KR102486284B1 - A method for regulating differentiation of germinal center B cells to plasma cells regulating glycogen synthase kinase 3 and composition therefor - Google Patents

Abstract

The present invention provides a method for regulating the differentiation of germinal center B cells into plasma cells by regulating glycogen synthase kinase 3, a composition for regulating the differentiation of germinal center B cells into plasma cells comprising glycogen synthase kinase 3 as an active ingredient, The present invention relates to a composition for promoting differentiation from germinal center B cells into plasma cells and a composition for increasing vaccine production comprising a glycogen synthase kinase 3 inhibitor as an active ingredient.

Description

A method for regulating differentiation of germinal center B cells to plasma cells by regulating glycogen synthase kinase 3 and a composition thereof

The present invention relates to methods and compositions for regulating differentiation of germinal center B cells into plasma cells by modulating glycogen synthase kinase 3.

The development of antibody secreting plasma cells (PCs) and memory B cells (MBCs) are key events in humoral immunity. Germinal center (GC)-experienced PCs secrete high-affinity class-switching antibodies that protect the host from invading pathogens, and MBC rapidly differentiates into PCs upon a second challenge with the same antigen, responsible for effective immunity.

Proper regulation of the generation of these effector B cells is essential for immune homeostasis. Therefore, elucidating the mechanisms underlying the GC response by which GC B cells are selected to determine their fate and directing their differentiation during the GC response is fundamental to 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 GCs, accompanied by somatic hypermutation to generate new versions of the B-cell receptor (BCR) with varying affinities. generate

CXCR4hiCD83lo (or CD86lo) DZ B cells with high BCR levels migrate to the LZ via the BCR downstream signaling pathway, where they compete with each other for binding to antigens displayed on the follicular dendritic cell surface and recruit them to a limited number of T follicle helpers (Tfh). present to the cell.

The LZ to DZ transition is in response to upregulated CXCR4 expression when CXCR4loCD83hi (or CD86hi) LZ B cells successfully associate with Tfh cells due to their high-affinity BCR receiving strong CD40 and IL-21 signals from Tfh cells. Occurs. 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 the function of the transcription factors c-Myc and Foxo1. Regulated recurrent cycling of positively selected GC B cells between the DZ and LZ is coupled with progeny generation of effector B cells, which is one of the key events of the GC response. This high-affinity-based selection model presupposes the existence of specific mechanisms by which GC B cells discriminate between weak and strong interactions between Tfh cells. However, the detailed mechanism has not yet been elucidated.

[Prior Patent Literature]

Korean Patent Publication No. 10-2018-0066263

The present invention was made in response to the above needs, and an object of the present invention is to investigate the differentiation mechanism from germinal center B cells into plasma cells.

In order to achieve the above object, the present invention provides a method for regulating the differentiation of germinal center B cells into plasma cells by regulating glycogen synthase kinase 3.

In one embodiment of the present invention, the method preferably promotes differentiation of germinal center B cells into plasma cells when glycogen synthase kinase 3 is inhibited, but is not limited thereto.

In another embodiment of the present invention, the glycogen synthase kinase 3 preferably consists of the amino acid sequence of SEQ ID NO: 1 (Glycogen synthase kinase-3 alpha) or SEQ ID NO: 2 (Glycogen synthase kinase-3 beta), but is not limited thereto. No.

In addition, the present invention provides a composition for regulating the differentiation of germinal center B cells into plasma cells, comprising glycogen synthase kinase 3 as an active ingredient.

In another embodiment of the present invention, the glycogen synthase kinase 3 preferably consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, but is not limited thereto.

In addition, the present invention provides a composition for promoting differentiation from germinal center B cells into plasma cells, comprising a glycogen synthase kinase 3 inhibitor as an active ingredient.

In the present invention, the glycogen synthase kinase 3 inhibitor is preferably a compound, antibody, or nucleic acid material, and the glycogen synthase kinase 3 inhibitor is more preferably CHIR99021, a compound represented by Formula 1 below, but is not limited thereto.

[Formula 1]

In addition, the present invention provides a composition for increasing vaccine production comprising a glycogen synthase kinase 3 inhibitor as an active ingredient.

In the present invention, the glycogen synthase kinase 3 inhibitor is preferably a compound, antibody, or nucleic acid material, and the glycogen synthase kinase 3 inhibitor is more preferably CHIR99021, a compound represented by Formula 1 below, but is not limited thereto.

[Formula 1]

The present invention will be described below.

B cells of the germinal center (hereinafter referred to as 'GC') are either plasma cells (hereinafter referred to as 'PC') or programmed to form memory B cells.

However, the exact mechanism underlying this process to complete the GC reaction is not clear.

We hypothesized that glycogen synthase kinase 3 (hereinafter referred to as 'GSK3') mediates c-Myc-dependent positive selection of GC B cells and post-GC processes, and to test this hypothesis, GC B cells were differentiated into PCs and the effect of GSK3 on LZ to DZ conversion was investigated. This is a result of positively selected GC B cells and essential events for PC formation. Our results can highlight the key role of GSK3 in the positive selection of GC B cells committed to a PC fate.

In the present invention, we show that genetic ablation and pharmacological inhibition of GSK3 in GC B cells promote cell fate decisions towards PC formation with acquisition of dark (DZ) B cell properties.

Mechanistically, upon stimulation with CD40L and IL-21, GSK3 inactivation synergistically induces the transcription factors Foxo1 and c-Myc, increasing levels of key transcription factors required for PC differentiation, including IRF4. These GSK3-mediated alterations of transcription factors in turn promoted DZ conversion and consequent PC fate commitment. Our results thus reveal an upstream master regulator that interprets the extrinsic signals of GC B cells to form PCs mediated by key transcription factors.

Hereinafter, the present invention will be described in detail.

CD40L and IL-21 mediated c-Myc induction is dependent on GSK3 in induced GC B cells

Since GSK3 directly phosphorylates c-Myc protein to promote its degradation, we hypothesized that GSK3 regulates cellular levels of c-Myc in GC B cells by acting downstream of CD40L and IL-21 signaling. To test this possibility, we in vitro induced GC (iGC) B cells by culturing for 4 days with naïve follicular B cells (FoB) on CD40L and BAFF expressing feeder cells (CD40LB) supplemented with IL-4. Created.

After stimulation of iGC B cells with CD40L and IL-21, GSK3 was shown to be phosphorylated at Ser9, indicating GSK3 inactivation upon CD40L and IL-21 stimulation (Fig. 1A). Importantly, c-Myc expression levels in iGC B cells varied with the amount of CD40L and IL-21 stimulation (Fig. 1B).

Therefore, it is very likely that GSK3 kinase activity is involved in CD40L- and IL-21-mediated determination of cellular levels of c-Myc in GC B cells.

To directly test this possibility, we retrovirally transduced iGC B cells expressing wild-type (GSK3 WT), constitutively active (GSK3 9A), or null iGC B cells generated from Gsk3 f/f. Gsk3f/f, which expresses the kinase-dead (GSK3 K85A) form of GSK3; Cg1Cre knockout (hereafter referred to as GSK3 KO) FoB cells were generated (Figure 1C).

GSK3 depletion resulted in synergistic induction of c-Myc compared to Gsk3 f/f iGC B cells (hereafter referred to as CTL) upon 6 h stimulation in CD40LB with supplemented IL-21 (Fig. 1D).

