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Laboratories of Immunology and Antibody Glycan Analysis, Institute for Nutrition Medicine, University of Lübeck & University Medical Center Schleswig Holstein, Lübeck, GermanyLaboratory of Glycodesign and Glycoanalytics, Institute for Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Charité–University Medicine Berlin, Berlin, Germany
Laboratories of Immunology and Antibody Glycan Analysis, Institute for Nutrition Medicine, University of Lübeck & University Medical Center Schleswig Holstein, Lübeck, GermanyDepartment of Anesthesiology and Intensive Care, University Medical Center Schleswig Holstein, Lübeck, Germany
Division of Clinical & Molecular Allergology, Research Center Borstel, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Borstel, GermanyInterdisciplinary Allergy Outpatient Clinic, Department of Internal Medicine, University of Lübeck, Lübeck, Germany
Division of Immunology, Allergy and Rheumatology, Department of Medicine, University of Cincinnati College of Medicine, Cincinnati, OhioDivision of Immunobiology, Cincinnati Children's Hospital Medical Center, Cincinnati, OhioDepartment of Medicine, Cincinnati Veterans Affairs Medical Center, Cincinnati, Ohio
In contrast, allergen-specific IgG antibodies, which are also induced by allergen-specific immunotherapies (AITs), can inhibit IgE-mediated anaphylaxis caused by low levels of allergen through allergen masking and cross-linking of FcεRI with the classical IgG inhibitory receptor FcγRIIb.
However, when allergen levels are high, IgG antibodies induced in untreated and AIT-treated allergic patients, as well as to medical drugs, also have the potential to mediate anaphylaxis by activating classical activating FcγRs on different immune cell types.
Systemic anaphylaxis in the mouse can be mediated largely through IgG1 and Fc gammaRIII. Assessment of the cardiopulmonary changes, mast cell degranulation, and death associated with active or IgE- or IgG1-dependent passive anaphylaxis.
However, the effects of IgG subclasses and Fc glycosylation patterns in allergy remain unclear.
We first compared the capacity of differently glycosylated forms of murine IgG1, a subclass that resembles AIT-induced human IgG4 in its limited ability to activate complement and classical activating FcγRs,
to inhibit IgE-mediated systemic anaphylaxis (Fig 1, B-E, and see Fig E1 and the Methods section in this article's Online Repository at www.jacionline.org). IgE-mediated anaphylaxis (assessed as decreased rectal temperature) was induced intravenously with 10 μg of IgE anti-2,4,6-trinitrophenyl (TNP) mAbs, followed by an intravenous challenge 24 hours later with 1 μg of TNP-coupled ovalbumin (TNP-OVA; Fig 1, B). Increasing doses of differently glycosylated murine IgG1 anti-TNP mAbs (clone H5; native = low-galactosylated, in vitro galactosylated, or in vitro galactosylated plus sialylated) decreased IgE-mediated hypothermia in an FcγRIIb-dependent manner (Fig 1, B-E, and see Fig E1).
Even though low-galactosylated IgG1 showed a tendency for more efficient inhibition (Fig 1, C; 3 μg of IgG1; not significant), possibly because of its higher affinity than sialylated IgG1 for FcγRIIb,
the IgG glycosylation pattern (Fig 1, D and E) and IgG subclass (studied by comparing IgG1, IgG2a, and IgG2b anti-TNP class-switch variant mAbs with identical V[D]J sequences; Fig E1, G, and data not shown)
was IgG subclass– and glycosylation-dependent (Fig 1, G and H, and see Fig E1). Desialylated plus degalactosylated IgG2a and IgG2b subclass anti-TNP switch variant mAbs induced more severe anaphylaxis than desialylated plus degalactosylated switch variant and low-galactosylated (H5) IgG1 mAbs (IgG2a = IgG2b > IgG1, see Fig E1).
IgG1-mediated anaphylaxis was inhibited by means of galactosylation and especially by means of additional sialylation (Fig 1, G); sialylation also significantly reduced the anaphylaxis potential of IgG2b and tended to reduce that of IgG2a (Fig 1, G). Sialylation even reduced the increased anaphylaxis potential of IgG1 in FcγRIIb-deficient mice (Fig 1, H),
suggesting the importance of additional/other inhibitory mechanisms of IgG1 sialylation, such as one dependent on the C-type lectin receptor SignR1 (specific ICAM-3 grabbing nonintegrin-related 1) (Fig 1, I).
