不同病毒载量慢性乙型肝炎患者树突状细胞B7-H1表达及对免疫功能的影响

目的：观察慢性乙型肝炎患者外周血树突状细胞表面共刺激分子B7-H1 的表达及对免疫功能的影响。方法：检测慢性乙型肝炎患者肝功能、HBV-DNA 水平,将患者分为高病毒载量高ALT 组（A 组）、高病毒载量低ALT 组（B 组）、低病毒载量组（C 组）及正常对照组（D 组）。流式细胞术检测各组患者外周血树突状细胞表面HLA-DR、CD80、CD86、CD83、CD1a、B7-H1 表达,酶联免疫吸附试验（ELISA）检测DC 培养上清液和混合淋巴细胞培养上清液中细胞因子IL-12、IL-10 水平。结果：慢性乙肝患者的树突状细胞膜表面分子HLA-DR、CD80、CD86、CD83、CD1a的表达均明显降低（A、B、C组与D组比较分别为42.3± 4.9 %、46.7± 7.0%、52.5± 6.3 %vs 94.5± 3.5 %;34.5± 5.3 %、39.9± 6.4 %、45.6± 5.2 %vs 90.6± 6.5 %;38.2± 8.6 %、36.1± 5.4 %、42.5± 6.8 % vs87.7± 5.1 %;28.3± 6.5 %、25.6± 3.4 %、33.5± 4.3% vs 82.6± 4.8 %;32.3± 5.8 %、29.3± 5.3 %、48.3± 4.9 % vs 68.2± 5.2 % P〈0.05）,B7-H1 表达水平明显升高（27.48± 21.4 %、21.83± 20.2 %、15.43± 10.32 %vs 4.23± 2.2%P〈0.05）。B7-H1 表达水平与ALT呈正相关,与IL-12 水平呈负相关。结论：慢性乙型肝炎患者树突状细胞功能低下,其机制可能与树突状细胞高表达B7-H1 有关。B7-H1 高表达抑制了淋巴细胞的功能,导致乙型肝炎病毒持续感染。     

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.     

不同病毒载量慢性乙型肝炎患者树突状细胞 B7-H1 表达及对 免疫功能的影响 *

摘要 目的： 观察慢性乙型肝炎患者外周血树突状细胞表面共刺激分子 B7-H1 的表达及对免疫功能的影响。方法： 检测慢性乙型 肝炎患者肝功能、 HBV-DNA 水平, 将患者分为高病毒载量高 ALT 组 （ A 组） 、 高病毒载量低 ALT 组 （B 组） 、低病毒载量组 （C 组） 及正常对照组 （D 组）。 流式细胞术检测各组患者外周血树突状细胞表面 HLA-DR、 CD80、 CD86、 CD83、 CD1a、 B7-H1 表达, 酶联免 疫吸附试验 （ELISA ） 检测 DC 培养上清液和混合淋巴细胞培养上清液中细胞因子 IL-12、 IL-10 水平。结果： 慢性乙肝患者的树突 状细胞膜表面分子 HLA-DR、 CD80、 CD86、 CD83、 CD1a 的表达均明显降低 (A、 B、 C 组与 D 组比较分别为 42.3± 4.9%、 46.7± 7.0 %、 52.5 ± 6.3 % vs 94.5± 3.5%; 34.5 ± 5.3%、 39.9 ± 6.4%、 45.6 ± 5.2 % vs 90.6± 6.5%; 38.2 ± 8.6%、 36.1 ± 5.4%、 42.5 ± 6.8 % vs 87.7 ± 5.1%; 28.3 ± 6.5%、 25.6 ± 3.4%、 33.5 ± 4.3% vs 82.6 ± 4.8%; 32.3 ± 5.8%、 29.3 ± 5.3%、 48.3 ± 4.9 % vs 68.2 ± 5.2 % P< 0.05), B7-H1 表达水平明显升高(27.48± 21.4%、 21.83± 20.2%、 15.43± 10.32 % vs 4.23± 2.2 % P<0.05)。B7-H1 表达水平与 ALT 呈正相关, 与 IL-12 水平呈负相关。 结论： 慢性乙型肝炎患者树突状细胞功能低下, 其机制可能与树突状细胞高表达 B7-H1 有关。 B7-H1 高表达抑制了淋巴细胞的功能, 导致乙型肝炎病毒持续感染。     

B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion

The B7 family members B7-1 and B7-2 interact with CD28 and constitute an essential T-cell co-stimulatory pathway in the initiation of antigen-specific humoral and cell-mediated immune response. Here, we describe a third member of the B7 family, called B7-H1 that does not bind CD28, cytotoxic T-lymphocyte A4 or ICOS (inducible co-stimulator). Ligation of B7-H1 co-stimulated T-cell responses to polyclonal stimuli and allogeneic antigens, and preferentially stimulated the production of interleukin-10. Interleukin-2, although produced in small amounts, was required for the effect of B7-H1 co-stimulation. Our studies thus define a previously unknown co-stimulatory molecule that may be involved in the negative regulation of cell-mediated immune responses.     

