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Chronic rhinosinusitis with nasal polyps is associated with local immunoglobulin hyperproduction and the presence of IgE antibodies against Staphylococcus aureus enterotoxins (SAEs). Aspirin-exacerbated respiratory disease is a severe form of chronic rhinosinusitis with nasal polyps in which nearly all patients express anti-SAEs.
We aimed to understand antibodies reactive to SAEs and determine whether they recognize SAEs through their complementarity-determining regions (CDRs) or framework regions.
Labeled staphylococcal enterotoxin (SE) A, SED, and SEE were used to isolate single SAE-specific B cells from the nasal polyps of 3 patients with aspirin-exacerbated respiratory disease by using fluorescence-activated cell sorting. Recombinant antibodies with “matched” heavy and light chains were cloned as IgG1, and those of high affinity for specific SAEs, assayed by means of ELISA and surface plasmon resonance, were recloned as IgE and antigen-binding fragments. IgE activities were tested in basophil degranulation assays.
Thirty-seven SAE-specific, IgG- or IgA-expressing B cells were isolated and yielded 6 anti-SAE clones, 2 each for SEA, SED, and SEE. Competition binding assays revealed that the anti-SEE antibodies recognize nonoverlapping epitopes in SEE. Unexpectedly, each anti-SEE mediated SEE-induced basophil degranulation, and IgG1 or antigen-binding fragments of each anti-SEE enhanced degranulation by the other anti-SEE.
SEEs can activate basophils by simultaneously binding as antigens in the conventional manner to CDRs and as superantigens to framework regions of anti-SEE IgE in anti-SEE IgE-FcεRI complexes. Anti-SEE IgG1s can enhance the activity of anti-SEE IgEs as conventional antibodies through CDRs or simultaneously as conventional antibodies and as “superantibodies” through CDRs and framework regions to SEEs in SEE–anti-SEE IgE-FcεRI complexes.
Staphylococcus aureus and its superantigens are implicated in the intense inflammatory processes of the upper and lower airways in patients with allergic diseases.1 These superantigens, in particular Staphylococcus aureus enterotoxins (SAEs), are strongly associated with chronic rhinosinusitis with nasal polyps (CRSwNP), particularly in the subpopulation of patients with aspirin-exacerbated respiratory disease (AERD), as well as those with allergic rhinitis, asthma, and atopic dermatitis.2, 3, 4, 5 SAEs are a family of structurally related proteins comprising different serological types, such as staphylococcal enterotoxin (SE) A, SEB, SEC, SED, and SEE (up to SEU) and toxic shock syndrome toxin 1 (TSST-1).6 SAEs are potent T-cell superantigens, causing polyclonal activation of up to 25% of certain T-cell populations by interacting with a common β-chain structural framework region in the T-cell receptor (TCR),7, 8 rather than the complementarity-determining region (CDR), which recognizes specific antigenic peptides bound to MHC. The activity of SEA and SED on B cells in vitro suggests that they can also act as B-cell superantigens by binding to the common structural framework regions in the immunoglobulin heavy-chain variable region (VH) domains shared by immunoglobulins with different CDRs,9, 10, 11 as previously shown for staphylococcal protein A (SpA).12, 13, 14 This might increase the polyclonality of the B-cell repertoire in patients with CRSwNP and allow S aureus to escape immune surveillance.15, 16
Nasal polyps in patients with CRSwNP are inflammatory outgrowths of the paranasal sinus mucosa, which are generally characterized by TH2 inflammation, local immunoglobulin production, and eosinophil infiltration driven by IL-5 and eotaxin.17, 18, 19, 20 Up to 100% of patients with AERD express anti-SAE IgEs in their nasal polyp homogenates and often have a higher prevalence of comorbid asthma and eosinophilic inflammation,17, 21, 22, 23 and IgEs from nasal polyps activate basophils in response to allergens and SEB in vitro.15 SEB acts as a human T-cell superantigen in vivo to cause the symptoms of atopic dermatitis.24 Basophils isolated from patients with atopic dermatitis with anti-SAE IgE in their sera, but not those from healthy control subjects, were responsive to SAEs.25 Although anti-SAE IgE can be detected in the circulation of patients with CRSwNP, B cells expressing this IgE are confined to the nasal mucosa, suggesting a role in sinonasal inflammation.17 Whether the SAEs bind to CDRs, framework regions, or both is addressed in the present work.
Antibody production in nasal polyps of patients with CRSwNP is driven by local activation and differentiation into plasma cells of B cells on exposure to various aeroallergens and microbial antigens.26, 27 Thus nasal polyps removed from patients with AERD provide a unique source of tissue to study how SAEs can shape the local antibody repertoire. In the first study of this kind, we used an efficient single-cell RT-PCR method to clone and express antibodies from single SAE-specific B cells isolated from the nasal polyps of 3 patients with AERD and tested their primary function in effector cell activation.
