Ontogeny of human IgE‐expressing B cells and plasma cells

Abstract Background IgE‐expressing (IgE+) plasma cells (PCs) provide a continuous source of allergen‐specific IgE that is central to allergic responses. The extreme sparsity of IgE+ cells in vivo has confined their study almost entirely to mouse models. Objective To characterize the development pathway of human IgE+ PCs and to determine the ontogeny of human IgE+ PCs. Methods To generate human IgE+ cells, we cultured tonsil B cells with IL‐4 and anti‐CD40. Using FACS and RT‐PCR, we examined the phenotype of generated IgE+ cells, the capacity of tonsil B‐cell subsets to generate IgE+ PCs and the class switching pathways involved. Results We have identified three phenotypic stages of IgE+ PC development pathway, namely (i) IgE+germinal centre (GC)‐like B cells, (ii) IgE+ PC‐like ‘plasmablasts’ and (iii) IgE+ PCs. The same phenotypic stages were also observed for IgG1+ cells. Total tonsil B cells give rise to IgE+ PCs by direct and sequential switching, whereas the isolated GC B‐cell fraction, the main source of IgE+ PCs, generates IgE+ PCs by sequential switching. PC differentiation of IgE+ cells is accompanied by the down‐regulation of surface expression of the short form of membrane IgE (mIgES), which is homologous to mouse mIgE, and the up‐regulation of the long form of mIgE (mIgEL), which is associated with an enhanced B‐cell survival and expressed in humans, but not in mice. Conclusion Generation of IgE+ PCs from tonsil GC B cells occurs mainly via sequential switching from IgG. The mIgEL/mIgES ratio may be implicated in survival of IgE+ B cells during PC differentiation and allergic disease.

IgE antibodies mediate the activation of IgE effector cells and antigen-presenting cells by allergen and hence are central to allergic disease (1,2). The increasing prevalence of allergic disease is alarming, yet little is known about the mechanisms of IgE regulation. The sparsity of IgE + B cells in vivo has hindered the attempts to investigate their development, particularly in the human system, while reliance on the results from mouse models often fails to predict the outcome of proposed therapies (3).
It is well established that T-cell helper type 2 (Th2) cytokines, IL-4 and/or IL-13, in association with CD40 cross-linking on B cells, promote class switch recombination (CSR) to IgE, which may be direct, from IgM to IgE, or sequential, via IgG (4). In vivo, CSR occurs in lymphoid tissues and at sites of inflammations (5,6). In lymphoid tissue, B-cell-T-cell interactions lead to B-cell proliferation and the formation of GCs, in which CSR is accompanied by somatic hypermutation (SHM) in the variable regions, culminating in affinity maturation and selection of the B cells of highest affinity for antigen, or allergen in the case of IgE (7,8). The selected cells may recycle via the T-cell compartment or differentiate into memory B cells and PCs to enter the circulation (9,10).
Recent studies in the mouse revealed that the fate of IgE + B cells is dramatically different from that of IgG1 + B cells, which express the most abundant and most thoroughly investigated isotype (11)(12)(13)(14)(15)(16). It was shown that although CSR to IgE is initiated in GCs, most of IgE + cells exhibited a PC phenotype and were excluded from the GCs (14). Likewise, other studies of IgE in the mouse showed that IgE responses are more transient than those of IgG1 and were predominantly directed into the PC lineage (13). It was also reported that CSR pathway leading to IgE + B cells determined their ultimate fate (16). Direct switching gave rise to IgE + GC cells with an impaired B-cell receptor (BCR) signalling, due to the low expression of the BCR, leading to cell death (16). This switching pathway was associated with the secretion of low-affinity IgE antibodies (16,17). In contrast, sequential switching generated IgE + PCs with elevated BCR expression and was associated with the secretion of high-affinity IgE antibodies (16,17). It was inferred that the inheritance of SHM and affinity maturation from IgG1 + B cells are needed for the generation of a memory IgE response (16,17).
The relevance of results in the mouse to human allergy has been questioned (18). For example, human IgE + B cells express two forms, one short and one long form, of mIgE, mIgE S and mIgE L (19,20). These mIgE isoforms arise from the alternative splicing of a common mRNA precursor, with mIgE L containing a longer extra-membrane proximal domain (EMPD) region, an additional 52-amino acid residue between the C-terminal Ig domain, Ce4 and the transmembrane M1 domain (19)(20)(21). Although nothing is yet known about the mechanisms that govern the relative expression of the two mIgE isoforms, there is evidence that the longer EMPD confers greater resistance to BCR-induced apoptosis (21,22).
We have previously characterized the capacity of various tonsil B-cell subsets to undergo CSR to IgE ex vivo (23). Using this ex vivo tonsil human B-cell culture system, we have now investigated the ontogeny of human IgE + PCs. We point out many similarities, but also important differences from studies in the mouse models that may illuminate the mechanisms in allergy.

