PD-L1 is upregulated by EBV-driven LMP1 through NF-κB pathway and correlates with poor prognosis in natural killer/T-cell lymphoma

Abstract

Background: Natural killer/T-cell lymphoma (NKTCL) is an Epstein-Barr virus (EBV)-associated, highly aggressive lymphoma. Treatment outcome remains sub-optimal, especially for advanced-stage or relapsed diseases. Programmed cell death receptor 1 (PD-1) and PD ligand 1 (PD-L1) have become promising therapeutic targets for various malignancies, but their role in the pathogenesis and their interactions with EBV in NKTCL remains to be investigated.

Methods: Expression of PD-L1 was measured in NK-92 (EBV-negative) and SNK-6 (EBV-positive) cells by western blot, quantitative real-time PCR and enzyme-linked immunosorbent assay, and flow cytometry, respectively. Latent membrane protein 1 (LMP1)-harboring lentiviral vectors were transfected into NK-92 cells to examine the correlation between LMP1 and PD-L1 expression. Proteins in the downstream pathways of LMP1 signaling were measured in NK-92 cells transfected with LMP1-harboring or negative control vectors as well as in SNK-6 cells. PD-L1 expression on tumor specimens and serum concentration of soluble PD-L1 were collected in a retrospective cohort of patients with Ann Arbor stage I~II NKTCL, and their prognostic significance were analyzed.

Results: Expression of PD-L1 was significantly higher in SNK-6 cells than in NK-92 cells, at both protein and mRNA levels. Expression of PD-L1 was remarkably upregulated in NK-92 cells transfected with LMP1-harboring lentiviral vectors compared with those transfected with negative control vectors. Proteins in the MAPK/NF-κB pathway were upregulated in LMP1-expressing NK-92 cells compared with the negative control. Selective inhibitors of those proteins induced significant downregulation of PD-L1 expression in LMP1-expressing NK-92 cells as well as in SNK-6 cells. Patients with a high concentration of serum soluble PD-L1 (≥3.4 ng/ml) or with a high percentage of PD-L1 expression in tumor specimens (≥38 %) exhibited significantly lower response rate to treatment and remarkably worse survival, compared with their counterparts. A high concentration of serum soluble PD-L1 and a high percentage of PD-L1 expression in tumor specimens were independent adverse prognostic factors among patients with stage I~II NKTCL.

Conclusions: PD-L1 expression positively correlated LMP1 expression in NKTCL, which was probably mediated by the MAPK/NF-κB pathway. PD-L1 expression in serum and tumor tissues has significant prognostic value for early-stage NKTCL.

Keywords: Epstein–Barr virus; Latent membrane protein 1; Natural killer/T-cell lymphoma; Programmed cell death receptor 1.

Fig. 1

Expression of PD-L1 in NK cell line NK-92 (EBV-negative) and NKTCL cell line SNK-6 (EBV-positive). The level of a PD-L1 protein detected by western blot, b PD-L1 mRNA detected by quantitative real-time PCR, c soluble PD-L1 protein in cell culture supernatant detected by ELISA, and d, e PD-L1 expression on cell surface detected by flow cytometry in NK-92 and SNK-6 cell lines, respectively. ^** P < 0.05

Fig. 2

Expression of PD-L1 was upregulated by LMP1 in NK-92 cells. a, b Infection efficiency of LV5-LMP1 vector (a) and LV5 vector (LV5-NC, b) in NK-92 cell line at a multiplicity of infection (MOI) of 100, 200, and 300, respectively. The level of c LMP1 and PD-L1 proteins detected by western blot, d LMP1 and e PD-L1 mRNA detected by quantitative real-time PCR, f soluble PD-L1 protein in cell culture supernatant detected by ELISA, and g, h PD-L1 expression on cell surface detected by flow cytometry in LMP1-expressing NK92 (LMP1) and negative control NK92 (NC) cell lines, respectively. ^** P < 0.05

Fig. 3

LMP1 upregulated PD-L1 expression through MAPK/NF-κB pathway in NK92 cells. a The expression level of p-Raf-B, Raf-B, p-p38, p38, p-JNK, JNK, p-ERK, ERK, and p65 in LMP1-expressing NK92 (LMP1) and negative control NK92 (NC) cell lines. b The expression level of p-Raf-B, Raf-B, p-p38, p38, p-JNK, JNK, p-ERK, ERK, p65, and PD-L1 in negative control NK92 (NC) or LMP1-expressing NK92 (LMP1) cell line treated with 0.1 μM SB590885, a selective B-Raf inhibitor for 1 h. c The expression level of p-ERK, ERK, p65, and PD-L1 in negative control NK92 (NC) or LMP1-expressing NK92 (LMP1) cell line treated with 20 μM PD98059, a selective ERK inhibitor for 1 h. d The expression level of p-p38, p38, p65, and PD-L1 in negative control NK92 (NC) or LMP1-expressing NK92 (LMP1) cell line treated with 10 μM SB203580, a selective p38 inhibitor for 1 h. e The expression level of p-JNK, JNK, p65, and PD-L1 in negative control NK92 (NC) or LMP1-expressing NK92 (LMP1) cell line treated with 20 μM SP600125, a selective JNK inhibitor for 1 h. f The expression level of p65 and PD-L1 in negative control NK92 (NC) or LMP1-expressing NK92 (LMP1) cell line treated with 100 μM pyrrolidine dithiocarbamate (PDTC), a selective inhibitor of NF-κB for 1 h

Fig. 4

a Concentration of serum soluble PD-L1 in patients with natural killer/T-cell lymphoma (NKTCL) and healthy individuals. b Correlation between concentration of serum soluble PD-L1 (ng/ml) and PD-L1 expression in tumor tissues (%) in patients with NKTCL

Fig. 5

a Progression-free survival (PFS) for NKTCL patients with a serum soluble PD-L1 of <3.4 and ≥3.4 ng/ml. b Overall survival (OS) for NKTCL patients with a serum soluble PD-L1 of <3.4 and ≥3.4 ng/ml. c PFS for NKTCL patients with a histological PD-L1 expression of <38 and ≥38 %. c OS for NKTCL patients with a histological PD-L1 expression of <38 and ≥38 %