B 5 r lsr 2

Splenocytes from female mice were pulsed with 1 µg/ml of the MHC-I-restricted OVA_257-264 peptide or HIV-1 gag peptide as a control before labeling with 5 or 0.5 µM of CFSE (Invitrogen, Merelbeke, Belgium), respectively. Labeled cells were mixed at a 1:1 ratio and a total of 1.5 × 10⁷ mixed cells were adoptively transferred into immunized mice 3 days after the second mRNA treatment. Forty eight hours later, splenocytes from mice were isolated and passed through 70 µm nylon strainers (BD Biosciences, San Diego, CA, USA) to obtain single-cell suspensions. Red blood cells were lysed using ACK red blood cell lysis buffer (BioWhittaker, Wakersville, MD, USA). Next, splenocytes were analyzed by flow cytometry. Percentage of antigen-specific killing was determined using the following formula: (1 − (%CFSE^hi cells/%CFSE^low cells)^{treated mice}/(%CFSE^hi cells/% CSFE^low cells)^{non-treated mice}) × 100. The experiments were performed on a triple-laser (B-V-R) LSR-II (Becton Dickinson, San Jose, CA, USA) and analyzed using the FlowJo software (Treestar, OR). Single cells were gated based on their SSC and FSC. Next CFSE-positive cells were selected. See Supplementary Fig. 5 for gating strategy.

Cells were plated 24 h before transfection in a 6- or 96-well plate at 10⁶ or 10⁴ cells/well, respectively. One million B16 cells were transfected with 1 µg of mRNA complexed with Lipofectamine® RNAiMAX (Life Technologies, Ghent; Belgium) diluted in OptiMem to obtain a total volume of 300 µl. The transfection mix was added to the cells and cells were incubated at 37 °C, 5% of CO₂ during a time period depending on the experiment. Transfection efficiency was evaluated by measuring uptake of Cy-5-labeled eGFP mRNA and the onset of translation of the transfected mRNA by determining GFP fluorescence at different time points after transfection using a triple-laser (B-V-R) LSR-II (Becton Dickinson, San Jose, CA, USA) flow cytometer. The flow cytometric data were analyzed with FlowJo (Treestar, OR).

Two days prior to mRNA immunization, 2 × 10⁶ OT-I or OT-II cells were purified and labeled with 5 µM of CFSE (Invitrogen, Merelbeke, Belgium) and subsequently adoptively transferred via i.v. injection into mice that had been s.c. inoculated with B16 cells. Four days after the mRNA treatment, draining lymph nodes were isolated and OT-I or OT-II cell division was analyzed by flow cytometry. Cells were stained with anti-CD16/CD32 (500× dilution) (BD Biosciences) to block Fc receptors followed by staining with Fixable Viability Dye (1000× dilution) (BD Biosciences), CD8 PE-Cy7 (200× dilution) (eBiosciences), CD3 efluor450 (200× dilution) (eBioscience), anti-CD19 allophycocyanin (APC; 200× dilution) (BD Biosciences), and MHC-I dextramer H-2 Kb/SINFEKL-PE (500× dilution) (Immundex). The experiments were performed on a triple-laser (B-V-R) LSR-II (Becton Dickinson, San Jose, CA, USA), and data were analyzed using the FlowJo software (Treestar, OR). Single cells were gated based on FSC and SSC. Living cells were selected and T cells gated for CD3⁺ CD19⁻ T cells. Within the CD8⁺ T cells or CD4⁺ T cells, OVA-specificity was gated by labeling with MHC-I SINFEKL-PE dextramer. See Supplementary Fig. 4 for the gating strategy.