Generation of Peptide MHC Class I Monomers and Multimers Through Ligand Exchange

Mireille Toebes1, Boris Rodenko1, Huib Ovaa1, Ton N.M. Schumacher1

1 The Netherlands Cancer Institute, Amsterdam, The Netherlands
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.16
DOI:  10.1002/0471142735.im1816s87
Online Posting Date:  November, 2009
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Abstract

The recognition of defined antigen‐MHC complexes by antigen‐specific T cells forms the molecular basis of T cell immunity. It has been shown that fluorescently labeled recombinant MHC tetramers can be utilized to detect antigen‐specific T cells by flow cytometry. Since this first description, MHC tetramers and other types of MHC multimers have become a core tool to monitor the development of disease‐ and therapy‐induced antigen‐specific T cell responses both in humans and in animal model systems. This unit describes a set of protocols that transform classical MHC multimer technology into a high‐throughput platform, allowing one to produce large collections of MHC class I molecules charged with different peptides. This technology is based on the development of conditional MHC ligands that can be triggered to self‐destruct while in the MHC‐bound state. Curr. Protoc. Immunol. 87:18.16.1‐18.16.20. © 2009 by John Wiley & Sons, Inc.

Keywords: HLA class I; MHC class I; tetramer; peptide; refolding; UV; exchange

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Bacterial Expression and Purification of HLA Class I Heavy Chains and Human β2m
  • Basic Protocol 2: Refolding, Biotinylation, and Purification of MHC Class I Complexes with Conditional Ligands
  • Basic Protocol 3: UV‐Mediated Peptide Exchange
  • Basic Protocol 4: Measuring Peptide‐Mediated MHC Stabilization by HLA ELISA
  • Basic Protocol 5: Multimerization of MHC Class I Molecules
  • Support Protocol 1: Synthesis of “J” and Conditional Ligands
  • Support Protocol 2: Expression and Purification of Biotin Ligase
  • Support Protocol 3: Determination of Biotinylation Efficiency
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Bacterial Expression and Purification of HLA Class I Heavy Chains and Human β2m

  Materials
  • Expression constructs (see recipe); vectors are available from the authors upon request, for academic purposes
  • Bacterial strain E. coli BL21 (DE3) pLysS (Novagen, cat. no. 69451)
  • LB agar plates (see recipe)
  • Carbenicillin (Sigma, cat. no. C 1389)
  • Chloramphenicol (Sigma, cat. no. C 0378)
  • LB liquid medium (see recipe)
  • 1 M isopropyl β‐D‐1‐thiogalactopyranoside (IPTG; Sigma, cat no I 5502)
  • Lysis buffer (see recipe)
  • Lysozyme (Sigma, cat. no. L 6876)
  • 1 M MgCl 2 stock (Aldrich, cat. no. 449172)
  • 100 mM MnCl 2 stock (Aldrich, cat. no. 244589)
  • 10 mg/ml DNase stock (see recipe)
  • Detergent buffer (see recipe)
  • Dry ice/ethanol bath
  • Wash solution: 0.5% (v/v) Triton X‐100 (Sigma, cat. no. T 9284)/1 mM EDTA in H 2O
  • Bacterial culture tubes
  • Incubator/orbital shaker
  • Spectrophotometer (for measuring OD at 600 nm)
  • Plastic cuvettes (Sarstedt, cat. no. 67.742)
  • 2‐liter Erlenmeyer flasks
  • Centrifuge and 250‐ml to 1‐liter buckets
  • 50‐ml conical polypropylene centrifuge tubes (BD Falcon)
  • End‐over‐end rotator
  • 30‐ml Corex centrifuge tubes (Gentaur, cat. no. 1‐8445; http://www.gentaur.com)
  • 18‐ to 22‐G needle with syringe
  • 1.5‐ml Sarstedt polypropylene microcentrifuge tubes with caps (Sarstedt, cat. no. 72692)

Basic Protocol 2: Refolding, Biotinylation, and Purification of MHC Class I Complexes with Conditional Ligands

