Peptide Induction of Surface Expression of Class I MHC

Ted Hansen1, Nancy Myers1

1 Washington University School of Medicine, St. Louis, Missouri
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 18.11
DOI:  10.1002/0471142735.im1811s57
Online Posting Date:  November, 2003
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Abstract

This unit describes a method for comparing the relative binding of different peptides to the same MHC class I (MHC‐I) molecule using live cells. Live cells expressing suboptimally loaded MHC‐I proteins are incubated with medium containing diluted amounts of synthetic peptides to be tested for binding to class I. After overnight incubation with peptide, surface class I expression is monitored by flow cytometry using an allele‐specific MAb. Relative binding affinity of peptide reliably correlates with the amount of surface induction of the class I molecule to which it specifically binds. The mechanistic basis of this assay is that surface MHC‐I molecules become conformationally unstable shortly after peptide dissociation. However, the binding of an exogenous peptide can stabilize the surface class I molecule, prevent conformational instability, and thus increase class I surface expression in an allele‐specific manner.

     
 
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Table of Contents

  • Basic Protocol 1: Peptide Binding to Class I Molecules as Measured by Surface Induction of Class I Expression on Live Cells Following Culture with Peptides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Peptide Binding to Class I Molecules as Measured by Surface Induction of Class I Expression on Live Cells Following Culture with Peptides

  Materials
  • Cells to be tested (e.g. T2/A2.1 TAP‐deficient cells expressing HLA‐A2, ATCC #CRL‐1992; also see Critical Parameters)
  • Complete DMEM or RPMI cell culture medium containing 10% FBS ( appendix 22)
  • Peptide(s) of interest (see Critical Parameters)
  • Fluorescently labeled MAb specific for MHC class I molecule of interest, suitable for flow cytometry (see, e.g., unit 5.3; also see Robinson et al., )
  • Refrigerated centrifuge with inserts for tissue culture tubes and 96‐well plates
  • 15 × 60–mm, 15 × 100–mm, 20 × 100–mm, or other size non–tissue culture treated petri dishes, flasks, or multiwell plates
  • 96‐well round‐bottom microtiter plate
  • Additional reagents and equipment for counting viable cells ( appendix 3B), and flow cytometry (Chapter 5; also see Robinson et al., )
NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.
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Figures

Videos

Literature Cited

Literature Cited
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