Upregulation of B7-H1 expression is associated with macrophage infiltration in hepatocellular carcinomas

Abstract

The overexpression of B7-H1 in hepatocellular carcinoma (HCC) mediates HCC immune escape and obstructs the immunotherapy based on tumor-specific CD8+ T cells. Tumor-associated macrophages (TAM) are a major component of cancer-related inflammation and play a central role in tumor promotion. To classify the mechanism underlying the overexpression of B7-H1 in HCC, we examined B7-H1 expression and TAM infiltration in 63 cases of human HCC samples using immunohistochemistry method and found that B7-H1 overexpression was associated with TAM infiltration in HCC tissues. Furthermore, B7-H1 expression was upregulated at both mRNA level and protein level in HCC cells (BEL-7402 and SMMC-7721) cocultured with macrophages in a transwell system. The upregulation of B7-H1 expression induced by macrophage was inhibited by blocking NF-κB or STAT3 signal pathways. These results suggest that overexpression of B7-H1 in HCC may be induced by inflammatory microenvironment involving macrophages and imply that anti-inflammation therapy might be preventive for immune escape and assistant for immunotherapy of HCC.

Fig. 1

B7-H1 overexpression was associated with TAM infiltration in HCC tissues. a Expression of B7-H1 and CD68 was tested in consecutive sections from 63 HCC patients using immunohistochemitry method. CD68-positive staining indicated the TAM infiltration. The strength of B7-H1 expression was classified into grade +, grade ++ and grade +++ using Image-Pro Plus 6.0 software. Three representative cases showed B7-H1 expression and TAM infiltration. Positive cells were stained brown (×200). Negative controls were performed by omitting the primary antibodies. b Statistical analysis of the relationship between B7-H1 expression and TAM infiltration. TAM number in HCC tissues with high expression of B7-H1 was significantly higher than that in HCC tissues with low expression of B7-H1. Data shown are expressed as means ± SE. Asterisk indicates P < 0.05. Double asterisk indicates P < 0.01. Triple asterisk indicates P < 0.001

Fig. 2

B7-H1 expression was upregulated in HCC cells by PMA-treated THP-1 cells. a B7-H1 mRNA expression was upregulated in HCC cells by PMA-treated THP-1 cells. After coculture for 6 and 24 h with PMA-treated THP-1 cells in a transwell system, BEL-7402 (left figure) and SMMC-7721 (right figure) cells were harvested, and B7-H1 mRNA expression was determined by RT-PCR. Lane 1, non-cocultured 6 h; lane 2, cocultured 6 h; lane 3, non-cocultured 24 h; lane 4, cocultured 24 h. Densitometric data shown are means ± SE of the results for three independent experiments. b B7-H1 protein expression was upregulated in HCC cells by PMA-treated THP-1 cells. HCC cells were cocultured with PMA-treated THP-1 cells in a transwell system. After 24 and 48 h of coculture, BEL-7402 (left figure) and SMMC-7721 (right figure) cells were harvested, and B7-H1 protein expression was determined by Western blot. Lane 1, non-cocultured 24 h; lane 2, cocultured 24 h; lane 3, non-cocultured 48 h; lane 4, cocultured 48 h. Densitometric data shown are means ± S.E of the results for three independent experiments. Asterisk indicates P < 0.05. Double asterisk indicates P < 0.01

Fig. 3

B7-H1 expression was upregulated in HCC cells by PMA-induced human macrophages After coculture for 12 and 24 h with PMA-induced human macrophages in a transwell system, BEL-7402 (a) and SMMC-7721 (b) cells were harvested, and B7-H1 mRNA expression was determined by RT-PCR. Lane 1, non-cocultured 12 h; lane 2, cocultured 12 h; lane 3, non-cocultured 24 h; lane 4, cocultured 24 h. Densitometric data shown are means ± S.E of the results for three independent experiments. Asterisk indicates P < 0.05. Double asterisk indicates P < 0.01

Fig. 4

The induction of B7-H1 expression was inhibited by blocking NF-κB pathway. BEL-7402 and SMMC-7721 cells were treated with PDTC (10 ng/mL) for one h before coculture with PMA-treated THP-1 cells. After 6 h of coculture, BEL-7402 (a) and SMMC-7721 (b) cells were harvested, and B7-H1 mRNA expression was determined by RT-PCR. Lane 1, non-cocultured; lane 2, cocultured; lane 3, PDTC-treated and cocultured. Densitometric data shown are the means ± SE of triplicate experiments. Asterisk indicates P < 0.05. Double asterisk indicates P < 0.01

Fig. 5

The induction of B7-H1 expression was inhibited by blocking STAT3 pathway. BEL-7402 and SMMC-7721 cells were treated with AG490 (20 ng/mL) for 24 h before coculture with PMA-treated THP-1 cells. After 6 h of coculture, BEL-7402 (a) and SMMC-7721 (b) cells were harvested, and B7-H1 mRNA expression was determined by RT-PCR. Lane 1, non-cocultured; lane 2, cocultured; lane 3, AG490-treated and cocultured. Densitometric data shown are the means ± SE of triplicate experiments. Asterisk indicates P < 0.05. Double asterisk indicates P < 0.01