High-throughput engineering and analysis of peptide binding to class II MHC

Class II major histocompatibility complex (MHC-II) proteins govern stimulation of adaptive immunity by presenting antigenic peptides to CD4+ T lymphocytes. Many allelic variants of MHC-II exist with implications in peptide presentation and immunity; thus, high-throughput experimental tools for rapid and quantitative analysis of peptide binding to MHC-II are needed. Here, we present an expression system wherein peptide and MHC-II are codisplayed on the surface of yeast in an intracellular association-dependent manner and assayed by flow cytometry. Accordingly, the relative binding of different peptides and/or MHC-II variants can be assayed by genetically manipulating either partner, enabling the application of directed evolution approaches for high-throughput characterization or engineering. We demonstrate the application of this tool to map the side-chain preference for peptides binding to HLA-DR1 and to evolve novel HLA-DR1 mutants with altered peptide-binding specificity.