In addition, reconstitution with GSK3 WT reduced the c-Myc expression level to that of RV-Mock-infected GSK3-null iGC B cells (Fig. 1D), indicating a regulatory role of GSK3 in determining cellular levels of c-Myc. suggests

in GC B cells. More importantly, introduction of GSK3 S9A resulted in c-Myc degradation in GSK3-null iGC B cells, whereas GSK3 K85A-expressing B cells did not induce c-Myc degradation under the same conditions (Fig. 1D).

Therefore, we conclude that regulating GSK3 kinase activity in response to CD40L and IL-21 stimulation is one of the essential regulatory modes determining the cellular level of c-Myc in GC B cells, suggesting that GSK3 plays a particularly important role in GC responses. post-GC B cell fate commitment and may play a regulatory role in the differentiation of positively selected GC B cells.

GSK3 deficiency promotes PC differentiation in vitro

Given that c-Myc expression is closely related to positive selection and PC generation in LZ GC B cells, we investigated the detailed role of GSK3 in GC B cells during post-GC B cell fate decisions and PC differentiation.

To better understand the role of GSK3 in post-GC B cells at the cellular and molecular level, we adopted the iGC B cell culture system. Effector B cells in this system can be generated by continuous iGC B cell culture and post iGC B cell culture.

iGC B cells were generated by 4 days of culture, followed by further culture for 4 days with IL-21 supplementation to induce the generation of CD138+ antibody-secreting cells (ASCs) (FIG. 2A).

Thus, we can functionally analyze GC B cell generation in effector B cell differentiation events, following post GC B cell fate determination and PC differentiation after GC B cell formation, which is not feasible using an in vivo system. .

We first generated iGC B cells from naive follicular B cells of CTL or GSK3 KO mice. iGC B cells from both CTL and GSK3 KO mice showed similar expression levels of FAS and GL-7 and generated similar numbers of iGC B cells at day 4 (Fig. 2B). Thus, GSK3 depletion had minimal effect on GC B cell differentiation itself.

To further evaluate the role of GSK3 in the post-GSK response, equal numbers of iGC B cells from CTL or GSK3 KO mice were treated post-iGC B cell culture.

Surprisingly, the frequency of CD138+ ASCs in induced plasmablasts (iPBs) from GSK3 KO mice was much higher than that in CTL iPBs after 4-day culture of iGC B cells (Figure 2C), and they were functional in secreted antibodies (Figure 2D). .

Since CD138+ ASC production is positively associated with proliferation, enhanced production of CD138+ ASCs may benefit proliferation of GSK3 KO iGC B cells. However, we could not detect a significant difference between CTL and GSK3KO iGC B cells in proliferation at day 3 (Figure 2E), suggesting that the enhanced generation of CD138+ ASCs is not a result of accelerated cell division. Instead, GSK3 deficiency could be an informative clue for iGC B cells to initiate PC differentiation. This is because IRF4 and Pax5 (key regulators of PC differentiation) were rapidly induced and downregulated for PC differentiation in iGC B cells derived from GSK3 KO mice, respectively, after 2 days of iGC B culture after PC differentiation in iGC B cells (Fig. 2F). , G).

In addition, Blimp-1 expression was highly upregulated from GSK3 KO iGC B cells on day 2 after iGC B cell culture (Fig. 2H), and CD138+ plasmablasts were rapidly generated (Fig. 2I). In summary, it is very likely that GSK3 deficiency functions as an internal cue to initiate PC differentiation, resulting in rapid ASC generation in vitro.

GSK3 deficiency in GC B cells lowers antibody production in vivo

To confirm that rapid PC differentiation occurs in the presence of GSK3 deficiency, we evaluated humoral immunity against antigens in GSK3 KO mice.

However, GSK3 KO mice were defective in low- and high-affinity NP-specific IgG1 antibody production after immunization with NP-OVA/Alum, whereas antigen-specific IgM antibody production was comparable to CTL mice.

This suggests that GSK3 expression in activated B cells is essential for ASC generation. In particular, these results may not be related to defects in GC formation or maintenance. This is because IgG1 + GC B cells were still generated in GSK3 KO mice after immunization with OVA/LPS/Alum, although they had ~50% lower GC B cell numbers than CTL mice.

Of note, GSK3 expression was effectively disrupted in IgG1+ and IgG1- FAS + CD38lo GC B cells. Instead, the generation of CD138+ ASCs may be severely affected by GSK3 deficiency, as few IgG1+ CD138+ ASCs were detected in GSK3 KO mice.

These results contrast with findings from the in vitro system described above and indicate that the presence of GSK3 in GC B cells is essential for maintaining the GC B cell pool and generating ASCs in vivo.

GSK3 deficiency and GSK3 inhibition are functionally distinct in GC B cells

We speculated that inhibition of GSK3 activity represents a physiological condition beyond GSK3 ablation, as GSK3 deficiency never occurs during the GC response. Alternatively, GSK3 activity can be regulated by signaling pathways triggered through external signals and by internal signaling regulators such as PKA and AKT in GC B cells.

Indeed, while the absolute number of iPBs was significantly reduced in GSK3 KO mice, the number of iPBs was similar to that of CTL mice when GSK3 kinase activity was inhibited with the specific pharmacological inhibitor CHIR99021 (Fig. 3A).

This finding provides insight reconciling paradoxical in vivo and in vitro results regarding the role of GSK3 in ASC generation, as impaired humoral immunity in GSK3 KO mice is most likely due to cell death in glucose and oxygen-limited GCs. do.

To evaluate this possibility, CTL or GSK3 KO iGC B cells were exposed to low glucose and hypoxic conditions during culture after iGC B. Indeed, iGC B cells deficient in GSK3 were highly susceptible to apoptosis under low glucose and hypoxic conditions (Fig. 3B, C).

However, CHIR99021-treated iGC B cells did not induce apoptosis under the same conditions (Fig. 3B, C).

This result is surprising because we hypothesized that inhibition of GSK3 activity should induce apoptosis similar to the GSK3-deficient condition in GC B cells by increasing metabolic demand and ROS production.

GSK3 inhibition via CHIR99021 also enlarged cell size (Fig. 3D) and increased glycolytic activity (Fig. 3E), which in turn increased ROS production (Fig. 3F), indicating increased metabolic demands.

These unexpected results strongly suggest that GSK3 deficiency and GSK3 inhibition in GC B cells may have different biological mechanisms, suggesting additional roles for GSK3 besides its main function as a metabolic checkpoint regulator.

GSK3 inhibition unlocks the PC differentiation program

We next tested whether pharmacological inhibition of GSK3 acts as a guidance signal for PC generation, as observed in GSK3 KO iGC B cells.

GSK3 inhibition during post-iGC B cell culture in the CD40LB system promoted the generation of functional CD138 + ASCs similar to GSK3 KO B cells (Fig. 4A, B).

Next, we investigated the kinetics of CD138+ cell emergence during 4 days of culture after iGC. There was no apparent difference in CD138+ frequencies during the first 2 days of culture. However, accelerated generation of CD138+ PCs apparently occurred later (Figure 4C).

Enhanced generation of CD138+ ASCs by CHIR99021 treatment may result from instructions to iGC B cells received during the first 2 days for their subsequent function to generate PCs or amplify CD138+ ASCs. To address this possibility, we inhibited the GSK3 activity of iGC B cells only for the first 2 days and then co-cultured them with control iGC B cells for 2 more days in the absence of GSK3 inhibitor (Fig. 4D). CHIR99021-pretreated cells for 2 days tended to form more CD138+ ASCs compared to cells generated by control cells in the absence of CHIR99021 (FIG. 4E). These data strongly suggest that inactive GSK3 in GC B cells functions as a guiding cue to generate PCs prior to ontogeny of CD138+ PCs, and we attributed this to an altered transcriptional network for PCs.

