IgE antibodies play a crucial role in allergic reactions, including systemic anaphylaxis, by binding to the high-affinity IgE Fc receptor FcεRI on mast cells and basophils and thereby inducing the release of inflammatory mediators.1Berings M. Karaaslan C. Altunbulakli C. Gevaert P. Akdis M. Bachert C. et al.Advances and highlights in allergen immunotherapy: on the way to sustained clinical and immunologic tolerance.J Allergy Clin Immunol. 2017; 140: 1250-1267Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar,2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,E1-E3 In contrast, allergen-specific IgG antibodies, induced also in response to allergen-specific immunotherapies, can suppress IgE-mediated anaphylaxis via allergen masking and particularly crosslinking FcεRI with the IgG inhibitory receptor FcγRIIB.1Berings M. Karaaslan C. Altunbulakli C. Gevaert P. Akdis M. Bachert C. et al.Advances and highlights in allergen immunotherapy: on the way to sustained clinical and immunologic tolerance.J Allergy Clin Immunol. 2017; 140: 1250-1267Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 3Finkelman F.D. Khodoun M.V. Strait R. Human IgE-independent systemic anaphylaxis.J Allergy Clin Immunol. 2016; 137: 1674-1680Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar,E1,E4-E6 However, when allergen levels are high, for example, medical drugs, IgG antibodies also have the potential to mediate anaphylaxis by crosslinking classical activating FcγRs, which is also controlled by FcγRIIB, on different innate immune cell types.2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 3Finkelman F.D. Khodoun M.V. Strait R. Human IgE-independent systemic anaphylaxis.J Allergy Clin Immunol. 2016; 137: 1674-1680Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 4Beutier H. Gillis C.M. Iannascoli B. Godon O. England P. Sibilano R. et al.IgG subclasses determine pathways of anaphylaxis in mice.J Allergy Clin Immunol. 2017; 139: 269-280.e7Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar,E5,E7-E14 Hence, analyzing or even enhancing the expression level of the inhibitory FcγRIIB might be a promising approach to predict or prevent IgG-mediated allergic reactions and also IgG-FcγRIIB–controlled-IgE-mediated allergic reactions. The intravenous immunoglobulin (IVIg), pooled human (hu) serum IgG from healthy donors, has been successfully used in high concentrations (1-2 g/kg) to treat patients with acute flares of inflammatory autoimmune diseases. Importantly, findings in animal autoimmune models have indicated that the therapeutic effect of IVIg/huIgG might be predominantly mediated via its Fc N-sialylated IgG subfraction (Fig 1, A).5Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1312) Google Scholar,6Maddur M.S. Kaveri S.V. Bayry J. Circulating normal IgG as stimulator of regulatory T cells: lessons from intravenous immunoglobulin.Trends Immunol. 2017; 38: 789-792Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,E15-E17 Elevating the fraction of sialylated bulk serum IgG antibodies to a certain critical level might be therefore sufficient to attenuate inflammatory autoimmune conditions.6Maddur M.S. Kaveri S.V. Bayry J. Circulating normal IgG as stimulator of regulatory T cells: lessons from intravenous immunoglobulin.Trends Immunol. 2017; 38: 789-792Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar,E15,E17 Functionally, it has been indicated in mice that sialylated huIgG antibodies with irrelevant specificities (or sialylated huIgG1 Fc portions) can interact with the sugar-binding C-type lectin receptor SIGN-R1 (specific intercellular adhesion molecule-3 (ICAM-3) grabbing nonintegrin-related 1) on marginal zone macrophages resulting in the expression of IL-33, which activates basophils to produce IL-4, which in turn upregulates FcγRIIB on effector macrophages in mice.5Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1312) Google Scholar,7Anthony R.M. Kobayashi T. Wermeling F. Ravetch J.V. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.Nature. 2011; 475: 110-113Crossref PubMed Scopus (461) Google Scholar,E15,E18 Here, we tested the capacity of high amounts of huIgG (1 g/kg) or lower amounts of highly sialylated murine (m) or huIgG subclass antibodies (10-50 mg/kg) with irrelevant specificities to enhance FcγRIIB expression on blood immune cells and to attenuate IgG-mediated anaphylaxis and IgG-FcγRIIB–controlled-IgE-mediated anaphylaxis in mice. IgG-mediated anaphylaxis was induced by intravenous injection of 200 μg of anti–2,4,6-trinitrophenyl (TNP) mIgG1 mAbs (clone H5),2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,8Oefner 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.J Allergy Clin Immunol. 