Abstract
IgE plays a pivotal role in allergic reactions and asthma through its ability to bind to the mast cell FcR for IgE (FcεRI). Current therapies to suppress such reactions include passive treatment with neutralizing Abs to IgE that block its binding to FcεRI. In theory, induction of immune tolerance in the B lymphocytes that carry IgE Ag receptors and give rise to IgE-secreting cells should provide longer term efficacy. However, recent data have suggested that such memory cells may lack cell surface IgE. Using a gene therapy approach, we show that a recombinant single-chain neutralizing anti-IgE could not only neutralize circulating IgE, but also reduce IgE+ B cell numbers and H chain transcripts. Therapeutic anti-IgE stimulated a calcium response in primary B cells or in a B cell line expressing membrane IgE and suppressed IgE secretion in vitro, suggesting that active signaling through membrane IgE likely promoted tolerance. Interestingly, upon subsequent challenge of anti-IgE-treated mice with an IgE cross-linking reagent capable of inducing activation of IgE-decorated mast cells, an anaphylaxis reaction was induced, apparently via a FcγRIII pathway involving recognition of anti-IgE Ab itself. These studies have important implications for the optimal design of safe and effective anti-IgE therapies and suggest that the IgE memory B cells may be targeted by such genetic Ab therapies.
Immunoglobulin E plays critical roles in allergic diseases including asthma, atopic dermatitis, and anaphylactic reactions (1). IgE binds to FcεRI α-chain expressed on mast cells and basophils (2). In people with Ag-specific IgE, contact with allergen induces IgE aggregation on mast cells, triggering their activation through the FcεRI signaling machinery. Activated mast cells release chemical mediators such as histamine that can induce life-threatening anaphylaxis. Two approaches can be considered to protect patients from allergic reactions. One is simply to remove IgE from the body. Alternatively, one could attempt to block the binding of IgE to mast cells, an approach that is currently used clinically with the anti-human IgE Ab omalizumab (3, 4).
In this study, we wished to investigate whether it was possible to not only block the interaction of IgE with FcRs, but also to suppress new IgE responses by providing a tolerogenic stimulus to developing or preexisting IgE memory B cells. In mammalian IgE H chain (IgHε) loci, there exist exons encoding not only secretory but also membrane forms of ε H chain. Although it has been suggested that, in the mouse, the memory B cells that give rise to IgE responses express surface IgG1 rather than membrane IgE (mIgE)3 (5), other studies point to a biologically active role of the mIgE as an Ag receptor (6, 7, 8). For example, germline truncations of the ε-chain membrane form result in reduced IgE titers, particularly in the secondary response (7). Moreover, exons encoding the membrane form of IgE are conserved over evolution. Previous studies have indicated it should be possible to suppress new IgE production by targeting membrane IgE on B cells (6, 8). We show here additional functional evidence that mIgE is expressed on the surface of some activated B cells and can transmit biologically relevant signals. Importantly, we show that mIgE can transmit tolerogenic signals to B cells upon binding of a single-chain anti-IgE delivered by a gene therapy approach.
Materials and Methods
Mice
Eight- to 12-wk-old BALB/c mice were used in most experiments. C.129S2-Fcer1atm1Knt/J (FcεRI−/−) mice were purchased from The Jackson Laboratory. Mice were bred and maintained in The Scripps Research Institute Animal Resources facility according to the Institutional Animal Care and Use guidelines.
Cells
293F cells were purchased from Invitrogen. Anti-IgE hybridoma cells R1E4 (9) and EM95 (10) were provided by Drs. D. Conrad (Virginia Commonwealth University, Richmond, VA) and F. Finkelman (University of Cincinnati, Cincinnati, OH). Anti-FcR hybridoma cells 2.4G2 (11) were purchased from American Type Culture Collection.
Development R1E4 single-chain variable fragment (scFv)
R1E4 variable region sequence was obtained using a 5′ RACE kit (Ambion) according to the manufacturer’s protocol. The following oligonucleotides were used for H chain and L chain 5′ RACE, respectively: 5′-AATAGCCCTTGACCAGGCATCC-3′ and 5′-CCAGTTGCTAAC TGTTCCGTGGA-3′.
