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Defining antigen-specific responses with human MHC class II tetramers

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1 Defining antigen-specific responses with human MHC class II tetramers
Jane H. Buckner, MD, Ursula Holzer, MD, Erik J. Novak, PhD, Helena Reijonen, PhD, William W. Kwok, PhD, Gerald T. Nepom, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 110, Issue 2, Pages (August 2002) DOI: /mai Copyright © 2002 Mosby, Inc. Terms and Conditions

2 Fig. 1 Schematic illustration of multimeric MHC-peptide molecules suitable for detection of antigen-specific T cells. B, Biotin; SA, streptavidin; PE, phycoerythrin. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2002 Mosby, Inc. Terms and Conditions

3 Fig. 2 Class II-peptide tetramers are highly specific for detection of antigen-responsive T cells. In the example shown, a human CD4+ T-cell clone specific for the influenza hemagglutinin peptide HA was incubated with fluorescent tetramers composed of HLA-DR1 molecules (HLA-DRB1*0101) complexed with the same peptide. Control tetramers consisted of the same HLA molecule containing an irrelevant peptide, in this case one derived from type II collagen. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2002 Mosby, Inc. Terms and Conditions

4 Fig. 3 Antigen-responsive T cells retain tetramer-binding properties during activation and expansion. HLA-DR4 (DRB1*0401)-restricted T-cell clones specific for the HA epitope were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated with specific antigen-HLA complexes and with anti-CD28 antibodies. Dividing cells are characterized by decreasing intensity of the carboxyfluorescein diacetate succinimidyl ester staining (upper panel) , and the number of cell divisions is noted for each fraction of the histogram. Tetramer binding (lower panel) is unchanged relative to cell division measurement with carboxyfluorescein diacetate succinimidyl ester. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2002 Mosby, Inc. Terms and Conditions

5 Fig. 4 Production of IL-4 by antigen-specific human CD4+ T cells stimulated with tetramers. Intracellular IL-4 was detected with fluorescent anti-IL-4 mAb, along with tetramer staining by peptide-HLA-DR4 complexes. Dose-dependent tetramer staining corresponds to acquisition of the intracellular IL-4 staining, indicating T-cell activation and IL-4 production by the antigen-specific signal conveyed by binding to the tetramer. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2002 Mosby, Inc. Terms and Conditions

6 Fig. 5 Peripheral blood T cells bind HLA class II tetramers. PBMCs were cultured with HA peptide for 6 days, followed by analysis with flow cytometry. Approximately 2% of the cells analyzed correspond to the CD4+ tetramer-positive subset responding to specific antigen. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2002 Mosby, Inc. Terms and Conditions

7 Fig. 6 Specific CD4+ T cells represent a range of antigen avidity characterized by differential tetramer staining. T-cell clones specific for the HA peptide were isolated by means of flow cytometry sorting of tetramer-positive T cells from peripheral blood. Examples of the clones recovered are shown, showing both high-avidity (left) and low-avidity (right) profiles represented within the antigen-specific CD4+ population. Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2002 Mosby, Inc. Terms and Conditions

8 Fig. 7 Detection of autoantigen-specific CD4+ T cells in peripheral blood. PBMCs from a diabetic patient were stimulated in vitro with hGAD65, followed by activation with specific HLA-DR4-peptide complexes containing the GAD peptide epitope. Flow cytometric analysis is shown, in which the activated T-cell population, defined by CD25-high and CD4-high markers, is analyzed with specific GAD-HLA-DR4 tetramers or control HLA-DR4 tetramers containing an irrelevant peptide. Detailed methods are described in the study by Reijonen et al.42 Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2002 Mosby, Inc. Terms and Conditions

9 Fig. 8 A general scheme for TGEM analysis of a complex protein-genome-proteome is shown, in which a peptide library if generated from computed predictive algorithms that identify putative HLA-binding peptides, followed by tetramer analysis of antigen-responsive T-cell populations. Pools of candidate peptides are simultaneously tested, and tetramer-positive staining identifies the immunodominant epitopes within the peptide library.23,49 Journal of Allergy and Clinical Immunology  , DOI: ( /mai ) Copyright © 2002 Mosby, Inc. Terms and Conditions

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