Dissection of the IgE and T-cell recognition of the major group 5 grass pollen allergen Phl p 5 Margarete Focke-Tejkl, PhD, Raffaela Campana, PhD, Renate.

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Dissection of the IgE and T-cell recognition of the major group 5 grass pollen allergen Phl p 5 Margarete Focke-Tejkl, PhD, Raffaela Campana, PhD, Renate Reininger, PhD, Christian Lupinek, MD, Katharina Blatt, MSc, Peter Valent, MD, Tea Pavkov-Keller, PhD, Walter Keller, PhD, Rudolf Valenta, MD Journal of Allergy and Clinical Immunology Volume 133, Issue 3, Pages 836-845.e11 (March 2014) DOI: 10.1016/j.jaci.2013.08.038 Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Mapping of the peptides onto a model of the 3-dimensional structure of Phl p 5. Peptides were highlighted in different colors (P1 aa 26-58, brown; P2 aa 59-91, blue; P3 aa 93-128, red; P4 aa 132-162, yellow; P5 aa 176-212, magenta; P6 aa 217-246: gray; P7 aa 252-283, green) in a ribbon (upper part) and surface representation of Phl p 5 (lower part). Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Allergenic activity of rPhl p 5 and a peptide mix as measured by upregulation of CD203c and CD63 expression on patients’ basophils. Blood samples were incubated with serial dilutions of rPhl p 5 (0.001-1 or 10 μg/mL; black bars), equimolar mixes of unconjugated (patients 25, 26, and 28) or KLH-coupled peptides (patients 30 and 31; gray bars), anti-IgE, or buffer (x-axes). The stimulation indices (SI) are displayed on the y-axes. Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Phl p 5- and peptide-specific lymphocyte proliferation and cytokine responses. PBMCs from 13 patients with grass pollen allergy were stimulated with equimolar concentrations of rPhl p 5, the individual peptides (P1-P7), the peptide mix (mix), or medium alone (med) (x-axes). Box plots (horizontal bars, medians ± SDs) of thymidine incorporations (cpm values) and cytokine levels (pg/mL) are shown on the y-axes. Statistically significant differences between medium and stimuli (**P < .01; *P < .05) and between Phl p 5 and peptides (▲▲P < .01; ▲P < .05) are indicated. Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Peptide-specific IgG antibodies inhibit allergen-induced basophil activation. Basophils from 4 patients with grass pollen allergy (patients 25, 30, 31, and 34) were exposed to different concentrations of Phl p 5 (1-0.0001 μg/mL) preincubated with peptide-specific rabbit IgG (squares, mix of anti-P1, -P2, -P5, and -P6) or preimmune IgG (dots). Response to anti-IgE is shown for 1 concentration (triangle). Allergen-induced upregulation of CD203c was calculated from mean fluorescence intensities obtained with stimulated (MFIstim) and unstimulated (MFIcontrol) cells, and is expressed as stimulation index (MFIstim : MFIcontrol) (SI) (y-axes). Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 CD analysis shows a comparison of rPhl p 5a and the 7 peptides. X-axis shows the molecular ellipticity and the y-axis the wave length. Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Position of peptides in a sequence alignment of group 5 allergens from different grasses. Comparison of the amino acid sequences of Phl p 5a and homologous grass allergens by using ClustalW. Each allergen sequence is preceded by its database entry code (Q40960: Phl p 5a, timothy grass; Q40963: Phl p 5b, timothy grass; Q40237: Lol p 5b, rye grass, Q9FPR0: Poa p 5, Kentucky bluegrass; Q93WP9 Dac g 5, Q9XF24: Lol p 5a, rye grass; CBG76811: Sec c 5, rye; BAK07309: Hor v 5, barley; Q70JP9: Tri a 5, wheat). Dashes indicate gaps, points represent identical amino acids. Colored frames indicate the positions of peptides 1 to 7 and their borders. Mean percentages of inhibition of patients’ (n = 9) IgE binding to Phl p 5 after preincubation of Phl p 5 with antipeptide antisera are displayed for each peptide. Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Illustration of the fluorescence-activated cell sorting strategy to detect allergen- and peptide-specific CD4+ cells by CFSE staining. PBMCs from a patient with grass pollen allergy were stimulated with rPhl p 5, peptides 1 and 4, or the peptide mix. A, The 7-AAD dead cell exclusion is shown. The gating on CD3+ (B) and on CD4+ (C) T cells is shown. Percentages of positive cells are displayed. FSC, Forward scatter. Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Reactivity of antipeptide IgG and anti-rPhl p 5 IgG with Phl p 5 by ELISA titration. OD values ± SDs corresponding to bound IgG (y-axis) are shown for 5 different dilutions (x-axis: 1:4 × 103, 1:16 ×103, 1:64 × 103, 1:256 × 103, 1:1024 × 103). Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Analysis of the interaction between rPhl p 5 and specific antisera with the use of surface plasmon resonance (BIAcore) measurements. Binding is expressed in response units (RUs) on the y-axis over time. A, The association and dissociation of the antisera (anti-rPhl p 5, black line; mix of all peptide antisera [anti-P1 to anti-P7], dark gray broken line; mix of anti-P1, -P2, -P5, and -P6 antisera, light gray broken line; antisera against individual peptides: P1, brown line, P2, blue line; P3, red line; P4, yellow line; P5, magenta line; P6, gray line; P7, green line) is shown over time (in seconds; x-axis). B, Shown is a magnification of the reactivity of the antipeptide antisera without the anti-rPhl p 5 antiserum. Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Specific inhibition of allergen-induced basophil activation by peptide antisera. Upregulation of CD203c expression on basophils (SI, stimulation index; y-axis) of a patient with birch pollen allergy by different concentrations of rBet v 1 (x-axis) in the presence of anti-P1, -P2, -P5, and -P6 antisera; a mix of the corresponding preimmune sera; or anti-Bet v 1 peptide antisera by buffer alone or by anti-IgE. Journal of Allergy and Clinical Immunology 2014 133, 836-845.e11DOI: (10.1016/j.jaci.2013.08.038) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions