Assessment of antigen immunogenicity formulated in minigenes transfected into antigen-presenting cells

Tumor cells exhibit deficient antigen presentation to T cells, significantly contributing to immune evasion and tumor genesis. Peptide pulsed Antigen-presenting cells (APCs) are commonly used in cancer immunotherapy to circumvent the defects of tumor cells in processing and presenting antigens to T lymphocytes. However, peptides do not always represent epitopes naturally processed by tumor cells, which might reduce the identification of actual immunogenic antigens. Minigenes encoding concatenated immunogenic tumor epitope sequences offer a promising alternative to select tumor antigens naturally processed and presented to T cells. Hence, using APCs transfected with minigenes might contribute to immunotherapy's effectiveness, avoiding non-naturally processed epitopes as vaccine candidates. This study evaluates APCs transfected with a minigene construct encoding HLA-A0201-restricted immunogenic antigens to stimulate antigen-specific CD8+ T lymphocytes in vitro. Artificial APCs (aAPCs) were also designed by co-transfecting the HEK293 cell line with plasmids encoding co-stimulatory molecules (CD80, CD83, CD137L) to assess CD8+ T cell activation efficiency, intracellular cytokine production, cytotoxic activity, activation and exhaustion marker expression. In this study, we successfully implemented a transfection methodology of HEK293 cells with a minigene encoding viral and tumor HLA-A * 0201 epitopes. These cells, used as aAPCs, allow studying the expansion and the phenotype of antigen-specific CD8+ T cells. However, our results indicate that epitope presentation alone is sufficient to activate CD8+ T cells, suggesting that the presence of co-stimulatory molecules may not be necessary for effective T cell activation. Considering that the use of HEK293 cells as aAPCs has yet to be explored and due to their high transfection efficiency with minigenes, the methodology implemented in this work enables their use to identify naturally processed immunogenic neoantigens. We believe our findings can contribute to selecting and designing personalized vaccines based on tumor neoantigens that are useful for cancer immunotherapy.