Identification of SARS-CoV-2 and of human fusion proteins specific TCRs via TCR repertoire sequencing

Adaptive immune responses, particularly T cell-mediated immunity, play a crucial role in controlling SARS-CoV-2 infection and influencing clinical outcomes. Lymphocyte counts and T cell functionality correlate with disease severity, highlighting the importance of early and effective T cell responses. Next-Generation Sequencing (NGS) of T cell receptor (TCR) repertoires has emerged as a high-resolution approach to characterize the diversity, clonality, and antigen specificity of T cell populations in both infectious and oncological contexts. This thesis investigates two complementary aspects of TCR-based immunity: the adaptive response to SARS-CoV-2 and the identification of tumor-specific TCRs targeting neoantigens from gene fusion events. In the first work package, peripheral blood samples from individuals in Emilia Romagna, Italy, who were either naturally exposed to SARS-CoV-2 or vaccinated with the Pfizer-BioNTech mRNA BNT162b2 vaccine, were analyzed. TCR beta repertoires were sequenced to compare clonality, diversity, and SARS-CoV-2 specificity between acute, convalescent, and vaccinated cohorts. Correlations between cell responses, serological anti-Spike and anti-N antibodies, and predicted HLA types were evaluated to define the interplay between cellular and humoral immunity. The second work package focused on identifying TCRs specific for fusion-derived tumor neoantigens in leukemia. Candidate fusion peptides were selected using RNA-Seq databases and computational prediction of MHC binding (NetMHCpan). Specific TCRs recognizing these peptides were identified through an in vitro stimulation protocol, activation markers, single-cell TCR sequencing, and NFAT-based fluorescent reporter assays. The expression and functionality of the isolated TCR sequences were assessed by expressing them in Jurkat cell lines. They will be further investigated in T cells for evaluation of anti-tumor reactivity. This work demonstrates the utility of NGS-based TCR profiling to dissect immune responses against viral infections and tumor neoantigens. It provides insights into the correlation between T cell diversity and functional immune outcomes, and lays the foundation for developing TCR-based immunotherapies.