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Background: Immune therapies are emerging as a critical component of cancer treatment, capable of delivering durable and potentially curative responses. While CAR-T cell therapy has proven effective for hematological malignancies, it faces challenges in treating solid tumors due to tumor antigen heterogeneity, an immunosuppressive tumor microenvironment, and physical barriers hindering CAR-T cell infiltration. NK cells, particularly adaptive NK (aNK) cells, offer a promising alternative due to their ability to recognize and kill tumor cells without prior sensitization and their resistance to immunosuppressive environments. Purpose: The study investigates the role of cytokines, specifically IL-21 and IL-15, in enhancing aNK cell expansion and activation using peripheral blood mononuclear cells (PBMCs) from healthy donors and tumor-infiltrating lymphocytes (TILs) from glioblastoma (GBM) patients. Methods: Buffy coats and GBM TILs were collected from Karolinska Hospital. NK cells were isolated and expanded in vitro with IL-15 and IL-21 cytokines and feeder cells (K562 and K562E). Furthermore, tumor lysate was added in the cultures to boost memory responses in aNK cells. NK cell functionality, cytotoxicity, and phenotyping was assessed using flow cytometry and statistical analysis (t-test and two-way ANOVA) used to validate the results. Further animal model was used to validate the cytotoxicity capacities of these cells against GBM tumors using a zebrafish model. Results: IL-21 drives the expansion of aNK better than IL-15 controls, data shown in PBMCs and TILs derived from GBM patients and IPLA OVCA patients. Additionally, the use of tumor lysate as a booster for restimulation further amplifies the cytotoxic capacity of aNK cells against autologous tumors. The zebrafish model validates this method, by decreasing the tumor size in zebrafish animals after 3 days of injection. Conclusion: The results demonstrate that IL-21 is essential for the specific expansion of aNK cells, enhancing their aggressiveness towards tumor cells. Additionally, tumor lysate significantly increases the cytotoxic efficacy of aNK cells upon restimulation with the same tumor cells. These findings suggest that IL-21 plays a crucial role in the specific expansion and activation of aNK cells, enhancing their aggressiveness towards tumor cells. By optimizing the expansion protocol, this method aims to advance the clinical application of aNK cells in immunotherapies for solid tumors, offering a potential solution to the limitations faced by current CAR-T therapies.
Almazán et al. (Thu,) studied this question.