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Plat-A cells (8×105 cells/well) were seeded 24h earlier than transfection in 6-well plates in 2 mL/well of infection medium (Plat-A medium without antibiotics). 20-24h later, once the cells had reached 70% confluence, 500μL/well of a mixture containing plasmids and Lipofectamine were added. The mixtures contained three μg/well of transgene carrying plasmid, 2 μg/well of pMD2.G and 10 μL/well of Lipofectamine 2000, and had been prepared in OPTIMEM medium in accordance with the manufacturer´s protocol. PMD2.G was a gift from Didier Trono (Addgene plasmid # 12259).

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Each insert was fused to the b1 SFV translation enhancer utilizing the 2A self-protease from foot and mouth illness virus as a linker. The inserts comprise a sign peptide from MHC class I, adopted by chosen 15-mer neoAgs situated in tandem and the MHC class I trafficking domain sequence . Inserts encoding the neoAgs had been obtained from Genscript and cloned into MSCV-IRES-Thy1.1 DEST and pBK-T-SFV-b12A utilizing BglII&SalI and Apa I sites, respectively. MSCV-IRES-Thy1.1 DEST was a gift from Anjana Rao (Addgene plasmid # 17442). Plasmids pMSCV-TMGP69-IRES-Thy1.1, pMSCV-TMGP73-IRES-Thy1.1 encoding the neoAgs and management plasmid (MSCV-IRES-Thy1.1 DEST) had been used to prepare retrovirus for transduction experiments. A needle tumor biopsy was extracted at diagnosis and preserved at -80°C in RNAlater.

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Prediction of binding to HLA class I molecules of mutated and wild kind peptides.

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