Antigen presentation and T cell activation by Coccidioides-Ag2/PRA-cDNA-transfected primary murine dendritic cells (78.21)

Abstract

Abstract The dendritic cells (DCs) have an important role in antigen presentation and regulation of host defense. Immunization with in vitro modified DCs has been shown to activate protective immune responses. Among various approaches used for modifying DCs, the nonviral genetic transfection of DCs is easy and safe as compared to viral-mediated transfection methods. In this study, we investigated antigen presentation and T cell stimulatory ability of nonvirally, gene-transfected primary murine DCs. We isolated primary murine bone marrow derived DCs and transfected isolated DCs with Ag2/PRA-cDNA (protective epitope of Coccidioides, a fungal pathogen) using TransIT-TKO (Mirus Bio, WI)- a nonviral transfection reagent. We found that upto 30-40% of primary murine DCs were transfected with the plasmid DNA carrying Ag2/PRA-cDNA. The transfected DCs remained viable and expressed the transgene (Ag2/PRA-cDNA)-encoded protein. Only moderate changes were observed in the immunophenotype of genetically-transfected DCs. The activation of T cells was studied by flow-cytometry after co-culture of naive splenic T cells with DCs. The T cells co-incubated with Ag2/PRA-cDNA transfected DCs showed upregulation of cell-surface CD25 and CD69 (activation markers). These results suggest that the primary DCs can be efficiently transfected in vitro using a non viral method; the transfected primary DCs can process and present the antigens and stimulate T cells.