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Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions

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1 Sialylation of IgG antibodies inhibits IgG-mediated allergic reactions
Alexandra Epp, MSc, Juliane Hobusch, MSc, Yannic C. Bartsch, MSc, Janina Petry, MSc, Gina-Maria Lilienthal, MSc, Carolien A.M. Koeleman, Simon Eschweiler, MSc, Christian Möbs, PhD, Ashley Hall, BS, Suzanne C. Morris, PhD, Dominique Braumann, MSc, Christine Engellenner, Josephine Bitterling, Johann Rahmöller, Alexei Leliavski, PhD, Robina Thurmann, MSc, Mattias Collin, PhD, Kelley W. Moremen, PhD, Richard T. Strait, MD, Véronique Blanchard, PhD, Arnd Petersen, PhD, Timo Gemoll, PhD, Jens K. Habermann, PhD, Frank Petersen, PhD, Andreas Nandy, PhD, Helga Kahlert, PhD, Michael Hertl, MD, Manfred Wuhrer, PhD, Wolfgang Pfützner, MD, Uta Jappe, MD, Fred D. Finkelman, MD, Marc Ehlers, PhD  Journal of Allergy and Clinical Immunology  Volume 141, Issue 1, Pages e8 (January 2018) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Effect 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. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Bet 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 Fig E2, 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 Fig E2. One of 2 independent ELISAs is shown. B and C, Percentage of agalactosylated (G0; Fig 1, B) and sialylated (Fig 1, 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 Filled areas indicate levels of agalactosylated (G0) or sialylated IgG autoantibodies in patients with rheumatoid arthritis (RA) for comparison.E14 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 Fig 1, 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. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Generation, 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. Fig 1, 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). Fig 1, E, Modification of the Fc glycan of IgG1 anti-TNP mAbs (clone H5) did not change affinity. Fig 1, 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 Fig 1, E and F. G, IgE-mediated anaphylaxis, as described in Fig 1, 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 Fig 1, 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 =  Symbols represent means. Pooled data are from independent experiments with n = 5 per group per experiment. *P < .05 and **P < .01. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Further 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 Fig 2, 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 Fig 2, 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 Fig 2, 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 K and L, Human neutrophil activation assay, as described in Fig 2, 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. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E2 Further 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 Fig 2, 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 Fig 2, 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 Fig 2, 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 K and L, Human neutrophil activation assay, as described in Fig 2, 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. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E3 Different 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 Fig 2, 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. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E4 Patients' characteristics.
Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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