Development of Immunoinformatics Methods for Improved Rational Identification of T cell Epitopes

The research projects presented in this thesis are centered around T cell specificity. T cells play a crucial role in maintaining health by eliminating intruding pathogens and malignant cell changes. This ability is granted via the T cell receptor (TCR), which interacts with peptides presented by MHC molecules on the surface of host cells. To ensure broad protection against any potential pathogen, the immune system has evolved to generate highly diverse TCRs which may recognize a wide range of targets. However, such a complex system is inevitably very challenging to study. Nevertheless, this thesis has been dedicated to investigate T cell specificity via popular experimental methods and develop immunoinformatic tools and analyses to enhance the yield of such methods.
A commonly used method for assaying T cell specificity is peptide-MHC (pMHC) multimer staining, which procures the distribution of T cells responding to given peptides of a panel. This method was applied to map SARS-CoV-2 epitopes across cohorts of infected and healthy individuals, in the first project of this thesis. We identified several immunodominant epitopes even in healthy individuals, which suggest strong influence of cross-reactive T cells primed for other, perhaps similar, antigens.
However, multimer staining only provides shallow insight into the complexity of TCR recognition of pMHCs. In order to truly understand the rules that govern T cell specificity, we employed singlecell sequencing, enabling the capture of TCRαβ-chains, the cognate pMHC provided by DNA-barcoded multimers, and hashing antibodies in the second project of the thesis. As single-cell data is polluted with multiple confounding factors, the key aim was to develop a method to efficiently remove noise and retain accurate pairing of TCR-pMHC.
In the third and final project, we benchmarked the previous project against a recently released method to learn the advantages and disadvantages of each approach. The two methods distinctively differ by their prioritization between specificity and sensitivity of detecting TCR-pMHC pairs.