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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
Analysis of the cellular requirements for the binding of exogenous peptides to MHC class II molecules
Insights
Cellular regulation of peptide binding to MHC class II molecules was investigated. Findings suggest peptide binding is influenced by cell membrane fluidity, with a shorter half-life than previously thought.
Area of Science:
- Immunology
- Cell Biology
Background:
- The precise cellular mechanisms governing peptide binding to MHC class II molecules remain unclear.
- Understanding this process is crucial for adaptive immunity and vaccine development.
Purpose of the Study:
- To quantitatively assess cellular requirements for exogenous peptide binding to MHC class II molecules.
- To investigate the regulatory mechanisms controlling peptide/MHC class II complex formation on the cell surface.
Main Methods:
- Development of a quantitative indirect fluorescence assay using mouse B lymphoma A20 cells.
- Utilized pharmacological agents (vinblastine, cycloheximide, chloroquine, azide) and physical agents to probe binding regulation.
- Analyzed temperature dependence to estimate the energy of activation for peptide binding.
Main Results:
- Assay specificity confirmed by lack of binding on MHC class II-negative cells and inhibition by competitor peptides or anti-MHC class II serum.
- Peptide/MHC class II complex half-life on A20 cells was found to be less than 3 hours.
- Peptide binding showed decreased energy of activation above 27°C, indicating a dependence on membrane fluidity.
Conclusions:
- Peptide binding to MHC class II molecules appears to be regulated at the cellular level.
- Cell membrane lipid fluidity may play a significant role in enhancing peptide binding to MHC class II molecules.
Abstract:
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 degrees C, suggesting that enhanced peptide binding to MHC class II molecules might depend on the fluidity of the cell membrane lipids.
