Malassezia sympodialis thioredoxin–specific T cells are highly cross-reactive to human thioredoxin in atopic dermatitis Hari Balaji, MSc, Annice Heratizadeh,

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Malassezia sympodialis thioredoxin–specific T cells are highly cross-reactive to human thioredoxin in atopic dermatitis Hari Balaji, MSc, Annice Heratizadeh, MD, Katja Wichmann, MD, Margarete Niebuhr, MD, Reto Crameri, PhD, Annika Scheynius, MD, PhD, Thomas Werfel, MD Journal of Allergy and Clinical Immunology Volume 128, Issue 1, Pages 92-99.e4 (July 2011) DOI: 10.1016/j.jaci.2011.02.043 Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 TCLs from patients with AD generated in the presence of Mala s 13 proliferate in the presence of Mala s 13 and hTrx. TCLs generated in the presence of Mala s 13 from the peripheral blood of patients with AD with (AD s) and without (AD ns) IgE sensitization to Malassezia species, patients with psoriasis, and healthy control subjects were stimulated with rMala s 13, rhTrx, rMala s 11, recombinant human MnSOD, and human serum albumin (HSA) for 5 days. Proliferation was assessed by means of incorporation of tritiated thymidine, and results are expressed as the SI. The counts per minute value for the medium control in this experiment was 672.5 ± 96.5 (mean ± SEM), which is indicative of efficient thymidine incorporation. Journal of Allergy and Clinical Immunology 2011 128, 92-99.e4DOI: (10.1016/j.jaci.2011.02.043) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 High degree of cross-reactivity of Mala s 13 with hTrx in TCCs. Forty-four TCCs generated from blood and 71 TCCs generated from positive APT lesions were specific for Mala s 13. The SIs for the antigen restimulation tests with TCCs performed with rMala s 13 (black bars) or with rhTrx (gray bars) are indicated. The horizontal line indicates the cutoff SI value of 2. A, Mala s 13–specific bTCCs generated from the blood of patients with AD sensitized to M sympodialis. B, Mala s 13–specific sTCCs generated from biopsy specimens of positive APT reactions to rMala s 13. C, Mala s 13–specific sTCCs generated from a biopsy specimen of a positive APT reaction to M sympodialis extract. Journal of Allergy and Clinical Immunology 2011 128, 92-99.e4DOI: (10.1016/j.jaci.2011.02.043) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Cytokine secretion profile of blood- and skin-derived Mala s 13–specific TCCs stimulated with hTrx. Forty-four bTCCs (A) and 85 sTCCs (B) shown to be hTrx reactive were stimulated with rhTrx for 5 days, and the supernatants were analyzed for various cytokines by means of ELISA. Journal of Allergy and Clinical Immunology 2011 128, 92-99.e4DOI: (10.1016/j.jaci.2011.02.043) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Human keratinocytes release thioredoxin (TRX). Human primary keratinocytes (n = 8) were stimulated with various cytokines, as described in the Methods section, and the supernatants were assayed for the release of hTrx by means of ELISA. ∗∗∗P < .001. Journal of Allergy and Clinical Immunology 2011 128, 92-99.e4DOI: (10.1016/j.jaci.2011.02.043) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Suppression of proliferation of Mala s 13/hTrx–specific TCCs in the presence of a blocking antibody against HLA-DR. Ten randomly selected Mala s 13–specific bTCCs (A) and 10 randomly selected Mala s 13–specific sTCCs (B) generated from positive APT reactions to rMala s 13 were restimulated with rhTrx in the presence or absence of a blocking antibody against HLA-DR. An isotype-matched control antibody was used as a negative control. ∗∗∗P < .001. Journal of Allergy and Clinical Immunology 2011 128, 92-99.e4DOI: (10.1016/j.jaci.2011.02.043) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions