Generation of tumor antigen-specific CD4+ and CD8+ T cells by simultaneous MHC-I and -II epitope presentation in vitro and in vivo

In recent years, activation of the patient's immune system to defend against tumors was demonstrated to be a promising alternative strategy to classical cancer treatments. Dendritic cells (DCs) can present antigens on MHC-II and -I leading to the activation of CD4+ or CD8+ T cells, respectively. Stimulated CD4+ T cells act as helper cells for cytotoxic CD8+ T cells to kill tumor cells by mediating strong proliferation and licensing DCs, increasing their presentation capacity. Tumors bearing MHC-II molecules can also be directly destroyed by cytotoxic CD4+ T cells. As an efficient anti-tumor response strongly depends on the interplay of DCs, CD4+ and CD8+ T cells, these three cell types are considered to be essential for successful immunotherapy design.In clinical trials, DCs were either endogenously or exogenously loaded with tumor antigens for the presentation on MHC-I rather MHC-II. As the antigen presentation pathways differ, a simultaneous loading of MHC-I and -II was suboptimal. To overcome this obstacle, we used a signaling sequence (CrossTAg) to force MHC-II cross-presentation of tumor-associated antigens (TAAs) encoded by in vitro transcribed (ivt) RNA. Subsequently, peripheral blood lymphocytes were primed by DCs expressing TAAs on MHC-I and -II enabling activation and interaction of CD4+ and CD8+ T cells. By this approach, we were able to induce and identify TAA-specific CD4+ and CD8+ T cells in the same experiment.In addition, superior induction efficiency of DCs loaded with CrossTAg-TAA-ivt RNA compared to conventional TAA-ivt RNA was shown in the humanized NOD/scid IL2Rgnull (NSG) mouse model. NSG mice were engrafted with human peripheral blood mononuclear cells (PBMC) and vaccinated twice with DCs either transfected with CrossTAg-TAA-ivt RNA or conventional TAA-ivt RNA. Reisolated PBMC of mice vaccinated with CrossTAg-TAA-ivt RNA transfected DCs showed higher numbers of antigen-specific CD8+ T cells and stronger activation/cytotoxic activity against tumor cells.These in vitro and in vivo data illustrate the benefits of loading DCs with CrossTAg-linked target antigens as CD8+ and CD4+ T cells can be induced side by side allowing interactions and T cell help.