Integrative single-cell transcriptomics and proteomics reveal an immunometabolic framework for MSC-exosome-mediated remodeling of expanded NK cells

BACKGROUND: Natural killer (NK) cells play a central role in anti-tumor immunity and immunosurveillance of senescence, yet their clinical performance is frequently limited by functional exhaustion during ex vivo expansion. Mesenchymal stem cell-derived exosomes (MSC-Exos) are increasingly recognized as immunomodulators, but their broader effects on NK cell fitness and functional states remain incompletely characterized. RESULTS: Here, we assessed MSC-Exos-mediated regulation of human NK cells using a standardized ex vivo priming platform integrated with single-cell transcriptomics and proteomic profiling. MSC-Exos significantly improved NK cell viability in a dose- and time-dependent manner while preserving a CD56⁺CD3⁻ NK-cell-enriched phenotype. MSC-Exos-treated NK cells showed enhanced cytotoxicity against K562 tumor cells and senescent fibroblasts. This phenotype was accompanied by increased expression of the activating receptors NKG2D and CD16, reduced LAG3 expression, and enhanced granzyme B expression and degranulation. Consistent with improved NK cell fitness, MSC-Exos treatment was also associated with upregulated expression of genes involved in NRF2-linked redox programs and improved mitochondrial readouts in NK cells. Single-cell analyses of MSC-Exos-treated NK cells revealed enhanced immune-effector programs and reduced inflammatory stress, while trajectory inference indicated that MSC-Exos may bias the NK cell state distribution toward more cytotoxic effector-like states. Proteomic profiling of MSC-Exos identified enrichment of FcγR-associated signaling components, supporting the hypothesis that exosomal composition may be related to the FcγR/CD16-associated transcriptional and phenotypic features observed in MSC-Exos-treated NK cells. CONCLUSIONS: Our data indicate that MSC-Exos improve NK cell viability and functional fitness during ex vivo expansion and bias NK cells toward a more effector-cytotoxic state. Together, these findings provide an immunometabolic framework for MSC-Exos-assisted NK cell manufacturing, while underscoring the need for further causal validation.