H chain and L chain genes were PCR amplified, respectively (T34 and T31 for L chain and T35 and T33 for H chain), and R1E4 scFv (pUb-R1E4) was generated with nested PCR. To generate a membrane-tethered single chain Ab, we subcloned this fragment into SpeI- and XmaI-digested pUb-187.1 plasmid (12). This strategy fuses the single chain Fv with rat-IgG1 hinge, CH2 and CH3 domains, followed by a C-terminal MHC class I transmembrane/intracellular domain. The primers were as follows: T34, 5′-ctattaattattacaggtgcctgtgcaGACATTGTCTTGACCCAGTCT-3′; T31, 5′-ACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACCCCGTTTCAATTCCAGCTTGGTGC- 3′; T35, 5′-GCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGTGACTCTGAAAGAGTCT-3′; and T33, 5′-cctcccgggtttct gggggctgttgtttcagcTGAGGAGACTGTGACCATGACTC-3′.
To decrease the antigenicity of R1E4, rat-IgG1 was replaced with mouse IgG1 and several amino acid reversion mutations were introduced to L chain framework regions (supplemental Fig. 14). To generate a secreted form in the final construct (pR1E4), the MHC transmembrane and cytoplasmic domains were excised. The specificity of recombinant single-chain membrane R1E4 to IgE was confirmed by the transient transfection to 293F cells and flow cytometry analysis of IgE binding (Fig. 1⇓B). All of the plasmids used in in vivo injection were purified with an EndoFree Plasmid Maxi kit (Qiagen). For recombinant R1E4 (rR1E4), pR1E4 Ab-coding sequences were inserted into the pIRES-Zeocin hrGFP plasmid (pIRES-pR1E4). pIRES-pR1E4 (3 μg) was transfected into 293F cells with Lipofectamine2000 (Invitrogen) and stable cells were established. From this supernatant, rR1E4 was purified with a rProtein A column (GE Healthcare).
Generation and characterization of recombinant neutralizing anti-IgE single-chain Ab construct. A, The gene construct encoding rR1E4 (anti-IgE) showing intron/exon structure and selected features. Introns are depicted as thin lines. R1E4 scFv express under the control of human ubiquitin promoter. The R1E4 scFv was fused to hinge and Fc-encoding exons of the mouse IgG1 H chain. For membrane-bound anti-IgE expression, H-2Kb MHC class I transmembrane-coding sequences were included downstream. B, Control or recombinant plasmids expressing the membrane form of single-chain R1E4 were transiently cotransfected with enhanced GFP (EGFP) plasmid into 293 cells and the ability of the cells to bind to IgE was analyzed by flow cytometry.
Quantitative PCR (qPCR)
Total RNA was purified from 2 to 3 million spleen cells of control or pR1E4-treated mice using a RNEasy Plus kit (Qiagen). Reverse transcription was performed with a QuanteTect Reverse Transcription Kit (Qiagen) per the manufacturer’s protocol. IgHε mRNA was quantitated using SYBR GreenER qPCR Supermix (Invitrogen) with 7900HT (Applied Biosystems) and normalized with CD19 mRNA. Oligonucleotide primers used for IgE detection were 5′-acactcggagatgcccagatc-3′ and 5′-ggagcaccgttttgatacaggtc-3′ and for CD19 detection, 5′-aggtcattgcaaggtcagcagtgtg-3′ and 5′-ggcgtcactttgaagaatctcctg-3′.
Flow cytometry analysis
Erythrocyte-depleted cells were suspended in ice-cold staining buffer (HBSS buffer including 0.5 mM EDTA, 0.05 mM sodium azide, and 0.5% BSA) with appropriately titrated Abs. The following Abs were used: CD45R/B220 (RA3-6B2; BD Biosciences) (Pacific Blue), FcεRIα (MAR-1; eBioscience) (PE), IgE (23G3; eBioscience, or EM95) (FITC, PE, Alexa Fluor 647), CD49b (HMa2; BD Biosciences) (PE, allophycocyanin; biotin), c-kit (2B8; eBioscience) (allophycocyanin; biotin), CD4 (GK1.5; BD Biosciences) (PerCP-Cy5.5), CD8 (53-6.7; BD Biosciences) (PerCP-Cy5.5), and strptavidin-PE-Cy7 (eBioscience). For intracellular IgE staining, cells were incubated during surface staining with unlabeled anti-IgE (EM95); after fixation and permeabilization using a Cytofix/Cytoperm kit (BD Biosciences), cells were stained with labeled EM95 conjugate. These Abs were purchased from eBioscience or BD Biosciences as indicated. Propidium iodide (Invitrogen) was included in some experiments to exclude dead cells. To calculate the total FcεRI expression level on basophils based on IgE-binding capacity, FcγRs were preblocked with 2.4G2 for 10 min and the cells further incubated with purified IgE (IgELa) at 10 μg/ml for 30 min. Cells were then washed twice with FACS buffer and bound IgE was quantitated with anti-IgE conjugate. Data collection was done on an LSR II flow cytometer (BD Biosciences) and was analyzed using FlowJo software (Tree Star).
