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

Moon Nian Lim^1*, Alvin Man Lung Chan², Pei Shan Poon², Kamal Shaik Fakiruddin¹，Noor Atiqah Fakharuzi¹, Qi Hao Looi², Ezalia Esa¹, Yuslina Mat Yusoff¹

1. Haematology Unit, Cancer Research Centre, Institute for Medical Research, National Institutes of Health (NIH), Ministry of Health Malaysia, 40170 Shah Alam, Selangor, Malaysia

2. My CytoHealth Sdn. Bhd, 5th Floor, Plaza Hamodal, Lot No. 15, Jalan 13/2, Section 13, 46200 Petaling Jaya, Selangor, Malaysia

*Corresponding author: Dr. Lim Moon Nian. This email address is being protected from spambots. You need JavaScript enabled to view it.

CITATION: Lim, M. N., Chan, A. M. L., Poon, P. S., Kamal Shaik, F., Fakharuzi, A., Looi, Q. H., Esa, E., & Mat Yusoff, Y. (2025). Combinations of interleukin 2/15/18 and interleukin 2/21 increased expansion and cytotoxicity of umbilical cord blood-derived natural killer cells against leukaemia. International Medical Research Journal, 11(2). https://doi.org/https://doi.org/10.63719/imrj.2025.11.02.005

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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 CD56^dim/bright and CD16^high/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 CD56^bright and cytotoxic CD56^dim 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.

KEYWORDS: cord blood, natural killer cell, cancer immunotherapy, cytokine, interleukin