A Study On Ex Vivo Expansion And Functions Of Highly Purified NK Cells From Human Peripheral Blood

[Objective] Adoptive immunotherapy using allogeneic natural killer (NK) cellsmay 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 and evaluate the changes in biological functions after Ex vivo Expansion.[Methods] First, we isolated NK cells from PBMNC by using miniMACS(Magnetic cell-selection) and NK Cell Isolation Kit Ⅱ (Miltenyi Biotec, Germany). The isolated cells were cultured in SCEM (Stemline Hematopoietic Stem Cell Expansion Medium, Sigma) supplemented with 10% human AB serum and different combinations of interleukin (IL)-2 and/or IL12, IL15 for 15 days. 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) the level of IFN-γ in the supernatants of NK cells culture were evaluated by ELISA; (3) perform and granzyme B mRNA expressions were assayed by competitive quantitative RT-PCR.[Results] The results showed that in group IL2+IL15 and IL2+IL15+IL12, cellswere expanded 50.46 ± 4.31 and 53.95 ± 6.72 fold, respectively, much more higher than others (P<0.01), but no significant difference between them (P>0.05). And the purity of CD3~- CD56~+NK cells was over 94% in all groups except the control. The cytotoxicity of expanded NK cells cultured with cytokines wassignificantly higher than the starting population at different E:T ratio (P<0.01), although the cytotoxicity of IL2+IL15+IL12 group was slightly higher than that of IL2+IL15 group, without significant difference (P>0.05). There was great increase in IFN-γ levels in the supernatants of NK cells culture in the presence of cytokines; IL2+IL15+IL12 group and IL2+IL12 group was significantly higher than others (P<0.01). The expressions of perform and granzyme B mRNA of expanded NK cells cultured with cytokines was significantly higher than the starting population (P<0.01), and with one accord to cytotoxicity.[Conclusion]: Our data suggest that high purity of NK cells could be efficientlyexpanded in culture with IL2+IL15, and its biological functions were enhanced in this condition..