Major Histocompatibility Complex (MHC) molecules are membrane proteins responsible for presenting peptides derived from self or foreign proteins to T lymphocytes, thereby activating a specific immune response. There are two main types: MHC class I and class II. MHC-I molecules (in humans, HLA-A, HLA-B, and HLA-C) present endogenous peptides originating from intracellular proteins, whereas MHC-II molecules (HLA-DR, HLA-DP, and HLA-DQ) present exogenous peptides derived from internalized or membrane proteins. The MHC genes are the most polymorphic in the human genome. This polymorphism is concentrated in the regions that form the peptide-binding groove, where the side chains of the ligand’s amino acids interact with specific cavities or pockets. The combination of these pockets defines the specificity of each allele and its repertoire of presented peptides. In class II MHC molecules, both the α and β chains contribute to polymorphism, although in HLA-DR the variability is mainly concentrated in the β chain, since the α chain is poorly polymorphic and does not affect peptide interaction. SARS-CoV-2 virus, the causative agent of COVID-19, is an enveloped, positive-sense single-stranded RNA zoonotic coronavirus. Its genome encodes four structural proteins: Spike (S), Envelope (E), Membrane (M), and Nucleocapsid (N). The S protein, responsible for receptor (ACE2) binding and membrane fusion, is the most immunogenic and the one that accumulates the most mutations, leading to variants such as Omicron (lineage B.1.1.529). Infection produces common symptoms such as fever, cough, and dyspnea, but it can become severe in elderly individuals or those with comorbidities. MHC molecules play an essential role in the immune response against SARS-CoV-2, and the peptides they present were key to vaccine design. Their identification requires purification of MHC–peptide complexes, isolation of the immunopeptidome, and analysis by mass spectrometry, a technique that has enabled the discovery of hundreds of thousands of ligands. Cysteine, an amino acid with a highly reactive thiol group, can undergo post-translational modifications (oxidation, disulfide bond formation, etc.). However, immunopeptidome studies have revealed a lower-than-expected number of cysteine-containing peptides, suggesting that their presence has been underestimated in conventional analyses. Thus, the main objective of this thesis was to optimize the detection of cysteine-containing peptides in HLA-DR–associated immunopeptidomes through a reduction and alkylation protocol that stabilizes this amino acid. Three lymphoid-derived cell lines (BEN, C1R, and Raji) were used, resulting in an enrichment of cysteine-containing peptides without affecting sample quality. In addition, most of the common HLA-DRB1 allotypes, along with HLA-DRB3*02:02 and HLA-DRB4*01:03, were cloned and expressed in C1R cells to generate stable lines. From these, immunopeptidomes treated with the new protocol were purified, which increased the number of detected cysteine-containing peptides and allowed the analysis of their contribution to the anchor motifs characteristic of each HLA-DR allotype. Finally, the methodology was applied to HEK293 cells transfected with the gene encoding the Spike protein from the Omicron variant of SARS-CoV-2. This approach enabled the identification of previously undescribed peptides derived from this viral protein, in both HLA-I and HLA-II molecules (after transfection with the CIITA gene). Overall, the results demonstrate that cysteine reduction and alkylation improve the detection and characterization of peptides containing this residue, providing a valuable tool for immunopeptidome research and for understanding the immune response against pathogens, such as SARS-CoV-2.
| Date of Award | 14 Jan 2026 |
|---|
| Original language | Spanish |
|---|
| Supervisor | Iñaki Alvarez (Director) & Manel Juan (Director) |
|---|