2012; 129: 1647-1655Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar,9Hess 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.J Clin Invest. 2013; 123: 3788-3796Crossref PubMed Scopus (88) Google Scholar,E19-E21 followed by intravenous challenge with 20 or 25 μg of TNP-coupled ovalbumin 30 minutes later to allow formation of immune complexes (Fig 1, B). We first verified key cellular and molecular players of anti-TNP mIgG1-mediated anaphylaxis. We confirmed that Gr-1–expressing cells (containing monocytes, neutrophils, and eosinophils) are critical for induction of anaphylaxis (see Fig E1, A and B, in this article’s Online Repository at www.jacionline.org).4Beutier H. Gillis C.M. Iannascoli B. Godon O. England P. Sibilano R. et al.IgG subclasses determine pathways of anaphylaxis in mice.J Allergy Clin Immunol. 2017; 139: 269-280.e7Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar To assess the role of activating and inhibitory Fcγ receptors, we tested mice deficient in the signaling receptor subunit, the FcR γ−chain, of activating FcγRs (Fcerg1−/−), or in FcγRIIB (Fcgr2b−/−), respectively. Notably, murine IgG1 interacts only with the activating FcγRIII/FcR γ−chain complex but not with FcγRI- or FcγRIV-containing complexes.E12 Indeed, FcR γ−chain-deficient mice were protected from IgG1-mediated anaphylaxis (Fig E1, C), whereas animals lacking the inhibitory receptor FcγRIIB showed exacerbated symptoms compared with controls (Fig E1, D).2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,4Beutier H. Gillis C.M. Iannascoli B. Godon O. England P. Sibilano R. et al.IgG subclasses determine pathways of anaphylaxis in mice.J Allergy Clin Immunol. 2017; 139: 269-280.e7Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar To test the effect of bulk IgG Fc sialylation on IgG-mediated anaphylaxis, we injected intraperitoneally high amounts of huIgG (IVIg; 1 g/kg) or intavenously lower amounts of highly galactosylated plus sialylated (sialylated; sial) versus desialylated plus degalactosylated (degal) mIgG1 mAbs (clone MOPC-21 [50 mg/kg]E22 or clone MRC OX-7 [10 mg/kg]9Hess 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.J Clin Invest. 2013; 123: 3788-3796Crossref PubMed Scopus (88) Google Scholar,E23) with irrelevant specificities 23.5 hours before induction of the 30-minute anti-TNP mIgG1-mediated anaphylaxis model described above (Fig 1, B and C; see Fig E2, A and B, in this article’s Online Repository at www.jacionline.org). The unspecific huIgG as well as sialylated mIgG1 antibodies attenuated the anti-TNP mIgG1-mediated anaphylaxis, whereas the degal mIgG1 antibodies had no effect (Fig 1, D and F, and Fig E2, B and C). Notably, the IgG sialylation-mediated inhibition required SIGN-R1 (Fig 1, E and F) and FcγRIIB (Fig 1, G), whereas SIGN-R1 had no influence on the anti-TNP mIgG1-mediated anaphylaxis itself (Fig E2, D). We could not revoke the SIGN-R1–dependent effect with anti–IL-4 or anti–IL-33R blocking antibodies (data not shown), suggesting further as yet unclear SIGN-R1–dependent inhibitory pathways. Interestingly, sialylated mIgG2a mAbs (clone C1.18.4; 50 mg/kg) with irrelevant specificitiesE24 failed to suppress the anti-TNP mIgG-mediated anaphylaxis (Fig 1, H, and Fig E2, E), further indicating that, in contrast to mIgG1 or mIgG2b,2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,8Oefner 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.J Allergy Clin Immunol. 2012; 129: 1647-1655Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar,9Hess 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.J Clin Invest. 