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Reshaping the Bet v 1 fold modulates TH polarization

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Presentation on theme: "Reshaping the Bet v 1 fold modulates TH polarization"— Presentation transcript:

1 Reshaping the Bet v 1 fold modulates TH polarization
Michael Wallner, PhD, Michael Hauser, PhD, Martin Himly, PhD, Nadja Zaborsky, PhD, Sonja Mutschlechner, PhD, Andrea Harrer, PhD, Claudia Asam, MSc, Ulrike Pichler, MSc, Ronald van Ree, PhD, Peter Briza, PhD, Josef Thalhamer, PhD, Barbara Bohle, PhD, Gernot Achatz, PhD, Fatima Ferreira, PhD  Journal of Allergy and Clinical Immunology  Volume 127, Issue 6, Pages e9 (June 2011) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 A, Circular dichroism spectra of Bet v 1 and BM4 at 20°C. Spectra are baseline corrected and presented as mean residue molar ellipticity. deg, Degree; MRW, mean residue weight. B, The FTIR spectra of Bet v 1 and BM4 are presented as second derivatives. Typical regions of secondary structure elements are highlighted (α-helix, blue; intramolecular β-sheet, red; intermolecular β-sheet, green). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Aggregation behavior of Bet v 1 and BM4 were analyzed by DLS (small panel) and gel filtration (large panel). Hydrodynamic radius and molecular weight of both proteins were determined by light scattering. HP-SEC, High performance-size exclusion chromatography; MW, molecular weight; RALS, Right-angle light scattering; RH, hydrodynamic radius; RI, refractive index. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Proliferative responses of PBMCs from donors with birch pollen allergy stimulated with increasing amounts of Bet v 1 or BM4. Symbols represent individual patients, bars medians. P values were calculated by paired-sample t test (∗P < .05). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 A, BALB/c mice were immunized 4 times with either Bet v 1 (filled circles) or BM4 (open circles; n = 8 per group). Bet v 1–specific antibody levels were determined by ELISA. B, ELISPOT analysis of splenocytes from immunized animals expressed as mean cytokine-secreting cells per 105 spleen cells ± SEM. P values were calculated with t tests (∗∗P < .01; ∗∗∗P < .001). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 BMDCs were pulsed with FITC or pHrodo-labeled Bet v 1 or BM4, stained for CD11c and CD86, and gated for CD11c expression. Representative fluorescence-activated cell-sorting profiles of 6 experiments per time point and antigen are shown. Numbers in the panels represent percentage values of analyzed cells (left panel). A time course analysis is presented as a line chart ± SEM (right panel). P values were calculated with t tests (∗P < .05; ∗∗P < .01). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 A, SDS-PAGE analysis of time-dependent endo-/lysosomal degradation of Bet v 1 and BM4. B, Percent degradation was determined densitometrically from SDS-PAGE bands and is presented as a line chart. C, Proteolytic fragments of Bet v 1 (black lines) or BM4 (red lines) were analyzed by mass spectrometry. Dominant T cell epitopes of Bet v 1 are indicated by vertical blue bars. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Primary sequence of BM4 compared to Bet v 1, isoform 0101, and Mal d 1, isoform The exchange sequence of BM4 is highlighted by a gray box. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 ANS binding to Bet v 1 and BM4 was analyzed with fluorescence spectroscopy. Data are presented in mean fluorescence units (RFU). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 IgE binding of BM4 was compared with Bet v 1 by ELISA detecting antigen-specific human serum IgE (n = 13; A), RBL assays using patients’ sera (n = 8; B), or basophil activation tests (n = 8; B). C, Four characteristic RBL titration curves are shown. Bet v 1 is represented as a black line and BM4 as a red line in the graphs. OVA, Ovalbumin. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 BALB/c mice were immunized 4 times with Bet v 1∗ (filled circle), A1-6 (filled triangle), or BM4 (open circle; n = 5 per group). Bet v 1∗–specific antibody levels were determined by ELISA. P values were calculated with t tests (∗P < .05). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 BALB/c mice were immunized 4 times with Bet v 1∗ (black bars), A1-6 (gray bars), or BM4 (white bars; n = 5 per group). ELISPOT analysis of splenocytes from immunized animals expressed as mean cytokine-secreting cells per 2 × 105 spleen cells ± SEM. P values were calculated with t tests (∗P < .05; ∗∗P < .01; ∗∗∗P < .001). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Bet v 1–coated ELISA plates were preincubated with IgE-depleted pooled mouse sera of animals immunized with Bet v 1 (n = 5) or BM4 (n = 5), and human serum IgE binding of 5 patients with birch pollen allergy was determined. The threshold was calculated by preincubation of Bet v 1 with pooled murine preimmune sera (n = 5). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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