Peripheral blood was donated from volunteers after receiving informed consent, and the study was approved by the ethics committee of the Second Affiliated Hospital of Nanhua University, Henyang, China.

Lymphocytes were separated and cultured in accordance with the studies of Pan et al (13) and Laport et al (14), with certain modifications. Peripheral blood was mixed 1:1 (V:V) with 0.9% physiological saline and used for Ficoll density gradient separation (LymphoPrep, PAA, Cölbe, Germany). Following centrifugation at 1,800 rpm for 20 min, the leukocyte layer was collected in fresh tubes. These cells were then washed twice with 0.9% physiological saline at 1,500 rpm for 7 min. Next, the lymphocyte was cultured in GT-T551 medium (Takara Biotechnology Co., Ltd., Dalian, China) with 1,000 U/ml γ-interferon (Beijing Biocoen Biotechnology Co., Ltd., Beijing, China). Ten percent autologous plasma was added on day 0, then 50 µg/ml CD3 monoclonal antibody (Skoda Biotechnology Co., Ltd., Beijing, China) and 100 U/ml interleukin 1α (IL-1α; PeproTech, Suzhou, China) were added on day 1, and 1,000 U/ml rhIL-2 (SL-PHARM, Beijing, China) and 2% autologous plasma was included in the medium from day 1 onward with the concentration calculated by a pocH-100i hemtology analyzer (Sysmex, Milton Keynes, UK). The cells were cultured at 37°C in 5% CO₂ until day 13.

The DC cells were cultured in vitro according to the studies of Miao et al and Pan et al with certain modifications (15,16). The lymphocyte separated from the peripheral blood was resuspended with 20 ml GT-T551 medium, and cultured for 3 h at 37°C in 5% CO₂. Finally, the adhered and suspended cells were separated and cultured as mononuclear cells and CIK cells, respectively. The mononuclear cells were cultured with 20 ml AIM-V medium (Invitrogen Life Technologies, Carlsbad, CA, USA) containing 10% autologous plasma, GM-CSF (0.2 µg/ml, Beijing Biocoen Biotechnology Co., Ltd, Beijing, China) and IL-4 (1 µg/ml, CELLBO Biotechnology Co., Ltd, Wuxi, China). Half of the medium was replaced with fresh medium supplemented with cytokines on day 3, and TNF-α (0.2 µg/ml, Beijing Biocoen Biotechnology Co., Ltd) was added on day 5 to induce maturation of the DCs. On day 7, the DCs were collected and co-cultured with CIK at 37°C in 5% CO₂ until day 13.

Following culture of CIKs and DC-CIKs for 13 days, 1 ml cell suspension was collected and centrifugated at 1,000 rpm for 10 min, then the precipitate was resuspended in 1 ml 0.9% physiological saline, centrifugated at 1,000 rpm for 10 min, then the precipitate was resuspended with 150 µl 0.9% physiological saline, and divided into two groups. APC mouse IgG1 (5 µl), FITC mouse IgG2α (5 µl), PE mouse IgG1 (5 µl) and PerCP-CyTM5.5 mouse IgG1 (1 µl) were added to one group to form the isotype control, and FITC mouse anti-human CD3 (5 µl), PE mouse anti-human CD4 (5 µl), PerCP-CyTM5.5 mouse anti-human CD8 (1 µl) and APC mouse anti-human CD56 (5 µl) were added to the second group to form the experimental group. The two groups were all incubated for 15 min at room temperature, then resuspended with 1 ml 0.9% physiological saline, and centrifugated at 1,000 rpm for 10 min. Finally, the precipitate was resuspended with 0.2 ml 0.9% physiological saline, and prepared for analysis using a BD Accuri C6 flow cytometer (BD Biosciences, Shanghai, China).

Following the culture of CIKs and DC-CIKs for 13 days, 1 ml cell suspension was collected and centrifugated at 1,000 rpm for 10 min, then the precipitate was resuspended in 1 ml 0.9% physiological saline and centrifugated at 1,000 rpm for 10 min. Next, the precipitate was resuspended and diluted with physiological saline to 1×10⁵ cells/ml, then the cell suspension was mixed with 0.4% trypan blue at 9:1 (V:V), and analyzed by Countstar (Inno-Alliance Biotech, Shanghai, China) within 3 min.

Hela cells as target cells were obtained at logarithmic growth phase and the concentration was adjusted to 1×10⁵ cells/ml. CIKs or DC-CIKs cultured for 13 days were used as effector cells, and then mixed with target cells at a proportion of 50:1 (effector cells to target cells). CIK or DC-CIK culture medium (10 ml) was collected, and following centrifugation at 1,000 rpm for 10 min, the precipitate was resuspended with GT-T551 containing 2% autologous plasma and diluted to 5×10⁶ cells/ml. The cells were divided into three groups: the effector-target group comprised 100 µl effector cells and target cells, respectively; the effector cells group comprised 100 µl effector cells and GT-T551 culture medium; and the target cells group comprised 100 µl target cells and GT-T551 culture medium. All groups were cultured at 37°C in 5% CO₂ for 24 h. There were five parallel tubes in every group. Ten microliters MTT (5 mg/ml) was added and cultured at 37°C in 5% CO₂ for 4 h. Following centrifugation at 2,000 rpm for 5 min, the precipitate was dissolved in 100 µl dimethyl sulfoxide, agitated for 15 min, and analyzed at an optical density (OD) of 490 nm. The killing rate was calculated as follows:

Rate = [1 - (OD_{effector-target cell well} - OD_{effector cell well}) / OD_{target cell well}] × 100%.

To determine the appropriate co-culture time of DCs and CIKs, the cell proliferation of CIKs was analyzed. From day 7, the CIKs were in a period of rapid proliferation (Fig. 1A), and the percentage of CD3⁺ T cells was over 90% (Fig. 1B). Of these, DCs were co-cultured with CIKs for 7 days. As shown in Fig. 2, the DC-CIKs were in rapid proliferation on day 7, but the cell quantity was lower than that of CIKs until day 11 and the DC-CIK quantity was significantly (1.17-fold) greater than that of CIKs on day 13.

Next, cell phenotype was analyzed by flow cytometry. As Fig. 3A reveals, the expression of CD3⁺, CD56⁺, CD3⁺CD4⁺, CD3⁺CD8⁺ and CD3⁺CD56⁺ T cells in DC-CIKs was higher than that in CIKs, and the expression of CD3⁺, CD3⁺CD4⁺ CD3⁺CD8⁺ and CD3⁺CD56⁺ T cells was significantly upregulated 1.02, 1.79, 1.26 and 2.44-fold, respectively.

For further analysis of the influence of DCs on CIKs in vitro, cell viability analysis and MTT were used. The CIK and DC-CIK cell viability was 96 and 98%, respectively (Fig. 4), and there was no significant difference between CIKs and DC-CIKs. In addition, MTT results revealed that the CIK and DC-CIK cell cytotoxicity in Hela cells was 58 and 80%, respectively, with a significant difference (Fig. 5).