These observations suggest that AIT protocols that promote sialylation of human IgG4 might optimally limit the possibility of IgG-mediated systemic anaphylaxis in the presence of higher allergen doses.
To evaluate this assumption, we analyzed how conventional AIT with birch pollen extract and the adjuvant aluminum hydroxide (alum, ALK-depot SQ; ALK-Abelló, Hørsholm, Denmark) affects the IgG subclass and glycosylation of anti–Bet v 1 (Betula verrucosa 1; the major birch pollen allergen) antibodies (see Fig E4).
In untreated patients Bet v 1–specific IgG4 titers were constantly low, whereas IgE and IgG1 titers increased during the pollen season (Fig 2, A, and see Fig E2). In contrast, during AIT, levels of Bet v 1–specific IgG1 increased in the first 12 months but decreased afterwards, whereas Bet v 1–specific IgG4 titers increased persistently (Fig 2, A, and see Fig E2).
However, the Fc glycosylation profile of Bet v 1–specific serum IgG antibodies from untreated and AIT-treated patients remained stable and was more highly galactosylated and sialylated than that of IgG autoantibodies from patients with rheumatoid arthritis (Fig 2, B and C, and see Fig E2).
The glycosylation profiles of the AIT-treated patients resembled those of 2 recently described patients with AIT who had received similar therapy with alum (Allergovit; Allergopharma, Reinbek, Germany)
Consistent with an inverse relationship between IgG sialylation and inflammatory potential, we found that desialylation of native Bet v 1–specific IgG from the sera of AIT-treated patients strongly increased its ability to activate neutrophils in vitro (Fig 2, B-E, and see Fig E2).
These observations suggest that conventional AIT with alum induces sialylated IgG(4) antibodies that probably have low potential to induce IgG-mediated allergic reactions. However, studies remain required to assess how Fc glycosylation modulates the effector functions of human IgG1 and IgG4 and how new AIT protocols with distinct adjuvants
will influence the human IgG subclass distribution and Fc glycosylation pattern and, consequently, the risk of IgG-mediated allergic reactions. To initiate such studies, we compared the effects of enriched complete Freund adjuvant (eCFA; highly inflammatory), alum, and monophosphoryl lipid A (MPLA; recently approved for AIT)
on IgG subclass and Fc glycosylation profiles in OVA-immunized mice (Fig 2, F, and see Fig E3). eCFA induced the highest IgG titer (eCFA > MPLA = alum; see Fig E3, C), but all 3 immunizations induced predominantly IgG1 (alum/94% > eCFA/81% > MPLA/65%), followed by IgG2b and hardly any IgG2c (IgG2 [IgG2b + IgG2c]: MPLA/35% > eCFA/19% > alum/6%; Fig 2, G, and see Fig E3, C), the functions of which depend on galactosylation (only IgG1) and sialylation (IgG1 and at least in part IgG2b; Fig 1, G).
In contrast to only small differences in the Fc glycosylation pattern between human IgG subclasses in the same sample,
Because alum and MPLA induced higher galactosylation and sialylation levels of both OVA-specific IgG1 and IgG2(b) than OVA-eCFA (Fig 2, H), MPLA, with the highest ratio of IgG2(b), induced the highest levels of total IgG galactosylation and sialylation, as determined by using HPLC glycan analysis (Fig 2, I, and see Fig E3). Consistently, only 100 μg of purified OVA-specific IgG antibodies from the OVA-eCFA group, but not from the OVA-MPLA group, induced IgG-mediated anaphylaxis (Fig 2, J).
Taken together, our data suggest that although IgG subclass and glycosylation patterns have relatively little effect on IgG antibody blocking of IgE-mediated anaphylaxis, increased sialylation of IgG(4) antibodies should decrease the risk of IgG-induced anaphylaxis in the presence of high allergen doses. Accordingly, it seems advisable to select adjuvants for new AIT protocols
for their ability to promote sialylated IgG(4) antibody responses.