Nasal polyps and sera were collected from 3 patients with AERD (HPK-014, HPK-016, and HPK-018) at the Royal National Throat, Nose and Ear Hospital, London, United Kingdom. The patients were male, with a mean age of 56 years. Only patient HPK-014 had positive skin prick test responses to aeroallergens. The ethical committee representing the Royal National Throat, Nose and Ear Hospital approved the study, and all patients provided written informed consent before the study commenced.
Detailed methods for processing sera and nasal polyp homogenates are available in the Methods section in this article's Online Repository at www.jacionline.org. All sera from blood samples and homogenates from nasal polyps were assayed for total IgE and specific IgE to SEA, SEB, SEC1, SED, SEE, and TSST-1 by using the UniCAP system (Phadia, Uppsala, Sweden; see Table E1 in this article's Online Repository at www.jacionline.org).
Single-cell suspensions from frozen nasal polyp samples were prepared by 1 hour of digestion with 1 mg/mL hyaluronidase (Sigma-Aldrich, St Louis, Mo) and 1 U/mL Liberase TL (Roche, Mannheim, Germany) at 37°C. Samples were stained for CD19, CD138, SEA, SED, and SEE. SAE-specific single B cells were sorted by means of fluorescence-activated cell sorting (FACS) into 96-well PCR plates for cDNA synthesis and immunoglobulin expression cloning as IgG1 (Fig 1). Full details of the cloning and expression strategy are available in Fig E1 and Table E2 in this article's Online Repository at www.jacionline.org.
SEA-, SED-, and SEE-specific antibodies were expressed in human embryonic kidney 293T cells (ATCC, Manassas, Va) as IgG1. Large-scale production of IgE, IgG1, and antigen-binding fragments (Fabs) was carried out with Expi293 cells (Invitrogen, Carlsbad, Calif). The IgG1 antibodies were purified by means of protein A–Sepharose (GE Healthcare, Pittsburgh, Pa), the IgE antibodies by omalizumab-coupled NHS-activated Sepharose (GE Healthcare), and the Fabs by LambdaFabSelect agarose (GE Healthcare). Anti-SAE IgG1 antibodies were identified in culture supernatants by means of routine ELISA (Fig 2), and their purity was estimated by means of SDS-PAGE under reducing conditions (see Fig E2 in this article's Online Repository at www.jacionline.org). Further details are available in the Methods section in this article's Online Repository.
Immunoglobulin gene sequences were analyzed by using the IMGT/V-Quest tool,28 allowing identification of their clonal signatures (unique CDR3 sequences); germline V, D, and J genes; and somatic mutations. Sequences are available in GenBank (http://www.ncbi.nlm.nih.gov/genbank), and their accession numbers are listed in Table E3 in this article's Online Repository at www.jacionline.org.
Surface plasmon resonance (SPR) with a Biacore T200 instrument (GE Healthcare) was performed to determine binding specificity, kinetics, and affinity of the antigen-antibody interactions. Further details are available in the Methods section in this article's Online Repository.
Degranulation of cells of the rat basophilic cell line RBL SX-38, which stably expresses the α, β, and γ chains of human FcεRI,29 was used as a routine method to measure the functionality of the SAE-specific antibodies. Further details are available in the Methods section in this article's Online Repository.
Three patients with AERD, HPK-014, HPK-016, and HPK-018, with titers of specific anti-SAEs in their serum or nasal polyp homogenate were studied (see Table E1). Specific IgEs to 6 common SAEs (SEA, SEB, SED, SEE, SEC, and TSST-1) were detected in all 3 homogenate samples and in the serum of patient HPK-018 and to 3 SAEs (SEA, SEE, and TSST-1) in the serum of patient HPK-14, whereas none were detected in the serum of patient HPK-016. Percentages of the anti-SAE IgEs were approximately 10-fold higher in the homogenates compared with the sera, ranging from 0.38% to 2.35% and 0.03% to 0.31%, respectively (see Table E1).
Viable SAE-positive B cells (SAE+CD19+CD138− 7-aminoactinomycin D [7-AAD]−) identified by means of FACS (Fig 1) were sorted into individual wells of 96-well plates. SAE-bound B cells accounted for 0.18%, 0.08%, and 0.25% of the nasal polyp B-cell population of patients HPK-014, HPK-016, and HPK-018, respectively (see Table E4 in this article's Online Repository at www.jacionline.org). Matched immunoglobulin VH and light-chain variable region (VL; κ or λ) DNA pairs were amplified from the cDNAs of 37 SAE+CD19+CD138− single B cells (18 from patient HPK-014, 6 from patient HPK-016, and 13 from patient HPK-018; see Fig E1). Of these 37 VH sequences obtained, 23 were from γ chains, and 14 were from α chains. No VH sequence was obtained from ε chains. The VH genes used in these 37 VH sequences consisted of 12 VH1 (32.43%), 13 VH3 (35.14%), 6 VH4 (16.22%), 5 VH5 (13.51%), and 1 VH6 (2.7%) sequences. Three (HPK-014_2D6, HPK-018_2A11, and HPK-018_2C10) of the 37 B cells expressed both a κ and a λ chain. Of 40 VL sequences obtained, 25 were from the λ chain, and 15 were from the κ chain. All 37 B cells represented different clones ascertained by their unique CDR-H3 sequences (BioProject ID: PRJNA347912; https://www.ncbi.nlm.nih.gov/bioproject). We expressed all 40 of the putative SAE-specific matched variable regions as human IgG1 antibodies.
