Fig 1: K528 is a versatile cell line for transgenic expression of MHC class I and II molecules from different mammalian species. (A) Phenotypic analysis of K528 cell line. K530 and Raji cells were used as controls in straining. The gating strategy is shown in Figure S2 ; similar gating strategies were applied to other panels below. Grey histograms show staining with isotype control antibodies. (B) K528 and K530 cells were transduced to express H2Kb with or without co-expressed mouse ß2M molecules. The expression vectors co-express mNeonGreen as a selection marker. Monoclonal cell lines with comparable mNeonGreen fluorescence intensities were selected for comparison. Cell surface expression of H2Kb molecules were detected with either mouse ß2M-independent (clone 5041.16.1) or -dependent (clone AF6-88.5) antibodies. (C) K528 cells were transduced to express single-chain trimer with disulfide trap (dtSCT) structures of H2Kb molecules with either VSV8 or OVA257 peptides. Surface expression of H2Kb and H2Kb molecules presenting OVA257 peptides were detected with relevant antibodies. (D) K528 cells were transduced to express IAb molecules. The resultant monoclonal cell line was further transduced with lentiviral vectors with expression cassettes Empty-IRES-mCD86 (K528-IAb), mCD74-IRES-mCD86 (K528-IAb-mCD74) or ?mCD74.Ea52-IRES-mCD86 (K528-IAb-?mCD74.Ea52) in which the core CLIP sequences were replaced with IAb presenting peptide Ea52 from IEd molecule. Monoclonal cell lines expressing comparable levels of mCD86 were selected for analysis. Surface and intracellular mCD74, surface IAb and Ea52/IAb complex were detected by FACS. (E) K528 cells expressing HLA-DQA1*05:01/DQB1*02:01 were transduced with lentiviral vectors with expression cassettes hCD74-IRES-mCD86 (hCD74) or Empty-IRES-mCD86 (Empty). Bulk cell lines after transduction were used for detection by FACS.