Disulfide linkage controls the affinity and stoichiometry of IgE Fcepsilon3-4 binding to FcepsilonRI

IgE antibodies cause long-term sensitization of tissue mast cells and blood basophils toward allergen-induced cross-linking and triggering of allergic inflammation. This persistence of IgE binding is due to its uniquely high affinity for the receptor FcepsilonRI and in particular its slow rate of dissociation once bound. The binding interface consists of two subsites, one contributed by each Cepsilon3 domain of IgE Fc in a 1:1 complex. We have investigated the contributions of Cepsilon3 disulfide linkage and glycosylation to the kinetics and affinity of binding of an Fc subfragment (Fcepsilon3-4) to a soluble receptor fragment (sFcepsilonRIalpha). In contrast to IgG Fc where deglycosylation abrogates receptor binding activity, the removal of the N-linked carbohydrate at Asn-394 in Fcepsilon3-4 only reduces binding affinity by a factor of 4, principally because of a faster off-rate. Removal of the inter-heavy chain disulfide bond unexpectedly resulted in a fragment with a much faster off-rate and the potential to form a complex with a 2:1 stoichiometry (sFcepsilonRIalpha:Fcepsilon3-4). This permitted the determination of the affinity of a single, natively folded Cepsilon3 domain for the first time. The low affinity Ka approximately 10(5)-10(6) m-1, similar to that determined previously for an isolated and partially folded Cepsilon3 domain, demonstrates that substantial reduction in affinity can be achieved by preventing the engagement of one of the two Cepsilon3 domains. Recent structural data indicate that conformational change in IgE is required to allow both Cepsilon3 domains to bind, and thus an allosteric inhibitor that prevents access to the second Cepsilon3 has the potential to reduce the ability of IgE to sensitize allergic effector cells.     

Catalytic folding of the Cepsilon3 domain by its high affinity receptor

The interaction of immunoglobulin E (IgE) with its cellular receptor FcepsilonRIalpha is a central regulator of allergy. Structural studies have identified the third domain (Cepsilon3) of the constant region of epsilon heavy chain as the receptor binding region. The isolated Cepsilon3 domain is a "molten globule" that becomes structured upon binding of the FcepsilonRIalpha ligand. In this study, fluorescence and nuclear magnetic resonance spectroscopies are used to characterise the role of soluble FcepsilonRIalpha in the folding of the monomeric Cepsilon3 domain of IgE. Soluble FcepsilonRIalpha is shown to display characteristic properties of a catalyst for the folding of Cepsilon3, with the rate of Cepsilon3 folding being dependent on the concentration of the receptor.     

Conformation of the isolated cepsilon3 domain of IgE and its complex with the high-affinity receptor, FcepsilonRI

Immunoglobulin E (IgE) exhibits a uniquely high affinity for its receptor, FcepsilonRI, on the surface of mast cells and basophils. Previous work has implicated the third domain of the constant region of the epsilon-heavy chain (Cepsilon3) in binding to FcepsilonRI, but the smallest fragment of IgE that is known to bind with full affinity is a covalent dimer of the Cepsilon3 and Cepsilon4 domains. We have expressed the isolated Cepsilon3 in Escherichia coli, measured its affinity for FcepsilonRI, and examined its conformation alone and in the complex with FcepsilonRI. Sedimentation equilibrium in the analytical centrifuge reveals that this product is a monomer. The kinetics of binding to an immobilized fragment of the FcepsilonRI alpha-chain, measured by surface plasmon resonance, yields an affinity constant K(a) = 5 x 10(6) M(-)(1), as compared with 4 x 10(9) M(-)(1) for IgE. The circular dichroism spectrum and measurements of fluorescence as a function of the concentration of a denaturant do not reveal any recognizable secondary structure or hydrophobic core. On binding to the FcepsilonRI alpha-chain fragment, there is no change in the circular dichroism spectrum, indicating that the conformation of Cepsilon3 is unchanged in the complex. Thus the isolated Cepsilon3 domain is sufficient for binding to FcepsilonRI, but with lower affinity than IgE. This may be due to the loss of its native immunoglobulin domain structure or to the requirement for two Cepsilon3 domains to constitute the complete binding site for FcepsilonRI or to a combination of these factors.     