2013; 123: 3788-3796Crossref PubMed Scopus (88) Google Scholar,E20,E21,E25,E26 effector functions of mIgG2a antibodies might be less dependent on Fc glycosylation.2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,E25,E27 Instead, in vitro galactosylated plus sialylated purified serum huIgG4 attenuated the anti-TNP mIgG1-induced anaphylaxis (Fig E2, F and G). We further observed that sole intraperitoneal injection of huIgG (IVIg; 1 g/kg) or intravenous injection of sialylated mIgG1 (clone MOPC-21; 50 mg/kg), but not IgG2a (clone C1.18.4, 50 mg/kg), mAbs upregulated FcγRIIB expression on blood classical monocytes and eosinophils, which was detected by flow cytometry 24 hours later (Fig 1, I and J). FcγRIIB expression on murine neutrophils was too low to identify significant differences (data not shown). Importantly, timing, rather than antigen specificity, is critical for inhibitory effects of sialylated mIgG1 antibodies. Indeed, when applied 1 day in advance, both mIgG1 antibodies with irrelevant specificity (Fig 1, F) as well as antigen-specific (ie, anti-TNP) mIgG1 antibodies (see Fig E3, A-C, in this article’s Online Repository at www.jacionline.org),2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar when sialylated, suppressed anti-TNP mIgG1-mediated anaphylaxis. In contrast, Fc sialylation had no significant effect on the ability of antigen-specific mIgG1 antibodies to induce anaphylactic reactions in the 30-minute model (Fig E3, D and E). We therefore conclude that this delay might be critical for modulating FcγRIIB expression in an IgG Fc sialylation–dependent manner. Similarly, sialylated mIgG1 (clone MOPC-21; 50 mg/kg) with irrelevant specificity enhanced the FcγRIIB-dependent2Epp A. Hobusch J. Bartsch Y.C. Petry J. Lilienthal G.M. Koeleman C.A.M. et al.Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions.J Allergy Clin Immunol. 2018; 141: 399-402.e8Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar inhibitory effect of antigen-specific mIgG1 antibodies in an IgE-mediated anaphylaxis model (Fig 2, A-C). These observations suggested that sialylated IgG with irrelevant specificity might also upregulate FcγRIIB expression on FcεRI-expressing blood cells. Indeed, we observed an upregulation of FcγRIIB on FcεRI-expressing blood basophils 24 hours after application of huIgG or sialylated muIgG1, but not sialylated mIgG2a antibodies, with irrelevant specificities, in a SignR1-dependent manner (Fig 2, D). Besides IVIg treatment, physiological levels of IgG Fc sialylation already vary between individuals in the blood,6Maddur M.S. Kaveri S.V. Bayry J. Circulating normal IgG as stimulator of regulatory T cells: lessons from intravenous immunoglobulin.Trends Immunol. 2017; 38: 789-792Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar which might in turn modulate the expression of Fcγ receptors on immune cells and, in turn, influence an individual’s susceptibility to IgG-mediated or IgG-FcγRIIB–controlled-IgE-mediated allergic reactions or other IgG antibody–mediated diseases. We therefore assessed in a pilot study serum IgG Fc glycosylation and the expression levels of the inhibitory receptor FcγRIIB and the activating FcγRIIA, FcγRIII(A+B), and FcεRI on peripheral human blood neutrophils, monocytes, basophils, and/or B cells of 36 volunteers (24 females and 12 males; for details, see this article’s Methods section in the Online Repository at www.jacionline.org). Because IgG sialylation levels are distinct between the 2 sexes, we analyzed male and female data separately. Intriguingly, we identified significant positive associations between the levels of serum IgG Fc sialylation and the expression levels of FcγRIIB on neutrophils in female donors and on basophils in male donors (Fig 2, E-G; see Tables E1 and E2 and Fig E4 in this article’s Online Repository at www.jacionline.org). Although a larger cohort of donors is required to clearly establish the connection between IgG sialylation and FcγRIIB expression in human leukocytes, these results are well in line with our findings in mice. The data suggest that natural or modified levels of blood IgG Fc N-sialylation regulate the expression level of the inhibitory receptor FcγRIIB on immune cells and might protect individuals via this way not only from inflammatory autoimmune conditions but also from IgG-mediated as well as IgG-FcγRIIB–controlled-IgE-mediated allergic reactions. Thus, IgG-inducing allergen-specific immunotherapy in patients suffering from IgE-mediated allergic diseases might be more promissing and successful in patients showing higher bulk serum IgG sialylation levels, which has to be investigated. Although this effect of bulk serum IgG Fc sialylation was dependent on SIGN-R1 in mice, we could not revoke this effect with anti–IL-4 or anti–IL-33R blocking antibodies, suggesting further as yet unclear SIGN-R1–dependent inhibitory pathways to upregulate FcγRIIB. Interestingly, this inhibitory effect of bulk serum IgG sialylation was mediated by mIgG1, but not mIgG2a antibodies, suggesting IgG subclass–specific