Inhibition of PI3K p110δ activity reduces IgE production in IL‐4 and anti‐CD40 stimulated human B cell cultures

Phosphoinositide 3‐kinase (PI3K) p110δ signalling negatively regulates the production of mouse IgE. However, there are disparities between the mouse and human IgE biology, and the role of PI3K p110δ in the production of human IgE is yet to be determined. To investigate the effect of PI3K p110δ inhibition in the production of human IgE we isolated human B cells from tonsil tissue and stimulated them with IL‐4 and anti‐CD40 antibody to induce class switching to IgE and IgG1 in the presence or absence of IC87114, a small molecule inhibitor of PI3K p110δ. Using FACS, RT‐PCR and ELISA we examined the effect of PI3K p110δ inhibition on IgE production and determined the mechanisms involved. Unlike in mice, we observed that PI3K p110δ inhibition significantly reduces the number of IgE+ switched cells and the amounts of secreted IgE in IL4 and anti‐CD40 cultures. However, the number of IgG1+ cells and secreted IgG1 were largely unaffected by PI3K p110δ inhibition. The expression levels of AID, ε and γ1 germinal transcripts or other factors involved in the regulation of CSR to IgE and IgG1 were also unaffected by IC87114. However, we found that IC87114 significantly decreases the proliferation of tonsil B cells stimulated with IL‐4 and anti‐CD40, specifically reducing the frequency of cells that had undergone 4 divisions or more. In addition, PI3K p110δ inhibition reduced the levels of IRF4 expression in IgE+ germinal centre‐like B cells leading to a block in plasma cell differentiation. In conclusion, PI3K p110δ signalling is required for the production of human IgE, which makes it a pharmacological target for the treatment of allergic disease.


INTRODUCTION
Immunoglobulin (IgE) antibodies play a fundamental role in the pathogenesis of allergic disease.They bind to the high-affinity IgE receptor (FcεRI) on mast cells and basophils, which when cross-linked by multivalent allergens, trigger cell activation and the release of various mediators of immediate hypersensitivity and allergic inflammation [1,2].Since its discovery, over half a century ago, significant progress has been made not only in understanding the immunological mechanisms associated with type 1 hypersensitivity reactions [1,2] but also in understanding the mechanisms involved in the regulation of IgE production [3][4][5][6][7][8][9].
The commitment of a B cell to undergo CSR to IgE is dependent on signals downstream of the tumour necrosis factor receptor superfamily member CD40, after its interaction with the CD40 ligand (CD40L) on the surface of T cells, and IL4/L13 binding to their receptors on B cells [2,10].These signals activate the nuclear factor-κ B (NFκB) and signal transducer and activator of transcription 6 (STAT6) transcription factors that act synergistically to regulate CSR to IgE [10][11][12].Both NFκB and STAT6 bind to the Iε promoter and initiate the synthesis of ε germline transcripts (εGLT) and make ε switch region available for genetic recombination.Furthermore, NFκB and STAT6 can also activate the expression of the enzyme activationinduced cytidine deaminase (AID) [13], an important regulator of CSR and SHM, which produces DNA breaks in the switch regions that precede the Cε exon [10].
Several molecular mechanisms have evolved to ensure rigorous regulation of IgE production [10,14].In mice, they also include signalling by PI3K p110δ [15][16][17], a member of the class IA PI3K catalytic isoforms, which is activated downstream of various receptors on B cells [18].Inhibition of the PI3K p110δ signalling in mouse B cells, both in vitro and in vivo, increases IgE production [15][16][17].This effect of PI3K p110δ inhibition was found to be associated with increased εGLT and AID expression levels, suggesting that under normal conditions PI3K p110δ signalling suppresses IgE production by repressing εGLT and AID expression.This was surprising since signalling by PI3K p110δ is also required for the production of type 2 cytokines such as IL4 and IL13 [19,20].It was later suggested that PI3K p110δ signalling limits IgE production in a B cell-intrinsic manner, a mechanism that involves the activation of Bcl6 [16], a known negative regulator of CSR to IgE [21].
However, the B cell regulatory systems of IgE production in humans differ from those in mice [7], and the role of PI3K p110δ signalling in the production of human IgE is yet to be determined.Here, we isolated human CD43 À tonsil B cells and stimulated CSR to IgE with IL4 and anti-CD40.To investigate the involvement of PI3K p110δ signalling in human IgE production, B cell cultures were treated with IC87114, a small molecular inhibitor of p110δ used to determine the role of PI3K p110δ signalling in IgE production by mouse B cells [15,17], and nemiralisib, a new generation of PI3K p110δ inhibitors [22].The data show that PI3K p110δ inhibition reduces IgE production in tonsil B cell cultures stimulated with IL-4 and anti-CD40 antibody.Interestingly, this effect was shown to be specific for IgE, with the production of IgG1 being largely unaffected.This result is in striking contrast with the effect of PI3K p110δ inhibition in mouse B cells [15][16][17]23].

