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Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions

      To the Editor:
      IgE antibodies can mediate allergic reactions, including systemic anaphylaxis, by activating the high-affinity FcεRI on mast cells and basophils, leading to release of inflammatory mediators.
      • Santos A.F.
      • James L.K.
      • Bahnson H.T.
      • Shamji M.H.
      • Couto-Francisco N.C.
      • Islam S.
      • et al.
      IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens.
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      • et al.
      A single glycan on IgE is indispensable for initiation of anaphylaxis.
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      • Finkelman F.D.
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      Advances and highlights in mechanisms of allergic disease in 2015.
      • Akdis C.A.
      • Akdis M.
      Advances in allergen immunotherapy: aiming for complete tolerance to allergens.
      • Finkelman F.D.
      • Khodoun M.V.
      • Strait R.
      Human IgE-independent systemic anaphylaxis.
      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.
      • Finkelman F.D.
      • Khodoun M.V.
      • Strait R.
      Human IgE-independent systemic anaphylaxis.
      • Strait R.T.
      • Morris S.C.
      • Finkelman F.D.
      IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking.
      • Santos A.F.
      • James L.K.
      • Bahnson H.T.
      • Shamji M.H.
      • Couto-Francisco N.C.
      • Islam S.
      • et al.
      IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens.
      • Zhu D.
      • Kepley C.L.
      • Zhang M.
      • Zhang K.
      • Saxon A.
      A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation.
      • Burton O.T.
      • Logsdon S.L.
      • Zhou J.S.
      • Medina-Tamayo J.
      • Abdel-Gadir A.
      • Noval Rivas M.
      • et al.
      Oral immunotherapy induces IgG antibodies that act through FcγRIIb to suppress IgE-mediated hypersensitivity.
      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.
      • Quakkelaar E.D.
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      • Loof N.M.
      • van Heiningen S.H.
      • et al.
      IgG-mediated anaphylaxis to a synthetic long peptide vaccine containing a B cell epitope can be avoided by slow-release formulation.
      • Bruhns P.
      • Jönsson F.
      Mouse and human FcR effector functions.
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      • Jönsson F.
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      • et al.
      Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice.
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      • Hirshman C.
      • et al.
      Association of protamine IgE and IgG antibodies with life-threatening reactions to intravenous protamine.
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      • Meyer D.
      • Heijnen I.A.
      • et al.
      Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice.
      • Miyajima I.
      • Dombrowicz D.
      • Martin T.R.
      • Ravetch J.V.
      • Kinet J.P.
      • Galli S.J.
      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.
      • Khodoun M.V.
      • Strait R.
      • Armstrong L.
      • Yanase N.
      • Finkelman F.D.
      Identification of markers that distinguish IgE- from IgG-mediated anaphylaxis.
      • Finkelman F.D.
      • Khodoun M.V.
      • Strait R.
      Human IgE-independent systemic anaphylaxis.
      • Strait R.T.
      • Morris S.C.
      • Finkelman F.D.
      IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking.
      • Beutier H.
      • Gillis C.M.
      • Iannascoli B.
      • Godon O.
      • England P.
      • Sibilano R.
      • et al.
      IgG subclasses determine pathways of anaphylaxis in mice.
      The effector functions of IgG antibodies depend on their subclass
      • Bruhns P.
      • Jönsson F.
      Mouse and human FcR effector functions.
      • Vidarsson G.
      • Dekkers G.
      • Rispens T.
      IgG subclasses and allotypes: from structure to effector functions.
      and the type of Fc N-glycosylation (Fig 1, A, and see Fig E1 in this article's Online Repository at www.jacionline.org). Agalactosylated IgG antibodies generally promote inflammation,
      • Scherer H.U.
      • van der Woude D.
      • Ioan-Facsinay A.
      • el Bannoudi H.
      • Trouw L.A.
      • Wang J.
      • et al.
      Glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid.
      • Ohmi Y.
      • Ise W.
      • Harazono A.
      • Takakura D.
      • Fukuyama H.
      • Baba Y.
      • et al.
      Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis.
      whereas galactosylation and terminal sialylation of IgG antibodies generally suppress inflammation.
      • Ohmi Y.
      • Ise W.
      • Harazono A.
      • Takakura D.
      • Fukuyama H.
      • Baba Y.
      • et al.
      Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis.
      • Kaneko Y.
      • Nimmerjahn F.
      • Ravetch J.V.
      Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.
