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Plat-A cells (8×105 cells/well) were seeded 24h before transfection in 6-well plates in 2 mL/well of infection medium (Plat-A medium without antibiotics). 20-24h later, as quickly as 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 have been prepared in OPTIMEM medium in accordance with the manufacturer´s protocol. PMD2.G was a present from Didier Trono (Addgene plasmid # 12259).

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Although TNBC has been described as a tumor with a mutational load greater than other breast tumors , it is well-known that, much like tumor infiltrates, there’s a broad spectrum of mutational burden between sufferers . Analyses of expression of mutated sequences revealed that about 65% of mutations have been positioned at expressed genes. A high variability concerning expression of mutated genes with potential neoAg-coding capacity has been reported in TNBC, starting from 35-50% in some instances , to greater than 80% in others . The pattern measurement and the filtering standards used to outline expression in the different research might account for these discrepancies, as properly as for the differences with our outcomes. By utilizing these mutations, we predicted HLA class I-restricted potential neoAgs encoded by mutated genes, observing a median of ninety eight neoAgs. Finally, to demonstrate recognition of processed antigens in the context of tumor cells, we prepared transfectants of MC703 cells expressing the neoAgs used for vaccination.

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