Objective: To investigate the influence of dendritic cells (DCs) on the prevalence and function of regulatory T cells (Tregs) in cytokine induced killer (CIK) cells.
Methods: The blood samples of 20 patients with solid tumors were collected. The peripheral mononuclear cells (PBMCs) were isolated. CIK cells were added into the culture fluid without CD(4)(+)CD(25)(+)T cells (CIK-Treg(del) cells) and the culture fluid of regular PBMCs respectively, and the proliferation and cytotoxicity of the CIK cells were detected by BrdU method and with the cells of human lung carcinoma, breast carcinoma, colon carcinoma, and lymphoma as target cells respectively. CD(4)(+)CD(25)(+)T cells were added into another culture fluid of CIK-Treg(del) cells at the proportions of 20:1, 10:1, and 5:1 respectively, then the proliferation and cytotoxicity of the CIK cells were detected as described above. Flow cytometry was used to detect the surface markers of CIK cells. To identify the influence of DCs on the anti-tumor activity of CIK, PBMCs were isolated from the patients with solid tumor to culture the DCs and CIK cells. Dendritic cells were harvested on day 7 and co-cultured with the CIK cells (DC+CIK cells). The frequency of Tregs in CIK was determined by flow cytometry. The cytotoxicity was examined by LDH assay. The levels of TGF-beta, IL-10, IFN-gamma, IL-2, and IL-6 were analyzed by ELISA.
Results: The rates of the main effector cells in CIK cells (CD(3)(+)CD(56)(+) cells) were 17% +/- 5% and 28% +/- 5% in the regular CIK cells and CIK-Treg(del) cells respectively. LDH method showed that the cytotoxicity towards tumor cells of the CIK-Treg(del) cells The rates of the main effector cells in CIK cells (CD(3)(+)CD(56)(+) cells) were 17% +/- 5% and 28% +/- 5% in the regular CIK cells and CIK-Treg(del) cells respectively. LDH method showed that the cytotoxicity towards tumor cells of the CIK-Treg(del) cells was higher than that of the regular CIK cells (P < 0.05), however, after the addition of selected cells, the cytotoxicity of the CIK-Treg(del) cells decreased. Flow cytometry showed that the proportions of CD(4)(+)CD(25)(+) Treg cells in the CIK cells and DC-CIK cells were 13% +/- 5% and 10% +/- 4% respectively (t = 3.977, P = 0.001). After the DC induction the cytotoxicity of CIK cells was significantly higher than that of the regular CIK cells. ELISA showed that after DC induction the levels of TGF-beta and IL-10 of the DC+CIK group were significantly lower than those of the regular CIK cells (t = 2.136, P = 0.046; and t = 2.965, P = 0.008), and the level of IFN-gamma was significantly higher in the DC+CIK group (t = 2.220, P = 0.039).
Conclusion: CD(4)(+)CD(25)(+) regulatory T cells inhibit the anti-tumor activity of CIK cells. The interaction between CIK cells and DCs is sufficient for the blockage of the properties of regulatory T cells. CIK cells have the desirable properties for immunotherapy approaches, especially after co-culture with DCs.