T-bet mediated anti-neoplastic effects of dendritic cell-cytokine induced killer cells in vitro

ABSTRACT

To investigate the molecular mechanism underlying T-bet mediated anti-neoplastic effects of cytokine induced killer [CIK] cells. Lymphocytes isolated from peripheral blood of leukemic children were induced with gamma- interferon [IFN- gamma], CD3McAb and interluki-2 [IL-2], and co-cultured with dendritic cells [DCs] to generate DC-CIK cells. The morphology and immunophenotype of these cells were determined by a light microscopy and flow cytometry, respectively. IL-2 and IFN- gamma levels released by DC-CIK cells were quantified by ELISA. Cytotoxicity of DC-CIK cells against leukemia cell lines was measured by MTT assay. FCM was used to detect CD4[+]CD25[+]Treg cells, while RT-PCR and Western blot were used to determine mRNA and protein expressions of Foxp3 and GATA3 in DC-CIK cells treated with T-bet monoclonal antibody. Induced DC-CIK cells were regular, round and transparent with variable cell volume and cellular aggregation. The main effector cells in this population were CD3[+]CD8[+] cells and CD3[+]CD56[+] cells. We demonstrated a time dependent increase in IL-2 and IFN- gamma levels after induction. DC-CIK cells were cytotoxic to B95 cells, Jhhan cells and M07e cells, with the highest cytotoxicity towards B95 cells. Treatment with mouse anti-human T-bet monoclonal antibody resulted in an increase in the proportion of CD4[+]CD25[+]Treg cells and elevation of Foxp3 and GATA3 mRNA and protein levels. DC-CIK cells induced with cytokines were strongly cytotoxic towards a number of cancer cell lines. Foxp3 and GATA3 were implicated in the T-bet mediated anti-neoplastic effects of DC-CIK cells via activation of the Th1 pathway and suppression of the Th2 and Treg pathways