(901) Cytokine training primes NK cells for enhanced antiviral activity against JC polyomavirus

Introduction/Rationale: JC polyomavirus (JCPyV) is a neurotropic DNA virus that causes progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease in immunocompromised individuals. There is currently no standard treatment option, highlighting the need for new immune-based approaches. Natural killer (NK) cells contribute to antiviral defense, and our lab previously showed NK-mediated control of JCPyV infection. Cytokine-induced memory-like (CIML) NK cells generated with IL-12, IL-15, and IL-18 display enhanced effector function in cancer settings, but their role in JCPyV infection is undefined.

Methods: NK cells from healthy donor PBMCs were stimulated for 16 h with IL-12, IL-15, and IL-18, followed by a 6–7-day rest. Fresh and IL-15–only NKs served as controls. CIML NKs were either unstimulated or restimulated with the same cytokine cocktail during co-culture with JCPyV-Turbo–infected SVGA cells. Viral control was measured by VP1 and T-antigen expression, and NK function by IFN-γ production and CD107a degranulation via flow cytometry. Exhaustion markers (PD-1, TIGIT, TIM-3) were also assessed.

Results: CIML NK cells showed enhanced IFN-γ and CD107a expression compared to fresh and IL-15–treated NKs, with modestly improved control of JCPyV infection. Restimulated CIML NKs exhibited the strongest IFN-γ and CD107a responses, indicating that these cells are primed for rapid recall and heightened activation upon secondary stimulation. CIML NKs also upregulated PD-1, TIGIT, and TIM-3, consistent with activation-linked exhaustion.

Conclusion: CIML NK cells enhance antiviral responses against JCPyV, and restimulation further amplifies their effector capacity, supporting a memory-like phenotype. These findings highlight the potential of trained NK cells as immunotherapy for persistent viral infections such as PML. Ongoing work uses transcript-level flow cytometry to determine whether checkpoint receptor upregulation reflects transient activation or functional exhaustion.