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. 2012 Jul;169(1):1-9.
doi: 10.1111/j.1365-2249.2012.04585.x.

Cytosine-phosphate-guanosine-DNA induces CD274 expression in human B cells and suppresses T helper type 2 cytokine production in pollen antigen-stimulated CD4-positive cells

S Kubo  1 , T Yamada, Y Osawa, Y Ito, N Narita, S Fujieda
Affiliations

Cytosine-phosphate-guanosine-DNA induces CD274 expression in human B cells and suppresses T helper type 2 cytokine production in pollen antigen-stimulated CD4-positive cells

S Kubo et al. Clin Exp Immunol. 2012 Jul.

Abstract

Co-stimulatory molecules are important for regulating T cell activation and immune response. CD274 [programmed death ligand 1 (PD-L1), B7-H1] has emerged as an important immune modulator that can block T cell receptor signalling. We have investigated whether PD-L1 and other co-stimulatory ligands could be expressed in human B cells stimulated by cytosine-phosphate-guanosine (CpG)-DNA. CpG-DNA strongly induced the co-inhibitory molecule ligand, PD-L1, of human B cells. Results show that nuclear factor-kappa B (NF-κB) signalling is involved directly in CpG-DNA-induced PD-L1 expression in human B cells. We sought to determine the effect of CpG-DNA-treated B cells on T helper type 2 (Th2) cytokine production in Cry j 1 (Japanese pollen antigen)-stimulated human CD4-positive cells from patients with seasonal allergic rhinitis caused by Japanese cedar pollen. CpG-DNA-treated B cells reduced Cry j 1-induced interleukin (IL)-5 and IL-13 production in CD4-positive cells. When the binding of PD-1 to PD-L1 was inhibited by PD-1-immunoglobulin (Ig), this chimera molecule reversed the previously described reductions in IL-5 and IL-13 production. In contrast, the CpG B-treated B cells increased both interferon (IFN)-γ and IL-12 production in the presence of Cry j 1-stimulated CD4-positive cells. CpG-DNA simultaneously reduced the expression of B7RP-1 [also known as inducible co-stimulator ligand (ICOSL), B7-H2] and the ligand of CD30 (CD30L). These results indicate that CpG-DNA induces co-inhibitory molecule ligand PD-L1 expression in human B cells and PD-L1 can suppress Th2 cytokine production in Cry j 1-stimulated CD4-positive cells, while CpG-DNA increased Th1 cytokine production and reduced the expression of co-stimulatory molecule ligands that can promote Th2 inflammatory responses.

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Figures

Fig. 1
Fig. 1
(a) Programmed death ligand 1 (PD-L1) expression induced by cytosine–phosphate–guanosine (CpG)-DNA in human B cells. Human B cells from peripheral blood mononuclear cells (PBMC) and Ramos 2G6 cells were incubated in the presence or absence of 1 µM oligodeoxynucleotide (ODN) 2216 (CpG-A) or ODN 2006 (CpG-B) for 6 h. The mRNA was reverse-transcribed to cDNA, which was then used for real-time polymerase chain reaction (PCR). All reactions were performed in triplicate. The results were normalized to the levels of β2 microglobulin mRNA. The data are presented as the mean ± standard error of the mean fold increase relative to the control (n = 6); *P < 0·05. (b) CpG-DNA induces PD-L1 surface expression on human B cells. Human B cells from PBMC were incubated in the presence or absence of 1 µM ODN 2006 (CpG-B) for 48 h. Cells were harvested and the expression of PD-L1 was analysed using a fluorescence activated cell sorter (FACS)Caliber. The open histogram indicates staining with isotype control, and the closed histogram indicates staining with allophycocyanin (APC)-anti-PD-L1 antibody. (c) Human B cells from PBMC were cultured with medium in the presence or absence of 1 µM ODN 2216 (CpG-A) or ODN 2006 (CpG-B) for 24 h. After the cells had been harvested, the samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted. The presence of PD-L1 and β-actin was monitored by Western blot/enhanced chemiluminescence (ECL) reaction using anti-PD-L1 antibody and anti-β-actin antibody. The positions of PD-L1 and β-actin are indicated by arrows.
Fig. 2
Fig. 2
Effect of cytosine–phosphate–guanosine (CpG)-DNA on expression levels of the ligand of co-stimulatory molecules; (a) B7-related protein-1 (B7RP-1), (b) CD30L. Human B cells from peripheral blood mononuclear cells (PBMC) were incubated in the presence or absence of 1 µM oligodeoxynucleotide (ODN) 2216 (CpG-A) or ODN 2006 (CpG-B) for 6 h. The mRNA was reverse-transcribed to cDNA, which was then used for real-time polymerase chain reaction (PCR). All reactions were performed in triplicate. The results were normalized to the levels of β2 microglobulin mRNA. Data are presented as the mean ± standard error of the mean fold increase relative to the control (n = 6); *P < 0·05.
Fig. 3
Fig. 3
The effect of cytosine–phosphate–guanosine (CpG)-DNA on nuclear factor (NF)-κB signalling related to programmed death ligand 1 (PD-L1) expression in human B cells. (a) Effects of signal transduction inhibitors on CpG-DNA-induced PD-L1 expression. Human B cells from peripheral blood mononuclear cells (PBMC) were pre-incubated with 10% dimethylsulphoxide (DMSO) (a vehicle control), 10 µM SP600125 [a c-Jun N-terminal kinase (JNK) inhibitor], 10 µM SB203580 [a p38 mitogen-activated protein kinase (MAPK) inhibitor], 10 µM U0126 [an extracellular signal-related kinase (ERK) inhibitor], 10 µM Bay 117082 (a NF-κB signalling inhibitor), 100 µM NEMO-binding domain (NBD) peptide (an NF-κB signalling inhibitor) or control NBD peptide for 30 min. The cells were then stimulated with 1 µM oligodeoxynucleotide (ODN) 2006 (CpG-B) for 6 h. The mRNA was reverse-transcribed to cDNA, which was then used for real-time polymerase chain reaction (PCR). All reactions were performed in triplicate. The results were normalized to the expression of β2 microglobulin mRNA. Data are expressed as the mean ± standard error of the mean fold increase relative to the control (n = 6); *P < 0·05. (b) The phosphorylation of inhibitor kappa B α(IκBα) induced by CpG-DNA. After pre-incubation with Bay 11-7082 (10 µM) or NBD peptide (100 µM) NF-κB signalling inhibitors for 30 min, the human B cells were stimulated with or without CpG-B (1 µM) for 30 min. Then the cells were harvested, lysed, applied to each lane and blotted with anti-phosphorylated IκBα antibody. The position of phosphorylated IκBα is indicated on the right by the arrow.
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