Ethics
Following full informed written consent, tonsils were obtained from patients undergoing routine tonsillectomies.The study was conducted at and in accordance with the recommendations of King's College London and Guy's and St Thomas's NHS Fundation Trust and the protocol was approved by the London Bridge Research Ethics Committee (REC number 08/H0804/94).

Flow cytometry
To determine the effect of the PI3K p110δ inhibition on STAT6 and NFκB phosphorylation levels, cells were harvested 24 h after culture with IL-4 and anti-CD40 and fixed with 2% paraformaldehyde (PFA), washed with PBS and permeabilised with 1 mL of ice-cold methanol for 20 min on ice.The permeabilised cells were then intracellularly stained for 1 h at room temperature (RT) with anti-STAT6 Tyr641 Alexa Fluor 647 (A15137E; Biolegend) and anti-NFκB p65 Ser536 Alexa Fluor 488 (93H1; Cell Signalling Technology).To determine the effect of the PI3K p110δ inhibition on CSR to IgE, cells were harvested on day 10 of culture and stained with a live/dead fixable stain dye (Life Technologies Ltd) and with anti-CD138 PE-Cy7 (MI15; BioLegend) followed by fixation with 2% paraformaldehyde and permeabilization with PBS containing 0.5% Triton X-100, and 0.5% saponin.The cells were then stained with anti-human IgE FITC (IgE21; eBioscience) and anti-IgG1-PE human (IS11-3B2.2.3; Miltenyi Biotec), as well as anti-IRF4 Alexa Fluor 647 (IRF4.3E4;BioLegend) or anti-AID Alexa Fluor 647 (EK2-5G9; BD Pharmingen) for 30 min at RT in the dark.Following the staining cells were washed and resuspended in FACS buffer and acquired with BD FACS Canto.

Proliferation
Following the CFSE labelling, performed using CellTrace™ CFSE Cell proliferation kit (C34554, Molecular Probes™ Invitrogen), the cells were cultured as above, with IL-4 and anti-CD40 antibody, in the presence or absence of 2 μM IC87114.After 10 days of culture, cells were harvested and acquired using FACSCanto.The data were analysed using FlowJo and the proliferative capacity of cultured tonsil B cells was determined based on the dilution of the CFSE labelling.

ELISA
Maxisorp plates (Nunc) were coated with polyclonal mouse anti-human IgE (Dako Agilent Technologies) or mouse anti-human IgG1 (BD Pharmingen) in pH 9.8 carbonate buffer overnight at 4 C. Unbound sites were blocked with 2.5% BSA (Sigma-Aldrich) for 2 h at RT and then washed 4 times with PBS + 0.05% Tween-20.Supernatants from day 10 cultures were then added at appropriate dilutions and plates incubated overnight at 4 C. Human serum IgE (NIBSC) or IgG1 (Sigma-Aldrich) were used to construct standard curves.Binding was detected by polyclonal goat anti-human IgE-HRP (Sigma-Aldrich) or mouse anti-human IgG HRP (BD Pharmingen) in 1% PBS/BSA for 2 h at 37 C.The colour reaction was developed using the 1-StepTM Ultra TMB-ELISA substrate solution (Thermo Scientific) and plates were read at 450 nm using the Labtech LT 4500 microplate reader.IgE and IgG1 concentrations were then calculated from the standard curve using Prism 9.2 software (GraphPad).

STATISTICAL ANALYSIS
Statistical analysis was performed using the One-Way ANOVA, with Tuky's correction unless otherwise stated.

