Journal of Allergy and Clinical Immunology Lifelong memory responses perpetuate humoral TH2 immunity and anaphylaxis in food allergy  Rodrigo Jiménez-Saiz, PhD, Derek K. Chu, MD, PhD, Talveer S. Mandur, MSc, Tina D. Walker, BSc, Melissa E. Gordon, MSc, Roopali Chaudhary, PhD, Joshua Koenig, BHSc, Sarah Saliba, BHSc, Heather J. Galipeau, PhD, Adam Utley, BSc, Irah L. King, PhD, Kelvin Lee, MD, Rachel Ettinger, PhD, Susan Waserman, MD, MSc, Roland Kolbeck, PhD, Manel Jordana, MD, PhD  Journal of Allergy and Clinical Immunology  DOI: 10.1016/j.jaci.2017.01.018 Copyright © 2017 The Authors Terms and Conditions

Fig 1 Duration of IgE-mediated clinical reactivity to food allergens. Separate groups of mice were orally sensitized to peanut (PN) and left unmanipulated until challenge 1, 3, 6, 12, or 15 months later. A and B, Anaphylaxis was assessed based on hypothermia (Fig 1, A) and hemoconcentration (Fig 1, B). C and D, Serum peanut-specific IgE (Fig 1, C) and IgG1 (Fig 1, D) before challenge. E, Correlation of serum peanut-specific immunoglobulins and clinical reactivity (hematocrit). Pooled data from 2 to 3 independent experiments and represented as means ± SEMs (n = 10-20). *P < .05 versus naive values. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig 2 Duration of reactivity caused by cell-bound IgE. A, Separate groups of IgE-deficient mice were passively sensitized to peanut (PN). B, Serum peanut-specific IgE after transfer. C and D, Recipient peritoneal IgE+ MCs. E and F, Recipient mice were challenged with peanut at different times after transfer, and clinical reactivity was evaluated based on hypothermia (Fig 2, E) and hemoconcentration (Fig 2, F). Pooled data from 2 to 3 independent experiments and presented as means ± SEMs (n= 3-5 per experiment). *P < .05 versus naive values. FMO, Fluorescence minus one. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig 3 Persistent IgE production requires GC formation by Bcl-6–competent B cells. A, Antigen (Ag)–specific GC B cells. B and C, GC activity in spleens (Fig 3, B) and mLNs (Fig 3, C) at different times after sensitization. D and E, Bcl-6B-cell KO and WT mice were sensitized to peanut (PN), and anaphylaxis was evaluated based on hypothermia (Fig 3, D) and hemoconcentration (Fig 3, E). F and G, Serum peanut-specific IgE (Fig 3, F) and IgG1 (Fig 3, G) levels at different times after sensitization. Representative data from 2 to 3 independent experiments and represented as means ± SEMs (n = 4-6 per experiment). *P < .05 versus naive values. FMO, Fluorescence minus one; n.s., nonsignificant. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig 4 Poor survival of CYC-resistant antigen-specific IgE+ PCs generated on sensitization to food allergens. A-C, Antigen-specific IgE+/IgG1+ PCs were identified in BM by using flow cytometry (Fig 4, A) and assessed at different times after sensitization (Fig 4, B and C). D, Separate groups of mice were sensitized to peanut (PN); treated with CYC, BZ, or a vehicle control (VEH); and later challenged. E, F, I, and J, Peanut-specific IgE/IgG1 with and without CYC (Fig 4, E and F) or BZ (Fig 4, I and J). G, H, K, and L, Anaphylaxis-induced hypothermia (Fig 4, G and K) and hemoconcentration (Fig 4, H and L) after challenge. Values are presented as means ± SEMs (n = 6-16 pooled from 5-8 [Fig 4, A-C] and 2-3 [Fig 4, E-L] experiments). *P < .05 versus naive values. n.s., Nonsignificant. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig 5 Allergen re-exposure reinvigorates IgE humoral immunity. A, Mice with peanut (PN) allergy treated with BZ were exposed to peanut either mucosally or systemically and challenged 2 weeks later. B and C, Serum peanut-specific IgE (Fig 5, B) and IgG1 (Fig 5, C). D and E, Anaphylaxis-induced hypothermia (Fig 5, D) and hemoconcentration (Fig 5, E) after challenge. Mice were sensitized and 8 months later were either left unmanipulated or re-exposed to oral allergen before analysis at 9 months. F-L, Gating strategy and identification of antigen-specific IgE+/IgG1+ memory B cells in LP of the small intestine, mLNs, and spleen. M-P, Antigen-specific PCs in BM (Fig 5, M and N) and serum antigen-specific IgE (Fig 5, O) and IgG1 (Fig 5, P). Values are presented as means ± SEMs (n = 9-15 pooled from 2-3 experiments). *P < .05 versus naive values. FMO, Fluorescence minus one; n.s., nonsignificant. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig 6 Memory responses to peanut at 15 months after sensitization. Mice were sensitized to peanut (PN), rested for 15 months, and then re-exposed to peanut either mucosally or systemically before challenge to peanut 2 weeks later. A and B, Serum peanut-specific IgE (Fig 6, A) and IgG1 (Fig 6, B) before challenge. C and D, Anaphylaxis-induced hypothermia (Fig 6, C) and hemoconcentration (Fig 6, D). E-G, Proliferation of memory B cells (Fig 6, E) and CD4 T cells (Fig 6, F) and plasmablasts (Fig 6, G) from spleens at 15 months. H and I, TH2 cytokines from culture supernatants (Fig 6, H and I). Values are presented as means ± SEMs (n = 8-15 pooled from 2-3 experiments). *P < .05 versus naive values. n.s., Nonsignificant. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig 7 IgE production on recall relies on memory B-cell responses and IL-4–producing CD4 T cells. A, Spleen cells from allergic mice were adoptively transferred into immunodeficient mice, and then recipients were exposed to oral allergen for 3 weeks. B and C, Serum antigen-specific IgE (Fig 7, B) and IgG1 (Fig 7, C). D and E, Anaphylaxis-induced hypothermia (Fig 7, D) and hemoconcentration (Fig 7, E). F and G, Proliferation of memory B cells (Fig 7, F) and plasmablasts (Fig 7, G) in the presence or absence of CD4 depletion, IL-4 neutralization, and/or CD40L blockade from spleens. Values are presented as means ± SEMs (n = 8-15 pooled from 2-3 experiments for Fig 7, A-D, and n = 15-20 pooled from 4 experiments for Fig 7, E and F). *P < .05 versus naive values for Fig 7, A-E; *P < .001 versus naive values for Fig 7, F and G. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig E1 Separate groups of mice were orally sensitized to peanut (PN) and left unmanipulated until challenge 1, 3, 6, 12, and 15 months later. A, Anaphylaxis was evaluated based on hypothermia. B, For clarity, the area under 39°C (AUC) over 40 minutes was calculated (see also Fig 1). Separate groups of mice were orally sensitized to peanut and OVA and left unmanipulated until challenge 1, 3, and 6 months later. C, Anaphylaxis was evaluated based on hypothermia. D and E, Sera were collected at different times after sensitization for OVA-IgE (Fig E1, D) and OVA-IgG1 (Fig E1, E) detection (see also Fig 2). Values are presented as means ± SEMs (n = 10-20 pooled from 2-3 experiments). *P < .05 versus naive values or as indicated. n.s., Nonsignificant. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig E2 Intracellular detection of IgE+ PCs (A) and assessment of antigen-specific IgG1+ PCs in the mLNs at 1 and 3 months after sensitization (B; see also Fig 3). Values are presented as means ± SEMs (n = 6-10 representative data from 2 independent experiments). *P < .05 vs naive values. FMO, Fluorescence minus one. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig E3 A-D, Effect of BZ treatment on PCs and antigen (Ag)−specific IgE+ PCs in the BM (Fig E3, A and B) and spleen (Fig E3, C and D). E-G, Effect of CYC treatment on total cell count (Fig E3, E) and number of B (Fig E3, F) and T (Fig E3, G) cells. H-J, Challenge of mice with peanut (PN) allergy after BZ treatment (Fig E3, H) did not impair clinical reactivity (Fig E3, I and J). K-M, BZ treatment immediately after peanut sensitization (Fig E3, K) did not affect clinical reactivity (Fig E3, L and M; see also Fig 3). Values are presented as means ± SEMs (n = 4-8 representative data of 2-3 independent experiments). *P < .05 versus naive values. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Fig E4 A and B, Extent of antibody-mediated CD4 T-cell depletion in the spleen (Fig E4, A) and LP (Fig E4, B) was evaluated by using flow cytometry. C and D, Administration of anti-CD40L blocking antibody during systemic sensitization to peanut abrogated clinical reactivity on challenge. E and F, Effect of anti−IL-4 and corresponding isotype control on plasmablast (Fig E4, E) or memory B-cell (Fig E4, F) proliferation was evaluated by using flow cytometry. Values are presented as means ± SEMs (n = 3-6 representative data of 2 independent experiments; see also Fig 5). *P < .001 versus naive values. Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions

Journal of Allergy and Clinical Immunology DOI: (10.1016/j.jaci.2017.01.018) Copyright © 2017 The Authors Terms and Conditions