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W4248949063 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract IgE can trigger potent allergic responses, yet the mechanisms regulating IgE production are poorly understood. Here we reveal that IgE+ B cells are constrained by chronic activity of the IgE B cell receptor (BCR). In the absence of cognate antigen, the IgE BCR promoted terminal differentiation of B cells into plasma cells (PCs) under cell culture conditions mimicking T cell help. This antigen-independent PC differentiation involved multiple IgE domains and Syk, CD19, BLNK, Btk, and IRF4. Disruption of BCR signaling in mice led to consistently exaggerated IgE+ germinal center (GC) B cell but variably increased PC responses. We were unable to confirm reports that the IgE BCR directly promoted intrinsic apoptosis. Instead, IgE+ GC B cells exhibited poor antigen presentation and prolonged cell cycles, suggesting reduced competition for T cell help. We propose that chronic BCR activity and access to T cell help play critical roles in regulating IgE responses. https://doi.org/10.7554/eLife.21238.001 eLife digest Antibodies are proteins that recognize and bind to specific molecules, and so help the immune system to defend the body against foreign substances that are potentially harmful. In some cases, harmless substances – such as pollen, dust or food – can trigger this response and lead to an allergic reaction. A type of antibody called immunoglobulin E (IgE) is particularly likely to trigger an allergic response. In general, immune cells called plasma cells produce antibodies and release them into the body. However, in B cells – the cells from which plasma cells develop – the antibodies remain on the surface of the cells. Here, the antibody acts as a “receptor” that allows the B cell to tell when its antibody has bound to a specific substance. Generally, B cells only activate when their B cell receptors bind to a specific substance. This binding triggers signals inside the cell that determine its fate – such as whether it will develop into a plasma cell. Recent studies have shown that B cells that have IgE on their surface (IgE+ B cells) are predisposed to develop rapidly into plasma cells. To investigate why this is the case, Yang et al. have now studied B cells both in cell culture and in mice. The results show that the IgE B cell receptor autonomously signals to the cell even when it is not bound to a specific substance, in a manner that differs from other types of B cell receptors. This increases the likelihood that the IgE+ B cell will develop into a plasma cell and limits the competitive fitness of IgE+ B cells. These findings provide new insights into how IgE responses are regulated by the B cell receptor. The next step will be to determine, at a molecular level, the basis for the autonomous signaling produced by the IgE B cell receptor when it is not bound to a specific substance. It will then be possible to investigate how this mechanism compares with the way that signals are normally transmitted when a B cell receptor binds to a specific substance. https://doi.org/10.7554/eLife.21238.002 Introduction Of all the antibody isotypes, immunoglobulin E (IgE) can elicit the most rapid immune responses in immediate-type hypersensitivity, contributing to the pathogenesis of numerous allergic diseases (Gould et al., 2003). IgE-mediated hypersensitivity responses are typically localized to specific tissues such as the skin, nose, lung, or intestine, whereas systemic responses can result in life-threatening anaphylaxis. Only a small fraction of individuals with allergic diseases will experience anaphylaxis, however, suggesting that IgE responses are normally restricted. Indeed, IgE is the least abundant antibody isotype in serum. While the availability of IgE in serum is limited in part by a short half-life and binding to Fc receptors, the production of secreted IgE appears to be tightly regulated (Geha et al., 2003). In order to understand the regulation of IgE production, recent attention has focused on IgE-expressing (IgE+) B cells. Little had been known about these cells due to their low abundance and technical difficulties in their detection. These challenges have largely