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

• Published in: Medit͡s︡inskai͡a︡ immunologii͡a
• Main Authors: Ruslan Vladimirovich Zhurikov, Elena Vyacheslavovna Abakushina, Kamilla Dmitrievna Balysheva, Maria Vladimirovna Beketova, Sergey Aleksandrovich Rumyantsev
• Format: Journal Article
• Language: Russian
• Published: St. Petersburg branch of the Russian Association of Allergologists and Clinical Immunologists 01.08.2019
• Subjects: adoptive therapy; natural killer cells; nk-cell cultivation; nk-cells markers; phenotype
• ISSN: 1563-0625; 2313-741X
• DOI: 10.15789/1563-0625-PCO-3068

Introduction. The development of new effective methods of activation and cultivation of natural killer cell in vitro led to advances in adoptive cancer immunotherapy. Aim. The aim of this study was to evaluate expression of activation markers on natural killer cells after cultivation and activation in vitro using flow cytometry. Material and methods. Periferal blood mononuclear cells were isolated from heparinized blood of 10 patients in a density gradient and were cultivated in RPMI-1640 medium (“Sigma-Aldrich”) with the addition of IL-2 and feeder cells TMDK562-15 for 14 days. Phenotyping of natural killer cells was carried out using an Image Stream MkII («Luminex») flow cytometer. We used a cytometry panel of 10 monoclonal antibodies against CD45/3/56/16 and against activation markers such as CD38/HLA-DR/CD25 (“BD Biosciences”). The cytotoxic activity of natural killers from patients against cancer cell lines AsPC-1, A-549, MDA-MB-231 and PC-3 was tested on 14th day of cultivation. Effector:target ratios were 5:1 and 10:1. Target cells were exposed to effector cells for 12 hours. Statistical analysis was carried out using “MS Excel 2016”, TIBCO STATISTICA 13. Results. At the beginning of cultivation, the percentage of natural killer cells in mononuclear cells was 13%. Expression levels of activation markers such as CD38/HLA-DR/CD25 were 7,6/0,7/0% accordingly. 14-day cultivation of mononuclear cells resulted in activation and expansion of certain subpopulations of lymphocytes.  Natural killer cells count increased 390-fold on 14th day of cultivation, percentage of natural killers was 86,5%. On the 14th day of cultivation, expression levels of activation markers such as CD38/HLA-DR/CD25 were 34,7/36,5/5% accordingly.  A study of the cytotoxic activity of natural killers against different cancer cell lines showed that after incubation of activated cells with target cells for 12 hours 100% of the latter were lysed. Donor cell viability was 98%; after 7 days of cultivation cell viability decreased to 81%; at the end of cultivation, cell viability was 95%.   Conclusion. The suggested method of cultivation of mononuclear cells increased proliferative activity and expression of activation markers of natural killer cells while allowed to maintain high viability of the cell population. High functional activity of cells after cultivation was confirmed in cytotoxicity tests against cancer cells.