B7-H3 Protein Expression Research Articles

Expression and regulation of B7‐H3 immunoregulatory receptor, in human mesothelial and mesothelioma cells: Immunotherapeutic implications

No treatment prolongs the survival of malignant mesothelioma (MM) patients. Since MM elicits anti-tumor host's immune responses, immunotherapy represents a promising strategy for its control. Immunomodulatory antibodies against components of the B7 family of immunomodulatory molecules that regulate T cell activation are being investigated in human malignancies including MM. The expression of B7-H3, a new component of the B7 family was investigated in primary cultures of human mesothelial cells (HMC) and in MM cell lines by flow cytometry and molecular analyses, and in MM tissues by immunohistochemistry. The role of DNA hypomethylating agents in modulating levels of B7-H3 expression in MM cells was also studied. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that B7-H3 mRNA was consistently detectable in mesothelial and MM cells investigated; however, real-time quantitative RT-PCR analyses showed highly heterogeneous levels of B7-H3 mRNA among investigated MM cells. The analysis of B7-H3 protein expression indicated that comparable levels of B7-H3 were expressed on both cell types. Treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine did not significantly affect the expression of B7-H3 mRNA in MM cells. In vivo, while B7-H3 was expressed in all 13 tumor biopsies of the epithelial variant, with high levels in 54% of cases, it was rarely detectable in spindle type MM in which 1/5 biopsies weakly expressed B7-H3. These findings suggest that B7-H3 is a promising target for new immunotherapeutic strategies in MM, with particular emphasis in the epithelial variant.

The influence of Cisplan on the expression of B7-H1 in retinoblastoma cells

Objective To observe the influence of cisplan on the expression of B7-H1 in retinoblastoma (RB) cells,and to investigate its mechanism. Methods Human RB cell line HXO-Rb44 cells were treated by 6 different concentrations of cisplan (0. 000, 0. 375, 0. 750, 1. 500, 3. 000, 6.000 μg/ml),and their B7-H1 mRNA expression was determined by the reverse-transcription polymerase chain reaction (RT-PCR) and fluorescence quantitative PCR (FQ-PCR);the B7-H1 protein expression was determined by immunofluorescence and flow cytometry. HXO-Rb44 cells were treated by 1.5 μg/ml cisplan for 0, 15, 30,60, 120 min, then the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) was detected by Western blot. Results The expression of B7-H1 mRNA and protein in the 0. 375, 0. 750, 1. 500,3. 000, 6. 000 μg/ml group were significantly higher than that of the blank control group (F= 395. 478,112.03;P=0. 000). Western blot showed that cisplan (1.5μg/ml) could activate ERK1/2 by increasing its phosphorylation in HXO-Rb44 cells. After cisplan treatment, the phosphorylation of ERK1/2 increased gradually and reached its peak at 30 min, and then went down gradually. Conclusion Cisplan can promote the expression of B7-H1 and activate ERK1/2 in RB cells. Key words: Retinoblastoma/drug therapy; Cisplatin/therapeutic use; Cisplatin/administration & dosage; Extracellular signal-regulated MAP kinases

B7–H3 Ligand Expression by Primary Breast Cancer and Associated With Regional Nodal Metastasis

: B7 ligand family members have been shown to be important immunoregulatory factors in host tumor immune responses. We hypothesized that B7-H3, a coinhibitory factor, is expressed by primary breast cancer cells and associated with metastasis to regional tumor-draining lymph nodes. : American Joint Committee on Cancer stage I to III primary breast cancers (n = 82) and normal breast specimens (n = 17) were assessed for B7-H3 expression using paraffin-embedded archival tissues. B7-H3 expression by breast cancer cells was assessed by a quantitative real-time reverse transcription-polymerase chain reaction, and B7-H3 protein expression was evaluated using immunohistochemistry. : B7-H3 mRNA expression was detected in 32 of 82 (39%) primary breast tumors but not in normal breast tissues (P = 0.0029). B7-H3 expression in primary tumors significantly correlated with increasing tumor size, American Joint Committee on Cancer stage, and lymphovascular invasion (P < 0.0001, P < 0.0001, P = 0.0071). B7-H3 expression was highly correlated to sentinel lymph node and overall number of lymph nodes with metastasis P = 0.003, and P = 0.004, respectively). In a multivariate analysis, B7-H3 mRNA expression of the primary tumor significantly predicted metastasis to regional lymph nodes (P = 0.021, and P = 0.023, respectively). Antibody staining analysis of paraffin-embedded archival tissue breast tumors and flow cytometry of breast cancer cell lines demonstrated B7-H3 protein expression. : B7-H3 protein expressed by primary breast cancer cells is a tumor progression factor and is associated with extent of regional nodal metastasis.

