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A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes
Published on: March 25, 2014
Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level
J A López1, I F Luescher, J C Cerottini
1Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland.
Insights
Researchers developed a method to track exogenous peptides binding to MHC class I molecules on single cells. This technique uses biotinylated peptides and flow cytometry to visualize peptide-MHC interactions, enabling detailed analysis of immune responses.
Area of Science:
- Immunology
- Molecular Biology
- Cell Biology
Background:
- Assessing exogenous peptide binding to cell surface MHC class I molecules is crucial for understanding immune responses.
- Existing methods lack the single-cell resolution needed to analyze these interactions directly.
- MHC class I molecules present antigenic peptides to cytotoxic T lymphocytes (CTLs).
Purpose of the Study:
- To develop a method for directly assessing exogenous peptide binding to cell surface MHC class I molecules at the single-cell level.
- To combine biotinylated peptide derivatives with immunofluorescence detection via flow cytometry.
- To analyze the specificity and kinetics of peptide-MHC interactions.
Main Methods:
- Synthesis and functional screening of biotinylated peptide derivatives of adenovirus 5 early region 1A peptide 234-243.
- Detection of bound biotinylated peptides using phycoerythrin-labeled streptavidin and flow cytometry.
- Competition assays with unlabeled peptides and analysis across various mouse strains and mutant MHC molecules.
Main Results:
- A C-terminally biotinylated peptide derivative showed efficient and specific binding to H-2Db MHC class I molecules on living cells.
- Binding was specific for Db alleles among over 20 tested class I alleles and reduced on Dbm13/14 mutant molecules.
- Flow cytometry allowed detection of peptide binding to distinct lymphocyte subpopulations, correlating with Db surface expression levels.
Conclusions:
- The developed method enables direct, single-cell level assessment of exogenous peptide binding to MHC class I molecules.
- This technique provides insights into the specificity and dynamics of peptide-MHC interactions in different cell types.
- The findings highlight the utility of biotinylated peptides and flow cytometry for studying antigen presentation and immune cell interactions.
Abstract:
To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.
