J Immunother Cancer 2024;12:e008817. doi:10.1136/jitc-2024-008817Open access PBMCs, TILs, and SKILs were screened for CD8+T cell recognition using the patient- specific peptide panels, loaded into barcode labeled pMHC- multimer complexes.27 In short, pMHCs and a short unique DNA barcode were both bound to a fluorochrome- labeled (PE: neo antigens, APC: viral antigens) dextran molecules, creating a DNA barcode- labeled pMHC I multimer, which is unique for each pMHC combination. The cells mentioned above were stained with a panel of these multimers in combination with a CD8 (BD Biosciences, RPA- T8) and CD3 (BD, clone SK7) anti- body. PE and APC fluorochrome- labeled CD8+T cells were sorted on the FACSAria (BD Biosciences). DNA barcodes bound to the sorted cells were hereafter amplified by PCR, as were a reference DNA barcode baseline sample from the collected pMHC multimer panel that the cells were stained with. Amplified barcodes from sorted cells and baseline were hereafter sequenced by PrimBio. Sequence results were uploaded to Barracoda27 for analysis, together with various information on primers, DNA barcodes, DNA barcode anno- tation for pMHC, and information on sample identification. Output files included log2 fold change of sorted barcodes compared with baseline barcodes and the related p value to determine significantly pMHC complexes among the sorted cells. Barcode- pMHC multimers were used to select pMHC, complexes possibly recognized by T cells in the pool (between 7 and 41 pMHC complexes) while this more restricted library of pMHC was included for analyses using combinatorial fluo- rochrome encoding of pMHCs as described above. RESULTS 12 patients were enrolled from January 2019 to October 2021.