Fine structure analysis of interaction of FcepsilonRI with IgE

The high affinity receptor for IgE (FcepsilonRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcepsilonRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the alpha-subunit (FcepsilonRIalpha). In this study, the IgE binding site of human FcepsilonRIalpha has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcepsilonRIalpha and the functionally distinct but structurally homologous low affinity receptor for IgG (FcgammaRIIa) have been used to localize two IgE binding regions of FcepsilonRIalpha to amino acid segments Tyr129-His134 and Lys154-Glu161. Both regions were capable of independently binding IgE upon placement into FcgammaRIIa. Molecular modeling of the three-dimensional structure of FcepsilonRIalpha-D2 has suggested that these binding regions correspond to the "exposed" C'-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129-His134 and Lys154-Glu161 regions of FcepsilonRIalpha was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159 decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116 in the B-C loop region, which lies adjacent to the C'-E and F-G loops, has suggested Trp113 also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcepsilonRIalpha-IgE interaction for the possible treatment of IgE-mediated allergic disease.     

The key role of protein flexibility in modulating IgE interactions

The interaction between IgE and its high affinity receptor (FcepsilonRI) is a critical step in the development of allergic responses. Detailed characterization of the IgE-FcepsilonRI interaction may offer insights into possible modes of inhibiting the interaction, which could thereby act as a potential therapy for allergy. In this study, NMR, CD, and fluorescence spectroscopies have been used to characterize structurally the Cepsilon3 domain of IgE and its interaction with other protein ligands, namely, Cepsilon2, Cepsilon4, sFcepsilonRIalpha, and CD23. We have shown that the recombinant Cepsilon3 domain exists alone in solution as a "molten globule." On interaction with sFcepsilonRIalpha, Cepsilon3 adopts a folded tertiary structure, as shown by the release of the fluorescent probe 8-anilinonaphthalene-1-sulfonate and by characteristic changes in the (1)H, (15)N heteronuclear single quantum coherence NMR spectrum. However, the interactions between the Cepsilon3 domain and Cepsilon2, Cepsilon4, or CD23 do not induce such folding and would therefore be expected to involve only local interaction surfaces. The conformational flexibility of the Cepsilon3 domain of the whole IgE molecule may play a role in allowing fine tuning of the affinity and specificity of IgE for a variety of different physiological ligands and may be involved in the conformational change of IgE postulated to occur on interaction with FcepsilonRI.     

A high-affinity natural autoantibody from human cord blood defines a physiologically relevant epitope on the FcepsilonRIalpha

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the alpha domain of the high-affinity IgE receptor (FcepsilonRIalpha) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcepsilonRIalpha Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcepsilonRIalpha had the same sequence. This single IgM Ab, named CBMalpha8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcepsilonRIalpha recognized by both CBMalpha8 and the previously isolated anti-FcepsilonRIalpha Abs from autoimmune and healthy donors. This common epitope on FcepsilonRIalpha coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMalpha8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcepsilonRIalpha Abs are rendered less likely to compete with IgE for binding to FcepsilonRIalpha.     

Membrane IgE binds and activates Fc epsilon RI in an antigen-independent manner

Interaction of secretory IgE with FcepsilonRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcepsilonRIalpha to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcepsilonRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cepsilon2-Cepsilon3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igalpha/Igbeta BCR accessory proteins), and both epsilonBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcepsilonRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca(2+) responses in the basophil cell line, while membrane IgE-FcepsilonRI complexes were detected by immunoprecipitation. FcepsilonRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcepsilonRI in several cellular entities suggests a possible membrane IgE-FcepsilonRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.     

Epitope analysis and primary structures of variable regions of anti-human FcepsilonRI monoclonal antibodies, and expression of the chimeric antibodies fused with human constant regions

The structural analysis of monoclonal antibodies (mAbs) against the alpha subunit of the high affinity IgE receptor (FcepsilonRIalpha) is an alternative approach to obtaining information for the design of inhibitors that will block complementary interaction between IgE and FcepsilonRIalpha and to analyzing the various biological effects induced by anti-FcepsilonRIalpha autoantibodies in chronic urticaria. In this study, epitopes for mouse anti-human FcepsilonRIalpha mAbs and primary structures of variable regions of the mAbs were analyzed. Three mAbs inhibitory for IgE-binding reacted to the deletion mutants of FcepsilonRIalpha containing the whole second immunoglobulin-like domain as well as IgE did. On the other hand, two uninhibitory mAbs reacted to those containing the whole first immunoglobulin-like domain. The cDNAs for variable regions of the five mAbs were cloned and sequenced. Two inhibitory mouse/human chimeric antibodies were expressed in COS7 cells and bound to Chinese hamster ovary transfectant cells expressing FcepsilonRI (CHO/alphabetagamma), and these inhibited the binding of IgE to CHO/alphabetagamma cells.     

