GSK3 Restrains Germinal Center B Cells to Form Plasma Cells

Jeonghyun Lee, Hyosung Park, Jiwon Lim, Hyung-Seung Jin, Yoon Park, Yu-Jin Jung, Hyun-Jeong Ko, Sung-Il Yoon, Geun-Shik Lee, Pyeung-Hyeun Kim, Sun Shim Choi, Changchun Xiao and Seung Goo Kang
J Immunol February 1, 2021, 206 (3) 481-493; DOI: https://doi.org/10.4049/jimmunol.2000908

Key Points

  • GSK3–c-Myc–IRF4 regulatory axis induces CD138+ Ab-secreting cell formation.

  • GSK3 inactivation facilitated the LZ to DZ transition via increasing Foxo1.

  • Spatiotemporal control of GSK3 activity may be a key regulator of the GC reaction.

Abstract

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.

This article is featured in Top Reads, p.453

Introduction

Development of Ab-secreting plasma cells (PCs) and memory B cells (MBCs) is a key event in humoral immunity. Germinal center (GC)–experienced PCs secrete high affinity class-switched Abs that protect the host against invading pathogens, and MBCs are responsible for effective immunization by their expedited differentiation into PCs upon a second challenge with the same Ag (1). Proper regulation in the generation of these effector B cells is essential for immune homeostasis. Therefore, elucidating the mechanism underlying the GC reaction by which GC B cells are selected to decide their fates and are instructed for differentiation during the GC reaction is fundamental knowledge for understanding the immune response and guiding vaccine development.

Selection of appropriate GC B cells occurs during cyclic re-entry between the light zone (LZ) and dark zone (DZ) of the GC, which is accompanied by somatic hypermutation to produce new versions of BCR with diverse affinities (2). CXCR4hiCD83lo (or CD86lo) DZ B cells with high levels of BCR move into the LZ through the BCR downstream signaling pathway (2, 3), where they compete with each other for binding to Ags displayed on the surface of follicular dendritic cells and present them to a limited number of T follicular helper (Tfh) cells. The LZ to DZ transition occurs in response to upregulated CXCR4 expression when CXCR4loCD83hi (or CD86hi) LZ B cells successfully engage with Tfh cells owing to high affinity BCR that receive strong CD40 and IL-21 signals from Tfh cells (1, 4). Cell cycle entry for proliferation of selected LZ B cells in the DZ also occurs during the LZ to DZ transition in a manner dependent on functions of the transcription factors c-Myc and Foxo1 (5–8). This regulated repetitive cycling of positively selected GC B cells between the DZ and LZ is coupled with the generation of a progeny of effector B cells as one of the key events of the GC reaction (9). This high affinity–based selection model presupposes the existence of a specific mechanism by which GC B cells discriminate between a weak or strong interaction among Tfh cells; however, the detailed mechanism remains to be elucidated.

A portion of the selected GC B cells are instructed to undergo an additional differentiation program for PC or MBC generation. This effector B cell differentiation program has been extensively studied, and it is now well established to involve the transcriptional control of Prdm1 encoding Blimp-1, a key transcription factor for PC function (10, 11). In this regard, the transcriptional regulatory network operated by several key transcription factors, including Bcl-6, Pax5, Bach2, IRF4, and PU.1, is essential by directly or indirectly regulating Prdm1 expression (12–15). In particular, the cellular IRF4 expression level is one of the most essential factors regulating PC fate determination (16). Bach2 was initially defined as a repressor of Prdm1 and is therefore known to suppress PC differentiation (17, 18); however, high levels of Bach2 instruct GC B cells to form MBCs independent of Bach2-mediated Prdm1 repression (19). Thus, IRF4 and Bach2 expression might be tightly linked for the affinity-based selection of GC B cells because Bcl6lo CD69hi high affinity LZ B cells express more IRF4 than Bcl6hi CD69hi low affinity LZ B cells, and high affinity LZ B cells preferentially form PCs (20). In addition, LZ B cells that receive relatively weak help from Tfh cells express high levels of Bach2, leading to the formation of MBC precursors (19). Thus, IRF4 and Bach2 appear to be two major mediators for interpreting extrinsic cues, including the strength of T cell help and BCR signaling, that ultimately determine the post-GC B cell fate.

According to the current high affinity–based selection model, GC B cells interpret external cues, whereas high affinity LZ B cells that are prone to becoming CD138+ PCs emigrate from the GC via the DZ (21, 22). In contrast, low affinity LZ B cells become precursors of CCR6+ MBCs and leave the GC via the LZ (23). These findings suggest that formation of PC progeny and the DZ transition of LZ B cells are tightly linked programs that are precisely regulated by modulating the cellular concentrations of c-Myc, Foxo1, IRF4, and Bach2. However, the specific mechanism by which GC B cells interpret these external cues and are instructed by means of regulating the expression levels of these key factors to decide their cell fates and control these distinct biological processes simultaneously is poorly understood.

The CD40/CD40L interaction is one of the key factors determining the strength of engagement with Tfh cells for GC B cell selection and instruction for differentiation. c-Myc is also induced by CD40 signaling in the presence of IL-21, which is also produced by Tfh cells (7), and its cellular level is tightly linked to the fate of GC B cells (8). Therefore, controlling the cellular level of c-Myc in GC B cells is a plausible mechanism for discriminating the strength of Tfh cell engagement to instruct selected GC B cells to become effector B cells. c-Myc, as an essential component of the GC reaction (24, 25), is one of the best-known targets of glycogen synthase kinase 3 (GSK3), a constitutively active multifaceted serine/threonine kinase that regulates diverse biological processes (26), which is enabled via the regulating stability of various protein substrates (27). A recent study revealed that phosphorylated GSK3, its inactive form, is present in the c-Myc+ cell fraction of GC B cells (28), suggesting that c-Myc protein expression levels are regulated by GSK3 in GC B cells. As c-Myc+ GC B cells are associated with positively selected GC B cells and are functionally linked to humoral immunity (7, 25), we hypothesized that GSK3 mediates the c-Myc–dependent positive selection of GC B cells and post-GC processes. To test this hypothesis, we examined the impact of GSK3 on the differentiation of GC B cells to PC and on the LZ to DZ transition, which is a consequence of positively selected GC B cells and a prerequisite event for PC formation. Our results can highlight the key roles of GSK3 in GC B cells committed to the PC fate.

Materials and Methods

Mice

Gsk3αf/f and Gsk3βf/f mice were provided by Dr. J. Woodgett (Mount Sinai Hospital and Lunenfeld-Tanenbaum Research Institute) and crossed with Cγ1Cre mice. cmycf/f and Cγ1Cre mice were provided by Changchun Xiao (The Scripps Research Institute). Eight- to ten-week-old mice were used in all experiments. All mice were maintained at Kangwon National University Animal Laboratory Center and used in accordance with the guidelines from the Animal Care and Use Committees of Kangwon National University.

Follicular B cell isolation

Splenocytes were collected from various experimental mice, and RBCs were lysed with Ammonium-Chloride-Potassium (ACK) Lysing Buffer (Life Technologies). RBC-lysed splenocytes were stained with anti-CD5 (PE), anti-CD9 (PE), anti-CD43 (PE), anti-CD93 (PE), and anti-Ter119 (PE) Abs for 20 min at 4°C. Stained splenocytes were washed with MACS buffer (0.5% BSA and 2 mM EDTA in Dulbecco's PBS) and incubated with anti-PE Beads (Miltenyi Biotec) for 20 min at 4°C. Follicular B cells (FoBs) were negatively isolated using an LS column (Miltenyi Biotec). Isolated CD21int CD23+ FoBs (>97%) were used for in vitro induced GC (iGC) B culture.