roles in mice. These findings might explain earlier observations of protective effects of IVIg in the context of allergyE27-E29 and might help to develop diagnostic, prognostic, and/or therapeutic tools for controlling IgG-mediated as well as IgG-FcγRIIB–controlled-IgE-mediated allergic reactions and IgG-dependent inflammatory disorders in general. We thank Mattias Collin for providing EndoS, Birgitta Heyman for the anti-TNP IgG1 (clone H5) hybridoma cells, Rudolf Manz for the anti–GR-1 mAbs (clone RB6-8C5), and Robina Thurmann and Kathleen Kuhrwahn for technical assistance. Human blood from healthy donors was collected with the approval of and in accordance with regulatory guidelines and ethical standards set by the University of Lübeck. Download .pptx (.11 MB) Help with pptx files Fig E1 Download .pptx (.19 MB) Help with pptx files Fig E2 Download .pptx (.13 MB) Help with pptx files Fig E3 Download .pptx (.96 MB) Help with pptx files Fig E4 Download .docx (.06 MB) Help with docx files Online Repository Download .docx (.07 MB) Help with docx files Table E1 Download .docx (.25 MB) Help with docx files Table E2

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.E1Santos 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.J Allergy Clin Immunol. 2015; 135: 1249-1256Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, E2Shade K.T. Platzer B. Washburn N. Mani V. Bartsch Y.C. Conroy M. et al.A single glycan on IgE is indispensable for initiation of anaphylaxis.J Exp Med. 2015; 212: 457-467Crossref PubMed Scopus (82) Google Scholar, E3Wawrzyniak P. Akdis C.A. Finkelman F.D. Rothenberg M.E. Advances and highlights in mechanisms of allergic disease in 2015.J Allergy Clin Immunol. 2016; 137: 1681-1696Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 1Akdis C.A. Akdis M. 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IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking.J Clin Invest. 2006; 116: 833-841Crossref PubMed Scopus (230) Google Scholar, E1Santos 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.J Allergy Clin Immunol. 2015; 135: 1249-1256Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, E11Bruhns P. Jönsson F. Mouse and human FcR effector functions.Immunol Rev. 2015; 268: 25-51Crossref PubMed Scopus (278) Google Scholar, E13Vidarsson G. Dekkers G. Rispens T. IgG subclasses and allotypes: from structure to effector functions.Front Immunol. 2014; 5: 520Crossref PubMed Scopus (1344) Google Scholar, E5Burton 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.J Allergy Clin Immunol. 2014; 134: 1310-1317.e6Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar 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,E16Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1379) Google Scholar 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)E20Strait 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.Nature. 2015; 517: 501-504Crossref PubMed Scopus (49) Google Scholar 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),2Finkelman F.D. Khodoun M.V. Strait R. Human IgE-independent systemic anaphylaxis.J Allergy Clin Immunol. 2016; 137: 1674-1680Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 3Strait 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.J Clin Invest. 2006; 116: 833-841Crossref PubMed Scopus (230) Google Scholar 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).4Beutier H. Gillis C.M. Iannascoli B. Godon O. England P. Sibilano R. et al.IgG subclasses determine pathways of anaphylaxis in mice.J Allergy Clin Immunol. 2017; 139: 269-280.e7Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar 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),4Beutier H. Gillis C.M. Iannascoli B. Godon O. England P. Sibilano R. et al.IgG subclasses determine pathways of anaphylaxis in mice.J Allergy Clin Immunol. 2017; 139: 269-280.e7Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar 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).7Collin M. Ehlers M. The carbohydrate switch between pathogenic and immunosuppressive antigen-specific antibodies.Exp Dermatol. 2013; 22: 511-514Crossref PubMed Scopus (64) Google Scholar, 8Pincetic 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.Nat Immunol. 2014; 15: 707-716Crossref PubMed Scopus (330) Google Scholar, E17Anthony R.M. Wermeling F. Karlsson M.C. Ravetch J.V. Identification of a receptor required for the anti-inflammatory activity of IVIG.Proc Natl Acad Sci U S A. 2008; 105: 19571-19578Crossref PubMed Scopus (433) Google Scholar, E18Anthony R.M. Kobayashi T. Wermeling F. Rav etch JV. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.Nature. 2011; 475: 110-113Crossref PubMed Scopus (481) Google Scholar 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).E21Möbs 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.J Immunol. 2010; 184: 2194-2203Crossref PubMed Scopus (108) Google Scholar, E22Gepp 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.J Allergy Clin Immunol. 2016; 137: 1600-1603.e1Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, E23Subbarayal 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.J Allergy Clin Immunol. 2015; 136: 1680-1682.e3Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 9Möbs 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.J Allergy Clin Immunol. 2012; 130: 1108-1116.e6Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar 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).1Akdis C.A. Akdis M. Advances in allergen immunotherapy: aiming for complete tolerance to allergens.Sci Transl Med. 2015; 7: 280ps6Crossref PubMed Scopus (90) Google Scholar, 9Möbs C. Ipsen H. Mayer L. Slotosch C. Petersen A. 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Prolonged immunization results in an IgG4-restricted response.J Immunol. 1983; 130: 722-726PubMed Google Scholar 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).E14Scherer 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.Arthritis Rheum. 2010; 62: 1620-1629Crossref PubMed Scopus (169) Google Scholar 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)5Oefner 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.J Allergy Clin Immunol. 2012; 129: 1647-1655Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar 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).E16Kaneko Y. Nimmerjahn F. Ravetch J.V. Anti-inflammatory activity of immunoglobulin G resulting from Fc sialylation.Science. 2006; 313: 670-673Crossref PubMed Scopus (1379) Google Scholar, E17Anthony R.M. Wermeling F. Karlsson M.C. Ravetch J.V. Identification of a receptor required for the anti-inflammatory activity of IVIG.Proc Natl Acad Sci U S A. 2008; 105: 19571-19578Crossref PubMed Scopus (433) Google Scholar, E18Anthony R.M. Kobayashi T. Wermeling F. Rav etch JV. Intravenous gammaglobulin suppresses inflammation through a novel T(H)2 pathway.Nature. 2011; 475: 110-113Crossref PubMed Scopus (481) Google Scholar 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 adjuvantsE25Larché M. Akdis C.A. Valenta R. Immunological mechanisms of allergen-specific immunotherapy.Nat Rev Immunol. 2006; 6: 761-771Crossref PubMed Scopus (634) Google Scholar, E26Pfaar O. Cazan D. Klimek L. Larenas-Linnemann D. Calderon M.A. Adjuvants for immunotherapy.Curr Opin Allergy Clin Immunol. 2012; 12: 648-657Crossref PubMed Scopus (54) Google Scholar, E27Patel 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.J Allergy Clin Immunol. 2014; 133 (e1-2): 121-129Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, E28Valenta 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.J Allergy Clin Immunol. 2016; 137: 351-357Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, E29Mahan A.E. Jennewein M.F. Suscovich T. Dionne K. Tedesco J. Chung A.W. et al.Antigen-specific antibody glycosylation is regulated via vaccination.PLoS Pathog. 2016; 12: e1005456Crossref PubMed Scopus (96) Google Scholar, 1Akdis C.A. Akdis M. Advances in allergen immunotherapy: aiming for complete tolerance to allergens.Sci Transl Med. 2015; 7: 280ps6Crossref PubMed Scopus (90) Google Scholar 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)1Akdis C.A. Akdis M. Advances in allergen immunotherapy: aiming for complete tolerance to allergens.Sci Transl Med. 2015; 7: 280ps6Crossref PubMed Scopus (90) Google Scholar, 6Hess C. Winkler A. Lorenz A.K. Holecska V. Blanchard V. 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Efficacy of a short course of specific immunotherapy in patients with allergic rhinoconjunctivitis to ragweed pollen.J Allergy Clin Immunol. 2014; 133 (e1-2): 121-129Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar 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,E30Bondt 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.J Proteome Res. 2013; 12: 4522-4531Crossref PubMed Scopus (117) Google Scholar, E31Pezer 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.Sci Rep. 2016; 6: 33198Crossref PubMed Scopus (18) Google Scholar 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).E32de 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.Front Immunol. 