BACKGROUND:Despite the efficacy of allergen-specific immunotherapy (AIT), the role of trained immunity and tolerance in this process has not been elucidated.OBJECTIVE:Here, we have performed a comprehensive longitudinal analysis of the systemic innate immune cell repertoire during the course of AIT.METHODS:Patients with allergy received standard preseasonal subcutaneous AIT with allergoids to birch and/or grass. Healthy controls were monitored without any intervention. Flow cytometry of innate lymphoid cell (ILC), natural killer cell, monocyte cell, and dendritic cell (DC) subsets was performed at baseline, 3 months (birch season), 6 months (grass seasons), and 12 months after the therapy in patients or at similar seasonal time points in controls. Additional analyses were performed in the third-year birch and grass season.RESULTS:We observed a durable decrease in group 2 ILCs and an increase of group 1 ILCs after AIT, with dynamic changes in their composition. We found that an expansion of CD127+CD25++ clusters caused observed shifts in the heterogeneity of group 1 ILCs. In addition, we observed development of CD127+CD25++c-Kit+ group 3 ILC clusters. Moreover, we found an increase in the number of intermediate monocytes in parallel with a reduction in nonclassical monocytes during the first year after AIT. Classical and intermediate monocytes presented significant heterogeneity in patients with allergy, but AIT reduced the HLA-DR++ clusters. Finally, an increase in plasmacytoid DCs and CD141+ myeloid DCs was observed in individuals with allergy, whereas the number of CD1c+ myeloid DCs was reduced during the first year of AIT.CONCLUSION:AIT induces changes in the composition and heterogeneity of circulating innate immune cells and brings them to the level observed in healthy individuals. Monitoring of ILCs, monocytes, and DCs during AIT might serve as a novel biomarker strategy.

The seventh "Future of the Allergists and Specific Immunotherapy (FASIT)" workshop held in 2019 provided a platform for global experts from academia, allergy clinics, regulatory authorities and industry to review current developments in the field of allergen immunotherapy (AIT). Key domains of the meeting included the following: (a) Biomarkers for AIT and allergic asthma; (b) visions for the future of AIT; (c) progress and data for AIT in asthma and the updates of GINA and EAACI Asthma Guidelines (separated for house dust mite SCIT, SLIT tablets and SLIT drops; patient populations) including a review of clinically relevant endpoints in AIT studies in asthma; (d) regulatory prerequisites such as the "Therapy Allergen Ordinance" in Germany; (e) optimization of trial design in AIT clinical research; (f) challenges planning and conducting phase III (field) studies and the future role of Allergen Exposure Chambers (AEC) in AIT product development from the regulatory point of view. We report a summary of panel discussions of all six domains and highlight unmet needs and possible solutions for the future.

A systematic nomenclature for allergens originated in the early 1980s, when few protein allergens had been described. A group of scientists led by Dr. David G. Marsh developed a nomenclature based on the Linnaean taxonomy, and further established the World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee in 1986. Its stated aim was to standardize the names given to the antigens (allergens) that caused IgE-mediated allergies in humans. The Sub-Committee first published a revised list of allergen names in 1986, which continued to grow with rare publications until 1994. Between 1994 and 2007 the database was a text table online, then converted to a more readily updated website. The allergen list became the Allergen Nomenclature database (www.allergen.org), which currently includes approximately 880 proteins from a wide variety of sources. The Sub-Committee includes experts on clinical and molecular allergology. They review submissions of allergen candidates, using evidence-based criteria developed by the Sub-Committee. The review process assesses the biochemical analysis and the proof of allergenicity submitted, and aims to assign allergen names prior to publication. The Sub-Committee maintains and revises the database, and addresses continuous challenges as new "omics" technologies provide increasing data about potential new allergens. Most journals publishing information on new allergens require an official allergen name, which involves submission of confidential data to the WHO/IUIS Allergen Nomenclature Sub-Committee, sufficient to demonstrate binding of IgE from allergic subjects to the purified protein.