Molecular characterization of human 4Ig-B7-H3, a member of the B7 family with four Ig-like domains

In an effort to characterize molecules with immunoregulatory potential, we raised mAbs to human dendritic cells. We selected an Ab that recognizes a molecule that is induced on monocytes differentiated in vitro toward dendritic cells. Retroviral expression cloning identified this molecule as B7-H3, a member of the B7 family described recently. In contrast to an earlier report, in which B7-H3 was described as a molecule consisting of two Ig-like domains, our cDNA encoded a type I membrane protein with four Ig-like domains, and the molecule identified by us was therefore named 4Ig-B7-H3. mRNA analysis as well as Western blotting experiments performed by us did not reveal evidence for a small B7-H3. B7-H3 is not expressed on peripheral blood lymphocytes, monocytes, or granulocytes. Upon in vitro stimulation, the expression of B7-H3 is induced on T cells, B cells, and NK cells. A number of different approaches were used to investigate the function of human B7-H3. In contrast to an earlier report, our data do not support a costimulatory role of B7-H3 in anti-CD3-mediated activation of the TCR-complex resulting in T cell proliferation and IFN-gamma production.     

Blockade of B7-H1 improves myeloid dendritic cell-mediated antitumor immunity

Suppression of dendritic cell function in cancer patients is thought to contribute to the inhibition of immune responses and disease progression. Molecular mechanisms of this suppression remain elusive, however. Here, we show that a fraction of blood monocyte-derived myeloid dendritic cells (MDCs) express B7-H1, a member of the B7 family, on the cell surface. B7-H1 could be further upregulated by tumor environmental factors. Consistent with this finding, virtually all MDCs isolated from the tissues or draining lymph nodes of ovarian carcinomas express B7-H1. Blockade of B7-H1 enhanced MDC-mediated T-cell activation and was accompanied by downregulation of T-cell interleukin (IL)-10 and upregulation of IL-2 and interferon (IFN)-gamma. T cells conditioned with the B7-H1-blocked MDCs had a more potent ability to inhibit autologous human ovarian carcinoma growth in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. Therefore, upregulation of B7-H1 on MDCs in the tumor microenvironment downregulates T-cell immunity. Blockade of B7-H1 represents one approach for cancer immunotherapy.     

B7-H3 is Overexpressed in Patients Suffering Osteosarcoma and Associated with Tumor Aggressiveness and Metastasis

B7-H3 is a member of the B7-family of co-stimulatory molecules, which has been shown to be broadly expressed in various tumor tissues, and which plays an important role in adaptive immune responses. The role of B7-H3 in osteosarcoma, however, remains unknown. In this study we used immunohistochemistry to analyze B7-H3 expression in 61 primary osteosarcoma tissues with case-matched adjacent normal tissues, and 37 osteochondroma and 20 bone fibrous dysplasia tissues. B7-H3 expression was expressed in 91.8% (56/61) of the osteosarcoma lesions, and the intensity of B7-H3 expression in osteosarcoma was significantly increased compared with adjacent normal tissues, osteochondroma and bone fibrous dysplasia tissues (p<0.001). Patients with high tumor B7-H3 levels had a significantly shorter survival time and recurrence time than patients with low tumor B7-H3 levels (p<0.001). Moreover, tumor B7-H3 expression inversely correlated with the number of tumor-infiltrating CD8⁺ T cells (p<0.05). In vitro, increasing expression of B7-H3 promotes osteosarcoma cell invasion, at least in part by upregulating matrix metalloproteinase-2 (MMP-2). In conclusion, our study provides the first evidence of B7-H3 expression in osteosarcoma cells as a potential mechanism controlling tumor immunity and invasive malignancy, and which is correlated with patients’ survival and metastasis.     

