Memory NKG2C+ NK cells develop from NKG2C+ transitional precursors and persist within secondary lymphoid organs

Abstract Background: Natural Killer (NK) cells are critical components of the innate immune system, providing rapid responses against virally infected and tumor cells. A specialized subset of these cells, characterized by the expression of the activating receptor NKG2C, plays an essential role in controlling human cytomegalovirus (HCMV) infections. The NKG2C/CD94 receptor complex specifically recognizes HLA-E molecules presenting viral peptides derived from the HCMV-encoded gpUL40 antigen, triggering NK cell activation. Upon engagement with these antigen-loaded HLA-E complexes, NKG2C+ NK cells acquire features reminiscent of adaptive immune cells, including enhanced functionality and the capacity for long-term persistence. As a result, HCMV-seropositive (HCMV+) individuals often harbor elevated levels of long-lived, memory NK cells. Despite growing recognition of their adaptive-like behavior, the signals that govern the development, differentiation, and long-term maintenance of Memory NK cells remain poorly understood. In this study, we identify a transcriptionally distinct subset of Memory NKG2C+ NK cells residing within secondary lymphoid organs. Through predictive cell fate trajectory analyses, we reveal that this memory population likely originates from a transitional NKG2C+ NKG2A+ subset. Methods & Results: Human spleen and matched lymph node samples were obtained from eight healthy adult donors, including four HCMV+ and four HCMV-seronegative (HCMV-) individuals. All donors tested positive for Epstein-Barr virus IgG, and the HCMV+ group included an equal distribution of male and female donors (n = 2 each). Nucleic acid testing confirmed the absence of hepatitis B virus, hepatitis C virus, and human immunodeficiency virus in all samples. Tissue specimens were obtained through the Versiti Organ Donor Center of Wisconsin and processed into single-cell suspensions. NK cells were isolated and subjected to droplet-based single-cell RNA sequencing (scRNA-seq). Transcriptomic data were analyzed using the Seurat R package, with filtering and clustering based on the number of uniquely expressed genes per cell. Single-cell trajectory analyses were performed using Monocle3 and RNA velocity to infer developmental dynamics. To identify and define the transcriptomic profile of Memory NKG2C+ NK cells, we first isolated all NKG2C-expressing NK cells by subsetting cells with a log₂-transformed expression level of KLRC2 greater than 0.3. Unbiased clustering analysis of these filtered cells revealed four transcriptionally distinct NKG2C+ NK cell subsets within the spleen and lymph node. Specifically, we identified 2,792 splenic and 1,843 lymph node NKG2C+ NK cells, which were integrated and clustered to define shared and tissue-specific populations. Among these, one subset found in both tissues exhibited elevated expression of memory-associated genes such as CD52, CD2, CD16, and TCF7, along with significantly reduced expression of FCER1G, NCR1, NCR3, and ZBTB16—features consistent with a memory-like phenotype. Notably, HCMV+ donors had significantly higher frequencies of this Memory NKG2C+ subset in both the spleen and lymph node. These findings suggest that secondary lymphoid organs contain a transcriptionally distinct population of Memory NKG2C+ NK cells, potentially shaped by prior HCMV exposure. To investigate the developmental trajectory of Memory NKG2C+ NK cells, we applied Monocle3 and RNA velocity analyses. Monocle3 revealed a clear progression from early to late differentiation states, which was mapped onto the UMAP plot. Notably, the NKG2A+NKG2C+ subset consistently emerged as the developmental origin, suggesting it may serve as a transitional precursor to the Memory NKG2C+ population. To further validate, we employed scVelo's dynamical RNA velocity modeling on NKG2C+ cells from individual donors. In HCMV+ donors, the analysis consistently demonstrated that NKG2A+NKG2C+ cells marked the starting point for all downstream NKG2C+ subsets and progressed toward the Memory NKG2C+ state. Collectively, these findings support a model in which HCMV exposure actively shapes NK cell fate by driving the differentiation of transitional NKG2A+NKG2C+ cells into long-lived, memory-like NKG2C+ NK cells. Conclusion: These findings uncover a potential developmental pathway underlying memory NK cell differentiation and establish a foundation for future strategies aimed at leveraging these cells in antiviral immunity and NK cell–based immunotherapies.