CD8⁺ T and NK Cell Crosstalk Preemptively Eliminates Tumor Antigen-Escape Variants

Roman V Uzhachenko^1,2Salvador González Ochoa^1,2Thanigaivelan Kanagasabai^1,2Harshana Rajakaruna^1,2Menaka Chanu Thounaojam^1,2,3Maria Teresa P. de Aquino^1,2,4Tanu Rana^1,2,5Lino Costa⁶Alexander Terekhov⁷William H Hofmeister⁷Thomas J Sayers⁸Claude Boyer⁹Alla Ivanova^1,2J Shawn Goodwin^1,2Anne-Marie Schmitt-Verhulst⁹Anil Shanker^1,2*

• ¹Department of Biochemistry, Cancer Biology, Neuroscience & Pharmacology, School of Medicine, Meharry Medical College, Nashville, United States
• ²Meharry Medical College, Nashville, Tennessee, United States
• ³Department of Biomedical Sciences, Meharry Medical College School of Graduate Studies, Nashville, United States
• ⁴Beckman Research Institute, City of Hope, Pasadena, California, United States
• ⁵Belmont University, Nashville, Tennessee, United States
• ⁶Center for Laser Applications, University of Tennessee Space Institute, Center for Laser Applications, University of Tennessee Space Institute,, Tullahoma, Tennessee, United States
• ⁷Center for Laser Applications, University of Tennessee Space Institute,, Tullahoma, United States
• ⁸Frederick National Laboratory for Cancer Research, National Cancer Institute at Frederick (NIH), Frederick, Maryland, United States
• ⁹Centre d'Immunologie de Marseille-Luminy, Aix Marseille Université UM2, Institut National de la Santé et de la Recherche Médicale U1104, Centre National de la Recherche Scientifique UMR7280, Marseille, France

The emergence of tumor antigen-escape variants poses a barrier to effective cancer immunotherapy by subverting immune effector functions and reshaping tumor–immune dynamics. To elucidate lymphocyte behavior during tumor progression, it is imperative to examine the complex functional interplay within immune cell networks. We investigated the effector functions and interactions between antigen-specific CD8⁺T-cells and natural killer (NK) cells in the context of tumor antigen-escape. Using distinct adoptive CD8⁺T-cell transfer protocols—administered either before tumor establishment (D–7) or post-establishment (D+1)—we assessed immune activation, proliferation, cytotoxicity, and memory differentiation by flow cytometry, bioluminescence imaging, and confocal microscopy, with emphasis on CD8⁺T–NK cell crosstalk. Pre-tumor CD8⁺T-cell administration (D–7) effectively suppressed the emergence of antigen-escape tumors in an antigen presentation-independent manner. In contrast, post-tumor CD8⁺T-cell transfer (D+1) led to the development of tumor variants resistant to antigen-specific cytolysis as assessed on day 25, despite T-cells retaining higher intrinsic cytotoxic potential than the D–7 T-cell cohort. Strikingly, NK cells emerged as key effectors in the D–7 group, controlling antigen-escape tumors. Mechanistically, CD8⁺T–NK cell interactions were mediated by pseudopodial intercellular channels, enabling bidirectional physical crosstalk and signaling that enhanced NK effector function, and promoted central memory CD8⁺T-cell differentiation. In silico analyses of human datasets identified candidate ligand-receptor pairs involved in this effector crosstalk, including CD200–CD200R, PDL1–PD1, and CD18/CD11a–DNAM1. These findings underscore the pivotal role of CD8⁺T–NK-cell cooperation in immunosurveillance against antigen-escape tumor variants and offer mechanistic insights to guide the development of next-generation immunotherapeutic strategies.