Isolation of human tonsil B cells
With informed written consent and ethical approval from Guy's Research Ethics Committee, we obtained human tonsils from donors undergoing routine tonsillectomies. Mononuclear cells were separated according to the density on a Ficoll gradient (GE Healthcare, Buckinghamshire, UK), and B cells were isolated using 2-aminoethylisothiouronium bromide-treated sheep red blood cells (TCS Biosciences Ltd, Buckingham, UK). B cells were >95% CD19 + as determined by flow cytometry analysis.

Cell cultures
To induce CSR to IgE, B cells were cultured as previously described (23). Briefly, 0.5 9 10 6 freshly isolated tonsil B cells were stimulated with IL-4 (200 IU/ml unless otherwise stated; R&D Europe Systems Ltd, Abingdon, UK) and anti-CD40 antibody (0.5 lg/ml unless otherwise stated; G28.5; American Type Culture Collection, Manassas, VA, USA) for up to 12 days.

FACS analysis
Surface and intracellular staining of IgE + cells was performed as previously described (23). A detailed account of FACS analysis, cell sorting, RNA isolation, qRT-PCR and switch circle transcript PCR experiments can be found in the supplemental methods (available on the Allergy website).

Human IgE + cells have three successive stages of differentiation into PCs
To induce CSR to IgE, we cultured freshly isolated human tonsil B cells with IL-4 and anti-CD40 antibody. When staining for intracellular IgE, we consistently observe two IgE + cell populations (Fig. 1A). We designate these IgE lo and IgE hi cells. Similarly, we observe two populations of IgG1 + cells, IgG1 lo and IgG1 hi cells. The ratio of IgE hi to IgE lo rose from day 7 to day 12 in culture, whereas that of IgG1 hi to IgG1 lo cells remained constant ( Fig. 1A and B). On profiling the surface markers of these cells, by FACS, we observed that both IgE hi and IgG1 hi express lower levels of CD20, Fas, IL-4R and Bcl-6 and higher levels of CD38, CD27 and Blimp-1 than IgE lo and IgG1 lo counterparts (Fig. 1C), indicating a more highly differentiated phenotype (24).
Two IgE + cell populations were observed after the immunization of mice and were characterized as IgE + GC cells and IgE + PCs (13). However, unlike in mice, when staining the stimulated human B cells for CD138, a surface marker for the fully differentiated PCs, we observed three IgE + cell populations: IgE lo CD138 À , IgE hi CD138 À and IgE hi CD138 + cells ( Fig. 2A).
To characterize in more detail the IgE + cell populations along their differentiation pathway into PCs, we used a fixation and permeabilization FACS procedure (25) to isolate RNA from sorted IgE lo CD138 À , IgE hi CD138 À and IgE hi CD138 + cells. For comparison, we also sorted the IgG1 + cell counterparts. cDNA generated from these cells was used to quantify the relative expression levels of selected transcription factors known to be involved in maintaining the GC reaction and the B-cell identity of the cells, or in inducing B-cell differentiation towards PCs (7,26). Figure 2B compares these expression levels with those on the unstimulated tonsil GC B-cell controls.
Bcl-6 and Pax-5, important for maintaining the B-cell identity and the GC phenotype (7,26,27), are abundantly expressed in the GC B cells and the IgE lo CD138 À and IgG1 lo CD138 À cells, but strongly down-regulated in both the IgE hi (IgE hi CD138 À and IgE hi CD138 + ) and IgG1 hi (IgG1 hi CD138 À and IgG1 hi CD138 + ) cells (Fig. 2B). The opposite pattern is seen for Blimp-1 expression, an important factor of PC differentiation and function (7,26,28). As the FACS-derived phenotype of these cells predicts, expression of the PC marker, CD138, is seen only in IgE hi CD138 + and IgG1 hi CD138 + cells (Fig. 2B). There were, moreover, no discernible phenotypic differences between IgE + and IgG1 + cells at different stages of differentiation. In addition, we find that as IgE + and IgG1 + cells differentiate, they down-regulate Ki-67 (Fig. S1), a marker of proliferation, and their cell cycle progression declines, with the majority of the IgE + and IgG1 + PCs being at the quiescent G 0 stage of the cell cycle (Fig. S2). In sum, these observations, consistent with those on the cell surface markers, indicate that IgE lo (IgE lo CD138 À ) cells have a phenotype with GC B cell-like characteristics, whereas the IgE hi cells represent a later stage of differentiation into a PC-like (IgE hi CD138 À ) 'plasmablast' phenotype, and only a small proportion appear to be fully differentiated PCs (IgE hi CD138 + ).

