B cells control maternofetal priming of allergy and tolerance in a murine model of allergic airway inflammation  Christine Happle, MD, PhD, Adan Chari Jirmo, PhD, Almut Meyer-Bahlburg, MD, Anika Habener, Heinz Gerd Hoymann, PhD, Christian Hennig, MD, Jelena Skuljec, PhD, Gesine Hansen, MD  Journal of Allergy and Clinical Immunology  Volume 141, Issue 2, Pages 685-696.e6 (February 2018) DOI: 10.1016/j.jaci.2017.03.051 Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Experimental scheme for evaluating the effects of maternal tolerization on the offspring's allergy risk. Murine mothers (WT or B-cell knockout) received 2 mucosal OVA doses and were consecutively mated. After weaning, offspring were tested in the OVA-based allergic airway inflammation model. In some experiments B cell–deficient mothers received WT B cells by means of tail vein injection 3 days before the first OVA dose. Ag, Antigen; AHR, airway hyperresponsiveness; KO, knockout; i.n., intranasal; i.p., intraperitoneal; Tx, transplantation. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Preconception mucosal antigen application to the dam reduces allergic airway inflammation in the offspring but aggravates airway inflammation in B-cell knockout (KO) mice, which is abolished after transfer of B cells to B cell–deficient mothers before antigen administration. A-C, Offspring of tolerized WT mothers display reduced lung inflammation and mucus production in hematoxylin and eosin (H&E)– and periodic acid–Schiff (PAS)–stained and paraffin-embedded lung slices (Fig 2, A), reduced airway inflammation in investigator-independent analysis of H&E-stained lung sections (Fig 2, B), and reduced total cell numbers and eosinophilia in BALF (Fig 2, C) compared with the offspring of naive WT dams. D-F, In B-cell knockout offspring, maternal OVA administration leads to aggravated lung inflammation and mucus production (Fig 2, D) and increased airway inflammation (Fig 2, E) in investigator-independent analysis of H&E-stained lung sections and increased total numbers and eosinophils in the BALF (Fig 2, F). G-I, B-cell transfer to B cell–deficient dams before antigen administration leads to reduced lung inflammation and mucus production (Fig 2, G), reduced airway inflammation in investigator-independent analysis of H&E-stained lung sections (Fig 2, H), and reduced total and eosinophil numbers in the BALF (Fig 2, I) compared with offspring of tolerized B-cell knockout mothers without B-cell transfer. Graphs display means + SEMs. Alum, Control group; B cell Tx, allergic offspring of OVA exposed and B cell–transplanted dams; eos, eosinophils; lym, lymphocytes; mac, macrophages; neu, neutrophils; Mova OVA, allergic offspring of OVA-exposed dams; OVA, allergic group. Groups: B cell KO, n ≥ 8 mice per experimental group (data from 1 [Fig 2, D and E] and 2 representative experiments of ≥3); B cell KO+Tx, n≥ 4 mice per experimental group (data from 1 [Fig 2, D and E] and 2 representative experiments of ≥3); WT, n ≥ 6 mice per experimental group (data from 1 [Fig 2, A and B] and 2 representative experiments of ≥3). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Preconception mucosal OVA administration reduces antigen-specific immunoglobulin and cytokine production in WT offspring, whereas it increases antigen-specific cytokine responses in offspring of B cell–deficient mice. This aggravation is partially abolished on B-cell transfer. A-D, Offspring of tolerized WT mice display reduced serum levels of OVA-specific IgE (Fig 3, A) and OVA-specific IgG1 (Fig 3, B) and diminished antigen-specific cytokine production from splenocytes (Fig 3, C) and bronchial lymph node cells (Fig 3, D). E-H, Offspring of OVA-exposed B cell–deficient mothers show no immunoglobulin production (Fig 3, E and F) but increased antigen-specific cytokine production by splenocytes (Fig 3, G) and bronchial