Hydrodynamic injection
Thirty micrograms of purified plasmid (pR1E4 or pUb control plasmid) was dissolved in 1.8 ml of TransIT-EE Delivery Solution (Mirus Bio) and injected via a tail vein. In the experiments depicted in Fig. 2⇓, 10 μg of a second plasmid driving human placental secreted alkaline phosphatase (pLIVE-SEAP; Mirus Bio) was coinjected, allowing one to monitor the efficiency of transfection by enzyme activity appearing in blood. All of the injected mice (five of five control and eight of eight pR1E4 treated) analyzed on day 13 after plasmid injection were alkaline phosphatase positive (data not shown).
Effect of in vivo expression of secreted form of chimeric single-chain anti-IgE on markers of IgE expression. Two-month-old BALB/c mice were given pR1E4 or control plasmid i.v. and analyzed 13 days later. A, Free serum IgE level measured on day 13 after plasmid treatment. B, Total serum IgE concentration before treatment (□) and 13 days after treatment (▪). C, qPCR comparison of relative IgHε mRNA levels in the spleen of control or pR1E4-treated mice at day 13 of treatment. D and E, Flow cytometry analysis of IgE bound to peritoneal mast cells in control and pR1E4-treated mice. Peritoneal mast cells were defined as c-kit+FcεRI+CD4−CD8−B220− cells. Mean fluorescence intensity (MFI) of binding by FcεRIα and anti-IgE Abs was monitored. F and G, Analysis of IgE bound to basophils in the spleen and bone marrow of control or treated individual mice. Basophils were identified as CD49b+FcεRI+CD4−CD8−B220−. H, Levels of surface IgE and FcεRI on basophils in the spleen (Sp) and bone marrow (BM) on day 13 after plasmid treatment. Results are means ± SD of eight mice receiving pR1E4 compared with five mice receiving empty vector. Statistical significance of differences between pR1E4-treated and control-treated mice was calculated using Student’s t test: *, p < 0.001 and #, p < 0.2.
IgE-eliciting immunizations
OVA (Sigma-Aldrich) was prepared with alum (Imject; Pierce) at a ratio of 10 μg of protein:100 μg of alum/mouse and was given i.p. Goat anti-mouse IgD (0.2 ml; eBioscience) was injected i.p.
IgE ELISA
An IgE ELISA quantitation kit was purchased from Bethyl and used according to the kit instructions. In experiments involving pR1E4, purified EM95 (anti-IgE) was coated on Nunc Maxisorp plates and bound serum IgE was detected with HRP- conjugated goat anti-mouse IgE (Bethyl). The presence of rR1E4 did not interfere with the total IgE concentration measurement because R1E4 and EM95 see nonoverlapping epitopes; R1E4 blocks IgE binding to FcεR1, whereas EM95 cannot. For quantitation of free IgE, rR1E4 was coated on the plate and bound IgE was detected with HRP-conjugated goat anti-mouse IgE. Color was developed with Ultra-TMB (Pierce).
Cell culture
Splenocytes (2 × 105/ml) were cultured at 37°C/5% CO2 in a final volume of 2.5 ml with 25 ng/ml IL-4, 1 μg/ml anti-CD40 (1C10; eBioscience) with or without 20 μg/ml rR1E4 in Advanced-RPMI 1640 medium supplemented with 5% FCS, 1× penicillin-streptomycin-glutamine (Life Technologies), 2 mM GlutaMAX-I (Life Technologies), and 55 μM 2-ME for 4 days. For Ca2+ mobilization analysis, splenocytes were cultured with 25 μg/ml IL-4 and 10 μg/ml anti-CD40. Culture supernatants were then tested for IgE concentration by ELISA; recovered cells were washed two times with FACS buffer before flow cytometry or Ca2+ mobilization analysis.
Calcium response
Cultured splenocytes were loaded with Fluo-4 (Invitrogen) per the manufacturer’s instructions. Calcium mobilization was induced by addition of 20 ng/ml mAbs at a cell concentration of 106/ml in volume of 0.5 ml. In some experiments, cells were preincubated with 10 μg/ml anti-mouse IgG1 Ab for 30 min before challenge and fluorescence analysis. Analysis was conducted using the FL1 channel of an LSR-II flow cytometer and analyzed with FlowJo software.