  Materials
  • Denaturing buffer: 8 M urea (Sigma, cat. no. U 5128)/100 mM Tris⋅Cl, pH 8 (see appendix 2A for Tris buffer)
  • HLA class I heavy chain and β2m proteins ( protocol 1)
  • Refolding buffer (see recipe)
  • Conditional ligand (see Table 18.16.1 and protocol 6)
  • Dimethylsulfoxide (DMSO)
  • Phenylmethylsulfonyl fluoride (PMSF; Sigma, cat. no. P 7626)
  • Phosphate‐buffered saline (PBS; appendix 2A), pH 7.4
  • Biotinylation solution (see recipe)
  • Glycerol (87% AR,, Bio‐Lab Ltd., cat. no. 071105; http://www.biolab‐chemicals.com/)
  • 1.5‐ml Sarstedt polypropylene microcentrifuge tubes with caps (Sarstedt, cat. no. 72692)
  • Amicon ultrafiltration membrane (MWCO 30 kDa) and pressure filtration cell with nitrogen source.
  • Centrifuge
  • 0.45‐µm low‐protein‐binding filters (size depending on refolding volume)
  • HPLC or FPLC system with gel‐filtration column, e.g., Phenomenex Biosep SEC S‐3000 column, 300 × 21.2 mm (Phenomenex, cat. no OOH‐2146‐PO; http://www.phenomenex.com); for analytical runs use BioSep‐SEC S‐3000 column 300 × 7.8 mm (Phenomenex, cat. no. OOH‐2146‐KO)
  • Amicon Ultra‐15 PLTK Ultracel‐PL Membrane, MWCO 30 KDa (Amicon, cat. no. UFC903008)
  • Amicon Ultra‐4 PLTK Ultracel‐PL Membrane, MWCO 30 KDa (Amicon, cat. no. UFC803024)

Basic Protocol 3: UV‐Mediated Peptide Exchange

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10× (500 µM) exchange peptide (peptide of interest; also see Critical Parameters) in PBS (see appendix 2A for PBS)
  • 10× (250 µg/ml or ∼5 µM) UV‐sensitive class I MHC molecules ( protocol 2)
  • 96‐well V‐bottom plates (polypropylene; Greiner Bio‐One, cat. #: 651201)
  • UV lamp: 366 nm UVItec tube light (Model LI215BLB; 505‐mm length × 140‐mm width × 117‐mm height) with two 15‐W 365‐nm black‐light blue tubes (http://www.uvitec.co.uk/)
  • Centrifuge with microtiter plate adapter

Basic Protocol 4: Measuring Peptide‐Mediated MHC Stabilization by HLA ELISA

  Materials
  • HLA ELISA kit (Sanquin Reagents, cat. no. M1924; http://www.sanquinreagents.com/) including:
    • Streptavidin
    • Coating buffer
    • Blocking buffer
    • HLA class I standard
    • Horseradish peroxidase (HRP)–conjugated anti‐human β2m antibody
    • 10× substrate buffer
    • 50× 2′,2′‐azino‐bis (3‐ethylbenzthiazoline‐6‐sulfonic acid) diammonium salt (ABTS) solution
    • 100× H 2O 2 solution
    • Stop buffer
  • Wash buffer: 0.05% Tween 20 (Sigma, cat. no. P 1379) in PBS (see appendix 2A for PBS)
  • HLA class I samples ( protocol 3)
  • 96‐well immuno plate (F96 CERT Maxisorp, cat. no. 439454,)
  • Absorbance plate reader
  • Additional reagents and equipment for ELISA (unit 2.1)

Basic Protocol 5: Multimerization of MHC Class I Molecules

  Materials
  • 1 mg/ml PE‐conjugated streptavidin solution (Molecular Probes, cat. no. S866) or 1 mg/ml APC‐conjugated streptavidin solution (Molecular Probes, S868)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Microtiter plates with exchanged MHC class I complexes, prepared according to protocol 3, step 3, containing 25 µg/ml of pMHC in 100 µl /well, corresponding to 2.5 µg or 0.05 nmol MHC class I per well