Expression of B7-H1 and B7-DC on the airway epithelium is enhanced by double-stranded RNA

Viral infection in the airway provokes various immune responses, including Th1 and Th2 responses, which are partly initiated by double-stranded RNA (dsRNA), a viral product for its replication. B7-H1 (PD-L1) and B7-DC (PD-L2) are B7-family molecules that bind to programmed death-1 (PD-1) on lymphocytes and are implicated in peripheral tolerance. We investigated the effect of dsRNA on the expression of B7-H1 and B7-DC on airway epithelial cell lines. B7-H1 and B7-DC were constitutively expressed on the cells, and their expression was profoundly upregulated by stimulation with an analog of viral dsRNA, polyinosinic-polycytidylic acid. B7-H1 and B7-DC were also upregulated by stimulation with IFN-gamma, IL-13, and the supernatant from T cell clones. A relatively high concentration of dexamethasone (1 microM) was required to suppress the upregulation of B7-H1 or B7-DC. These results suggest that epithelial B7-H1 and B7-DC play a role in virus-associated immune responses in the airways.     

Murine B7-H3 is a negative regulator of T cells

T cell activation is regulated by the innate immune system through positive and negative costimulatory molecules. B7-H3 is a novel B7-like molecule with a putative receptor on activated T cells. Human B7-H3 was first described as a positive costimulator, most potently inducing IFN-gamma production and cellular immunity. In this study we examined the expression and function of mouse B7-H3. B7-H3 is mostly expressed on professional APCs; its expression on dendritic cells appears to be up-regulated by LPS. In contrast to human B7-H3, we found that mouse B7-H3 protein inhibited T cell activation and effector cytokine production. An antagonistic mAb to B7-H3 enhanced T cell proliferation in vitro and led to exacerbated experimental autoimmune encephalomyelitis in vivo. Therefore, mouse B7-H3 serves as a negative regulator of T cell activation and function.     

B7-H1 up-regulation on myeloid dendritic cells significantly suppresses T cell immune function in patients with chronic hepatitis B

Although dysfunctional dendritic cells contribute to inadequate adaptive immunity in chronic hepatitis B (CHB), underlying molecular mechanisms remain largely undefined. In this study, we examined B7-H1 expression on circulating myeloid dendritic cells (mDCs) in 46 CHB patients, 10 autoimmune hepatitis patients, and 10 healthy subjects as control. We found that B7-H1 expression is significantly up-regulated on circulating mDCs of CHB and autoimmune hepatitis patients compared with healthy individuals. The B7-H1 up-regulation was significantly correlated with an elevation of serum alanine aminotransaminase levels and plasma viral load. In addition, in vitro, both IFN-alpha and IFN-gamma could strongly stimulate mDCs to express B7-H1. More importantly, elevated B7-H1 expression is also closely associated with the suppression of T cell immune function. In vitro blockade of B7-H1 signaling could not only down-regulate IL-10 and up-regulate IL-12 production by mDCs, but also enhance mDC-mediated allostimulatory capacity and cytokine production of T cells. Blockade of B7-H1 signaling could improve hepatitis B c Ag-pulsed monocyte-derived DC-induced IFN-gamma production by autologous hepatitis B virus-specific T cells. These new findings suggested that chronic inflammation may contribute to B7-H1 up-regulation on mDCs in CHB patients, which potentially cause defective hepatitis B virus-specific T cell function and viral persistence. Our findings further support the notion that the blockade of B7-H1 may represent a novel therapeutic approach for this disease.     

Characterization of mouse and human B7-H3 genes

T cell activation and immune function are regulated by costimulatory molecules of the B7 superfamily. Human B7-H3 is a recent addition to this family and has been shown to mediate T cell proliferation and IFN-gamma production. In this work we describe the identification of the mouse B7-H3 homolog, which is ubiquitously expressed in a variety of tissues. Activated CD4 and CD8 T cells express a putative receptor that can be recognized by soluble mouse B7-H3-Ig molecules. While the mouse B7-H3 gene was found to contain a single copy, we discovered a novel isoform of human B7-H3 (named as B7-H3b hereafter) with four Ig-like domains that results from gene duplication and differential splicing. B7-H3b is the major isoform expressed in several tissues. This structural information suggests a genetic variation of the B7-H3 gene in mammalian species.     

B7-H1-induced apoptosis as a mechanism of immune privilege of corneal allografts

The programmed death-1 (PD-1) costimulatory pathway has been demonstrated to play a role in the regulation of immune responses and peripheral tolerance. We investigated the role of this pathway in establishing an immune privilege status of corneal allografts in mice. B7-H1, but not B7-DC or PD-1, was expressed constitutively in the eye, i.e., cornea, iris-ciliary body, and retina. After corneal allografting, PD-1(+)CD4(+) T cells infiltrated and adhered with B7-H1(+) corneal endothelium. Blockade of PD-1 or B7-H1, but not B7-DC, led to accelerated corneal allograft rejection. In B7-H1-expressing corneal allografts, apoptosis of the infiltrating PD-1(+)CD4(+) or CD8(+) T cells was observed, after which there was allograft acceptance. In contrast, B7-H1 blockade suppressed apoptosis of infiltrating PD-1(+) T cells, which led to allograft rejection. In vitro, destruction of corneal endothelial cells by alloreactive T cells was enhanced when the cornea was pretreated with anti-B7-H1 Ab. This is the first demonstration that the constitutive expression of B7-H1 plays a critical role in corneal allograft survival. B7-H1 expressed on corneal endothelial cells maintains long-term acceptance of the corneal allografts by inducing apoptosis of effector T cells within the cornea.     

