Fig 4. In vitro evaluation of Tcj2 mRNA showed the translation and antigen presentation of the mRNA construct, as well as the activation of SIINFEKL-specific CD8+ T cells.

DC2.4 cells were transfected with mRNA with Lipofectamine MessengerMAX, or without mRNA (transfection control). After a 24-hour incubation, cells were subjected to analysis. A) Schematic representation of Tcj2 mRNA construct. B) Cell viability measured after transfection. C) Detection of the translated FLAG-tag sequence by intracellular flow cytometry staining using a FLAG-specific antibody. D) Presentation of SIINFEKL on surface MHC-I (H-2K^b) measured by flow cytometry using an antibody specific for the combination of SIINFEKL presented by H-2K^b. E) Cytokines secreted by C57BL/6J “normal” or SIINFEKL specific OT-1 CD8+ T cells after co-culture with transfected cells. DC2.4 cells were transfected for 24 hours and then counted and seeded. Splenocytes were added in a ratio of 1:10 (DC2.4: splenocytes) and the co-culture was incubated for 24 hours before the supernatant was collected and analyzed for cytokines by Luminex. Figure prepared with Biorender.com. From all experiments, mean and standard deviations are shown from triplicate experiments.