Support Protocol 1: Synthesis of “J” and Conditional Ligands

  Materials
  • Fluorenylmethylchloroformate (Fmoc‐chloride; Sigma‐Aldrich, cat. no. 160512)
  • Dioxane (Merck, cat. no. 103115)
  • 3‐amino‐3‐(2‐nitro)phenyl‐propionic acid (Lancaster Synthesis, cat. no. B22176; http://www.alfa.com/)
  • 10% (w/w) sodium carbonate
  • Diethyl ether
  • 2 M HCl
  • 100‐ml addition funnel
  • 500‐ml round‐bottom flask
  • 2‐liter separatory funnel
  • P3 sintered‐glass filter
  • 2‐liter Erlenmeyer flask suitable for vacuum filtration
  • 40°C vacuum oven
  • Additional reagents and equipment for Fmoc‐peptide synthesis methodology (Wellings and Atherton, )

Support Protocol 2: Expression and Purification of Biotin Ligase

  Materials
  • Expression construct: BirA biotin ligase cloned in pET21B vector (amino acids 1‐ 321, in frame with a His‐tag). M w 36.4 kDa
  • Bacterial strain E. coli BL21 (DE3) pLysS (Novagen, cat. no. 69451)
  • LB agar plates (see recipe)
  • Carbenicillin (Sigma, cat. no. C 1389)
  • Chloramphenicol (Sigma, cat. no. C 0378)
  • LB liquid medium (see recipe)
  • 1 M isopropyl β‐D‐1‐thiogalactopyranoside (IPTG; Sigma, cat no I 5502)
  • 20 mM Tris⋅Cl, pH 8 ( appendix 2A)/100 mM NaCl
  • Talon Co2+‐resin (Clontech, cat. no. 8901‐2)
  • 5× equilibration/wash buffer: 100 mM Tris⋅Cl, pH 8 ( appendix 2A) containing 50 mM NaCl
  • 5 mM imidazole (Sigma, cat. no. I 5513) in 20 mM Tris⋅Cl, pH 8 ( appendix 2A)/100 mM NaCl
  • 10 mM imidazole (Sigma, cat. no. I 5513) in 20 mM Tris⋅Cl, pH 8 ( appendix 2A)/100 mM NaCl
  • 50 mM imidazole (Sigma, cat. no. I 5513) in 20 mM Tris⋅Cl, pH 8 ( appendix 2A)/100 mM NaCl
  • 2‐mercaptoethanol
  • Glycerol
  • BirA substrate (optional): peptide GGGLNDIFEAQKIEWH (mol. wt.1813)
  • 10× ligase buffer (optional): 200 mM Tris⋅Cl, pH 7.5 ( appendix 2A)/50 mM MgCl 2
  • 500 mM ATP (optional)
  • Trifluoracetic acid (TFA)
  • Acetonitrile (CH 3CN)
  • Incubator/orbital shaker at 37°C and 30°C
  • Centrifuge
  • 250‐ml to 1‐liter centrifuge buckets
  • Spectrophotometer (for measuring OD at 600 nm and 280 nm)
  • Plastic and quartz spectrophotometer cuvettes
  • 50‐ml conical polypropylene centrifuge tubes
  • Probe sonicator
  • 2‐ml gravity flow column with end cap (Clontech)
  • HPLC system with Waters Delta‐Pak, C18, 100 Å, 15 µm, 3.9 × 300 mm reversed‐phase column (optional)
  • Additional reagents and equipment for batch/gravity affinity column purification (http://www.clontech.com/images/pt/PT1320‐1.pdf) and SDS‐PAGE (unit 8.4)

Support Protocol 3: Determination of Biotinylation Efficiency

  Materials
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10× (250 µg/ml, ∼ 5 mM) UV‐sensitive MHC monomer ( protocol 2)
  • 1 mg/ml PE‐conjugated streptavidin (Invitrogen Molecular Probes, cat. no. S866) or APC‐conjugated streptavidin (cat. no. S868, Molecular Probes)
  • 1.5‐ml polypropylene microcentrifuge tubes with caps (Sarstedt, cat. no. 72692, Sarstedt)
  • HPLC system with Phenomenex Biosep SEC S‐3000 column, 300 × 21.2 mm (Phenomenex, cat. no. OOH‐2146‐PO; http://www.phenomenex.com) and 200‐µl injection loop
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Figures

