T-Bet Mediated Anti-Neoplastic Effects of Dendritic Cell-Cytokine Induced Killer Cells in vitro.

Objective: To investigate the molecular mechanism underlying T-bet mediated anti-neoplastic effects of cytokine induced killer (CIK) cells.

Methods: Lymphocytes isolated from peripheral blood of leukemic children were induced with γ- interferon (IFN-γ), 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-γ 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.

Findings: 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-γ 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.

Conclusion: 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.