All 40 antibodies, expressed as IgG1, were assayed for the 6 SAEs detected in nasal polyp homogenates by means of ELISA. Only SEA-, SED-, and SEE-specific antibodies could be identified, which is consistent with screening the B cells for only these specificities. Two anti-SEA antibodies, HPK-018_1B6 (IgA1, IGHV1-46) and HPK-016_1F3 (IgG1, IGHV1-69); 2 anti-SED antibodies, HPK-014_1A4 (IgA2, IGHV3-34) and HPK-018_2D6 (IgA1, IGHV3-49); and 2 anti-SEE antibodies, HPK-014_1G2 (IgA1, IGHV3-30) and HPK-016_203 (IgG4, IGHV5-51), were identified (Fig 2). The germline immunoglobulin variable genes and isotypes used by those 6 antibodies are shown in Table E3. We refer to these antibodies below as the anti-SEA antibodies 1B6 and 1F3, the anti-SED antibodies 1A4 and 2D6, and the anti-SEE antibodies 1G2 and 203.
The V(D)J rearrangement and CDR signatures of the 6 anti-SAE antibodies are shown in Table E3. A higher frequency of λ light chains (5/6) was found when compared with that in human peripheral blood B cells (1/5).30 Sequence alignments of 6 antibodies against their most closely related germline VH and VL gene sequences revealed a relatively high frequency of amino acid replacements in framework regions of VH and VL chains (see Fig E3 in this article's Online Repository at www.jacionline.org).
ELISA was used to determine the specificity of the purified recombinant IgG1 antibodies with the SAEs (Fig 3, A-C, and results not shown). The 1G2 and 203 antibodies were specific for SEE, although both displayed cross-reactivity with SEC1 (27% amino acid sequence homology with SEE) to a different extent rather than the more homologous SEA and SED (81% and 54% sequence homology with SEE, respectively).31 1F3 was specific for SEA but was not cross-reactive with SEE. All 3 antibodies exhibited high affinity for their cognate SAEs, demonstrating low nanomolar binding affinities, as measured from the kinetics of interaction with specific SAEs by using SPR (Fig 3, D-F).
The γ1 chain constant region of the IgG1 antibodies was substituted with the human ε constant region and the resulting IgEs were expressed and purified to examine the activities of the corresponding anti-SAE IgEs (see Fig E2, B). Basophil degranulation activities of the 6 anti-SAE IgEs in the presence of the cognate SAE are shown in Fig 4.
Conventional antigen binding through CDRs to 2 identical receptor-bound monoclonal IgE molecules, each of which recognizes a single epitope in a monomeric antigen, should not induce cross-linking of FcεRI on the surfaces of basophils to cause basophil degranulation. The anti-SEAs (Fig 4, A) and the anti-SEDs (Fig 4, B) behaved as expected, whereas both anti-SEE IgEs, 1G2 and 203, were active in this assay (Fig 4, C). Basophil activation would require either 2 identical epitopes in the SEE because it formed a dimer like SED32 or 2 independent epitopes in an SEE molecule if it were a monomer. A recent crystal structure of SEE in a complex with TCR revealed a monomeric structure.33 Thus we conclude that SEE has 2 distinct and nonoverlapping epitopes that explains this activity; this is illustrated and discussed below. SEC1 did not trigger 1G2 or 203 IgE-mediated basophil degranulation, although both show cross-reactivity to SEC1 (Fig 4, D).
To map the epitopes of anti-SEE 1G2 and 203 antibodies on SEE, we carried out sandwich binding assays using ELISA and SPR. ELISA revealed that saturation of the 1G2 binding site, which was achieved through addition of SEE to immobilized 1G2 Fab and IgE, did not prevent binding of 203 IgG1 (see Fig E4 in this article's Online Repository at www.jacionline.org). Also, SPR revealed that binding of saturating concentrations of 1G2 IgG1 to immobilized SEE did not prevent the simultaneous binding of soluble 203 IgG1 (Fig 5, A). The reverse was also observed: after saturating all of the 203 binding sites on immobilized SEE, 1G2 IgG1 could still bind with high affinity (Fig 5, B). Both ELISA and SPR assays confirmed that the 2 anti-SEE antibodies bind to nonoverlapping epitopes on SEE.
