Peptide Induction of Surface Expression of Class I MHC

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.