Comprehensive longitudinal profiling identifies differential frequency, epitope specificity, and effector function of CD8⁺ T-cells across COVID-19 disease severities.

Abstract

CD8⁺ T-cells are crucial for controlling and resolving SARS-CoV-2 infection, yet their epitope specificity and relationship to COVID-19 disease severity remain incompletely understood. We performed comprehensive longitudinal profiling of antigen-specific CD8⁺ T-cell populations using DNA-barcoded peptide-HLA multimers, analysing 553 SARS-CoV-2 epitopes across globally prevalent HLA alleles in patients with mild and severe COVID-19. Functional and phenotypic characterisation was performed using multidimensional single-cell analysis and detailed cytokine profiling. The impact of post-infection COVID-19 vaccination on T-cell memory was also assessed. Severe and mild COVID-19 were associated with robust yet distinct patterns of CD8⁺ T-cell activation. In the acute phase, severe disease was characterised by a broader T-cell repertoire (139 unique epitopes) with a median frequency of 1.4% (IQR 0.2-5.0) and a high-frequency of immunodominant epitope-specific T-cells that exhibited reduced cytotoxic profile. In contrast, patients with mild COVID-19 mounted responses against a more limited set of epitopes (98 unique epitopes), partially overlapping with those observed in severe disease, with a median T-cell frequency of 0.7% (IQR 0-1.9) and displayed a stronger cytotoxic phenotype and functional state. Over time, the memory T-cell compartment contracted to a restricted subset of immunodominant epitopes in the two patient groups and COVID-19 vaccination further enhanced frequencies of spike-specific T-cells independent of prior disease severity. These findings delineate the epitope-specific frequency, function, and persistence of antigen-specific T-cell populations during SARS-CoV-2 infection, highlighting how differential activation, rather than magnitude alone, shapes immune outcomes across disease severities and other viral infections. This work was supported by the Independent Research Fund Denmark (DFF-Sapere Aude, 2066-00044B), the EU Horizon Europe REACT project (101057129), the European Research Council (ERC) Starting Grant MIMIC (101045517), and the Danish National Research Foundation (DNRF170).