Anaphylaxis
One hundred micrograms of EM95 was given per mouse retro-orbitally after anesthesia; rectal temperature was monitored with a RET-3 probe (Physitemp Instruments) for 60 min. In some experiments, 0.5 mg of 2.4G2 (anti-FcγRII/III) Ab was given to each mouse 24 h before challenge. In the case of rR1E4 injection, 10 μg of 2,4,6-trinitrophenyl (TNP)-BSA and 100 μg of IgE anti-TNP (IgELa) were given per mouse and 50 μg of rR1E4 was given retro-orbitally; rectal temperature was monitored for 60 min.
Results
Development of recombinant single-chain anti-IgE
In contrast to passive anti-IgE administration, anti-IgE delivery via gene therapy has the potential to provide permanent therapy of IgE-mediated disease. To study the effects of anti-IgE gene therapy in the mouse (an intensively studied model for human allergy, asthma, and anaphylaxis (13, 14, 15)), we generated a chimeric single-chain anti-IgE gene based on neutralizing rat anti-mouse IgE mAb R1E4 (9). R1E4, like omalizumab (Genetech) in the human system, is unable to activate mast cells via IgE bound to FcεRI, but binds free IgE. R1E4 Ig H and L chain variable region codons were cloned, sequenced, and joined together with a short linker sequence, yielding a scFv gene (Fig. 1⇑A). The scFv gene was placed upstream of mouse IgG1 hinge and membrane proximal codons. Plasmids driving plasma membrane expression under the control of the human ubiquitin promoter (16) were prepared and validated (Fig. 1⇑B). A modified plasmid, called pR1E4, encoding a secreted version of the rR1E4 chimeric protein (rR1E4) was generated. The biological effects of in vivo expression of plasmids encoding membrane or secreted proteins was tested by the hydrodynamic (naked DNA) injection method, which leads to transient expression in the liver. Because tolerance of cognate B cells to protein Ags can be induced either by membrane expression on the liver (17) or by soluble protein (18, 19, 20), we also compared in vivo efficacy of plasmids encoding membrane and secreted forms of recombinant anti-IgE.
Neutralizing serum IgE in vivo
pR1E4 plasmid given to BALB/c mice led to single-chain anti-IgE secretion lasting at least 30 days that diminished at later times (data not shown). At day 13 after pR1E4 injection, levels of free IgE were reduced to ∼1% of those of control mice (Fig. 2⇑A). There was also a marked decline in the levels of total IgE, which includes both free IgE and IgE-rR1E4 complexes (Fig. 2⇑B). Plasmid encoding membrane-bound single-chain anti-IgE was also effective in reducing IgE levels, although the effect was less long-lasting (supplemental Fig. 2). For unknown reasons, plasmids encoding membrane-bound single-chain constructs were apparently somewhat toxic to expressing liver cells, even if they lacked specificity for IgE (data not shown). Therefore, additional experiments focused on the effects of the soluble form. Importantly, RNA analysis of the spleens of mice treated with pR1E4 revealed a >99% reduction in the levels of IgHε mRNA (Fig. 2⇑C). Consistent with the reduced IgE levels in pR1E4-treated mice, c-kit+ peritoneal mast cells and CD49b+ basophils in the bone marrow and spleens of anti-IgE-treated mice lacked detectable surface IgE, indicating that their FcεRs had lost bound IgE (Fig. 2⇑, D–H). Basophils were still present in these tissues as indicated by FcεRIα/CD49b double staining (Fig. 2⇑G). In treated mice, FcεRI expression levels in basophils appeared higher than in control plasmid-treated mice, however, total IgE-binding capacity of basophils was somewhat reduced (supplemental Fig. 3, A and B). This discrepancy was probably because anti-FcεRI mAb MAR-1 binds more tightly to FcεRI lacking bound IgE than to FcεRI carrying IgE (supplemental Fig. 3C). Reduced levels of bioactive IgE in pR1E4-treated mice did not lower the percentages or absolute numbers of mast cells and basophils (Table I⇓). Overall, these data support the conclusions that 1) gene therapy with recombinant single-chain anti-IgE is feasible and can effectively neutralize circulating IgE; 2) reduced levels of bioactive IgE have surprisingly minimal negative effects on mast cell and basophil survival, at least at the time point tested, and 3) recombinant single-chain anti-IgE treatment could actively suppress new IgE production by B cells.
Effects of pR1E4 treatment on basophil and mast cell numbers in selected tissuesa
Recombinant anti-IgE treatment of preimmunized mice
We next determined whether pR1E4 could be used to treat and suppress ongoing, established IgE responses. Basal serum IgE levels in mice were quite low (∼500 ng/ml in BALB/c mice and under 100 ng/ml in C57BL/6 mice). To evaluate the anti-IgE treatment, we used two different model responses: OVA immunization with alum adjuvant (OVA/alum), which is commonly used to elicit an Ag-specific IgE response, and anti-IgD treatment, a nonspecific B cell activator which elicits a stronger, but polyclonal IgE response (21, 22).
Ten days after receiving IgE-eliciting stimulus, mice were treated with pR1E4 or control plasmids and then analyzed 12 days later, i.e., 22 days after immunization (Fig. 3⇓). OVA/alum immunization increased total serum IgE levels manyfold above preimmune levels, a response that was essentially fully suppressed by treatment with pR1E4 (Fig. 3⇓A). IgHε mRNA qPCR analysis showed that pR1E4 treatment reduced IgHε mRNA levels in the spleen by 99% (Fig. 3⇓B). pR1E4 treatment also reduced the levels of the low-affinity IgE receptor CD23 (23) on follicular B cells by ∼50% (supplemental Fig. 4, A and B). We conclude that in mice with ongoing IgE responses, treatment with the soluble recombinant anti-IgE-producing plasmid pR1E4 could significantly (p = 0.0023) suppress levels of serum IgE, new IgE synthesis, and B cell-expressed CD23.
Effects of pR1E4 plasmid treatment on ongoing IgE responses induced by OVA/alum immunization or goat anti-mouse IgD treatment. Two-month-old BALB/c mice injected i.p. 10 days previously (day −10) with either 10 μg of OVA/alum or 200 μl of goat anti-mouse IgD serum were treated with control or pR1E4 plasmids i.v. on day 0 and analyzed on days 0 and 12 for suppression of the IgE response. A, Total serum IgE levels on day 0 (▪) and day 12 (□). B, Splenic IgHε mRNA levels assessed by qPCR. C–G, Flow cytometry analysis of IgE+ B cells in spleens of mice injected with anti-IgD and subsequently treated with control or pR1E4 plasmid. C, Analysis of the frequencies of B220high and B220lowIgE+ B cells among CD4−CD8−c-kit−CD49b− gated viable spleen cells. D, Comparison of CD138 and IgD expression of B220low and B220high populations in control plasmid and pR1E4-treated mice, as gated in C. Left, B220low cells from control plasmid-treated mouse; center, B220high cells from control plasmid-treated mouse; and right, B220high cells of pR1E4-treated mouse. E, Analysis of treated and control anti-IgD-injected mice for the presence of cells that expressed high levels of intracellular IgE. F, Summary of pR1E4-induced reductions in IgE+B220high and IgE+B220low subsets using the analysis in C. G, Quantitation of pR1E4-induced reduction in surface B220highIgE+IgD− B cells as in D. Significance was calculated using Student’s t test. *, p < 0.01 and #, p < 0.05. n = 4–6 mice/group.
Treatment of mice after anti-IgD injection served as a more stringent test of the ability to tolerize ongoing responses because IgE responses were >10-fold further enhanced over the OVA/alum-induced response. Anti-IgD injection induced ∼10 μg/ml IgE in sera in control mice, which could be markedly (99%) suppressed by subsequent in vivo expression of pR1E4 (Fig. 3⇑A). However, IgHε mRNA qPCR analysis revealed that mice given pR1E4 after anti-IgD treatment still had high levels 12 days later, suggesting that IgE mRNA-producing cells remained in the spleen (Fig. 3⇑B).