Mantle Cell Lymphoma Cells Express B7 Family Molecules and B7-H1 Expression Are up-Regulated After LPS Exposure Via MEK-Dependent and PI3K/Akt-Dependent Pathways In Mantle Cell Lymphoma Cells

AbstractAbstract 3114Mantle cell lymphoma (MCL) is a distinct subtype of B-cell non-Hodgkin lymphomas characterized by a specific t(11;14) (q13;q32) translocation, causing over-expression of cyclin D1. Recent studies demonstrated that B7 family molecules were not only expressed on antigen presenting cells but also on various hematopoietic malignancies, solid tumors and infiltrating immune cells and may play important roles in tumor immunology. Many cytokines could upregulate the expression of B7 molecules, however, the molecular mechanism of regulating expressions of B7 molecules is still unknown. In this study, we analyzed B7 family molecule expression on cultured mantle cell lymphoma cells and primary MCL cells by RT-PCR. We confirmed that B7 family molecules were detected in mantle cell lymphoma cell lines SP53, Jeko-1 and Mino and primary MCL cells. We next used LPS to activate TLR4 signaling pathway.Although B7 family molecules were detected in mantle cell lymphoma cells, only B7-H1 expression was greatly enhanced after LPS exposure. B7-H1 mRNA was constitutively expressed on MCL cells and was further up-regulated after LPS stimulation in dose and time dependent manners. Western blot also indicated that total and surface B7-H1 protein expression were up-regulated after stimulated by LPS. Flow cytometry demonstrated that surface B7-H1 protein expression were up-regulated after stimulated by LPS. When we knockdown TLR4, LPS stimulation did not up-regulate B7-H1 expression. To clarify the signaling pathways of LPS induced B7-H1 up-regulation in MCL cells, we incubated the SP53 and Jeko-1 cells with LPS after pretreatment with different signal transduction pathway inhibitors for 1h and detect the activity of each inhibitor. Pretreatment and coincubation of SP53 and Jeko-1 cells with the MEK1/2 inhibitor UO126 and PI3K/Akt inhibitor LY294002 strongly reduced LPS induced B7-H1 expression, indicating that the MEK1/2 and PI3K/Akt pathway are crucial for B7-H1 expression in MCL cells. We also tested whether JNK or p38, respectively, are involved in controlling the expression of B7-H1 in MCL cells. Blocking of JNK and p38 MAPK with specific inhibitors reduced B7-H1 mRNA expression, but B7-H1 protein expression was not obvious, which may be regulated after transcriptional factors. To confirm that LPS induced B7-H1 expression through a MEK and PI3K/Akt pathway pathways in MCL cells, we stimulated SP53 and Jeko-1 with LPS and analyzed the phosphorylation of ERK1/2, p38, and JNK at different time. ERK1/2, p38, and JNK phosphorylation were significantly up-regulated following LPS treatment. We confirmed that pretreatment of the cells with these specific inhibitors inhibited LPS-induced phosphorylation of ERK1/2, p38 MAPK, and JNK, respectively. In conclusion, our study demonstrated that mantle cell lymphoma cells express B7 family molecules. B7-H1 expression were up-regulated after LPS exposure via MEK-dependent and PI3K/Akt-dependent in mantle cell lymphoma cells. Disclosures:No relevant conflicts of interest to declare.

Functional significance of B7-H1 expressed by human uveal melanoma cells

B7-H1, a recently described B7 family member, has been reported to negatively regulate T-cell function in most cancer cells. In this study, we sought to investigate B7-H1 expression in four uveal melanoma (UM) cells (OCM1, SP6.5, OM431 and VUP) to determine the functional significance of B7-H1 expression in T-cell immune response. Using flow cytometry (FCM), we demonstrated that SP6.5 cells had high B7-H1 protein expression, while the other three UM cell lines had none. However, all four UM cell lines expressed B7-H1 mRNA, as confirmed by reverse transcription-polymerase chain reaction. In co-culture experiments using B7-H1-expressing UM cells with T-cells, FCM to determine CD69 expression in T-cells revealed that SP6.5 cell-related B7-H1 inhibited T-cell activation. This effect was eliminated by B7-H1-targeted RNA interference. An Annexin V/PI double staining assay further showed that B7-H1 expressed by SP6.5 cells did not increase the apoptosis of T-cells, though it was found in a variety of other solid tumors. In conclusion, all the UM cell lines constitutively expressed B7-H1 mRNA, while B7-H1 protein was expressed at different levels. UM-related B7-H1 expression negatively regulated T-cell immune response through the inhibition of T-cell activation, and not through the promotion of T-cell apoptosis. This provides new insight into anti-tumor immunity against B7-H1-expressing UM cells.