      • Anthony R.M.
      • Wermeling F.
      • Karlsson M.C.
      • Ravetch J.V.
      Identification of a receptor required for the anti-inflammatory activity of IVIG.
      • Anthony R.M.
      • Kobayashi T.
      • Wermeling F.
      Rav etch JV. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.
      • Karsten C.M.
      • Pandey M.K.
      • Figge J.
      • Kilchenstein R.
      • Taylor P.R.
      • Rosas M.
      • et al.
      Anti-inflammatory activity of IgG1 mediated by Fc galactosylation and association of Fc gamma RIIB and dectin-1.
      • Oefner C.M.
      • Winkler A.
      • Hess C.
      • Lorenz A.K.
      • Holecska V.
      • Huxdorf M.
      • et al.
      Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs.
      • Hess C.
      • Winkler A.
      • Lorenz A.K.
      • Holecska V.
      • Blanchard V.
      • Eiglmeier S.
      • et al.
      T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.
      • Collin M.
      • Ehlers M.
      The carbohydrate switch between pathogenic and immunosuppressive antigen-specific antibodies.
      • Pincetic A.
      • Bournazos S.
      • DiLillo D.J.
      • Maamary J.
      • Wang T.T.
      • Dahan R.
      • et al.
      Type I and type II Fc receptors regulate innate and adaptive immunity.
      However, the effects of IgG subclasses and Fc glycosylation patterns in allergy remain unclear.
      Figure thumbnail gr1
      Fig 1Effect of differently glycosylated IgG subclass antibodies on IgE- and IgG-mediated murine anaphylaxis. A, The conserved biantennary N-glycan (4 N-acetylglucosamines [dark blue] and 3 mannoses [green]) at Asn 297 in the IgG Fc part can be modified by fucose (red), bisecting GlcNAc (light blue), galactose (G; yellow), and sialic acid (S; magenta). The cleavage site of EndoS used for IgG glycan analysis is depicted. B, Experimental design of IgE-mediated anaphylaxis as done in Fig 1, D and E. C, Fc glycosylation profiles of the differently glycosylated murine IgG1 anti-TNP mAbs (clone H5) used in the murine experiments. Native = low-galactosylated (low-gal), in vitro galactosylated (gal), or galactosylated plus sialylated (sial). D and E, Inhibition of IgE-mediated temperature decrease by differently glycosylated IgG1 anti-TNP mAbs (H5) in wild-type (Fig 1, D) and FcγRIIb-deficient (Fig 1, E) mice (in each graph: no IgG, n = 8-10; low-gal, n = 8-10; gal, n = 5; sial, n = 5). Symbols represent means. One of 2 independent experiments is shown. ns, Not significant. F, Experimental design of IgG-mediated anaphylaxis as done in Fig 1, G-I. G-I, Decrease in body temperature induced with differently glycosylated IgG subclass anti-TNP mAbs (IgG1 [clone H5] or IgG2a and IgG2b switch variants [sv]) in wild-type (Fig 1, G-I) or FcγRIIb-deficient (Fig 1, H) mice without (Fig 1, G-I) or with α-SignR1 treatment (Fig 1, I). de-gal, In vitro desialylated plus degalactosylated. Fig 1, G, Pooled data (n = 10-15) from independent experiments with 5 per group per experiment. Fig 1, H and I, One of 2 independent experiments is shown. **P < .01 and ***P < .001. i.v., Intravenous.
      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,
      • Strait R.T.
      • Morris S.C.
      • Finkelman F.D.
      IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking.
      • Santos A.F.
      • James L.K.
      • Bahnson H.T.
      • Shamji M.H.
      • Couto-Francisco N.C.
      • Islam S.
      • et al.
      IgG4 inhibits peanut-induced basophil and mast cell activation in peanut-tolerant children sensitized to peanut major allergens.
      • Bruhns P.
      • Jönsson F.
      Mouse and human FcR effector functions.
      • Vidarsson G.
      • Dekkers G.
      • Rispens T.
      IgG subclasses and allotypes: from structure to effector functions.
      • Burton O.T.
      • Logsdon S.L.
      • Zhou J.S.
      • Medina-Tamayo J.
      • Abdel-Gadir A.
      • Noval Rivas M.
      • et al.
      Oral immunotherapy induces IgG antibodies that act through FcγRIIb to suppress IgE-mediated hypersensitivity.
      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,
      • Kaneko Y.
      • Nimmerjahn F.
      • Ravetch J.V.
      Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.
      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)
      • Strait R.T.
      • Posgai M.T.
      • Mahler A.
      • Barasa N.
      • Jacob C.O.
      • Köhl J.
      • et al.
      IgG1 protects against renal disease in a mouse model of cryoglobulinaemia.
      had only a slight effect on the extent of inhibition.
      In contrast, the severity of IgG-mediated systemic anaphylaxis, which required challenge with a higher antigen dose (20 μg),
      • Finkelman F.D.
      • Khodoun M.V.
      • Strait R.
      Human IgE-independent systemic anaphylaxis.
      • Strait R.T.
      • Morris S.C.
      • Finkelman F.D.
      IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking.
      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).
      • Beutier H.
      • Gillis C.M.
      • Iannascoli B.
      • Godon O.
      • England P.
      • Sibilano R.
