Impact of stabilized IVT RNA constructs on T-cell stimulation in vivo and in vitro. (A) Increase of antigen-specific peptide/MHC complexes by using stabilized IVT RNA constructs. Cells were electroporated with Sec-SIINFEKL-A(67)ACUAG RNA or Sec-SIINFEKL-2β-globinUTR-A(120) RNA (EL4 cells, 10 pmol, 50 pmol; C57Bl/J6 immature BMDCs in triplicate, 150 pmol). Electroporation with buffer only was used as control. Cells were stained for SIINFEKL/K^b-complexes with 25D1.16 antibody. Concentrations of SIINFEKL peptide were calculated from the mean fluorescence values of viable cells using a peptide titration as standard curve. Data for BMDCs are shown as the mean ± SEM of 3 experiments. (B) Improved in vivo T-cell expansion with stabilized IVT RNA constructs. TCR transgenic CD8⁺ OT-I cells (1 × 10⁵) were adoptively transferred to C57Bl/J6 mice. BMDCs of C57Bl/J6 mice were transfected with 50 pmol RNA (Sec-SIINFEKL-A(67)ACUAG, Sec-SIINFEKL-2β-globinUTR-A(120) or control RNA), matured for 16 hours with poly(I:C) (50 μg/mL), and injected intraperitoneally 1 day after T-cell transfer (n = 3). Peripheral blood was taken at day 4 and stained for SIINFEKL tetramer–positive CD8⁺ T cells. Dot plots show CD8⁺ T cells, and the numbers given represent the percentages of tetramer-positive CD8⁺ T cells. (C) Improved in vitro expansion of human T cells with stabilized IVT RNA constructs. CD8⁺ and CD4⁺ lymphocytes from HCMV-seropositive healthy donors were cocultivated with autologous DCs transfected with Sec-pp65-A(67)ACUAG RNA or Sec-pp65-2β-globinUTR-A(120) RNA, pp65 peptide pool (1.75 μg/mL) as positive control, or control RNA (data not shown). After expansion for 7 days, each effector cell population (4 × 10⁴/well) was tested in IFN-γ ELISpot on autologous DCs (3 × 10⁴/well) loaded with pp65 peptide pool or an irrelevant peptide pool (1.75 μg/mL). Graphs represent the mean ± SEM spot number of triplicates.