Cryptosporidium parvumInduces B7‐H1 Expression in Cholangiocytes by Down‐Regulating MicroRNA‐513

Expression of B7 costimulatory molecules represents an important compartment of immune response of epithelial cells after microbial infection. We report here that the protozoan parasite Cryptosporidium parvum induced B7-H1 expression in cultured human cholangiocytes. Induced expression of B7-H1 was identified in cells after exposure to infective C. parvum parasite or parasite lysate. Interestingly, the level of microRNA-513 (miR-513) was reduced in cells after exposure to C. parvum, which resulted in a relief of 3' untranslated region-mediated translational suppression of B7-H1. Overexpression of miR-513 through transfection of miR-513 precursor inhibited C. parvum-induced B7-H1 protein expression. Moreover, enhanced apoptotic cell death was identified in activated human T cells after coculture with C. parvum-infected cholangiocytes. The apoptosis of activated T cells was partially blocked by a neutralizing antibody to B7-H1 or transfection of cholangiocytes with miR-513 precursor. These data suggest a role of miR-513 in regulating B7-H1 expression by cholangiocytes in response to C. parvum infection.

Abstract C243: B7-H4 protein expression in invasive ductal carcinoma and its association with tumor progression

Abstract High levels of B7-H4 (DD-O110) protein expression have been previously observed in malignant ductal epithelium of the breast but the mechanisms that mediate B7-H4 up-regulation in tumor progression have not yet been determined. An objective of our research has been to explore the differences in the levels of B7-H4 expression that underlie the transition from benign ductal epithelium to ductal carcinoma in situ (DCIS) and to the earliest stages of invasive ductal carcinoma. B7-H4 is a member of the B7 family of proteins which has been found to play a role in antigen-specific immune response regulation, including the regulation of T cell activation. The potential role of B7-H4 in tumor progression, or in shielding breast cancers from immune surveillance mechanisms, remains relatively unexplored. Previous immunohistochemical (IHC) studies have evaluated only a small subset of available B7-H4 antibody epitopes. The aim of the current study is to determine if there is differential expression of specific B7-H4 epitopes that are associated with benign versus proliferative and malignant breast disease. A panel of monoclonal antibodies to defined B7-H4 epitopes (diaDexus, Inc.) was evaluated in normal breast tissues versus invasive ductal carcinoma, using indirect immunoperoxidase methods, in sections of formalin fixed tissues. The patterns of B7-H4 localization of each antibody were compared with the patterns observed with the previously reported A57.1 antibody (Mugler et al., 2007). B7-H4 IHC scores were determined based on the intensity of staining (0 to 3+), the proportion of positive cells, and the cellular pattern of distribution (apical membranous versus circumferential membranous plus cytoplasmic). Preliminary data suggest that certain B7-H4 epitopes appear to have enhanced cancer selectivity, reflected by differential expression between normal ductal cells versus ductal carcinoma cells. Additionally, these studies confirm that there are differences in the subcellular distribution of B7-H4 expression, with low level, apical membranous expression in benign ductal epithelium and high level circumferential membranous expression in most well to moderately-differentiated ductal carcinomas. Furthermore, there appears to be a marked reduction in the levels of B7-H4 expression in some poorly differentiated carcinomas, despite normal levels of expression in adjacent benign ductal epithelial cells. These results suggest that B7-H4 overexpression may play a role in the progression from benign proliferative disease to low grade carcinomas. Further studies are indicated to refine the association of B7-H4 with tumor histologic grade and stage, and to elucidate the potential role of this protein in shielding breast carcinomas from cell-mediated immune surveillance. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C243.