PI3K p110δ inhibition reduces CSR of human tonsil B cells to IgE in vitro
Studies in mice have shown that CSR to IgE can be enhanced both in vivo and in vitro when the activity of PI3K p110δ is inhibited [15][16][17].This effect of p110δ inhibition is not exclusive to CSR to IgE, with CSR to IgG1 also being enhanced in mouse B cells [15,17,23].To investigate whether CSR to IgE and IgG1 in human B cells is also affected by the inhibition of the p110δ signalling, as in the mouse system, the isolated CD43 À tonsil B cells were cultured with IL-4 and anti-CD40 and different concentrations of the IC87114.After 10 days of culture, we observed an IC87114 dose-dependent reduction in the frequency of IgE + cells (Figure 1a, b).While the number of IgE + cells in cultures treated with 0.125 and 0.25 μM of IC87114 were not significantly different to the IL-4 and anti-CD40 cultures only, cultures treated with 0.5, 1, and 2 μM of IC87114 had a significant reduction in the number of IgE + cells.Similar effects of IC87114 on IgE + cells were also observed on day 7 of the culture (Figure 1a, b).When quantifying the amounts of secreted IgE antibody in culture supernatants we observed that the effects of IC87114 on secreted IgE were much more drastic (Figure 1d).Indeed, cultures treated with 1 and 2 μM of IC87114 had almost 80% less secreted IgE compared to the IL-4 and anti-CD40 cultures only.Furthermore, we can also observe a significant reduction of the secreted IgE in cultures treated with 0.25 μM of IC87114.We find that the cell viability in these cultures was unaffected suggesting that the reduction of IgE production by the PI3K p110δ inhibition was not due to reduced cell survival in our cultures (Figure S1).Conversely, IC87114 had a negligible effect on the number of IgG1 + cells, and we could only observe a significant IgG1 + cell reduction in cultures treated with 2 μM of IC87114 (Figure 1a, c).
Similar effects of IC87114 on CSR were also observed in naïve B cell cultures stimulated with IL-4 and anti-CD40 (Figure 1f, g).The effects of IC87114 on IgE production were also confirmed using nemiralisib, one of the most selective and potent PI3K p110δ currently in development [22].As can be seen in Figure 2, the percentages of IgE + cells and the amounts of secreted IgE in cultures As can be seen in Figure 3a, b, 24 h post culture with IL-4 and anti-CD40, the phosphorylation levels of STAT6 and NFκB were unaffected by the inhibition of PI3K p110δ with 2 μM IC87114.However, previous mouse studies have reported that IC87114 regulates CSR to IgE and IgG1 by upregulating the expression of AID, εGLT and γ1GLT [15,17].Therefore, to confirm that the class switch signals downstream of the IL-4R and CD40 in human B cells are unaffected by the p110δ PI3K inhibition we also evaluated the expression levels of AID, εGLT, and γ1GLT on day 5 of the cell culture.The RT-PCR data showed that the treatment of the cell culture with 2 μM IC87114 has no significant effect in the expression levels of AID, εGLT and γ1GLT (Figure 3c).The levels of AID protein expression are also unaffected by the inhibition of PI3K p110δ (Figure 3d, e).This is consistent with the unchanged STAT6 and NFκB phosphorylation levels and suggests that unlike in the mouse system, the effect of PI3K p110δ inhibition on CSR of human B cells to IgE and IgG1 occurs independently of class switch signals downstream of the IL-4R and CD40.

PI3K p110δ inhibition does not affect the expression levels of BCL6 in human B cells
Previously, it was reported that PI3K p110δ regulates CSR of mouse B cells to IgE in a B cell-intrinsic manner by modulating BCL6 expression [16].BCL6 is a transcription factor that is important for GC reaction but also is known to compete with STAT6 at the εGLT binding site leading to the repression of its transcription and that of CSR to IgE [10,21].Therefore, in mice, the reduction of the BCL6 by the inhibition of p110δ can lead to enhanced CSR to IgE [16].
To test if this is also true in the human B cells, we evaluated the expression levels of BCL6 by RT-PCR.As can be seen from Figure 4, we did not observe any changes in the BCL6 expression levels between cell cultures that were untreated and those treated with 2 μM IC87114.
Overall, this data, and that from above, suggests that there are fundamental differences in the mechanisms through which PI3K p110δ signalling regulates CSR to IgE in human and mouse B cells.

