Related Experiment Video
Updated: Aug 9, 2026
A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
Peptide binding to class I MHC on living cells and quantitation of complexes required for CTL lysis
E R Christinck1, M A Luscher, B H Barber
1Department of Biochemistry, University of Toronto, Canada.
Insights
CD8+ T cells recognize antigen peptides presented by MHC class I molecules. This study reveals rapid, reversible peptide binding to H-2Db molecules on cells, with few complexes needed for T-cell recognition.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Class I major histocompatibility complex (MHC) molecules present antigenic peptides to CD8+ T lymphocytes.
- Previous studies indicated peptide binding to MHC class I molecules occurs at nonphysiological temperatures.
- The kinetics and equilibrium of peptide binding to MHC class I on viable cells at physiological temperatures were not well characterized.
Purpose of the Study:
- To determine the kinetic and equilibrium parameters of influenza nucleoprotein peptide binding to the murine H-2Db molecule on intact, viable cells at 37°C.
- To investigate the relationship between peptide-MHC complex formation and T-cell recognition.
- To establish the minimum number of peptide-MHC complexes required to sensitize target cells for lysis by cytotoxic T-lymphocytes.
Main Methods:
- Utilized radiolabelled influenza nucleoprotein peptides (NP-Y365-380 and shorter analogues).
- Measured binding parameters to the murine H-2Db molecule on intact, viable cells at 37°C.
- Determined the minimum number of class I-peptide complexes for target cell sensitization using cytotoxic T-lymphocyte assays.
Main Results:
- Peptide binding to H-2Db molecules on viable cells at 37°C is rapid and reversible, with dissociation constants in the nanomolar to micromolar range.
- Only a fraction (approximately 10%) of cell-surface Db molecules are capable of binding these peptides.
- As few as 200 class I-peptide complexes per cell (less than 0.08% of surface Db molecules) are sufficient to sensitize target cells for lysis by cytotoxic T-lymphocytes.
Conclusions:
- Peptide binding to MHC class I molecules on viable cells at physiological temperatures is a dynamic and typical ligand-receptor interaction.
- A small number of stable peptide-MHC class I complexes are sufficient for effective T-cell recognition and target cell lysis.
- These findings provide critical insights into the molecular basis of T-cell mediated immunity and antigen presentation.
Abstract:
Antigenic peptides are presented to CD8+T lymphocytes by class I major histocompatibility complex (MHC) molecules. Peptides specifically bind to purified class I molecules in vitro, and to class I molecules on cells at nonphysiological temperatures. We report here the kinetic and equilibrium parameters for the binding of radiolabelled influenza nucleoprotein peptides (NP-Y365-380 and shorter homologues) to the murine H-2Db molecule on intact, viable cells at 37 degrees C. In contrast to earlier reports, we show that peptide binding is rapid and reversible, with dissociation constants ranging from nanomolar to micromolar, suggestive of typical ligand-receptor interactions. Only 10% of cell-surface Db molecules can bind these peptides. To address the relationship between peptide binding and T-cell recognition of the antigen-MHC complex, we determined the minimum number of complexes required to sensitize a target cell for lysis by class I-restricted cytotoxic T-lymphocytes. Our data indicate that EL4 thymoma cells (H-2b) can be sensitized for lysis by cytotoxic T-lymphocytes when as few as 200 class I-peptide complexes (less than 0.08% of surface Db molecules) are present per cell.
