A Study On Ex Vivo Expansion And Function Of NK Cells From Human Peripheral Blood By Cytokines IL2, 15 And Autologous DC Cells

[Objective] Adoptive immunotherapy using allogeneic natural killer (NK) cells may prove useful in recipients after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) and in patients with solid tumor. But it has been limited by the inability to obtain sufficient numbers of pure NK cells. The goal of our study was to optimize the expansion of high purity NK cells from human peripheral blood by cytokines IL2,15 and autologous DC Cells, and evaluate the changes in biological functions after ex vivo expansion.[Methods] First, we isolated CD14+cells and NK cells from PBMNC by using miniMACS (Magnetic cell-selection) (Miltenyi Biotec, Germany). DCs were induced f rom CD14~+cells by cytokines GM-CSF and IL4 for 5 days. The isolated NK cells were cultured in SCGM supplemented with 5% human AB serum and combination of interleukin IL2 and IL15 for 5 days, then DCs come from CD14~+cells was add to NK cells at different ratio. Cultures were fed with fresh media and cytokines every 3 days, and were evaluated for cell expansion, phenotype, and biological functions at the end of the culture period. The biological functions of NK cells were detected by 3 ways:(1) cytotoxicity to target K562 cells; (2) IFN-y, perforin and granzyme B mRNA expressions were assayed by realtime-PCR; (3)Expression of KIR3DL1 (CD158e) and NKG2D of NK cells were analyzed by flow cytometry. stimulate NK cells proliferation and strengthen NK cells vitality. When NK/DCs ratio were 10 to 1,5 tol,2 to l,or 1 to 1 but separated by transwell, cells were expanded 168±64.4,170.5±82.6,244.8±148 and 70.8±17.5 fold, respectively. But when NK/DCs ratio was 2 to 1, CD3~-CD56+NK cells purity dropping significantly, while in other groups was over 92%. The cytotoxicity of expanded NK cells cultured with DCs was significantly higher than the no DCs control, and NK/DCs ratio 10:1 group was slightly higher than other groups. The expressions of IFN-Y, perforin and granzyme B mRNA of expanded NK cells was significantly higher than the starting population (P<0.01). In NK/DCs ratio≤2:1 groups, IFN-Y mRNA expression level were higher, and in NK/DCs ratio≥5:1 group, perforin and granzyme B mRNA expression level were higher. After culture with cytokins for 15 days, CD158e~+NK cells number drop 10%, while there was no change in NKG2D expression. Using a luciferase reporter assay, we found miRNA 146b maybe regulate KIR3DL1 expression.[Conclusion]:Our data suggest that high purity NK cells could be efficiently expanded in culture with IL2+IL15 and autologous DCs at 10:1 NK/DCs ratio, and its biological functions were enhanced in this condition.The miRNA 146b maybe regulate KIR3DL1 expression..