lymph node cells (Fig 3, H). I-L, In offspring of B cell–transplanted and OVA-exposed μMT mice, absent immunoglobulins (Fig 3, I and J) and reduced antigen-specific cytokine production after in vitro splenocyte (Fig 3, K) and bronchial lymph node cell (Fig 3, L) restimulation with OVA are observed. Graphs display means + SEMs. Alum, Control group; B cell Tx, allergic offspring of OVA-exposed and B cell–transplanted dams; eos, eosinophils; lym, lymphocytes; mac, macrophages; Mova OVA, allergic offspring of OVA-exposed dams; neu, neutrophils; OVA, allergic group. Groups: B cell KO, n ≥ 7 mice per experimental group from 2 (Fig 3, F and G) and n ≥ 3 mice per experimental group from 1 representative experiment (Fig 3, E and H); B cell KO +Tx, n ≥ 7 mice per experimental group from 2 (Fig 3, K) and n ≥ 3 mice per experimental group from 1 representative experiment (Fig 3, I-L); WT, n ≥ 11 mice per experimental group from 2 (Fig 3, A-C) and n ≥ 4 mice per experimental group from 1 representative experiment (Fig 3, D). *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Defective Treg cell priming and in utero antigen (Ag) transfer in B cell–deficient mice. A, Reduced Treg cell frequency in 2-week-old B cell–deficient pups (frequency of CD4+CD25hiFoxp3+ T cells, n ≥ 26 mice per experimental group; data are from 3 experiments). B, Defective antigen-specific Treg cell priming in 2-week-old B cell–deficient pups after antigen re-exposure of the mother during lactation (frequency of antigen-specific OT-II CD4+Foxp3+ T cells in recipient pups (n ≥ 6 mice per group; data are from 2 experiments). C, Reduced in vivo intrauterine transfer of fluoOVA to CD11c/CD11b+ fetal DCs in B cell–deficient mice (frequency of OVA+CD11c/CD11b+ fetal splenic and thymic DCs, means ± SEMs of 2 experiments, each including n ≥ 5 fetuses per group). D, OVA-specific IgG (ELISA, 1 representative experiment including n ≥ 5 fetuses per group) in WT compared with B cell–deficient AF. n.d., Not determined. E, Transfer of higher amounts of OVA in the AF of WT mice that received OVA before conception compared with AF of equally treated B cell–deficient mice (AF, Western blotting, 1 representative experiment with pooled AF of n ≥ 5 fetuses per group). F, Priming of antigen-specific CD4+CD25hiFoxp3+ Treg cells by fetal DCs after coincubation with AF of naive WT dams (Ctrl.), OVA-exposed, tolerized WT dams (WT TOL), and OVA-exposed B cell–deficient mothers (KO TOL). G and H, CD4+CD25hiFoxp3+ Treg cells to CD4+CD25hiFoxp3− effector T cell ratio in the different coincubation settings (Ctrl: control AF, WT: AF of OVA-exposed WT dams, KO: OVA-exposed B cell–deficient mothers, Med: medium alone, OVA-IC, and OVA alone). KO, B cell–deficient mice. All bars display means + SEMs. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Increased in vitro uptake of IC by fetal DCs. A and B, Flow cytometric gating (Fig 5, A) and frequency (Fig 5, B) of adult versus fetal CD16/32 FcγRhi DCs. FSC, Forward scatter. C-E, Uptake of fluoOVA in CD11b+CD11c+ fetal and adult DCs after in vitro incubation with OVA-IC (Fig 5, C and D, percentage of fluoOVA IC+ DCs in total CD11b+CD11c+ DCs, data from ≥3 mice per group, 1 representative experiment). E, Uptake in specific DC subsets (6 mice/group, 2 experiments). F and G, Uptake of OVA-IC by total fetal DCs after injection of OVA alone (OVA) or OVA-IC (IC) to B cell–deficient pregnant mothers (means of 2 experiments, each including n ≥ 5 fetuses per group). H and I, Antigen uptake in human adult (black and white bar) versus cord blood (dotted dark grey and dotted light bar) DCs after incubation with OVA or OVA-IC (Fig 5, H) and ratio of OVA IC/OVA antigen uptake (Fig 5, I) from individual donors (data from n = 7 donors per group, data from 3 experiments). All bars display means + SEMs. KO, Knockout. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 B cell–deficient mice lack CD5+ regulatory B cells but still can be tolerized in a murine model of allergic airway inflammation. A, Lacking population of CD19+CD5+ B cells in the spleens of B cell–deficient mice (1 experiment with 19-26 mice per group). B, Experimental scheme. i.n., Intranasal; i.p., intraperitoneal. C, Reduced BALF cell counts and BALF eosinophils (eos), macrophages (mac), lymphocytes (lym), and neutrophils (neu). D, Reduced lung inflammation and mucus production (periodic acid–Schiff [PAS]/hematoxylin and eosin [H&E] staining of paraffin-embedded lung slices) in tolerized compared with allergic B-cell knockout mice. E, Reduced concentration of cytokines in cell-culture supernatants after antigen-specific restimulation of bronchial lymph node cells (1 representative experiment with 2-6 mice per group, means + SEMs). allergic, OVA-allergic group; control, control group; tolerized, OVA-tolerized group. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Increased airway hyperresponsiveness in offspring of B cell–deficient mice that received OVA mucosally before mating. Aggravated airway hyperreagibility in B-cell knockout mice after maternal OVA administration is shown. ED, Effective dose to elicit significant increase of lung resistance (RL) in invasive lung function measurements (1 experiment with n ≥ 12 mice per experimental group). MCh, Methacholine. **P < .005. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Proallergenic effect of maternal immunization in B cell–deficient mice is antigen specific. A, Experimental scheme. B, BALF total cell count and BALF eosinophils (eos), macrophages (mac), lymphocytes (lym), and neutrophils (neu). C, Lung inflammation and mucus production, as evaluated by using hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining. D, Antigen-specific cytokine production. control, Control group; mother naive, BLG-allergic offspring of naive dams; mother OVA, BLG-allergic offspring of OVA-tolerized dams. Data are means + SEMs (n ≥ 4 mice per experimental group from 2 experiments). n.s., Not significant. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Successful engraftment of B cells in B cell–deficient recipient mice. A and B, Increased spleen size (Fig E4, A) and detection of B220+IgD+ cells in bone marrow by means of flow cytometry (Fig E4, B) of transplanted B cell–deficient mice more than 2 months after transplantation and mating. C, Total and OVA-specific IgM production, as assessed by using ELISpot assays of splenocytes. D and E, OVA-specific IgG (Fig E4, D) but no OVA-specific IgE (Fig E4, E) production after B-cell transfer (1-3 representative mice from ≥2 experiments with n ≥ 5 mice per group). KO, Knockout; n.d., not determined; Tx, transplantation. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Detection of OVA in the AF of treated WT and B cell–deficient mice: increased OVA amounts in the AF of WT mice that received OVA before conception compared with AF of equally treated B cell–deficient mice (full unedited Western Blot). KO, Knockout. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Donor individual data for IC uptake in human adult versus cord blood DCs, including information on atopy status. A, Antigen uptake in human adult versus cord blood DCs after incubation with OVA or OVA-IC, as shown in Fig 5. Individual donors can be tracked by respective data point formatting: ▴, mild atopic dermatitis; ♦, allergic rhinitis (all other donors are nonatopic). B, ratio of OVA-IC/OVA antigen uptake from individual donors with the same individual data point markings as in Fig E6, A (n = 7 donors per group, 3 independent experiments). All bars display means + SEMs. **P < .01. Journal of Allergy and Clinical Immunology 2018 141, 685-696.e6DOI: (10.1016/j.jaci.2017.03.051) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions