Analysis of the Cellular Requirements for the Binding of Exogenous Peptides to MHC Class II Molecules

The cellular mechanism regulating the binding of exogenous peptides to MHC class II molecule is still an object of controversy. In order to study the cellular requirements of peptide binding we have set up an indirect fluorescence assay that enables us to detect quantitatively peptide/MHC class II complexes on the cell surface of the mouse B lymphoma A20. The absence of binding on several MHC class II-negative cell lines and the inhibition of binding in the presence of competitor peptides or in the presence of a polyclonal serum against MHC class II molecules confirmed the specificity of the assay. A panel of pharmacological and physical agents was then used to determine the mechanism of this regulation. Binding was not significantly affected by vinblastine or cycloheximide and was affected only to a small extent by chloroquine or azide. In contrast to the long half-life previously reported for soluble complexes, we found that the half-life of a peptide/MHC class II complex expressed on A20 was shorter than 3 hr, suggesting that peptide binding might be regulated at the cellular level. The energy of activation of peptide binding, estimated from the temperature dependence of the rate of peptide binding, was decreased above 27°C, suggesting that enhanced peptide binding to MHC class II molecules might depend on the fluidity of the cell membrane lipids.