NK cells are key effectors of innate immunity and produce enhanced amounts of cytokines upon restimulation after short-term stimulation with IL-12, IL-15, and IL-18. These cytokine-induced memory-like NK cells (CIML NK) are promising tools for adoptive immunotherapy. In addition to cytokines, signals from feeder cells also influence NK cell function and can drive phenotypic reprogramming.

This study aimed to use high-dimensional protein analysis to characterize the polyfunctional heterogeneity of conventional NK (cNK) cells by comparing their stimulation with K562-mbIL21-4-1BBL feeder cells alone or in combination with a cytokine cocktail.

Human NK cells from peripheral blood donors were cultured for 7 days under distinct conditions: IL-15-stimulated cNK, cNK + feeder cells, IL-12 + IL-15 + IL-18-stimulated CIML NK, and IL-12 + IL-15 + IL-18-stimulated CIML NK + feeder cells. On day 7, all groups except the cNK unstimulated control were restimulated with IL-12 and IL-15 for 5 hours and analyzed by multiparametric flow cytometry, followed by dimensionality reduction using t-SNE and UMAP.

High-dimensional clustering revealed a progression from quiescent cNKs to highly activated, proliferating CD56bright subsets following feeder cells or IL-12, IL-15, and IL-18 stimulation. Among all groups, the combination of IL-12 + IL-15 + IL-18 stimulation with feeder cells yielded the most robust phenotype, with the highest expression of IFN-γ, CD107a, CD69, Ki-67, NKG2C, NKG2A, and NKG2D. Co-expression analysis revealed an enrichment of IFN-γ⁺ cells also expressing TNF-α, CD107a, Ki-67, and CD69, forming a distinct multifunctional cluster consistent with a memory-like phenotype. NKG2D and NKG2C were broadly expressed across conditions, but IL-12 + IL-15 + IL-18-stimulated CIML NKs in the presence of feeder cells showed a more stable activation profile. NKG2A expression was elevated in feeder-free CIML NKs compared to cNKs and was further upregulated upon feeder co-culture. CD56/CD16-defined subsets showed distinct responses to stimulation. Phenotypically, the IL-12 + IL-15 + IL-18-stimulated CIML NK + feeder selectively expanded the CD56brightCD16bright subset, the most functionally potent subset observed, while the CD56brightCD16dim population, predominant in control cNK, was nearly absent. Additionally, a CD56dimCD16dim subset emerged uniquely in IL-12 + IL-15 + IL-18-stimulated groups, with or without feeder support, displaying a less functional profile, and was characterized by lower Ki-67 expression. Interestingly, cNKs cultured with feeder cells alone exhibited a phenotype closely resembling that of CIML NKs, suggesting that feeder-derived signals alone can partially reprogram cNKs toward a memory-like state.

These results underscore the synergy between cytokine-driven and contact-dependent signals, supporting an alternative approach to generate memory-like NK cells with a robust polyfunctional profile for immunotherapy.