To test this hypothesis and gain more mechanistic insights into GSK3-mediated alterations of cell fate, we investigated the gene expression of GSK3-inactivated post-iGC B cells cultured for 36 h with GSK3 inhibitors in a post-iGC B culture system. profiling was performed. We then compared transcriptome changes compared to DMSO-treated control cells.

The gene expression profile of GSK3-suppressed post-iGC B cells was clearly distinct from that of the DMSO control group.

We identified 407 differentially expressed genes [DEGs; Q value <0.05, fold change (|fc|) ≥ 2] were identified (Fig. 4F).

These 407 GSK3-responsive genes were involved in several biological processes, including B-cell activation, B-cell differentiation, antigen presentation to major histocompatibility class II (MHCII), and apoptosis.

Importantly, expression of transcriptional regulators of effector B cell differentiation was altered for PC input with reduced Bach2, Bcl6 and Pax5 and induced Irf4, Xbp1 and Prdm1 expression (Fig. 4G).

These results strongly suggest that inactive GSK3 functions as an internal cue to alter the transcriptional program for rapidly occurring PC differentiation before being directed to PC progenitors by CD138 expression.

GSK3-induced PC differentiation depends on IRF4 and Bach2 expression

Among the plasma cell regulatory genes altered by GSK3 inhibition (Figure 4G), increased Irf4 expression is functionally responsible for inactive GSK3-mediated increased CD138+ ASC generation, as cellular levels of IRF4 are a key mediator of cell fate decisions towards post-GSK B PC differentiation. was considered responsible.

To test this hypothesis, we reduced cellular levels of IRF4 through retroviral introduction of Irf4-targeting short hairpin RNA (shRNA) in GSK3-suppressed iGC B cells and then investigated its effect on CD138+ ASC generation.

Indeed, GSK3-mediated increased CD138 + ASC production was effectively eliminated.

Bach2 is another key mediator of post-GC B cell fate commitment, and its expression guides post-GC B cells to MBC while preventing PC differentiation.

As shown in Figure 4G, Bach2 expression was also downregulated in iGC B cells after GSK3 inhibition, thus occurring slightly earlier than Irf4 induction upon GSK3 inhibition.

suggesting that reduced Bach2 expression may also be responsible for enhanced CD138+ ASC generation.

To validate this possibility, introduction of a Bach2-expressing retroviral vector into CHIR99021-treated iGC B cells almost completely prevented CD138 + ASC generation. These results indicate that GSK3 inactivation stimulates a well-established transcriptional program of PC fate commitment.

c-Myc is involved in GSK3-mediated synergistic IRF4 induction

As c-Myc, a substrate of GSK3, is a key regulator of GC responses, it can mediate positive selection of GC B cells.

Since c-Myc regulates one-third of all genes involved in various biological processes and has been proposed to directly control Pax5 and Irf4 expression in B cells, we investigated c-Myc as a transcriptional reprogramming initiated by inactive GSK3. was considered an important medium for

To test 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 lasted for 24 h (Fig. 5A).

Next, we compared gene expression profiles between c-Myc+ GC B cells and GSK3-suppressed iGC B cells, as we inferred that these gene expression profiles would be shared if c-Myc was responsible for GSK3-mediated altered gene expression.

We reanalyzed a published data set of c-Myc+ and c-Myc-GC B cells sorted from immunized mice and found 425 DEGs (Q value <0.05, |fc| ≥ 2).

Of the 407 GSK3-responsive genes identified in previous experiments, 8.8% of the DEGs (14 out of 134 up-regulated genes and 22 out of 274 down-regulated genes) overlapped with c-Myc-responsive genes (Fig. 5B).

However, 86% (31/36) of overlapping genes were positively correlated with c-Myc expression (Fig. 5C), suggesting that c-Myc may partially contribute to GSK3-mediated alterations in gene expression profiles. .

Importantly, both Irf4 and Bach2 were included in the positively correlated genes (Fig. 5C). These results suggest that c-Myc is not the sole responsible factor for GSK3-mediated altered gene expression, but may directly or indirectly affect GSK3-mediated GC B cell fate through Irf4 induction and Bach2 inhibition.

To test whether c-Myc indeed regulates Irf4 and Bach2 in post-GC B cells, we generated cmyc f/f iGC B cells, then retroviral introduction of Cre recombinase to induce c-Myc in post-iGC B cells. inhibited Myc function.

In virus-infected post-iGC B cells, c-Myc function was efficiently eliminated as indicated by smaller cell sizes than controls (Fig. 5D). This resulted in abrogation of Irf4 induction (Fig. 5E) and thus reduced cellular levels of IRF4 in GSK3-suppressed post-iGC B cells (Fig. 5F). However, Bach2 inhibition was only partially dependent on c-Myc (Fig. 5G).

These results strongly suggest that the cellular IRF4 expression level that allows GC B cells to commit to PC progenitors is determined through the GSK3-c-Myc regulatory axis.

Inactive GSK3 promotes LZ to DZ conversion in GC B cells

Cyclic journey between LZ and DZ is a key feature of positively selected GC B cells and is related to cell fate commitment after GC B.

It has been shown that selected LZ B cells form PC progenitors, further differentiate in the DZ, and exit the GC from there. Thus, transcriptional reprogramming to PC fate commitment by GSK3 inactivation involves LZ to DZ conversion and may also be regulated by GSK3.

First, we observed reduced expression of Cd83 and Cd86 in GSK3-suppressed post-iGC B cells (Fig. 4F).

Cd83 and Cd86 are characteristic genes of LZ B cells, and loss of expression by CXCR4 upregulation is required for DZ metastasis. These data prompted us to investigate whether CXCR4 is upregulated by GSK3 inhibition during the post-iGC B cell culture period, as it is important for localization in the DZ. Indeed, GSK3 inactivation stimulated Cxcr4 induction in post-iGC B cell cultures (Figure 6A) and increased the generation of CXCR4hi CD83lo DZ-like B cells (Figure 6B).

As Foxo1 is one of the key regulators of the LZ to DZ transition, we reasoned that GSK3 could control the LZ to DZ transition by regulating Foxo1 expression in post-iGC B cells. Indeed, inactive GSK3-mediated DZ-like B cell generation was accompanied by synergistic induction of Foxo1 (Fig. 6C). However, Foxo1 gene expression was not altered by GSK3 inactivation (Fig. 6D), indicating that GSK3 can control Foxo1 stability and thus the LZ to DZ transition in GC B cells.

Next, we reanalyzed the gene expression profiles of LZ and DZ B cells and found that 315 genes were differentially expressed (Q value <0.05, |fc| ≥ 2). Of these, 48 genes were common DEGs with GSK3-responsive genes, and the gene expression profile of DZ B cells clustered closely with that of iGC B cells after CHIR99021 treatment (Fig. 6E, F). In addition, 25 genes among 48 common DEGs (Table S4) were LZ or DZ B cell signature genes. Altogether, these results strongly suggest that LZ to DZ conversion, another functional hallmark of positively selected post-GC B cells, is promoted by GSK3 inactivation.

As can be seen through the present invention, it can be seen that differentiation into plasma cells is promoted by regulating GSK3 protein activity, and this can be expected that GSK3 will play a significant role in enhancing the differentiation and survival of plasma cells.