      • et al.
      IgG subclasses determine pathways of anaphylaxis in mice.
      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),
      • Beutier H.
      • Gillis C.M.
      • Iannascoli B.
      • Godon O.
      • England P.
      • Sibilano R.
      • et al.
      IgG subclasses determine pathways of anaphylaxis in mice.
      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).
      • Collin M.
      • Ehlers M.
      The carbohydrate switch between pathogenic and immunosuppressive antigen-specific antibodies.
      • Pincetic A.
      • Bournazos S.
      • DiLillo D.J.
      • Maamary J.
      • Wang T.T.
      • Dahan R.
      • et al.
      Type I and type II Fc receptors regulate innate and adaptive immunity.
      • Anthony R.M.
      • Wermeling F.
      • Karlsson M.C.
      • Ravetch J.V.
      Identification of a receptor required for the anti-inflammatory activity of IVIG.
      • Anthony R.M.
      • Kobayashi T.
      • Wermeling F.
      Rav etch JV. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.
      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).
      • Möbs C.
      • Slotosch C.
      • Löffler H.
      • Jakob T.
      • Hertl M.
      • Pfützner W.
      Birch pollen immunotherapy leads to differential induction of regulatory T cells and delayed helper T cell immune deviation.
      • Gepp B.
      • Lengger N.
      • Möbs C.
      • Pfützner W.
      • Radauer C.
      • Bohle B.
      • et al.
      Monitoring the epitope recognition profiles of IgE, IgG1, and IgG4 during birch pollen immunotherapy.
      • Subbarayal B.
      • Schiller D.
      • Möbs C.
      • Pfützner W.
      • Jahn-Schmid B.
      • Gepp B.
      • et al.
      The diversity of Bet v 1-specific IgG4 antibodies remains mostly constant during the course of birch pollen immunotherapy.
      • Möbs C.
      • Ipsen H.
      • Mayer L.
      • Slotosch C.
      • Petersen A.
      • Würtzen P.A.
      • et al.
      Birch pollen immunotherapy results in long-term loss of Bet v 1-specific TH2 responses, transient TR1 activation, and synthesis of IgE-blocking antibodies.
      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).
      • Akdis C.A.
      • Akdis M.
      Advances in allergen immunotherapy: aiming for complete tolerance to allergens.
      • Möbs C.
      • Ipsen H.
      • Mayer L.
      • Slotosch C.
      • Petersen A.
      • Würtzen P.A.
      • et al.
      Birch pollen immunotherapy results in long-term loss of Bet v 1-specific TH2 responses, transient TR1 activation, and synthesis of IgE-blocking antibodies.
      • Möbs C.
      • Slotosch C.
      • Löffler H.
      • Jakob T.
      • Hertl M.
      • Pfützner W.
      Birch pollen immunotherapy leads to differential induction of regulatory T cells and delayed helper T cell immune deviation.
      • Gepp B.
      • Lengger N.
      • Möbs C.
      • Pfützner W.
      • Radauer C.
      • Bohle B.
      • et al.
      Monitoring the epitope recognition profiles of IgE, IgG1, and IgG4 during birch pollen immunotherapy.
      • Aalberse R.C.
      • van der Gaag R.
      • van Leeuwen J.
      Serologic aspects of IgG4 antibodies. I. Prolonged immunization results in an IgG4-restricted response.
      Figure thumbnail gr2
      Fig 2Bet v 1–specific serum IgG Fc glycosylation of untreated and treated allergic patients and influence of different adjuvants on IgG Fc glycosylation. A, Serum titers of Bet v 1–specific IgG1 and IgG4 from untreated subjects (season, n = 8; no season, n = 6 + 11 [AIT-treated, month 0]) and AIT-treated (n = 11) patients with birch pollen allergy. Black line, mean; gray columns, pollen season. Green data points depict the 5 AIT-treated patients who were selected for the glycan analysis in Fig 2, B and C, and , whereas red data points depict the 3 samples (patient 5 at months 12 [5-12], 5-36, and 21-18) that were chosen for in vitro desialylation and neutrophil activation in Fig 2, B-E, and . One of 2 independent ELISAs is shown. B and C, Percentage of agalactosylated (G0; , B) and sialylated (, C) glycans from purified Bet v 1–specific IgG antibodies of untreated (season, n = 5; no season, n = 5 + 5 [AIT-treated, month 0]) and the 5 selected AIT-treated patients and from IVIG and purified native and in vitro desialylated (de-sial) total serum IgG from patient samples 5-12, 5-36, and 21-18. Filled areas indicate levels of agalactosylated (G0) or sialylated IgG autoantibodies in patients with rheumatoid arthritis (RA) for comparison.
      • Scherer H.U.
      • van der Woude D.
      • Ioan-Facsinay A.
      • el Bannoudi H.
      • Trouw L.A.
      • Wang J.
      • et al.
      Glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid.