Inhibition of the PI3K p110δ activity reduces the proliferative capacity of human B cells stimulated to undergo CSR to IgE
PI3K signalling is important for B cell activation [18,25].Thus, inhibition of PI3K p110δ signalling may affect the activation of the cultured B cells leading to reduced IgE production.To address this, we FACS sorted activated (CD38 + ) B cells from freshly isolated tonsils B cells and cultured them for 7 days with IL-4 and anti-CD40.As seen in Figure 5a, b, PI3K p110δ inhibition has a significant impact on CSR to IgE, confirming that the PI3K p110δ inhibitors reduce IgE production regardless of the B cell activation status.
PI3K signalling is also known to be crucial for B cell proliferation [18,26], which is required for CSR [27,28].For CSR to IgE to happen B cells are required to divide a minimum of 5 times, whereas CSR to IgG1 requires only 2 cell divisions [27,28].Mouse studies investigating the role of p110δ PI3K in IgE production did not report any significant effect of the p110δ PI3K inhibition on the IL-4 and anti-CD40 induced B cell proliferation [15,17,23].To determine if the p110δ PI3K inhibition reduces the proliferative capacity of the cultured tonsil B cells, thus, reducing the CSR of human B cells to IgE, we first labelled the isolated tonsil B cells with CFSE and analysed cell division after 10 days of culture.The representative FACS histograms and cumulative data (Figure 5c, d) show that the number of proliferating B cells in cell cultures treated with 2 μM IC87114 was reduced by half.Considering that the minimum number [5] of cell divisions required for CSR to IgE to occur, we also investigated the number of cell divisions by measuring the halving of the CFSE intensity.We find that the number of B cells undergoing 4 cell divisions, or more is significantly reduced when cell cultures were treated with 2 μM IC87114 (Figure 5e, f).In contrast, the number of B cells undergoing 2 and 3 divisions, which are decisive for CSR to IgG1 [22] was slightly, but not significantly, reduced.
The data suggest that the modulation of the B cell proliferation is the main mechanism through which PI3K p110δ signalling regulates CSR to IgE in human B cells stimulated with IL-4 and anti-CD40.

Inhibition of PI3K p110δ blocks IgE + PC differentiation by reducing IRF4 protein expression
Previous mouse studies have demonstrated that membrane IgE expression on IgE + B cells promotes their swift differentiation into IgE-secreting PCs independently of antigen, and this process involved PI3K signalling [3][4][5].Using flow cytometry and transcriptomic analysis we have previously characterized three populations of IgE + and IgG1 + cells in our cultures, a GC-like B cell (IgE lo CD138 À and IgG1 lo CD138 À ), a PC-like 'plasmablast' (IgE hi CD138 À and IgG1 hi CD138 À ) and a PC (IgE hi CD138 + and IgG1 hi CD138 + ) population [8,29].Furthermore, we found that human IgE + B cells, just as the mouse IgE + B cells, are more prone to differentiation into PCs than IgG1 + B cells [8].To evaluate the contribution of the PI3K p110δ signalling to the PC differentiation of human IgE + B cells we examined the proportions of these three different cell populations within the gated IgE + cells.As can be seen in Figure 6a, b, inhibition of the PI3K p110δ signalling leads to an increase in the proportions of the IgE + GC-like B cells while the proportions of the differentiated IgE + plasmablast (PB) and IgE + PCs are decreased.We observed that IgE + PCs were particularly sensitive to inhibition of the PI3K p110δ as they were significantly reduced by even  the lowest dose of the inhibitor and were almost undetectable in cultures with 1 and 2 μM of IC87114, suggesting that PI3K p110δ signalling is essential for the PC differentiation of human IgE + B cells.In contrast, we only observed an effect on IgG1 + PCs in cultures treated with 2 μM IC87114 (Figure 6a, c).We have previously reported that IgE + GC-like B cells express higher levels of IRF4 than IgG1 + GC-like B cells [29].Given that IRF4 contributes to the antigen-independent PC differentiation of mouse IgE + B cells downstream of the PI3K signalling [3,5] we examined the effect of IC87114 on the levels of IRF4 expression in the IgE + and IgG1 + GC-like B stage of differentiation.We observed that the inhibition of PI3K p110δ reduced the levels of IRF4 expression in human IgE + GC-like B cells by about 50%, whereas in IgG1 + GC-like B cells we observed only a slight reduction in IRF4 expression, which was not significant.Taken together, these data suggest that PI3K p110δ signalling via the modulation of IRF4 expression is essential for the PC differentiation of human IgE + B cells.