1 is a diagram showing that CD40L and IL-21-mediated c-Myc expression is dependent on GSK3 kinase activity.
(A) Immunoblot analysis (left) and bar graph (right) of phosphorylated GSK3 in iGC B cells upon stimulation with agonistic CD40 antibody (25ug/ml) and IL-21 (50ng/ml) (n = 3).
(B) Immunoblot analysis of c-Myc in iGC B cells stimulated with functional CD40 antibody and IL-21 at different concentrations as described.
(C) Scheme of experiments to reconstitute GSK3 into GSK3-null iGC B cells.
(D) c-Myc, GSK3 of reconstituted Thy1.1+ iGC B cells upon stimulation for 6 h in CD40LB with IL-21 (50 ng/ml)? and GSK3? Immunoblot analysis for phosphorylation.
Wild-type or mutant GSK3-expressing retrovirus (RV)-infected Thy1.1 + iGC B cells were isolated and stimulated. Data from immunoblot analysis shown are representative of three independent experiments.
Figure 2 shows that the absence of GSK3 in iGC B cells promotes CD138 + ASC generation.
(A) Experimental scheme of in vitro iGC B cell and post iGC B cell culture.
(B) Flow cytometry analysis of FAS+ GL7+ iGC B cells (left) and absolute numbers of FAS+ GL7+ iGC B cells (right) in the in vitro culture system (n = 7).
(C) Frequency of CD138+ ASCs at day 4 post iGC B cell culture (n = 4).
(D) iPB-secreted IgG1 levels determined by ELISA (n = 3).
(E) Proliferation of CTV-labeled iGC B cells at day 3 post iGC B cell culture. Representative data from three independent experiments are shown.
Intracellular staining and mean fluorescence intensity (MFI) of IRF4 (F), Pax5 (G) and Blimp-1 (H) on day 2 post iGC B cell culture (n = 4).
(I) Flow cytometry analysis of CD19+CD138+ cells (left) and percentage of CD19+CD138+ cells (right) at day 2 post iGC B cell culture (n = 5).
Figure 3 shows that GSK3 inactivation increases the metabolic demand of iGC B cells.
(A) Absolute numbers of CTL, CHIR99021 (3 μM) treated CTL or GSK3KO CD19+ iPBs at day 4 post iGC B cell culture (n = 4).
Flow cytometric analysis (B) and frequency (C) of Annexin V + PI + apoptotic post-iGC B cells at day 1 under low-glucose and hypoxic conditions (n = 5).
(D) Cell size of post-iGC B cells at day 1 of post-iGC B cell culture. Representative data from three independent experiments are shown.
(E) Basal and maximal glycolytic activity of post iGC B cells at day 1.5 (n = 3).
(F) Levels of mitochondrial ROS in post-iGC B cells at day 1 (n = 3).
Figure 4 shows that GSK3 inactivation alters the transcriptional network for ASC generation.
(A) Flow cytometric analysis (left) and frequency (right) of CD19+CD138+ cells in iPBs (n = 4) at day 4 post iGC B cell culture.
(B) iPB-secreted IgG1 levels determined by ELISA (n = 6).
(C) Frequency of CD138+ cells on day 2 or day 4 post iGC B cell culture (n = 3).
(D) Experimental scheme of co-culture with DMSO or CHIR99021 (3 μM) pre-exposed post iGC B cells.
(E) Flow cytometric analysis of CD138+ cells on day 2 or day 4 of the co-culture experiment. Representative data from three independent experiments are shown.
(F) Volcano plot of gene expression profiles from cells after DMSO or CHIR99021-treated iGC B at day 1.5 post iGC B cell culture.
(G) Clustered heat map of selected effector B cell differentiation regulatory genes.
Figure 5 is a diagram showing that 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. Bar graphs represent relative c-Myc expression levels normalized to β-actin levels (n = 3).
(B) Common DEGs of GSK3 responsive gene and c-Myc responsive gene analyzed by two different criteria. Numbers indicate the amount of genes.
(C) Number and list of positively correlated overlapping genes among common DEGs.
(DG) iGC B cells generated from cmyc f/f FoB cells were infected with control or Cre recombinase-encoding retroviruses. Virus-infected iGC B cells were used for post-iGC B cell culture.
(D) Cell size of cmyc f/f post-iGC B cells at day 1 of post-iGC B culture.
(E) RT-PCR analysis of Irf4 in cmyc f/f post iGC B cells at day 1.5 of post iGC B culture (n = 3).
(F) Cellular levels of IRF4 in cmyc f/f post-iGC B cells at day 1.5 of post-iGC B culture in the presence of CHIR99021 (n = 5).
(G) RT-PCR analysis of Bach2 in cmyc f/f post-iGC B cells at day 1 of post-iGC B culture (n = 3).
6 is a diagram showing that the conversion of LZ to DZ 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 dark zone (DZ; CXCR4hiCD83lo) or light zone (LZ; CXCR4loCD83hi)-like B cells on day 2 or 4 post iGC B culture (n = 3).
(C) Immunoblot analysis of Foxo1 expression in iGC B and post-iGC B cells at indicated time points. Bar graph represents Foxo1 expression levels normalized to β-actin levels (n = 3).
(D) RT-PCR analysis of Foxo1 on day 2 of post-iGC B culture (n = 4).
(E) Clustered heatmap of 48 common DEGs of LZ/DZ-DEG and GSK3 responsive genes. Red indicates DZ or LZ signature genes.
(F) Principal component analysis plot of the 48 common DEGs.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are described with the intention of illustrating the present invention, and the scope of the present invention is not construed as being limited by the following examples.

The Gsk3 f/f and Gsk3 f/f mice used in the present invention were prepared by Dr. J. Woodgett (Mount Sinai Hospital & Lunenfeld-Tanenbaum Research Institute) and crossed with Cγ1Cre mice. cmyc f/f and Cγ1Cre mice were provided by Changchun Xiao (The Scripps Research Institute). Mice aged 8–10 weeks were used for all experiments. All mice were managed by the Animal Research Center of Kangwon National University and used according to the guidelines of the Animal Care and Use Committee of Kangwon National University.

Example 1: Follicular B cell isolation

Splenocytes were collected from various experimental mice and red blood cells were disrupted with ACK lysis buffer (Gibco). Red blood cell disrupted splenocytes were stained with anti-CD5 (PE), anti-CD9 (PE), anti-CD43 (PE), anti-CD93 (PE) and anti-Ter119 (PE) antibodies for 20 minutes at 4°C. It became. Stained splenocytes were washed with MACS buffer [0.5% bovine serum albumin (BSA), 2 mM ethylenediaminetetraacetic acid in DPBS] and incubated with anti-PE beads (Miltenyi Biotec) for 20 min at 4 °C. Follicular B cells were negatively isolated using LS columns (Miltenyi Biotec). Isolated CD21int CD23 + follicular B cells (>97%) were used for iGC B culture in vitro.

Example 2: In vitro iGC B cell and post iGC B cell culture

CD40LB cells were maintained in Dulbecco's modified Eagle's medium Glutamax (Gibco) with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco). CD40LB cells were irradiated at 100 Gy using the Gammacell 40 Exactor (Best Theratronics) and then used as feeder cells for in vitro iGC B cell and post-iGC B cell cultures.

To generate iGC B cells, isolated follicular B cells (2 x 10 ⁵ ) were cultured from irradiated CD40LB cells with complete RPMI1640-Glutamax medium (Gibco) supplemented with murine IL-4 (1 ng/ml, Peprotech). were incubated for 4 days.

iGC B cells were harvested and purified by depletion of CD40LB cells using biotin anti-H-2Kd (Biolegend) and anti-biotin beads (Miltenyi Biotec) with an LS column. Purified iGC B cells were cultured without CD40LB for 12 h in complete RPMI medium at 37 °C for resting.

