MHC‐Peptide Tetramers to Visualize Antigen‐Specific T Cells

John D. Altman1, Mark M. Davis2

1 Emory University School of Medicine, Atlanta, Georgia, 2 Stanford University School of Medicine and The Howard Hughes Medical Institute, Palo Alto, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 17.3
DOI:  10.1002/0471142735.im1703s53
Online Posting Date:  May, 2003
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Abstract

Mature T lymphocytes of the CD8 or CD4 classes bear T cell receptors (TCR) that are specific for a molecular complex consisting of a major histocompatibility complex class I or II (MHC class I or II) molecule bound to a unique self or foreign peptide. Until recently, methods for monitoring the T cell immune response to a viral or tumor antigen were restricted primarily to functional assays based on limiting dilution analysis, because the lack of specific molecular reagents to identify clonal T cells obviated approaches to identify and enumerate specific T cells. Development of efficient methods to express and refold MHC class I molecules with synthetic peptides coincided with identification of specific protein sequences that provide the substrate for enzymatic biotinylation. This combination has led to the development of a straightforward method for generating synthetic TCR ligands, making them tetravalent to provide increased avidity, and labeling them through a streptavidin moiety with useful fluorescent tags such as fluorescein or phycoerythrin. This unit describes the preparation of MHC class I/peptide tetramers in detail, including bacterial expression and refolding of the MHC class I light chain, 2-microglobulin (2m), as well as the formation of a complex consisting of the MHC class I heavy chain of interest, 2m, and a chosen peptide.

     
 
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Table of Contents

  • Unit Introduction
  • Strategic Planning
  • Basic Protocol 1: Preparation of Inclusion Bodies of MHC Class I Subunits with a BirA Substrate Peptide Tag
  • Basic Protocol 2: Refolding of MHC Class I Peptide Complexes
  • Support Protocol 1: MHC Determination by ELISA
  • Support Protocol 2: Concentration of Folded Protein by Ultrafiltration and Purification by Size-Exclusion Chromatography
  • Basic Protocol 3: Enzymatic Biotinylation, Purification, and Analysis of Efficiency of Biotinylation
  • Basic Protocol 4: Multimerization of Biotinylated Monomers with Streptavidin
  • Basic Protocol 5: Use of Labeled MHC Class I Tetramers for Flow Cytometric Enumeration of Specific TCR-Bearing Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Inclusion Bodies of MHC Class I Subunits with a BirA Substrate Peptide Tag

 Materials
  • E. coli expression strain—e.g., BL21(DE3); see Strategic Planning
  • MHC-BSP expression vector (see Strategic Planning)
  • Selective LB plates and medium (see recipe)
  • 20% (v/v) glycerol/LB medium with appropriate antibiotic, ice-cold
  • Dry ice/methanol bath or liquid nitrogen
  • 20% (w/v) glucose, sterile
  • Nonselective LB medium (unit 10.3)
  • 1× and 2× SDS-PAGE/sample buffer: prepare as described in unit 8.4, except use only 10 mM (0.31 g) DTT; dilute as necessary in H2O
  • 400 mM isopropyl--d-thiogalactopyranoside (IPTG) in H2O: filter sterilize and store up to several months in 1-ml aliquots at –20°C
  • Resuspension buffer (see recipe)
  • 50 mg/ml lysozyme
  • 1.0 M MgCl2
  • 2 mg/ml DNase I in 50 (v/v) glycerol/75 mM NaCl
  • Triton X-100
  • 1 M dithiothreitol (DTT)
  • Inclusion body wash buffer with and without Triton X-100 (see recipe)
  • Urea solution, fresh (see recipe)
  • 15-ml snap-cap tubes
  • Cryotubes
  • 1- and 2-liter baffled flasks
  • 1-liter centrifuge bottle
  • Beckman Avanti-J20 centrifuge and JLA-8.100 rotor or equivalent
  • 50-ml screw-cap polypropylene centrifuge tubes
  • 100- and 1000-ml polypropylene beaker
  • 0.5-in. stir bar
  • Lab jack
  • Horn-type sonicator
  • 25 × 89–mm polyallomer tubes
  • Beckman GS-15R centrifuge and FO 630 rotor or equivalent
  • Teflon or glass stirring rod
  • Additional reagents and equipment for monitoring growth with a spectrophotometer (unit 10.3), SDS-PAGE (unit 8.1), and estimating extinction coefficients (Gill and von Hippel, 1989)

Basic Protocol 2: Refolding of MHC Class I Peptide Complexes

 Materials
  • Folding buffer (see recipe)
  • Gluathione, oxidized and reduced
  • 100 mM PMSF in 2-propanol
  • MHC class I heavy chain and 2m (see Basic Protocol 1)
  • Injection buffer (see recipe)
  • Peptide
  • DMSO (optional)
  • 10°C water bath
  • 5-ml syringe and 26-G needle

Support Protocol 1: MHC Determination by ELISA

 Materials
  • G25 PD-10 columns (Amersham Pharmacia Biotech)
  • 1× PBS (appendix 2)
  • Folding reaction (see Basic Protocol 2)
  • 5 µg/ml W6/32 monoclonal antibody (ATCC# HB-95) in 1× PBS (appendix 2)
  • ELISA blocking buffer (see recipe)
  • HLA-A2, properly folded
  • Unknown MHC sample (see Basic Protocol 1)
  • ELISA wash buffer: 0.05% Tween-20 in PBS
  • Rabbit anti-2m (Boehringer-Mannheim)
  • Goat anti-rabbit IgG–HRP conjugate adsorbed against human, mouse, and rat serum proteins (Jackson Immunoresearch)
  • 10 mg/ml 2,2¢-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) diammonium salt
  • Hydrogen peroxide
  • Citrate buffer (see recipe)
  • Immulon-4 ELISA plates (Dynex Technologies)
NOTE: If the secondary antibody is directly conjugated to HRP, omit the azide from the ELISA blocking buffer (steps , , and ).

