Comparison of the allergic and nonallergic CD4+ T-cell responses to the major dog allergen Can f 1 Suvi Parviainen, MSc, Antti Taivainen, MD, PhD, Aino.

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Comparison of the allergic and nonallergic CD4+ T-cell responses to the major dog allergen Can f 1 Suvi Parviainen, MSc, Antti Taivainen, MD, PhD, Aino Liukko, BM, Anssi Nieminen, MSc, Marja Rytkönen-Nissinen, PhD, Tuure Kinnunen, MD, PhD, Tuomas Virtanen, MD, PhD Journal of Allergy and Clinical Immunology Volume 126, Issue 2, Pages 406-408.e4 (August 2010) DOI: 10.1016/j.jaci.2010.06.005 Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Detection of antigen-specific proliferation of peripheral blood CD4+ T cells by the CFSE dilution assay after a 7-day culture. A representative example of antigen-specific responses from a subject with allergy (A). Proliferative responses of peripheral blood CD4+ T cells from subjects with (●) and without allergy (○) on stimulation with 100 μg/mL Can f 1 before (B) and after depletion of CD25hi cells (C), and in the presence of an isotype control or anti–IL-10 antibody (10 μg/mL; D). Results are expressed as the percentage of CD4+CFSElow cells. SK, Streptokinase. Journal of Allergy and Clinical Immunology 2010 126, 406-408.e4DOI: (10.1016/j.jaci.2010.06.005) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 The frequencies and functional characteristics of long-term Can f 1–specific memory TCLs. A, The number of Can f 1–specific TCLs generated from subjects with (●) and without allergy (○; per 30 replicate wells seeded). The functional TCR avidity (B) and cytokine production (C) of the TCLs. Functional avidity was evaluated by assessing EC50 values from the titration curves of the TCLs. Cytokine production was measured from the supernatants of cultures stimulated with 100 μg/mL Can f 1. Journal of Allergy and Clinical Immunology 2010 126, 406-408.e4DOI: (10.1016/j.jaci.2010.06.005) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Proliferative responses of peripheral blood CD4+ T cells from subjects with (●) and without allergy (○) on stimulation with 200 U/mL streptokinase (SK) before (A) and after depletion of CD25hi cells (B) and in the presence of an isotype control or anti–IL-10 antibody (10 μg/mL; C). Results are expressed as the percentage of CD4+CFSElow cells. Journal of Allergy and Clinical Immunology 2010 126, 406-408.e4DOI: (10.1016/j.jaci.2010.06.005) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Identification of CD4+CD25+Foxp3+ Treg cells in PBMCs and their depletion. CD4+CD25–, CD4+CD25int, and CD4+CD25hi cells were identified as shown (A). CD4+CD25hi cells were defined as those cells with a higher CD25 expression than that on the CD4– population. Foxp3 expression within each population was assessed by intracellular staining. Depletion of CD25hi by anti-CD25–coated magnetic beads (B). The percentages of CD4+CD25+Foxp3+(C) and CD4+CD25hiFoxp3+(D) cells in the PBMCs of subjects with (●) and without allergy (○) before and after CD25 depletion. Journal of Allergy and Clinical Immunology 2010 126, 406-408.e4DOI: (10.1016/j.jaci.2010.06.005) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Production of IL-4 and IL-5 by higher-avidity (EC50 ≤ 2 μg/mL, ●) and low-avidity (EC50 > 2 μg/mL, ○) TCLs from subjects with allergy. Cytokine production was measured from the supernatants of cultures stimulated with 100 μg/mL Can f 1. Journal of Allergy and Clinical Immunology 2010 126, 406-408.e4DOI: (10.1016/j.jaci.2010.06.005) Copyright © 2010 American Academy of Allergy, Asthma & Immunology Terms and Conditions