For post-iGC B cell culture, resting iGC B cells (2 × 10 ⁵ cells per 60 mm culture dish or 2 × 10 ⁴ cells per well of a 12-well plate) were irradiated with irradiated CD40LB cells for 1, 1.5, 2 or 4 days. were cultured in complete RPMI medium containing murine IL-21 (10 ng/ml, Peprotech) and used for further analysis after CD40LB depletion.

For in vitro proliferation and co-culture experiments, iGC B cells or post-iGC B cells were labeled with carboxyfluorescein succinimidyl ester (CFSE; 5 μM, Invitrogen) or CellTrace Violet (CTV; 5 μM, Invitrogen) according to manufacturer instructions and post-iGC B cells culture conditions were used.

For quantification of secreted IgG1 antibodies from iPBs, post-iGC B cells were harvested on day 4 of post-iGC B cell culture and further cultured for 24 h in complete RPMI1640-Glutamax medium without feeder cells (1 x 10 per well). ⁵ cells/96-well plate), culture media were subjected to enzyme-linked immunosorbent assay (ELISA).

For immunoblot analysis, rested iGC B cells (1 x 10 ⁶ cells per well of a 48-well plate) were incubated at various concentrations for 1 hour (phosphorylation of GSK3 or agonistic CD40 antibody (BioXcell) and murine IL-21 for 4 hours). (Peprotech).Activated iGCB cells were infected with Thy1.1-expressing retroviruses encoding wild-type or mutant forms of GSK3, and then Thy1.1 + cells were infected with anti-Thy1.1(PE) (Biolegend) and anti-Thy1.1 (Biolegend). -PE microbeads (Miltenyi Biotec) were used to purify by magnetically activated cell sorting.Rested Thy1.1+ cells (1 x 10 ⁶ cells per well of a 48-well plate) were stimulated on CD40LB feeder cells for 6 hours; Immunoblot analysis was performed.

Example 3: Immunization

Gsk3 f/f; Gsk3 f/f; Cg1Cre and control Gsk3 f/f; 50 μl prepared by mixing 25 μl of Immject Alum (Thermo Fisher Scientific) with 25 μl of 1x PBS containing 50 μg of ovalbumin (Sigma-Aldrich) and 5 μg of LPS (Sigma-Aldrich) for Gsk3 f/f) mice. of ovalbumin/alum/LPS was injected subcutaneously.

Mice were sacrificed 10 days after immunization for evaluation of GC responses by flow cytometry. To perform ELISA of NP-specific antibodies, mice were immunized subcutaneously with NP-OVA/Alum prepared by mixing 25 μl of Immject Alum (Thermo Fisher Scientific) and 25 μl of 1x PBS containing 50 μg of NP-Ovalbumin (Biosearch). Serum was collected weekly up to 21 days after immunization.

Example 4: Flow Cytometry

Intracellular expression of IRF4, Blimp1 and Pax5 was measured by flow cytometry.

Post-iGC B cells were fixed for 1 hour at 4 °C with fixation/permeabilization buffer (eBioscience). The staining antibody was diluted in Permeabilization Buffer (eBioscience) and intracellular staining was performed at 37 °C for 30 min. The following antibodies were also used: anti-IRF4 (PE), anti-Blimp1 (PE) and anti-Pax5 (PE).

For surface marker staining, the following antibodies were used for flow cytometric analysis: anti-CD19 (PE/cy7), anti-CD138 (APC), anti-CD83 (PE), anti-CXCR4 (Biotin), streptavidin (APC), anti -FAS (PE), anti-FAS (BV510), anti-CD38 (PE/cy7) and anti-B220 (APC/cy7).

For mitochondrial ROS measurement, post iGC B cells on day 1 of culture were first stained for surface markers and then with mitoSOX (5 μM, Invitrogen) at 37 °C in a 5% CO2 incubator.

Example 5: ELISA

ELISA plates were coated with NP1-4-BSA or NP20-BSA (10 ug/ml in PBS, Biosearch Technologies) to quantify total or high affinity NP-specific antibodies, respectively.

Blocking was performed for 2 h at 37 °C in PBS with 0.5% BSA. Serum collected from mice was diluted and loaded onto plates for incubation at 37 °C for 2 h. Plates were washed 3 times with PBS containing 0.05% Tween (PBS-T). Biotin-conjugated primary antibodies 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-horseradish peroxidase (HRP) (Biolegend) was added and the plate was 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-tetra methyl benzidine dihydrochloride hydrate (TMB) solution (BD Bioscience). Reactions were stopped with 0.5 M HCl solution and then quantified on a Synergy H1 microplate reader (BioTek). For quantification of secreted IgG1 from iPBs, ELISA plates were coated with purified goat anti-mouse Ig kappa and lambda light chain antibodies (2.5 ug/ml in PBS, Southernbiotech) and biotin-conjugated anti-mouse IgG1 antibody (Southernbiotech) was used for detection.

Example 6: ELISPOT (Enzyme-linked immunospot) analysis

ELISPOT assay was performed for detection of IgG1 + ASC. Multi-screen membrane filtration 96-well plates (EMD Millipore, cat # MAIPSWU10) were coated with goat anti-mouse IgG1 (2.4 μg/ml, Southernbiotech) overnight at 4 °C.

Blocking was performed with 1% gelatin in PBS for 2 h at 37 °C. After washing three times with PBS-T, iPB was added to each well and incubated at 37 °C for 4 hours. Plates were washed three times with PBS-T and incubated with biotinylated anti-mouse IgG1 (1:1000, Southernbiotech) for 1 hour followed by streptavidin-HRP (1:1000, Biolegend) for 2 hours. After three washing steps, color developed in 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 dimethylformide. Spots were manually counted under a stereomicroscope.

Example 7: Cell death assay

Purified iGC B cells were cultured under hypoxia (4% O2, 37 °C, 5% CO2) and low glucose (1 mM) for one day post iGC B cell culture conditions. Cells were harvested and cell death was quantified using the Annexin V/PI Cell Kill Kit (eBioscience) according to the manufacturer's instructions. Briefly, cells were incubated with Annexin V (1 μl in 20 μl buffer, 1:20) at 4 °C for 20 min, followed by propidium iodide (PI; 1 μl in 40 μl buffer, 1:40) 5 times at room temperature. incubated in the dark.

Example 8: Immunoblot analysis

Sorted GC B cells, purified iGC B cells, and post-iGC B cells were washed with PBS and incubated on ice in 1x RIPA buffer (Cell Signaling Technology) supplemented with 1x phosphatase inhibitor cocktail (GenDEPOT) and 1x protease inhibitor cocktail (GenDEPOT). It was disrupted by incubating for 30 minutes on top.

The cell lysate was separated on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and transferred to a polyvinylidenefluoride membrane (GE Healthcare).

Blots were performed at 4 °C with primary antibodies diluted in 2.5% (wt/vol) skim milk (Cell Signaling Technologies) and 1x TBS-T buffer (10 mM Tris-HCl, pH 8.0 and 150 mM NaCl, 0.1% Tween-20). Incubated overnight.

All membranes were scanned using an ImageQuant LAS 500 (GE Healthcare) and images were analyzed with ImageJ (NIH Software). Immunoblotting was performed with anti-c-Myc (Cell Signaling Technologies), anti-FOXO1 (Cell Signaling Technologies), anti-pGSK3β (Cell Signaling Technologies), anti-GSK3β (Santacruz) and β-actin-HRP (Santa Cruz Biotechnology) It became. Anti-rabbit IgG-HRP (Cell Signaling Technologies) was used as the secondary antibody.