CNS expression of B7-H1 regulates pro-inflammatory cytokine production and alters severity of Theiler's virus-induced demyelinating disease

The CNS is a unique organ due to its limited capacity for immune surveillance. As macrophages of the CNS, microglia represent a population originally known for the ability to assist neuronal stability, are now appreciated for their role in initiating and regulating immune responses in the brain. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease is a mouse model of multiple sclerosis (MS). In response to TMEV infection in vitro, microglia produce high levels of inflammatory cytokines and chemokines, and are efficient antigen-presenting cells (APCs) for activating CD4(+) T cells. However, the regulatory function of microglia and other CNS-infiltrating APCs in response to TMEV in vivo remains unclear. Here we demonstrate that microglia increase expression of proliferating cell nuclear antigen (PCNA), and phenotypically express high levels of major histocompatibility complex (MHC)-Class I and II in response to acute infection with TMEV in SJL/J mice. Microglia increase expression of the inhibitory co-stimulatory molecule, B7-H1 as early as day 5 post-infection, while CNS-infiltrating CD11b(+)CD11c(-)CD45(HIGH) monocytes/macrophages and CD11b(+)CD11c(+)CD45(HIGH) dendritic cells upregulate expression of B7-H1 by day 3 post-infection. Utilizing a neutralizing antibody, we demonstrate that B7-H1 negatively regulates TMEV-specific ex vivo production of interferon (IFN)-γ, interleukin (IL)-17, IL-10, and IL-2 from CD4(+) and CD8(+) T cells. In vivo blockade of B7-H1 in SJL/J mice significantly exacerbates clinical disease symptoms during the chronic autoimmune stage of TMEV-IDD, but only has minimal effects on viral clearance. Collectively, these results suggest that CNS expression of B7-H1 regulates activation of TMEV-specific T cells, which affects protection against TMEV-IDD.     

Differential binding properties of B7-H1 and B7-DC to programmed death-1

Programmed death-1 (PD-1) is a negative regulatory receptor expressed on activated T and B cells. Two ligands for PD-1, B7-H1 (PD-L1) and B7-DC (PD-L2), have been identified, but their binding properties have not been characterized yet. In this study, we generated soluble Ig fusion proteins of these molecules and examined the kinetics and relative affinities of the interactions between B7-H1 or B7-DC and PD-1 by flow cytometry and surface plasmon resonance. The interaction of B7-DC/PD-1 exhibited a 2-6-fold higher affinity and had different association/dissociation kinetics compared with the interaction of B7-H1/PD-1. Our results suggest that the differential binding properties of B7-H1 and B7-DC may be responsible for differential contributions of these two PD-1 ligands to immune responses.     

Clinical significance and regulation of the costimulatory molecule B7-H3 in human colorectal carcinoma

B7-H3, a member of the B7-family molecules, plays an important role in adaptive immune responses, and was shown to either promote or inhibit T-cell responses in various experimental systems. B7-H3 was expressed in some human cancers and correlated with poor outcome of cancer patients. However, its exact role in cancer is not known. In the present study, we studied the expression of B7-H3 in the pathologic specimens of 102 patients treated for colorectal carcinoma (CRC) by immunohistochemistry. Strong B7-H3 expression was found in cancer tissues from 54.3% CRC patients, while minimal expression was found in adjacent normal colorectal tissues. Higher B7-H3 expression in tumor positively correlated with a more advanced tumor grade. In addition, consistent with a role of B7-H3 in suppressing tumor immune surveillance, the expression of B7-H3 in cancer cells negatively correlated with the intensity of tumor infiltrating T lymphocytes in both tumor nest and tumor stroma. Furthermore, we found that the level of soluble B7-H3 in sera from CRC patients was higher than healthy donors. TNF-α, an important cancer-promoting inflammatory molecule, was subsequently found to significantly increase the release of soluble B7-H3 in colon cancer cell lines. Therefore, our data suggest that both soluble and membranous B7-H3 proteins are involved in colon cancer progression and evasion of cancer immune surveillance.     