The different anti-SEE isotypes, IgE and IgG1, were tested in pairwise combinations in basophil degranulation assays to characterize their interplay. As expected, because of competition for epitope binding to SEE, the 1G2 IgG1 inhibited SEE-induced degranulation of basophils sensitized with the 1G2 IgE, and the 203 IgG1 inhibited degranulation of basophils sensitized with the 203 IgE (Fig 6, A).34, 35 As also expected, the IgG1 that recognized the nonoverlapping epitope on IgE enhanced degranulation (Fig 7, A) by cross-linking 2 SEE molecules bound to adjacent receptor-bound IgE molecules; this is illustrated below in Fig 5, B. The 1F3 antibody, which is specific for SEA, had no such effects, demonstrating that antigen specificity is crucial for the modulation of IgE activity by IgG1.
We repeated the pairwise assays by using the 2 anti-SEE IgG1 Fabs (Fig 6, B). As expected, the Fabs exhibited the same inhibitory activity as the whole IgG1 antibodies (because of competitive inhibition) when Fab and IgE were of the same specificity. Cross-linking of adjacent IgE-SEE complexes on the basophil surface cannot occur with a monomeric Fab, even one that binds to the nonoverlapping epitope. Paradoxically, the Fab recognizing the nonoverlapping epitope retains the enhancing activity of the whole IgG1 (Fig 6, B).
Fig 7, C, illustrates the mechanism by which receptor cross-linking can occur. We suggest that high-affinity binding of receptor-bound anti-SEE IgE to SEE through its CDRs to the epitope represented in green (Fig 7, C) facilitates subsequent low-affinity binding through a site on SEE, which is represented in red, that binds to a framework region of an adjacent IgG (or Fab) that recognizes a nonoverlapping SEE epitope, which is represented in blue. We call such an antibody, in principle of any isotype that acts synergistically to enhance IgE effector functions, a “superantibody.”
Several human mAbs against specific allergens have been obtained from allergic patients by means of phage display,36, 37, 38, 39, 40 EBV transformation of PBMCs,41 or single-cell RT-PCR.34 In addition, many groups have generated anti-SEB mAbs by using either mouse hybridomas or phage display and pursued their use as therapeutic agents.42, 43 In earlier work we produced a recombinant allergen (Phl p 7)–specific antibody with the native (“matched”) heavy and light chain by using single-cell PCR.34 To the best of our knowledge, the present work is the first report of recombinant anti-SAE antibodies containing matched heavy and light chains cloned by using single-cell RT-PCR. We produced 6 anti-SAE antibodies that bind to SAEs with high affinity: 2B6 and 1F3 for SEA, 1A4 and 2D6 for SED, and 1G2 and 203 for SEE. The VH regions of 1F3 and 203 were derived from single IgG-expressing B cells and 1B6, 1A4, 1G2, and 2D6 from single IgA-expressing B cells, which were present in nasal polyps from 3 patients with CRSwNP (see Table E3). The antibodies were first expressed as IgG1 and then converted to IgE and Fab.
We began with the hypothesis that SAEs can act as B-cell superantigens, as well as T-cell superantigens. Indeed, this suggestion has often been made before. It is consistent with the evidence that SEA and SED bind weakly to immunoglobulins bearing VH3 and VH4, respectively.10, 11 Several authors have demonstrated the overabundance of VH5 in IgE, suggesting that such a B-cell superantigen can recognize members of this VH family.44, 45, 46, 47 Low-affinity and promiscuous binding to the TCR variable β (Vβ) chains are characteristics of T-cell superantigens, which include SEA, SED, and, notably, SEE.7, 8 SEE binds to 6 different human Vβ chains.48 We have shown that SEE can indeed behave as a B-cell superantigen by interacting with IgE molecules both conventionally through CDRs and unconventionally through framework regions (Fig 7, A).
ELISA assays used to select the 6 antibodies described in the present study (Fig 3) would not have detected weak binding of SAEs to the framework regions. However, strong binding of SAEs to the CDRs in the context of a cell (Fig 2) would facilitate weak binding to the framework regions because of an increase in avidity. Binding of SAEs to the framework regions as well as CDRs of BCRs can contribute to somatic hypermutation and affinity maturation of antibodies. Coker et al46 found 75% of mutational hot spots in the framework regions of IgE and IgA expressed by B cells in the nasal mucosa, which are often exposed to S aureus commensal infections.46 Superantigen-driven selection at the stage of local affinity maturation might explain the high frequency of mutations in the framework regions of our cloned anti-SAEs (see Fig E3).
