Spike-specific de novo CD8+ T cell clones exhibit robust effector phenotypes in the context of hybrid immunity 2252473

Abstract Introduction Repeated SARS-CoV-2 exposures through infections and vaccinations shape memory CD8+ T cell responses. While exposure history is known to affect CD8+ T cell differentiation, its impact on the TCR repertoire and phenotype remains unclear. Here, we analyze the TCR repertoires of SARS-CoV-2 spike-specific memory CD8+ T cells and their phenotypes in clone-dependent manner under various exposure conditions. Methods Two cohorts were enrolled: 1) individuals with infection followed by three doses of the mRNA vaccine (BNT162b2), and 2) individuals with three doses of the mRNA vaccine followed by Omicron BA.1 breakthrough infection. Antigen-reactive T cell enrichment (ARTE)-based multi-omic sequencing of spike-specific CD8+ T cells was conducted to analyze single-cell gene, protein, and TCR expressions. Results TCR repertoire analysis demonstrated that repeated antigenic exposures under hybrid immunity conditions boosted not only pre-existing “old” clones but also triggered the emergence of de novo clones. Additionally, analysis using the distance-based TCR clustering method (TCRdist) showed that de novo clones occupied a distinct region from old clones within the TCR similarity space. Phenotypic profiling showed that old clones, after repeated antigenic exposures, displayed more differentiated states, while de novo clones were less differentiated effector memory cells. Conclusion Our findings demonstrate that repeated antigenic exposures through infections and vaccinations promote differentiation of pre-existing clones. However, de novo clones can arise under hybrid immunity conditions and may functionally replace terminally differentiated pre-existing old memory clones. Funding Source n/a Topic Categories Viral Immunology (VIR)