Enhanced Cytotoxicity Of Natural Killer Cells By Mature Dendritic Cells Pulsed With Apoptotic Lung Adenocarcinoma Cells

Dendritic cells (DCs) are potent antigen presenting cells (APCs), and DC maturation is now accepted as a crucial component of the induction of most adaptive immune response. Recent interest has focused on interactions between natural killer (NK) cells and DCs. There is considerable controversy as to whether necrotic (lysate) or apoptotic tumor cells serve as the superior source of multiple tumor associated antigens (TAAs) to pulse DCs for immunotherapeutic application.ObjectiveTo prepare DCs from healthy human peripheral blood, which were induced mature by pulsed with apoptotic tumor cells comparing to necrotic tumor cells (freeze-thawed lysates, heat shocked lysates, DDP-induced lysates); and investigate the enhanced cytotoxicity of NK cells by mature DCs in vitro.Methods1. Peripheral blood monocyte-derived DCs culture and identification.Peripheral blood mononuclear cells (PBMC) were isolated from the peripheral blood of healthy donors by density gradient centrifugation. Briefly, PBMC were incubated in tissue culture flasks in 5% CO2 at 37℃in RPMI 1640 medium supplemented with 10% FBS. After incubation 2h, non-adherent cells were removed gently, and adherent cells were cultured for 7 days in mixed medium containing rhGM-CSF 1000U/ml and rhIL-4 50ng/ml to generate immature DCs. In 7th day, no adherent cells were harvested and calculated using trypan blue staining. On day 6, immature DCs were incubated with apoptotic tumor cells and necrotic tumor cells (freeze-thawed tumor lysates, heat-shocked tumor lystats, DDP-induced tumor lysates) for 24h. On day 7 of their preparation to assess the phenotype of immature or mature DCs, DCs were collected, stained and analyzed by flow cytometry. Antibodies used to phenotype of the cells were anti-HLA-DR-FITC, anti-CD80-FITC. For staining, 10⁵ cell were suspended in 100μl of PBS and were incubated with 10μl of antibodies for 20 min on ice. Flow cytometric analysis was performed on a FACS-calibur.2. Cytokine assaysThe supermatants of DCs were collected, and the concentrations of IL-12, were measured by using standard ELISA. Cytokine release was reported as mean pictograms of IL-12±SD from triplicate samples.3. Cytotoxicity assayThe DCs or its supernatants were collected and cocultured with autologous NK cells for 12h. Standard 4h-⁵¹Cr release assays were used to determine the cytotoxicity of NK cells in vitro. A suspension of single K562 cell labeled with ⁵¹Cr 1×10⁴/well were incubation with NK cells at E:T ratio ranging from 5:1 to 20:1 in round-bottomed 96-well micro titer plates; After 4h, removed supernatants, and calculated by y-scintilator.4. Neutralizing experimentIL-12mAb and IFN-γmAb was cocultured with the supernatants of mature DCs before cytotoxicity assay.5. StatisticsDates were analyzed by the student t test, whereby a P of less than 0.05 indicated that the value of test sample was significantly different from that of the relevant controls.ResultsA larger number of DCs were obtained from peripheral blood of healthy donors and showed specific morphologic and phenotypic characteristics. The DCs were strongly positive for HLA-DR and expressed the costimulatory molecules CD80 after pulsed with apoptotic tumor cells. Mature DCs culture supernatants contained 289pg/ml of IL-12 at day 7. A549 apoptosis induced by DDP in vitro can induced DCs mature, meanwhile the later have specific phenotype and cytokines. Both matured DCs and its supernatants could stimulate NK cells markedly to lyses target cells in standard 4h-⁵¹Cr release assay. And matured DCs could secrete IL-12 and other cytokines to enhance NK cytolysis against K562. Conclusion1. The mature dendritic cells can be obtained by pulsed with apoptotic lung adenocarcinoma cells.2. The killing ability of NK cells activated by mature DCs was strong,the same as it supernatant.3. The mature DCs could secrete IL-12 and other cytokine to enhance NK cells lysis against K562.