Mutagenesis within human FcepsilonRIalpha differentially affects human and murine IgE binding

Soluble fragments of the alpha-chain of FcepsilonRI, the high-affinity receptor for IgE, compete with membrane-bound receptors for IgE and may thus provide a means to combat allergic responses. Mutagenesis within FcepsilonRIalpha is used in this study, in conjunction with the crystal structure of the FcepsilonRIalpha/IgE complex, to define the relative importance of specific residues within human FcepsilonRIalpha for IgE binding. We have also compared the effects of these mutants on binding to both human and mouse IgE, with a view to evaluating the mouse as an appropriate model for the analysis of future agents designed to mimic the human FcepsilonRIalpha and attenuate allergic disease. Three residues within the C-C' region of the FcepsilonRIalpha2 domain and two residues within the alpha2 proximal loops of the alpha1 domain were selected for mutagenesis and tested in binding assays with human and mouse IgE. All three alpha2 mutations (K117D, W130A, and Y131A) reduced the affinity of human IgE binding to different extents, but K117D had a far more pronounced effect on mouse IgE binding, and although Y131A had little effect, W130A modestly enhanced binding to mouse IgE. The mutations in alpha1 (R15A and F17A) diminished binding to both human and mouse IgE, with these effects most likely caused by disruption of the alpha1/alpha2 interface. Our results demonstrate that the effects of mutations in human FcepsilonRIalpha on mouse IgE binding, and hence the inhibitory properties of human receptor-based peptides assayed in rodent models of allergy, may not necessarily reflect their activity in a human IgE-based system.     

Inhibition of IgG1 and IgE production by stimulation of the B cell CTLA-4 receptor

Although a large amount of information is available on the activity of CTLA-4 in T cells, the role of this receptor in B cells has not been previously characterized. Our results show that CD40 or LPS stimulation in the presence of IL-4 induces CTLA-4 expression in purified B cells; the maximum level is reached in both membrane and intracellular compartments after 48-72 h. Engagement of the B cell CTLA-4 by immobilized mAb inhibits IgG1 and IgE production and reduces the frequency of IgG1- and IgE-expressing B cells. Cepsilon and Cgamma(1) germline mRNA expression as well as NF-kappaB and STAT6 activation, events required for isotype switching, are also inhibited by CTLA-4 engagement. Together these findings show the critical role of CTLA-4 in the control of IL-4-driven isotype switching and suggest new approaches for modulating immediate-type hypersensitivity responses.     

An intermediate pH unfolding transition abrogates the ability of IgE to interact with its high affinity receptor FcepsilonRIalpha

The interaction between IgE-Fc (Fcepsilon) and its high affinity receptor FcepsilonRI on the surface of mast cells and basophils is a key event in allergen-induced allergic inflammation. Recently, several therapeutic strategies have been developed based on this interaction, and some include Fcepsilon-containing moieties. Unlike well characterized IgG therapeutics, the stability and folding properties of IgE are not well understood. Here, we present comparative biophysical analyses of the pH stability and thermostability of Fcepsilon and IgG1-Fc (Fcgamma). Fcepsilon was found to be significantly less stable than Fcgamma under all pH and NaCl conditions tested. Additionally, the Cepsilon3Cepsilon4 domains of Fcepsilon were shown to become intrinsically unfolded at pH values below 5.0. The interaction between Fcepsilon and an Fcgamma-FcepsilonRIalpha fusion protein was studied between pH 4.5 and 7.4 using circular dichroism and a combination of differential scanning calorimetry and isothermal titration calorimetry. Under neutral pH conditions, the apparent affinity of Fcepsilon for the dimeric fusion protein was extremely high compared with published values for the monomeric receptor (KD < 10(-12) m). Titration to pH 6.0 did not significantly change the binding affinity, and titration to pH 5.5 only modestly attenuated affinity. At pH values below 5.0, the receptor binding domains of Fcepsilon unfolded, and interaction of Fcepsilon with the Fcgamma-FcepsilonRIalpha fusion protein was abrogated. The unusual pH sensitivity of Fcepsilon may play a role in antigen-dependent regulation of receptor-bound, non-circulating IgE.     

