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Natural protective immunity against grass pollen allergy is maintained by a diverse spectrum of response types  Almedina Kurtaj, MSc, Christoph Hillebrand,

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Presentation on theme: "Natural protective immunity against grass pollen allergy is maintained by a diverse spectrum of response types  Almedina Kurtaj, MSc, Christoph Hillebrand,"— Presentation transcript:

1 Natural protective immunity against grass pollen allergy is maintained by a diverse spectrum of response types  Almedina Kurtaj, MSc, Christoph Hillebrand, MSc, Gerda Fichtinger, BSc, Eva Hattinger, MSc, Melanie Lietzenmayer, BSc, Yoan Machado, PhD, Sandra Scheiblhofer, PhD, Angelika Stoecklinger, PhD, Theresa Thalhamer, PhD, Susanne Suessner, MSc, Martin Danzer, MSc, Sabine Keplinger, MSc, Johannes Weinberger, PhD, Susanne Schaller, MSc, Stephan Winkler, PhD, Christian Gabriel, MD, Josef Thalhamer, PhD, Richard Weiss, PhD  Journal of Allergy and Clinical Immunology  Volume 140, Issue 6, Pages e11 (December 2017) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Phl p 5–specific antibody responses in allergic and nonallergic donors. A, Percentages of IgG1 and IgG4 responders and nonresponders among nonallergic farmers (n = 114), nonallergic townspeople (n = 114), and allergic subjects (n = 11). B-D, Titers of Phl p 5–specific IgG1 and IgG4 (Fig 1, B), IgG1/IgG4 ratios (Fig 1, C), and IgA antibodies (Fig 1, D) in nonallergic farmers (white box), nonallergic townspeople (gray box), and allergic subjects (dark gray box). The dashed line indicates the detection limit. *P < .05. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Phl p 5–specific T-cell responses in allergic and nonallergic donors. A, Numbers of IFN-γ–, IL-10–, and IL-4–secreting cells in nonallergic farmers (n = 60), nonallergic townspeople (n = 60), and allergic subjects (n = 6). SFU, Spot-forming units. B, Intracellular expression of T-bet, GATA-3, RORγt, and FoxP3 in nonallergic farmers (n = 24) and nonallergic townspeople (n = 24). C, Percentage of TH1, TH2, TR1 and nonpolarized responses. D-G, TH1 (Fig 2, D), TH2 (Fig 2, E), TH9/TH17/TH22 (Fig 2, F), and TR1 (Fig 2, G) cytokine secretion in culture supernatants (farmers, n = 60; townspeople, n = 60; and allergic subjects, n = 6). Results are shown as means ± SEMs. *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 In vitro expansion of allergen-specific memory CD4+ T cells. An in vitro and antigen-specific expansion is required to study the low-frequency population of allergen-specific T cells in nonallergic subjects. A, Scheme of in vitro expansion of allergen-specific memory CD4+ T cells. B, Gating strategy, isotype control, and representative dot plots showing expression of CD45RO and L-selectin (CD62L) of viable CD3+CD4+ T cells before expansion and after the resting phase. Dot plots illustrate log fluorescence intensity for all markers. C, Comparison of T cells at different stages of maturation before expansion (open boxes) and after the resting phase (gray boxes; n = 26). Results are shown as percentages of CD3+CD4+Ki67+ cells. D, Antigen specificity of expanded cells from nonallergic subjects (n = 5) was confirmed by measuring proliferation after restimulation with Phl p 5 or the irrelevant antigen KLH. Results are shown as stimulation indices. The dashed line indicates the proliferation cutoff. Identical symbols correspond to the same subject. The horizontal line indicates the mean. Statistically significant differences were determined by using the paired Student t test. TCM, Central memory T cells; TEFF, effector T cells; TEM, effector memory T cells; TN, naive T cells. *P < .05 and ***P < .001. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Clonotype analysis of in vitro–expanded T cells. As an indicator of clonal expansion of circulating memory T cells from peripheral blood of the donors, the T-cell receptor repertoire of 1 nonallergic farmer (donor 1) and 1 nonallergic townsperson (donor 2) was analyzed before and after in vitro expansion. Tables show percentage values of the top 10 clonotypes based on complementarity-determining region 3 (CDR3) sequence before expansion, after expansion, and after resting phase. Color code indicates the same amino acid (AA) sequences at different time points. The curve diagram summarizes the frequency (percentage) of the top 10 clonotypes before expansion (dashed line), after expansion (dotted line), and after the resting phase (solid line). Most of the top 10 clones found after the resting phase were present already in the top 10 clones before the resting phase. Thus they apparently represent Phl p 5–specific central memory T cells that survived in the absence of antigen. In addition to the clear trend of clonal expansion, data indicate that clonal proliferation of memory T cells after stimulation with Phl p 5 is limited to a few dominant clones per subject. A, Donor 1, primer set 1; B, donor 1, primer set 2; C, donor 2, primer set 1; D, donor 2, primer set 2. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 TH2 cytokine secretion in nonallergic subjects is compensated by increased levels of proinflammatory cytokines. TH1/TH9/TH17/TH22-related cytokine levels in cultures from nonallergic farmers (circles, n = 8) and nonallergic townspeople (squares, n = 11) secreting increased levels of TH2 cytokines (sum of IL-4/IL-5/IL-13 = >90 pg/mL) and allergic subjects (triangles, n = 6) were determined after 72 hours of restimulation with rPhl p 5 by using a human cytokine 9-plex panel (IL-4, IL-5, IL-9, IL-10, IL-13, IL-17A, IFN-γ, TNF-α, and GM-CSF) and a quantitative luminometric ELISA (IL-2, IL-21, and IL-22). Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 Cytokine secretion pattern of compensatory cytokines in nonallergic farmers. The relative contribution of non-TH2 cytokines to the total amount of proinflammatory or regulatory cytokines is shown as a pie chart. Amounts of cytokines in supernatants are displayed as picograms per milliliter. Shown are the 8 farmers with the highest secretion of TH2 cytokines (sum of IL-4, IL-5, and IL-13). Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 Cytokine secretion pattern of compensatory cytokines in nonallergic townspeople. The relative contribution of non-TH2 cytokines to the total amount of proinflammatory or regulatory cytokines is shown as a pie chart. Amounts of cytokines in supernatants are displayed as picograms per milliliter. Shown are the 11 townspeople with the highest secretion of TH2 cytokines (sum of IL-4, IL-5, and IL-13). Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E6 Correlogram of farmers (A), townspeople (B), and allergic donors (C). Correlation matrices were assembled with data of 60 donors from each nonallergic group and 6 allergic donors, comprising a complete data set of the results from antibody, ELISpot, and cytokine measurements. Transcription factor data were only available for nonallergic donors (n = 22 for farmers and n = 24 for townspeople). The size of the circles and colors in the correlation plots represent the degree of correlation (Spearman rho) between 2 features. The significance level was set to less than .05. Nonsignificant coefficients were left blank. Journal of Allergy and Clinical Immunology  , e11DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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