To identify the source of the IgE mRNA, we analyzed splenocytes from anti-IgD-treated mice for the presence of mIgE+ B cells (Fig. 3⇑, C–G). In control plasmid-treated mice, B220high and B220lowIgE+ B cells were detected (Fig. 3⇑C). B220high B cells were mostly IgD+CD138− cells and possibly represented background staining of naive B cells (Fig. 3⇑D, center). B220low B cells were mostly IgD−CD138+ or CD138− cells, indicating that they were IgE preplasma cells or B220low memory B cells (Fig. 3⇑D, left). In pR1E4-treated mice, B220highIgE+ B cells were detected, but most B220lowIgE+ B cells were lost. The remaining B220highIgE+ B cells in pR1E4-treated mice were IgD+CD138− (Fig. 3⇑D), suggesting that pR1E4 blocked development of IgE memory B cells and preplasma cells. The frequency of the B220lowIgE+ fraction declined in pR1E4-treated mice (p = 0.0024; Fig. 3⇑F). Intracellular staining for IgE (preblocked for surface staining) was conducted, revealing that plasma cells remained in the spleens of pR1E4-treated mice (Fig. 3⇑E). Total numbers of B220low/intracellular IgE+ cells were on average lower in pR1E4-treated mice compared with control, but not to a statistically significant extent. We conclude that in anti-IgD- stimulated mice pR1E4 could neutralize circulating IgE and suppress the numbers of mIgE+ B cells, but could not suppress fully developed IgE plasma cells.
Anti-IgE can block IgE secretion in vitro
To determine mechanistically how soluble rR1E4 affects IgE B cells, we assessed the effects of R1E4 mAb on cultured naive B cells induced to switch to IgE by the addition of IL-4 and anti-CD40. Although the binding of R1E4 has been mapped to the ε CH3 domain (9), it was not clear whether this epitope would be accessible on mIgE+ B cells because on B cells mIg is oriented differently than in solution and is associated with Igα/β signal transducers. We found that mIgE+ B cells could be clearly identified with both R1E4 IgG and rR1E4 Ab (Fig. 4⇓A). Moreover, inclusion of rR1E4 both inhibited the secretion of IgE in the supernatant and also reduced the frequency of CD138+ and CD138−IgE+ B cells emerging in the cultures (Fig. 4⇓, B–D). Ca2+ signaling analysis indicated that rR1E4 Ab could induce Ca2+ mobilization in mIgE+ B cells upon supercross-linking by a second Ab (rat anti-mouse IgG1; Fig. 4⇓E). These data indicate that anti-IgE can block the development of IgE plasma cells in vitro and may in part explain the in vivo effects of this treatment.
Ability of rR1E4 anti-IgE protein to affect the biological responses of IgE+ B cells generated in tissue culture. A, BALB/c spleen cells were induced to undergo IgE class switch by culture at 2 × 105 cells/ml for 4 days with IL-4 (25 ng/ml) and anti-CD40 (10 μg/ml); binding by rR1E4 was examined. Right panel shows staining with rR1E4 (10 μg/ml) followed by FITC anti-mouse IgG1 and Alexa Fluor 647 anti-IgE (EM95). B–D, Spleen cells (2 × 105 cells/ml) were stimulated for 4 days with IL-4 (25 ng/ml) and anti-CD40 (1 μg/ml) with or without 20 μg of rR1E4. B, IgE levels in culture supernatants on day 4 as measured by ELISA. C, Analysis of IgE+ B cell and IgE+ plasma cell frequencies at day 4 of culture, as determined by staining for intracellular IgE, B220, and CD138. D, Summary of rR1E4-induced changes in IgE subpopulations as shown in C. E, rR1E4-induced calcium flux in fluo-4-loaded B cells recovered at day 4 of culture. Top panel, B cells were treated with either anti-Igκ or rR1E4 alone. Lower panel, Cells were precultured with anti-IgG1 alone for 30 min (to obscure the response of IgG1 cells) and then stimulated with either anti-IgG1 or rR1E4, which is supercross-linked by free anti-IgG1. Results presented in B and D represent the means and SDs of four separate experiments. *, p < 10−4 and #, p < 0.05.
Anaphylaxis reaction
The ability of in vivo pR1E4 treatment to suppress allergic responses was tested by challenging mice on day 13 after treatment with an activating anti-IgE, EM95, which has the ability to induce mast cell degranulation and anaphylactic manifestations. Surprisingly, and notwithstanding their suppressed IgE levels, pR1E4-treated mice challenged in this way showed a systemic anaphylaxis reaction as indicated by rapidly lowered body temperature (Fig. 5⇓A). Recently, an alternative pathway for anaphylaxis has been elucidated in which IgG1-immune complexes activate FcγRIII on basophils, releasing platelet-activating factor (15, 24, 25, 26, 27). To test the possibility that this pathway was triggered by clustering of anti-IgE Abs upon challenge, pR1E4-treated or control mice were given FcγRII/III-blocking mAb 2.4G2 (28) 1 day before challenge. Indeed, anaphylaxis was blocked in pR1E4-treated but not in control mice receiving 2.4G2 (Fig. 5⇓B). To test whether mouse IgG1 R1E4 could induce anaphylaxis directly in a FcεRI-independent manner, we preinjected TNP-BSA and IgE anti-TNP into FcεRIα−/− mice and challenged them 1 h later with rR1E4. As shown in Fig. 5⇓C, mice that received rR1E4 manifested an anaphylaxis reaction, indicating that rR1E4 itself triggered the reaction through FcγRs. Collectively, these data indicate that pR1E4 treatment suppresses FcεRI-mediated anaphylaxis, but permits and in fact promotes FcγRIII-mediated anaphylaxis.
