Background/Objectives: Systemic administration of recombinant cytokines for immunomodulation in cancer and chronic infection is limited by toxicity and poor spatial control. Messenger RNA (mRNA)-encoded cytokines offer a programmable alternative, but the immunological consequences of interferon-β (IFN-β) mRNA delivery in primary human immune cells, and its compatibility with co-delivered antigen-encoding mRNA, remain incompletely defined. This study evaluated whether nucleoside-modified, mRNA-encoded human IFN-β can function as a multifunctional immunomodulator with pro-apoptotic activity and timing-dependent immunostimulatory properties. Methods: N1-methylpseudouridine-modified IFN-β mRNA was transfected into HEK293T and HepG2 cell lines and into primary human monocyte-derived dendritic cells (MDDCs) and M2-polarized macrophages (MDMs) using Lipofectamine MessengerMAX, with OVA mRNA as a non-adjuvant control. Apoptosis, interferon-stimulated CXCL10 secretion, activation marker expression, and cytokine profiles were assessed by flow cytometry, ELISA, Western blot, and cytometric bead array. Adjuvant activity was evaluated in MDDCs co-transfected with IFN-β and antigen (EGFP or OVA) mRNA under four temporal delivery regimens. Results: IFN-β mRNA induced time-dependent apoptosis, most pronounced in HepG2 cells, and CXCL10 secretion in both cell lines. In primary immune cells, IFN-β mRNA activated MDDCs, increased pro-inflammatory cytokine secretion, and repolarized M2-like macrophages toward a pro-inflammatory phenotype. In co-transfection experiments, IFN-β mRNA enhanced MDDC activation across regimens, but antigen expression was preserved only when antigen mRNA was delivered before IFN-β mRNA. Conclusions: mRNA-encoded IFN-β combines direct pro-apoptotic activity with programmable, timing-dependent immunostimulatory effects, supporting its development as a component of next-generation mRNA-based immunotherapies and vaccines.
