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Optimizing the process of nucleofection for professional antigen presenting cells.

Mullins CS, Wegner T, Klar E, Classen CF, Linnebacher M - BMC Res Notes (2015)

Bottom Line: In this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression.And no differences with regard to nucleofectability were observed between the two cell types.Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Immunotherapy, Department of General Surgery, University Hospital Rostock, Schillingallee 35, 18057, Rostock, Germany. christina.mullins@uni-rostock.de.

ABSTRACT

Background: In times of rapidly increasing numbers of immunological approaches entering the clinics, antigen delivery becomes a pivotal process. The genuine way of rendering antigen presenting cells (APC) antigen specific, largely influences the outcome of the immune response. Short peptides bear the demerit of HLA restriction, whereas the proper way of delivery for long peptide sequences is currently a matter of debate. Electroporation is a reliable method for antigen delivery, especially using nucleic acids. The nucleofection process is based on this approach with the twist of further ensuring delivery also into the nucleus. Beside the form of antigen, the type of APC used for immune response induction may be crucial. Dendritic cells (DC) are by far the most commonly used APC; however B cells have entered this field as well and have gained wide acceptance.

Results: In this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression. APC were transfected either with plasmid DNA containing the reporter gene green fluorescent protein (GFP) or directly with in vitro-transcribed (IVT) GPF mRNA as a surrogate antigen. Out of nearly 100 different nucleofection programs tested, the top five for each cell type were identified and validated using cells from cancer patients. Flow cytometric analyses of transfected cells determining GFP expression and viability revealed a reverse correlation of efficiency and viability. Finally, donor dependant variances were analyzed.

Conclusion: In summary, nucleofection of both DC and B cells is feasible with plasmid DNA and IVT mRNA. And no differences with regard to nucleofectability were observed between the two cell types. Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.

No MeSH data available.


Related in: MedlinePlus

Top 10 DC nucleofection programs. The percentage of GFP positive cells (green) as well as viability (blue) of immature DC with plasmid DNA for the ten most potent nucleofection programs are represented, the average percentage (+standard deviation) for three donors is given
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Fig2: Top 10 DC nucleofection programs. The percentage of GFP positive cells (green) as well as viability (blue) of immature DC with plasmid DNA for the ten most potent nucleofection programs are represented, the average percentage (+standard deviation) for three donors is given

Mentions: In a first step, we assessed the differentiation stage at which nucleofection of monocyte derived DC would best be applied. Therefore, we analyzed the nucleofection efficacy—as measured by green fluorescent protein (GFP) positive cells post nucleofection with pmaxGFP plasmid—in monocytes, immature and mature DC (Fig. 1). Nucleofection was most efficient in immature DC with all three programs tested (U-022, V-001 and X001) and viability tended to be highest in immature DC as well (Fig. 1; Additional file 1: Figure S1). In a next step, nucleofection of immature DC with 98 different programs was performed to determine optimal settings (see Additional file 2: Table S1 for a detailed list). The percentage of vital GFP positive cells with the top ten programs ranged from 30 % (±4) to 45 % (±8) with a mean viability rate of 51 % (Fig. 2).Fig. 1


Optimizing the process of nucleofection for professional antigen presenting cells.

Mullins CS, Wegner T, Klar E, Classen CF, Linnebacher M - BMC Res Notes (2015)

Top 10 DC nucleofection programs. The percentage of GFP positive cells (green) as well as viability (blue) of immature DC with plasmid DNA for the ten most potent nucleofection programs are represented, the average percentage (+standard deviation) for three donors is given
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4581479&req=5

Fig2: Top 10 DC nucleofection programs. The percentage of GFP positive cells (green) as well as viability (blue) of immature DC with plasmid DNA for the ten most potent nucleofection programs are represented, the average percentage (+standard deviation) for three donors is given
Mentions: In a first step, we assessed the differentiation stage at which nucleofection of monocyte derived DC would best be applied. Therefore, we analyzed the nucleofection efficacy—as measured by green fluorescent protein (GFP) positive cells post nucleofection with pmaxGFP plasmid—in monocytes, immature and mature DC (Fig. 1). Nucleofection was most efficient in immature DC with all three programs tested (U-022, V-001 and X001) and viability tended to be highest in immature DC as well (Fig. 1; Additional file 1: Figure S1). In a next step, nucleofection of immature DC with 98 different programs was performed to determine optimal settings (see Additional file 2: Table S1 for a detailed list). The percentage of vital GFP positive cells with the top ten programs ranged from 30 % (±4) to 45 % (±8) with a mean viability rate of 51 % (Fig. 2).Fig. 1

Bottom Line: In this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression.And no differences with regard to nucleofectability were observed between the two cell types.Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Immunotherapy, Department of General Surgery, University Hospital Rostock, Schillingallee 35, 18057, Rostock, Germany. christina.mullins@uni-rostock.de.

ABSTRACT

Background: In times of rapidly increasing numbers of immunological approaches entering the clinics, antigen delivery becomes a pivotal process. The genuine way of rendering antigen presenting cells (APC) antigen specific, largely influences the outcome of the immune response. Short peptides bear the demerit of HLA restriction, whereas the proper way of delivery for long peptide sequences is currently a matter of debate. Electroporation is a reliable method for antigen delivery, especially using nucleic acids. The nucleofection process is based on this approach with the twist of further ensuring delivery also into the nucleus. Beside the form of antigen, the type of APC used for immune response induction may be crucial. Dendritic cells (DC) are by far the most commonly used APC; however B cells have entered this field as well and have gained wide acceptance.

Results: In this study, we compared B cells to DC with regard to nucleofection efficiency and intensity of resulting antigen expression. APC were transfected either with plasmid DNA containing the reporter gene green fluorescent protein (GFP) or directly with in vitro-transcribed (IVT) GPF mRNA as a surrogate antigen. Out of nearly 100 different nucleofection programs tested, the top five for each cell type were identified and validated using cells from cancer patients. Flow cytometric analyses of transfected cells determining GFP expression and viability revealed a reverse correlation of efficiency and viability. Finally, donor dependant variances were analyzed.

Conclusion: In summary, nucleofection of both DC and B cells is feasible with plasmid DNA and IVT mRNA. And no differences with regard to nucleofectability were observed between the two cell types. Using IVT mRNA omits the danger of genomic integration and plasmid DNA constructs permit a more potent and longer lasting antigen expression.

No MeSH data available.


Related in: MedlinePlus