In vitro iGC B cell and post-iGC B cell culture

CD40LB cells were maintained in DMEM GlutaMAX (Life Technologies) with 10% FBS and 1% penicillin/streptomycin (Life Technologies). CD40LB cells were irradiated at 100 Gy using Gammacell 40 Exactor (Best Theratronics) before use as feeder cells of in vitro iGC B cell and post-iGC B cell cultures as described previously (29). To generate iGC B cells, isolated FoBs (2 × 105) were cultured on irradiated CD40LB cells with complete RPMI 1640 GlutaMAX medium (Life Technologies) supplemented with murine IL-4 (1 ng/ml; PeproTech) for 4 d. The iGC B cells were harvested and purified by depleting CD40LB cells using biotin anti–H-2Kd (BioLegend) and anti-biotin beads (Miltenyi Biotec) with an LS column. The purified iGC B cells were then cultured without CD40LB for 12 h in complete RPMI medium at 37°C for resting. For post-iGC B cell culture, the rested iGC B cells (2 × 105 cells per 60-mm culture dish or 2 × 104 cells per well of a 12-well plate) were cultured with complete RPMI medium containing murine IL-21 (10 ng/ml, PeproTech) on irradiated CD40LB cells for 1, 1.5, 2, or 4 d and used for further analysis after CD40LB depletion. For in vitro proliferation and coculture experiments, iGC B cells or post-iGC B cells were labeled with CFSE (5 μM; Invitrogen) or CellTrace Violet (5 μM; Invitrogen), according to the manufacturer instructions, and used in the iGC B cell or post-iGC B cell culture condition. For quantification of IgG1 Abs secreted from induced plasmablasts (iPBs), post-iGC B cells were harvested at day 4 of post-iGC B cell culture and incubated further for 24 h in complete RPMI 1640 GlutaMAX medium without feeder cells (1 × 105 cells per well of a 96-well plate), and the culture medium was subjected to ELISA.

For the immunoblot assay, rested iGC B cells (1 × 106 cells per well of a 48-well plate) were stimulated for 1 h (phosphorylation of GSK3β) or for 4 h (c-Myc) with agonistic anti-CD40 Ab (Bio X Cell) and murine IL-21 (PeproTech) at various concentrations. Activated iGC B cells were infected with a Thy1.1-expressing retrovirus (RV) encoding wild-type or mutant forms of GSK3β, and then Thy1.1+ cells were positively purified via MACS isolation with anti-Thy1.1 (PE) (BioLegend) and anti-PE microbead (Miltenyi Biotec). Rested Thy1.1+ cells (1 × 106 cells per well of a 48-well plate) were then stimulated on CD40LB feeder cells for 6 h and subjected to the immunoblot assay.

Immunization

Gsk3αf/f;Gsk3βf/f;Cg1Cre and control (Gsk3αf/f;Gsk3βf/f) mice were s.c. injected with 50 μl of OVA/alum/LPS prepared by mixing 25 μl of Imject Alum (Thermo Fisher Scientific) and 25 μl of 1× PBS containing 50 μg of OVA (Sigma-Aldrich) and 5 μg of LPS (Sigma-Aldrich). The mice were sacrificed on day 10 after immunization for assessment of the GC reaction by flow cytometry. To perform ELISA of NP-specific Abs, the mice were immunized s.c. with NP-OVA/Alum prepared by mixing 25 μl of Imject Alum (Thermo Fisher Scientific) and 25 μl of 1× PBS containing 50 μg of NP-OVA (Biosearch Technologies). Serum was collected every week after immunization up to day 21.

Flow cytometry analysis

Intracellular expression of IRF4, Blimp1, and Pax5 was measured by flow cytometry. Post-iGC B cells were fixed for 1 h at 4°C with Fixation/Permeabilization Buffer (eBioscience). Staining Abs were diluted in Permeabilization Buffer (eBioscience), and intracellular staining was performed at 37°C for 30 min. The following Abs were also used: anti-IRF4 (PE), anti-Blimp1 (PE), and anti-Pax5 (PE). For surface marker staining, the following Abs were used for flow cytometry: anti-CD19 (PE/cy7), anti-CD138 (allophycocyanin), anti-CD83 (PE), anti-CXCR4 (biotin), streptavidin (allophycocyanin), anti-FAS (PE), anti-FAS (Brilliant Violet 510), anti-CD38 (PE/cy7), and anti-B220 (allophycocyanin/cy7). For mitochondrial reactive oxygen species (ROS) measurement, post-iGC B cells at day 1 of culture were first stained for surface markers, followed by staining with MitoSOX (5 μM; Invitrogen) at 37°C in a 5% CO2 incubator. Detailed information of resources, including the list of Abs used in this study, are in the Supplemental Table I.

ELISA

ELISA plates were coated with NP1–4–BSA or NP20–BSA (10 μg/ml in PBS; Biosearch Technologies) for quantification of total or high affinity NP-specific Ab, respectively. Blocking was performed at 37°C for 2 h in PBS with 0.5% BSA. Sera collected from the mice were diluted and loaded on the plate for incubation for 2 h at 37°C. The plates were washed three times with PBS containing 0.05% Tween (PBS-T). Biotin-conjugated primary Abs against mouse IgM and IgG1 (SouthernBiotech) were added to the wells and further incubated for 1 h at 37°C in PBS with 0.5% BSA. After three washing steps, streptavidin–HRP (BioLegend) was added, and the plates were incubated again for 1 h at 37°C in PBS with 0.5% BSA. After incubation, the wells were washed three times with PBS-T and developed with 5 ml of 3,30,5,50-tetramethylbenzidine dihydrochloride hydrate solution (BD Biosciences). The reaction was stopped with 0.5 M HCl solution, followed by quantification on a Synergy H1 Microplate Reader (BioTek Instruments). For quantification of secreted IgG1 from iPBs, purified goat anti-mouse Ig κ and λ L chain Ab (2.5 μg/ml in PBS; SouthernBiotech) was coated on the ELISA plates, and biotin-conjugated anti-mouse IgG1 Ab (SouthernBiotech) was used for detection.

ELISPOT assay

ELISPOT assays were performed for the detection of IgG1+ Ab-secreting cells (ASCs). Multiscreen membrane filtration 96-well plates (catalog no. MAIPSWU10; EMD Millipore) were coated with goat anti-mouse IgG1 (2.4 μg/ml; SouthernBiotech) at 4°C overnight. Blocking was performed for 2 h at 37°C with 1% gelatin in PBS. After washing three times with PBS-T, iPBs were loaded into each well and incubated for 4 h at 37°C. The plates were washed with PBS-T three times and incubated with biotinylated anti-mouse IgG1 (1:1000; SouthernBiotech) for 1 h, followed by incubation with streptavidin–HRP (1:1000; BioLegend) for 2 h. After three washing steps, the color was developed in 3-amino-9-ethylcarbazole (AEC) solution (acetate buffer [pH 5.1] + AEC Stock + 3% H2O2); AEC stock was prepared by dissolving 3-amino-9-ethyl carbazole (Sigma-Aldrich) in dimethyl formide. The spots were counted manually under a stereoscopic microscope.

Cell death assay

Purified iGC B cells were incubated in the post-iGC B cell culture condition under hypoxia (4% O2, 37°C, and 5% CO2) and low glucose (1 mM) for 1 d. The cells were harvested, and apoptosis was quantified using an Annexin V/propidium iodide (PI) apoptosis kit (eBioscience), according to the manufacturer’s instructions. In brief, the cells were incubated with Annexin V (1 μl in 20 μl buffer, 1:20) for 20 min at 4°C and then with PI (1 μl in 40 μl buffer, 1:40) for 5 min in the dark at room temperature.