Expression of B7-H1 protein in human pancreatic carcinoma tissues and its clinical significance

B7-H1, a member of B7 family, is expressed in tumor cells and has emerged as an important immune modulator capable of suppressing host immunity by inhibiting T cells function. This study was to probe into the correlation between the expression level of B7-H1 protein in pancreatic carcinoma tissues and clinicopathological characteristics and prognosis. The expression of B7-H1 was measured in 40 cases of pancreatic carcinoma tissues and 10 cases of normal corresponding paracarcinoma tissues by immunohistochemistry. The relationship between the expression level of B7-H1 and clinicopathological characteristics and survival was analyzed. The positive rate of B7-H1 was significantly higher in the tumor tissues [45.00% (18/40)] than in the normal corresponding paracarcinoma tissues [0(0/10)] (P<0.05); moreover, B7-H1 expression was significantly associated with the staging of tumor and preoperative serum CA19-9 level (P<0.05). The multivariate cox proportional hazards regression analysis of prognostic factors for overall survival and relapse-free survival showed that the expression of B7-H1 was an independent factor for poor prognosis. B7-H1 protein was expressed in human pancreatic carcinoma tissues, and was associated with the prognosis.

Interferon-γ and Tumor Necrosis Factor-α Induce An Immunoinhibitory Molecule, B7-H1, Via NfκB Activation in Blasts of Myelodysplastic Syndromes.

Abstract 2766Poster Board II-742 (Introduction)B7-H1 (CD274) is a costimulatory/coinhibitory molecule that plays a crucial role in T cell regulation in various immune responses. B7-H1 expression is detected not only on professional antigen-presenting cells but also on some tumor cells. It has been reported that B7-H1 molecules on tumor cells inhibit the proliferation of tumor-specific cytotoxic T-lymphocytes and are associated with poor prognosis in patients with some tumors such as renal and breast cancers. In this study, we investigated whether functional B7-H1 molecules are expressed on blasts from myelodysplastic syndromes (MDS) and, if so, are associated with pathophysiology in specific immune evasion in MDS. (Materials and methods)(1) First, we analyzed the expression of B7-H1 mRNA and protein in MDS cell lines (OIH-1, SKM-1, and F36P) using reverse transcription-PCR and flow cytometry (FCM), respectively. Although F36P cells alone expressed B7-H1 molecules at mRNA and protein levels, interferon (IFN)-γ and more efficiently a combination of IFN-γ and tumor necrosis factor (TNF)-α enhanced or induced B7-H1 expression in all of these cell lines. Notably, the combination of IFN-γ and TNF-α induced nuclear factor (NF) γB activation of MDS cell lines, and the blocking of NFκB activation by pyrrolidine dithiocarbamate (PDTC) or NFκB inhibitor peptide turned off the B7-H1 induction. (2) To investigate whether B7-H1+ MDS blasts have a proliferative advantage, we compared the cell cycle and colony formation between B7-H1+ and B7-H1– cell fractions in F36P cells. B7-H1+ cells had fewer G0/G1-phase cells and more G2/M-phase cells compared with B7-H1– cells. Furthermore, purified B7-H1+ cells formed more colonies than B7-H1– cells in the colony-forming assay using the methylcellulose. (3) We investigated the immunomodulatory effects of B7-H1+ blasts on T cells. When normal CD3+ T cells were cultured with or without irradiated B7-H1+ F36P cells, the B7-H1+ F36P cells had markedly increased percentages of T cells showing apoptosis. The blocking antibody for B7-H1 or PD-1 inhibited the T-cell apoptosis induced by B7-H1+ cells, decreased the percentage of caspase-3+ cells in T cells, and increased T-cell proliferation. (4) We then examined B7-H1 expression on blasts from 41 MDS patients, 20 patients with acute myeloid leukemia transformed from MDS (AML-MDS), and 10 hematologically normal individuals using FCM. B7-H1+ cases, in whom more than 5% of blasts expressed B7-H1 molecules, were observed in 6 of 14 (43%) patients with refractory anemia with excess blasts (RAEB), 6 of 12 (50%) with RAEB in transformation (RAEB-t), and 4 of 20 (20%) AML-MDS patients, but in none of 15 RA patients and normal individuals. Similar to the data obtained using MDS cell lines, B7-H1 expression on blasts purified from MDS patients was induced by stimulation with IFN-γ or TNF-α, and more strikingly by their combination. PDTC inhibited the induction of B7-H1 expression. The percentages of G0/G1-phase cells were lower and those of G2/M-phase cells higher in B7-H1+ blasts than in B7-H1– blasts from patients. Furthermore, in allogeneic coculture, in which B7-H1+ blasts from an MDS patient and CD3+ T cells from healthy volunteers were cultured, T-cell proliferation was significantly augmented by blocking B7-H1 molecules. (5) Finally, we investigated the expression of PD-1, a counterreceptor of B7-H1, on circulating T cells in 34 MDS patients. PD-1 expression on CD3+, CD4+, and CD8+ T cells in MDS patients was significantly higher than that in healthy volunteers. (CONCLUSIONS)Taking the results together, in MDS: 1) B7-H1 expression on blasts occurs more often in the advanced disease stages; 2) B7-H1 expression on blasts may be induced by cytokines derived from the bone marrow environment via NFκB activation; 3) cell cycle and proliferation were more active in B7-H1+ blasts than in B7-H1– blasts; and 4) B7-H1 molecules on blasts suppress T-cell immunity. These findings indicate that B7-H1 molecules on blasts may be involved in the pathophysiology of MDS. Disclosures:No relevant conflicts of interest to declare.

