Techniques to improve the direct ex vivo detection of low frequency antigen-specific CD8+ T cells with peptide-major histocompatibility complex class I tetramers

Authors

  • Pratip K. Chattopadhyay,

    Corresponding author
    1. Immunotechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
    • Pratip K. Chattopadhyay, Immunotechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Mary Land 20892===

      David A. Price, Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Cardiff CF14 4XN, UK===

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    • PKC and JJM contributed equally to this work.

  • J. Joseph Melenhorst,

    1. Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Mary Land 20892
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    • PKC and JJM contributed equally to this work.

  • Kristin Ladell,

    1. Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdom
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  • Emma Gostick,

    1. Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdom
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  • Phillip Scheinberg,

    1. Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Mary Land 20892
    2. Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Mary Land 20892
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  • A. John Barrett,

    1. Hematology Branch, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, Mary Land 20892
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  • Linda Wooldridge,

    1. Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdom
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  • Mario Roederer,

    1. Immunotechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892
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  • Andrew K. Sewell,

    1. Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdom
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  • David A. Price

    Corresponding author
    1. Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Cardiff CF14 4XN, United Kingdom
    2. Human Immunology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Mary Land 20892
    • Pratip K. Chattopadhyay, Immunotechnology Section, Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Mary Land 20892===

      David A. Price, Department of Medical Biochemistry and Immunology, Cardiff University School of Medicine, Cardiff CF14 4XN, UK===

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  • This article is based, in part, on a talk given by David Price at the 3rd MASIR meeting, La Plagne, France.

Abstract

The ability to quantify and characterize antigen-specific CD8+ T cells irrespective of functional readouts using fluorochrome-conjugated peptide-major histocompatibility complex class I (pMHCI) tetramers in conjunction with flow cytometry has transformed our understanding of cellular immune responses over the past decade. In the case of prevalent CD8+ T cell populations that engage cognate pMHCI tetramers with high avidities, direct ex vivo identification and subsequent data interpretation is relatively straightforward. However, the accurate identification of low frequency antigen-specific CD8+ T cell populations can be complicated, especially in situations where T cell receptor-mediated tetramer binding occurs at low avidities. Here, we highlight a few simple techniques that can be employed to improve the visual resolution, and hence the accurate quantification, of tetramer binding CD8+ T cell populations by flow cytometry. These methodological modifications enhance signal intensity, especially in the case of specific CD8+ T cell populations that bind cognate antigen with low avidities, minimize background noise, and enable improved discrimination of true pMHCI tetramer binding events from nonspecific uptake. © 2008 International Society for Advancement of Cytometry

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