共刺激因子B7-H1和B7-H3蛋白表达与胆囊癌病例特征相关

本研究选取2015年3月至2017年5月在我院保存的胆囊癌标本55例,同选取癌旁正常组织(距癌边缘>5 cm)作为对照,采用免疫组化染色检测B7-H1和B7-H3蛋白表达,分析B7-H1和B7-H3表达与胆囊癌临床病理特征和预后的关系,探讨共刺激因子B7-H1和B7-H3蛋白在胆囊癌中的表达及意义。研究结果表明:胆囊癌组织B7-H1和B7-H3蛋白阳性表达率分别为76.36%和63.63%,明显高于癌旁组织(p<0.05);B7-H1蛋白表达与胆囊癌TNM分期、淋巴结转移和侵袭深度有关(p<0.05);B7-H3蛋白表达与胆囊癌TNM分期、分化程度、淋巴结转移和侵袭深度有关(p<0.05);胆囊癌组织中B7-H1与B7-H3蛋白表达呈正相关(rs=0.516, p<0.05);B7-H1和B7-H3表达双阳性者和B7-H1或B7-H3单一表达阳性者中位总生存时间分别为20个月和21个月,明显低于B7-H1和B7-H3表达双阴性者组,差异比较有统计学意义(p<0.05)。本研究结论认为:B7-H1和B7-H3蛋白表达与胆囊癌病理特征有关系,两者间有一定相关性,且与患者预后有关。     

B7-H4 enhances oncogenicity and inhibits apoptosis in pancreatic cancer cells

B7-H4 is expressed in a variety of tumor cells and functions as a negative regulator of T cells. However, clarification is needed as to whether B7-H4 mediates tumorigenesis through mechanisms, such as apoptosis, in addition to mediating tumor immune escape. We investigate the mechanisms involved in enhanced oncogenicity and the inhibition of apoptosis by B7-H4 in pancreatic cancer cells. Short interfering RNAs (siRNAs) specific for B7-H4 were evaluated for their ability to knockdown B7-H4 mRNA and protein expression in pancreatic cancer cells and the most effective siRNA was selected for investigating the effect of B7-H4 gene silencing in a number of functional assays. The inhibition of B7-H4 increased cell-cell adhesion and decreased the formation of pseudopodia. It also increased the expression of E-cadherin and decreased the expression of vimentin and CD44. B7-H4 siRNA inhibited cell proliferation, colony formation and migration of pancreatic cancer cells. Moreover, increased apoptosis in pancreatic cancer cells following B7-H4 silencing was demonstrated in vitro by using flow cytometry and in a xenograft tumor model and was associated with increased caspase activity and decreased Erk1/2 phosphorylation both in vitro and in vivo. Loss of B7-H4 function thus prevents tumor growth through many processes, including the induction of apoptosis and inhibition of the Erk1/2 signaling pathway indicating that B7-H4 is a cancer promoter and a potentially important therapeutic target. B7-H4 inhibition might offer an exciting opportunity to inhibit the progression of human pancreatic cancers.     

Increased B7-H1 expression on dendritic cells correlates with programmed death 1 expression on T cells in simian immunodeficiency virus-infected macaques and may contribute to T cell dysfunction and disease progression

Suppression of dendritic cell (DC) function in HIV-1 infection is thought to contribute to inhibition of immune responses and disease progression, but the mechanism of this suppression remains undetermined. Using the rhesus macaque model, we show B7-H1 (programmed death [PD]-L1) is expressed on lymphoid and mucosal DCs (both myeloid DCs and plasmacytoid DCs), and its expression significantly increases after SIV infection. Meanwhile, its receptor, PD-1, is upregulated on T cells in both peripheral and mucosal tissues and maintained at high levels on SIV-specific CD8(+) T cell clones in chronic infection. However, both B7-H1 and PD-1 expression in SIV controllers was similar to that of controls. Expression of B7-H1 on both peripheral myeloid DCs and plasmacytoid DCs positively correlated with levels of PD-1 on circulating CD4(+) and CD8(+) T cells, viremia, and declining peripheral CD4(+) T cell levels in SIV-infected macaques. Importantly, blocking DC B7-H1 interaction with PD-1(+) T cells could restore SIV-specific CD4(+) and CD8(+) T cell function as evidenced by increased cytokine secretion and proliferative capacity. Combined, the results indicate that interaction of B7-H1-PD-1 between APCs and T cells correlates with impairment of CD4(+) Th cells and CTL responses in vivo, and all are associated with disease progression in SIV infection. Blockade of this pathway may have therapeutic implications for HIV-infected patients.