been overcome by the generation of fluorescent IgE reporter mice as well as improved technical methods (He et al., 2013; Talay et al., 2012a; Wesemann et al., 2011; Yang et al., 2012). Initial studies of IgE+ B cells in mice have revealed several key differences from B cells expressing IgG1, the other major isotype induced in type 2 immune responses. IgE+ B cells appeared only transiently and at low frequencies in germinal centers (GCs) (He et al., 2013; Talay et al., 2012b; Yang et al., 2012). These structures form during immune responses in lymphoid tissues and are major sites for antibody affinity maturation as well as the generation of long-lived plasma cells (PCs) and memory B cells (Allen et al., 2007a; Victora and Nussenzweig, 2012). Consistent with the limited participation of IgE+ B cells in GCs, IgE+ responses typically exhibit reduced affinity maturation compared with IgG1+ responses, and most of the affinity maturation that does occur requires an IgG1+ B cell intermediate (Erazo et al., 2007; He et al., 2013; Xiong et al., 2012; Yang et al., 2012). In addition, we observed a relative paucity of long-lived IgE+ PCs (Yang et al., 2012) and other groups reported that memory IgE responses were largely initiated by non-IgE-expressing B cells (He et al., 2013; Katona et al., 1991; Turqueti-Neves et al., 2015). Taken together, it appears that IgE+ B cells undergo an abortive GC phase that limits downstream IgE responses. In contrast, a larger proportion of IgE+ B cells were observed to have a PC phenotype compared with IgG1+ B cells in several studies (note that here we use a broad definition of PCs to refer to all antibody secreting cells, including plasmablasts) (Erazo et al., 2007; He et al., 2013; Laffleur et al., 2015; Yang et al., 2012). We reported that this observation could be recapitulated in cell culture of primary mouse B cells, suggesting the increased PC differentiation of IgE+ B cells was B cell intrinsic (Yang et al., 2012). We proposed that the propensity of IgE+ B cells to undergo terminal differentiation into PCs may directly contribute to the low frequency and disappearance of IgE+ B cells from GCs (Yang et al., 2012). Another group proposed that IgE+ GC B cells exhibit diminished BCR signaling and undergo increased rates of apoptosis (He et al., 2013). Both intrinsic and extrinsic mechanisms could account for the distinct features of IgE+ B cells. A likely candidate for intrinsic regulation is the expression of the IgE B cell receptor (BCR). Each isotype of BCR has a different constant region sequence which may confer different signaling capabilities. IgG BCRs can promote enhanced responses compared with IgM BCRs, most notably enhanced PC differentiation in recall responses (Martin and Goodnow, 2002). This is thought to be due, at least in part, to the extended intracellular cytoplasmic tail of the IgG BCR, compared with the short three amino acid sequence (KVK) in IgM (Martin and Goodnow, 2002). A conserved tyrosine motif in the cytoplasmic tail of the IgG BCR, which was reported to be primarily responsible for its differential signaling, is also present in the IgE BCR (Engels et al., 2009). The cytoplasmic tail of the IgE BCR has also been implicated in promoting apoptosis through binding the mitochondrial protein Hax1 (Laffleur et al., 2015). Here we show that the IgE BCR is a major determinant of IgE+ B cell fate. In the presence of T cell help signals, the IgE BCR promoted PC differentiation. This cell fate predisposition occurred in the absence of cognate antigen, whereas the IgG1 BCR promoted PC differentiation only in the presence of cognate antigen. Multiple domains of the BCRs were responsible for the difference between the IgE and IgG1 isotypes. The propensity of the IgE BCR to induce antigen-independent PC differentiation was associated with a weak, constitutive activity of the IgE BCR. Genetic or pharmacological perturbations in BCR signaling led to reduced PC differentiation of IgE+ B cells in the absence of cognate antigen in cell culture. In immunized mice, reductions in BCR signaling led to consistently increased IgE+ GC B cell responses with variable effects on PCs. The effects of BCR signaling on IgE+ GC B cell responses could not be explained by