Immunoblot analysis

Sorted GC B cells, purified iGC B cells, and post-iGC B cells were washed with PBS and lysed in 1× RIPA buffer (Cell Signaling Technology) supplemented with 1× phosphatase inhibitor mixture (GenDEPOT) and 1× protease inhibitor mixture (GenDEPOT) by incubating for 30 min on ice. Cell lysates were resolved on NaDodSO4–PAGE gels and transferred onto a polyvinylidene fluoride membrane (GE Healthcare). Blots were incubated overnight at 4°C with diluted primary Abs in 2.5% (wt/vol) skim milk (Cell Signaling Technology) and 1× TBS-T buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, and 0.1% Tween-20). All membranes were scanned using ImageQuant LAS 500 (GE Healthcare), and images were analyzed by ImageJ (National Institutes of Health Software). Immunoblotting was performed with the following primary Abs: anti–c-Myc (Cell Signaling Technology), anti-FOXO1 (Cell Signaling Technology), anti-pGSK3β (Cell Signaling Technology), anti-GSK3αβ (Santa Cruz Biotechnology), anti-Cre (Santa Cruz Biotechnology), and anti–β-actin–HRP (Santa Cruz Biotechnology). Anti-rabbit IgG–HRP (Cell Signaling Technology) was used as the secondary Ab.

Real-time PCR

Total RNA was extracted using TRIzol Reagent (Invitrogen) and subjected to cDNA synthesis using iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad Laboratories), according to the manufacturer’s instructions. RT-PCR was performed using Maxima SYBR Green/ROX RT-PCR Master Mix (Thermo Fisher Scientific) on an Applied Biosystems 7500 system (Thermo Fisher Scientific). Gene expression levels were normalized to the level of control gene encoding β-actin. Primer designations are listed in the Supplemental Table I.

Real-time metabolic assays

The oxygen consumption rate and extracellular acidification rate were measured from post-iGC B cells (1.5 × 105 cells/well) in nonbuffered RPMI medium (pH 7.4) (Agilent Technologies), containing 1 mM HEPES, without phenol red, sodium bicarbonate, glucose, l-glutamine, and sodium pyruvate, following the manufacturer’s instructions. All metabolic assays were performed with XFp Extracellular Flux Analyzer (Agilent Technologies). Data were analyzed by Wave (Agilent Technologies software).

Plasmids and retroviral gene transduction

Cre recombinase (MSCV-Cre, plasmid no. 320824; Addgene) and cDNA of murine Bach2 provided by Jeehee Youn (Hanyang University) were inserted into a Thy-1.1-expressing retroviral vector (plasmid no. 17442; Addgene). For short hairpin RNA (shRNA) expression, annealed shRNA primers encoding control or Irf4-specific shRNA were amplified with linker primers containing restriction enzyme sites and inserted into a pLMP-GFP vector. S-Eco packing cells were transfected by jetPRIME Transfection Kit (Polyplus), and retroviral supernatants were collected 48 h after transfection. For retroviral infection, day 3–cultured iGC B cells were subjected to spin infection with the retroviral supernatant supplemented with polybrene (8 μg/ml; MilliporeSigma) at 1500 × g for 90 min at 30°C, followed by one more day of culture in the iGC B cell culture system. The RV-infected iGC B cells were harvested and used as the post-iGC B cell culture system. For GSK3β reconstitution, murine Gsk3β was amplified from cDNA of iGC B cells and cloned into a Thy-1.1–expressing retroviral vector (plasmid no. 17442; Addgene). Site-directed mutagenesis was performed to generate retroviral vectors expressing mutant forms of GSK3β with QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies), according to the manufacturer’s instructions. All of the plasmids used in the experiments were sequenced and confirmed. Primer designations are listed in Supplemental Table I.

Cell cycle analysis

The cell cycle distribution was analyzed by flow cytometry after PI incorporation. In brief, harvested post-iGC B cells were washed with PBS and fixed with ice-cold 70% ethanol. The cells were then washed twice with PBS and incubated with RNase (100 μg/ml; Worthington Biochemical) diluted in PBS for 1 h at 37°C in a non-CO2 incubator. The cells were washed again with PBS and incubated with PI solution (50 μg/ml; Sigma-Aldrich) and immediately analyzed with FACSVerse Flow Cytometer (BD Biosciences).

Cell sorting

For immunoblotting of GSK3 expression, single cells of the draining lymph nodes were prepared from immunized mice and stained for GC B cells. Both IgG1+ FAS+CD38low and IgG1–FAS+CD38low GC B cells were sorted and subjected to the immunoblot assay. To perform ELISA and ELISPOT of IRF4 knockdown or Bach2 overexpression, virus-infected GFP+CD19+ (IRF4 knockdown or control) or Thy1.1+CD19+ (Bach2 overexpression or control) cells were sorted. For RNA sequencing analysis, CD19+ post-iGC B cells treated with CHIR99021 or DMSO for 36 h were sorted and subjected to total RNA isolation. All sorting processes were performed using a FACSAria II flow cytometer (BD Biosciences).

RNA sequencing

Total RNA was isolated from sorted post-iGC B cells using the RNAeasy Kit (QIAGEN), according to the manufacturer’s instructions. Illumina TruSeq Stranded mRNA Sample Prep Kit (no. RS-122-2101; Illumina, San Diego, CA) was used for library construction. Indexed libraries were then submitted to an Illumina NovaSeq platform (Illumina), and paired-end (2 × 100 bp) sequencing was performed by Macrogen. For analysis of RNA sequencing data, alignment and annotation were processed with TopHat2 (30). Fasta and gtf files of the Mus musculus genome (version mm10 from University of California, Santa Cruz) were used as the reference for alignment and annotation. Using the same Fasta and gtf files, Cufflinks, Cuffnorm, and Cuffdiff were subsequently conducted for calculating the fragments per kb of transcript per million (FPKM) values, normalizing library sizes, and identifying differently expressed genes (DEGs) (31), respectively. After excluding genes with 0 FPKM in all samples, log (FPKM+1) values were used for downstream analysis (32). After quantile normalization performed within DMSO/CHIR and DZ/LZ, significant DEGs were estimated according to Q value <0.05 and fold change (|fc|) ≥2. All plots were constructed with R (v.3.5.3) (the R Foundation for Statistical Computing, Vienna, Austria). Gene Ontology analysis was performed using Database for Annotation, Visualization, and Integrated Discovery (v6.8) (33). Gene Ontology terms with low p values were only selected from the biological process category to construct Supplemental Fig. 2C, 2D. To identify common significant DEGs between the DMSO/CHIR and LZ/DZ comparisons, the list of DEGs of each experiment was overlapped, and a total of 48 common DEGs obtained from 12 samples were used for constructing the heat map and principal components analysis plot.

Published gene expression data

For the DZ and LZ analysis, FastQ files of Foxo1(+/+) DZ and Foxo1(+/+) (34) were downloaded from the Sequence Read Archive: accession no. SRP096690. Single-end reads were processed with the same pipeline described above. The list of c-Myc–responsive genes was obtained from Supplemental Table III of Dominguez-Sola et al. (25). The public CEL files of GFPMYC+ and GFPMYC– were downloaded from Gene Expression Omnibus: accession no. GSE38304.

Statistical analysis

The p values were determined using a two-tailed Student t test. Statistical significance was judged at p < 0.05. All data points and ‘‘n’’ values represent biological replicates. GraphPad Prism 7 software was used for statistical analysis.

Data availability

The Gene Expression Omnibus accession number for the RNA sequencing data reported in this paper is GSE144361 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?&acc=GSE144361).

Results

CD40L and IL-21–mediated c-Myc induction depends on GSK3 in iGC B cells

Because GSK3 directly phosphorylates c-Myc protein and thus expedites its degradation (35), we presumed that GSK3 acts downstream of CD40L and IL-21 signaling to control the cellular level of c-Myc in GC B cells. To test this possibility, we generated in vitro iGC B cells by 4 d of culture with naive FoB on CD40L- and BAFF-expressing feeder cells (CD40LB) supplemented with IL-4 (29). After stimulating iGC B cells with CD40L and IL-21, GSK3β was found to be phosphorylated at S9, indicating GSK3β inactivation upon CD40L and IL-21 stimulation (Fig. 1A). Importantly, the c-Myc expression level in iGC B cells was dependent on the amount of CD40L and IL-21 stimuli (Fig. 1B). Therefore, it is very likely that GSK3 kinase activity is responsible for the CD40L- and IL-21–mediated determination of the cellular level of c-Myc in GC B cells.