Maintenance of GC B cells contributes to increased yields of IgE + PCs
In IL-4-and anti-CD40-stimulated tonsil B-cell cultures, B cells from the GC compartments are the main sources of IgE + cells (23). By comparison with na€ ıve and memory B cells, these cells have very high rates of cell death, but also display an elevated expression of IL-4R and CD40 (23). We investigated the significance of these differences by stimulating human tonsil B cells with different IL-4 and anti-CD40 concentrations. Reducing the IL-4 concentration from 200 IU/ml (the level customarily used to stimulate CSR to IgE) to 100 IU/ml reduced the percentage of IgE + cells (Fig. 3A,B), revealing that CSR to IgE is sensitive to the IL-4 concentration within this range of to 400 IU/ml caused no further accretion of IgE + cells, but engendered a threefold increase in the proportion of IgE + PCs (Fig. 3A,B,C). In contrast, these changes in IL-4 concentrations did not affect IgG1 + cells (Fig. 3A,B,C). Neither IgE + nor IgG1 + cells evinced any response to changes in anti-CD40 concentration (Fig. 3A,B,C). We also noted a significant reduction in the percentage of live cells cultured with 100 IU/ ml of IL-4, and a marked increase in cultures with 400 IU/ml of IL-4 ( Fig. 3D).
This observation ledbecause GC B cells are the main source of IgE + cells (23)to the conjecture that the action of IL-4 may be restricted to the B cells from the GC compartments. To address this, we sorted tonsil B cells into na€ ıve (CD27 À CD38 À CD77 À ), memory (CD27 + CD38 À CD77 À ), early GC (CD27 À CD38 + CD77 + ) and GC (CD27 + CD38 + CD77 + ) B cells (23) (Fig. S3) and cultured these cells with IL-4 and anti-CD40 (Fig. 3E). Again, unlike anti-CD40, IL-4 exerted a concentration-dependent effect on GC-derived B-cell cultures, but not on na€ ıve and memory B-cell cultures (Fig. 3E). The percentage of live cells in GC B-cell cultures rose when the concentration of IL-4 was increased from 200 to 400 IU/ ml (Fig. 3E). We conclude that IL-4 contributes to the maintenance of the GC B cells and that the maintenance of these cells in culture results in higher yields of IgE + PCs.

Switched IgE + cells from GC B-cell cultures undergo rapid differentiation into IgE + PCs
The above results suggest that the GC environment may favour the generation of IgE + PCs. To confirm this, we     (Fig. 4A). IgG1 + cells, by contrast, despite their similar pattern of differentiation throughout the different B-cell cultures (Fig. 4A), were predominantly IgG1 lo (0.06 AE 0.06-0.44 AE 0.19).
Examining the expression of Blimp-1 and Xbp-1, two important factors in PC differentiation and function (7,26,28), before culture, we found that eGC and GC B-cell fractions expressed significantly higher levels of Blimp-1 (Figs 4B and S4) and Xbp-1 (Fig. 4B) than the other fractions. We hypothesized that the higher levels of Blimp-1 and Xbp-1 expression in eGC and GC B cells might predispose newly switched IgE + cells, derived from these cells, towards the PC lineage. We therefore examined the PC differentiation of IgE + cells in each of the sorted B-cell cultures (Fig. 4C).
To account for the differences in CSR to IgE in these four different tonsil B-cell fractions and the variability among the different tonsil B-cell cultures (23), we measured the yields of IgE + PCs as a fraction of all IgE + cells in each culture (Fig. 4D). We found that the GC B-cell culture yielded the highest proportion of IgE + PCs and that na€ ıve B-cell cultures yielded the lowest (Fig. 4C,D). Yet, despite the higher expression of Blimp-1 and Xbp-1 in eGC compared to the memory cells, the two cell cultures yielded similar proportions of IgE + PCs (Fig. 4C,D). The yields of IgE + PCs in the unfractionated B-cell cultures were between those in na€ ıve and memory/eGC B-cell cultures (Fig. 4C,D). These results show that elevated Blimp-1 and Xbp-1 expression cannot fully account for the rapid rate of PC differentiation.
The difference in the yields of IgG1 + PCs between the various B-cell cultures showed a similar pattern to the yields of IgE + PCs (Fig. 4C,D). However, despite this, we find that the yields of IgG1 + PCs were much lower than those of IgE + PCs. This is also evident in na€ ıve B-cell cultures, which have no IgG1 + cells at the start of the culture (23), where the yields of IgE + PCs are twice those of IgG1 + PCs (Fig. S5). The data demonstrate that IgE + B cells have a much higher frequency of PC differentiation than IgG1 + cells.