2017; 8: 608Crossref PubMed Scopus (34) Google Scholar 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 protocols1Akdis C.A. Akdis M. Advances in allergen immunotherapy: aiming for complete tolerance to allergens.Sci Transl Med. 2015; 7: 280ps6Crossref PubMed Scopus (90) Google Scholar, E25Larché M. Akdis C.A. Valenta R. Immunological mechanisms of allergen-specific immunotherapy.Nat Rev Immunol. 2006; 6: 761-771Crossref PubMed Scopus (634) Google Scholar, E26Pfaar O. Cazan D. Klimek L. Larenas-Linnemann D. Calderon M.A. Adjuvants for immunotherapy.Curr Opin Allergy Clin Immunol. 2012; 12: 648-657Crossref PubMed Scopus (54) Google Scholar 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 patientsE21Möbs 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.J Immunol. 2010; 184: 2194-2203Crossref PubMed Scopus (108) Google Scholar, E22Gepp 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.J Allergy Clin Immunol. 2016; 137: 1600-1603.e1Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, E23Subbarayal 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.J Allergy Clin Immunol. 2015; 136: 1680-1682.e3Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, E33Möbs 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.J Allergy Clin Immunol. 2012; 130: 1108-1116.e6Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar 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−/−

Immediate hypersensitivity reactions are induced by the interaction of allergens with specific IgE antibodies bound via FcεRI to mast cells and basophils. While these specific IgE antibodies are needed to trigger such reactions, not all individuals harboring IgE exhibit symptoms of allergy. The lack of responsiveness seen in some subjects correlates with the presence of IgG antibodies of the same specificity. In cell culture studies and in vivo animal models of food allergy and anaphylaxis such IgG antibodies have been shown to exert suppression via FcγRIIb. However, the reported absence of this inhibitory receptor on primary mast cells derived from human skin has raised questions about the role of IgG-mediated inhibition of immediate hypersensitivity in human subjects. Here, we tested the hypothesis that mast cell FcγRIIb expression might be tissue specific. Utilizing a combination of flow cytometry, quantitative PCR, and immunofluorescence staining of mast cells derived from the tissues of humanized mice, human skin, or in fixed paraffin-embedded sections of human tissues, we confirm that FcγRIIb is absent from dermal mast cells but is expressed by mast cells throughout the gastrointestinal tract. IgE-induced systemic anaphylaxis in humanized mice is strongly inhibited by antigen-specific IgG. These findings support the concept that IgG, signaling via FcγRIIb, plays a physiological role in suppressing hypersensitivity reactions.

The aim of this review will be to familiarize the reader with the general area of antibody (Ab) glycosylation and to summarize the known functional roles of glycosylation and how glycan structure can contribute to various disease states with emphasis on allergic disease.

Antibodies can protect from Plasmodium falciparum (Pf) infection and clinical malaria disease. However, in the absence of constant reexposure, serum immunoglobulin (Ig) levels rapidly decline and full protection from clinical symptoms is lost, suggesting that B cell memory is functionally impaired. We show at the single cell level that natural Pf infection induces the development of classical memory B cells (CM) and atypical memory B cells (AtM) that produce broadly neutralizing antibodies against blood stage Pf parasites. CM and AtM contribute to anti-Pf serum IgG production, but only AtM show signs of active antibody secretion. AtM and CM were also different in their IgG gene repertoire, suggesting that they develop from different precursors. The findings provide direct evidence that natural Pf infection leads to the development of protective memory B cell antibody responses and suggest that constant immune activation rather than impaired memory function leads to the accumulation of AtM in malaria. Understanding the memory B cell response to natural Pf infection may be key to the development of a malaria vaccine that induces long-lived protection.