Avian IgY binds to a monocyte receptor with IgG-like kinetics despite an IgE-like structure

An ancestor of avian IgY was the evolutionary precursor of mammalian IgG and IgE, and present day chicken IgY performs the function of human IgG despite having the domain structure of human IgE. The kinetics of IgY binding to its receptor on a chicken monocyte cell line, MQ-NCSU, were measured, the first time that the binding of a non-mammalian antibody to a non-mammalian cell has been investigated (k(+1) = 1.14 +/- 0.46 x 10(5) mol(-1)sec(-1), k(-1) = 2.30 +/- 0.14 x 10(-3) s(-1), and K(a) = 4.95 x 10(7) m(-1)). This is a lower affinity than that recorded for mammalian IgE-high affinity receptor interactions (Ka approximately 10(10) m(-1)) but is within the range of mammalian IgG-high affinity receptor interactions (human: Ka approximately 10(8)-10(9) m(-1) mouse: Ka approximately 10(7)-10(8) m(-1). IgE has an extra pair of immunoglobulin domains when compared with IgG. Their presence reduces the dissociation rate of IgE from its receptor 20-fold, thus contributing to the high affinity of IgE. To assess the effect of the equivalent domains on the kinetics of IgY binding, IgY-Fc fragments with and without this domain were cloned and expressed in mammalian cells. In contrast to IgE, their presence in IgY has little effect on the association rate and no effect on dissociation. Whatever the function of this extra domain pair in avian IgY, it has persisted for at least 310 million years and has been co-opted in mammalian IgE to generate a uniquely slow dissociation rate and high affinity.     

TLR9- and FcepsilonRI-mediated responses oppose one another in plasmacytoid dendritic cells by down-regulating receptor expression

Plasmacytoid dendritic cells (pDC) express not only TLR9 molecules through which ligation with CpG DNA favors Th1 responses but also possess IgE receptors (FcepsilonRI) implicated in allergen presentation and induction of Th2 responses. This dichotomy prompted an investigation to determine whether TLR9- and IgE receptor-mediated responses oppose one another in pDC by affecting receptor expression and associated functional responses. Results showed that IgE cross-linking reduced TLR9 in pDC and inhibited the capacity of these cells to secrete IFN-alpha when stimulated with the CpG oligodeoxynucleotide (ODN)-2216. In contrast, an approximately 15-fold reduction in FcepsilonRIalpha mRNA and a loss in surface protein were seen in pDC first exposed to TLR9 ligation with ODN-2216. Results indicated that type I IFNs partly mediated this effect, as rIFN-alpha also caused a significant approximately 4-fold reduction in FcepsilonRIalpha mRNA. Finally, this reduction in FcepsilonRIalpha mediated by ODN-2216 correlated with a selective suppression of allergen-induced CD4+ T cell proliferation, but not of responses resulting from tetanus toxoid. Overall, these results imply mechanisms by which specific innate and IgE-dependent immune responses counterregulate one another at the dendritic cell level and may have significant impact on whether an ensuing response is either of Th1 or Th2 in nature.     

Down-regulation of human basophil IgE and FC epsilon RI alpha surface densities and mediator release by anti-IgE-infusions is reversible in vitro and in vivo

Previously, infusions of an anti-IgE mAb (rhumAb-E25) in subjects decreased serum IgE levels, basophil IgE and FcepsilonRIalpha surface density, and polyclonal anti-IgE and Ag-induced basophil histamine release responses. We hypothesized that these effects would be reversed in vivo by discontinuation of infusions and in vitro by exposing basophils to IgE. Subjects received rhumAb-E25 biweekly for 46 wk. Blood samples taken 0-52 wk after rhumAb-E25 were analyzed for serum IgE and basophil expression of IgE, FcepsilonRIalpha, and CD32. Basophil numbers were unaffected by infusions. Eight weeks after infusions, free IgE levels rose in vivo but did not reach baseline. Basophil IgE and FcepsilonRIalpha rose in parallel with free IgE while CD32 was stable. FcepsilonRI densities, measured by acid elution, returned to 80% of baseline, whereas histamine release responses returned to baseline. Basophils cultured with or without IgE or IgG were analyzed for expression of IgE, FcepsilonRIalpha, and CD32. By 7 days with IgE, expression of IgE and FcepsilonRIalpha rose significantly, whereas cultures without IgE declined. IgE culture did not effect CD32. IgG culture did not effect expression of any marker. The present results strongly suggest that free IgE levels regulate FcepsilonRIalpha expression on basophils.     

