Fig. 2: EpiScan pooled screening allows for high-throughput MHC class I ligand discovery.

a, Schematic representation of the screening procedure. A pool of random oligonucleotides encoding 9-mer peptides was cloned into the EpiScan lentiviral vector and introduced into EpiScan cells expressing a single HLA allele. Cells expressing exogenous peptides binding MHC class I that hence exhibited elevated cell surface MHC class I levels were isolated by FACS, and the identity of the peptides was revealed by next-generation sequencing. The left dot plot displays two separate samples; light blue dots represent negative-control EpiScan cells before transduction, while red dots show EpiScan cells expressing the library of exogenous peptides; MOI, multiplicity of infection; gDNA, genomic DNA; NGS, next-generation sequencing. b,c, EpiScan screens recapitulate known binding preferences for common MHC class I alleles. Logo plots summarize the sequences of the MHC class I ligands identified by EpiScan (b); for comparison, analogous logo plots based on MHC class I ligands identified by MS⁸ are shown in c. d, Histograms show cell surface MHC class I levels on EpiScan cells expressing the indicated HLA alleles with (left) or without (right) SPP. e, Logo plot summarizing the composition of the HLA-A*02:01 ligands identified by EpiScan screens using SPP-deficient EpiScan cells.