Reverse signaling via PD-L1 supports malignant cell growth and survival in classical Hodgkin lymphoma

Abstract

Treatment with programmed death-1 (PD-1) blocking antibodies results in high overall response rates in refractory and relapsed classical Hodgkin lymphoma (cHL) patients, indicating that PD-1/PD-1 ligand interactions are integral to progression of this disease. Given the genetically driven increased PD-L1/2 expression in HL, we hypothesized that reverse signaling through PD-1 ligands may be a potential mechanism contributing to the growth and survival of Hodgkin Reed-Sternberg (HRS) cells in cHL. Our data show that engagement of PD-L1 using an agonistic monoclonal antibody increases cell survival and proliferation and reduces apoptosis in HL cell lines. We show that HL patients have significantly higher serum levels of soluble PD-1 than healthy controls, and find that both membrane-bound and soluble forms of PD-1 are able to induce PD-L1 reverse signaling in HL cell lines. PD-L1 signaling, which is associated with activation of the MAPK pathway and increased mitochondrial oxygen consumption, is reversed by PD-1 blockade. In summary, our data identify inhibition of reverse signaling through PD-L1 as an additional mechanism that accounts for clinical responses to PD-1 blockade in cHL.

Fig. 1. Stimulation of the HL cell lines with PD-L1 antibody increases cell survival and proliferation and reduces apoptosis.

HL cell lines (HL-428, HL-1236, HL-HDLM2, and HL-KMH2) were starved overnight and plated over either isotype control (IgG1) antibody- or PD-L1 antibody-coated (10 μg/ml) plates for 48 h. a Flow-cytometry analysis showing Annexin/PI staining on four different HL cell lines. b Bar graphs showing the proliferation rate of HL cell lines, in response to incubation with isotype control (blue) or PD-L1 antibody (red). c Apoptotic protein array illustrating the differences between apoptotic proteins and phospho-proteins in the HL-428 cells that are incubated with isotype control or PD-L1 antibody (left panel). Bar graphs show the quantitative analysis of the protein dots using ImageJ (right). d Flow-cytometry analysis (left) displays the intracellular staining with p-ERK and p-P38 antibodies in two HL cell lines incubated with isotype control or PD-L1 antibodies. Bar graphs (right) represent the percentage of the cells that are positive for p-P38 and p-ERK in four different cell lines

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