FIGURE 1.
FIGURE 1.

CD40L- and IL-21–mediated c-Myc expression depends on GSK3β kinase activity. (A) Immunoblot analysis (left) and bar graph (right) of phosphorylated GSK3β in iGC B cells upon agonistic anti-CD40 Ab (25 μg/ml) and IL-21 (50 ng/ml) stimulation (n = 3). (B) Immunoblot analysis of c-Myc in stimulated iGC B cells with agonistic anti-CD40 Ab and IL-21 at different concentrations as indicated. (C) Experimental scheme of GSK3β reconstitution into GSK3β-null iGC B cells. (D) Immunoblot analysis for c-Myc, GSK3β, and GSK3β phosphorylation of reconstituted Thy1.1+ iGC B cells upon 6-h stimulation on the CD40LB with IL-21 (50 ng/ml). Wild-type or mutant forms of GSK3β-expressing RV-infected Thy1.1+ iGC B cells were isolated and subjected for stimulation. Data of the immunoblot assay shown are representative of three independent experiments. RT-PCR analysis of cmyc expression in iGC B cells upon agonistic anti-CD40 Ab (25 μg/ml) and IL-21 (50 ng/ml) stimulation for 4 h (E) (n = 3) and in GSK3 KO iGC B cells cultured for 6 h with CD40LB cells under stimulation with IL-21 (10 ng/ml) (F) (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

To test this possibility directly, we generated iGC B cells expressing wild-type (GSK3βWT), constitutively active (GSK3βS9A), or kinase-dead (GSK3βK85A) forms of GSK3β by means of retroviral transduction into GSK3-null iGC B cells generated from Gsk3αf/f; Gsk3βf/f; Cg1Cre knockout (GSK3 KO) FoBs (Fig. 1C). GSK3 deficiency resulted in the synergistic induction of c-Myc compared with Gsk3αf/f; Gsk3βf/f (hereafter referred to as control) iGC B cells upon 6-h stimulation on the CD40LB with supplemented IL-21 (Fig. 1D). Moreover, reconstitution with GSK3βWT reduced the c-Myc expression level to that of the RV mock-infected GSK3-null iGC B cells (Fig. 1D), suggesting a regulatory role of GSK3β regarding determination of the cellular level of c-Myc in GC B cells. More importantly, GSK3βS9A introduction resulted in the degradation of c-Myc in GSK3-null iGC B cells, whereas GSK3βK85A-expressing B cells did not induce c-Myc degradation under the same condition (Fig. 1D). However, the contribution of transcriptional activation to the GSK3-mediated c-Myc protein induction was marginal in iGC B cells (Fig. 1E, 1F). Therefore, we concluded that modulating GSK3 kinase activity upon CD40L and IL-21 stimulation is one of the essential posttranscriptional regulatory modes to determine the cellular level of c-Myc in GC B cells, suggesting that GSK3 might play regulatory roles during the GC reaction, particularly in post-GC B cell fate commitment and the differentiation of positively selected GC B cells.

GSK3 deficiency expedites PC differentiation in vitro

Given the findings that c-Myc expression is tightly linked to the positive selection of LZ GC B cells and PC generation (7, 8, 25), we next explored the detailed roles of GSK3 in GC B cells during the post-GC B cell fate decision and PC differentiation. To better understand the roles of GSK3 in the post-GC B cells at the cellular and molecular level, we adopted the iGC B cell culture system (29). In this system, effector B cells can be generated by consecutive iGC B cell culture and post-iGC B cell culture. iGC B cells are generated by 4 d of culture, which were then cultured for 4 more days with IL-21 supplementation to induce the generation of CD138+ ASCs (Fig. 2A). Therefore, we could functionally dissect GC B cell generation from effector B cell differentiation events and could follow the post-GC B cell fate decision and PC differentiation after GC B cell formation, which is not feasible using an in vivo system.

FIGURE 2.
FIGURE 2.

GSK3 absence in iGC B cells expedites CD138+ ASC generation. (A) Experimental scheme of in vitro iGC B cell and post-iGC B cell culture. (B) Frequency of CD138+ ASCs at day 4 of post-iGC B cell culture (n = 4). (C) iPB-secreted IgG1 levels determined by ELISA (n = 3). (D) Proliferation of CellTrace Violet–labeled iGC B cells at day 3 of post-iGC B cell culture. Representative data from three independent experiments are shown. Intracellular staining and mean fluorescence intensity (MFI) of IRF4 (E), Pax5 (F), and Blimp-1 (G) in CD19+CD138– cells at day 2 of post-iGC B cell culture (n = 4). (H) Flow cytometry analysis of CD19+ CD138+ cells (left) and percentage of CD19+CD138+ cells on day 2 post-iGC B cell culture (right) (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

Given the incomplete control of Cre expression with the Cg1Cre system (36), we first evaluated the timing and the extent of Cre-mediated deletion of Gsk3 during the first round of iGC B culture. Cre was expressed as of day 2 followed by diminished GSK3 expression, with profound depletion observed at day 4 of iGC B culture (Supplemental Fig. 1A). Importantly, Cre was expressed in both IgG1+ and IgG1– iGC B cells of GSK3 KO cells after 4 d of iGC B cell culture (Supplemental Fig. 1B). Although less Cre was detected in the IgG1– population than that of IgG1+ iGC B cells but GSK3 was efficiently deleted in both populations (Supplemental Fig. 1B), suggesting that GSK3 is depleted in iGC B cells regardless of IgG1 positivity, at least in our experimental system. Proliferation and class switching were observed as of day 3 of culture with this system, whereas the extent of proliferation and class switching to IgG1+ was not affected by the absence of GSK3 during iGC B culture (Supplemental Fig. 1C, 1D). Absolute numbers of CD19+ iGC B cells in the GSK3 KO group were also similar to those of the control group during iGC B cell culture (Supplemental Fig. 1E). In addition, the iGC B cells from both control and GSK3 KO mice showed comparable expression levels of FAS and GL-7 (Supplemental Fig. 1F, left). Although there was a trend of a lower number of FAS+GL7+ iGC B cells under GSK3 deficiency compared with that of the control at day 4, the difference was not statistically significant (Supplemental Fig. 1F, right). Therefore, we concluded that GSK3 depletion had a minimal impact on iGC B cell differentiation per se.

To further evaluate the role of GSK3 in the post-GC reaction, the same numbers of iGC B cells of control or GSK3 KO mice were subjected to post-iGC B cell culture. To our surprise, the frequency of CD138+ ASCs in iPBs of GSK3 KO mice was much higher than that in control iPBs after 4-d culture of iGC B cells (Fig. 2B), and they were functional in secreting Abs (Fig. 2C). As CD138+ ASCs generation is positively associated with proliferation (37), enhanced generation of CD138+ ASCs could be advantageous in the proliferation of GSK3 KO iGC B cells. However, we were unable to detect a significant difference between control and GSK3KO iGC B cells in proliferation at day 3 (Fig. 2D), suggesting that enhanced generation of CD138+ ASCs is not a consequence of accelerated cell division. Instead, GSK3 deficiency might be an instructive cue to iGC B cells to initiate PC differentiation because IRF4 and Pax5 (as key modulators of PC differentiation) were rapidly induced and downregulated, respectively, in favor of PC differentiation in CD19+CD138– post-iGC B cells from GSK3 KO mice at 2 d post-iGC B culture (Fig. 2E, 2F). In addition, Blimp-1 expression was highly upregulated (Fig. 2G), and CD138+ plasmablasts were rapidly generated from GSK3 KO iGC B cells at day 2 post-iGC B cell culture (Fig. 2H). In summary, it is very likely that GSK3 deficiency functions as an internal cue to initiate PC differentiation, resulting in expedited ASC generation in vitro.

