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

Gholamin M, Moaven O, Farshchian M, Mahmoudi M, Sankian M, Memar B, Forghani MN, Malekzadeh R, Rajabi-Mashhadi MT, Abbaszadegan MR - BMC Cancer (2010)

Bottom Line: 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.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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Human Genetics, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran.

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.

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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%.
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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%.

Mentions: In order to establish the optimized gene delivery into monocyte-derived DCs, the transfection efficacy of in vitro transcribed GFP mRNA into DCs was first confirmed. Successful GFP expression, as a reporter gene, was assessed by fluorescent microscopy and FACS analyses. The average viability percentage of the transfected cell population was 94.5% as compared to 97.9% in mock cells and the average transfection efficiency was 87.8%. The flow cytometry results of patient 1 are presented in Figure 1. The kinetic of GFP expression was analyzed afterwards and an approximately 30% expression was detected 96 h after electroporation.


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

Gholamin M, Moaven O, Farshchian M, Mahmoudi M, Sankian M, Memar B, Forghani MN, Malekzadeh R, Rajabi-Mashhadi MT, Abbaszadegan MR - BMC Cancer (2010)

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%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902443&req=5

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%.
Mentions: In order to establish the optimized gene delivery into monocyte-derived DCs, the transfection efficacy of in vitro transcribed GFP mRNA into DCs was first confirmed. Successful GFP expression, as a reporter gene, was assessed by fluorescent microscopy and FACS analyses. The average viability percentage of the transfected cell population was 94.5% as compared to 97.9% in mock cells and the average transfection efficiency was 87.8%. The flow cytometry results of patient 1 are presented in Figure 1. The kinetic of GFP expression was analyzed afterwards and an approximately 30% expression was detected 96 h after electroporation.

Bottom Line: 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.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).

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Human Genetics, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences (MUMS), Mashhad, Iran.

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.

Show MeSH
Related in: MedlinePlus