      D and E, Human neutrophil activation assay: experimental setup (Fig 2, D) and reactive oxygen species (ROS) production (Fig 2, E) after activation with native or in vitro desialylated Bet v 1–specific IgG antibodies of patient 5 (P5 m36). Black line, No IgG. One of 2 independent ROS assays is shown. F-J, Effect of distinct adjuvants (eCFA, alum, or MPLA) on induction of OVA-specific serum IgG antibodies. Fig 2, F, Experimental design. Fig 2, G, IgG1 and IgG2 (IgG2b + IgG2c; neither can be distinguished by means of glycopeptide analysis because of the comparable peptide sequence) frequencies in purified OVA-specific IgG antibodies, as determined by using glycopeptide analysis. H and I, IgG1 and IgG2 or total IgG Fc sialylation profiles of pooled and purified OVA-specific IgG antibodies, as determined by glycopeptide (Fig 2, H) or total IgG glycan (Fig 2, I) analysis. J, IgG-mediated anaphylaxis as described in , F, with 100 μg of pooled and purified OVA-specific serum IgG antibodies (n = 4-5 per group). Symbols represent means. *P < .05, **P < .01, and ***P < .001.
      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).
      • Scherer H.U.
      • van der Woude D.
      • Ioan-Facsinay A.
      • el Bannoudi H.
      • Trouw L.A.
      • Wang J.
      • et al.
      Glycan profiling of anti-citrullinated protein antibodies isolated from human serum and synovial fluid.
      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)
      • Oefner C.M.
      • Winkler A.
      • Hess C.
      • Lorenz A.K.
      • Holecska V.
      • Huxdorf M.
      • et al.
      Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs.
      and of those in therapeutic intravenous immunoglobulin (IVIG), which have Fc sialylation–dependent anti-inflammatory properties (Fig 2, B and C, and see Fig E2).
      • Kaneko Y.
      • Nimmerjahn F.
      • Ravetch J.V.
      Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.
      • Anthony R.M.
      • Wermeling F.
      • Karlsson M.C.
      • Ravetch J.V.
      Identification of a receptor required for the anti-inflammatory activity of IVIG.
      • Anthony R.M.
      • Kobayashi T.
      • Wermeling F.
      Rav etch JV. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.
      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
      • Larché M.
      • Akdis C.A.
      • Valenta R.
      Immunological mechanisms of allergen-specific immunotherapy.
      • Pfaar O.
      • Cazan D.
      • Klimek L.
      • Larenas-Linnemann D.
      • Calderon M.A.
      Adjuvants for immunotherapy.
      • Patel P.
      • Holdich T.
      • Fischer von Weikersthal-Drachenberg K.J.
      • Huber B.
      Efficacy of a short course of specific immunotherapy in patients with allergic rhinoconjunctivitis to ragweed pollen.
      • Valenta R.
      • Campana R.
      • Focke-Tejkl M.
      • Niederberger V.
      Vaccine development for allergen-specific immunotherapy based on recombinant allergens and synthetic allergen peptides: lessons from the past and novel mechanisms of action for the future.
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      • Dionne K.
      • Tedesco J.
      • Chung A.W.
      • et al.
      Antigen-specific antibody glycosylation is regulated via vaccination.
      • Akdis C.A.
      • Akdis M.
      Advances in allergen immunotherapy: aiming for complete tolerance to allergens.
      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)
      • Akdis C.A.
      • Akdis M.
      Advances in allergen immunotherapy: aiming for complete tolerance to allergens.
      • Hess C.
      • Winkler A.
      • Lorenz A.K.
      • Holecska V.
      • Blanchard V.
      • Eiglmeier S.
      • et al.
      T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.
      • Pfaar O.
      • Cazan D.
      • Klimek L.
      • Larenas-Linnemann D.
      • Calderon M.A.
      Adjuvants for immunotherapy.
      • Patel P.
      • Holdich T.
      • Fischer von Weikersthal-Drachenberg K.J.
      • Huber B.
      Efficacy of a short course of specific immunotherapy in patients with allergic rhinoconjunctivitis to ragweed pollen.
      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,
      • Bondt A.
      • Selman M.H.
      • Deelder A.M.
      • Hazes J.M.
      • Willemsen S.P.
      • Wuhrer M.
      • et al.
      Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation.
      • Pezer M.
      • Stambuk J.
      • Perica M.
      • Razdorov G.
      • Banic I.
      • Vuckovic F.
      • et al.
      Effects of allergic diseases and age on the composition of serum IgG glycome in children.
      glycopeptide analysis confirmed that murine IgG2 (IgG2b and IgG2c) was, on average, much more highly galactosylated and sialylated than IgG1 (Fig 2, H, and see Fig E3).
      • de Haan N.
      • Reiding K.R.
      • Krištić J.
      • Ederveen A.L.H.
      • Lauc G.
      • Wuhrer M.
      The N-glycosylation of mouse IgG-Fc differs between IgG subclasses and strains.
      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
      • Akdis C.A.
      • Akdis M.
      Advances in allergen immunotherapy: aiming for complete tolerance to allergens.
      • Larché M.
      • Akdis C.A.
      • Valenta R.
      Immunological mechanisms of allergen-specific immunotherapy.
      • Pfaar O.
      • Cazan D.
      • Klimek L.
      • Larenas-Linnemann D.
      • Calderon M.A.
      Adjuvants for immunotherapy.
      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).

      Methods

       Human samples

      Serum samples of 7 patients (P2, P5, P7, P8, and P11-P13) from a previously published cohort of 15 patients
      • Möbs C.
      • Slotosch C.
      • Löffler H.
      • Jakob T.
      • Hertl M.
      • Pfützner W.
      Birch pollen immunotherapy leads to differential induction of regulatory T cells and delayed helper T cell immune deviation.
      • Gepp B.
      • Lengger N.
      • Möbs C.
      • Pfützner W.
      • Radauer C.
      • Bohle B.
      • et al.
      Monitoring the epitope recognition profiles of IgE, IgG1, and IgG4 during birch pollen immunotherapy.
      • Subbarayal B.
      • Schiller D.
      • Möbs C.
      • Pfützner W.
      • Jahn-Schmid B.
      • Gepp B.
      • et al.
      The diversity of Bet v 1-specific IgG4 antibodies remains mostly constant during the course of birch pollen immunotherapy.
      • Möbs C.
      • Ipsen H.
      • Mayer L.
      • Slotosch C.
      • Petersen A.
      • Würtzen P.A.
      • et al.
      Birch pollen immunotherapy results in long-term loss of Bet v 1-specific TH2 responses, transient TR1 activation, and synthesis of IgE-blocking antibodies.
      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.

       Mice

      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.
      • Takai T.
      • Ono M.
      • Hikida M.
      • Ohmori H.
      • Ravetch J.V.
      Augmented humoral and anaphylactic responses in Fc gamma RII-deficient mice.
      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.

       Reagents

      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).
      • Hess C.
      • Winkler A.
      • Lorenz A.K.
      • Holecska V.
      • Blanchard V.
      • Eiglmeier S.
      • et al.
      T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.
      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).

