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. 2010 Oct 13;6(7):639-48.
doi: 10.7150/ijbs.6.639.

Comparative Analysis of Cytotoxic T Lymphocyte Response Induced by Dendritic Cells Loaded With Hepatocellular Carcinoma -Derived RNA or Cell Lysate

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Free PMC article

Comparative Analysis of Cytotoxic T Lymphocyte Response Induced by Dendritic Cells Loaded With Hepatocellular Carcinoma -Derived RNA or Cell Lysate

Ke Pan et al. Int J Biol Sci. .
Free PMC article

Abstract

The choice of the tumor antigen preparation used for dendritic cell (DC) loading is important for optimizing DC vaccines. In the present study, we compared DCs pulsed with hepatocellular carcinoma (HCC) total RNA or cell lysates for their capacity to activate T cells. We showed here that HCC total RNA pulsed-DCs induced effector T lymphocyte responses which showed higher killing ability to HCC cell lines, as well as higher frequency of IFN-γ producing of CD4+ and CD8+ T cells when compared with lysate pulsed-DCs. Both of RNA and lysate loading did not influence the changes of mature DC phenotype and the capacity of inducing T cell proliferation. However, HCC lysate loading significantly inhibited the production of inflammatory cytokines IL-12p70, IFN-γ and enhanced the secretion of anti-inflammatory cytokines IL-10 of mature DCs. Our results indicated that DCs loaded with HCC RNA are superior to that loaded with lysate in priming anti-HCC CTL response, suggesting that total RNA may be a better choice for DCs-based HCC immunotherapy.

Keywords: Dendrtic cells; cytotoxic T cells; hepatocellular carcinoma; tumor lysate; tumor total RNA.

Conflict of interest statement

Conflict of Interest: The authors declare that they do not have any conflict of interest with respect to this manuscript.

Figures

Figure 1
Figure 1
Tumor cell killing by T cells stimulated with tumor antigen-pulsed DCs. Autologeous nonadherent PBMCs were cocultured with DCs pulsed with HCC total RNA or lysate. After 5 days of incubation, cytotoxic activity of T cells against Hep-G2, Hep-3B, Huh-7 and SMMC-7721 was evaluated using a LDH release assay at different E:T ratios. *p<0.05.
Figure 2
Figure 2
Intracellular IFN-γ production of CD4+ and CD8+ T-cells stimulated with DC pulsed with HCC total RNA or lysate. Autologeous nonadherent PBMCs were cocultured with RNA-DCs or lysate-DCs for 5 days. And then, T cells were collected and accessed for intracellular expression of IFN-γ in CD4+ and CD8+ T cells using flow cytometry. Original histograms are shown with percentage of dual-positive cells. *p<0.05.
Figure 3
Figure 3
Analysis of DC phenotype by flow cytometry. Different group of DCs were harvested and processed for staining with a panel of mAb as indicated. The staining of the indicated surface marker is shown and the percentage of positive cells is stated. The MFI value are also indicated.
Figure 4
Figure 4
Effects of HCC total RNA or lysate loading on the cytokines release of mature DCs. Immature DCs were incubated with RNA (black bar) or lysate (white bar) for 3 hour and then stimulated with OK-432 (0.1 KE/ml) for 2 days. The supernatant were collected for TNF-α, IL-12p70, IFN-γ, IP-10, IL-10 and TGF-β production determine by using ELISA method. The supernatant come from immature DC and OK-432-stimulated DC without loading any antigen are as control (stripe bar). Data are means ± SD of triplicates from one experiment. *p<0.05 (RNA loading compared with lysate loading).
Figure 5
Figure 5
The stimulatory capacity of DCs pulsed with HCC total RNA or lysate. Analysis of T cells proliferation. Autologous T lymphocytes labeled with CFSE were mixed with RNA-DCs or lysate-DCs at a ratio of 10:1 (T cells to DCs) and cocultured for 5 days. T cell proliferation was performed using flow cytometry and the percentage of proliferated T cells is stated.

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