Inhibition of antigen-induced mediator release from IgE-sensitized cells by a monoclonal anti-Fc epsilon RI alpha-chain receptor antibody: implications for the involvement of the membrane-proximal alpha-chain region in Fc epsilon RI-mediated cell activation

The interaction between human IgE and its high affinity receptor, FcepsilonRI, is a critical event in mediating the allergic response. Aggregation of the alpha-chain of FcepsilonRI (FcepsilonRIalpha) occurs via cross-linking of receptor-bound IgE by Ag, resulting in cell activation and the release of mediators of hypersensitivity. Recently, we mapped the epitopes of two anti-FcepsilonRIalpha mAbs, 15/1 and 5H5F8. In contrast to 15/1, mAb 5H5F8 does not inhibit IgE binding to FcepsilonRIalpha. Here we demonstrate both 5H5F8 binding to FcepsilonRI(+) cells as well as a high level of IgE binding to 5H5F8-saturated cells. At the same time 5H5F8 strongly inhibits hexosaminidase release and Ca(2+) flux after Ag triggering from human IgE-sensitized RBL-2H3 cells stably transfected with human FcepsilonRIalpha. Further, 5H5F8 and its Fab inhibit sulfidoleukotriene and histamine release from primary human peripheral blood leukocytes, including cells bearing endogenous IGE: Furthermore, we confirm that 5H5F8 maps to a linear peptide sequence in close proximity to the cell membrane. Two chemically synthesized peptides containing the 5H5F8 epitope sequence PREKY were selected for detailed analysis of 5H5F8 and 5H5F8 Fab binding and were found to produce K(d) values of similar magnitude to that observed for binding to recombinant FcepsilonRIalpha. These peptides may prove useful as targets for the identification of antagonists of FcepsilonRIalpha-mediated biological activity. Moreover, our data indicate that FcepsilonRIalpha-mediated activation may involve a novel alpha-chain epitope in an early step of the cell-triggering pathway leading to cellular activation.     

Different stabilities of the structurally related receptors for IgE and IgG on the cell surface are determined by length of the stalk region in their alpha-chains

A variant of the high affinity IgE receptor FcepsilonRI, which is composed of alpha- and gamma-chains without the beta-chain, is expressed on human APC, such as dendritic cells, and has been suggested to facilitate Ag uptake through IgE and hence to facilitate Ag presentation to T cells. The level of FcepsilonRI on these cells is correlated with the serum IgE concentration, suggesting IgE mediates the up-regulation of the alphagamma2-type FcepsilonRI. The IgE-mediated FcepsilonRI up-regulation on mast cells and basophils has been shown to enhance the ability of these cells to release chemical mediators and cytokines that are responsible for allergic inflammatory reactions. Here, to elucidate the mechanism controlling FcepsilonRI expression, we compared two structurally related Ig receptors, human FcepsilonRI and FcgammaRIIIA, which carry different alpha-chains but the same gamma-chains. The half-life of FcepsilonRI on the cell surface was short unless it bound IgE, whereas FcgammaRIIIA was stably expressed without IgG binding. Shuffling of the non Ig-binding portions of the FcepsilonRIalpha and FcgammaRIIIAalpha chains revealed that the stalk region was critical in determining the difference in their stability and ligand-induced up-regulation. Unexpectedly, analyses with added or deleted amino acids in the stalk region strongly suggested that the length rather than the amino acid sequence of the stalk region was of major importance in determining the different stabilities of FcepsilonRI and FcgammaRIIIA on the cell surface. This finding provides new insights into the mechanism regulating surface FcepsilonRI expression.     

Direct expression of the extracellular portion of human FcepsilonRIalpha chain as inclusion bodies in Escherichia coli

The extracellular portion of the alpha chain of the human high-affinity IgE receptor (FcepsilonRIalpha) was expressed as inclusion bodies in Escherichia coli. In immunoblot analysis, two bands were reactive to human IgE and mouse anti-human FcepsilonRIalpha monoclonal antibodies. N-terminal sequencing showed that the two bands were equivalent to the soluble FcepsilonRIalpha with a methionine residue at the N-terminus (Met-1-172) and 23-172, in which the N-terminal 22 residues of the soluble FcepsilonRIalpha have been removed, possibly by degradation in E. coli cells. IgE-binding to CHO cells expressing FcepsilonRI was inhibited by the addition of the recombinant products prepared by the refolding procedure from inclusion bodies. The system for the expression of soluble human FcepsilonRIalpha in E. coli presented in this study and its further improvement would be useful for the production of the protein as a potent therapeutic and for analysis of the IgE-FcepsilonRIalpha interaction.     

IL-27 induces the production of IgG1 by human B cells

It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human na?ve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to na?ve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by na?ve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.     

Molecular basis for nonanaphylactogenicity of a monoclonal anti-IgE antibody

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.