Example 9: Real-Time Polymerase Chain Reaction (PCR)

Total RNA was extracted using Trizol Reagent (Invitrogen) and cDNA synthesis was performed using the iScript gDNA clear cDNA synthesis kit (Bio-Rad) 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). Primer assignments are listed in Table 1.

Example 10: Real-time Metabolic Analysis

Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in unbuffered XP RPMI medium, pH 7.4 (Agilent Technologies) with 1 mM HEPES and without phenol red, sodium bicarbonate, glucose, L-glutamine and sodium pyruvate. Post iGC B cells (1.5 x 10 ⁵ cells/well) were measured according to the manufacturer's instructions.

All metabolic analyzes were performed with an XFp Extracellular Flux Analyzer (Agilent Technologies). Data were analyzed with Wave (Agilent software).

Example 11: Plasmid and retroviral gene introduction

Cre recombinase (MSCV-Cre, Addgene plasmid # 320824) and mouse Bach2 cDNA provided by Jihee Yoon (Hanyang University) were inserted into a Thy-1.1 expressing retroviral vector (Addgene plasmid # 17442).

For shRNA expression, annealed shRNA primers encoding control or Irf4-specific shRNAs were amplified with linker primers containing restriction enzyme sites and inserted into the pLMP-GFP vector.

S-Eco packing cells were transfected with the JetPrime transfection kit (Polyplus) and retroviral supernatants were collected 48 h after transfection. For retroviral infection, iGC B cells cultured for 3 days were spin-infected with retroviral supernatant supplemented with polybrene (8 μg/ml, Merck Milipore) at 1500 xg at 30 °C for 90 min, followed by another day of iGC B infection. Cultivation was carried out in a cell culture system.

Retrovirus-infected iGC B cells were harvested and used as a post-iGC B cell culture system. For GSK3 reconstruction, murine Gsk3 was amplified from cDNA of iGCB cells and cloned into a Thy-1.1-expressing retroviral vector (Addgene plasmid #17442). Site-directed mutagenesis was performed to generate retroviral vectors expressing mutant forms of GSK3 using the QuickChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent Technologies) according to the manufacturer's instructions. All plasmids used in the experiments were sequenced and verified. Primer assignments are listed in Table 1.

Example 12: Cell cycle analysis

Cell cycle distribution was analyzed by flow cytometry after PI incorporation.

Briefly, harvested post-iGC B cells were washed with PBS and fixed with cold 70% ethanol. Cells were then washed twice with PBS and incubated with RNase (100 μg/ml, Worthington Biochemical) diluted in PBS for 1 hour at 37 °C in a non-CO2 incubator. Cells were washed again with PBS, incubated with PI solution (50 μg/ml, Sigma Aldrich) and immediately analyzed by FACSVerse™ flow cytometer (BD Biosciences).

Example 13: Cell Sorting

For immunoblotting of GSK3 expression, single cells from draining lymph nodes from immunized mice were prepared and stained for GC B cells. IgG1+ FAS+ CD38low and IgG1- FAS+ CD38low GC B cells were sorted and subjected to immunoblot analysis.

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 hours were sorted and subjected to total RNA isolation. All sorting procedures were performed using a FACSAria II flow cytometer (BD Biosciences).

Example 14: RNA sequencing

Total RNA was isolated from sorted post-iGC B cells using the RNAeasy kit (QIAGEN) according to the manufacturer's instructions. The Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA, #RS-122-2101) was used for library construction. The indexed libraries were then submitted to the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA, USA) and paired-end (2 x 100bp) sequencing was performed by Macrogen Incorporated.

Alignment and annotation were performed with TopHat2 (54) for RNA-sequencing data analysis. Fasta and gtf files of the Mus musculus genome (version mm10 from UCSC) were used as references for alignment and annotation.

Using the same Fasta and gtf files, Cufflinks, Cuffnorm and Cuffdiff were performed to calculate fragments per kilobase of parts per million (FPKM) values, normalize library size, and identify DEGs, respectively.

After excluding genes with an FPKM of 0 in all samples, the log (FPKM + 1) values were used for downstream analysis. After performing quantile normalization within DMSO/CHIR and DZ/LZ, significant DEGs were estimated according to Q values <0.05 and |fc|≥ 2.

All plots were constructed in R (v.3.5.3) (R Foundation for Statistical Computing, Vienna, Austria). Gene Ontology (GO) analysis was performed using DAVID (v6.8). GO terms with low P values were selected only from the biological process category to construct Fig. S2C,D.

DEG lists from each experiment were overlaid to identify common significant DEGs between the DMSO/CHIR and LZ/DZ comparisons, and 48 common DEGs from a total of 12 samples were used for heat map and principal component analysis (PCA) plot construction. .

For DZ and LZ analysis in this study, Foxo1 (+/+) DZ and Foxo1 (+/+) FastQ files were downloaded from the Sequence Read Archive (SRA).

SRP096690. Single-ended reads were processed with the same pipeline as described above. A list of c-Myc responsive genes was obtained from Supplementary Table 2 from Dominguez-Sola et al. Publication CEL files of GFPMYC+ and GFPMYC− were downloaded from Gene Expression Omnibus (GEO); section number GSE38304.

In the present study, P values were determined using a two-tailed Student's t-test. Statistical significance was judged at P<0.05. All data points and 'n' values represent biological repeats. GraphPad Prism 7 software was used for statistical analysis.

RESOURCE SOURCE IDENTIFIER Antibodies PE anti-mouse CD5 antibody Biolegend Cat#100608 PE anti-mouse CD9 antibody Biolegend Cat#124806 PE anti-mouse CD93 (AA4.1, early B lineage) Antibody Biolegend Cat#136504 PE Rat Anti-Mouse CD43 BD Biosciences Cat#553271 PE anti-mouse TER-119/Erythroid Cells Antibody Biolegend Cat#116208 PE anti-mouse CD23 Antibody Biolegend Cat#101608 PE anti-rat CD90/mouse CD90.1 (Thy-1.1) Antibody Biolegend Cat#202524 FITC anti-mouse CD21/CD35 (CR2/CR1) Antibody Biolegend Cat#123408 PE Rat Anti-Mouse CD83 BD Biosciences Cat#558205 PE/Cy7 anti-mouse CD19 antibody Biolegend Cat#115520 PE/Cy7 anti-mouse CD38 Antibody Biolegend Cat#102718 APC Rat anti-Mouse CD138 BD Biosciences Cat#558626 APC anti-mouse CD19 Antibody Biolegend Cat#115512 APC anti-mouse IgG1 Antibody Biolegend Cat#406610 CD184 (CXCR4) Monoclonal Antibody (2B11), APC eBioscience Cat#17-9991-82 APC/Cy7 anti-mouse/human CD45R/B220 Antibody Biolegend Cat#103224 Pacific Blue?? anti-rat CD90/mouse CD90.1 (Thy-1.1) Antibody Biolegend Cat#202522 Brilliant Violet 421?? anti-mouse CD86 Antibody Biolegend Cat#105031 Brilliant Violet 510?? Streptavidin Biolegend Cat#405233 CD184 (CXCR4) Monoclonal Antibody (2B11), Biotin eBioscience Cat#13-9991-82 Alexa Fluor® 647 Rat Anti-Mouse T- and B-Cell Activation Antigen BD Biosciences Cat#561529 APC Streptavidin Biolegend Cat#405207 BV510 Hamster Anti-Mouse CD95 BD Biosciences Cat#563646 PE Hamster Anti-Mouse CD95 Biolegend Cat#554258 IRF4 Monoclonal Antibody (3E4), PE eBioscience Cat#12-9858-82 PAX5 Monoclonal Antibody (1H9), PE eBioscience Cat#12-9918-80 PE anti-mouse Blimp-1 Antibody Biolegend Cat#150005 PE anti-rat CD90/mouse CD90.1 (Thy-1.1) Antibody Biolegend Cat#202524 Biotin anti-mouse H-2Kd Antibody Biolegend Cat#116604 Anti-PE MicroBeads, lyophilized Miltenyi Biotec Cat#130-097-054 Anti-Biotin MicroBeads Miltenyi Biotec Cat#130-104-454 InVivoMab anti-mouse CD40L (CD154) BioXcell Cat#BE0017-1 InVivoMab Armenian hamster IgG isotype control, anti-glutathione S-transferase BioXcell Cat#BE0260 InVivoMAb anti-mouse CD40 BioXcell Car#BE0016-2 FoxO1 (C29H4) Rabbit mAb Cell Signaling Technology Cat#2880 GAPDH antibody (0411) Santacruz Cat#sc47724 GSK-3ab antibody (0011-A) Santacruz Cat#sc7291 c-Myc (D84C12) Rabbit mAb Cell Signaling Technology Cat#5605

