Application of Interleukin-12 Expressing Dendritic Cells for the Treatment of Animal Model of Leukemia

Tumor Cell Lines and Animals.

RL♂1 is a cell line derived from radiation-induced T-cell leukemia in a Balb/c mouse and was kindly provided by Dr. Lih-Hwa Hwang (College of Medicine, National Taiwan University). The cells were maintained in Balb/c mice by in vivo passage. Briefly, RL♂1 (1 × 10⁶ cells/mouse) were injected intraperitoneally (i.p.) into Balb/c mice. Cells were harvested 10–14 days after inoculation and stored in liquid nitrogen until use. Upon infection, cells were thawed and grown in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS; Gibco Laboratories, Grand Island, NY). Typically, animals injected s.c. with 1 × 10⁵ of RL♂1 died 40–50 days after inoculation, due to outgrowth of the tumor. Male Balb/c mice aged 6–8 and 10–12 weeks were used in these experiments. They were purchased from the animal center of the National Taiwan University Hospital (Taipei, Taiwan).

Preparation of Tumor Cell Lysate.

RL♂1 cells were re-suspended at 3.5 × 10⁸ cells/ml in Hank’s balance salt solution and disrupted by four freeze/thaw cycles (alternating dry ice/37°C water bath). The tumor cell lysates were centrifuged at 600 × g for 10 min and the supernatant was collected and centrifuged again at 13,000 × g for 60 min. The final supernatant was collected, filtered through a 0.2 μ m filter, and aliquoted into freezing vials to be stored at − 70°C until needed. The supernatant was used as sources of soluble TCL.

Generation of Dendritic Cells from Bone Marrow Cultures.

DCs were prepared as described previously (29–31). Briefly, erythrocyte-depleted mouse bone marrow cells were cultured in RPMI-1640 medium supplemented with 5% FCS with murine rGM-CSF (500 U/ml) and rIL-4 (1000 U/ml) (Pepro Tech Inc., Rocky Hill, NJ) for 8 days. The purity of the DCs ( > 70%) were collected and analyzed by flow cytometry examining the expression of MHC class II, CD80, CD86, CD11c, DEC-205, and 33D1.

Cytokine-Expressing Adenovirus Vectors.

Cytokine- and green fluorescence protein (GFP)-expressing adenovirus vectors were obtained. Construction of the single-chain IL-12 gene was performed as previously described (17, 18). The p40 and p35 subunits of the murine IL-12 gene were connected by a linker that generated the single-chain IL-12 gene. Mouse IL-12 (AdIL-12) gene was inserted into the pAdhPGK vector. The pAdhPGK vector carried no cytokine transgene as a control (Ad-mock). The viral stocks were prepared by 293 cells transfected with IL-12 expressing or mock vector. We collected the supernatant of vector transfected 293 cells after amplification. The cells were lysed by repeated thawing and freezing. After CsCl purification and dialysis, the viral titer was titrated by standard protocol. The final viral titer was 10¹² pfu/ml. We infected 5 × 10⁵ DCs with different titer of virus and got the optimized titer of virus infection—1.5 × 10⁷ pfu. So the MOI equals to 30. The transduction efficiency of adenovirus was from 63% to 80% (mean ± SD is 70% ± 6.46) analyzed by flow cytometry in 5 different independent experiments.

Preparation of Engineered Dendritic Cells.

DCs were cultured as described above. At day 4, DCs were seeded into 24-well culture plate with 5 × 10⁵ cell number. TCL was added at 50 ug/well into the culture plate. The next day, AdIL-12 with MOI of 30 were added into the wells with TCL-pulsed DCs. DCs that were transfected with adenoviral vectors after feeding with TCL were defined as engineered DCs. On day 6, these engineered DCs were collected for experiments.

ELISA for the Determination of Mice IL-12 Cytokines.

To measure the amounts of cytokine secreted from the engineered DCs, commercially available enzyme-linked immunosorbent assay (ELISA) kits for mIL-12 p70 (R&D, UK) were used. Procedures followed were described in the manufacturer’s instructions. The concentration of cytokines in the sample was obtained by interpolation from the standard curve.

