Quantitating MHC Class I Ligand Production and Presentation Using TCR-Like Antibodies

Abstract

Accurately determining the number of peptide–MHC class I complexes on the cell surface is necessary when evaluating cellular processes or pharmaceuticals that alter the antigen presentation machinery. Here I describe a quantitative flow cytometry application for determining the number of peptide–MHC complexes on the surface of cells grown in tissue culture that express an endogenous protein from which the peptide is derived. The procedure requires a monoclonal antibody with the ability to distinguish MHC class I molecules presenting the peptide of interest from other peptide–MHC complexes. Fluorescence signal measured on antibody-labeled cells can be compared to fluorescent-calibrated beads to determine the relative number of antibodies bound to the cell surface and hence the number of specific peptide–MHC complexes expressed by the cell. As new monoclonal antibodies with TCR-like specificity for peptide–MHC complexes are created, this method will be helpful in quantifying the exact numbers of complexes generated by cell types and relating these numbers to physiological outcomes of T cell activation.