DISCUSSION
Mast cells lacking the PI3K p110δ activity have a considerable reduction in IgE-FcεRI mediated degranulation and release of proinflammatory mediators [30,31].Inhibition of the PI3K p110δ signalling in mouse models of asthma is also associated with reduced Th2 cytokines as well as reduced lung cellular influx, including tissue esonophilia, airway mucus production and airway hyperresponsiveness [32][33][34].These studies confirm PI3K p110δ as a potential target for the treatment of allergic disease.Surprisingly, however, other mouse studies have reported that PI3K p110δ signalling reduces CSR to IgE, thus its inhibition enhances IgE production [15][16][17].In this study, we investigated the effect of PI3K p110δ inhibition in cultures of human tonsil B cells stimulated to undergo CSR to IgE.We find that the inhibition of the PI3K p110δ signalling in these human B cell cultures reduces IgE production.This contradiction between the human and mouse responses to PI3K p110δ inhibition highlights the differences in their B cell regulatory systems of IgE production.PI3K p110δ is activated downstream of various receptors on B cells and plays critical roles in the development and function of B cells [18,[35][36][37].Several studies have also implicated PI3K signalling in the regulation of CSR [15,17,23,38].The first line of evidence came from mouse B cells lacking PTEN, a phosphatase that negatively regulates PI3K activity [38].These PTEN-deficient mouse B cells, which have enhanced PI3K signalling, fail to undergo CSR both in vivo and in vitro.IC87114 reverses the effect of PTEN deficiency on CSR, suggesting that CSR is predominantly regulated by the PI3K p110δ isoform.Furthermore, inhibition of PI3K p110δ was able to also enhance CSR in wild-type mouse B cells, suggesting that under normal circumstances PI3K p110δ negatively regulates CSR.In the case of mouse IgE and IgG1, PI3K p110δ negatively regulates their IL4 and anti-CD40 induced CSR in vitro by downregulating the levels of AID as well as that of εGLT and γ1GLT, respectively [15,17].Interestingly, our data from the human B cell cultures were inconsistent with these observations made in the mouse systems.IC87114 treatment leads to a significant reduction in the number of IgE + cells in the IL-4 and anti-CD40 stimulated tonsil B cell cultures.The effect of IC87114 on IgG1 + cells was not as marked, and a significant reduction was observed only with the highest concentration of 2 μM IC87114.It has also been reported that upon immunization with T cell-dependent antigens, both total and antigen-specific IgE titters are increased in mice treated with IC87114 and those with B cell-deficient PI3K p110δ (CD19 Cre p110δ flox/flox ) [15,39].[Correction added on 04 September 2023, after first online publication: the term 'dramatically reduced' has been changed to 'increased' in the preceding sentence.]In contrast, the titers of IgG1 and IgM are unaffected, suggesting that the PI3K p110δ selectively regulates IgE production in vivo.Further investigations indicated that PI3K p110δ regulates CSR to IgE in a B cell-intrinsic manner by modulating the expression levels of BCL6 [16].However, we did not observe an effect of IC87114 on the class switch signals downstream of IL4R and CD40, and the levels of BCL6 expression were unchanged, suggesting that the mechanisms through which PI3K signalling regulates CSR to IgE differ between the human and the mouse systems.
PI3K p110δ signalling plays a key role in B cell proliferation as it regulates the expression levels of different components of the cell cycle machinery [30,36,37].Our findings show that the regulation of the B cell proliferative capacity is the key mechanism through which PI3K p110δ signalling regulates CSR to IgE in our IL4 and anti-CD40 stimulated human B cell cultures.Indeed, IC87114 significantly reduces the frequency of cells that have undergone 4 or more cell divisions.This contradicts the previous observations made in mice, which showed that the proliferation of mouse B cells, stimulated with IL-4 and anti-CD40, is unaffected by the PI3K p110δ inhibition [3,15,17,23].Furthermore, the effect of the IC87114 on the number of B cells undergoing 4 or more cell divisions may explain why we see a more pronounced effect on CSR to IgE, which requires a minimum of 5 divisions, compared to CSR to IgG1, which only requires two cell divisions.
