Analysis of clonal NK cell populations using single-cell transcriptomics data

Natural killer (NK) cells represent a critical component of antiviral immunity, demonstrating remarkable adaptability during infections such as human cytomegalovirus (hCMV) and SARS-CoV-2. Recent advances in single-cell transcriptomics have revealed the clonally expanding NK cell populations with distinct functional profiles, blurring the traditional boundaries between innate and adaptive immunity. However, the functional heterogeneity and immunological significance of these clones remain incompletely understood. Our aim was to dissect clonal NK cell heterogeneity using published scRNA-seq datasets from hCMV-seropositive, seronegative, and COVID-19 patients, focusing on cluster-specific gene expression patterns. Our computational pipeline employed Seurat-based integration and high-resolution clustering of datasets from hCMV-seropositive (n = 5) and seronegative (n = 2) donors, along with COVID-19 patients (n = 2). We analyzed datasets using Seurat 5 in R. Quality-controlled data were normalized (SCTransform), integrated (batch-corrected), and clustered (UMAP). Differential gene expression (Wilcoxon test, log2FC 0.25, p-adj 0.05) and annotation were performed. In hCMV-seropositive individuals, we identified 12 transcriptionally distinct NK cell clusters exhibiting KLRC2 (NKG2C)-dependent organization, with specific clones showing either enhanced cytotoxic potential (marked by GZMB/GZMA upregulation), or unique inhibitory receptor profiles (variable KIR expression patterns). The hCMV-seronegative cohort displayed a simpler clonal structure with 9 clusters showing reduced KIR diversity but maintained distinct effector gene signatures. Analysis of COVID-19 patients revealed divergent clonal patterns: one patient showed reduced KLRC2 variability with prominent KLRC1 (NKG2A) expression, while another exhibited KIR heterogeneity without KLRC2 variation. Our analysis reveals distinct clonal NK cell populations in hCMV and COVID-19 contexts, characterized by divergent expression of activating and inhibitory receptors. These findings demonstrate infection-specific dynamics of clonal NK cell populations, highlighting their adaptive potential through differential receptor expression in antiviral responses.