The murine IgG1, IgG2a, and IgG2b anti-TNP hybridoma switch variants were a gift from Lucien Aarden (Amsterdam, The Netherlands), and the murine IgG1 anti-TNP (clone H5) hybridoma cell line was from Birgitta Heyman (Uppsala, Sweden).
Serum samples of 7 patients (P2, P5, P7, P8, and P11-P13) from a previously published cohort of 15 patients
and 4 additional patients (P19-P22) who completed a 3-year subcutaneous AIT with birch pollen extract containing alum (ALK-depot SQ; ALK-Abelló) were investigated. Before treatment, all 11 patients had allergic symptoms to birch pollen (rhinoconjunctival symptoms with or without asthma) and were characterized by a positive skin prick test response with birch pollen extract (ALK Prick SQ; ALK-Abelló), total serum IgE concentrations, and serum IgE reactivity against both birch pollen extract and Bet v 1 (kUA/L; Phadia ImmunoCAP System; Thermo Fisher, Uppsala, Sweden; Fig 2, and Figs E2 and E4). The studies were approved by the Ethics Committee of the Medical Faculty of Philipps University, Marburg, Germany; all patients provided written informed consent to participate in the trials. Additionally, sera from untreated patients with moderate-to-severe birch pollen allergy symptoms were collected during (n = 8) or outside (n = 8) the birch pollen season (Fig E4) after patients provided written informed consent (Ethics Committee of the Medical Faculty of the University of Lübeck; approval no. 12-042). Bet v 1–specific IgE, IgG, IgG1, and IgG4 titers of untreated (season, n = 8; no season, n = 6) and all treated patients and Fc glycosylation profiles of purified Bet v 1–specific IgG antibodies from 10 of the 16 untreated patients during (n = 5) or outside (n = 5) the birch pollen season and 5 randomly selected treated patients (P2, P11, P13, P19, and P21) at different time points (Fig 2 and Figs E2 and E4) were analyzed.
All mice were bred and maintained in the specific pathogen-free facilities at the University of Lübeck or the Cincinnati Children's Research Foundation, and all experiments were done with approval of and in accordance with regulatory guidelines and ethical standards set by the University of Lübeck and the Ministry of Schleswig-Holstein, Germany, or the Institutional Animal Care and Use Committee of Cincinnati Children's Hospital Medical Center, respectively. BALB/c and C57BL/6 wild-type mice were purchased from Charles River Laboratories (Malvern, Pa). Fcgr2b−/− mice had been backcrossed for a minimum of 10 generations to the BALB/c or C57BL/6 background.
Only 8- to 12-week-old female mice were analyzed in the experiments. Mice were randomly assigned to groups, but a specific randomization program was not used.
TNP-coupled BSA (TNP-BSA), BSA, and TNP-OVA were purchased from Biosearch Technologies (Novato, Calif), and OVA was purchased from Sigma-Aldrich (St Louis, Mo). Incomplete Freund adjuvant was purchased from Sigma-Aldrich. eCFA was prepared by adding heat-killed Mycobacterium tuberculosis H37 RA (DIFCO Laboratories, Oxford, United Kingdom) to incomplete Freund adjuvant (5 mg of Mycobacterium tuberculosis/mL).
Alum was purchased from Thermo Scientific (Waltham, Mass), MPLA was derived from Salmonella minnesota R595 (MPLA-SM; # vac-mpla; InvivoGen, San Diego, Calif) and IVIG (Intracet from Biotest [Boca Raton, Fla]; pooled serum IgG of healthy donors used in high concentrations [2 g/kg] to treat patients with acute flares of autoimmune disease).
were authenticated by using an antigen-specific IgG subclass ELISA and grown in 0.03% Primatone RL/UF (175#DR from Kerry Biosciences, Tralee, Ireland) for antibody production. The IgG hybridoma cell lines had negative test results for Mycoplasma species contamination. IgG mAbs were purified from cell culture medium with Protein G–Sepharose (GE Healthcare, Little Chalfont, United Kingdom), and IgE anti-TNP mAbs were purified with TNP-BSA coupled to cyanogen bromide (CNBr)–activated Sepharose 4B (GE Healthcare) prepared in our laboratory. Antibody integrity was verified by using SDS-PAGE, and anti-TNP reactivity was tested with ELISA. IgG Fc glycan structures were analyzed by means of HPLC.