IgE + PCs can be generated by both direct and sequential CSR
Previous studies in the mouse demonstrated that sequential CSR to IgE from an IgG1 + B cell is required for the generation of high-affinity IgE antibodies (17). A follow-up report suggested that direct CSR from IgM to IgE generates IgE + GC cells and sequential CSR from IgG to IgE leads to IgE + PCs (16). To determine the relative importance of the CSR pathways in the generation of IgE + cells in our total B-cell cultures, we examined switch circle transcripts (SCTs) by a nested PCR. Analysis of SCTs shows that both IgM to IgE (Ie-Cl) and IgG to IgE (Ie-Cc) SCTs were present in IgE lo CD138 À , IgE hi CD138 À and IgE hi CD138 + cells (Fig. 5A), revealing that both direct and sequential CSR can give rise to IgE + GC B cells and IgE + PCs.
Next, we investigated the CSR pathways that generate IgE + PCs in the cultures of B cells from the GC and na€ ıve compartments. We found that in GC B-cell cultures, both Ie-Cl and Ie-Cc SCTs were present in CD138 À cells, but only Ie-Cc SCTs in the CD138 + cells (Fig. 5B). In contrast, in na€ ıve B-cell cultures, we detected both Ie-Cl and Ie-Cc SCTs in the CD138 À and CD138 + cells (Fig. 5C). Overall, these data demonstrate that in human tonsil B-cell cultures, both direct and sequential CSR can give rise to IgE + cells at all stages of differentiation. However, the generation of IgE + PCs from the GC-derived B cells appears to be selective, favouring sequential CSR to IgE. This may reflect the process of affinity maturation in IgG1 + GC B cells in vivo, leading to greater survival of cells expressing high-affinity B cells, coordinated with CSR, and resulting in apoptosis of the lessfit GC cells during ex vivo culture (9,16,17,29,30).

Modulation of surface IgE expression along the differentiation pathway of IgE + cells
In mouse IgG1 + GC B cells, surface IgG1 is expressed at a 20-fold higher level than in IgG1 + PCs, whereas the opposite is true for the IgE + cells (13, 16). When examining this phenomenon in human B cells, it is necessary to recall that there are two isoforms of human mIgE, mIgE L and mIgE S , the latter being homologous to the one in the mouse. Stimulated human B cells express a preponderance of the mIgE L isoform (19)(20)(21). To determine the surface expression of the two mIgE isoforms along the differentiation pathway of human IgE + B cells, we used a combination of different anti-IgE antibodies. Staining with either a polyclonal anti-IgE, which recognizes all forms of IgE, or the anti-IgE omalizumab, which recognizes only free IgE (31), revealed that surface mIgE expression was higher in IgE hi than in IgE lo cells (Fig. 6A). The similarity of staining by these two antibodies excludes the possibility that free exogenous IgE is binding to the low-affinity IgE receptor, CD23, expressed on the B-cell membrane, consistent with our observation that surface expression of membrane CD23 is lower on IgE hi cells relative to IgE lo cells (Fig. 6B).
Next, we determined the surface expressions of mIgE and mIgG1 at successive stages of IgE + and IgG1 + cell differentiation, with the aid of, respectively, anti-IgE omalizumab and a polyclonal anti-IgG1 (Fig. 6C,D). It can be seen that surface mIgG1 is down-regulated, and surface mIgE is upregulated in the course of PC differentiation in humans, as in mouse (13, 16). Importantly, however, staining with anti-mIgE L , which recognizes only the mIgE L (32), reveals a reduction in the surface mIgE on IgE hi cells relative to that on IgE lo cells (Fig. 6A). This result was confirmed by the concomitant reduction in surface mIgE L along the differentiation pathway of IgE + cells into PCs (Fig. 6E,F).
In sum, our data provide evidence that the down-regulation of surface mIgE L is compensated by the up-regulation of

