Victoria M. Leb, MSc, Beatrice Jahn-Schmid, PhD, Klaus G

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Presentation transcript:

Molecular and functional analysis of the antigen receptor of Art v 1–specific helper T lymphocytes Victoria M. Leb, MSc, Beatrice Jahn-Schmid, PhD, Klaus G. Schmetterer, MSc, Hans J. Kueng, MSc, Daniela Haiderer, MSc, Alina Neunkirchner, MSc, Gottfried F. Fischer, MD, Karl Nissler, PhD, Arnulf Hartl, PhD, Josef Thalhamer, PhD, Barbara Bohle, PhD, Brian Seed, PhD, Winfried F. Pickl, MD Journal of Allergy and Clinical Immunology Volume 121, Issue 1, Pages 64-71 (January 2008) DOI: 10.1016/j.jaci.2007.10.006 Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Nucleotide and predicted amino acid sequence of the HLA-DRB1*0101–restricted TCR of the Art v 1–specific T-cell clone SSR20. A, TCR α-chain sequence. B, TCR β-chain sequence. Codon numbering and nomenclature are according to the international immunogenetics information system.29 Only the 5′-ends of the constant-region sequences are shown. Complementarity-determining regions are boxed. Journal of Allergy and Clinical Immunology 2008 121, 64-71DOI: (10.1016/j.jaci.2007.10.006) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Expression pattern of endogenous TCR, Art v 1–specific tg TCR, and accessory molecules on WT, bulk TCR tg, and TCR tg single-cell clones (nos. 3, 5, and 7) of Jurkat T cells are shown. Binding of specific mAbs or Art v 1–specific tetramers (HLA-DRB1*0101/Art v 119-36; bold lines) is compared with control antibody binding (thin lines). Two-parameter dot-plot analysis of Jurkat cells stained with TCR Vβ8 and TCR Vβ18 mAbs (right panel). Data are representative of 2 independently performed experiments. Journal of Allergy and Clinical Immunology 2008 121, 64-71DOI: (10.1016/j.jaci.2007.10.006) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 The Art v 1–specific TCR in tg Jurkat T cells is functionally active. A, IL-2 promoter activity of Art v 1 TCR tg or WT Jurkat T cells on coculture with or without HLA-DRB1*0101+ EBV B cells and the indicated peptides or with medium alone. B, Similar to Fig 3, A, but supplemented with SEE. Data show mean values ± SDs of triplicate cultures representative of several independently performed experiments. Journal of Allergy and Clinical Immunology 2008 121, 64-71DOI: (10.1016/j.jaci.2007.10.006) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Art v 1–specific TCR tg Jurkat T cells recognize Ii-encoded allergenic peptides presented by aAPCs. A, IL-2 promoter activity of TCR tg Jurkat T cells (bulk) cocultured with 293 cells expressing HLA-DRA*0101, HLA-DRB1*0101, CD80::GPI, CD54::GPI, cathepsin S, and the indicated forms of Ii or controls. Coculture with Raji B cells, PHA plus phorbol 12-myristate 13-acetate (PMA), or medium served as a control. B, Similar to Fig 4, A, but supplemented with SEE. Expression levels of HLA-DR1 (C) or Ii (D) in 293 aAPC transfectants (bold lines) superimposed by negative control staining (thin lines) is also shown. Journal of Allergy and Clinical Immunology 2008 121, 64-71DOI: (10.1016/j.jaci.2007.10.006) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Efficient stimulation of Art v 1–specific Jurkat T cells requires costimulation. IL-2 promoter activity of TCR tg Jurkat T cells incubated with 293 cells cotransfected with DRA*0101, DRB1*0101, Ii::Art v 125-34, CD54, CD80, green fluorescent protein (GFP), or control plasmid alone or combined as indicated or phorbol 12-myristate 13-acetate (PMA) plus PHA. Data show mean values ± SDs of triplicate cultures representative of 3 independently performed experiments. Journal of Allergy and Clinical Immunology 2008 121, 64-71DOI: (10.1016/j.jaci.2007.10.006) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 TCR tg PB T cells proliferate on challenge with Ii::Art v 125-36–expressing aAPCs. A, Expression levels of TCR Vβ18 and CD3 on control and tg PB T cells. Numbers indicate percentage of CD3+ TCR Vβ18+ cells. B, Proliferation of human PBMCs transduced with Art v 1–specific TCR cocultured with aAPCs coexpressing the indicated molecules or alternatively with microbeads coated with CD3 plus CD28 mAbs or with medium alone. Data show mean values ± SDs of triplicate cultures corrected by mean values (2.6 kcpm; range, 0-8.2 kcpm) of green fluorescent protein (GFP)–transduced tg T cells. Data are representative of several independently performed experiments. Journal of Allergy and Clinical Immunology 2008 121, 64-71DOI: (10.1016/j.jaci.2007.10.006) Copyright © 2008 American Academy of Allergy, Asthma & Immunology Terms and Conditions