       mAbs

      The IgE anti-TNP (clone IgEL a2; ATCC-TIB-142)
      • Rudolph A.K.
      • Burrows P.D.
      • Wabl M.R.
      Thirteen hybridomas secreting hapten-specific immunoglobulin E from mice with Iga or Igb heavy chain haplotype.
      and IgG1 anti-TNP (clone H5)
      • Hess C.
      • Winkler A.
      • Lorenz A.K.
      • Holecska V.
      • Blanchard V.
      • Eiglmeier S.
      • et al.
      T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.
      • Wernersson S.
      • Karlsson M.C.
      • Dahlström J.
      • Mattsson R.
      • Verbeek J.S.
      • Heyman B.
      IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice.
      hybridoma cells and the murine IgG1, IgG2a, and IgG2b anti-TNP hybridoma switch variants with identical V(D)J sequences
      • Strait R.T.
      • Posgai M.T.
      • Mahler A.
      • Barasa N.
      • Jacob C.O.
      • Köhl J.
      • et al.
      IgG1 protects against renal disease in a mouse model of cryoglobulinaemia.
      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.
      • Kaneko Y.
      • Nimmerjahn F.
      • Ravetch J.V.
      Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.
      • Hess C.
      • Winkler A.
      • Lorenz A.K.
      • Holecska V.
      • Blanchard V.
      • Eiglmeier S.
      • et al.
      T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.
      • Oefner C.M.
      • Winkler A.
      • Hess C.
      • Lorenz A.K.
      • Holecska V.
      • Huxdorf M.
      • et al.
      Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs.
      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.
      • Barb A.W.
      • Meng L.
      • Gao Z.
      • Johnson R.W.
      • Moremen K.W.
      • Prestegard J.H.
      NMR characterization of immunoglobulin G Fc glycan motion on enzymatic sialylation.
      • Meng L.
      • Forouhar F.
      • Thieker D.
      • Gao Z.
      • Ramiah A.
      • Moniz H.
      • et al.
      Enzymatic basis for N-glycan sialylation: structure of rat α2,6-sialyltransferase (ST6GAL1) reveals conserved and unique features for glycan sialylation.
      The anti-TNP reactivity of differently glycosylated antibodies was tested by using ELISA. IgG Fc N-glycosylation was analyzed by using HPLC.

       Glycan analysis of purified IgG antibodies by using HPLC

      N-glycans were isolated from purified IgG samples by means of hydrolysis with recombinant endoglycosidase S (EndoS) from Streptococcus pyogenes.
      • Hess C.
      • Winkler A.
      • Lorenz A.K.
      • Holecska V.
      • Blanchard V.
      • Eiglmeier S.
      • et al.
      T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.
      • Oefner C.M.
      • Winkler A.
      • Hess C.
      • Lorenz A.K.
      • Holecska V.
      • Huxdorf M.
      • et al.
      Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs.
      • Collin M.
      • Olsén A.
      Effect of SpeB and EndoS from Streptococcus pyogenes on human immunoglobulins.
      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)
      • Packer N.H.
      • Lawson M.A.
      • Jardine D.R.
      • Redmond J.W.
      A general approach to desalting oligosaccharides released from glycoproteins.
      and labeled with anthranilamide (Sigma-Aldrich), as previously described.
      • Bigge J.C.
      • Patel T.P.
      • Bruce J.A.
      • Goulding P.N.
      • Charles S.M.
      • Parekh R.B.
      Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid.
      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.
      • Hess C.
      • Winkler A.
      • Lorenz A.K.
      • Holecska V.
      • Blanchard V.
      • Eiglmeier S.
      • et al.
      T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.
      • Oefner C.M.
      • Winkler A.
      • Hess C.
      • Lorenz A.K.
      • Holecska V.
      • Huxdorf M.
      • et al.
      Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs.
      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,
      • Bondt A.
      • Selman M.H.
      • Deelder A.M.
      • Hazes J.M.
      • Willemsen S.P.
      • Wuhrer M.
      • et al.
      Association between galactosylation of immunoglobulin G and improvement of rheumatoid arthritis during pregnancy is independent of sialylation.
      • Pezer M.
      • Stambuk J.
      • Perica M.
      • Razdorov G.
      • Banic I.
      • Vuckovic F.
      • et al.
      Effects of allergic diseases and age on the composition of serum IgG glycome in children.
      the Fc glycosylation pattern of murine IgG subclass antibodies in the same sample highly differ.
      • de Haan N.
      • Reiding K.R.
      • Krištić J.
      • Ederveen A.L.H.
      • Lauc G.
      • Wuhrer M.
      The N-glycosylation of mouse IgG-Fc differs between IgG subclasses and strains.
      Accordingly, polyclonal murine IgG Fc subclass glycopeptides were analyzed, as described previously.
      • Sonneveld M.E.
      • Koelewijn J.
      • de Haas M.
      • Admiraal J.
      • Plomp R.
      • Koeleman C.A.
      • et al.
      Antigen specificity determines anti-red blood cell IgG-Fc alloantibody glycosylation and thereby severity of haemolytic disease of the fetus and newborn.
      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,
      • de Haan N.
      • Reiding K.R.
      • Krištić J.
      • Ederveen A.L.H.
      • Lauc G.
      • Wuhrer M.
      The N-glycosylation of mouse IgG-Fc differs between IgG subclasses and strains.
      and IgG1 versus IgG2 ratios and their summarized G0, G1, G2, G1S1, G2S1, and G2S2 forms were calculated, as described above. Glycans without fucose or with bisecting GlcNAc were hardly detected.