In Vivo Immunization and Tumor Challenge.

Balb/c mice were immunized intravenously (i.v.) in the tail vein with engineered DCs twice at seven-day intervals. To study the preventive effect, the mice were re-challenged one week after the last immunization with a lethal dose of 1 × 10⁴ RL♂1 viable tumor cells by i.v. injection into the tail vein. Alternatively, the mice immunization with 5 × 10³ RL♂1 viable tumor cells by i.v. injection into the tail vein, and then treated with engineered DCs at the same day to determine the therapeutic effects. During the follow-up period, the mice were monitored for the survival time.

Cell-Mediated Cytotoxicity Assays.

Spleens isolated from the Balb/c mice received different treatments seven days after challenging with tumors. In vitro stimulation was performed by incubating 5 × 10⁶ splenocytes with 1 × 10⁵ irradiated RL♂1 cells per well in 24-well plates for 5 days at 37°C in the presence of 10 U/ml recombinant human IL-2. Target cells were allowed to be incubated with ⁵¹Cr for 1 hour, and then washed extensively with RPMI medium. The labeled target cells (1 × 10⁴) were mixed with effector cells at effector/target (E/T) ratios of 100:1, 50:1, 10:1, and 5:1 in 96-well U-bottomed plates. The mixtures were incubated at 37°C for 4 hours, and the released ⁵¹Cr radioactivity was measured in 100-ml aliquots of the supernatants. The percentage of specific lysis was calculated using the following formula:

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Each assay was performed in triplicate, and spontaneous release was < 20% of the maximal release by detergent in all assays.

Winn Test.

Spleen cells were isolated from mice that were treated with DCs transfected with AdIL-12 or Ad-mock after being pulsed with TCL or DCs alone. One week after the second immunization of engineered DCs from untreated mice, naïve mice were injected s.c. with mixtures of 1 × 10⁷ splenocytes described above and 10⁶ parental RL♂1 cells. During the follow-up period, the mice were tracked for tumor size and survival time. Primary tumor areas were measured twice a week using mechanical calipers. Tumor volumes were calculated by the formula v = πab ² /6, where a is the longest diameter and b is the next longest diameter perpendicular to a (19, 20).

Statistical Analysis.

All of the results were expressed as the mean ± standard error of the mean (SEM) and evaluated with a one-way analysis of variance (ANOVA), followed by an unpaired two-tailed Student’s t test for comparison between the two groups.

Transfection Efficiency of Dendritic Cells with Adenoviral IL-12.

DCs transfected with adenoviral vectors after being pulsed with TCL were analyzed by flow cytometry and determined by ELISA. To determine the efficiency of the adenoviral vector transfection, we used adenoviral-GFP vectors that expressed fluorescent proteins. The control cells were normalized between 10¹ and 10² in fluorescence intensity. The transduction efficiency of adenovirus is from 63% to 80% (mean ± SD is 70% ± 6.46) analyzed by flow cytometry in 5 different independent experiments. As Figure 1A shows, 74% of the cells expressed fluorescent proteins compared to DCs alone. Different doses of adenoviral vectors were tried during the DC transfections, and we finalized an “optimal dose” of 30 MOI (data not shown). Every transfection was monitored for cell viability by adding trypan blue and scoring live vs. dead cells in a haemocytometer. The viability of DCs transfected with adenoviral vector was as much as 99% with no difference among different groups. The cell culture supernatants of engineered DCs were collected to determine IL-12 expression level by ELISA. Both TCL pulsed-DCs and DCs alone infected with AdIL-12 expressed significantly higher level of IL-12 cytokines than those with DCs (Fig. 1B). Interestingly, IL-12 expression levels were significantly higher when pulsed with TCL than DCs infected with AdIL-12 (Fig. 1B). This suggested that the engineered DCs made by AdIL-12 transfection and pulsed with TCL had good transfection efficiency and higher IL-12 expression levels.