Discussion
Most of the recent insights into the biology of IgE have come from studies in the mouse (13-17, 33). The primary aim of our work was to elucidate the developmental pathway of IgE + PCs in the human system. A secondary aim was to compare the results obtained using tonsil B cells with those reported recently from various mouse models.
The developmental pathway of human IgE + cells can be resolved into three easily distinguishable stages, characterized as (i) IgE + GC-like (IgE lo CD138 À ), (ii) IgE + PC-like "plasmablasts" (IgE hi CD138 À ) and (iii) IgE + PCs (IgE hi CD138 + ). The same developmental sequence was seen to prevail in the human tonsil IgG1 + cells. In contrast, mouse in vivo and ex vivo studies show the existence of only two distinct IgE + (IgE lo CD138 À and IgE hi CD138 + ) and IgG1 + (IgG1 lo CD138 À and IgG1 hi CD138 + ) cell populations (13).
In the mouse, nascent IgE + cells appeared to differentiate more swiftly into PCs than IgG1 + cells (13, 14), although this may have been their only route to survival (16,33). Indeed, PC differentiation was the predominant fate of the mouse IgE + cells in vivo as well as ex vivo (13, 14). Similarly, we show that in the human system, a greater proportion of IgE + cells, compared with their IgG1 + counterparts, differentiate into PCs. The mechanisms contributing to the apparent propensity of IgE + cells to differentiate into the PC lineage remain unclear.
The transgenic Blimp-1-deficient mouse B cells undergo CSR to IgE, but fail to differentiate into PCs (13), confirming that Blimp-1 is not required for the CSR, but only for PC differentiation. However, Blimp-1 expression in our IgE + and IgG1 + cells is similar and therefore does not account for their differences in PC differentiation.
Both mIgG1 and mIgE have cytoplasmic tails that contribute to their enhanced signalling capacity (34)(35)(36)(37)(38). However, mIgE has a unique motif (YANIL-motif) within its cytoplasmic tail, which is not found in the cytoplasmic tails of IgG isotypes, and binds proteins such as HS-1 and HAX-1 (39). This could potentially explain the enhanced PC differentiation of IgE + cells. We have previously shown that CD23 plays an important role in IgE synthesis (1). Therefore, the propensity of IgE + cells towards the PC lineage could also result from the activity of CD23.
An important way in which the mouse and human systems diverge is that whereas mouse IgE hi cells are mainly IgE + PCs (13, 14), only a minor fraction of IgE hi cells in total tonsil B cells become CD138 + PCs. It is possible that in these cultures, IgE + PCs are generated, but fail to survive. However, we have demonstrated that increased IL-4 concentration improved the yields of IgE + PCs by maintaining the GC B cells. This implies that GC B cells are important for the generation of IgE + PCs and that IL-4 is essential not only for CSR to IgE but also for the maintenance of GC B cells, which have very high rates of cell death (23).
The high proportion of IgE + cells that differentiated into IgE + PCs at the end of the GC B-cell cultures demonstrates the importance of GCs in the IgE + PC generation. Yet, despite the different levels of Blimp-1 and Xbp-1 expression, eGC and memory B-cell cultures display similar IgE + cell propensity to PC differentiation, implying that other factors are important, for example the response to cytokines that affect the balance between cell division and cell death. In contrast, the majority of IgE + switched cells in na€ ıve B-cell cultures, which undergo similar levels of CSR to IgE (23) and express similar levels of Blimp-1 and Xbp-1 to memory B cells, undergo a lower rate of PC differentiation. In vivo memory B cells are generated in GCs following SHM and affinity maturation, resulting in cells with high-affinity BCRs (9,10). The expression of a high-affinity BCR is associated with the capacity for PC differentiation in the mouse (29,30).
A novel observation in the mouse was the inheritance of IgE memory from IgG + cells (13,14,16) and the different effects of direct and sequential switching on the fate of IgE + cells (16,17). The kinetics of B-cell development revealed the relatively poor survival of IgE + GC cells, attributed to their low level of mIgE expression, and impaired BCR signalling (13, 16). The conclusion was that IgE + GC cells fail to undergo the canonical B-cell differentiation programme that potentiates IgG1 memory immune responses (16). The fate of the mouse IgE + cells was determined by their switching pathway; direct switching from IgM + cells generated only a transient population of IgE + GC cells, whereas switching from IgG1 + cells generated IgE + PCs, accompanied by the up-regulation of mIgE (16).