Support Protocol 2: Concentration of Folded Protein by Ultrafiltration and Purification by Size-Exclusion Chromatography

 Materials
  • Folding reaction (see Basic Protocol 2)
  • 1× PBS (appendix 2)
  • Sephadex G25 PD-10 columns
  • Sephacryl S300 26/60 column
  • 20 mM Tris×Cl, pH 8.0 (appendix 2)/150 mM NaCl (optional)
  • Avanti J-25 centrifuge and JLA-16.250 rotor with 250-ml polypropylene tubes
  • 0.45-µm filter
  • Amicon 8400 stirred cell equipped with Biomax-30 membranes (Millipore) and appropriate N2 source
  • 25-ml pipet
  • 17 × 100–mm polypropylene tubes
  • Ultrafree-15 centrifugal concentrator with 10-kDA MWCO (Millipore)
  • Additional reagents and equipment for estimating extinction coefficients (Gill and von Hippel, 1989)

Basic Protocol 3: Enzymatic Biotinylation, Purification, and Analysis of Efficiency of Biotinylation

 Materials
  • 1 M Tris×Cl, pH 7.5 (appendix 2)
  • 5 M NaCl
  • 1 M MgCl2
  • 100 mM biotin in 200 mM Tris base (pH not adjusted): store indefinitely at –80°C
  • 100 mM ATP: adjust to pH 7.0 using 5 M NaOH; store up to 6 months at –80°C
  • 100 mM PMSF in 2-propanol: store up to 1 month at room temperature
  • 1 mg/ml leupeptin
  • 1 mg/ml pepstatin in methanol
  • MHC (see Support Protocol 2 step )
  • BirA (Avidity)
  • 20 mM Tris×Cl, pH 8.0 (appendix 2)
  • MonoQ 5/5 column (Amersham Pharmacia Biotech)
  • Buffer A: 20 mM Tris×Cl, pH 8.0
  • Buffer B: 20 mM Tris×Cl, pH 8.0/500 mM NaCl
  • PBS containing protease inhibitors (see recipe)
  • Liquid nitrogen
  • 0.8 mg/ml streptavidin in PBS
  • 1× PBS (appendix 2)
  • 2× SDS/sample buffer: prepare as described in unit 8.4, but omit DTT
  • ELISA blocking buffer (see recipe)
  • 50% (w/v) slurry of washed streptavidin-agarose beads (e.g., Sigma )
  • Control beads: Sepharose 4B beads (Sigma) or equivalent
  • Screw-cap microcentrifuge tubes
  • 10-kDA-MWCO Ultrafree-15 centrifugal concentrator (Millipore) or equivalent
  • Additional reagents and equipment for estimating extinction coefficients (Gill and von Hippel, 1989), analysis by SDS-PAGE (unit 8.4), staining with Coomassie blue (unit 8.9), and MHC ELISA (see Support Protocol 1)
    NOTE: If the secondary antibody is directly conjugated to HRP, omit the azide from the ELISA blocking buffer (steps , , and ).

Basic Protocol 4: Multimerization of Biotinylated Monomers with Streptavidin

 Materials
  • Biotinylated MHC stock, purified (see Basic Protocol 3)
  • Streptavidin-PE (Molecular Probes)
  • Amber polypropylene tube
  • Box with cover
  • Additional reagents and equipment for calculating molar quantities of biotinylated MHC (see Basic Protocol 3)

Basic Protocol 5: Use of Labeled MHC Class I Tetramers for Flow Cytometric Enumeration of Specific TCR-Bearing Cells

 Materials
  • MHC tetramer (see Strategic Planning)
  • FACS buffer, ice-cold: 2% (w/v) FBS/0.1% (w/v) sodium azide in PBS (appendix 2)
  • Staining reagents
  • 1% paraformaldehyde (PFA) in PBS
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Figures

  •  FigureFigure 17.3.1 Expression vectors for the soluble domain of MHC class I heavy chains fused to BSP.
    View Image
  •  FigureFigure 17.3.2 Example of gating procedures that can be applied to FACS data. Shown is the CD8 response in Mamu-A*01-positive rhesus macaques to the P11C epitope from SIV gag. See text for details.
    View Image
  •  FigureFigure 17.3.3 Profile of gel-filtration (size-exclusion) chromatography to purify refolded peptide/MHC class I/2M complexes.
    View Image
  •  FigureFigure 17.3.4 FACS analysis of a cross-titration a Kb/SIINFEKL APC tetramer and an anti-CD8 monoclonal antibody (CT-CD8a). The concentration of the tetramer is decreased in successive columns from left to right, while the concentration of CD8 antibody is decreased in successive rows from top to bottom.
    View Image

Videos

Literature Cited

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