GSK3 deficiency in GC B cells results in compromised Ab production in vivo

To confirm the findings that expedited PC differentiation occurs under a condition of GSK3 deficiency, we evaluated humoral immunity against Ags in GSK3 KO mice. However, the GSK3 KO mice were defective in the production of both low and high affinity NP-specific IgG1 Abs after immunization with NP-OVA/Alum, whereas Ag-specific IgM Ab production was comparable with that in control mice (Fig. 3A), suggesting that GSK3 expression in B cells is essential for ASC generation. The generation of FAS+CD38lo GC B cells was affected in GSK3 KO mice after immunization with OVA/LPS/Alum, with approximately one third the GC B cell count of that found in control mice, whereas the frequency of IgG1+ GC B cells was relatively comparable between the GSK3 KO and control groups (Fig. 3B, 3C). Importantly, GSK3 expression was effectively abolished in both the IgG1+ and IgG1– FAS+CD38lo GC B cells of GSK3 KO mice (Fig. 3D), indicating that GSK3 expression was completely absent in all FAS+CD38lo GC B cells. Depletion of GSK3 in IgG1– GC B cells might account for the much lower amount of other NP-specific high affinity IgG subclass Abs such as IgG2b or IgG3 than those detected in the control group after 21 d of immunization (data not shown). Notably, the generation of CD138+ ASCs was more severely affected by GSK3 deficiency than that of GCs, as IgG1+ CD138+ ASCs were scarcely detected in GSK3 KO mice (Fig. 3E), implying more GSK3 dependency in sustaining the pool of CD138+ ASCs than GC B cells. These results are in contrast to the findings from the in vitro system described above, indicating that the presence of GSK3 in GC B cells is essential for sustaining both the GC B cell and ASC pools in vivo.

FIGURE 3.
FIGURE 3.

GSK3 expression in GC B cells is indispensable for ASC generation. (A) NP-OVA/alum–immunized control (n = 5) or GSK3 KO (n = 4) mice were bled at the indicated time points, and the amounts of NP-specific IgG1 and IgM were determined by ELISA. (B and C) Flow cytometry analysis of GC B (B220+FAS+CD38lo) cells and IgG1+ GC B cells in the draining lymph nodes at 10 d after OVA/Alum/LPS immunization; percentage and absolute numbers are indicated. (D) Immunoblot analysis of GSK3α and GSK3β in GC B cells. (E) Percentage and absolute number of IgG1+CD138+ cells in the peripheral lymph nodes at 10 d after OVA/Alum/LPS immunization (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

GSK3 inhibition degree affects the susceptibility of iGC B cells to apoptosis

In a previous study, GSK3-null B cells showed increased metabolic demands, as indicated by an increased cell size and enhanced glycolysis; thus, the cells were highly susceptible to ROS-induced apoptosis in a glucose-limited environment (28). Indeed, we found that the absolute numbers of CD19+ iPBs of GSK3 KO mice were profoundly lower than those of the control group at the late stage of post-iGC B culture (Fig. 4A), whereas the frequency of CD138+ cells in GSK3 KO mice was much higher than that of the control group (Fig. 4B). Notably, this difference did not appear to be related to a proliferation defect of GSK3 KO post-iGC B cells (Fig. 2D). Importantly, the number of CD138+ iPBs was higher in the GSK3 KO group than that of the control group at day 2 of culture but then declined at a late stage of culture when the cell death might have occurred (Fig. 4C) and therefore did not reflect a much higher frequency of CD138+ cells. These results imply that expedited cell death occurred in the absence of GSK3 during the late stage of post-iGC B culture. Therefore, we speculated that the compromised humoral immunity in GSK3 KO mice is mainly due to the expedited cell death in GCs in a condition of limited glucose and oxygen (28, 38).

FIGURE 4.
FIGURE 4.

GSK3 inactivation increases the metabolic demand in iGC B cells. (A) Absolute numbers of CD19+ iPBs (A), and the frequencies (B) and absolute numbers (C) of CD19+CD138+ iPCs of the control or GSK3 KO group during 4 d of post-iGC B culture (n = 4). (D) Flow cytometry analysis and (E) frequency of Annexin V+PI+ apoptotic post-iGC B cells at day 1 in low-glucose and -hypoxia conditions (n = 5). (F) Absolute numbers of CD19+ iPBs of control or CHIR99021 (3 μM)–treated control CD19+ iPBs at day 4 of post-iGC B cell culture (n = 4). (G) Cell size of post-iGC B cells at day 1 of post-iGC B cell culture. Representative data from three independent experiments are shown. (H) Basal and maximum glycolytic activity of post-iGC B cells at day 1.5 (n = 3). (I) Level of mitochondrial ROS in post-iGC B cells at day 1 (n = 3). (J) Flow cytometry analysis and (K) frequency of Annexin V+PI+ apoptotic post-iGC B cells at day 1 in low-glucose and -hypoxia conditions treated with high concentrations of CHIR99021 (n = 3). (L) Absolute numbers of CD19+ iPBs of control or CHIR99021 (10 or 30 μM)–treated control CD19+ iPBs at day 4 of post-iGC B cell culture (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

To evaluate this possibility, control or GSK3 KO iGC B cells were exposed to low-glucose and -hypoxia conditions during post-iGC B culture. Indeed, GSK3-deficient iGC B cells were very susceptible to apoptosis in low-glucose and -hypoxia conditions (Fig. 4D, 4E). However, a specific pharmacological inhibitor, CHIR99021 (39)–treated iGC B cells did not induce apoptosis in the same conditions and eventually generated a comparable number of CD19+ iPBs to that produced in the control group after 4 d of post-iGC B culture (Fig. 4F). This result was initially striking because we presumed that GSK3 activity inhibition should result in increased metabolic demands and ROS production and thus induce apoptosis similar to a state of GSK3 deficiency in GC B cells (28). GSK3 inhibition via CHIR99021 also resulted in cell size enlargement (Fig. 4G) and elevated the glycolytic activity (Fig. 4H), eventually increasing ROS production (Fig. 4I), indicating increased metabolic demands. These unexpected results prompted us to examine the possibility of the incomplete inhibition of GSK3 by 3 μM of CHIR99021, which differs from the GSK3 KO condition. Interestingly, treatment with a 10 or 30 μM of CHIR99021 resulted in increased susceptibility against cell death (Fig. 4J, 4K) and a lower absolute number of CD19+ iPBs (Fig. 4L), suggesting that the extent of GSK3 inhibition (depending on concentration) is positively correlated to the susceptibility of iGC B cells to cell death.

Taken together, these results suggest that the greater susceptibility to cell death of GC B cells in the absence of kinase activity is likely one of the main reasons contributing to the compromised humoral immunity observed in GSK3 KO mice and can also explain the apparent paradoxical in vivo and in vitro results regarding the role of GSK3 in ASC generation.