Immunization and purification of OVA-specific IgG antibodies
Eight- to 10-week-old C57BL/6 mice were immunized intraperitoneally, as indicated in Fig 2, F, and Fig E3. Serum samples were collected on day 14, and pooled OVA-specific serum IgG antibodies were purified with OVA coupled to CNBr-activated Sepharose 4B (GE Healthcare) prepared in our laboratory. Enrichment of OVA-specific IgG antibodies was verified by using ELISA. IgG subclass distribution and Fc glycan structures were analyzed by means of glycopeptide and IgG glycan HPLC analysis. The potential of the pooled and purified IgG anti-OVA antibodies from distinct groups was analyzed in the IgG-mediated anaphylaxis model.
Recombinant Bet v 1
Recombinant Bet v 1-A (Bet v 1.0101; www.allergen.org) was expressed in Escherichia coli BL21 cells (Merck Millipore, Darmstadt, Germany) in the form of inclusion bodies. Inclusion bodies were solubilized with 6 mol/L guanidine hydrochloride, and Bet v 1 was subsequently refolded by means of rapid dilution in sodium phosphate buffer (pH 7.2). Folded Bet v 1 was purified by means of hydrophobic interaction chromatography (phenyl Sepharose 6FF high sub; GE Healthcare) and size exclusion chromatography (Superdex 75; GE Healthcare) and finally formulated in 18 mmol/L sodium phosphate, 135 mmol/L sodium chloride, and 10% glycerol.
Purification of Bet v 1–specific IgG antibodies
Human serum IgG was purified with Protein G–Sepharose (GE Healthcare). Bet v 1–specific IgG was enriched with Bet v 1 coupled to CNBr-activated Sepharose 4B (GE Healthcare) prepared in our laboratory (Fig E2). Enrichment of Bet v 1–specific IgG and exclusion of tetanus toxin-specific IgG was verified by using ELISA (Fig E2). IgG Fc glycan structures were analyzed by using HPLC.
TNP- and OVA-reactive ELISA
ELISA plates were coated with 10 μg/mL TNP-BSA or OVA with or without the indicated concentrations of BSA to measure the reactivity or affinity of the indicated IgG subclass anti-TNP mAbs or (purified) serum IgG anti-OVA antibodies. Bound antibodies were detected with horseradish peroxidase–coupled polyclonal goat anti-mouse IgG-, IgG1-, IgG2c- (the isoform of IgG2a in C57BL/6 mice), IgG2b-, or IgE-specific antibodies purchased from Bethyl Laboratories (Montgomery, Tex). After incubation with the 3,3′,5,5′-tetramethylbenzidine substrate (BD Biosciences, San Diego, Calif) or in Fig E1 with Femto Substrate (Thermo Fisher Scientific), OD was measured at 450 nm or 425 nm, respectively.
Bet v 1– and tetanus toxin–reactive ELISA
ELISA plates were coated with 10 μg/mL Bet v 1 or 2.5 Lf/mL tetanus toxin (National Institute for Biological Standards and Control, Potters Bar, United Kingdom) to measure Bet v 1– or tetanus toxin–specific IgE, IgG, IgG1, or IgG4 levels. Unless indicated otherwise, plates were incubated with 1:100 diluted serum or plasma, and bound antibodies were detected with anti-human IgG (clone HP-6017, mouse IgG2a) or IgG1 (clone HP-6001, mouse IgG2b) and horseradish peroxidase–conjugated polyclonal goat anti-mouse IgG2a or anti-mouse IgG2b secondary antibodies, respectively; horseradish peroxidase–conjugated anti-human IgG4 (clone HP-6025, mouse IgG1); or horseradish peroxidase–conjugated polyclonal anti-human IgE (all antibodies from Bethyl Laboratories).
