Eeffect Of Dendritic Cells Co-cultured With Cytokine Induced Killer Cells On Cytotoxicity Against B Cell Lymphoma

ObjectiveMalignant lymphoma is a group of highly heterogeneous hematopoietic malignancies, which is originated from lymph nodes or lymph tissue, and the incidence has an increasing trend. In recent years, with new drugs, especially targeted drugs have been developed, the level of treatment of malignant lymphoma has made substantial progress, and the complete remission (CR) rate of the disease has reached50%-70%, or even higher. However, only a part of the patients receive long-term disease-free survival in the end, and a substantial proportion of patients with disease recurrence, which was mainly due to the presence of minimal residual disease. Adoptive cellular immunotherapy(ACI) is to the patients with autologous or allogeneic immune cells which have anti-tumor activity, and can be directly or indirectly mediated anti-tumor effect, and kill the residual tumor cells in vivo more effectively, which can achieve to eliminate the tumor cells and prevent tumor cell metastasis and recurrence.cytokine-induced killer (CIK) cells are generated by culturing peripheral blood lymphocytes (PBL) with interferon-γ (INF-γ)、 monoclonal antibody against CD3(anti-CD3) and IL-2in a particular time schedule. CIK cells exhibit potent, non-MHC-restricted cytolytic activities against tumor cells. Dendritic cells (DC) is currently considered the most powerful antigen-presenting cells, and can present tumor antigen to T lymphocytes effectively. In recent years, CIK cells co-cultured with DC showed stronger antitumor activity in vitro and in vivo against hematopoietic neoplastic cells.Their cytotoxicity against B-NHL (B-cell non-Hodgkin lymphoma), however, has not been fully investigated in vitro. The study is to investigate the variation of cell number secreting cytokines、 immune phenotype and killing activity for NHL cells of DC-CIK cell with the incubation time in the cells culture period of the healthy population, to find better immune active cells through comparing with the anti-tumor activity of CIK cell; and compared with cell number、secreting cytokines、immune phenotype and killing activity for Riji cells of DC-CIK cell of NHL patients on day15of culture, to clear the differences between the healthy individual and the NHL patients; and investigate the variation of killing activity of DC-CIK cell against different B-NHL cells. This would be the preparation for better clinical application of allogeneic DC-CIK cells immunotherapy.Methods1. Take use of conventional mononuclear cell extraction method to extract the mononuclear cells in peripheral blood50ml of20healthy volunteers and10NHL patients, and then cultivate the cells with5%CO2at37℃for2hour DCs were induced from suspended cells, and CIK cells were induced from adherent cells. After9days of nurturing, two kinds of cells were co-cultured. CIK cells were cultured alone as controls.2. Determing the cell morphology and cell number viability in the cells culture period with inverted microscope and cell counter specifically.3. Determing the immune phenotype of DC cells on day9of culture and CIK cells on days12、15of culture with flow cytometry.4. Determing the secreting cytokines on day15of culture with Enzyme Linked Immunosorbent Assay.5. Determing the killing activity of CIK cells and DC-CIK cells against NHL cells with the MTT colorimetry when the ratio of effective cells totarget cells was5:1、10:1and20:1. Results1. The proliferation capability and the killing activity against Riji cells of DC-CIK cells are significantly higher than that of CIK cells. Compared with CIK cells, DC-CIK cells significantly enhanced the antitumor activity and increased TNF-α、IFN-γ and IL-12secretion, and there are more CD3+CD56+cells and CD3+CD8+cells in the DC co-cultured with CIK cells.2. Comparing healthy volunteers and NHL patients, on day15of culture, the killing activity of DC-CIK cells against Riji cell appeared a significant difference; the percentage of CD3+CD56+cells and CD3+CD8+cells, the secretion of TNF-α、 IFN-γ and IL-12are increase. Cultured NK cells from healthy volunteers were better than NHL patients.3. Healthy population’s DC-CIK cells had strong cytotoxicity on different B-NHL cells (SU-DHL4cells, Raji cells KARPAS422cells), and enhanced the killing effect with the increasing ratio of effective cells to target cells.ConclusionThe proliferation capability and the killing activity against Riji cells of DC-CIK cells are significantly higher than that of CIK cells. Comparing healthy volunteers and NHL patients, on day15of culture, the killing activity of Healthy population’s DC-CIK cells against Riji cell appeared a significant higher, and Healthy population’s DC-CIK cells had strong cytotoxicity on different B-NHL cells.