Stability Screening of Arrays of Major Histocompatibility Complexes on Combinatorially Encoded Flow Cytometry Beads

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.Class I MHC molecules are crucially tasked with presenting repertoires of peptides at the cell surface for inspection by CD8 ϩ 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. The rational design of vaccines and immunotherapeutics depends on the accurate identification and characterization of those peptide antigens capable of precipitating a robust immune response. Such efforts are confronted with both an overwhelming diversity of potentially immunogenic peptide sequences and the polymorphism of the MHC system whereby each allelic product 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-MHC (pMHC) 4 molecules to stimulate a specific population of T cells or ascertain the specificity and affinity of the peptide for a particular MHC variant (1-4). Cell-free biophysical methods that employ soluble complexes purified to homogeneity have the advantage of directly measuring the molecular interaction between the peptide and MHC without the confounding effects of simultaneous expression of multiple MHC variants in a cellular context. 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), (5-7) murine MHC, (5, 8 -11), and class II MHC molecules (12) and have been exploited for an ELISA-based MHC stability assay (6, 13). Nevertheless, methods that employ cell lines defective in antigen presentation, where the exogenous addition of peptide can measurably restore MHC surface expression, such as the murine RMA-S (14, 15) or human T2 lines transfected with an MHC of choice...

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