CD8⁺ T-NK cell crosstalk establishes preemptive immunosurveillance to eliminate antigen-escape tumors

Tumor antigen-escape variants undermine immunotherapy by subverting lymphocyte effector functions and reshaping tumor-immune dynamics. It is essential to delineate functional interplay within immune networks during tumor progression. We investigated whether homeostatic crosstalk between CD8⁺T cells and natural killer (NK) cells preempts tumor antigen-escape.

Adoptive CD8⁺T cell transfers were administered before (D_-7, homeostatic pre-priming) or after (D₊₁) tumor establishment in Rag1^-/- and Rag1^-/-γc^-/- mice. Antigen presentation, immune activation, proliferation, cytotoxicity, and memory were quantified by flow cytometry, live bioluminescence and confocal imaging. Monoculture, co-culture, and a 3D silica nanofiber carpet mimicking basement-membrane-like topography modeled intercellular interactions. Signaling arrays and motion metrics (Speed-Distance Index, deceleration) were conducted. Human ligand-receptor pairs engaged in CD8⁺T-NK crosstalk were probed in silico.

Pre-tumor D_-7 CD8⁺T cell transfer completely suppressed antigen-escape tumors with NK cells as major effectors showing elevated CD25, CD69, CD107a, and GzmB, marking activated and effector phenotype, and promoting central-memory CD62L⁺CD44⁺CD8⁺T_CM precursors. By contrast, post-tumor D₊₁-transferred CD8⁺T cells allowed emergence of tumor variants resistant to antigen-specific cytolysis as assessed on day 25, despite those T cells retaining higher intrinsic cytotoxic capacity than the D_-7 T cell cohort. Mechanistically, CD8⁺T and NK cells formed stable contacts through pseudopodial intercellular nanotubes enabling bidirectional membrane exchange and signaling via STAT, Akt, and mTOR pathways, augmenting NK effector function and promoting CD8⁺T_CM differentiation. In silico analysis identified human ligand-receptor pairs engaged in CD8⁺T-NK adhesion, stimulatory and regulatory axes, including CD200-CD200R, PD-L1-PD-1, and CD18/CD11a-DNAM-1 (CD226). Together, data support a three-phase model of preemptive immunosurveillance initiated by early CD8⁺T-NK crosstalk.

Homeostatic conditioning and effector cooperativity between CD8⁺T and NK cells protect against tumor immune escape. The findings uncover a mechanistic axis of preemptive immunosurveillance that lays the foundation for next-generation preventive immunotherapies to control antigen-escape tumors.