Evaluation of the effects of different interleukin combinations on proliferation and cytotoxicity of natural killer cells

Background. One of the approaches to expand natural killer (NK) and NKT cells for subsequent CAR (chimeric antigen receptor) therapy is cultivation in the presence of certain cytokines and/or feeder cells.

Aim. To evaluate the effect of combinations of interleukin (IL): IL-2 + IL-15, IL-2 + IL-15 + IL-21 or IL-2 alone on NK and NKT cells during long-term cultivation in the presence of autologous feeder cells, which were non-irradiated T lymphocytes activated by monoclonal antibodies (mAbs).

Materials and methods. Peripheral blood and leukocyte-platelet concentrate were used as a source of peripheral blood mononuclear cell. After peripheral blood mononuclear cell isolation, the lymphocyte culture was activated using adsorbed mAbs to CD3 and CD28 receptors. Then, activated lymphocytes were cultured in the presence of IL-2 + IL-15, IL-2 + IL-15 + IL-21, or IL-2 alone, periodically assessing the increase in the total number of cells, expression of surface markers, and cytotoxic activity.

Results. Using the combination of IL-2 + IL-15 + IL-21, the greatest increase in cells and maximum cytotoxicity were observed; the proportion of NKT cells (CD3⁺CD56⁺) by the end of the cultivation was also the highest compared to other cultivation modes and amounted to 59 %. In the presence of a combination of IL-2 and IL-15, lower cytotoxicity of the cultured lymphocytes was determined compared to other experimental options, which may be due to exhaustion of the cell population. Based on the literature data, the addition of IL-21 to the culture medium probably neutralizes this negative effect of IL-15.

Conclusion. Based on the dynamics of proliferation, receptor expression and cytotoxicity of cultured lymphocytes, it can be concluded that the cytokine combination consisting of IL-2, IL-15 and IL-21 is most effective for enriching the NKT cell population upon stimulation with mAb and in the presence of autologous activated T lymphocytes. For the most effective generation of the NK cell population, it is necessary to use other feeder cells, probably of tumor origin, or other expansion protocols.

1. Poorebrahim M., Abazari M.F., Sadeghi S. et al. Genetically modified immune cells targeting tumor antigens. Pharmacol Ther 2020;214:107603. DOI: 10.1016/j.pharmthera.2020.107603

2. Wang X., Yang X., Yuan X. et al. Chimeric antigen receptor-engineered NK cells: new weapons of cancer immunotherapy with great potential. Exp Hematol Oncol 2022;11(1):85. DOI: 10.1186/s40164-022-00341-7

3. Abramson J.S., Palomba M.L., Gordon L.I. et al. Lisocabtagene maraleucel for patients with relapsed or refractory large B-cell lymphomas (TRANSCEND NHL 001): a multicentre seamless design study. Lancet 2020;396(10254):839–52. DOI: 10.1016/S0140-6736(20)31366-0

4. Maalej K.M., Merhi M., Inchakalody V.P. et al. CAR-cell therapy in the era of solid tumor treatment: current challenges and emerging therapeutic advances. Mol Cancer 2023;22(1):20. DOI: 10.1186/s12943-023-01723-z

5. Wang E., Cesano A., Butterfield L.H. et al. Improving the therapeutic index in adoptive cell therapy: key factors that impact efficacy. J Immunother Cancer 2020;8(2):e001619. DOI: 10.1136/jitc-2020-001619

6. Liu Z., Zhou Z., Dang Q. et al. Immunosuppression in tumor immune microenvironment and its optimization from CAR-T cell therapy. Theranostics 2022;12(14):6273–90. DOI: 10.7150/thno.76854

7. Shah N., Li L., McCarty J. et al. Phase I study of cord blood-derived natural killer cells combined with autologous stem cell transplantation in multiple myeloma. Br J Haematol 2017;177(3):457–66. DOI: 10.1111/bjh.14570

