Efficient Preparation And Characterization Of Cytotoxic Activity Of Human Peripheral Blood Derived Natural Killer Cells

Objectives:Natural killer cells(NK cells)are pivotal effector lymphocytes,which are characterized by innate immune response to invade pathogenic microorganisms from outside and abnormal(mechanical damage,programmed apoptosis and canceration)cells in the body.They play an immune role in tumor immunosurveillance without specific activation and sensitization.At the same time,NK cell-based cytotherapy in clinical applications also shows bright prospects.However,the content of NK cells,around 5%-20%,is quite low in human peripheral blood(PB).In addition,the large-scale clinical application of NK cell-based tumor immunotherapy has been impeded partially due to the deficiency of large-scale,clinical-grade NK cells with high purity and activity,which has become the great obstacle in the fundmental research and clinical application.The purpose of this study is not only to employ the cytokine combinations for the large-scale preparation of NK cells and to fulfill the requirment in clinical treatment,but also to ensure that the obtained NK cells have high cellular vitality and cytotoxic activity.Contents:(1)To establish an efficient in vitro culture system for the preparation of human peripheral blood derived NK(PB-NK)cells based on cytokine combinations.(2)To evaluate the effect of cytokines on the expansion of PB-NK cells.(3)To verifiy the detailed function of cytokines for the generation of total PB-NK cells and the subsets with specific attributes,together with the distribution of T lymphocyte subsets.(4)To dissect the influence of the indicated cytokines to the cell cycle and apoptosis of PB-NK cells.(5)To evaluate the cytotoxic activity of the cytokine cocktail-programmed PB-NK cells against the tumor cell line K562 cells.Methods:(1)The enriched human PBMCs were cultured in basal medium with various combination of cytokine cocktail addition(IL-2,IL-12,IL-15,IL-18,IL-21)(as the experimental groups)or single IL-2 stimulation(as the control group),for the induction of PB-NK cells.At day 14 of the induction,PB-NK cells were detected and identified by flow cytometry and immunofluorescence staining.(2)The biological characteristics of PB-NK cells cultured with the aforementioned cytokine cocktails were verified by morphological observation and electron microscopy imaging.(3)The cell vitality of the indicated PB-NK cells were analyzed and evaluated by cell population doubling(Pd)assay,propidium iodide(PI)DNA staining,Annexin V/7-AAD double staining.(4)The PB-NK cells were co-cultured with K562 cells for 4 hours,and then the quantification of cytotoxic activity of the indicated PB-NK cells was accomplished based on the expression of CD 107a on the PB-NK cell surface and the remaining amount of K562 cells.Results:(1)Compared with the control group with single IL-2 stimulation,the experimental groups with the combination of other cytokines including IL-12,IL-15,IL-18 and IL-21 could promote PB-NK cell proliferation to varying degrees.We found that PB-NK cells induced by IL-2,IL-15,IL-18(denote as 3IL-15)and IL-2,IL-18,IL-21(denote as 3IL-21)could significantly promote the growth of PB-NK cells.(2)As to the percentage of induced PB-NK cells with the indicated cytokine cocktail,we found that a higher percentage of PB-NK cells in the 3IL-15 group with the expression of CD56,CD 16,and NKG2D compared with those in the 3IL-21 group.Meanwhile,the distribution of T cells in the 3IL-18 group was significantly lower than that in the 3IL-21 group,and the major subset of T lymphocytes was CD8+T cells rather than CD4+T cells.(3)with the aid of cell apoptosis and cell cycle analysis,we found that the proportion of PB-NK cells in the 3IL-15 group was lower but the PB-NK cells actived by 3IL-15 in the S phase and the G2/M phase was significantly higher than those by 3IL-21 stimulation.(4)According to the coculture experiment of PB-NK cells and K562 cells,we found that the expression of CD 107a on the surface of NK cells was up-regulated with the decrease of E:T ratio.Notably,PB-NK cells in the 3IL-15 group revealed better cytotoxic activity.Conclusion:In this study,with the aid of cytokine cocktail-based programming,we found the 3IL-15 or 3IL-18 combination showed higher efficacy upon PB-NK cell generation from PBMCs over the other 14 kinds of co-stimulating options.Therewith,by conducting multifaceted analyses including NK cell expansion,cellular phenotype,together with cytotoxic activity evaluated by the up-regulation of CD107a,we found the derived NK cells by 3IL-15 co-stimulation showed superiority in cytotoxicity in vitro.Collectively,on the bais of cytokine cocktails,we have established a high-efficient strategy for large-scale generation of NK cells from PBMCs,which will provide overwhiling new references for NK cell-based cytotherapy in future.