Single-cell transcriptomics reveals dynamics of natural killer cell expansion in a feeder cell-free culture of peripheral blood mononuclear cells—implications for immunotherapy

Natural killer (NK) cell therapies hold great promise for cancer treatment; however, donor-to-donor heterogeneity in the ex vivo expansion process remains a critical bottleneck in their supply. This study aimed to identify factors influencing donor variability in a 2-week-long ex vivo NK cell expansion from peripheral blood mononuclear cells, analyzed across three donors. Single-cell transcriptomics was applied to investigate the distribution of cell types and phenotypes, as well as trajectory inference and differential gene expression. Our results identified that several factors were associated with the variability in the final NK cell fraction and expansion, and that their influence was prevalent between culture days 3 and 8. Compared to a high final NK cell expansion, a culture with a low final NK cell expansion exhibited an upregulation of some stress and inflammatory genes and an increase in one specific subcluster of NK cells already on culture day 3. It showed a low score of CD56^Bright CD16^- phenotype and a high score of CD56^Dim CD16⁺ phenotype. It also had a decreased presence of cytotoxic CD8⁺ Tm cells. Among the observed subclusters of CD8⁺ Tm cells, it exhibited a higher presence of a subcluster associated with a less differentiated and less cytotoxic phenotype, as well as a lower prevalence of a subcluster associated with chemokine and cytotoxic genes. Finally, it had a major expansion of one of the CD8⁺ Tm cells subclusters annotated as NK-like T cell and characterized by a high CCR5 mRNA expression, while the levels of CCL3, CCL4 and CCL5 mRNA were downregulated. The present findings point toward a potential link between CCL signaling and improved NK cell expansion performance, including possible markers for further investigations, and suggest future strategies to increase the final NK cell fraction and expansion based on donor-specific markers.