MicroRNA miR-29 Modulates Expression of Immunoinhibitory Molecule B7-H3: Potential Implications for Immune Based Therapy of Human Solid Tumors

B7-H3, a surface immunomodulatory glycoprotein, inhibits natural killer cells and T cells. The monoclonal antibody (mAb) 8H9 is specific for 4Ig-B7-H3, the long and principal form of B7-H3. Early results from radioimmunotherapy using 8H9 have shown promise in patients with metastatic solid tumors to the central nervous system. Whereas B7-H3 transcript was ubiquitously expressed in a wide spectrum of human solid tumors as well as human normal tissues, B7-H3 protein was preferentially expressed only in tumor tissues. By quantitative reverse transcription-PCR, all three isoforms of microRNA miR-29 (a, b, and c) were highly expressed in normal tissues. However, they were down-regulated in a broad spectrum of solid tumors, including neuroblastoma, sarcomas, brain tumors, and tumor cell lines. B7-H3 protein expression was inversely correlated with miR-29 levels in both cell lines and tumor tissues tested. Using luciferase reporter assay, miR-29a was shown to directly target B7-H3 3' untranslated region, and knock-in and knockdown of miR-29a led to down-regulation and up-regulation, respectively, of B7-H3 protein expression. The ability of miR-29 to control B7-H3 protein expression has implications in immune escape by solid tumors. Differential modulation of this key immunoinhibitory molecule in tumor versus normal tissues may advance both cell-mediated immunotherapy and antibody-based targeted strategies using the B7-H3-specific mAb 8H9.

Local Expression of B7-H4 by Recombinant Adenovirus Transduction in Mouse Islets Prolongs Allograft Survival

Allogeneic pancreatic islet transplantation has the potential to cure type 1 diabetes. One of the barriers to islet transplantation is the alloreactive T-cell response between donors and recipients. Costimulatory molecules, which play a major role in the regulation of the immune response to antigens during graft rejection, may be used to inhibit allograft destruction. B7-H4 is one such member in the costimulatory family, which has established negative regulatory function of T-cell responses. To determine whether local expression of B7-H4 protein can protect beta cells from damage in islet allotransplantation, we have constructed a recombinant adenovirus expressing a B7-H4 complementary deoxyribonucleic acid (Ad-B7-H4). To study the in vivo effects of B7-H4 expression on islet graft survival, adenovirus-transduced islets from donor Balb/c mice were transplanted into streptozotocin-diabetic C57BL/6 mice (n=12). Expression of B7-H4 in islets by Ad-B7-H4 transduction at an optimized condition did not inhibit glucose-stimulated insulin secretion of the treated islets. The recipient mice transplanted with Ad-B7-H4-transduced islets established euglycemia for a longer time (mean 56.5 days), compared with control mice transplanted with Ad-LacZ-transduced islets (mean 14.5 days, [n=12, P<0.001]). Splenocytes isolated from the recipients of Ad-B7-H4-transduced islets showed hyporesponsiveness to alloantigenic stimulation, compared with control recipients. CD45 and insulin staining of the graft transplanted with Ad-B7-H4-transduced islets indicated the preservation of beta cells and decrease of infiltrating immune cells. Local expression of B7-H4 prolongs islet allograft survival in vivo, suggesting translational potential for beta-cell replacement with reduced immune injury.

