Phenotypic characteristics of natural killer cells after their generation and activation in vitro

The development of new effective methods for in vitro activation and culturing of natural killer (NK) cells led to advances in adoptive cancer immunotherapy. Our aim was to evaluate expression of activation markers on natural killer cells after cultivation and activation in vitro using flow cytometry. Peripheral blood mononuclear cells were isolated from heparinized blood of 10 donors in a density gradient and were cultivated in RPMI-1640 medium (Sigma-Aldrich, Germany) supplied with IL-2 and TMDK562-15 feeder cells for 14 days. Phenotyping of the NK cells was carried out using an Image Stream MkII (Luminex) flow cytometer. We used a cytometry panel containing 10 monoclonal antibodies against СD45, СD3, СD56, СD16, and against cell activation markers, i.e., CD38/HLA-DR/CD25 (BD Biosciences). Cytotoxic activity of the donor-derived NK cells was tested towards cancer cell lines AsPC-1, A-549, MDA-MB-231 and PC-3 on 14th day of cultivation. The effector-to-target ratios were 5:1 and 10:1. The target cells were exposed to effector cells for 12 hours. Statistical analysis was performed using MS Excel 2016, TIBCO STATISTICA 13. Prior to cultivation, the percentage of natural killer cells in mononuclear cells was 12%. Of those, activation markers (CD38/HLA-DR/CD25) were expressed, accordingly, on 7.6%, 0.7%, 0% cells, Cultivation of mononuclear cells for 14 days resulted in activation and expansion of distinct lymphocyte subpopulations. NK cell counts increased 390-fold on 14th day of cultivation, with percentage of natural killers reaching 86.5%. On the 14th day of cultivation, expression levels of activation markers (CD38/HLA-DR/CD25) were 34.7%, 36.5%, 5%, accordingly. Studying the cytotoxic activity of NK cells against different cancer cell lines showed that, after 12-h incubation of activated cells with target cells, 100% of cancer cells have been lysed. The donor cells were viable by 98%. After 7 days of cultivation, the cell viability decreased to 81%; at the end of cultures, their viability was 95%. The suggested cultivation technique for mononuclear cells cause an increase of proliferative activity and expression of activation markers of NK cells, thus allowing to maintain high viability of the cell population. High functional activity of cultured NK cells was confirmed in cytotoxicity tests against cancer cells.

1. Abakushina E.V. Recombinant cell line TMDK562-15 capable of activation and proliferation of human NK-cells. Patent RU 2803178 C1, 07.09.2023.

2. Abakushina E.V. Flow cytometry method for evaluation of NK-cells and their activity. Klinichescheskaya laboratornaya diagnostika = Russian Clinical Laboratory Diagnostics, 2015, Vol. 60, no. 11, pp. 37-44. (In Russ.)

3. Abakushina E.V., Kozlov I.G. NK-cells immunotherapy in treatment of oncological diseases. Rossiyskiy immunologicheskiy zhurnal = Russian Journal of Immunology, 2016, Vol. 10, no. 2, pp. 131-142. (In Russ.)

4. Abakushina E.V., Marizina U.V., Kaprin A.D. Morphofunctional Characteristics of human lymphocytes after in vitro. Byulleten eksperimentalnoy biologii i meditsiny = Bulletin of Experimental Biology and Medicine, 2016, Vol. 161, no. 5, pp. 731-735. (In Russ.)

5. Bald T., Krummel M.F., Smyth M.J., Barry K.C. The NK cell–cancer cycle: advances and new challenges in NK cell–based immunotherapies. Nat. Immunol., 2020, Vol. 21, no. 8, pp. 835-847.

6. Kiessling, R., Klein E., Wigzell H. “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur. J. Immunol., 1975, vol. 5, no. 2, pp. 112-117.

7. Klimpel G.R., Niesel D.W., Klimpel K.D. Natural cytotoxic effector cell activity against Shigella flexneri-infected HeLa cells. J. Immunol., 1986, Vol. 136, no. 3, pp 1081-1086.

8. Lamers-Kok N., Panella D., Georgoudaki A.M., Liu H., Özkazanc D., Kučerová L., Duru A.D., Spanholtz J., Raimo M. Natural killer cells in clinical development as non-engineered, engineered, and combination therapies. J. Hematol. Oncol., 2022, Vol. 15, no. 1, 164. doi: 10.1186/s13045-022-01382-5.

9. Macfarlan R.I., Burns W.H., White D.O. Two cytotoxic cells in peritoneal cavity of virus-infected mice: antibody-dependent macrophages and nonspecific killer cells. J. Immunol., 1977, Vol. 119, no. 5, pp. 1569-1574.

10. Melaiu O., Lucarini V., Cifaldi L., Fruci D. Influence of the Tumor Microenvironment on NK Cell Function in Solid Tumors. Front. Immunol., 2019, Vol. 10, 3038. doi: 10.3389/fimmu.2019.03038.

11. Ortaldo J.R., Oldham R.K., Cannon G.C., Herberman R.B. Specificity of natural cytotoxic reactivity of normal human lymphocytes against a myeloid leukemia cell line. J. Natl Cancer Inst., 1977, Vol. 59, no. 1, pp. 77-82.

12. Shimasaki N., Jain A., Campana D. NK cells for cancer immunotherapy. Nat. Rev. Drug Discov., 2020, Vol. 19, pp. 200-218.

13. Voigt J., Malone D.F.G., Dias J., Leeansyah E., Björkström N.K., Ljunggren H.-G., Gröbe L., Klawonn F., Heyner M., Sandberg J.K. Proteome analysis of human CD56neg NK cells reveals a homogeneous phenotype surprisingly similar to CD56dim NK cells. Eur. J. Immunol., 2018, Vol. 48, pp. 1456-1469.