differential rates of apoptosis, as we found no evidence that the IgE BCR directly promotes apoptosis. Instead, BCR signaling slowed the cell cycle progression of IgE+ GC B cells, and low IgE BCR expression limited antigen uptake and presentation. Thus, IgE B cell responses are restrained by a predisposition toward early PC differentiation, prolonged cell cycles, and limited access to T cell help, leading to reduced affinity maturation and memory cell generation. Results The IgE BCR promotes antigen-independent PC differentiation Several studies in mice have observed that a larger fraction of IgE+ cells have a PC phenotype compared with IgG1+ cells after immunization (Erazo et al., 2007; He et al., 2013; Yang et al., 2012). We previously reported that this in vivo observation could be recapitulated in primary B cell cultures with anti-CD40 antibodies and IL-4 (Yang et al., 2012), which promote class switch recombination (CSR) to IgE and IgG1. In these B cell cultures, a substantially greater fraction of IgE+ cells were PCs compared with IgG1+ cells. This result was confirmed again here using CD138 (Syndecan-1) as a marker of PCs (Figure 1A and B). We found that a larger fraction of IgE+ cells than IgG1+ cells had a PC phenotype regardless of the concentration of anti-CD40 antibody, although we noted that stronger CD40 stimulation was inhibitory toward PC differentiation (Figure 1A and B). We also previously established that the increased PC differentiation occurs in IgE+ B cells that have undergone the same number of cell divisions as IgG1+ B cells (Yang et al., 2012). These observations established a strong correlation between CSR to IgE and PC differentiation, but the cause of this correlation was unknown. Notably, these culture conditions mimic T cell help but do not stimulate the BCR. We hypothesized that the IgE BCR itself promotes PC differentiation in the absence of cognate antigen. We sought to test this model by ectopically expressing the IgE BCR in primary B cells. Figure 1 with 2 supplements see all Download asset Open asset The IgE BCR promotes antigen-independent PC differentiation of mouse B cells in culture. (A and B) Representative flow cytometry (A) and quantification (B) of PC differentiation (CD138+) from wild-type B cells cultured for 4 d with IL-4 and anti-CD40 (αCD40). Cells were pre-gated as IgD–IgM– and with a broad B220+ gate. (C) Diagram of the retroviral construct for the ectopic expression of BCR heavy chains of various isotypes. A detailed vector diagram is provided in Figure 1—figure supplement 1. (D) Frequency of PCs (CD138+) among AID-deficient B cells ectopically expressing different BCR isotypes or Thy1.1 (Thy1) as a control. Cells were cultured for a total of 4 d with anti-CD40 and IL-4 and were retrovirally transduced on d 1. (E) Diagram of the retroviral construct for the ectopic expression of both heavy and light chains of BCRs specific for TNP. (F) Frequency of PCs (CD138+) among AID-deficient B cells ectopically expressing different isotypes of TNP-specific BCRs in the absence or presence of TNP-OVA antigen (Ag). Cells were cultured for 4d with anti-CD40 and IL-4, were retrovirally transduced on d 1, and antigen was added on d 2. Similar data were obtained with B1-8flox/+ Cγ1Cre/+ B cells (Figure 1—figure supplement 2). Transduced cells for (D) and (F) were identified as Cerulean+. Dots represent data points from individual experiments. Bars represent the mean. LTR, long terminal repeat, which on the 3’ end is self-inactivating (white x); ψ+, extended packaging signal; VH and Vκ, coding sequences for the variable region of the heavy and light chains, respectively; CH and Cκ, coding sequences for constant regions of the heavy chain and light chain, respectively; M1, M2, coding sequences for the M1 and M2 exons; n.s., not significant; **p<0.01; ***p<0.001; ****p<0.0001 (t-tests with the Holm-Sidak correction for multiple comparisons (B,F), or one-way ANOVA with Dunnett’s post-test comparing each heavy chain to the Thy1.1 control sample (D)). https://doi.org/10.7554/eLife.21238.003 In order to determine whether the IgE BCR directly promoted PC differentiation, we developed an approach to ectopically express the IgE BCR versus other BCR isotypes. We chose