Modulation of allergen-specific T-lymphocyte function by virus-like particles decorated with HLA class II molecules Victoria M. Leb, MSc, Beatrice Jahn-Schmid,

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Presentation transcript:

Modulation of allergen-specific T-lymphocyte function by virus-like particles decorated with HLA class II molecules Victoria M. Leb, MSc, Beatrice Jahn-Schmid, PhD, Hans J. Kueng, MSc, Klaus G. Schmetterer, MSc, Daniela Haiderer, MSc, Alina Neunkirchner, MSc, Gottfried F. Fischer, MD, Arnulf Hartl, PhD, Josef Thalhamer, PhD, Peter Steinberger, PhD, Barbara Bohle, PhD, Brian Seed, PhD, Winfried F. Pickl, MD Journal of Allergy and Clinical Immunology Volume 124, Issue 1, Pages 121-128 (July 2009) DOI: 10.1016/j.jaci.2009.04.008 Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Expression levels of HLA class II molecules, Ii constructs, and CD80 on 293 producer cells. The 293 cells transiently transfected with the indicated constructs were subjected to surface (HLA-DR, CD80) or intracellular (Ii) staining followed by flow-cytometric analysis. Histograms compare nonbinding control (thin lines) and specific mAb (thick lines) binding. Data show one representative experiment (n ≥ 4). Numbers indicate geometric mean fluorescence intensity. Journal of Allergy and Clinical Immunology 2009 124, 121-128DOI: (10.1016/j.jaci.2009.04.008) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Biochemical characteristics of HLA class II–expressing VLPs. The 293 cells were transfected with the indicated constructs or control plasmid (GFP). Cell lysates and corresponding cell culture supernatants (VLPs) were analyzed by SDS-PAGE followed by immunoblotting (IB) with HLA class II or CD80 mAbs. Vesicle induction and loading were controlled by detection of viral core protein p30Gag. Data show one representative experiment (n = 5). Journal of Allergy and Clinical Immunology 2009 124, 121-128DOI: (10.1016/j.jaci.2009.04.008) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 HLA class II/allergen peptide–decorated VLPs activate TCR tg Jurkat T cells. Art v 1–specific TCR tg Jurkat T cells harboring an IL-2 promoter/enhancer driving luciferase expression were coincubated with VLPs (A) or 293 producer cells (B) expressing the indicated molecules. PMA/PHA was used as positive and GFP transfection as negative control. Data show mean + SD of triplicate cultures of 1 representative experiment (n = 3). ∗∗∗P < .001, Student t test, paired, 2-tailed. C, Vesicle induction and loading were controlled by detection of viral core protein p30Gag. AU, Arbitrary units. Journal of Allergy and Clinical Immunology 2009 124, 121-128DOI: (10.1016/j.jaci.2009.04.008) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Art v 1–specific VLPs activate allergen-specific T-cell clones and TCR tg PB T cells. VLPs decorated with the indicated molecules were incubated with an Art v 1–specific T-cell clone (A) or TCR tg PB T lymphocytes (B). CD3/CD28 mAb–coated microbeads or culture medium alone served as positive and negative controls, respectively. Cell proliferation is shown (mean + SD of triplicates; n = 3). ∗∗P < .01, ∗∗∗P < .001, Student t test, paired, 2-tailed. C, Bulk TCR tg PB T cells (12 × 106) stimulated 3 times (arrows) with Art v 1–specific VLPs and supplemented with IL-2 were cultured for 24 days, and total cell numbers were determined at different time points (squares). Insert compares flow-cytometric characteristics of starting culture (day 0) with cells obtained after 24 days. Numbers of CD3+/TCRVβ18+ cells (∼2% in PB T cells) are indicated. Data show one representative experiment (n = 6). kcpm, Kilo counts per minute. Journal of Allergy and Clinical Immunology 2009 124, 121-128DOI: (10.1016/j.jaci.2009.04.008) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Induction of allergen-specific unresponsiveness by VLPs. Proliferation of TCR tg PB T cells from an individual with mugwort pollen allergy (A) and an individual without allergy (B) cultured with indicated primary stimuli for 4 days is shown (gray bars). Proliferation of T cells obtained on culture with indicated stimuli for 10 days, washed, and recultured with CD3/CD28 mAb–coated microbeads in the absence (white bars) or presence (black bars) of exogenous IL-2 (500 U/mL) is demonstrated. Data show one representative experiment (n = 4). ∗∗∗P < .001, ∗∗P < .001, Student t test, paired, 2-tailed. kcpm, Kilo counts per minute. Journal of Allergy and Clinical Immunology 2009 124, 121-128DOI: (10.1016/j.jaci.2009.04.008) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Nature of the costimulatory molecule present on VLPs affects the cytokine profile of allergen-specific T cells. (A and B) Cytokine profiles and (C and D) proliferation of TCR tg allergen-specific PB T cells from an individual with mugwort pollen allergy (A and C) and an individual without allergy (B and D) upon incubation with VLPs expressing the indicated combinations of molecules. One representative experiment is shown (n = 5). ∗∗∗P < .001, ∗∗P < .001, Student t test, paired, 2-tailed. kcpm, Kilo counts per minute; conc, concentration; n.d., not determined. Journal of Allergy and Clinical Immunology 2009 124, 121-128DOI: (10.1016/j.jaci.2009.04.008) Copyright © 2009 American Academy of Allergy, Asthma & Immunology Terms and Conditions