MSC-exosome-driven NK-cell expansion and effector reprogramming

Natural killer (NK) cells are central to immune surveillance against malignancies and age-related cellular senescence. However, their clinical translation is hindered by functional exhaustion and senescence during ex vivo expansion. Mesenchymal stem cell-derived exosomes (MSC Exos), noted for their minimal immunogenicity and robust immunomodulatory potential, have emerged as key regulators of immune cell fate. Nevertheless, a comprehensive understanding of their global impact on human NK cell proliferation and effector function remains elusive. In this study, we employed a standardized ex vivo expansion and exosome-priming system to systematically evaluate the regulatory effects of MSC-Exos on peripheral blood-derived NK cells via flow cytometry, cytotoxicity assays, single cell transcriptomics, and proteomic profiling. MSC Exos significantly accelerated NK cell proliferation in a dose- and time-dependent manner while maintaining the integrity of the CD56+CD3- phenotype. Functionally, MSC-Exos augmented NK mediated cytotoxicity against tumor cells K562 and senescent fibroblasts. This potentiation was characterized by the upregulation of activating receptors (NKG2D, CD16, NKG2C), concomitant downregulation of the inhibitory receptor LAG3, enhanced degranulation, and increased granzyme B secretion. Mechanistically, MSC-Exos optimized mitochondrial performance and bolstered NRF2-mediated redox homeostasis. Single-cell transcriptomic landscapes revealed that MSC Exos prioritized translational machinery and immune-effector pathways, intensified granule-mediated cytolysis, and mitigated IFNG,TNF-driven inflammatory stress while reprogramming metabolic networks. Subpopulation trajectory analysis demonstrated that MSC-Exos facilitated the transition of NK cells from a regulatory state toward a potent cytotoxic effector phenotype. Furthermore, proteomic profiling identified an enrichment of FcgR related signaling components within MSC-Exos, suggesting a potential role in enhancing antibody-dependent cellular cytotoxicity (ADCC).Collectively, our findings demonstrate that MSC-Exos bolster NK cell expansion and effector maturation through coordinated immuno-metabolic reprogramming, offering a strategic approach to enhancing the therapeutic efficacy of NK cell-based immunotherapies.