Effect Of Dendritic Cells On In Vitro Expansion And Function Of Autologous Natural Killer Cells

Objects: To investigate the effect and mechanism of dendritic cells(DCs) on in vitro expansion and function of autologous natural killer(NK) cells.Methods: We expanded NK cells from peripheral blood mononuclear cells(PBMCs) of healthy volunteers. PBMCs were cultured in stem cell growth medium(SCGM) or RPMI1640, supplemented with different cytokine cocktail of group A: rhIL-2, group B:rhIL-2, rhIL-15 and SCF, group C: rhIL-2, rhIL-15, SCF and CD3, respectively. After 7 days, PBMCs from the same health volunteers who donated twice(first for NK cell cultures and then for DCs cultures) was incubated with A23187 in RPMI1640 to induce DCs. DCs was identified by the expression of CD1a and CD83 by flow cytometry. NK cells under different cytokines and medium was cultured with autologous DCs in the ratio of 1 to 1 or 1 to 5.Totol cells of every group were counted on days 7, 14and 21 to calculate the expansion of NK cells;Flow cytometry was used to assay the expression of CD3, CD16/56 on the surface of NK cells of different groups on days 7, 14and 21;Expanded NK cells' cytotoxic function against K562 cells was assayed by MTT method. TNF- α and IL-12p70 were detected in culture supernatants by sandwich ELISA.Results: On condition of SCGM, no difference in the expansion rates or cytotoxic function of group A, B or C on the culture medium of SCGM(P>0.05). But NK cells expansion and cytotoxic capacity were improved after mixed with autolgous DCs. Furthermore, when mixed DCs with NK cells , the higher ratio of DCs to NK cells it was ,the higher expansion and cytotoxic capacity it was. On day 14, the expansion rate of group A , group A5:1, group A1:1 wasl7.26 ± 1.58 , 29.43 ± 3.13 and 22.23 ± 2.91,respectively.The expansion rate of group A5:1 was much higher than that of the other two groups (p<0.05). The expression rate of CD3-, CD56+/CD16+ on the surface of group A, group A5:1,, group A1:1 was (36.8±5.1) , (60.4±8.1), and (53.4±8.1) % while the expression rates of group A5:1 was the highest(P<0.05). The cytotoxic capacity against K562 cells of group A, group A5:1, group A1:1 was (68.5±3.4)%, (88.7±4.4) % and (82.4±6.3) % .The cytotoxic capacity of group As:i was higher than that of group A or group A1:1(P<0.05). When NK cells were cultured in RPMI1640, the expansion rates of every group was lower than 7.Compared with cells that were cultured in SCGM, the difference was significant(P<0.05). TNF- α and IL-12p70 level in culture supernatants when DCs and NK cells were mixed in the ratio of 5 to 1 were much higher than those inculture supernatants of DC^ NK cells alone or in culture supernatants when DCs and NK cells were mixed in the ratio of 1 to l(P<0.05).Conclusion: Human NK cells in vitro culture can be expanded effectively in SCGM. The expansion and cytotoxic capacity of NK cells can be improved by DCs and it depended on the mixed ratio of DCs to NK cells.The elevated expansion of NK cells by DCs is relative to IL-12 produced by DCs.The enhanced cytotoxic capacity of NK cells is associated with TNF- a secreted by NK cells.