retroviral transduction, which is a robust method to express genes in primary B cells (Wu et al., 1987). However, initial experiments with standard retroviral vectors were hampered by variable expression. We therefore engineered a retroviral vector using the EF1α promoter (Figure 1C, Figure 1—figure supplement 1), which we found gave much more uniform and robust expression. A heavy chain variable region specific for 2,4,6-trinitrophenyl (TNP) was linked to the heavy chain constant regions of each BCR isotype (Figure 1C). As a reporter of transduction, Cerulean (Rizzo et al., 2004), a derivative of cyan fluorescent protein, was placed upstream of the heavy chain, linked by a 2A sequence (de Felipe et al., 2006) which allows translationally-linked expression of multiple proteins from a single transcript (Figure 1C and Figure 1—figure supplement 1). With this optimized retroviral vector, we expressed BCRs of various isotypes in B cells from AID-deficient (Aicda–/–) mice that cannot undergo class switch recombination (Muramatsu et al., 2000). B cell proliferation was again stimulated with anti-CD40 and IL-4. Transduced cells were identified as Cerulean+ and PC differentiation was measured by the expression of the marker CD138 (Syndecan-1). Strikingly, among all isotypes tested, the IgE BCR promoted the highest frequency of PC differentiation in the absence of cognate antigen (Figure 1D). The IgM and IgG1 BCRs did not promote PC differentiation in the absence of cognate antigen, whereas the IgD and IgA BCRs had intermediate effects (Figure 1D). Since the IgG1 BCR has been reported to be much more efficient than IgM at inducing PC differentiation in vivo (Martin and Goodnow, 2002), we modified our system to test antigen-dependent effects. Specifically, we generated retroviral constructs encoding both the light chain and heavy chain from a TNP-specific monoclonal antibody, in which the heavy chain variable region was linked to the constant regions of various heavy chain isotypes (Figure 1E). These TNP-specific BCR constructs were then transduced into AID-deficient B cells. Upon the addition of TNP-ovalbumin (OVA), the TNP-specific IgG1 BCR promoted robust PC differentiation, similar to the IgE BCR (Figure 1F). The addition of TNP-OVA also resulted in a more moderate increase in PC differentiation in cells transduced with TNP-specific IgM BCRs, with intermediate results with IgD BCRs (Figure 1F). However, the addition of TNP-OVA did not cause a further increase in PC differentiation in cells transduced with TNP-specific IgE or IgA BCRs (Figure 1F). Taken together, these data suggest that the IgE BCR is a strong inducer of PC differentiation in an antigen-independent manner, mimicking the behavior of an antigen-engaged IgG1 BCR. Multiple domains of the IgE BCR contribute to antigen-independent PC differentiation Having established that the IgE BCR promotes antigen-independent PC differentiation which can be recapitulated by ectopic expression, we sought to determine which domain(s) of IgE were responsible for this activity by domain swap experiments with IgG1. Initial efforts focused on the intracellular cytoplasmic tail (CT) region, which is thought to be responsible for major differences in signaling among BCR isotypes (Wienands and Engels, 2016). Surprisingly, antigen-independent PC differentiation was unaffected when the IgE CT was replaced with that of IgG1 (Figure 2A). The transmembrane (TM) region of the BCR is thought to mediate association with the signaling adapters Igα and Igβ (Reth, 1992), yet swapping the TM region also had no impact on antigen-independent PC differentiation (Figure 2A). Membrane BCRs also contain a short extracellular segment proximal to the membrane that is unique to each isotype, known as the membrane Ig isotype-specific (migis) segment or the extracellular membrane proximal domain (Davis et al., 1991; Major et al., 1996). The expression of constructs in which the migis of IgE was swapped with that of IgG1 led to a profound loss of antigen-independent PC differentiation (Figure 2A). In reverse swaps, in which these domains of IgE were introduced into IgG1, the migis enhanced antigen-independent PC differentiation, but more striking results were seen when the IgE migis