Due to the real-world limitations of the experimental methods used in this study, we have not obtained definitive evidence for the proposed interactions (Fig 7). First, IgE-expressing antibodies were not among the 40 recombinant antibodies we expressed; this was not surprising because of the extremely low abundance of IgE- compared with IgG- and IgA-expressing B cells.49 Second, although we have shown that IgG-expressing B cells undergo local switching to IgE in nasal polyps from patients with CRSwNP and in the respiratory tract mucosa in patients with allergic diseases,50, 51, 52, 53 1G2 and 203 antibodies came from different patients. We would have needed IgE related to at least 1 of them and both antibody isotypes from the same subject to prove the existence of superantibodies in this AERD cohort. Nevertheless, in unpublished work using next-generation sequencing of B-cell repertoires in nasal polyps, we have observed as many as 17/10,000 IgE sequences related to IgG or IgA. In the B-cell repertoire of patient HPK-014, we were able to identify IgE relatives of 2 recombinant IgG1 antibodies (A: HPK-014_1A6 and B: HPK_014 2A8; see Fig E5 in this article's Online Repository at www.jacionline.org), although these 2 IgG1 clones were not among the 6 clones that exhibited high affinity for the SAEs (Fig 3).
One of the antibodies, 203, was originally an IgG4 and, interestingly, expresses IGHV5-51, the only of 2 VH5 family members that is overexpressed in IgE (Wu et al, unpublished results) and a candidate for binding in a superantigen-like manner.46 The other antibody, 1G2, was originally an IgA1 and expresses a member of the VH3 family (IGHV3-30, see Table E3). The CDRs differ between 203 and 1G2, and therefore it might be that SEE binds promiscuously to both VH5 and VH3 framework regions. Structural studies are required to define the epitopes in SEE.
It was previously shown by using x-ray crystallography that SpA is recognized by a rheumatoid factor antibody in a superantigen-dependent manner, in which SpA interacts with the framework residues of the VH3 domain.14 We believe the anti-SEE superantibodies presented here might be the first antibodies shown to have both conventional antigen- and superantigen-binding sites and thus to have superantibody activity.
The ability of the anti-SEE IgG1 and its Fabs to inhibit the activity of basophils sensitized by the related IgE resembles the blocking activities of IgG4 or IgA in specific allergen immunotherapy.34, 35, 54 Blocking IgG antibodies have been developed by many groups for therapeutic use as antitoxins. Nearly all antitoxin antibodies and all isolated anti-SEB antibodies are blocking, as opposed to enhancing, antibodies.42, 43, 55 SAE vaccination has been investigated, with encouraging success for efficacy in treating infections with S aureus.56, 57 Whether anti-SEE IgGs from the present study might be similarly efficacious could be determined, but a combination of anti-SAEs might be even more efficacious than any single anti-SAE.
To the best of our knowledge, 1G2 and 203 are the first examples of enhancing anti-SAE antibodies. However, there is a precedent in the case of the antibody BAB2 against a dominant birch pollen allergen, Bet v 1.58 BAB2 and, notably, its Fab enhanced binding of Bet v 1 to IgE and caused immediate-type hypersensitivity reactions in human skin. The authors suggested that enhancing antibodies could account for the failure of specific allergen immunotherapy.58, 59, 60 Although enhancing antibodies are relatively rare, they might be important because their activity might “win” in competition with an excess of blocking antibodies.
In previous studies it has been demonstrated that certain antibodies against a specific allergen are more frequently observed than others in atopic patients; such allergens, that is, Der p 1 and Der p 2 in patients with house dust mite allergy, are said to be dominant allergens.61, 62 These allergens usually have multiple epitopes, allowing more extensive cross-linking of the IgE-FcεRI complexes on effector cells. In this case noncompetitive allergen-specific IgG or IgA, which are likely present in much higher concentrations than IgE, might be able to enhance cell activation if the configuration of the 2 sites and the resulting distance between cross-linked complexes is favorable (Fig 7, B).
SEE was first documented in a case of food poisoning.63 It was later identified as a 26.4-kDa single-chain protein belonging to the group III superantigens (the SEA superfamily), having strong homology to SEA and SED, as stated above. SEE has been shown to be a potent enterotoxin and a polyclonal activator of T cells64, 65 and binds promiscuously to multiple human TCR Vβ chains.48 Anti-SEE IgE was demonstrated in 16.7% of nasal polyps compared with 0% of control tissue, as well as in all nasal polyp samples from all 3 patients with CRSwNP in the present study.17 Sequence comparison of SEA, SEC1, SED, and SEE and inspection of the SEA and SEE crystal structures32, 33 suggest that certain surface-exposed residues could alter the electrostatic potential or shape of the enterotoxin surface, rendering SEE and SEC1 different from SEA and SED and thereby contributing to the cross-reactivity of the 203 antibody.