An important feature of both mouse and human IgE + B cells is their tendency to differentiate into the PC lineage [6,8,9,29].The drastic reduction in IgE secretion in our cultures treated with IC87114 suggested that in addition to inhibiting the CSR to human IgE, the loss of PI3K p110δ signalling might also inhibit the generation of IgEproducing PCs.Indeed, our observations that the proportions of IgE + GC-like B cells are increased whereas those of IgE + PBs and PCs are reduced in IC87114 cultures confirms that PC differentiation of human IgE + B cells is regulated by the PI3K p110δ signalling.In fact, in some cultures with IC87114, we could not detect any IgE + PCs, suggesting that PI3K p110δ inhibition completely blocks IgE + PC differentiation.Our results show that IRF4, which mediates the antigen-independent PC differentiation of mouse IgE + B cells downstream of the PI3K/Akt/mTOR pathway [3,5], is significantly reduced in human IgE + GC-like B cells when PI3K p110δ is inhibited.Thus, we conclude that PI3K p110δ signalling regulates the PC differentiation of IgE + B cells by modulating their IRF4 expression levels.In contrast, PC differentiation of IgG1 + B cells was largely unaffected, and we could only observe a significant reduction in the IgG1 + PC proportions when cultures were treated with 2 μM of IC87114.In addition, we only observed a slight reduction in the levels of IRF4 expression in IgG1 + GC-like B cells.This may be explained by the fact that IRF4 expression is significantly higher in IgE + GC-like B cells compared to IgG1 + GC-like B cells [29].Previous studies have also shown that PI3K signalling plays an important role in PC survival [40].Thus, although PI3K p110δ inhibition had no effect on the overall viability of the cultured cells it may still affect the survival of IgE + and IgG1 + PCs in our cultures.Our future studies will aim to evaluate the contribution of PI3K p110δ signalling to the survival of human IgE + and IgG1 + PCs.
Overall, our data have demonstrated that the regulation of IgE production by PI3K p110δ differs between the mouse and the human systems.In human B cell cultures, the PI3K p110δ regulates IgE production by modulating the proliferative capacity of the IL-4 and anti-CD40 stimulated B cells.In addition, PI3K p110δ regulates the IgE + PC differentiation via the modulation of IRF4 expression.These data highlight the therapeutic potential of small molecule inhibitors of PI3K p110δ in the treatment of allergic disease.
-CD40 + 0•125µM IC87114 IL4 + anti-CD40 + 0•25µM IC87114 IL4 + anti-CD40 + 0•5µM IC87114 IL4 + anti-CD40 + 1µM IC87114 IL4 + anti-CD40 + 2µM IC87114 F I G U R E 1 IC87114 reduces class switching of human B cells to IgE.IL4 and anti-CD40 stimulated cells were harvested from day 7 and 10 of the culture containing different concentrations of IC87114 and intracellularly stained for IgE and IgG1.(a) Flow cytometry dot plots showing the intracellularly stained IgE + and IgG1 + cells from day 10 of the culture.The percentages of total IgE + cells (b) and IgG1 + cells (c) from day 7 and 10 of the culture relative to the IL-4 and anti-CD40 only cultures.Secreted IgE (d) and IgG1(e) analysed by ELISA.Data show the amounts of secreted IgE and IgG1 [ng/mL] made relative to the IL-4 and anti-CD40 only cultures.The percentages of total IgE + cells (f) and IgG1 + cells (g) from naïve B cell cultures made relative to the IL-4 and anti-CD40 only cultures.
F I G U R E 2 Inhibition of PI3K p110δ with nemiralisib selectively reduces class switching to IgE.Purified CD43-tonsil B cell cultures were stimulated with IL-4 and anti-CD40 alone or in the presence of 1 nM and 10 nM nemiralisib.(a) On day 10 of the culture, cells were harvested and intracellularly stained for IgE and IgG1.The percentages of total IgE + cells (b) and IgG1 + cells (c) from several experiments were made relative to the IL-4 and anti-CD40 only cultures.Secreted IgE (d) and IgG1(e) analysed by ELISA show the amounts of secreted IgE and IgG1 [ng/mL] made relative to the IL-4 and anti-CD40 only cultures.[Correction added on 04 September 2023, after first online publication: in Figure 2b image, 'four star (****)' has been replaced with 'one star (*)', which indicate the statistical difference between black and blue.]containing 1 and 10 nM nemiralisib are selectively reduced.These observations are in striking contrast to those reported by the mouse studies and suggest that PI3K p110δ regulation of CSR to IgE and IgG1 differs between the mouse and the human system.PI3K p110δ inhibition does not affect the class switch signals downstream of the IL-4R and CD40To identify the mechanism by which PI3K p110δ regulates CSR to IgE and IgG1 in our cultured tonsil B cells, we first examined the expression levels of phosphorylated and thus activated, STAT6 and NFκB.Considering that 2 μM of IC87114 showed the strongest effect on both IgE + and IgG1 + cells we investigated the effect of this IC87114 concentration on the regulators of class switching to IgE and IgG1.
U R E 3 IC87114 has no effect on the class switch signalling downstream of IL-4R and CD40.(a) After 24 h of IL-4 and anti-CD40 stimulation, B cells were fixed, permeabilized and subsequently stained with anti-STAT6 phospho (Tyr641) and anti-NFκB p65 (Ser536).(b) The data show the fold change in median fluorescence intensity (MFI) of anti-STAT6 phospho (Tyr641) and anti-NFκB p65 (Ser536) relative to IL-4 and anti-CD40 only cultures.(c) The expressions of AID, εGLT and γ1GLT were quantified with qRT-PCR from RNA isolated from day 5 of the tonsillar B cell culture.Gene expression was normalized with 18 s rRNA.(d) Histograms showing the expression of AID protein on day 5 of the cultured B cells with IL4 and anti-CD40 (black line) and treated with 2 μM IC87114 (red line).(e) Data show the fold change in median fluorescence intensity (MFI) of anti-AID stained cells relative to the IL-4 and anti-CD40 culture condition.