was combined with the IgE TM and CT, but not with the TM alone, suggesting that the CT may also be able to contribute to antigen-independent PC differentiation (Figure 2A). Taken together, the IgE migis region appears to be necessary, but not sufficient, for antigen-independent PC differentiation mediated by the IgE BCR. Full antigen-independent activity of the IgE BCR required the IgE migis region to be combined with either the extracellular domains of IgE or the CT of IgE. Figure 2 with 1 supplement see all Download asset Open asset Contribution of different domains of the IgE BCR to antigen-independent PC differentiation. Cells were retrovirally transduced with constructs in which the domains of IgE (orange) and IgG1 (blue) were swapped as illustrated (see legend in upper-right for numbered constructs in (B, D, and E)). Primary B cells were cultured with anti-CD40 and IL-4 in (A–C and F–G). (A) Frequency of PC differentiation (CD138+) among transduced AID-deficient B cells. (B) Representative flow cytometry of the surface abundance of IgM in transduced AID-deficient B cells. Numbers in the plots show the gMFI of surface IgM. (C) Inverse correlation of cell surface IgM with PC differentiation (CD138+) in transduced AID-deficient B cells. Each dot represents the results from B cells transduced with a distinct chimeric BCR from the constructs shown in (A); dots in orange represent chimeric BCRs with the migis derived from IgE; dots in blue represent chimeric BCRs with the migis derived from IgG1. (D) Representative flow cytometry of surface BCR (λ light chain) expression on transduced J558L cells. The lower panels depict cells that had been stably transduced with Cd79a (Igα). (E) Quantification of surface BCR (λ light chain) expression on transduced (Cerulean+) J558L cells (Cd79a–). (F and G) Frequency of PC differentiation (CD138+) among transduced B1-8flox/+ Cγ1Cre/+ B cells. In (G) the individual CH domains of IgE and IgG1 were swapped as illustrated. Surface BCR and Cerulean reporter expression are provided in Figure 2—figure supplement 1. Transduced cells were identified as Cerulean+ (A–E) with the addition of IgM–IgD– (F–G). Dots represent data points from individual experiments, except in (C) where the data are from a single experiment representative of three independent experiments. Bars represent the mean. VH, coding sequence for the variable region of the heavy chain; CH, coding sequence for the constant region of the heavy chain; TM, transmembrane region; CT, cytoplasmic tail; gMFI, geometric mean fluorescence intensity; n.s., not significant. **p<0.01, ***p<0.001, ****p<0.0001 (for each group of related constructs, one-way ANOVA with Dunnett’s post-test comparing each construct to the leftmost parent construct). https://doi.org/10.7554/eLife.21238.006 In the course of these experiments, we also measured the surface expression of IgM, which remains genetically encoded in the AID-deficient B cells that we transduced with IgE versus IgG1 BCRs. We observed that IgM was downmodulated in B cells transduced with IgE (Figure 2B, construct 1) but not with IgG1 (Figure 2B, construct 3). The IgM downmodulation was dependent on the migis region, as revealed by transducing constructs with the migis regions swapped (Figure 2B, constructs 2 and 4). Indeed, in cells transduced with each of the constructs shown in Figure 2A, all of the constructs that contained the IgE migis resulted in downmodulation of surface IgM, whereas the constructs that contained the IgG1 migis did not (Figure 2C). We hypothesized that this IgM downmodulation might reflect competition of the transduced BCRs versus IgM for binding to Igα. It was reported that similar to IgM, the IgE BCR depends on binding to Igα for export from the endoplasmic reticulum, whereas the IgG BCR does not (Venkitaraman et al., 1991). This difference among isotypes had been attributed to the TM domain (Reth, 1992; Venkitaraman et al., 1991), but we considered whether the migis, which is proximal to the membrane, could also be involved in Igα association. We transduced the IgE versus IgG1 BCRs, or constructs in which the migis regions were swapped, into J558L cells, which lack Igα (Hombach et al., 1990). For comparison, we transduced the BCRs into