In vitro desialylation and/or degalactosylation of IgG antibodies
Purified (native) IgG antibodies were desialylated with sialidase A (#GK80040; ProZyme, Hayward, Calif) or additionally degalactosylated with β(1-4)-galactosidase (Streptococcus pneumoniae; #GKX-5014, ProZyme). Anti-TNP and anti–Bet v 1 reactivities of differently glycosylated antibodies were analyzed by means of ELISA. IgG Fc N-glycosylation was analyzed by using HPLC.
In vitro galactosylation and/or sialylation of IgG antibodies
In vitro galactosylation and sialylation of purified IgG antibodies was performed in a 2-step procedure, as previously described.
Briefly, purified (native) antibodies were galactosylated with human β1,4-galactosyltransferase and UDP-galactose or subsequently additionally sialylated with human α2,6-sialyltransferase and CMP–sialic acid (substrates and transferases were purchased from Calbiochem [Nottingham, United Kingdom] or Roche [Basel, Switzerland]), or α2,6-sialyltransferase was produced, as previously described.
EndoS cleaves the Fc N-glycans of IgG antibodies at the chitobiose core between the first and second N-acetylglucosamine (GlcNAc; Fig 1, A, and Fig E1). The resulting N-glycans were purified by using solid-phase extraction with homemade Carbograph graphitized carbon columns (Fisher Scientific, Hampton, NH)
The hydrophilic interaction liquid chromatography–HPLC with labeled glycans was performed on a Dionex Ultimate 3000 (Thermo Fischer Scientific, Waltham, Mass) by using an Xbridge XP BEH Glycan column (2.5 μm, 100 × 4.6 mm i.d.; Waters, Milford, Mass). Peak identity was confirmed by analyzing the collected peak fractions by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as previously described.
Glycans with human or murine sialic acids (human N-acetylneuraminic acid or murine N-glycolylneuraminic acid) had different retention times. Based on the terminal sugar moiety, peaks were assigned to one of the following 9 groups: G0 + bisecting GlcNAc, G0 − bisecting GlcNAc, G1 + bisecting GlcNAc, G1 − bisecting GlcNAc, G2 + bisecting GlcNAc, G2 − bisecting GlcNAc, G1S1, G2S1, and G2S2. Peaks containing both sialic acid and bisecting GlcNAc were not detected. Calculated proportions of the bisecting GlcNAc versions of G0, G1, and G2 were added to percentages of the G0, G1, and G2 versions without bisecting GlcNAc, respectively, to present 6 groups totaling 100%: G0, G1, G2, G1S1, G2S1, and G2S2 (Figs 1 and 2 and Fig E1, Fig E2, Fig E3). Mouse IgG antibodies rarely have a bisecting GlcNAc, whereas 10% to 15% of human IgG antibodies have a bisecting GlcNAc. Because more Fc glycans of total serum IgG from untreated C57BL/6 mice are sialylated than pooled serum IgG from healthy human donors (IVIG), percentages of sialylation of murine and human IgG antibodies cannot be directly compared.
IgG Fc subclass glycopeptide analysis
In contrast to only small differences in Fc glycosylation patterns between human IgG subclasses in the same sample,
In short, OVA-specific IgG antibodies were cleaved with trypsin, and IgG1 and IgG2 (IgG2b and IgG2c, both cannot be distinguished because of the comparable peptide sequence) Fc glycopeptides were analyzed by using nano–liquid chromatography mass spectrometry. Glycopeptide signals were assigned and quantified,
). Mice were treated with various amounts of IgG subclass anti-TNP mAbs 22.5 hours later and challenged intravenously with 1 μg of TNP-OVA 1.5 hours later. Changes in body core/rectal temperature were measured to assess the severity of systemic anaphylaxis (Physitemp BAT-12R thermometer; Science Products GmbH, Hofheim, Germany).
Two hundred micrograms of IgG subclass anti-TNP or 100 μg of purified OVA-specific IgG antibodies were injected intravenously into female C57BL/6 mice. Twenty-four hours later, the mice were challenged intravenously with 20 μg of TNP-OVA. Anaphylaxis severity was measured by determining the changes in the body core/rectal temperature.
The α-SignR1 (clone 22D1) mAb was purchased from BioXCell (West Lebanon, NH). One hundred micrograms of this antibody was injected intravenously 1 hour before IgG subclass anti-TNP antibody injection to induce SignR1 internalization.