F
I G U R E 4 PI3K p110δ inhibition does not affect the expression levels of BCL6 in IL-4 and anti-CD40 stimulated human B cells.The expressions of BCL6 was quantified with qRT-PCR from RNA isolated from day 5 of the tonsillar B cell culture.Gene expression was normalized with 18 s rRNA.

IL4
U R E 5 IC87114 inhibits the proliferative capacity of the IL-4 and anti-CD40 stimulated B cells.(a) The FACS sorted CD38 + B cells were cultured for 7 days with IL-4 and anti-CD40 alone or with 2 μM IC87114.After the culture, cells were harvested and intracellularly stained for IgE and IgG1.(b) The percentages of total IgE + cells and IgG1 + cells made relative to the IL-4 and anti-CD40 only cultures.To determine the effect of PI3K p110δ inhibition on proliferation, the isolated CD43 À B cells were stained with CFSE and cultured for 10 days with IL-4 and anti-CD40 alone or with 2 μM IC87114.(c) The FACS histograms show the percentage of proliferating B cells after 10 days of culture, whereas (d) shows the fold change in proliferation relative to the IL-4 and anti-CD40 only culture.The statistical comparisons were performed using the paired t-test.**** p < 0.0001.(e) The FACS plots show the number of divisions and the percentage of cells at each division that the IL-4 and anti-CD40 stimulated B cells, with or without IC87114, were undergoing.Based on the CFSE dilution, six numbers of cell divisions (D1-D6+) were identified and gated.(f) Graph shows the frequency of B cells at divisions 1 to 6+ (D1 to D6+).[Correction added on 04 September 2023, after first online publication: in Figure 5b image, 'three star (***)' has been added.]

F
I G U R E 6 PI3K p110δ inhibition blocks the PC differentiation of IgE + B cells.IL4 and anti-CD40 stimulated cells were harvested from day 10 cultures containing different concentrations of IC87114 and surface stained for CD138 and intracellularly for IgE and IgG1.(a) The proportions of GC-like B cell (IgE lo CD138 À and IgG1 lo CD138 À ), PC-like 'plasmablast' (IgE hi CD138 À and IgG1 hi CD138 À ) and PC (IgE hi CD138 + and IgG1 hi CD138 + ) populations were determined within the gated IgE + and IgG1 + cells.The percentages of the three different IgE + cells (b) and IgG1 + cells (c) were made relative to the IL-4 and anti-CD40 only cultures.(d) Expression levels of IRF4 in IgE + and IgG1 + GC-like B cells as determine by flow cytometry.(e) Data show the fold change in median fluorescence intensity (MFI) of anti-IRF4 stained cells relative to the IL-4 and anti-CD40 culture condition.Statistical analysis was performed using the paired t-test.