Ankur Saikia

• Major histocompatibility class I (MHC-I) molecules are key to our body's immune defence against pathogens and tumors by presenting the cytosolic peptidome to cytotoxic T lymphocytes (CTLs). MHC-I/peptide complexes are presented on the cell surface, and recognition by CTLs results in a kill signal and the destruction of aberrant cells. In this thesis, I have described the use of small molecule-assisted refolding of MHC-I proteins for generating their empty, peptide-receptive forms. I have also developed alternative methods for peptide exchange, thermostability and peptide affinity measurements, which can contribute significantly to the understanding of MHC-I selection mechanism and developing reagents for clinical applications. The classical refolding of MHC-I in vitro has always required full-length peptides, whereas empty forms were nearly impossible to obtain. We demonstrated that both wild type and disulfide bond-stabilized MHC-I molecules can be folded with an excess of an allotype specific dipeptide. To discover specific dipeptides, I have developed a competitive enzyme-linked immunosorbent assay and used it to screen dipeptides and tripeptides that modulate MHC-I refolding. The folded dsMHC-I molecules can later be stripped of dipeptides in a washing process to generate the stable empty forms. Such empty dsMHC-I molecules can be used in rapid multimer generation. We can also use these empty molecules to fish out peptides from tumor tissues or infected cells before characterizing them using mass spectrometry. We can also compare the peptide affinities from in-vitro assays by monitoring direct binding of the peptide to empty dsMHC-I molecules. We have successfully developed a new assay with potential clinical applications. Taken together, in the future, we will be able to accelerate the process of neoepitope discovery and provide efficient, and robust solutions to accelerate immunotherapeutic treatments.