More than IL-2: Optimizing feeder-free NK cell function through cytokine synergy

Abstract Introduction Natural killer (NK) cells represent a promising therapeutic immune cell sources for the treatment of hematological malignancies. Unlike allogeneic CAR-T cell therapies, which are associated with risks such as graft vs. host disease (GvHD), NK cell-based therapies offer a safer, allogeneic “off-the-shelf” alternative. However, NK cells exhibit inherent biological variability between individual donors, which may negatively impact their functionality. Therefore, strategies to harness primary NK cells for clinical applications need to be improved. The aim of our study is to identify the most convenient and effective feeder-free ex vivo culture conditions for expanding NK cells without impairing their cytolytic activity. We conducted a comprehensive priming screen using different cytokines and their combinations. Our in-depth analysis identified optimal interleukin (IL) combinations, such as IL-2, IL-15, and IL-21, which significantly enhanced cell expansion, activation status, and the antitumor activity of primary NK cells. Methods Peripheral blood NK cells were isolated from healthy donors using magnetic based untouched NK cell isolation method. Isolated cells were cultured for four weeks in Miltenyi NK MACS® medium, supplemented with one of three interleukin combinations: IL-2 + IL-15, IL-15 + IL-21, or IL-2 + IL-15 + IL-21. The study was conducted under feeder-free conditions. NK cell cytotoxicity was evaluated weekly using a Calcein AM–based assay, against chronic myeloid leukemia cell line K562 and multiple myeloma cell line MM.1S serving as target cells. Degranulation was assessed by flow cytometry using CD107a surface expression. NK phenotyping using flow cytometry was evaluated each week. In parallel, cell proliferation and fold expansion were also monitored weekly to assess the impact of each cytokine combination in supporting NK cell growth and function over time. Results NK cells isolated from healthy donors exhibited inherent variability in baseline cytotoxicity and expansion potential. Throughout the four-week feeder-free culture period, cytotoxic capacity peaked between weeks 3 and 4 (days 14 to 21). At week 3, NK cells cultured with IL-2 + IL-15 + IL-21 showed significantly higher cytotoxicity compared to those cultured with IL-2 + IL-15 (p 0.05) against K562 cells. Against the MM.1S cell line, there were statistically significant differences between cytokine conditions (p 0.05), with both IL-2 + IL-15 + IL-21 and IL-15 + IL-21 resulting in superior cytotoxic responses compared to IL-2 + IL-15. Peak expansion was observed around day 14, with IL-2 + IL-15 supporting the lowest fold expansion relative to the other two conditions. Furthermore, degranulation capacity of NK cells upon tumor cell sensitization, was higher in IL-15–containing cultures, particularly in the IL-15 + IL-21 and IL-2 + IL-15 + IL-21 groups, compared to IL-2 + IL-15 alone. Conclusions To enhance ex vivo NK cell expansion and activation, ongoing efforts are directed at optimizing not only cytokine combinations but also their concentrations and timing. Although IL-2 + IL-15 remains the standard cytokine combination for feeder-free NK cell culture, our findings suggest alternative priming cytokine combinations beyond the classical IL-2 or IL-15 cytokines. In particular, the use of IL-15 + IL-21 in the presence or absence of IL-2 provides superior outcomes. These combinations significantly improved NK cell cytotoxicity, degranulation, and proliferation, highlighting their potential for clinical-scale, feeder-free NK cell production with optimal functional properties across diverse donors.