Identification of Human Antigen‐Specific T Cells Using MHC Class I and Class II Tetramers

Lori A. Krueger1, C. Thomas Nugent1, Johannes Hampl1

1 Beckman Coulter Inc., San Diego, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 6.18
DOI:  10.1002/0471142956.cy0618s30
Online Posting Date:  November, 2004
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Abstract

Major histocompatibility complex (MHC) tetramers typically consist of a fluorophore‐streptavidin complex and biotinylated soluble MHC molecules carrying a peptide of interest. Tetramers bind to T cell receptors (TCR) that recognize the MHC molecule/peptide combination with high specificity. Native MHC molecules are expressed as cell‐surface glycoproteins capable of binding a variety of peptides generated from the degradation of self and non‐self proteins for display to T cells. The human MHC gene locus is highly polymorphic, with >800 class I and >500 class II alleles currently identified. This heterogeneity contributes to the uniqueness of each person's immune system. This unit describes procedures for labeling CD8+ T cells with MHC class I tetramers and CD4+ T cells with MHC class II tetramers. The protocols can be used for detecting and enumerating human antigen‐specific T cells. Both CD8+ and CD4+ antigen‐specific T cells are rare events and require that sufficient numbers of cells be evaluated. To minimize nonspecific tetramer binding contributed by irrelevant cell populations, a cumulative gating strategy using positive selection and/or exclusion gating is described.

Keywords: MHC tetramers; antigen‐specific T cells; flow cytometry

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Identification of MHC Class I Antigen‐Specific T Cells
  • Basic Protocol 2: Identification of MHC Class II Antigen‐Specific T Lymphocyes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Identification of MHC Class I Antigen‐Specific T Cells

  Materials
  • Whole blood collected into EDTA or heparin anticoagulant (unit 5.1)
  • Labeling buffer: e.g., PBS ( appendix 2A)
  • Anti‐CD8‐FITC (e.g., Beckman Coulter)
  • Anti‐CD3‐PE‐Cy5 (e.g., Beckman Coulter)
  • Irrelevant (negative) MHC class I tetramer conjugated to PE (Beckman Coulter)
  • Specific MHC class I tetramer conjugated to PE (Beckman Coulter)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.5% paraformaldehyde (see recipe) or 0.5% formalin in PBS
  • 12 × 75–mm polypropylene test tubes
  • Centrifuge capable of 400 × g
  • Flow cytometer
  • Additional reagents and equipment for preparing PBMCs and lysis of erythrocytes (unit 5.1) and titering antibodies (unit 4.1)

Basic Protocol 2: Identification of MHC Class II Antigen‐Specific T Lymphocyes

  Materials
  • Whole blood collected into EDTA or heparin anticoagulant (unit 5.1) or ex vivo–expanded lymphocytes
  • Culture medium for ex vivo–expanded lymphocytes (see recipe)
  • Irrelevant MHC class II tetramer conjugated to PE (Beckman Coulter)
  • Specific MHC class II tetramer conjugated to PE (Beckman Coulter)
  • Anti‐CD3‐FITC (e.g., Beckman Coulter)
  • Anti‐CD4‐ECD (e.g., Beckman Coulter)
  • Anti‐CD8‐PE‐Cy5 (e.g., Beckman Coulter)
  • Anti‐CD13‐PE‐Cy5 (e.g., Beckman Coulter)
  • Anti‐CD19‐PE‐Cy5 (e.g., Beckman Coulter)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.5% paraformaldehyde (see recipe) or 0.5% formalin in PBS
  • 12 × 75–mm polypropylene test tubes
  • 5% CO 2 incubator, equilibrated at 37°C
  • Centrifuge capable of 400 × g
  • Flow cytometer
  • Additional reagents and equipment for preparing PBMC and lysis of erythrocytes (unit 5.1) and titering antibodies (unit 4.1)
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Figures

Videos

Literature Cited

Literature Cited
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