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
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 17.3
DOI:  10.1002/cpim.14
Online Posting Date:  November, 2016
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Mature T lymphocytes of the CD8 or CD4 classes bear αβ 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 antigen‐specific T cell immune responses 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 allophycocyanin or R‐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, β2‐microglobulin (β2m), as well as the formation of a complex consisting of the MHC class I heavy chain of interest, β2m, and a chosen peptide. © 2016 by John Wiley & Sons, Inc.

Keywords: MHC tetramers; T cells; flow cytometry

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Table of Contents

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

  • 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 H 2O
  • 400 mM isopropyl‐β‐d‐thiogalactopyranoside (IPTG) in H 2O: 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 MgCl 2
  • 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; Engebrecht et al., ), SDS‐PAGE (unit 8.4; Gallagher, ), and estimating extinction coefficients (Gill and von Hippel, )

Basic Protocol 2: Refolding of MHC Class I Peptide Complexes

  • Folding buffer (see recipe)
  • Gluathione, oxidized and reduced
  • 100 mM PMSF in 2‐propanol
  • MHC class I heavy chain and β2m (see 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

  • G25 PD‐10 columns (Amersham Pharmacia Biotech)
  • 1× PBS ( appendix 2A)
  • Folding reaction (see protocol 2)
  • 5 μg/ml W6/32 monoclonal antibody (ATCC# HB‐95) in 1× PBS ( appendix 2A)
  • ELISA blocking buffer (see recipe)
  • HLA‐A2, properly folded
  • Unknown MHC sample (see protocol 1)
  • ELISA wash buffer: 0.05% Tween‐20 in PBS
  • Rabbit anti‐β2m (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 3, 4, and 8).

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

  • Folding reaction (see protocol 2)
  • 1× PBS ( appendix 2A)
  • Sephadex G25 PD‐10 columns
  • Sephacryl S300 26/60 column
  • 20 mM Tris·Cl, pH 8.0 ( appendix 22)/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 N 2 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, )

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

  • 1 M Tris·Cl, pH 7.5 ( appendix 2A)
  • 5 M NaCl
  • 1 M MgCl 2
  • 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 protocol 4, step 9)
  • BirA (Avidity)
  • 20 mM Tris·Cl, pH 8.0 ( appendix 2A)
  • 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 2A)
  • 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, ), analysis by SDS‐PAGE (unit 8.4; Gallagher, ), staining with Coomassie blue (unit 8.9; Sasse and Gallagher, ), and MHC ELISA (see protocol 3)
NOTE: If the secondary antibody is directly conjugated to HRP, omit the azide from the ELISA blocking buffer (steps 3, 4, and 8).

Basic Protocol 4: Multimerization of Biotinylated Monomers with Streptavidin

  • Biotinylated MHC stock, purified (see protocol 5)
  • Streptavidin‐PE (Life Technologies, Prozyme, or others)
  • Amber polypropylene tube
  • Box with cover
  • Additional reagents and equipment for calculating molar quantities of biotinylated MHC (see protocol 5)

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

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