To generate plate-bound, immobilized immune complexes, 50 μg/mL Bet v 1 in 0.05 mmol/L carbonate/bicarbonate buffer (pH 9.6) was coated on Lumitrac 600 96-well plates (Greiner Bio-one, Frickenhausen, Germany) for 1 hour, followed by an 18-hour incubation with desialylated or native purified serum IgG (200 μg/200 μL per well). Freshly isolated neutrophils in chemiluminescence medium (RPMI medium without phenol red and sodium bicarbonate containing 20 nmol/L HEPES and 0.06 mmol/L Luminol; 2 × 106 cells/mL) were seeded in the plates with the immobilized immune complexes (2 × 105 cells per well). The sum of intracellular and extracellular reactive oxygen species (ROS), a sign of neutrophil activation, was measured (duplicate measurements) by using luminol-amplified chemiluminescence for 1.5 hours at 37°C with FluoStar Omega (BMG Labtech, Ortenberg, Germany).
Analysis of OVA injection data and ROS data (area under the curve) was performed with the Student t test. Body core/rectal temperature data (area under the curve) were analyzed by using 1-way ANOVA. Longitudinal analyses of the Bet v 1–specific antibody titers, skin prick tests, and clinical scores were performed by using the Wilcoxon signed-rank test. All experiments were not blinded. If not stated otherwise, data were expressed as means ± SEMs. In all experiments normal distribution was assumed.
Advances in allergen immunotherapy: aiming for complete tolerance to allergens.
Supported by the German Research foundation (grants nos. EH 221/8-1, International Research Training Group [iGRK] 1911, GRK 1727, CRU 303 and Excellence cluster 306 to M.E., iGRK1911 to F.D.F., as well as MO 2076/3-1, HE 1602/10-1, PF 344/3-1 and SFB/TR22 to C.M, M.H. and W.P.), the Else-Kröner-Fresenius Foundation (2014_A91 to M.E.), the U.S. Department of Veterans Affairs Merit Award to F.D.F., the National Institutes of Health (R01 AI072040 to F.D.F., as well as GM103390 and GM107012 to K.W.M.), and the Food Allergy Research and Education (FARE; to F.D.F.).
Disclosure of potential conflict of interest: A. Epp's, C. Möbs', J. Rahmöller's, A. Petersen's, M. Hertl's, W. Pfützner's, and M. Ehlers' institutions received grants (MO2076/3-1, HE1602/10-1, PF344/3-1, SFB/TR22, EH221/8-1, iRTG1911) from the German Research Foundation for this work. J. Hobusch's, Y. C. Bartsch's, S. Eschweiler's, D. Braumann's, and A. Leliavski's, and M. Ehlers' institution received grants (RTG1727, CRU303, and Excellence Cluster 306) from the German Research Foundation for other works. J. Petry's, G. M. Lilienthal's, and M. Ehlers' institution received a grant (2014_A91) from the Else Kroener-Fresenius-Foundation for other works. C. Engellenner's and F. Petersen's institutions received grants from the German Research Foundation and the Federal Ministry of Research and Education for other works. R. Thurmann and M. Ehlers are employed by the University of Luebeck. K. W. Moremen's institution received grants GM 103390 and GM 107012 from the US National Institutes of Health for this work and is employed by the University of Georgia. M. Collin received consultant fees from Genovis AB for this work and received consultancy fees, has patents with, and received royalties from Hansa Medical AB for other works, and his institution received grants from HansaMedical AB for other works. A. Nandy and H. Kahlert are employed by and have patents pending with Allergopharma GmbH & Co KG for other works and A. Nandy had stock options from Merck KGaA. U. Jappe's institution received grants from the Federal Ministry of Research and Education, the German Research Foundation, and the Federal Ministry of Economy and Technology for other works; and she is a consultant for Fraunhofer Zukunftsstiftung and received travel expenses from ALK-Abelló. F. D. Finkelman received grants from the National Institutes of Health (R01 AI072040), the Food Allergy Research and Education, and the US Department of Veterans Affairs Merit Award and personal fees from Vedanta Bioscience for other works and is employed by the University of Cincinnati. The rest of the authors declare that they have no relevant conflicts of interest.