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. 2019 Dec 19;9:436. doi: 10.3389/fcimb.2019.00436

Figure 4.

Figure 4

Binding interactions of recombinant soluble chPD-1/PD-L1 with surface chPD-1/PD-L1 on COS cells. (A) Representative EVOS images illustrating chPD-1 and chPD-L1 interactions. Images were taken using an EVOS digital microscope. Giemsa staining was carried out on COS cells transfected with surface expression constructs pKW06-chPD-1and pKW06-chPD-L1. Rosette formation was observed when surface pKW06-chPD-L1 interacts with soluble chPD-1-human-IgG1Fc fusion protein (red arrows) and surface pKW06-chPD-1 with soluble chPD-L1-human-IgG1Fc fusion protein (red arrows). No rosettes was observed in surface pKW06-chPD-1 with soluble chPD-1-human-IgG1Fc fusion protein interaction, surface pKW06-chPD-L1 with soluble chPD-L1-human-IgG1Fc fusion protein interaction, and untransfected cells. Scale bar represents 50 μm. (B) Percentage of soluble chPD-1/PD-L1-human-IgG1Fc fusion proteins interactions with surface pKW06-chPD-1/PD-L1 were calculated by counting 10 randomly selected fields by scoring COS cells with 5 or more beads as positive. On surface pKW06-chPD-1 and pKW06-chPD-L1 transfected COS cells, 34.89 ± 6.8% of chPD-L1-human-IgG1Fc and 33.79 ± 10.3% of chPD-1-human-IgG1Fc showed clear rosette structures, respectively. Data are represented as means of three independent biological experiments ± standard deviation (error bars). (C) Representative confocal photo micrographs illustrating clear rosette (white arrows) formation during chPD-1 and chPD-L1 interactions. Red fluorescence visualizes Dynabeads®. Scale bar represents 40 μm.