Induction of cytotoxic T lymphocytes primed with tumor RNA-loaded dendritic cells in esophageal squamous cell carcinoma: preliminary step for DC vaccine design

Abstract

Background: Dendritic cells (DC) are potent antigen presenting cells with the ability to prime naïve T cells and convert them to cytotoxic T-lymphocytes (CTL). We evaluated the capability of autologous DCs transfected with total tumor and normal RNA to induce cytotoxic CTL as the preliminary step to design a DC-based vaccine in the esophageal squamous cell carcinoma (ESCC).

Methods: Monocytes-derived DCs were electroporated with either total tumor RNA or normal RNA. T cells were then primed with tumor RNA transfected DCs and lytic effects of the generated CTL were measured with Cytotoxicity assay and IFN-gamma Release Elispot assay.

Results: Cytotoxicity was induced against DCs loaded with tumoral RNA (%24.8 +/- 5.2 SEM) while in normal RNA-loaded DCs, it was minimal (%6.1 +/- 2.4 SEM) and significantly lower (p < 0.05). INF-gamma secretion was more than 2-folds higher in tumoral RNA-loaded DCs when compared with normal RNA-loaded DCs (p < 0.05).

Conclusion: Electroporating DCs with tumor RNA generated tumor antigen presenting cells which in turn enhanced cytotoxic effects of the T cells against ESCC. This may be a useful autologous ex vivo screening tool for confirming the lytic effects of primed T cells on tumors and evaluate probable further adverse effects on noncancerous tissues. These data provide crucial preliminary information to establish a total tumor RNA-pulsed DC vaccine therapy of ESCC.

Figure 1

mRNA transfection of DC. GFP mRNA delivery into DCs of patient 1 with electroporation (500 V, 300 μs). FACS analysis of transfected DC and Mock as a control is shown. Viability percentage was 85.7% in transfected cells as compared with 93.9% in control Mock. Transfection efficiency was 79.8%.

Figure 2

Cytotoxicity assay results representing cytotoxic activity of CTLs induced by DC/tumor-mRNA. Cytotoxicity assay was performed against DC/Tumor mRNA, DC/Normal mRNA and DC/Mock as targets at various effector:target (E:T) ratios assessed by Calcein-AM release assay. Experiments were repeated three times, and representative data of similar results are shown. For example, in the patient 3 cytotoxicity against DC/Tumor-mRNA, DC/Normal mRNA and DC/Mock were 22%, 2% and -1% respectively. *p < 0.05 (Tumor vs. Normal); ** p < 0.05 (Tumor vs. Mock).

Figure 3

INF-γ secretion Elispot assay results. INF-γ spots are counted per 10⁵ T cells. Results are recorded as mean number of INF-γ spots with 95% confidence interval (CI) indicated on the bars. Specific T cell activation and INF-γ secretion was more than 2-folds higher in DC/Tumor-mRNA when compared to DC/Normal-mRNA in both patients (p < 0.05).