Basophils were activated to release granular mediators by allergens and SEB when sensitized by IgE in homogenates from the nasal polyps of patients with CRSwNP in vitro,15 indicating the functionality of IgE antibodies in nasal polyps. Thus the putative anti-SEE IgEs 1G2 and 203 in patients HPK_014 and HPK_016, together with the potential IgG4 or IgA1 precursors, could have activated a variety of effector cells and contribute to the proinflammatory environment in the patients' nasal polyps. This is compatible with evidence from the work of Gevaert et al66 showing that omalizumab is therapeutic in patients with CRSwNP, although an earlier study did not show efficacy.67 It should also be noted that IgG and IgA superantibody activities are independent of isotypes.
In summary, our results provide proof of concept that SAES can act as B-cell superantigens and that anti-SAE antibodies of any isotype can behave as superantibodies. We have isolated antibodies with potential superantibody activity from nasal polyps of patients with AERD, a highly inflammatory TH2 disease. However, similar antibodies can occur in patients with allergic diseases and asthma associated with commensal S aureus infections and dominant allergens.
Anti-SAE IgE antibodies alone or together with IgG1 can contribute to the pathogenesis of CRSwNP and allergic disease.
Supported by the Medical Research Council (grant no. G1100090), the London Law Trust (to L.K.J.), Medical Research Council (Y.-C.B. Wu), the National Institute for Health Research (NIHR) Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London (to H.J.G., B.J.S., and J.M.M.), and the Wellcome Trust for support of the King's Biomolecular Spectroscopy Facility (085944). The views expressed are those of the authors and not necessarily those of the National Health Service, the NIHR, or the Department of Health.
Disclosure of potential conflict of interest: J.-B. Chen has received grants and support for travel to meetings for the study of other purposes from the Genomics Research Center, Academia Sinica. V. J. Lund has received consultancy fees from GlaxoSmithKline, Actelion, Vifor, Johnson & Johnson, and Navigent and payment for lectures from Neilmed and MSD. The rest of the authors declare that they have no relevant conflicts of interest.
Blood samples were allowed to clot at room temperature for 1 hour and centrifuged at 2000g for 20 minutes, and serum was separated and stored at −70°C. PBS containing protease inhibitor cocktail (Roche) was added to snap-frozen nasal polyp specimens at 1 mL per 0.1 g of tissue. Tissues were homogenized with a TissueLyser (Qiagen, Hilden, Germany) for 2 minutes at 30 Hz until disrupted, homogenates were centrifuged at 15,000g for 20 minutes at 4°C, and supernatants were collected and stored at −70°C.
After dispersion by means of enzymatic digestion of nasal polyps, cells were washed with complete RPMI medium (10% FBS in RPMI medium) to remove the enzymes, filtered through a 40-mm cell strainer, and finally resuspended at a concentration of 1 × 106 cells/mL in FACS buffer (5% normal goat serum [Invitrogen, Carlsbad, Calif] and 2 mmol/L EDTA in PBS). Then cells were stained with phycoerythrin-coupled anti-CD138 (BioLegend, San Diego, Calif), allophycocyanin-coupled anti-CD19 (BD Biosciences, San Jose, Calif), 7-AAD (eBioscience, San Diego, Calif), and biotinylated SAEs (Toxin Technology, Sarasota, Fla) in FACS buffer. Biotinylated SEA, SED, and SEE were simultaneously used for labeling cells each at a concentration of 10 μg/mL and detected by means of FACS with streptavidin–Alexa Fluor 488 (Invitrogen) at a dilution of 1:1000.
Single SAE+CD19+CD138−7-AAD− B cells were directly sorted into 96-well PCR plates (Fermentas, Vilnius, Lithuania) containing 18 μL/well of ice-cold 1× RT buffer (Invitrogen) containing 0.1% Triton-X 100, 10 mmol/L dithiothreitol, 300 ng of random hexamers (Invitrogen), 0.5 mmol/L dNTP mix (Fermentas), and 40 U of Ribolock RNase Inhibitor (Fermentas) on a FACSAria II cell sorter (BD Biosciences). Fifty units of Superscript III reverse transcriptase (Invitrogen) was added, and then reverse transcription was performed at 42°C for 10 minutes, 25°C for 10 minutes, 50°C for 60 minutes, and 85°C for 5 minutes.
Primer nucleotide sequences to cover all known V-region genes and reverse primers specific for κ and λ constant regions were as previously described.E1 Reverse primers for γ, α, and ε constant regions were Cγ1 (5′-GGAAGGTGTGCACGCCGCTGGTC-3′), Cε1 (5′-GGTTTTGTTGTCGACCCAGTCTG-3′), and Cα1 (5′-TGGGAAGTTTCTGGCGGTCACG-3′) as primary primers for the first-round PCR and Cγ2 (5′-GTTCGGGGAAGTAGTCCTTGAC-3′), Cα2 (5′-GTCCGCTTTCGCTCCAGGTCACACT-3′), and Cε2 (5′-TGGCATAGTGACCAGAGAGCGTG-3′) as nested primers for the second-round PCR, respectively. All PCRs were carried out by proofreading Phusion DNA polymerase (Fermentas). Second-round PCR products were sequenced after purification by using the QIAquick PCR Purification Kit (Qiagen).