Videos

Literature Cited

   Altman, J.D., Moss, P.A., Goulder, P.J., Barouch, D.H., McHeyzer‐Williams, M.G., Bell, J.I., McMichael, A.J., and Davis, M.M. 1996. Phenotypic analysis of antigen‐specific T lymphocytes. Science 274:94‐96.
   Bakker, A.H. and Schumacher, T.N. 2005. MHC multimer technology: Current status and future prospects. Curr. Opin. Immunol. 17:428‐433.
   Bakker, A.H., Hoppes, R., Linnemann, C., Toebes, M., Rodenko, B., Berkers, C.R., Hadrup, S.R., van Esch, W.J., Heemskerk, M.H., Ovaa, H., and Schumacher, T.N. 2008. Conditional MHC class I ligands and peptide exchange technology for the human MHC gene products HLA‐A1, ‐A3, ‐A11, and ‐B7. Proc. Natl. Acad. Sci. U.S.A. 105:3825‐3830.
   Celie, P.H., Toebes, M., Rodenko, B., Ovaa, H., Perrakis, A., and Schumacher, T.N. 2009. UV‐induced ligand exchange in MHC class I protein crystals. J. Am. Chem. Soc. 131:12298‐12304.
   Frickel, E.M., Sahoo, N., Hopp, J., Gubbels, M.J., Craver, M.P., Knoll, L.J., Ploegh, H.L., and Grotenbreg, G.M. 2008. Parasite stage–specific recognition of endogenous Toxoplasma gondii–derived CD8(+) T cell epitopes. J. Infect. Dis. 198:1625‐1633.
   Garboczi, D.N., Hung, D.T., and Wiley, D.C. 1992. HLA‐A2‐peptide complexes: Refolding and crystallization of molecules expressed in Escherichia coli and complexed with single antigenic peptides. Proc. Natl. Acad. Sci. U.S.A. 89:3429‐3433.
   Grotenbreg, G.M., Roan, N.R., Guillen, E., Meijers, R., Wang, J.H., Bell, G.W., Starnbach, M.N., and Ploegh, H.L. 2008. Discovery of CD8+ T cell epitopes in Chlamydia trachomatis infection through use of caged class I MHC tetramers. Proc. Natl. Acad. Sci. U.S.A. 105:3831‐3836.
   Hadrup, S.R., Bakker, A.H., Shu, C.J., Andersen, R.S., Van Veluw, J., Thor Straten, P., Blank, C., Haanen, J.B., Heemskerk, M.H., and Schumacher, T.N. 2009. Parallel detection of antigen‐specific T‐cell responses by multidimensional encoding of peptide‐Major Histocompatibility Complexes. Nat. Methods 6:520‐526.
   Rodenko, B., Toebes, M., Hadrup, S.R., van Esch, W.J., Molenaar, A.M., Schumacher, T.N., and Ovaa, H. 2006. Generation of peptide‐MHC class I complexes through UV‐mediated ligand exchange. Nat. Protoc. 1:1120‐1132.
   Toebes, M., Coccoris, M., Bins, A., Rodenko, B., Gomez, R., Nieuwkoop, N.J., van de Kasteele, W., Rimmelzwaan, G.F., Haanen, J.B., Ovaa, H., and Schumacher, T.N. 2006. Design and use of conditional MHC class I ligands. Nat. Med. 12:246‐251.
   Wellings, D.A. and Atherton, E. 1997. Standard Fmoc protocols. Methods Enzymol. 289:44‐67.
Key Reference
   Toebes et al., 2006. See above.
  The first description of conditional MHC class I complexes and the use of such complexes to monitor antigen‐specific T cell responses in a high‐throughput fashion.
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