(Ser9) (D85E12) XP® Rabbit mAbPhospho-GSK-3

(Ser9) (D85E12) XP® Rabbit mAb Cell Signaling Technology Cat#5558 Anti-mouse IgG, HRP-linked Antibody Cell Signaling Technology Cat#7076 Anti-rabbit IgG, HRP-linked Antibody Cell Signaling Technology Cat#7074

-Actin Antibody (C4) Santacruz Cat#sc47778 Goat Anti-Mouse IgM, Human ads-BIOT Southernbiotech Cat#1020-08 Goat Anti-Mouse IgG1, Human ads-BIOT Southernbiotech Cat#1070-08 HRP Streptavidin Biolegend Cat#405210 Mouse IgM-UNLB Southernbiotech Cat#0101-01 Mouse IgG1-UNLB Southernbiotech Cat#0102-01 Goat Anti-Mouse IgG1-UNLB Southernbiotech Cat#1071-01 Goat Anti-Mouse Kappa - UNLB Southernbiotech Cat#1050-01 Goat Anti-Mouse Lambda-UNLB Southernbiotech Cat#1060-01 Chemicals and Recombinant Proteins CHIR 99021 Tocris Cat#4423 NP-OVAL Biosearch Cat#N-5051-10 Albumin from chicken egg white Sigma Aldrich Cat#A5253 Lipopolysaccharides from Escherichia coli O55:B5 Sigma Aldrich Cat#L2880 Imject?? Alum Adjuvant Thermo Scientific Cat#77161 Recombinant Murine IL-4 Peprotech Cat#214-14 Recombinant Murine IL-21 Peprotech Cat#210-21 Ribonuclease A, DNase & Protease Free Worthinton Cat#LS002131 RIPA Buffer (10X) Cell Signaling Technology Cat#9806 TRIzol?? Reagent invitrogen Cat#15596018 NP-BSA (Bovine Serum Albumin), Ratio > 20 Biosearch Cat#N-5050H-100 NP-BSA (Bovine Serum Albumin), Ratio 1-4 Biosearch Cat#N-5050XL-100 RPMI 1640 Medium, GlutaMAX?? Supplement Gibco Cat#61870127 Fatal Bovine Serum Gemini bioproducts Cat#100-500 Lot#A51E14S Gentamicin (10 mg/mL) Gibco Cat#15710064 Penicillin-Streptomycin (10,000 U/mL) Gibco Cat#15140122 HEPES (1M) Gibco Cat#15630080 2-Mercaptoethanol Gibco Cat#21985023 ACK Lysing Buffer Gibco Cat#A1049201 Maxima SYBR Green/ROX qPCR Master Mix (2X) Thermo Scientific Cat#K0222 Propidium iodide Sigma Aldrich Cat#P4170 Agilent Seahorse XF base medium Agilent Cat#102353-100 TMB Substrate Reagent Set BD Biosciences Cat#555214 SuperLumia ECL Plus HRP Substrate Kit ABBKINE Cat#K22030 Critical Commercial Assays CellTrace?? Violet Cell Proliferation Kit, for flow cytometry invitrogen Cat#C34557 CellTrace?? CFSE Cell Proliferation Kit - For Flow Cytometry invitrogen Cat#C34554 MitoSOX?? Red Mitochondrial Superoxide Indicator, for live-cell imaging invitrogen Cat#M36008 Annexin V Apoptosis Detection kit APC eBioscience Cat#88-8007-74 RNeasy Plus Micro Kit Qiagen Cat#74034 Foxp3 staining kit set eBioscience Cat#00-5523-00 iScript gDNA clear cDNA Synthesis Kit Biorad Cat#BR172-5035 Agilent Seahorse XF Glycolysis Stress Test Kit Agilent Cat#103020-100 QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Academic) Agilent Cat#210513 Oligonucleotides Primer: IRF4 Forward: the present invention 5′-CGTTACCACTCCAGATTACCAC-3′ N/A Primer: IRF4 Reverse: the present invention N/A 5′-TCTTCCAAAGAGAAAGGGCAG-3′ Primer: Bach2 Forward: the present invention N/A 5′-AGCTAACCTCAGAACAGTTGG-3′ Primer: Bach2 Reverse: the present invention N/A 5'-GCACACCAGTTTCCGTATTTC-3' shRNA primer: IRF4 Forward: the present invention N/A 5′-TGCTGTTGACAGTGAGCGATGTATTACTTTGCTCAACAAATAGTGAAGCCACAGATGTA-3′ shRNA primer: IRF4 Reverse: the present invention N/A 5'-TCCGAGGCAGTAGGCACTGTATTACTTTGCTCAACAAATACATCTGTGGCTTCACTA-3' Overexpression primer: Bach2 Forward: the present invention N/A 5′-ATCGAGATCTATGTCTGTGGATGAGAAG-3′ Overexpression primer: Bach2 Reverse: the present invention N/A 5′-CGATACGCGTCTAGGCATAATCTTTCCTG-3′ shRNA linker primer: XhoI Forward: the present invention N/A 5′-CAGAAGGCTCGAGAAGGTATATTGCTGTTGACAGT-3′ shRNA linker primer: EcoRI Reverse: the present invention N/A 5′-CTAAAGTAGCCCCTTGAATTCCGAGGCAGTAGGCA-3′ Overexpression primer: GSK3b Forward: the present invention N/A 5′-ATGCAGATCTCATCATGTCGGGGCGACCGAG-3′ Overexpression primer: GSK3b Reverse: the present invention N/A 5′-ATGCGTCGACCTGTTCAGGTGGAGTTGGAAG-3′ mutatuon primer: GSK3b S9A Forward: the present invention N/A 5′-GACCGAGAACCACCGCCTTTGCGGAGAGC-3′ mutatuon primer: GSK3b S9A Reverse: the present invention N/A 5′-GCTCTCCGCAAAGGCGGTGGTTCTCGGTC-3′ mutatuon primer: GSK3b K85A Forward: the present invention N/A 5′-TGGAGAACTGGTTGCCATCGCGAAAGTTCTACAGGACAAG-3′ mutatuon primer: GSK3b K85A Reverse: the present invention N/A 5'-CTTGTCCTGTAGAACTTTCGCGATGGCAACCAGTTCTCCA-3'

