Polymorphisms of major histocompatibility complex (MHC) and molecular mechanisms of their antigen-presenting specificity and promiscuity have great impact on T cell-mediated immune responses and related diseases. Challenges in elucidating the characteristics of antigenic peptide binding by MHC motivate the development of high throughput experimental tools to quantitatively analyze interactions between hundreds of MHC allelic proteins and various peptide sequences. We demonstrated such a method by co-displaying target peptides and class II MHC (MHC-II) on the yeast surface in an intracellular association-dependent manner. The optimized yeast co-display system enabled quantitative mapping of side-chain preferences and general motifs for peptides binding to MHC-II by site-directed mutagenesis or peptide library screening, and also allowed rapid tailoring of MHC-II peptide binding specificity by directed evolution approaches, which derived MHC-II allelic mutants with altered peptide binding specificity or hyper-promiscuity. Comparison of these experimentally engineered mutants with naturally discovered MHC-II proteins recovered valuable information about structure-function relationship in the evolutionary mechanisms for polymorphic MHC-II molecules, which could direct future immunotherapeutic innovation
