Characterization of MHC Class I and Class II-associated Peptides

Major Histocompatibility Complex (MHC) molecules play a crucial role in the immune system by presenting peptides to T-cells, allowing for the recognition of infected or abnormal cells. This dissertation presents three projects focused on the identification of peptides presented through the MHC class I and/or class II pathways, contributing to a better understanding of immune recognition and potential therapeutic applications. The first project focuses on the verification of peptide presentation using a combination of chromatographic and tandem mass spectrometry (MS2) data. This project involved ensuring that the correct synthetic peptides—either the wild-type ovalbumin peptide (OvaWT) or its trimethylated lysine variant (OvaK7m3)—were bound to MHC molecules. By applying rigorous analytical techniques, including the RMA-S stabilization assay, this study confirmed that the expected peptides were presented on MHC molecules. This validation is critical for ensuring the reliability of MHC-peptide interaction studies, paving the way for further exploration of post-translational modifications and their effects on immune responses. The second project aimed to characterize the MHC class I peptide binding motifs of Golden Syrian hamsters, an emerging model organism for COVID-19 research. This study was essential for identifying viral epitopes, particularly from the spike protein of SARS-CoV-2, that could be presented by hamster MHC molecules. By elucidating these binding motifs, the project provides a foundation for developing predictive algorithms to identify immunogenic peptides from viral proteins in this species. This work enhances the use of hamsters in immunological studies, especially for infectious disease research and vaccine development. The third project investigates the presentation of phosphopeptides on MHC class I molecules in head and neck squamous cell carcinoma (HNSCC) cells. Phosphorylation is a key regulator of cellular processes, and in cancerous cells, phosphopeptides can be presented on MHC molecules due to disruptions in normal phosphatase activity, particularly by SET and CIP2A proteins. Using advanced sample preparation techniques, including hydrophilic interaction chromatography (HILIC) and immobilized metal affinity chromatography (IMAC), along with LC-MS/MS analysis, 27 phosphopeptides were identified in HNSCC cells. Many of these phosphopeptides were also found in other cancerous and infected cells, suggesting their potential as universal vaccine candidates for both cancer and infectious diseases. Together, these three projects provide significant insights into MHC peptide presentation, with applications ranging from the study of post-translational modifications in model systems to the identification of viral epitopes and cancer-specific antigens. These findings have broad implications for the development of immunotherapies and vaccines.