Phenotypic Alterations of CD8+ and CD8- CD56+ NK Cell Subsets in Moderate and Severe COVID-19 Disease

The COVID-19 pandemic has created a global health challenge. Severe cases are associated with immune system dysfunction, which can lead to uncontrolled inflammation. There are no published data on the specific roles of natural killer (NK)-cell subsets and immune checkpoint (IC) molecules in disease severity. Thirty-five patients diagnosed with COVID-19 and 14 healthy controls were involved in the study. From peripheral blood, CD56dim and CD56bright cell subsets were analyzed by flow cytometry for the expression of IC molecules (T-cell immunoglobulin and ITIM domain [TIGIT], CD226, and PD-1), activation markers (CD69), and cytotoxic potential (CD107a degranulation, granzymes, and perforin content). In COVID-19 patients, the proportion of CD8- CD56dim cells was significantly higher than the CD8+ subset. Inhibitory receptors TIGIT and PD-1 exhibited significantly higher relative expression in CD8+ CD56dim cells compared to their CD8- counterparts across infected groups, an effect particularly pronounced in deceased patients. Conversely, activating CD226 expression was reduced in the CD8- CD56dim subset only in severe cases. Functional assays revealed significantly elevated CD107a and CD69 expression in CD56dim cells of patients versus controls. Notably, CD8- CD56bright cells from deceased patients demonstrated enhanced CD107a expression and elevated perforin content, suggesting a shift toward hyperactivation. The preferential upregulation of inhibitory checkpoint molecules on the highly active CD8+ subset may reflect a compensatory response to immune activation, whereas the enhanced activation of CD8- NK-cell subsets was associated with disease severity and mortality. Similarly, the heightened cytotoxic profile of CD8- CD56bright cells observed in fatal cases may reflect immune dysregulation associated with severe COVID-19.