Influence of autologous dendritic cells on cytokine-induced killer cell proliferation, cell phenotype and antitumor activity in vitro

Abstract

Dendritic cell (DCs) are essential antigen processing and presentation cells that play a key role in the immune response. In this study, DCs were co-cultured with cytokine-induced killer cells (DC-CIKs) in vitro to detect changes in cell proliferation, cell phenotype and cell cytotoxicity. The results revealed that the DCs were suitable for co-culture with CIKs at day 7, and that cell quantity of DC-CIKs was lower than that of CIKs until day 11, but it was significantly improved to 1.17-fold that of CIKs at day 13. Flow cytometry was used to detect the cell phenotype of CIKs and DC-CIKs. Compared with CIKs at day 13, the percentage of CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD3⁺CD56⁺ T cells in DC-CIKs was significantly improved 1.02, 1.79, 1.26 and 2.44-fold, respectively. In addition, trypan blue staining analysis demonstrated that the cell viability of CIKs and DC-CIKs was 96% and 98%, respectively. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) analysis verified that CIK and DC-CIK cytotoxicity in Hela cells was 58% and 80%, respectively, with a significant difference. Taken together, our results indicate that the cell proliferation, cell phenotype and antitumor activity of CIKs were all enhanced following co-culture with DCs in vitro. These results are likely to be useful for DC-CIK application in antitumor therapies.

Keywords: MTT; cytokine-induced killer cells; dendritic cells; flow cytometry.

Figure 1.

Proliferation activity analysis of cytokine-induced killer cells (CIKs). (A) Change in cell quantity at day 3, 5, 7, 9, 11 and 13. (B) Expression of CD3⁺ T cells in CIKs at day 3, 5, 7, 9, 11 and 13. Bars represent means ± standard deviation (n=5).

Figure 2.

Detection of cell quantity in cytokine-induced killer cells (CIKs) and dendritic cell (DC)-CIKs at various times. Cell quantity was measured at day 3, 5, 7, 9, 11 and 13, and the DCs were co-cultured with CIKs on day 7. Bars represent means ± standard deviation (n=5). *P<0.05.

Figure 3.

Flow cytometry analysis of the cell phenotype in cytokine-induced killer cells (CIKs) and dendritic cell (DC)-CIKs. (A) Bar graph demonstrating percentage of different cell phenotypes between CIKs and DC-CIKs cultured at day 13. (B) Percentage of different phenotype T cells measured by flow cytometry. Lanes 1–3 represent CD3⁺CD4⁺, CD3⁺CD8⁺ and CD3⁺CD56⁺ phenotype, respectively. Bars represent means ± standard deviation (n=5). *P<0.05; **P<0.01.

Figure 4.

Cell viability analysis. Cells (900 µl cell suspension) were dyed with 100 µl 0.4% trypan blue at room temperature for 3 min, then a 10 µl sample was analyzed by Countstar. Those with bright color are living cells. CIKs, cytokine-induced killer cells; DC, dendritic cells.

Figure 5.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis of cytotoxicity of cytokine-induced killer cells (CIKs) and dendritic cell (DC)-CIKs in Hela cells. Bars represent means ± standard deviation (n=5). *P<0.05.