Systemic anaphylaxis reactions in rR1E4-treated mice. A, Untreated BALB/c (wild-type (WT)) mice or pR1E4-treated (Sec) mice were challenged with 100 μg of activating anti-IgE mAb EM95 and rectal temperatures were measured over 60 min. B, Mice received 0.5 mg of Fc-blocking mAb 2.4G2 twenty-four hours before challenge with 100 μg of anti-IgE EM95. Untreated BALB/c (WT + Fc), pR1E4 treated (Sec + Fc). C, FcεRIα−/− mice received 10 μg of TNP-BSA and 100 μg of anti-TNP IgE (IgELa) 1 h before challenge with 50 μg of rR1E4. Shown are means and SDs of the results obtained with the following numbers of mice per group. All groups included three mice, except pR1E4-treated mice in A which included four mice.
Discussion
Although it has been known that anti-IgE Abs that block IgE-FcεRI interactions prevent acute allergic reactions, it has not been clear that such Abs have strong effects on mIgE+ B cells nor that a gene therapy approach to their application would be possible. Our data support earlier work of Haba and Nisonoff (6) who demonstrated that high doses of syngeneic anti-IgE could reduce IgE Ab-forming responses when given just before immunization. We show here that expression of an anti-IgE single-chain fusion construct can drive prolonged expression of a presumably dimeric anti-IgE protein in vivo and that this recombinant anti-IgE can block IgE binding to mast cells and basophils and suppress new IgE production. Because rR1E4 Ab was able to bind to mIgE+ B cells in vitro and to trigger an altered in vitro response of these cells, we conclude that it can trigger signals in mIgE+ B cells. Omalizumab (Xolair) neutralizes circulating IgE in humans, providing relief from allergic symptoms, apparently without reducing the underlying IgE stimulus. In a human trial of Xolair, the serum concentration of free IgE dropped rapidly (to 13.9 ng/ml), whereas the total IgE concentration increased over time (to >1000 ng/ml for 120 days) (29). By contrast, in the present study pR1E4 treatment suppressed both free IgE and total IgE and RNA analysis indicated that it also suppressed new IgE synthesis. These results may suggest that Xolair fails to suppress mIgE+ B cells. If this is the case, our data suggest the possibility that more effective anti-IgE mAbs may be found with the potential for longer-term benefit. Such Abs would ideally share Xolair’s ability to block IgE-FcεRI interactions, but also be able to suppress IgE+ B cells. Our preliminary studies indicate that Xolair is able to trigger Ca2+ mobilization in a B cell line carrying human mIgE. However, it may be that Xolair promotes rather than inhibits IgE production because of the quality of this signal or through its ability to generate high-order immune complexes.
We presume that in vivo expression of rR1E4 mediates negative regulation of developing memory IgE B cells, probably by induction of apoptosis. However, the numbers of mIgE+ B cells were too low to directly demonstrate this. Indeed, there is some controversy over whether mIgE+ B cells stably exist in vivo (5). Several studies demonstrate that IgE class switching often occurs through an IgG1 intermediate (30, 31, 32, 33). On the other hand, there are clear indications that B cells giving rise to IgE responses must carry mIgE and functionally signal through mIgE, at least for a short time. The membrane exons and protein sequences of mammalian IgEs are well conserved and similar to other membrane Igs (34). Moreover, mutations or truncations of the membrane exons of IgE severely inhibit IgE responses, particularly secondary responses (7). The membrane form of ε H chain mRNA may be poorly expressed owing to inefficient polyadenylation signals (35), suggesting that IgE memory B cells may express relatively low mIgE+ levels. Nevertheless, Abs directed to the membrane form of IgE can have toleragenic effects (8). In the present studies, we detected mIgE+ cells and showed that their numbers increased in appropriately immunized mice. Moreover, mIgE+ cells were specifically reduced in pR1E4-treated mice (Fig. 3⇑C), indicating that negative regulation of developing IgE B cells by IgE-reactive ligands is possible.
