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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

Time kinetic of B cell nucleofection with IVT mRNA. The percentage of GFP positive B cells 4, 8 and 20 h post nucleofection with IVT mRNA using the five most potent nucleofection programs are represented
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Fig6: Time kinetic of B cell nucleofection with IVT mRNA. The percentage of GFP positive B cells 4, 8 and 20 h post nucleofection with IVT mRNA using the five most potent nucleofection programs are represented

Mentions: The risk of stable integration into the host genome makes DNA less favorable with regard to clinical approaches. The nucleofection of B cells using IVT mRNA was thus investigated next. Therefore, two patient derived B cell lines were analyzed using the top five programs as determined with plasmid DNA. The efficacy ranged from 38 % (±27) to 48 % (±25) with a mean viability of 34 % (Fig. 5). Contrary to what is described in literature [22] and thus to some extend surprising, the efficacy was not higher for IVT mRNA compared to plasmid DNA. We thus performed a time kinetics analysis; assessing the percentage of vital GFP positive cells 4, 8, and 20 h post nucleofection (Fig. 6). GFP expression was well detectable already 4 h after nucleofection, peaked at 8 h and started decreasing thereafter but was still detectable 20 h post nucleofection (Fig. 6). Even longer expression periods have been described for IVT mRNA-nucleofected GFP of DC [22, 23]. Although (anti)gene transduction efficiency is by far not the only factor determining the overall antigen presentation capacity of APC, enhanced efficiency of (anti)gene delivery is likely to improve antigen processing and presentation resulting in increased levels of induced immune responses (reviewed by Garg and colleagues in [24]).Fig. 5


Optimizing the process of nucleofection for professional antigen presenting cells.

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

Time kinetic of B cell nucleofection with IVT mRNA. The percentage of GFP positive B cells 4, 8 and 20 h post nucleofection with IVT mRNA using the five most potent nucleofection programs are represented
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig6: Time kinetic of B cell nucleofection with IVT mRNA. The percentage of GFP positive B cells 4, 8 and 20 h post nucleofection with IVT mRNA using the five most potent nucleofection programs are represented
Mentions: The risk of stable integration into the host genome makes DNA less favorable with regard to clinical approaches. The nucleofection of B cells using IVT mRNA was thus investigated next. Therefore, two patient derived B cell lines were analyzed using the top five programs as determined with plasmid DNA. The efficacy ranged from 38 % (±27) to 48 % (±25) with a mean viability of 34 % (Fig. 5). Contrary to what is described in literature [22] and thus to some extend surprising, the efficacy was not higher for IVT mRNA compared to plasmid DNA. We thus performed a time kinetics analysis; assessing the percentage of vital GFP positive cells 4, 8, and 20 h post nucleofection (Fig. 6). GFP expression was well detectable already 4 h after nucleofection, peaked at 8 h and started decreasing thereafter but was still detectable 20 h post nucleofection (Fig. 6). Even longer expression periods have been described for IVT mRNA-nucleofected GFP of DC [22, 23]. Although (anti)gene transduction efficiency is by far not the only factor determining the overall antigen presentation capacity of APC, enhanced efficiency of (anti)gene delivery is likely to improve antigen processing and presentation resulting in increased levels of induced immune responses (reviewed by Garg and colleagues in [24]).Fig. 5

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