Splicing patterns in SF3B1 mutated uveal melanoma generate shared immunogenic tumor-specific neo-epitopes [scTCR-seq]

Purpose: In this study, we show that mutations of the splicing factor SF3B1 in uveal melanoma (UM) generate immunogenic neo-antigens. Overall design: Thawed PBMC from patient UM1 were stained with PE and APC tetramers loaded with peptide 37, then tetramer positive cells were positively enriched using anti-APC and anti-PE microbeads (Miltenyi Biotech), stained with CD3 A700 and CD8 FITC and finally with DAPI. The positive fraction was sorted in a FACS ARIA (BD). To combine 5 tetramer positive populations from 2 donors (14, 17, 26 and 37 for UM2 and 18 for UM3) the tetramers were prepared using 5 different TotalSeq-PE streptavidins (BioLegend) and 1 classical fluorochrome-streptavidin (APC, PE-CF594, PE-Cy5, PE-Cy7), PE-CF594 was used for UM3 who had only one population sorted. The PBMC were stained with the 4 pairs of tetramers for patient UM2 and 1 pair of tetramers for patient UM3, enriched with anti PE-microbeads (Milteny Biotech), stained with CD3 A700 and CD8 FITC and DAPI and sorted separately. The cells were then counted, mixed and loaded onto a Chromium controller using Chromium next GEM Single Cell V(D)J reagent kit with feature barcoding technology according to manufacturer's instructions