Increased programmed death-ligand-1 expression in human gastric epithelial cells in Helicobacter pylori infection

Abstract

B7-H1 [programmed death-ligand-1 (PD-L1)] is a B7-family member that binds to programmed death-1 (PD-1). Recently, deficiency of PD-L1 has been demonstrated to result in accelerated gastric epithelial cell damage in gastritis, and PD-L1 is suggested to play a critical role in regulating T cell homeostasis. Here, we aimed to gain more insight into gastric PD-L1 expression, regulation and function during Helicobacter pylori infection. PD-L1 expression in human gastric epithelial cells was analysed using Western blotting, quantitative polymerase chain reaction and fluorescence activated cell sorter analysis. Furthermore, co-culture experiments of human gastric epithelial cells with primary human T cells or Jurkat T cells were conducted. PD-L1 expression in primary human gastric epithelial cells was strongly enhanced by H. pylori infection and activated T cells, and augmented markedly by further stimulation with interferon-γ or tumour necrosis factor-α. Moreover, PD-L1 expression in gastric epithelial cells significantly induced apoptosis of T cells. Our results indicate that a novel bidirectional interaction between human gastric epithelial cells and lymphocytes modulates PD-L1 expression in human gastric epithelial cells, contributing to the unique immunological properties of the stomach.

Fig. 1

Western blot of programmed death-ligand-1 (PD-L1) expression using mouse anti-human PD-L1 monoclonal antibody (mAb). (a) Lane 1, primary gastric epithelial cell; lane 2, human gastric adenocarcinoma (AGS); lane 3, gastric biopsy samples from patient negative to Helicobacter pylori infection; lane 4, gastric biopsy samples from H. pylori-infected patient. α-Tubulin was used as an internal control. (b) Densitometric scan was performed using BioRad's Quantity One software. Percentage of adjacent volume was determined for each band of PD-L1 and α-tubulin. Ratio of percentage of adjacent volume was plotted. *P < 0·05 compared to control.

Fig. 2

Programmed death-ligand-1 (PD-L1) expression of human gastric epithelial cells after Helicobacter pylori infection. (a) Human gastric adenocarcinoma (AGS) cells were infected with H. pylori and RNA was isolated 24 and 48 h after infection. Non-infected AGS cells served as controls. (b) Primary human gastric epithelial cells were infected with H. pylori and RNA was isolated 24 and 48 h after infection. Non-infected primary human gastric epithelial cells served as controls. PD-L1 mRNA expression was quantified in relation to β-actin expression by real-time polymerase chain reaction. *P < 0·05 compared to control.

Fig. 3

Programmed death-ligand-1 (PD-L1) expression of gastric epithelial cells after co-incubation with T cells. Human gastric adenocarcinoma (AGS) cells (a) and primary human gastric epithelial cells (b) were co-cultured with resting cells or activated Jurkat T cells as well as naive or activated primary human T cells (PHTC), respectively. Data represent PD-L1/β-actin ratio on gastric epithelial cell. Furthermore, Transwell co-cultures were established with AGS cells and activated Jurkat T cells or activated primary human T cells (PHTC), respectively (c). PD-L1 mRNA expression in AGS cells and primary human gastric epithelial cells was quantified in relation to β-actin expression by real-time polymerase chain reaction. AGS cells and primary human gastric epithelial cells in monoculture served as controls.*P < 0·05 compared to control.

Fig. 5

Effects of interferon (IFN)-γ and tumour necrosis factor (TNF)-α on programmed death-ligand-1 (PD-L1) expression in primary human gastric epithelial cells. Primary human gastric epithelial cells were stimulated with IFN-γ (100 U/ml) and/or TNF-α (40 ng/ml) for 24 h (for PD-L1 mRNA analysis) or 48 h (for analysis of PD-L1 surface expression), respectively. (a) PD-L1 mRNA expression was quantified in relation to β-actin expression by real-time polymerase chain reaction. (b) Expression of cell surface PD-L1 determined by fluorescence activated cell sorter (FACS) analysis (open profiles, anti-PD-L1 antibody; filled profiles, isotype control). *P < 0·05 compared to control.

Fig. 4

Effects of interferon (IFN)-γ and tumour necrosis factor (TNF)-α on programmed death-ligand-1 (PD-L1) expression. Human gastric adenocarcinoma (AGS) were stimulated with the indicated concentrations of IFN-γ (a) and TNF-α (c) for 24 h, and PD-L1 expression was analysed. Time–course of IFN-γ (b) and TNF-α (d) induced PD-L1 mRNA-expression. At the indicated time intervals after stimulation RNA was isolated and PD-L1 mRNA expression was quantified in relation to β-actin expression by real-time polymerase chain reaction. Data represent means ± standard deviation from three separate experiments.

Fig. 6

Expression of programmed death-ligand-1 (PD-L1) in gastric epithelial cell-mediated T cell apoptosis. Human gastric adenocarcinoma (AGS) cells (1 × 10⁶) were activated by addition of interferon (IFN)-γ (100 U/ml) and/or tumour necrosis factor (TNF)-α (40 ng/ml) in the presence or absence of blocking antibodies to PD-L1 (10 µg/ml). Unbound antibody was removed prior to establishment of co-cultures with Jurkat T cells (5 × 10⁶). After 8 h of co-culture, apoptosis in Jurkat T cells was examined by fluorescence activated cell sorter (FACS) analysis using annexin V/PI-staining. The percentages of apoptotic T cells (annexin V⁺/PI⁺) are indicated. One of three independent experiments is depicted. For positive controls, cells were permeabilized with saponin 0·1% for 1 h.