Fig 2. Screening PD-L1 mutants using a high-throughput microbead binding FACS assay.

A) Schematic of the microbead FACS binding assay. B) Representative control microbead experiment. Cells expressing either mCherry alone (-control) or PD-L1 mCherry were queried with microbeads conjugated with control Fc, PD-1 Fc or B7-1 Fc fusion protein. The FACS data was gated for all live cells and shows binding of both PD-1 and B7-1 coated microbeads (upper right quadrant) to cells expressing wild type PD-L1. C) FACS microbead binding data for a panel of 54 PD-L1 mutants. Data shows the fraction of mCherry positive cells (PD-L1 expressing) bound to microbeads coated in either PD-1 (Blue) or B7-1 (Red) with binding normalized to wild type. PD-1 and B7-1 binding was done in parallel triplicate experiments with error bars representing the standard deviation.