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. Advances in allergen immunotherapy: aiming for complete tolerance to allergens.Sci Transl Med. 2015; 7: 280ps6Crossref PubMed Scopus (90) Google Scholar, 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 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.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, 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, E4Zhu 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.Nat Med. 2002; 8: 518-521Crossref PubMed Scopus (180) 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 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.E10Quakkelaar E.D. Fransen M.F. van Maren W.W. Vaneman J. 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.J Immunol. 2014; 192: 5813-5820Crossref PubMed Scopus (11) Google Scholar, E11Bruhns P. Jönsson F. Mouse and human FcR effector functions.Immunol Rev. 2015; 268: 25-51Crossref PubMed Scopus (278) Google Scholar, E12Gillis C.M. Jönsson F. Mancardi D.A. Tu N. Beutier H. Van Rooijen N. et al.Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice.J Allergy Clin Immunol. 2017; 139: 1253-1265.e14Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, E6Weiss M.E. Nyhan D. Peng Z.K. Horrow J.C. Lowenstein E. Hirshman C. et al.Association of protamine IgE and IgG antibodies with life-threatening reactions to intravenous protamine.N Engl J Med. 1989; 320: 886-892Crossref PubMed Scopus (158) Google Scholar, E7Hazenbos W.L. Gessner J.E. Hofhuis F.M. Kuipers H. Meyer D. Heijnen I.A. et al.Impaired IgG-dependent anaphylaxis and Arthus reaction in Fc gamma RIII (CD16) deficient mice.Immunity. 1996; 5: 181-188Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, E8Miyajima 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.J Clin Invest. 1997; 99: 901-914Crossref PubMed Scopus (310) Google Scholar, E9Khodoun M.V. Strait R. Armstrong L. Yanase N. Finkelman F.D. Identification of markers that distinguish IgE- from IgG-mediated anaphylaxis.Proc Natl Acad Sci U S A. 2011; 108: 12413-12418Crossref PubMed Scopus (103) Google Scholar, 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, 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 The effector functions of IgG antibodies depend on their subclassE11Bruhns 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 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,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, E15Ohmi Y. Ise W. Harazono A. Takakura D. Fukuyama H. Baba Y. et al.Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis.Nat Commun. 2016; 7: 11205Crossref PubMed Scopus (115) Google Scholar whereas galactosylation and terminal sialylation of IgG antibodies generally suppress inflammation.E15Ohmi Y. Ise W. Harazono A. Takakura D. Fukuyama H. Baba Y. et al.Sialylation converts arthritogenic IgG into inhibitors of collagen-induced arthritis.Nat Commun. 2016; 7: 11205Crossref PubMed Scopus (115) Google Scholar, 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, E19Karsten 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.Nat Med. 2012; 18: 1401-1406Crossref PubMed Scopus (315) Google Scholar, 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, 6Hess 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 (60) Google Scholar, 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 However, the effects of IgG subclasses and Fc glycosylation patterns in allergy remain unclear. We first compared the capacity of differently glycosylated forms of murine IgG1, a subclass that resembles AIT-induced human IgG4 in its limited ability to activate complement and classical activating FcγRs,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, 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. 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, 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, E24Aalberse R.C. van der Gaag R. van Leeuwen J. Serologic aspects of IgG4 antibodies. I. 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. Eiglmeier S. et al.T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies.J Clin Invest. 2013; 123: 3788-3796Crossref PubMed Scopus (60) 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 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−/−

Allergic patients display abnormal immune responses to harmless antigens, including abnormal Th2 polarization and induction of allergen-specific T and B cells, resulting in development of allergy instead of tolerance. Allergen-specific immunotherapy (AIT) is currently the only causative treatment of allergic disorders. Yet, understanding of the underlying differences in allergen-specific responses between allergy and tolerance is lacking. We investigated whole-genome transcriptomics of circulating birch and grass specific CD4+ T cells in patients before and during AIT, as well in non-allergic healthy controls. Detailed immunophenotyping with flow cytometry and CD4+ MHC class II tetramer staining with low RNA/single cell next generation sequencing were performed. At baseline, out of the season, there were more allergen-specific CD4+ T cells in allergic patients than in controls. At this time, over 1500 genes were differently expressed in allergen-specific CD4+T cells in patients, but they were less upregulated in signal transduction, inflammatory response and coagulation categories as compared to controls. During AIT we noted further increase of allergen-specific CD4+ cells with subsequent gene expression change. Finally, we found increase in allergen-specific Treg cells in patients upon AIT, but not in tolerant controls. Yet, at early AIT time points, allergen-specific Treg cells displayed gene profiles suggesting their insufficient functions. In summary, in vivo allergen exposure causes profound changes in the transcriptomic profiles of allergen-specific T cells. These gene profiles seem to be deficient at baseline in allergic patients, but AIT is skewing them into the tolerant controls levels.

Allergens produced by recombinant DNA technology have the ability to improve allergy diagnosis and are used as reference standards for analytical methods. In addition, the use of recombinant allergens in specific immunotherapy has long been considered potentially superior compared with the use of conventional extracts. The advantages are clear: a complex natural substance that is difficult to characterize is replaced by only those components relevant for treatment, which furthermore can be reproduced in pharmaceutical quality. The challenges faced here include selecting the relevant allergen molecules and establishing a manufacturing that meets all the regulatory requirements for marketing authorization. In addition to unmodified recombinant allergens, hypoallergenic variants with lower IgE reactivity can also be made by genetic engineering. Proof of concept has been demonstrated for both these approaches in clinical trials.