Purified second-round PCR products were used as a PCR template for amplifying DNA inserts by V-region– and J-region–specific primers containing an overhang sequence (Table E2), which are homologous to the bases at one end of adjacent DNA fragments of pIgG1(κ) or pIgG1(λ) vectors.E2 Purified DraIII/NheI- and BspEI/NheI-digested DNA fragments from pIgG1(κ) and pIgG1(λ) vectors were used as a template for amplifying “backbone insert” by CL forward primers, SL-Cκ+ or SL-Cλ+, and the IgG leader reverse primer SL-IgG Leader- (Fig E1 and Table E2), respectively. All PCR products above were synthesized by using Phusion DNA polymerase and purified with the QIAquick PCR Purification Kit. The linear pIgG1(κ) and pIgG1(λ) vectors were prepared by means of double digestion with DraIII/NheI and BspEI/NheI, respectively, and purified by means of agarose gel electrophoresis and gel extraction (gel purification kit, Qiagen). The assembly of antibody expression vector was carried out by means of homologous recombination with a Geneart Seamless Assembly Kit (Invitrogen) according to vendor's instructions as shown in Fig E1.
Briefly, 20 ng of paired VH and VL DNA segments, 50 ng of backbone insert, and 100 ng of linear vector were combined with Seamless enzyme mix and incubated at room temperature for 30 minutes. The assembly products were immediately transformed into competent E coli (One Shot Top 10, Invitrogen), and transformants were analyzed for the presence of DNA inserts by using colony PCR. The plasmid DNA of positive clones was purified and sequenced to ensure no base mutations and correct recombination.
Human embryonic kidney 293T cells (ATCC) were seeded in 6-well plates to produce cloned antibodies on a small scale for validating their binding specificities. Transient transfections were performed at 80% cell confluency by linear polyethylenimine with an average molecular weight of 25 kDa (Polysciences, Warrington, Pa) as a transfection reagent. The cells were cultured for 2 days before the supernatants were harvested and analyzed by means of ELISA for SAE binding reactivity. The concentrations of recombinant IgG1 antibodies released into the supernatants of transfected 293T cells were measured by means of ELISA with human IgG Quantitation Kits (Bethyl Laboratories, Montgomery, Tex). The γ1 constant regions of SAE-specific antibody expression vectors were replaced by the ε constant region or the CH1 domain of the γ1 constant region, to construct IgE and Fab expression vectors, respectively.
Microtiter plates were coated with 2 μg/mL of each SEA, SEB, SEC1, SED, SEE, and TSST-1 (Toxin Technology) or ovalbumin (50 μL/well) overnight at 4°C and blocked with 200 μL of assay diluent (0.5% BSA, 0.05% Tween-20, and 0.01% thimerosal in PBS). Bound IgG1 antibodies were detected by using horseradish peroxidase–conjugated goat anti-human IgG Fc antibody (Jackson ImmunoResearch, West Grove, Pa) and developed with NeA-Blue TMB substrate (Clinical Science Products, Mansfield, Mass).
RBL SX-38 cells were typically grown in complete MEM medium, as previously described.E3 One day before a planned experiment, RBL SX-38 cells were seeded at 6 × 105 cells/mL (500 μL/well) in 48-well plates. After an overnight incubation, 250 μL of complete MEM medium containing SAE-specific IgE at 1 μg/mL was added to sensitize the cells. After a 2-hour incubation at 37°C, each well was washed twice with 500 μL of prewarmed Tyrode buffer (135 mmol/L NaCl, 5 mmol/L KCl, 5.6 mmol/L glucose, 1.8 mmol/L CaCl2, 1 mmol/L MgCl2, 20 mmol/L HEPES, and 0.5 mg/mL BSA, pH 7.3) and then 250 μL of Tyrode buffer containing SAEs at 1 μg/mL or polyclonal anti-human IgE antibody (Bethyl Laboratories) at 3 μg/mL or 1% Triton X-100 was added to the cells. After a 30-minute incubation in a 37°C incubator, culture media were collected and subjected to centrifugation at 300g for 5 minutes at room temperature. Fifty microliters of cleared supernatants were transferred to each well of a 96-well black OptiPlate (PerkinElmer, Waltham, Mass), and then 50 μL of substrate solution (80 μmol/L 4-methylumbelliferyl-N-acetyl-D-glucosaminide [Sigma-Aldrich] in 0.1 mol/L citric acid buffer, pH 4.5) was added to each well. The tape-sealed plate was incubated at 37°C for 1 hour, and the reaction was terminated by adding 100 μL of glycine buffer (0.2 mol/L glycine and 0.2 mol/L NaCl, pH 10.7). The resulting fluorescence (excitation, 355 nm; emission, 460 nm) was measured with a Victor 3 fluorescence reader (PerkinElmer). Measured values were expressed as percentages relative to the value of release (100%) obtained by lysing cells with 1% Triton X-100.