Inhibition of GSK3 unlocked the PC differentiation program

GSK3 activity is regulated by several types of posttranscriptional modifications (40–42), which may in turn be modulated by signaling pathways triggered via external cues and by internal signaling modulators such as PKA (43) and AKT (44) in GC B cells. Thus, it is possible that GSK3 activity is not regulated in a binary fashion, but rather the level of activity is adjusted to various extents according to the modification type. However, the specific extent of GSK3 inhibition during the GC reaction remains unclear. This suggests the existence of additional roles for GSK3 besides its main function as a metabolic checkpoint regulator revealed in studies under a GSK3-depleted condition (28). To explore such additional roles of GSK3, we next tested whether pharmacological inhibition of GSK3 with CHIR99021 also acts as an instructive cue for PC generation, as observed in GSK3 KO iGC B cells. GSK3 inhibition during post-iGC B cell culture in the CD40LB system facilitated functional CD138+ ASCs generation, even with high concentrations of the inhibitor (Fig. 5A, 5B); nevertheless, we used a concentration of 3 μM of CHIR99021 for the following experiments. Next, we examined the kinetics of CD138+ cell appearance during 4 d of post-iGC culture. There was no prominent difference in the CD138+ frequency during the first 2 d of culture; however, the facilitated generation of CD138+ PCs clearly occurred subsequently (Fig. 5C). Enhanced generation of CD138+ ASCs by CHIR99021 treatment possibly occurs because of instructions for iGC B cells received during the first 2 d for generating PCs or for subsequent functioning to amplify CD138+ ASCs. To address these possibilities, we inhibited the GSK3 activity of iGC B cells only for the first 2 d, which were then cocultured with the control iGC B cells for 2 more days in the absence of the GSK3 inhibitor (Fig. 5D). CHIR99021-pretreated cells for 2 d still tended to form more CD138+ ASCs compared with those generated by control cells in the absence of CHIR99021 (Fig. 5E). These data strongly suggest that GSK3 inhibition in GC B cells serves as a signal for generating PCs before the ontogeny of CD138+ PCs, and we consider this to be due to altered transcriptional networks in favor of PCs.

FIGURE 5.
FIGURE 5.

GSK3 inactivation alters transcriptional networks in favor of ASC generation. (A) Flow cytometry analysis (left) and frequency of CD19+CD138+ cells (right) of iPBs (n = 4) at day 4 of post-iGC B cell culture. (B) iPB-secreted IgG1 levels determined by ELISA (n = 6). (C) Frequency of CD138+ cells at day 2 or day 4 of post-iGC B cell culture (n = 3). (D) Experimental scheme of coculture with DMSO or CHIR99021 (3 μM) pre-exposed post-iGC B cells. (E) Flow cytometry analysis of CD138+ cells at day 2 or day 4 of coculture experiments. Representative data from three independent experiments are shown. (F) Volcano plots of gene expression profiles from DMSO- or CHIR99021-treated post-iGC B cells at day 1.5 of post-iGC B cell culture. (G) Clustered heat map of selected effector B cell differentiation regulatory genes. *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

To test this hypothesis and gain more mechanistic insights into the GSK3-mediated alteration of cell fate, we performed gene expression profiling of GSK3-inactivated post-iGC B cells cultured for 36 h with a GSK3 inhibitor in the post-iGC B culture system and then compared the transcriptome changes relative to DMSO-treated control cells. Gene expression profiles of GSK3-inhibited post-iGC B cells were clearly distinguished from those of the DMSO control group (Supplemental Fig. 2A, 2B). We identified 407 DEGs (Q value <0.05, |fc| ≥ 2), including 134 upregulated genes and 273 downregulated genes (Fig. 5F, Supplemental Table II). These 407 GSK3-responsive genes were involved in several biological processes, including B cell activation, B cell differentiation, Ag presentation to MHC class II, and apoptosis (Supplemental Fig. 2C). Importantly, the expression of transcriptional regulators of effector B cell differentiation was altered toward favoring the commitment of PCs with diminished Bach2, Bcl6, and Pax5 and induced Irf4, Xbp1, and Prdm1 expression (Fig. 5G). Together, these results strongly suggest that GSK3 activity functions as an internal cue to alter the transcriptional program against PC differentiation, which occurs rapidly before indication as PC precursors by CD138 expression.

GSK3-induced PC differentiation depends on IRF4 and Bach2 expression

Among the PC regulatory genes altered by GSK3 inhibition (Fig. 5G), increased Irf4 expression was considered to be functionally responsible for inactive GSK3-mediated enhanced CD138+ ASC generation because the cellular level of IRF4 is a key mediator of the post-GC B cell fate decision to PC differentiation (16). To validate this hypothesis, we reduced the cellular level of IRF4 through retroviral introduction of shRNA targeting Irf4 in GSK3-inhibited iGC B cells (Supplemental Fig. 3A) and then examined the impact on CD138+ ASC generation. Indeed, GSK3-mediated enhanced generation of CD138+ ASCs was effectively abolished (Supplemental Fig. 3B, 3C).

Bach2 is another key mediator of post-GC B cell fate commitment, and its expression guides post-GC B cells to MBC while simultaneously preventing PC differentiation (17, 19). As shown in Fig. 5G, Bach2 expression was also downregulated in GSK3-inhibited post-iGC B cells and thus occurred slightly earlier than Irf4 induction upon GSK3 inhibition (Supplemental Fig. 3D), suggesting that diminished Bach2 expression also might be responsible for enhanced CD138+ ASC generation. To validate this possibility, we introduced a Bach2-expressing retroviral vector into CHIR99021-treated iGC B cells, which almost completely prevented CD138+ ASC generation (Supplemental Fig. 3E, 3F). Together, these results indicate that GSK3 inactivation stimulates the well-established transcriptional programs of PC fate commitment.

c-Myc is responsible for GSK3-mediated synergistic IRF4 induction

c-Myc, a substrate of GSK3, is a key regulator of the GC reaction (24, 25) and thus may mediate the positive selection of GC B cells (3). As c-Myc regulates one third of all genes involved in diverse biological processes and has been suggested to directly control Pax5 (45) and Irf4 expression in B cells (46), we reasoned that c-Myc is an important mediator of the transcriptional reprograming initiated by inactive GSK3. To validate this hypothesis, we first examined c-Myc expression levels in GSK3-inactivated post-iGC B cells and found synergistic induction of c-Myc at 12 h after stimulation, which lasted for 24 h (Fig. 6A); however, this synergistic c-Myc induction was not caused by transcriptional activation (Fig. 6B). Next, we compared the gene expression profiles between c-Myc+ GC B cells and GSK3-inhibited post-iGC B cells because we reasoned that these gene expression profiles would be shared if c-Myc is responsible for GSK3-mediated altered gene expression. We thus reanalyzed the published dataset of c-Myc+ and c-Myc– GC B cells sorted from immunized mice (25) and found 425 DEGs (Q value <0.05, |fc| ≥ 2; Supplemental Table III). Among the 407 GSK3-responsive genes identified in the previous experiment, 8.8% of the DEGs (14 of 134 upregulated genes and 22 of 274 downregulated genes) overlapped with c-Myc–responsive genes (Fig. 6C). However, 86% of the overlapped genes (31/36) were positively correlated with c-Myc expression (Fig. 6D), suggesting that c-Myc might partly contribute to the GSK3-mediated alteration of gene expression profiles. Importantly, both Irf4 and Bach2 were included among the positively correlated genes (Fig. 6D). These results suggest that c-Myc is not the sole responsible factor of GSK3-mediated altered gene expression but might affect GSK3-mediated GC B cell fate commitment specifically through Irf4 induction and Bach2 suppression, either directly or indirectly.