       Experimental passive IgE-mediated systemic anaphylaxis

      On day 0, female mice were primed intravenously with 10 μg of IgE anti-TNP (clone IgEL a2; ATCC-TIB-142
      • Rudolph A.K.
      • Burrows P.D.
      • Wabl M.R.
      Thirteen hybridomas secreting hapten-specific immunoglobulin E from mice with Iga or Igb heavy chain haplotype.
      ). 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).

       Experimental passive IgG-mediated systemic anaphylaxis

      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.

       α-SignR1 treatment

      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.
      • Anthony R.M.
      • Wermeling F.
      • Karlsson M.C.
      • Ravetch J.V.
      Identification of a receptor required for the anti-inflammatory activity of IVIG.

       Human neutrophil activation assay

      Human neutrophils were isolated from heparinized peripheral blood from healthy adult donors, as previously described.
      • Esmann L.
      • Idel C.
      • Sarkar A.
      • Hellberg L.
      • Behnen M.
      • Möller S.
      • et al.
      Phagocytosis of apoptotic cells by neutrophil granulocytes: diminished proinflammatory neutrophil functions in the presence of apoptotic cells.
      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).

       Statistical analysis

      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.
      Figure thumbnail fx1
      Fig E1Generation, analysis, and function of differently glycosylated murine IgG subclass antibodies. A, Conserved IgG biantennary N-glycan at Asn297 can be modified by fucose (red), bisecting GlcNAc (light blue), galactose (G; yellow), and sialic acid (S; magenta). B, Exemplary HPLC glycan peaks of in vitro desialylated plus degalactosylated (de-gal), galactosylated (gal), or galactosylated plus sialylated (sial) IgG2a anti-TNP mAbs (switch variant [sv]). C, Fc glycosylation profiles of the differently glycosylated murine IgG1, IgG2a, and IgG2b anti-TNP switch variants that were used in the murine experiments. Native = low-galactosylated (low-gal). D-F, Anti-TNP IgG1 affinity ELISA. , D, Experimental setup. ELISA plates were coated with TNP-BSA alone or with increasing amounts of BSA, as indicated as ratios (TNP-BSA/BSA = 1:0, 1:10 or 1:1000). , E, Modification of the Fc glycan of IgG1 anti-TNP mAbs (clone H5) did not change affinity. , F, IgG1 anti-TNP clone H5 has a higher affinity than the IgG1 anti-TNP switch variant. One of 2 independent ELISA experiments is shown in , E and F. G, IgE-mediated anaphylaxis, as described in , B, with 10 μg of low-gal IgG subclass anti-TNP mAbs (switch variants) in C57BL/6 wild-type or FcγRIIb-deficient mice. One of 2 independent experiments is shown. H, IgG-mediated anaphylaxis, as described in , F and G, with low-gal (H5) or de-gal (switch variants) IgG subclass anti-TNP mAbs in wild-type mice. For each IgG subclass antibody, n = 10-15. Symbols represent means. Pooled data are from independent experiments with n = 5 per group per experiment. *P < .05 and **P < .01.
      Figure thumbnail fx2aj
      Fig E2Further characterization and Fc glycan analysis of Bet v 1–specific IgG antibodies from untreated and AIT-treated patients. A and B, Skin prick test results (Fig E2, A) and clinical scores (Fig E2, B) of the 11 AIT-treated patients (in months). C and D, Serum titers of Bet v 1–specific IgE (Fig E2, C) and IgG (Fig E2, D) from untreated (season, n = 8; no season, n = 6 + 11 [AIT treated, moth 0]) and AIT-treated (n = 11) patients with birch pollen allergy. Black line, Mean; gray columns, pollen season. One of 2 independent ELISAs is shown. Green data points depict 5 AIT-treated patients who were selected for glycan analysis in , B and C, and Fig E2, I and J, whereas red data points depict 3 samples (patient 5 at month 12 [5-12], 5-36, and 21-18) that were chosen for in vitro desialylation and neutrophil activation in , B-E, and Fig E2, I, K, and L. E, IgG4/IgG1 ratios (OD [IgG4]/OD [IgG1]) as calculated from IgG1 and IgG4 ELISA data in , A. F, Protocol for purification and EndoS treatment of Bet v 1–specific serum IgG antibodies. G and H, Amplification and purification