Purified and concentrated immunoglobulin solutions were quantified with a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, Del) by using absorbance at 280 nm, and the protein concentration was calculated with the extinction coefficient of each antibody, which was predicted by conducting the computation with a ProtParam program (http://web.expasy.org/protparam/). Biotin-labeled SEE and SEA were immobilized on a streptavidin-coated sensor chip (GE Healthcare). Binding of SAE-specific IgG1 antibodies was measured at 25°C by using a 3-minute association phase and a 10-minute dissociation phase (unless otherwise specified in the figure legend) in a concentration series ranging from 250 to 16 nmol/L. One antibody was injected for 3 minutes, followed by a 3-minute injection of the second antibody or buffer, to detect simultaneous binding of 2 antibodies. After regeneration with glycine-HCl (pH 2.5), the experiment was performed in reverse. Standard double-referencing data subtraction methods were used with a control surface (biotin alone).
|Patient ID||Total IgE||Serum||Total IgE||Nasal polyp homogenate|
|HPK-014||89.3||0.17 (0.19%)†||—||—||0.28 (0.31%)||—||0.16 (0.18%)||26.1||0.25 (0.96%)||0.12 (0.46%)||0.1 (0.38%)||0.13 (0.5%)||0.13 (0.5%)||0.21 (0.8%)|
|HPK-016||53.2||—||—||—||—||—||—||8.1||0.11 (1.36%)||0.11 (1.36%)||0.08 (0.99%)||0.09 (1.11%)||0.11 (1.36%)||0.19 (2.35%)|
|HPK-018||317||0.09 (0.03%)||0.81 (0.26%)||0.78 (0.25%)||0.13 (0.04%)||0.05 (0.02%)||0.07 (0.02%)||48||0.59 (1.23%)||0.24 (0.5%)||0.31 (0.65%)||0.27 (0.56%)||0.32 (0.67%)||0.28 (0.58%)|
|VH cloning PCR|
|Vκ cloning PCR|
|Vλ cloning PCR|
|JH cloning PCR|
|Jκ cloning PCR|
|Jλ cloning PCR|
|Backbone insert cloning PCR|
Homologous sequences to the bases at one end of the adjacent DNA fragment are displayed in lower case.
|Clone name||Isotype||Accession (VH)||IGHV||IGHD||IGHJ||CDR-H3 amino acids||Accession (VL)||IGK(L)V||IGK(L)J||CDR-L3 amino acids|
|HPK-014_1A4||A (α2)||KT369789||3-23||3-9||3||CAKKRRYDAVTGYLFDMW||KT369790||4-1 (κ)||2||CQQFYSPAPYTF|
|HPK-014_1G2||A (α1)||KT369791||3-30||6-13||4||CARDRAQGIVAAAGTSFGYW||KT369792||3-25 (λ)||2||CQSGDTSGFDPDVAF|
|HPK-016_203||G (γ4)||KT369793||5-51||2-15||4||CVRHCSGGRCSSAPLDSW||KT369794||6-57 (λ)||3||CLSHDTTNFVF|
|HPK-018_1B6||A (α1)||KT369795||1-46||2-8||3||CAYWGADGFDIW||KT369796||1-44 (λ)||3||CAVWDDSLSAWVF|
|HPK-018_1F3||G (γ1)||KT369797||1-69||3-9||4||CARGMTGYVGAYDYW||KT369798||1-44 (λ)||3||CASWDDRLNGPVF|
|HPK-018_2D6||A (α1)||KT369799||3-49||2-2||4||CTTYCSSTSCQIDYW||KT369800||2-14 (λ)||1||CSSYTGSSGYVV|
The VH and VL DNA sequences of each clone are available in GenBank (http://www.ncbi.nlm.nih.gov/genbank), and accession numbers are indicated in the table.
IGHV, Immunoglobulin heavy-chain variable region; IGKV, immunoglobulin kappa-chain variable region; IGLV, immunoglobulin lambda-chain variable region.
|Lymphocytes||10.1% (255,337)||7.42% (157,133)||17.2% (313,277)|
|Live cells (7-AAD−)†||94.3% (239,878)||87.1% (136,067)||95.3% (297,159)|
|Live B cells (CD19+7-AAD−)†||20.7% (52,717)||21.1% (33,024)||29.4% (91,607)|
|SAE+ cells (SAE+CD138−)‡||0.18% (92)||0.08% (26)||0.25% (225)|
|Plasma cells (SAE−CD138+)‡||2.98% (1,573)||0.1% (34)||0.37% (339)|