In contrast to the results in the mouse, we observe that both direct and sequential CSR can give rise to IgE + GC B Figure 5 Both direct and sequential switching generate IgE + GC B cells and IgE + PCs. (A) RNA from the sorted IgE lo CD138 À , IgE hi CD138 À and IgE hi CD138 + was used for the detection of Ie-Cl SCT (167 bp; direct switching) and Ie-Cc SCT (202 bp; sequential switching) by nested PCR. The PCRs were standardized by using equal amounts of RNA for the cDNA synthesis. CD138 À and CD138 + cells were sorted from the IL-4-and anti-CD40-stimulated cultures of enriched GC (CD38 + ) B cells (B) and na€ ıve B-cell cultures (C), and RNA was isolated and used for the analysis of IgE switching pathway as above. As a positive control, cDNA from an IL-4-and anti-CD40-stimulated B-cell culture that yielded high percentages of IgE + cells was used, and as a negative control, dH 2   IgE lo CD138 À , IgE hi CD138 À , IgE hi CD138 + and IgG1 lo CD138 À , IgG1 hi CD138 À , IgG1 hi CD138 + , respectively. (D) Summarized surface expression levels of mIgE and mIgG1 at different stages of differentiation into PCs. Expression levels (MFI) were made relative to the levels on the IgE lo -and IgG1 lo -gated cells. (E) The histogram shows the surface expression levels of mIgE L on IgE lo CD138 À (black line), IgE hi CD138 À (blue line) and IgE hi CD138 + (red line). (F) Summarized surface expression levels of mIgE L made relative to the expression levels on IgE lo CD138 À -gated cells. Data represent the mean AE SD. *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA, Dunnett's test). cells and IgE + PCs in our tonsil B-cell cultures. This is supported by studies on a chimeric mouse model containing the human M1 0 sequence inserted into a murine e gene reporter construct (12,15). Their IgE + GC B cells were longer-lived than in other mouse models and able to differentiate into both IgE + memory B cells and IgE + PCs. Memory IgE + B cells and IgE + PCs can also be detected in the peripheral blood of humans (40). We and others have attributed this to the M1 0 sequence, which protects against apoptosis (11,12,15,18).
Furthermore, we have also presented evidence of only sequential CSR in IgE + PCs generated from the human GCderived B-cell cultures. The major implication of sequential CSR is the probability of affinity maturation of the IgG + Bcell precursors, by analogy to observations in the mouse (14,16,17). It may follow that sequential CSR is the predominant route to IgE in allergic disease (41). This is supported by the relative frequency of Ie-Cc and Ie-Cl transcripts in nasal biopsies from allergic rhinitis patients (42) and bronchial biopsies from asthma patients (43).
Our experiments also reveal the modulation of the surface mIgE L and mIgE S expression during PC differentiation. Earlier studies have shown that mIgE L and mIgE S are the predominant isoforms in the IL-4-and anti-CD40-stimulated and unstimulated peripheral blood lymphocytes, respectively (19)(20)(21). Therefore, the expression levels of the two mIgE isoforms during the PC differentiation may reflect the levels of signalling and proliferation induced by the IL-4 and anti-CD40 stimulations. Nonetheless, the above considerations suggest that the regulation of mIgE L expression is important for IgE homeostasis in the human system. Expression of this isoform is unique to humans and could well be one reason why allergic disease occurs naturally in humans, but apparently not in mice (18). These data are of particular interest because the EMPD of surface mIgE L is a validated antibody target for immunotherapy of allergic disease, now under development in several laboratories with a view to therapeutic application (32,44,45).
In summary, using a tonsil ex vivo human system, we have investigated the ontogeny of IgE + B cells and IgE + PCs. We believe that our results have a direct relevance to the discovery of novel targets for the treatment of allergy.