Transfection of dendritic cells (DC) with in vitro transcribed RNA was shown to be a highly efficient method to generate antigen specific T cells, probably due to the induction of a polyclonal T cell response directed against multiple antigens presented on different HLA allels. However, the experimental evidence of this assumption remains to be demonstrated. To accomplish this, we used monocyte derived DC that were electroporated with RNA coding for the CMV pp65 antigen. The induction and expansion of antigen specific CD8+ and CD4+ T cells was assessed using a pannel of peptides derived from this antigen and presented on HLA-A2, -A1, -A11, -A24, -B35 and -B7 in IFN-g ELISPOT, 51Cr-release and proliferation assays. Autologous DC generated from CMV positive healthy donors were pulsed with peptides or transfected with pp65 RNA and utilized as stimulators. Autologous purified CD8+ and CD4+ lymphocytes were used as effector cells. By applying this approach we found that transfection of DC with pp65 RNA induces an expansion of polyclonal CD8+ mediated T cell responses that recognized peptide antigens presented on different HLA molecules. These in vitro generated cytotoxic T cells were able to efficiently lyse DC pulsed with pp65 derived peptides or transfected with the cognate RNA in an antigen specific manner after several in vitro restimulations. Furthermore, this experimental approach allowed the identification of the immunodominace of T cell epitopes presented upon RNA transfection. The HLA-2 directed responses were more pronounced as compared to the HLA-A1, -A11, -A24 or -B35 allels. In contrast, in 7 out of 7 HLA-A2 and HLA-B7 positive donors B7-peptides elicited a stronger T cell response than the A2-peptide, indicating the immunodominance of HLA-B7 epitopes. Interestingly, transfection of DC with pp65 RNA resulted in the induction of CD4+ antigen specific T cells that produced IFN-g and proliferated upon stimulation with transfected DC. In the next set of experiments we analyzed the possible induction of CMV specific T cells that recognize epitopes deduced from different antigens. Co-transfection of DC with a mixture of RNAs coding for the CMV pp65 and IE1 antigens elicited polyclonal T lymphocytes specific for peptides derived from both antigens. More importantly, polyclonal cytotoxic T cells could be elicited in peripheral blood of 2 out of 3 CMV negative donors demonstrating the efficiency of this approach. Our results demonstrate that DC transfected with RNA can elicit polyclonal T cell responses and have implications for the development of immunotherapeutic strategies to target viral or tumor associated antigens.

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