J558L cells in which we had stably transduced Igα. Remarkably, the IgE migis made BCR surface expression completely dependent on Igα, whereas the IgG1 migis permitted substantial surface BCR localization in the absence of Igα (Figure 2D and E). These data indicate that the migis region is a major site of interaction with Igα. In order to determine whether residual IgM expression in AID-deficient B cells was affecting our analysis of IgE versus IgG1 BCR domains, we repeated our experiments in B cells in which the pre-existing IgM BCR is deleted, analogous to natural CSR. Specifically, we made use of mice with a loxP-flanked B1-8 Ig heavy chain variable region allele (B1-8flox), which could be deleted with Cre recombinase (Lam et al., 1997). These mice were bred to mice carrying Cγ1 (Ighg1)-Cre (Casola et al., 2006), which is efficiently induced by anti-CD40 and IL-4, resulting in Cre-mediated deletion of the existing B1-8flox BCRs. We retrovirally transduced these cells with new BCRs close in time to the deletion of the existing BCR, mimicking natural CSR. Ectopic expression of BCRs of different isotypes in B1-8flox/+ Cγ1Cre/+ B cells gave similar results to AID-deficient B cells, with IgE promoting a high frequency of antigen-independent PC differentiation, whereas IgG1 promoted antigen-dependent PC differentiation (Figure 1—figure supplement 2). In domain swap experiments in B1-8flox/+ Cγ1Cre/+ cells, the IgE migis region still contributed to antigen-independent PC differentiation, but played a less prominent role than in the AID-deficient B cells, presumably since no IgM was present to compete for Igα (Figure 2F). Specifically, in IgE BCR constructs with the IgG1 migis, antigen-independent PC differentiation was reduced compared with constructs with the IgE migis, but was still elevated compared with the full IgG1 BCR, suggesting only a partial requirement of the migis region for antigen-independent PC differentiation. In reverse domain swaps in which regions of the IgG1 BCR were substituted with their counterparts in IgE, we observed a synergistic contribution of both the IgE migis and IgE CT to antigen-independent PC differentiation (Figure 2F). However, when the IgG1 extracellular domains were coupled with the IgE migis, IgE TM, and IgE CT, the frequency of antigen-independent PC differentiation was intermediate between that of the full IgE BCR and IgG1 BCRs (Figure 2F), again suggesting a contribution of the IgE extracellular domains, which were further explored below. Taken together, these data indicated that the IgE migis and CT both contributed to, but could not fully account for, the antigen-independent activity of the IgE BCR. We therefore tested the contribution of the extracellular domains of the IgE BCR to antigen-independent PC differentiation. The IgE BCR has four extracellular constant region domains (CH1-4), whereas the IgG1 BCR has three domains (CH1-3), with the second domain (CH2) of IgE replaced by a hinge in IgG1 (Gould et al., 2003). As expected, in the context of the IgE migis and CT, which we established above were major contributors to antigen-independent PC differentiation, there was no significant effect of swapping the extracellular domains, although there was a trend suggesting a contribution of the IgE CH2 and CH3 domains (Figure 2G). We therefore considered whether a contribution of the extracellular domains could be further revealed in hybrid constructs containing the IgG1 CT, to remove the contribution of the IgE CT. Indeed, both the IgE CH2 and IgE CH3 reproducibly contributed to antigen-independent PC differentiation, as revealed by transducing hybrid constructs in which these domains had been swapped with the IgG1 hinge and CH2, respectively (Figure 2G). Thus, multiple parts of the IgE molecule, specifically the CH2, CH3, migis, and CT, all contribute to antigen-independent PC differentiation, making this BCR distinct from the IgG1 BCR. In order to further validate the results from our isotype and domain swap BCR comparisons, we measured the surface expression of the ectopically-expressed BCRs. The surface expression of the transduced IgE and IgG1 BCRs were equivalent to each other, as measured with an antibody to the light chain which pairs with these