Potential role of extracellular vesicle–mediated antigen presentation in Helicobacter pylori hypersensitivity during eradication therapy Takamasa Ito,

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Potential role of extracellular vesicle–mediated antigen presentation in Helicobacter pylori hypersensitivity during eradication therapy Takamasa Ito, MD, Takashi Shiromizu, PhD, Shunsuke Ohnishi, MD, PhD, Shotaro Suzuki, PhD, Katsuhiro Mabe, MD, PhD, Akito Hasegawa, MD, Hideyuki Ujiie, MD, PhD, Yasuyuki Fujita, MD, PhD, Yuichi Sato, MD, PhD, Shuji Terai, MD, PhD, Mototsugu Kato, MD, PhD, Masahiro Asaka, MD, PhD, Takeshi Tomonaga, MD, PhD, Hiroshi Shimizu, MD, PhD, Riichiro Abe, MD, PhD Journal of Allergy and Clinical Immunology Volume 142, Issue 2, Pages 672-676.e12 (August 2018) DOI: 10.1016/j.jaci.2018.02.046 Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Clinical features and analysis of ELISA, LTT, and ELISpot results in patients with skin reactions after H pylori (HP) eradication therapy. A, Skin manifestations of patients with skin eruptions after H pylori eradication therapy (patients 2 and 13; patients with negative drug LTT responses). A maculopapular rash developed on the chest and back. B, Clinical course of patients with skin eruptions after H pylori eradication therapy assessed as the mean percentage of skin area affected in each patient. C, Cytokine levels in cell-culture supernatants from PBMCs exposed to H pylori, E coli, or PBS for 24 hours. PBMCs were sampled from patients with and without skin eruption (SE) after H pylori eradication therapy and healthy volunteer control subjects. Supernatants were assayed for multiple cytokines by using the Bio-Plex Human Cytokine 27-Plex panel (Bio-Rad Laboratories, Hercules, Calif). Values are means of 3 independent experiments. *P < .05. D, Proliferation of T cells in the PBS, drug, or H pylori groups was measured by using an LTT. Results are presented as an SI. E, IFN-γ secretion from PBMCs was measured by using an ELISpot in response to H pylori, drugs, or PBS. Data are from patients with skin eruptions after H pylori eradication therapy. The number of spots per well is indicated next to the corresponding well image. Journal of Allergy and Clinical Immunology 2018 142, 672-676.e12DOI: (10.1016/j.jaci.2018.02.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Expression of CD154 on CD4+ T cells after incubation of patients' PBMCs stimulated with H pylori (HP) or H pylori peptides containing EVs. A, Flow cytometric analysis of CD154 expression by CD4+ T cells from a patient with skin eruptions after H pylori treatment after 24 hours of culture with drugs or H pylori (shaded area; isotype control). B, CD154 expression in CD4+ T cells from patients with and without skin eruptions after H pylori treatment after 24 hours of culture with PBS, drugs, or H pylori (n = 11). ≥ 3 days, Skin eruptions develop 3 or more days after completing H pylori eradication treatment; ≤ 2 days, skin eruptions develop 2 days or less after completing H pylori eradication treatment. *P < .05 and **P < .01. C, Flow cytometric analysis of CD154 expression in CD4+ T cells from a patient with skin eruptions after H pylori eradication (patient 1) after 24 hours of culture with EVs from MDDCs cultured with PBS, eradication drugs, or H pylori. D, Number of patients with negative drug LTT responses or patients with positive drug LTT responses with skin eruptions after H pylori treatment. E, Numbers of patients with a positive result for any immunologic test for H pylori and those with a positive result for any immunologic test for drugs. Journal of Allergy and Clinical Immunology 2018 142, 672-676.e12DOI: (10.1016/j.jaci.2018.02.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Histopathologic findings for patients with negative drug LTT responses and patients with positive drug LTT responses. Histologic analysis of 5 patients with negative drug LTT responses (A) and 15 patients with positive drug LTT responses (B) exhibited vacuolar interface dermatitis and superficial perivascular infiltrate of lymphocytes with hematoxylin and eosin staining. Original magnification ×100. Journal of Allergy and Clinical Immunology 2018 142, 672-676.e12DOI: (10.1016/j.jaci.2018.02.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Scanning electron microscope micrograph of whole mounted EVs purified from H pylori–exposed MDDC cultures. Journal of Allergy and Clinical Immunology 2018 142, 672-676.e12DOI: (10.1016/j.jaci.2018.02.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Flow cytometric analysis of MDDC-derived EVs. MDDC-derived EVs were analyzed for CD9, CD81, HLA-ABC, and HLA-DR by using flow cytometry. Blue histograms, MDDC-derived EVs; red histograms, isotype control. Journal of Allergy and Clinical Immunology 2018 142, 672-676.e12DOI: (10.1016/j.jaci.2018.02.046) Copyright © 2018 American Academy of Allergy, Asthma & Immunology Terms and Conditions