Fig. 2. Both membrane-bound and soluble forms of PD-1 induce the PD-L1 reverse signaling and nivolumab treatment counteracts this effect in HL cells.

a Effect of membrane-bound PD-1 on ERK phosphorylation. Dox-inducible PD-1 expressing HEK293 cells were treated with Dox (2 μg/ml) for 48 h and then cocultured with starved GFP + HL-428 or HL-1236 cells for 24 h. Cells were sorted and GFP + cells were collected, followed by western blot analysis using p-ERK and total ERK antibodies. Actin staining shows the protein loading control. b Soluble PD-1 is increased in the serum of the HL patients. Serum samples were obtained from HL patients and normal donors and used to determine the concentration of soluble PD-1. As shown in the dot plot, the concentration of soluble PD-1 is significantly higher in HL patients than normal samples (****p < 0.0001, normal serum: n = 17; HL serum: n = 47). c Effect of soluble PD-1 on mitochondrial respiration. HL cell lines were starved overnight and then incubated with recombinant Fc-chimera active PD-1 protein (10 μg/ml) in the presence r absence of Nivolumab. An equal number of the cells were plated on the microplates and used for mitochondrial stress analysis using XFe96 seahorse analyzer and data were analyzed using Wave software. The plot represents oxygen consumption rate (OCR) at baseline and over time following oligomycin, FCCP, and rotenone injection in the HL-1236 cell line. Bar graphs show basal OCR and spare respiratory capacity in three different HL lines