FIGURE 6.
FIGURE 6.

c-Myc is a mediator of GSK3-mediated IRF4 induction. (A) Immunoblot analysis of c-Myc in iGC B cells and post-iGC B cells at the indicated time points. The bar graph indicates the relative c-Myc expression level normalized to the level of β-actin (n = 3). (B) RT-PCR analysis of c-myc expression in iGC B cells and post-iGC B cells at the indicated time points (n = 3). (C) Common DEGs of GSK3-responsive genes and c-Myc–responsive genes analyzed with two different criteria. Numbers indicate the quantity of genes. (D) Numbers and list of positively correlated overlapped genes among the common DEGs. (E–H) iGC B cells generated from cmycf/f FoBs were infected with a control or Cre recombinase-encoding RV. The virus-infected iGC B cells were then used for post-iGC B cell culture. (E) Cell size of cmycf/f post-iGC B cells at day 1 of post-iGC B culture. (F) RT-PCR analysis of Irf4 in cmycf/f post-iGC B cells at day 1.5 of post-iGC B culture (n = 3). (G) Cellular level of IRF4 in cmycf/f post-iGC B cells at day 1.5 of post-iGC B culture in the presence of CHIR99021 (n = 5). (H) RT-PCR analysis of Bach2 in cmycf/f post-iGC B cells at day 1 of post-iGC B culture (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

To test if c-Myc indeed regulates Irf4 and Bach2 in post-GC B cells, we generated cmycf/f iGC B cells and then repressed c-Myc function in post-iGC B cells via retroviral introduction of Cre recombinase. c-Myc function in virus-infected post-iGC B cells was efficiently abrogated, as indicated by a smaller cell size than that of the control (Fig. 6E). This resulted in abrogation of Irf4 induction (Fig. 6F) and thus reduced the cellular level of IRF4 in GSK3-inhibited post-iGC B cells (Fig. 6G). Of note, the Irf4 expression level was comparable between the control cmycf/f;Cre– and cmycf/f;Cre+ groups, indicating that the dependency of c-Myc for Irf4 induction seems to occur only in the context of GSK3 inhibition. Bach2 suppression was only partly dependent on c-Myc (Fig. 6H). Together, these results suggest that cellular IRF4 expression levels enabling GC B cells to be committed as PC precursors is determined via the GSK3–c-Myc regulatory axis.

Inactive GSK3 facilitates the LZ to DZ transition of GC B cells

The cyclic itinerary between the LZ and DZ is a key characteristic of positively selected GC B cells and is linked to post-GC B cell fate commitment. Previous studies have revealed that selected LZ B cells form PC precursors and differentiate further in the DZ and then egress the GCs from there (22, 23). Therefore, it is highly possible that transcriptional reprograming for PC fate commitment by GSK3 inactivation is accompanied by the LZ to DZ transition and may also be regulated by GSK3. First, we observed diminished expression of Cd83 and Cd86 in GSK3-inhibited post-iGC B cells (Fig. 5F). Cd83 and Cd86 are characteristic genes of LZ B cells, and loss of their expression with CXCR4 upregulation is required for the DZ transition. These data prompted us to examine if CXCR4 is upregulated by GSK3 inhibition during the post-iGC B cell culture period because it is important for positioning in the DZ. Indeed, GSK3 inactivation stimulated Cxcr4 induction in post-iGC B cell culture (Fig. 7A) and enhanced the generation of CXCR4hi CD83lo DZ-like B cells (Fig. 7B). As Foxo1 is one of the key regulators of the LZ to DZ transition (5, 6), we reasoned that GSK3 might regulate Foxo1 expression in post-iGC B cells and thus regulate the LZ to DZ transition. Indeed, inactive GSK3-mediated DZ-like B cell generation was accompanied by the synergistic induction of Foxo1 (Fig. 7C). However, Foxo1 gene expression was not altered by GSK3 inactivation (Fig. 7D), indicating that GSK3 might control Foxo1 stability and thus regulate the LZ to DZ transition of GC B cells.

FIGURE 7.
FIGURE 7.

The LZ to DZ transition is regulated by GSK3 activity. (A) RT-PCR analysis of Cxcr4 expression in post-iGC B cells at day 2 (n = 4). (B) Frequency of DZ (CXCR4hiCD83lo)– or LZ (CXCR4loCD83hi) –like B cells at day 2 or day 4 of post-iGC B culture (n = 3). (C) Immunoblot analysis of Foxo1 expression in iGC B and post-iGC B cells at the indicated time points. The bar graph indicates the Foxo1 expression level normalized to the level of β-actin (n = 3). (D) RT-PCR analysis of Foxo1 at day 2 of post-iGC B culture (n = 4). (E) Clustered heat map of 48 common DEGs of LZ/DZ–DEGs and GSK3-responsive genes. Red indicates DZ or LZ signature genes. (F) Principal components analysis plot of the 48 common DEGs. *p < 0.05, **p < 0.01, ***p < 0.001. n.s., not significant.

Next, we reanalyzed the previously published gene expression profiles of LZ and DZ B cells (34) and found that 315 genes were differentially expressed (Q value <0.05, |fc| ≥ 2; Supplemental Table IV). Among them, 48 genes were common DEGs with the GSK3-responsive genes, and the gene expression profiles of DZ B cells closely clustered with that of CHIR99021-treated post-iGC B cells (Fig. 7E, 7F). In addition, 25 genes among the 48 common DEGs (Supplemental Table V) were LZ or DZ B cell signature genes identified in a previous study (47). Altogether, these results strongly suggest that the LZ to DZ transition, as another functional feature of positively selected post-GC B cells, is also facilitated by GSK3 inactivation.

Discussion

The mechanisms by which high affinity BCR-possessing GC B cells are selected and determination of their post-GC fates as PCs or MBCs, which are fundamental characteristics of humoral immunity, have been studied extensively. In this regard, cognate engagement of Tfh cells with LZ B cells was the first mechanism to be recognized (48). According to the widely accepted model, CD40 signaling that occurs during engagement with Tfh cells is the most critical factor for selection, and longer interactions serve as an external cue for the PC fate decision (20, 49). Selected LZ B cells undergo rapid proliferation with a DZ transition or alteration of gene expression regulatory networks and thus enter another round of selection or become effector B cells, respectively. The molecular events in selected GC B cells are gradually being elucidated by revealing the key transcription factors, such as c-Myc, IRF4, and Bach2, and their respective roles. In particular, cellular expression levels of these factors are thought to determine the fates of post-GC B cells. However, the intracellular mediators interpreting these external cues to determine the cellular expression level of key mediators have been unknown. In this study, we demonstrate that inhibition of GSK3 activity in iGC B cells in the presence of CD40L and IL-21 resulted in alteration of gene expression regulation to favor PCs, and the LZ to DZ transition via synergistic upregulation of c-Myc, IRF4, and Foxo1 suggested that GSK3 inactivation is a critical intracellular cue for positive selection of GC B cells as precursors of PCs.

The presence of GSK3 in GC B cells might be essential for ASC generation because genetically GSK3-ablated GC B cells were unable to form ASCs in vivo even though they were activated and formed GCs. According to a recent report, GSK3 also restricts GC B cell proliferation by suppressing metabolic activities such as glycolysis and mitochondrial biogenesis, which are critical for GC function (28). Therefore, enhanced GC reaction and Ab production should be expected in the context of GSK3 inactivation in GC B cells. However, constitutive GSK3 inactivation, such as through the specific deletion of both Gsk3α and Gsk3β in GC B cells, resulted in cell death in a metabolically challenging environment, including under glucose-limited and -hypoxic conditions (28). These experimental results strongly indicate the requirement for the spatial and temporal regulation of GSK3 activity in GC B cells and its critical roles in the GC reaction. External cues enabling the phosphorylation of GSK3, such as Ags and Tfh cells, are enriched in the LZ but are scarce in the DZ. Therefore, spatial and temporal regulation of GSK3 activity in GC B cells might be possible during the cyclic re-entry between the LZ and DZ.