8. Khawar M.B., Sun H. CAR-NK cells: from natural basis to design for kill. Front Immunol 2021;12:707542. DOI: 10.3389/fimmu.2021.707542

9. Peng L., Sferruzza G., Yang L. et al. CAR-T and CAR-NK as cellular cancer immunotherapy for solid tumors. Cell Mol Immunol 2024;21(10):1089–108. DOI: 10.1038/s41423-024-01207-0

10. Shimasaki N., Jain A., Campana D. NK cells for cancer immunotherapy. Nat Rev Drug Discov 2020;19(3):200–18. DOI: 10.1038/s41573-019-0052-1

11. Wolf B.J., Choi J.E., Exley M.A. Novel approaches to exploiting invariant NKT cells in cancer immunotherapy. Front Immunol 2018;9:384. DOI: 10.3389/fimmu.2018.00384

12. Peighambarzadeh F., Najafalizadeh A., Esmaeil N. et al. Optimization of in vitro expansion and activation of human natural killer cells against breast cancer cell line. Avicenna J Med Biotechnol 2020;12(1):17–23.

13. Sivori S., Pende D., Quatrini L. et al. NK cells and ILCs in tumor immunotherapy. Mol Aspects Med 2021;80:100870. DOI: 10.1016/j.mam.2020.100870

14. Koehl U., Brehm C., Huenecke S. et al. Clinical grade purification and expansion of NK cell products for an optimized manufacturing protocol. Front Oncol 2013;3:118. DOI: 10.3389/fonc.2013.00118

15. Conlon K.C., Lugli E., Welles H.C. et al. Redistribution, hyperproliferation, activation of natural killer cells and CD8 T cells, and cytokine production during first-in-human clinical trial of recombinant human interleukin-15 in patients with cancer. J Clin Oncol 2015;33(1):74–82. DOI: 10.1200/JCO.2014.57.3329

16. McMichael E.L., Jaime-Ramirez A.C., Guenterberg K.D. et al. IL-21 enhances natural killer cell response to cetuximab-coated pancreatic tumor cells. Clin Cancer Res 2017;23(2):489–502. DOI: 10.1158/1078-0432.CCR-16-0004

17. Sivonen M., Sirviö K.A., Wojciechowski S. et al. Cytokines impact natural killer cell phenotype and functionality against glioblastoma in vitro. Front Immunol 2023;14:1227064. DOI: 10.3389/fimmu.2023.1227064

18. Koh E.K., Lee H.R., Son W.C. et al. Antitumor effects of NK cells expanded by activation pre-processing of autologous feeder cells before irradiation in colorectal cancer. Oncol Lett 2023;25(6):232. DOI: 10.3892/ol.2023.13818

19. Troshina E.A. The role of cytokines in the processes of adaptive integration of immune and neuroendocrine reactions of the human body. Problemy endokrinologii = Problems of Endocrinology 2021;67(2):4–9. (In Russ.). DOI: 10.14341/probl12744

20. Leonard W.J., Wan C.K. IL-21 Signaling in immunity. F1000Res 2016;5:F1000 Faculty Rev-224. DOI: 10.12688/f1000research.7634.1

21. Shanley M., Daher M., Dou J. et al. Interleukin-21 engineering enhances NK cell activity against glioblastoma via CEBPD. Cancer Cell 2024;42(8):1450–66.e11. DOI: 10.1016/j.ccell.2024.07.007

22. Liu Y., Dang Y., Zhang C. et al. IL-21-armored B7H3 CAR-iNKT cells exert potent antitumor effects. iScience 2023;27(1):108597. DOI: 10.1016/j.isci.2023.108597

23. Felices M., Lenvik A.J., McElmurry R. et al. Continuous treatment with IL-15 exhausts human NK cells via a metabolic defect. JCI Insight 2018;3(3):e96219. DOI: 10.1172/jci.insight.96219