Combinations of interleukin 2/15/18 and interleukin 2/21 increased expansion and cytotoxicity of umbilical cord blood-derived natural killer cells against leukaemia

Abstract

The use of cytokines to expand natural killer (NK) cells offers both promising opportunities and notable challenges in the field of immunotherapy. The selection and optimisation of cytokines are critical to achieving efficient expansion of NK cells. Therefore, this study aimed to evaluate different cytokine combinations for the expansion and activation of cord blood-derived NK cells. Briefly, various interleukins (IL) combinations were tested: Group A (100 IU/mL IL-2 only), Group B: (100 IU/mL IL-2 and 100 IU/mL IL-15), Group C (100 IU/mL IL-2, 100 IU/mL IL-15 and 20 ng/mL IL-18) and Group D (100 IU/mL IL-2, 10 ng/mL IL-21). The NK cells were subsequently characterised by flow cytometry for CD56dim/bright and CD16high/lo subpopulations, as well as for their activating and inhibitory receptors. The cytotoxic effect of the different NK cell groups towards luciferase-expressing leukaemia cell line (Nalm6) and the secretion levels of tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) were also assessed. NK cells treated with various combinations of cytokines demonstrated a higher proliferation rate and increased expression of CD56+CD16+ compared to the untreated (UT) group. Further subpopulation analysis revealed that groups B, C and D promoted the expansion of both cytokine-producing CD56bright and cytotoxic CD56dim NK cell subsets. Notably, only groups C and D exhibited significantly elevated levels of TNF-α and IFN-γ secretion. These groups also showed higher specific lysis (%) of Nalm6 cells (p<0.0001). The combination of IL-2 with either IL-15 and IL-18 or IL-21 synergistically enhanced the expansion, activation, and cytotoxic function of NK cells, highlighting their potential application in immunotherapeutic strategies.