Human PDCD1 Stable Cell Line-HEK293T

Treatment of patients with refractory and relapsed classical Hodgkin lymphoma (cHL) with programmed death-1 (PD-1 or PDCD1) blocking antibodies resulted in high overall response rates, suggesting that PD-1/PD-1 ligand interactions are integral to the progression of this disease. Given the genetically driven increase in PD-L1/2 expression in HL, researchers hypothesized that reverse signaling through PD-1 ligands may be a potential mechanism for promoting growth and survival of Hodgkin-Reed-Sternberg (HRS) cells in cHL. Here, data show that binding to PD-L1 with an agonist monoclonal antibody increased cell survival and proliferation and reduced apoptosis in HL cell lines. Researchers found that soluble PD-1 levels were significantly higher in the serum of HL patients than in healthy controls, and that both membrane-bound and soluble forms of PD-1 induced PD-L1 reverse signaling in HL cell lines. PD-L1 signaling was associated with MAPK pathway activation and increased mitochondrial oxygen consumption, which was reversed by PD-1 blockade. Together, these data suggest that inhibition of PD-L1 reverse signaling is an additional mechanism underlying clinical response to PD-1 blockade in patients with classical Hodgkin lymphoma (cHL).

The researchers hypothesized that the interaction between the PD-1 ligand and the PD-1 receptor would generate a signal similar to that seen with agonist anti-PD-L1 antibodies. They first tested the stimulatory effects of membrane-bound PD-1 and evaluated the efficacy of the blocking antibody nivolumab in counteracting such effects. Doxycycline-inducible PD-1 overexpressing HEK293 cells were constructed and used as membrane-bound receptors for PD-L1. Coculture of HL-428 and HL-1236 cells with PD-1 overexpressing HEK293 cells resulted in decreased p-ERK protein in HL cells, whereas nivolumab treatment reversed this effect and increased p-ERK to normal levels (Figure 1). Given that decreased ERK and P38 phosphorylation are associated with pro-survival signaling, these data are consistent with flow cytometry results showing decreased p-ERK levels after incubation with agonist anti-PD-L1 antibodies and confirm that PD-L1 reverse signaling is also a result of PD-L1/PD-1 interactions. Furthermore, treatment with the anti-PD-1 antibody nivolumab neutralizes this signaling process.

Figure 1. Effect of membrane-bound PD-1 on ERK phosphorylation. (Jalali S, et al., 2019)