Multiomics Dissection Reveals Natural Killer Cell–CD8+ T-cell Cooperation in Shaping an Immunostimulatory Tumor Microenvironment

Abstract Immune evasion remains a major barrier to effective immunotherapy in cancer. Coordinated activation of different immune cell types shapes immune responses within the tumor microenvironment (TME). In this study, we uncovered a spatiotemporal synergy between cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in tumors with low T-cell infiltration. An integrative multiomics approach combining spatial transcriptomics, single-cell RNA sequencing, and high-dimensional flow cytometry demonstrated that vaccination, capable of activating both innate and adaptive immunity, rapidly reshapes the TME. Early infiltration of IRF8+KLRG1+ NK cells established a chemokine-rich niche together with myeloid cells that promoted the recruitment of antigen-primed CXCR3+ CTLs. The accompanying inflammatory monocyte recruitment and dendritic cell activation led to the coordinated structural and chemotactic remodeling of the TME. Functionally, NK–CTL clustering at the tumor margin was associated with enhanced cytotoxic activity and sustained immune engagement. These spatially organized immune interactions involved CCR5–CCL5 and CXCR3–CXCL9 signaling pathways that coordinate communication between innate and adaptive immune compartments. Together, these findings reveal a previously unrecognized NK–CTL cooperative program that promotes the transition of poorly infiltrated tumors toward an inflamed, immune-responsive state. More broadly, this study illustrates how spatially resolved multiomics approaches can uncover immune interactions and provides a conceptual framework for designing next-generation immunotherapies that mobilize coordinated innate and adaptive immunity. Significance: Early infiltration of NK cells followed by T-cell accumulation reshapes the tumor microenvironment to promote antitumor immunity following vaccination, providing a mechanistic rationale for the development of effective vaccine-based immunotherapy strategies.