heavy chains (Figure 2—figure supplement 1A). We also compared the surface expression of the transduced BCRs with the endogenous BCRs in normal B cells that had been induced to undergo natural CSR to IgE versus IgG1. We observed that the surface expression of the transduced BCRs was overlapping with the surface expression of normal endogenous IgE BCRs, but less than that of normal endogenous IgG1 BCRs (Figure 2—figure supplement 1A). This difference was due to the fact that membrane IgE normally has lower expression than membrane IgG1 (Figure 2—figure supplement 1A), as previously reported (He et al., 2013). Therefore, in the transduction system we did not ‘overexpress’ the BCRs but rather achieved a surface abundance similar to normal membrane IgE. We also noted that the induction of PC differentiation by the IgE BCR occurred over the entire range of surface BCR expression, indicating that small changes in surface expression would be unlikely to impact our results (Figure 2—figure supplement 1B). All domain swap constructs had equivalent expression of the Cerulean reporter and achieved measurable surface IgE and/or IgG1 expression within approximately a 4-fold range (Figure 2—figure supplement 1C). We therefore conclude that our system allowed a fair comparison of IgE versus IgG1 BCR domains for the ability to promote antigen-independent PC differentiation. Constitutive activity of the IgE BCR We next sought to determine whether the antigen-independent PC differentiation mediated by IgE BCR was due to antigen-independent BCR signaling that differed from the IgG1 BCR. Initial attempts to look at phosphorylation of the downstream signaling adapters such as Syk, Btk, Erk, and Akt, by phosflow failed to show striking differences between IgE+ and IgG1+ B cells (data not shown), presumably because many of these phosphorylation events are transient and can only be observed within minutes of strong acute stimulation, whereas the antigen-independent activity may be weaker and constitutive. We therefore considered cumulative readouts of BCR activity. We found that a larger fraction of IgE+ B cells than IgG1+ B cells expressed the activation marker CD69 (Figure 3A), which was particularly apparent with low concentrations of anti-CD40 antibody, since strong CD40 stimulation could itself promote CD69 upregulation (data not shown). Recently, it has been reported that Nur77 upregulation is a readout that is very sensitive to antigen-receptor signaling but only weakly induced by CD40 stimulation (Zikherman et al., 2012). IgE+ B cells had higher constitutive Nur77 expression than IgG1+ B cells, as revealed by a Nur77-GFP reporter (Figure 3B). The elevated CD69 and Nur77 expression suggest that IgE+ B cells have higher constitutive BCR activity than IgG1+ B cells. Figure 3 Download asset Open asset The IgE BCR exhibits differential constitutive activity compared with the IgG1 BCR. (A and B) Primary B cells were cultured for 4 d with IL-4 and anti-CD40 followed by flow cytometric evaluation of IgE+ and IgG1+ cells as in Figure 1A. Representative flow cytometry (left) and quantification (right) of the frequency of cells that were CD69+ (A) or Nur77-GFP+ (B). Dots represent individual samples pooled from four (A) or five (B) experiments. (C–H) TIRF microscopy of J558L cells that were transduced with the IgE or IgG1 BCRs together with Igα-YFP (‘BCR’) and stained to show the plasma membrane (PM). (C and D). Representative TIRF microcopy images of single cells (C) and individual BCR clusters (D). Scale bars, 1.5 µm. (E and F) Quantification of the fluorescence intensity (E) and size (F) of BCR microclusters. Dots indicate individual measurements. (G and H) Characterization of the Brownian diffusion coefficient of IgE and IgG1 BCRs by single molecule tracking, displayed as mean squared displacement (MSD) versus time (G) and cumulative probability distribution (H) plots. Bars show the mean (A, B, and F) or the geometric mean (E). Error bars (G) indicate the SEM. **p<0.01, ***p<0.00" @default.
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W4248949063 title "Decision letter: Regulation of B cell fate by chronic activity of the IgE B cell receptor" @default.
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