of Bet v 1–specific IgG antibodies was verified through anti–Bet v 1 ELISA (Fig E2, G) and anti–tetanus toxin ELISA (Fig E2, H) to document the exclusion of IgG antibodies that lack Bet v 1 specificity, here exemplified for patient P2 (month 36). I and J, Percentages of different glycans (G0, G1, G2, G1S1, G2S1, and G2S2) from summarized (Fig E2, I) and single human (Fig E2, J) samples from purified Bet v 1–specific IgG antibodies of untreated (season, n = 5; no season, n = 5 + 5 [AIT treated, month 0]) and the 5 randomly selected AIT-treated patients and from IVIG and purified native and in vitro desialylated total serum IgG from patient samples 5-12, 5-36, and 21-18. K and L, Human neutrophil activation assay, as described in , D and E. Experimental setup (Fig E2, K) and ROS production (Fig E2, L) after activation with native or in vitro desialylated (de-sial) Bet v 1–specific IgG antibodies of patient samples P5 m12 and P21 m18. Black line, No IgG. One of at least 2 independent ROS assays is shown for each patient. *P < .05, **P < .01, and ***P < .001.
      Figure thumbnail fx2kl
      Fig E2Further characterization and Fc glycan analysis of Bet v 1–specific IgG antibodies from untreated and AIT-treated patients. A and B, Skin prick test results (Fig E2, A) and clinical scores (Fig E2, B) of the 11 AIT-treated patients (in months). C and D, Serum titers of Bet v 1–specific IgE (Fig E2, C) and IgG (Fig E2, D) from untreated (season, n = 8; no season, n = 6 + 11 [AIT treated, moth 0]) and AIT-treated (n = 11) patients with birch pollen allergy. Black line, Mean; gray columns, pollen season. One of 2 independent ELISAs is shown. Green data points depict 5 AIT-treated patients who were selected for glycan analysis in , B and C, and Fig E2, I and J, whereas red data points depict 3 samples (patient 5 at month 12 [5-12], 5-36, and 21-18) that were chosen for in vitro desialylation and neutrophil activation in , B-E, and Fig E2, I, K, and L. E, IgG4/IgG1 ratios (OD [IgG4]/OD [IgG1]) as calculated from IgG1 and IgG4 ELISA data in , A. F, Protocol for purification and EndoS treatment of Bet v 1–specific serum IgG antibodies. G and H, Amplification and purification of Bet v 1–specific IgG antibodies was verified through anti–Bet v 1 ELISA (Fig E2, G) and anti–tetanus toxin ELISA (Fig E2, H) to document the exclusion of IgG antibodies that lack Bet v 1 specificity, here exemplified for patient P2 (month 36). I and J, Percentages of different glycans (G0, G1, G2, G1S1, G2S1, and G2S2) from summarized (Fig E2, I) and single human (Fig E2, J) samples from purified Bet v 1–specific IgG antibodies of untreated (season, n = 5; no season, n = 5 + 5 [AIT treated, month 0]) and the 5 randomly selected AIT-treated patients and from IVIG and purified native and in vitro desialylated total serum IgG from patient samples 5-12, 5-36, and 21-18. K and L, Human neutrophil activation assay, as described in , D and E. Experimental setup (Fig E2, K) and ROS production (Fig E2, L) after activation with native or in vitro desialylated (de-sial) Bet v 1–specific IgG antibodies of patient samples P5 m12 and P21 m18. Black line, No IgG. One of at least 2 independent ROS assays is shown for each patient. *P < .05, **P < .01, and ***P < .001.
      Figure thumbnail fx3
      Fig E3Different adjuvants induce distinct IgG subclass distributions and IgG Fc glycosylation patterns. A, Induction of differently glycosylated OVA-specific serum IgG antibodies with distinct adjuvants (eCFA, alum, or MPLA), as described in , F-J. i.p., Intraperitoneal. B, Design of the IgG anti-OVA antibody ELISA, as used in Fig E3, C. C, Serum IgG (subclass) anti-OVA antibody distribution, as analyzed by means of ELISA (n = 5-10 for each group). Each ELISA represents one of 2 independent experiments. D and E, IgG1 and IgG2 (IgG2b + IgG2c; Fig E3, D) or total IgG Fc glycosylation (Fig E3, E) of all (G0, G1, G2, G1S1, G2S1, and G2S2) or only G0 forms of purified OVA-specific IgG antibodies, as determined by using glycopeptide (Fig E3, D) or total IgG glycan HPLC (Fig E3, E) analysis. **P < .01 and ***P < .001.

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