The Biological Standardization Programme of the European Directorate for Quality of Medicines and Healthcare (EDQM) aims at the establishment of well-characterized reference standards based on recombinant allergens and validated assays for the quantification of major allergen content. The objective of this study was to examine the detailed physicochemical and immunological characterization of recombinant Phl p 5.0109, the second available allergen reference standard.Recombinant Phl p 5.0109 PP5ar06007 was produced under GMP conditions and analyzed by an array of physicochemical and immunological methods for identity, quantity, homogeneity, and folding stability in bulk solution, as well as thermal denaturation, aggregation state, and biological activity when formulated for long-time storage.PP5ar06007 revealed as a highly homogeneous, monomeric, well-folded preparation of rPhl p 5.0109, as documented by mass spectrometry, SDS-PAGE, isoelectric focusing, size-exclusion chromatography with light scattering, circular dichroism, and infrared spectroscopy. Upon storage at +4°C, PP5ar06007 retained the monomeric state for at least 2 months. A protein quantity of 1.56 ± 0.03 mg/ml was determined by amino acid analysis in PP5ar06007, and its biological activity was shown to be comparable to natural Phl p 5 in terms of basophil activation and T-cell reactivity.Recombinant Phl p 5.0109 PP5ar06007 was characterized extensively at the physicochemical and immunological level. It revealed to be a highly stable, monomeric, and immunologically equivalent of its natural counterpart. PP5ar06007 is now available as European Pharmacopoeia allergen reference standard for grass pollen products.

Die molekulare Allergiediagnostik (komponentenaufgelöste Diagnostik, „component resolved diagnostic“, CRD) erlaubt bei Verdacht auf Gräser- und Baumpollenallergie die Identifikation der verantwortlichen Allergenquelle durch den Nachweis von spezifischem IgE. Insbesondere kann mit geeigneten Markerallergenen eine echte Sensibilisierung gegen Baum- oder Gräserpollen von der Kreuzreaktivität durch Pollenpanallergene (Profilin und Polcalcine) unterschieden und die fehlende analytische Spezifität von Allergenextrakten überwunden werden. Vor allem bei scheinbar polysensibilisierten Patienten mit Reaktionen auf zahlreiche Pollenextrakte ermöglicht die CRD somit eine allergenspezifische Diagnose unabhängig von den Panallergenen sowie eine fundierte Entscheidung für oder gegen eine spezifische Immuntherapie und ihre Zusammensetzung.

Biotechnologisch hergestellte rekombinante Allergene können die molekulare Allergiediagnostik verbessern und werden als Referenzstandards für analytische Methoden eingesetzt. Daneben wurde der Einsatz von rekombinanten Allergenen auch in der spezifischen Immuntherapie schon seit Langem als mögliche Verbesserung gegenüber der Verwendung konventioneller Extrakte gesehen. Die Vorteile liegen auf der Hand: Ein schwer zu beschreibender, komplexer Naturstoff wird ersetzt durch die relevanten therapieentscheidenden Bestandteile, die in höchster Qualität reproduzierbar hergestellt werden können. Herausforderungen sind dabei die Auswahl der notwendigen Allergenmoleküle und die Etablierung einer Herstellung, die allen regulatorischen Anforderungen der Zulassung entspricht. Neben unveränderten rekombinanten Allergenen lassen sich biotechnologisch auch hypoallergene Varianten mit erniedrigter IgE-Reaktivität herstellen; für beide Konzepte wurde in klinischen Studien bereits das „Proof of Concept“ gezeigt.

Detection of specific IgE using component resolved diagnostics (CRD) identifies the underlying allergen source in suspected cases of tree and grass pollen allergy. Suitable marker allergens can be used to distinguish genuine sensitization to tree or grass pollen from cross-reactivity to pollen panallergens (e. g. profilin and polcalcins) and to overcome the lack of analytical specificity of natural allergen extracts. In patients reacting with a variety of pollen extracts suspected of polysensitization, CRD allows allergen specific diagnosis regardless of the confounding effect of panallergenic cross-reactivity and administration of tailored, specific immunotherapy. In this article, allergens indicating specific sensitization to grass and tree pollen are described. Allergens defined as marker allergens for tree and grass pollen allergy are Bet v 1 (birch pollen major allergen) for birch, beech and other trees from the Fagales order, Ole e 1 (olive pollen major allergen) for olive and other trees including ash from the Oleaceae family, Pla a 1 (major allergen of the London plane tree) for plane trees, Cry j 1 (major allergen of the Japanese cedar), Cup a 1 (major allergen of the Arizona cypress) for cypress trees and Phl p 1 und 5 (Timothy grass major allergens) for sweet grasses including rye. Grass and tree pollen allergens with serological and clinical cross-reactivity to a great number of allergen sources are also identified as possible confounding factors in allergen specific diagnosis with natural extracts. Structured diagnostic procedures for clinical routine work are proposed.

The IUIS Allergen Nomenclature Sub-Committee, under the auspices of the World Health Organization and the International Union of Immunological Societies, maintains the systematic nomenclature of allergenic proteins and publishes a database of approved allergen names on its Web site, www.allergen.org. In this paper, we summarize updates of allergen names approved at the meetings of the committee in 2011 through 2013. These changes reflect recent progress in identification, cloning, and sequencing of allergens. The goals of this update were to increase consistency in the classification of allergens, isoallergens, and variants and in the incorporation of the evolutionary classification of proteins into allergen nomenclature, while keeping changes of established names to a minimum in the interest of continuity. Allergens for which names have been updated include respiratory allergens from birch and ragweed pollen, midge larvae, and horse dander; food allergens from peanut, cow's milk, and tomato; and cereal grain allergens. The IUIS Allergen Nomenclature Sub-Committee encourages researchers to use these updated allergen names in future publications.