Table 1 is information on the materials used in the present invention

<110> KNU-Industry Cooperation Foundation <120> A method for regulating differentiation of germinal center B cells to plasma cells regulating glycogen synthase kinase 3 and composition therefor <130> P20-0118HS <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 490 <212> PRT 213 <213> <400> 1 Met Ser Gly Gly Gly Pro Ser Gly Gly Gly Pro Gly Gly Ser Gly Arg 1 5 10 15 Ala Arg Thr Ser Ser Phe Ala Glu Pro Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly Gly Pro Gly Gly Ser Ala Ser Gly Pro Gly Gly Thr Gly Gly 35 40 45 Gly Lys Ala Ser Val Gly Ala Met Gly Gly Gly Val Gly Ala Ser Ser 50 55 60 Ser Gly Gly Gly Pro Ser Gly Ser Gly Gly Gly Gly Ser Gly Gly Pro 65 70 75 80 Gly Ala Gly Thr Ser Phe Pro Pro Pro Gly Val Lys Leu Gly Arg Asp 85 90 95 Ser Gly Lys Val Thr Thr Val Val Ala Thr Val Gly Gln Gly Pro Glu 100 105 110 Arg Ser Gln Glu Val Ala Tyr Thr Asp Ile Lys Val Ile Gly Asn Gly 115 120 125 Ser Phe Gly Val Val Tyr Gln Ala Arg Leu Ala Glu Thr Arg Glu Leu 130 135 140 Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Lys Asn Arg Glu 145 150 155 160 Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn Ile Val Arg Leu Arg 165 170 175 Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu Leu Tyr Leu Asn 180 185 190 Leu Val Leu Glu Tyr Val Pro Glu Thr Val Tyr Arg Val Ala Arg His 195 200 205 Phe Thr Lys Ala Lys Leu Ile Thr Pro Ile Ile Tyr Ile Lys Val Tyr 210 215 220 Met Tyr Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Gln Gly Val 225 230 235 240 Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Val Asp Pro Asp Thr 245 250 255 Ala Val Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val Arg 260 265 270 Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala Pro 275 280 285 Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val Trp 290 295 300 Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile Phe 305 310 315 320 Pro Gly Asp Ser Gly Val Asp Gln Leu Val Glu Ile Ile Lys Val Leu 325 330 335 Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr Thr 340 345 350 Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp Thr Lys Val Phe 355 360 365 Lys Ser Ser Lys Thr Pro Pro Glu Ala Ile Ala Leu Cys Ser Ser Leu 370 375 380 Leu Glu Tyr Thr Pro Ser Ser Arg Leu Ser Pro Leu Glu Ala Cys Ala 385 390 395 400 His Ser Phe Phe Asp Glu Leu Arg Arg Leu Gly Ala Gln Leu Pro Asn 405 410 415 Asp Arg Pro Leu Pro Pro Leu Phe Asn Phe Ser Pro Gly Glu Leu Ser 420 425 430 Ile Gln Pro Ser Leu Asn Ala Ile Leu Ile Pro Pro His Leu Arg Ser 435 440 445 Pro Ala Gly Pro Ala Ser Pro Leu Thr Thr Ser Tyr Asn Pro Ser Ser 450 455 460 Gln Ala Leu Thr Glu Ala Gln Thr Gly Gln Asp Trp Gln Pro Ser Asp 465 470 475 480 Ala Thr Thr Ala Thr Leu Ala Ser Ser Ser 485 490 <210> 2 <211> 420 <212> PRT 213 <213> <400> 2 Met Ser Gly Arg Pro Arg Thr Thr Ser Phe Ala Glu Ser Cys Lys Pro 1 5 10 15 Val Gln Gln Pro Ser Ala Phe Gly Ser Met Lys Val Ser Arg Asp Lys 20 25 30 Asp Gly Ser Lys Val Thr Thr Val Val Ala Thr Pro Gly Gln Gly Pro 35 40 45 Asp Arg Pro Gln Glu Val Ser Tyr Thr Asp Thr Lys Val Ile Gly Asn 50 55 60 Gly Ser Phe Gly Val Val Tyr Gln Ala Lys Leu Cys Asp Ser Gly Glu 65 70 75 80 Leu Val Ala Ile Lys Lys Val Leu Gln Asp Lys Arg Phe Lys Asn Arg 85 90 95 Glu Leu Gln Ile Met Arg Lys Leu Asp His Cys Asn Ile Val Arg Leu 100 105 110 Arg Tyr Phe Phe Tyr Ser Ser Gly Glu Lys Lys Asp Glu Val Tyr Leu 115 120 125 Asn Leu Val Leu Asp Tyr Val Pro Glu Thr Val Tyr Arg Val Ala Arg 130 135 140 His Tyr Ser Arg Ala Lys Gln Thr Leu Pro Val Ile Tyr Val Lys Leu 145 150 155 160 Tyr Met Tyr Gln Leu Phe Arg Ser Leu Ala Tyr Ile His Ser Phe Gly 165 170 175 Ile Cys His Arg Asp Ile Lys Pro Gln Asn Leu Leu Leu Asp Pro Asp 180 185 190 Thr Ala Val Leu Lys Leu Cys Asp Phe Gly Ser Ala Lys Gln Leu Val 195 200 205 Arg Gly Glu Pro Asn Val Ser Tyr Ile Cys Ser Arg Tyr Tyr Arg Ala 210 215 220 Pro Glu Leu Ile Phe Gly Ala Thr Asp Tyr Thr Ser Ser Ile Asp Val 225 230 235 240 Trp Ser Ala Gly Cys Val Leu Ala Glu Leu Leu Leu Gly Gln Pro Ile 245 250 255 Phe Pro Gly Asp Ser Gly Val Asp Gln Leu Val Glu Ile Ile Lys Val 260 265 270 Leu Gly Thr Pro Thr Arg Glu Gln Ile Arg Glu Met Asn Pro Asn Tyr 275 280 285 Thr Glu Phe Lys Phe Pro Gln Ile Lys Ala His Pro Trp Thr Lys Val 290 295 300 Phe Arg Pro Arg Thr Pro Pro Glu Ala Ile Ala Leu Cys Ser Arg Leu 305 310 315 320 Leu Glu Tyr Thr Pro Thr Ala Arg Leu Thr Pro Leu Glu Ala Cys Ala 325 330 335 His Ser Phe Phe Asp Glu Leu Arg Asp Pro Asn Val Lys Leu Pro Asn 340 345 350 Gly Arg Asp Thr Pro Ala Leu Phe Asn Phe Thr Thr Gln Glu Leu Ser 355 360 365 Ser Asn Pro Pro Leu Ala Thr Ile Leu Ile Pro Pro His Ala Arg Ile 370 375 380 Gln Ala Ala Ala Ser Pro Pro Ala Asn Ala Thr Ala Ala Ser Asp Thr 385 390 395 400 Asn Ala Gly Asp Arg Gly Gln Thr Asn Asn Ala Ala Ser Ala Ser Ala 405 410 415 Ser Asn Ser Thr 420

Claims (11)

delete delete delete delete delete delete delete In vitro, by treating germinal center (GC) B cells with the compound of Formula 1 for 2 days, the expression of Bach2, Bcl6 and Pax5 in the cells was reduced and the expression of Xbp1 and Prdm1 was induced in the cells compared to the case where the compound was not treated. How to.

[Formula 1]
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