The potency of IgE in allergic reactions is a result of the extraordinarily high affinity of FcεRI for monomeric IgE (Ka = 1010 M−1) combined with the powerful biological consequences of FcεRI ligation on mast cells and basophils (2). But the levels of IgE in blood were very low compared with other types of Igs, facilitating the effectiveness of treatment with passively administered IgE-neutralizing Abs and, as we show here, gene therapy using recombinant anti-IgE plasmid. We expressed the anti-IgE-neutralizing Ab as scFv fusion protein with a naked DNA injection method. The merit of this method is that gene expression is potentially long-lasting and it also permits the expression of membrane-bound proteins in vivo. Importantly, soluble rR1E4 expression lasted >3 wk after plasmid injection and could completely neutralize serum IgE, as measured by the level of free IgE and the levels of IgE bound to mast cells and basophils. Although convenient for proof of principle, plasmid injection provides only transient expression. Other modes of gene transfer, including retroviral transduction, should ultimately be more effective and practical in a clinical setting and may provide permanent IgE suppression. Long-term anti-IgE gene expression is predicted to not only suppress new plasma cell formation but also to neutralize IgE secreted by long-lived plasma cells that are no longer subject to regulation by surface Ig.
An unexpected finding was that even when IgE was neutralized mice still underwent anaphylaxis upon challenge with an activating anti-IgE stimulus, presumably through a FcγRIII-mediated pathway triggered by cross-linking IgE-rR1E4 complexes. We also showed that rR1E4 administration induced anaphylaxis in mice with IgE-Ag complexes. A comparable reaction may occur in a small fraction of anti-IgE-treated patients. According to the U.S. Food and Drug Administration (http://www.fda.gov/Cder/drug/InfoSheets/HCP/omalizumabHCP.htm), the frequency of anaphylaxis attributed to Xolair use in patients was ≥0.2%. Of reported cases, 39% occurred after the first dose of Xolair, while only 19% occurred with the second dose, indicating the cause of anaphylaxis was not anti-Id (anti-Xolair) Ab. Xolair is a humanized IgG1 mAb that makes complexes with IgE at various ratios, the dominant complex being a trimer (36). If patients have both IgE and IgG Abs against the same Ag or IgE-bound foreign Ag itself, Xolair infusion may form higher order immune complexes and induce anaphylaxis through clustering of FcγRIII (Fig. 6⇓). Conceivably, such reactions may be influenced by polymorphisms in FcγRIII and FcγRII (37, 38). To prevent anaphylaxis, mutagenesis of therapeutic anti-IgE to prevent FcγRIII binding may be desirable. Alternatively, expression of soluble monovalent or membrane- tethered anti-IgE may be used. Overall, our data suggest several ways that anti-IgE therapy can be improved to facilitate safety and longer-term effectiveness. We also show the feasibility of providing the Ab by gene therapy, which may provide a strategy to permanently suppress IgE reactions.
Schematic of how IgE-anti-IgE complexes may promote FcγR activation. A, Depicted are IgE, anti-IgE, and a bivalent Ag. B, IgG anti-IgE Abs such as Xolair carry Fc portions able to interact with FcγRs. Upon interaction with IgE, anti-IgE may form small complexes that block binding of IgE to FcεRI and that are of too low valency to bind strongly to FcγRs. C, Higher order complexes of IgE:anti-IgE formed by cross-linking with an additional ligand, such as the IgE’s cognate Ag (allergen) shown here, permits multipoint binding to FcγRs.
Acknowledgments
We thank Dr. Fred D. Finkelman and Dr. Daniel Conrad for providing R1E4 and EM95 hybridoma cells. We thank Patrick Skog for technical assistance.
Disclosures
The authors have no financial conflict of interest.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵1 This work was supported by National Institutes of Health Grant R21AI069866 and by a Pfizer Postdoctoral Fellowship (to T.O.).
↵2 Address correspondence and reprint requests to Dr. David Nemazee, Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address: nemazee{at}scripps.edu
↵3 Abbreviations used in this paper: mIgE, membrane IgE; scFv, single-chain variable fragment; qPCR, quantitative PCR; TNP, 2,4,6-trinitrophenyl.
↵4 The online version of this article contains supplemental material.
- Received January 30, 2009.
- Accepted April 10, 2009.
- Copyright © 2009 by The American Association of Immunologists, Inc.