Fundamental Study On The Cytotoxicity Of The Freeze-thaw Tumor Cell Lysate-loaded DC Co-cultured With CIK Cells Against RB-Y79Cells

Objectives:In our study, peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured dendritic cells (DC) and cytokine-induced killer (CIK) cells with different cytokines, then loaded tumor cell lysate on DC and co-cultured with CIK cells.(1) To observe the changes of the cell morphology, cell phenotype and proliferation when the complete tumor antigen-loaded DC (DC-Ag) co-cultured with CIK cells.(2) To investigate the cytotoxicity of DC-Ag-CIK cells against RB-Y79cells, and the synergetic antitumor activity of DC-Ag-CIK cells combined with Carboplatin against RB-Y79cells.(3) To explore the possible mechanism of their synergetic cytotoxicity which DC-Ag-CIK combined with Carboplatin against RB-Y79cells.Methods:(1) We observed the proliferation and biological activity of tumor antigen-loaded DCand CIK cells in vitro. PBMCs from healthy volunteers were cultured DC and CIK cells with different cytokines. DCs were incubated in the presence of the tumor lysates from the day3of DC maturation and co-cultured with CIK cells on day7, which divided into CIK group, DC-CIK group and DC-Ag-CIK group. On day7, observing the cell morphology and proliferation of DC and CIK cells by inverted microscope in the three groups. Cell phenotype of DC and CIK cells were analysed using flow cytometry on day7and day15. IL-6and IL-10levels in the media culture were quantitatively measured by using a CBA Cytokine Kit according to manufacture’s instructions on day15in three groups(2) Detection of antitumor activity assay. The cytotoxic activity of the Carboplatin against RB-Y79cells and the hTERT-RPE1cells (normal retinal pigment epithelial cells) were checked by PI assay; the cytotoxicity of CIK cells, DC-CIK cells and DC-Ag-CIK cells against RB-Y79cells, hTERT-RPE1cells and RB-resistant (RB-R) cells were checked by CFSE/PI assay; as well as effector cells combined with Carboplatin against RB-Y79cells and the cytotoxicity CIK cells against RB-Y79cells in different culture media were also checked by CFSE/PI assay; detecting the cytotoxicity of Carboplatin and DC-Ag-CIK cells against RB-Y79cells in different order application.(3) A RB-Y79-GFP stable cell line was created by transfection with the GFP fluorescent protein. Dynamic antitumor processes were observed using a Leica immunofluorescence microscope which Carboplatin and/or DC-Ag-CIK cells against the RB-GFP cells. The expression of Annexin V protein were checked by using immunofluorescence staining and flow cytometry when Carboplatin and/or DC-Ag-CIK cells against RB cells.Results:(1) DC cells showed a typical dendritic-like structure in the cytokines medium, and CIK cells showed depicting irregular shape and gathered totether. On day15, the CIK cell number of DC-Ag-CIK group and DC-CIK group was significantly higher than the CIK group (P<0.01). IL-6levels increased when CIK cells were growth in presence of DC cells or DC-Ag respectively, compared with levels of secreted IL-6in CIK culture alone (P<0.01). In contrast, secreted IL-10level was lower in DC-Ag-CIK cells co-culture compared with CIK or CIK co-cultured with DC cells (P<0.01). After7days in culture, a FCM assay detected values for the CD80, CD83and CD86significantly higher compared with the cells on day3, respectively (P<0.01). On day15, the cell phenotype of CIK cells in each groups were significantly higher than the CIK cells on day7(P<0.01), On day15, the proportion of CD3+CD56+and CD3+CD8+cells were higher when the CIK cells were co-cultured with DC-Ag cells (P<0.01).(2) Higher effector cells:target cells (E:T) ratios resulted in significantly higher cytotoxicity throughout all experimental conditions, the cytotoxicity was significantly higher when they were in the presence of DC-Ag than CIK cells and DC-CIK cells (P<0.05). Effector cells had no cytotoxicity against normal retinal pigment epithelial cells hTERT-RPE1the cells. No significant cytotoxicity was detected on hTERT-RPE1cells in all the assayed groups (P>0.05). No significant differences were found on the CIK cytotoxic activity on RB-Y79cells between any of the3culture medium (P>0.05). Effector cells cytotoxicity on RB-Y79cells was higher when Carboplatin was present in the culture than the cytotoxicity of the drug alone or the effector cells alone (P<0.01). Effector cells cytotoxicity on Carboplatin resistant RB cells higher when Carboplatin was present in the culture than the cytotoxicity of the effector cells alone (P<0.01). A significantly greater effect (71.16±4.65%) was observed in the double Carboplatin incubation alternated with DC-Ag-CIK cells than in any of the other comparable treatments (P<0.01).(3) The green fluorescence disappeared when the RB-GFP cells were dead, at the same time the dead RB cells were stained red by PI. Apoptotic RB cells exposed for a shorter time to DC-Ag-CIK cells were added to the Carboplatin than Carboplatin alone or DC-Ag-CIK cells alone. This percentage of early apoptotic cells was higher when RB cells were co-cultured in the presence of DC-Ag-CIK cells than DC-CIK cells and CIK cells (P<0.01). The percentage of Annexin V positive cells increased by preconditioning RB with Carboplatin and incubated with DC-Ag-CIK than Carboplatin alone or DC-Ag-CIK cells alone (P<0.01).Conclusions:(1) CIK maturation and differentiation were enhanced by co-culturing CIK with DC-Ag. Moreover, the co-culture improved CIK proliferation rate by increasing IL-6and decreasing IL-10levels in the culture medium.(2) The DC-Ag-CIK cells were substantially more cytotoxic against RB cells than DC-CIK and CIK cells alone. This increment in the CIK cytotoxic activity when co-cultured with DC pulsed with the Ag is probably due to both, an increment of the proportion of T lymphocyte expressing CD3+CD56+plus a larger number of cells actively differentiating and proliferating. The use of DC-Ag-CIK cells had little effect on normal retina cells.(3) Effector cells combined with Carboplatin have an significant synergistic antitumor activity against RB-Y79cells and RB-resistant cells. The sequential treatment with a low dose of Carboplatin followed by DC-Ag-CIK cells also has a obvious synergetic effect on RB cells mortality.(4) DC-Ag-CIK cells combined with Carboplatin treatment can increase cytotoxicity and shorten the time on their cytotoxic activity, the high cell mortality is probably due to increased early apoptosis.