Yeast display platform for expression of linear peptide epitopes to assess peptide-MHC-II binding in high-throughput

Yeast display can serve as a powerful tool to assess peptide-MHC (pMHC) and pMHC-TCR binding. However, this approach is often limited by the need to optimize MHC proteins for yeast surface expression, which can be laborious and may not yield productive results. Here we present a second-generation yeast display platform for class II MHC molecules (MHC-II) which decouples MHC-II expression from yeast-expressed peptides, referred to as "peptide display". Peptide display obviates the need for yeast-specific MHC optimizations and increases the scale of MHC-II alleles available for use in yeast display screens. Because MHC identity is separated from the peptide library, a further benefit of this platform is the ability to assess a single library of peptides against any MHC-II. We demonstrate the utility of the peptide display platform across MHC-II proteins, screening HLA-DR, HLA-DP, and HLA-DQ alleles. We further explore parameters of selections, including reagent dependencies, MHC avidity, and use of competitor peptides. This approach presents an advance in throughput and accessibility of screening peptide-MHC-II binding.