Early CD8 + T and NK cell interactions orchestrate preemptive immunosurveillance to potentiate optimal cytotoxicity that blocks tumor antigen-escape variants

Background: Tumor antigen-escape variants pose a major barrier to immunotherapy by disrupting lymphocyte effector pathways and reconfiguring tumor-immune landscapes. A deeper understanding of immune networks during tumor development is required. We assessed whether homeostatic CD8⁺ T cell-NK cell interactions can preemptively block tumor antigen escape. Methods: Adoptive CD8⁺ T cell transfers were performed either before tumor implantation (D -7 , homeostatic pre-priming) or after tumor establishment (D +1 ) in Rag1⁻ / ⁻ and Rag1⁻ / ⁻gammac⁻ / ⁻ mice. Antigen presentation, immune activation, proliferation, cytotoxicity, and memory differentiation were quantified using multiparameter flow cytometry, live bioluminescence imaging, and high-resolution confocal microscopy. Intercellular interactions were modeled through monoculture, co-culture, and a 3D silica nanofiber carpet that recapitulates basement-membrane-like architecture. Phospho-signaling arrays and cellular motion metrics (Speed-Distance Index, deceleration) were employed to assess activation dynamics and coordination. Human ligand-receptor pairs implicated in CD8⁺ T-NK cell crosstalk were identified through in silico analyses. Results and Discussion: Our study shows that pre-tumor (D -7 ) CD8⁺ T cell transfer establishes a preemptive immune surveillance network by orchestrating NK cell activation and effector function. These early T cells enhance NK cytotoxicity (CD25, CD69, CD107a, T-bet, GzmB) and promote CD62L⁺CD44⁺ central-memory CD8⁺ T (T CM ) precursors, providing immediate and long-term protection. Spatial analyses reveal that early T cells reposition NK cells toward tumors, increasing synapse formation and infiltration effects absent in post-tumor (D +1 ) transfers that allow antigen-loss variants despite potent T cell cytotoxicity. Mechanistically, CD8⁺ T-NK interactions via pseudopodial nanotubes enable bidirectional membrane/vesicle exchange and coordinated STAT, Akt, AMPK, and mTOR signaling, enhancing NK metabolic fitness, mitochondrial potential, and T CM differentiation. In silico analyses identify conserved human adhesion and co-signaling networks (CD200-CD200R, PD-L1-PD-1, CD18/CD11a-DNAM-1, TIGIT-PVR, NTB-A/SLAM) regulating adhesion, activation thresholds, and cooperative effector functions, with translational relevance. Precise molecular signaling remains under investigation. Conclusion: Early CD8⁺ T-NK crosstalk establishes preemptive immunosurveillance by integrating metabolic, cytokine, and adhesion signals to potentiate optimal cytotoxicity blocking tumor antigen escape. This axis represents a targetable checkpoint and a framework for next-generation preventive immunotherapies against antigen-loss tumors.