The quantity of CD40 signaling provided by Tfh cells is the primary external cue for selection and cell fate commitment in LZ B cells, whereas BCR was considered to be dispensable for directly altering the GC B cell fate because the BCR signaling pathway is uncoupled from Ag recognition by the high phosphatase activity in GC B cells (50). Instead, BCR confers the fitness of GC B cells during the selection process through Ag recognition, uptake, and processing for presentation to Tfh cells (51, 52); owing to its higher affinity, BCR increases the possibility for a cognate interaction with Tfh cells. Indeed, without BCR-mediated signals, CD40L- and BAFF-expressing feeder cells (CD40LB) along with IL-21 stimulate iGC B cells to upregulate c-Myc and IRF4, which were sufficient for differentiation to CD138+ ASCs in our experimental system. However, we found the synergistic induction of c-Myc in response to a pharmacological inhibitor of GSK3, even though GSK3 phosphorylation on S9 was observed in this culture system. This is a very interesting finding because it suggests the possible presence of additional regulatory signals for GSK3 activity in GC B cells besides those downstream of CD40 and supports recent reports that readdressed the role of BCR in the GC reaction (3, 53). According to these reports, GC B cells can execute BCR-mediated signals, which coordinate with the functions downstream of CD40 as intracellular cues for the positive selection of GC B cells, as indicated by the synergistic induction of c-Myc and IRF4, which is the same phenotypic feature observed from the results of GSK3 inhibition in iGC B cells on CD40LB plus IL-21. Therefore, our results support the essential role of BCR-mediated signaling in GC B cells, and it is very likely that GSK3 is one of the key downstream mediators of BCR engagement via the PI3K–AKT signaling pathway during the selection of GC B cells (3, 54).

Under the same experimental condition, the gene expression levels of various transcription factors involved in the GC B cell fate decision were altered toward the PC fate along with the acquisition of a DZ B cell phenotype, which might be a consequence of positively selected high affinity BCR-bearing LZ B cells. The fate-determined GC B cells should exit the repetitive recycle between the LZ and DZ, leading to further differentiation into effector B cells. Although little is known about the mechanistic details of the GC exit, a higher cellular concentration of IRF4 is believed to act as a “driver” of GC exit (55). In line with this notion, repressed cellular levels of IRF4 eventually abrogated all of the GSK3 inactivation-mediated features of selection in our experimental system. Therefore, enhanced IRF4 might account for the GSK3 inactivation-mediated features of selection. In this context, the GSK3–c-Myc regulatory axis may play an essential role in the synergistic induction of IRF4 because c-Myc can bind directly to the enhancer of Irf4, located in intron 1 (46), and the effect of GSK3 inactivation-mediated IRF4 upregulation was abrogated by c-Myc deletion at the transcription level. Thus, the GSK3–c-Myc–IRF4 regulatory axis in GC B cells might explain why synergistic c-Myc upregulation is a hallmark of positively selected GC B cells for PCs.

Foxo1 is required for compartmentalization of the LZ and DZ, and phosphorylation-mediated regulation of its cellular localization is tightly linked to the cyclic re-entry between the LZ and DZ (5, 6, 34). BCR–PI3K signaling might phosphorylate Foxo1 and relocalize it into the cytosol from the nucleus, ultimately driving the DZ to LZ transition (3). In the LZ, positively selected LZ B cells may presumably have nuclear-localized dephosphorylated Foxo1 and thus upregulate CXCR4 for the LZ to DZ transition. However, the mechanisms by which Foxo1 relocalizes to the nucleus are not well documented. In this study, we found that GSK3 inactivation in the presence of CD40 signals facilitated the generation of DZ-like CXCR4hiCD83lo cells, accompanied by increased cellular levels of Foxo1. Plausibly, GSK3-mediated enhanced Foxo1 is a mechanism by which Foxo1 relocalizes to the nucleus and thus initiates the LZ to DZ transition. Moreover, several LZ or DZ signature genes were altered in the direction from LZ B cells to DZ B cells by GSK3 inactivation. Therefore, GSK3 activation status might be one of the important cues regulating the LZ to DZ transition.

As the post-GC B cell fate commitment to PC or MBC precursors is mutually exclusive, it is plausible that GSK3 inactivation-mediated intracellular cues simultaneously function toward inducing PC generation and suppressing MBC generation. Indeed, we found that GSK3 inactivation in the presence of CD40 signals resulted in the suppression of Bach2 and Ccr6 expression, which are key mediators and signature genes of precursor MBCs, respectively (19, 23). Repression of Bach2 transcription might be critical for GSK3 inactivation-mediated PC generation because overexpression of Bach2 completely abrogated GSK3 inactivation-mediated PC differentiation, presumably because of the inhibition of Irf4 expression. This indicates that Bach2 downregulation is essential for the generation of PCs. Comparative analysis of the transcriptome showed an inverse correlation between c-Myc and Bach2, suggesting the suppressive role of c-Myc in Bach2 expression in the same manner as it acts on Pax5 (45). However, this may not be the case because transcriptional repression of Bach2 was also observed in the absence of c-Myc. These results indicate the presence of an additional regulatory mode of GSK3-mediated Bach2 repression independent of c-Myc, which is critical for the post-GC B cell fate commitment to PCs. One possibility is involvement of the ERK1/2–Elk1–Bach2 regulatory axis (56, 57) because GSK3 was suggested to suppress ERK1/2 activity (58).

Taken together, our study extends the role of GSK3 in the GC reaction beyond its function as metabolic checkpoint regulator but also as a critical regulator of the PC commitment of GC B cells. Inactive GSK3 acts as a signal for GC B cells to determine the cell fate of the progeny as PCs through the GSK3–c-Myc–IRF4 regulatory axis, at least in part. In addition, inactive GSK3 regulates the LZ to DZ transition via stabilizing Foxo1. Importantly, these features are all involved in the positive selection and fate commitment of GC B cells and are regulated by a single factor, GSK3 activity. In addition, it should be noted that most malignant B cells are derived from the GC reaction (59), and prolonged GSK3 inactivation is linked to apoptosis. This represents an additional layer of the regulatory mode of the GC reaction to eliminate metabolically overpowered GC B cells that are susceptible to becoming malignant B cells (28). Indeed, aberrant regulation of the GSK3–c-Myc–IRF4 regulatory axis might be responsible for the survival of lymphoma and multiple myeloma cells (46, 60). A recent genome-wide association study identified two single nucleotide polymorphisms in GSKA and GSKB with implications on leukocyte cell numbers (61), suggesting its roles in regulating the proliferation of B cells in humans as well.

Taken all together, our data strongly suggest that the spatial and temporal control of GSK3 activity is one of the key regulatory modes of the GC reaction. Therefore, future studies on the detailed molecular regulatory mode upstream and downstream of GSK3 in GC B cells will provide a comprehensive understanding of the GC reaction.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank James R. Woodgett (Mount Sinai Hospital and the Lunenfeld-Tanenbaum Research Institute) for providing the Gsk3αf/f;Gsk3βf/f mice, the members of the Y.-J.J. laboratory for assistance in using the irradiator, the H.-J.K. laboratory for use of their flow cytometer, Jeehee Youn (Hanyang University) for providing murine Bach2 cDNA, and Jee Ho Lee, Hyun-Young Jin, Yesol Kim, and Mihyun Park for discussion and critical reading of the manuscript.

Footnotes

  • The sequences presented in this article have been submitted to the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144361) under accession number GSE144361.

  • This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT, and Future Planning (NRF-2015R1C1A1A01052387 and NRF-2018R1D1A1B07050262 to S.G.K., NRF-2017M3A9G7073033 to S.S.C., NRF-2016R1A4A1010115 to P.-H.K., and NRF-2015M2B2A6028602 to Y.-J.J.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    AEC
    3-amino-9-ethylcarbazole
    ASC
    Ab-secreting cell
    DEG
    differently expressed gene
    DZ
    dark zone
    fc
    fold change
    FoB
    follicular B cell
    FPKM
    fragment per kb of transcript per million
    GC
    germinal center
    GSK3
    glycogen synthase kinase 3
    GSK3 KO
    Gsk3αf/f; Gsk3βf/f; Cg1Cre knockout
    iGC
    induced GC
    iPB
    induced plasmablast
    LZ
    light zone
    MBC
    memory B cell
    PBS-T
    PBS containing 0.05% Tween
    PC
    plasma cell
    PI
    propidium iodide
    ROS
    reactive oxygen species
    RV
    retrovirus
    shRNA
    short hairpin RNA
    Tfh
    T follicular helper.

  • Received August 3, 2020.
  • Accepted November 13, 2020.

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