(437a) Cytotoxic Effects of Cytokine Treated Natural Killer Cells on Human Pediatric Malignant Brain Rhabdoid Tumor Cells

Background: Natural killer (NK) cells have an innate ability to recognize and eliminate cancer cells and pathogens. They employ activating and inhibitory receptors such as killer and leukocyte immunoglobulin like-receptors (KIRs and LILRs) to identify their targets. Upon recognition and interaction with the target, NK cells get stimulated and use various strategies to destroy the target. These activated NK cells increase the expression of fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), release perforin and granzymes, and secrete various cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. An atypical teratoid rhabdoid tumor (ATRT) is a type of highly malignant brain tumor primarily diagnosed in young children. NK cell immunotherapy was effective against acute myeloid leukemia and myeloma, especially when stimulated with proliferative cytokines. This study focuses on targeting ATRT cells using the NK cell immunotherapy approach and compares the cytotoxicity of NK-92mi cells exposed to interleukins with the cytotoxicity of unexposed NK92mi cells.

Methods: The NK-92 mi cells were exposed to 20 ng/mL of IL-2, IL-12, IL-15, IL-18, and IL-21 for 24 hours prior to the co-culture with ATRT cells. The co-cultures were maintained for a certain time, after which the target cells (CHLA-02 and CHLA-05) were isolated from the effector cells (IL-exposed NK92mi and IL-unexposed NK92mi) to perform each experiment. The killing potential of NK-92mi cells was measured using a calcein-based cytotoxicity test and the viability of ATRT cells was tested using a cell proliferation assay. Moreover, the expression of cellular markers and cytokines was studied using flow cytometry.

Results: The IL-exposed NK-92mi cells had drastically higher expression of IL-6 (62.7%), compared to the unexposed NK-92mi cells. This suggests possible cytokine release syndrome in NK cells due to exposure to the combination of interleukins. The expression of CD56 increased in IL-exposed (11.0%) versus unexposed (2.5%) cells showing possible deviation towards the less cytotoxic CD56+ subtype of NK cells. The intracellular release of cytolytic cytokines such as IFN-gamma and TNF-alpha also decreased when NK-92mi cells were exposed to the combination of interleukins. Especially the IFN-gamma expression in IL-exposed NK cells was half of the expression in unexposed NK cells. In addition, NK92mi cells displayed ratio-dependent lysis of both CHLA-02 and CHLA-05 in both IL-exposed and unexposed states. Furthermore, CHLA-05 was found to be more vulnerable to cytolysis by NK cells compared to CHLA-02.

Conclusion: This study signifies the therapeutic potential of NK cells towards ATRT cells. The cell cytotoxicity data indicated that the efficacy of NK cell immunotherapy varies within subtypes of ATRT and is dependent on effector to target ratio. Moreover, the quantitative cytokine expression data implied that the cytokine stimulation of NK cells might slightly improve the lysis potential of NK cells, however, it can trigger possible cytokine release syndrome, hence compromising the safety of NK cell immunotherapy.

(Supported by Florida Department of Health grants, 9LA01 and 21L10, to Q.-X.A.S. and Y.L.)