Abstract
The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.
Highlights
Class I MHC molecules are crucially tasked with presenting repertoires of peptides at the cell surface for inspection by CD8ϩ T cells, thereby allowing the immune system to respond to degradation products of proteins that are indicative of exposure to infectious disease or cellular transformation
Uct has a distinct peptide-binding motif that dictates their interactions. This has led to the development of a myriad of assays to probe either the ability of peptide-MHC4 molecules to stimulate a specific population of T cells or ascertain the specificity and affinity of the peptide for a particular MHC variant [1,2,3,4]
Limitations on the expeditious and high throughput generation of collections of recombinantly produced pMHC products have largely been resolved with the introduction of conditional ligands for class I human leukocyte antigens (HLAs), [5,6,7] murine MHC, (5, 8 –11), and class II MHC molecules [12] and have been exploited for an ELISA-based MHC stability assay [6, 13]
Summary
Synthetic peptides used for epitope screening and the FITClabeled A*02:01 competitor peptide, FLPSD(K-FITC)FPSV were obtained from GenScript (Piscataway, NJ) with a purity of Ͼ70% and Ͼ90%, respectively. Conditional ligands that incorporate the photocleavable 3-amino-3-(2-nitro)phenyl-propanoic acid residue (J), SV9-P7* (FAPGNY-J-AL) for H-2Kb and H-2Db [8], and GILGFVF-J-L for A*02:01 [5] were synthesized in-house by standard Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based solid phase peptide synthesis. The identity and purity of the conditional ligands was confirmed by LC/MS analysis. All of the lyophilized powders were diluted to 10 mg mlϪ1 in 20% H2O, 80% Me2SO, and 10 mM tris(2-carboxyethyl)phosphine and stored at Ϫ80 °C until further use. Peptide concentrations used for determination of IC50 values were calculated by assuming quantitative synthetic yield
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