Production and loading of IVT- mRNA

The mRNA expression vectors pGEM4Z/A64 containing full-length coding sequences for enhanced Green Fluorescent Protein (EGFP) (pGEM4Z/EGFP/A64) or Influenza matrix protein M1 (FMP) (pGEM4Z/FMP/A64) were kindly provided by Professor Eli Gilboa.⁵⁵ WT1 antigen IVT-mRNA was produced from pGEM4Z/WT1/A64 or purchased from CureVac.⁸ Plasmids were linearized with SpeI (New England Biolabs, R0133S) and IVT was performed using a mMessage Machine T7 Ultra (Life Technologies, AM1345) as described previously.⁵⁶ The quantity and quality of mRNA were analyzed using a Bioanalyzer 2000 (Agilent). BDC or Mo-DC (1–2 × 10⁶) were transfected with 10 µg IVT-mRNA mRNA using an Amaxa Nucleofector 4D with P3 primary cell buffer (Lonza V4XP-3024). Samples that were transfected in the absence of mRNA (mock) were used as described.

Specific FMP HLA-A*0201 antigen presentation was measured by transfecting cells with FMP IVT-mRNA or irrelevant WT1 IVT-mRNA. FMP antigen presentation was measured kinetically by staining cells with HLA-A*0201 FMP mAb. The median fluorescence intensity of the control IVT-mRNA was subtracted from the FMP-IVT transfected sample to determine specific MHC I FMP presentation.