti-CD19, -B220, -IgG1, -IgM, -IgD, -CD43, -CD93, -CD21, -CD23, -CD5, -FAS, -CL-7, -CD3ε, -CXCR4, or anti-CD86) conjugated with different fluoresce proteins. All antibodies were purchased from BD, eBioscience or BioLegend. Labeled cells were detected by 5 lasers Fortessa (BD), all data were analyzed with FlowJo software. Cell sorting was performed by using FACSAria III Cell Sorter (BD), following the protocols provided by BD.

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.

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.

To detect the PTEN function in GCBs, we generated the Pten^fl/flCγ1^Cre/+ mice by breeding Pten^fl/fl mice to Cγ1-Cre mice. In Pten^fl/flCγ1^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/flAicda^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/flCγ1^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/flCγ1^Cre/+ versus Pten^+/+Cγ1^Cre/+ control mice (Figure 1C).

Pten^fl/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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 expressing GCBs were almost lost in SRBC immunized Pten^fl/flCγ1^Cre/+ mice GC (Figures 3C,D). Similar results were also obtained in the immunized Pten^fl/flCγ1^Cre/+ and Pten^+/+Cγ1^Cre/+ mice by the utilization of alum adjuvant-precipitate BSA (Figures S1A,B in Supplementary Material). These results suggested that PTEN deletion in IgG1⁺ B cells significantly damaged the CSR within GCs.

To further verify the above observation of the impaired CSR within the GC of Pten^fl/flCγ1^Cre/+ mice, we purified the splenic B cells and performed an in vitro CSR assay following the published protocols (13, 16). Clearly, B cells from Pten^fl/flCγ1^Cre/+ mice failed to undergo CSR to form IgG1-BCR expressing B cells in the presence of lipopolysaccharide (LPS) alone or LPS plus IL-4 after 4 days of stimulation, respectively (Figures S2A,B in Supplementary Material). Similar results were acquired in the presence of other CSR-driven reagents of anti-CD40 antibodies or anti-CD40 plus IL-4 (Figures S2A,B in Supplementary Material).

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/flCγ1^Cre/+ mice upon the immunization by both SRBC and VLP (Figures 4A–C). Moreover, we observed that the number of DZ B cells in Pten^fl/flCγ1^Cre/+ mice GCs was dramatically decreased, consistent with a recent study showing that upon FoxO1 ablation or induction of PI3K activity, GCs lost their DZ, owing at least partly to downregulation of the chemokine receptor CXCR4 (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/flCγ1^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/flCγ1^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/flCγ1^Cre/+ mice exhibited an increased levels of GCBs than the control Pten^+/+Cγ1^Cre/+ mice upon immunization (Figure 3A; Figure S1B in Supplementary Material).

Our observations suggested that Cγ1 transcription-mediated PTEN KO impairs antibody responses, CSR activity, as well as the DZ and LZ compartmentalization and SHM within the GC. We next investigated the molecular mechanism accounting for the lack of CSR and SHM in the B cells from Pten^fl/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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/flCγ1^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.