MicroRNA-513 Regulates B7-H1 Translation and Is Involved in IFN-γ-Induced B7-H1 Expression in Cholangiocytes

Biliary epithelial cells (cholangiocytes) respond to proinflammatory cytokines such as IFN-gamma and actively participate in the regulation of biliary inflammatory response in the liver. B7-H1 (also known as CD274 or PD-L1) is a member of the B7 costimulatory molecules and plays a critical immunoregulatory role in cell-mediated immune responses. In this study, we show that resting human cholangiocytes in culture express B7-H1 mRNA, but not B7-H1 protein. IFN-gamma induces B7-H1 protein expression and alters the microRNA (miRNA) expression profile in cholangiocytes. Of those IFN-gamma-down-regulated miRNAs, we identified microRNA-513 (miR-513) with complementarity to the 3'-untranslated region of B7-H1 mRNA. Targeting of the B7-H1 3'-untranslated region by miR-513 results in translational repression. Transfection of cholangiocytes with an antisense oligonucleotide to miR-513 induces B7-H1 protein expression. Additionally, transfection of miR-513 precursor decreases IFN-gamma-induced B7-H1 protein expression and consequently influences B7-H1-associated apoptotic cell death in cocultured Jurkat cells. Thus, miR-513 regulates B7-H1 translation and is involved in IFN-gamma-induced B7-H1 expression in human cholangiocytes, suggesting a role for miRNA-mediated gene silencing in the regulation of cholangiocyte response to IFN-gamma.

Differentiation-Induced Post-Transcriptional Control of B7-H1 in Human Trophoblast Cells

Trophoblast expression of immunomodulatory proteins in the human placenta is among the mechanisms that are critical for ensuring lymphocyte tolerance to the semi-allogeneic fetus. High levels of B7-H1 on trophoblast cells together with the known role of this protein in establishment of peripheral tolerance suggest that B7-H1 mediates immunological protection of the placenta during gestation. In this study, we investigated the molecular mechanisms of regulation of B7-H1 in trophoblast cells by epidermal growth factor (EGF), a key regulator of trophoblast cell differentiation. EGF increased B7-H1 protein levels within 24 h and mRNA levels within 4 h of the initiation of treatment; by 24 h B7-H1 mRNA levels were similar between control and EGF-treated cells. Analysis of two different potential promoter regions revealed strong promoter activity in response to IFN-γ. In contrast, no promoter activity could be induced by EGF, suggesting that this cytokine regulates B7-H1 expression post-transcriptionally in trophoblast cells. EGF-induced B7-H1 protein expression was completely blocked in the presence of inhibitors of the PI3Kinase/Akt/mTOR pathway, a pathway known to regulate gene expression at the translational level. Finally, analysis of monosomal and polysomal mRNA fractions of untreated and EGF-treated term trophoblast cells revealed that EGF induces a shift towards the translatable fractions and away from the untranslated fractions. These results highlight a novel mechanism for regulation of B7 family proteins in the placenta.

B7-H3 Ligand Expression by Prostate Cancer: A Novel Marker of Prognosis and Potential Target for Therapy

B7 coregulatory ligands can be aberrantly expressed in human disease. In the context of cancer, these ligands may act as antigen-specific inhibitors of T-cell-mediated antitumoral immunity. We recently reported that B7-H1 expression by carcinomas of the kidney and bladder portends aggressive disease and diminished survival. The expression of these proteins in prostate cancer, however, has not been investigated. We evaluated B7-H3 and B7-H1 protein expression in the pathologic specimens of 338 men treated for clinically localized prostate cancer between 1995 and 1998 with radical retropubic prostatectomy. Expression levels of B7-H3 in prostate cancer were correlated with pathologic indicators of aggressive cancer as well as clinical outcome. We report that B7-H3 is uniformly and aberrantly expressed by adenocarcinomas of the prostate, high-grade prostatic intraepithelial neoplasia, and four prostate cancer cell lines, whereas B7-H1 is rarely expressed. B7-H3 is expressed by benign prostatic epithelia, although at a more reduced level relative to neoplastic tissue. Increasing levels of B7-H3 intensity correlate with worsening clinicopathologic features of prostate cancer. Marked B7-H3 intensity, present in 67 (19.8%) specimens, confers a >4-fold increased risk of cancer progression after surgery (risk ratio, 4.42; P < 0.001). A survey of normal tissues revealed that B7-H3 is expressed within the liver, urothelium, and fetal kidney. In summary, B7-H3 is aberrantly expressed in all prostate cancers and represents an independent predictor of cancer progression following surgery. Moreover, B7-H3 encompasses a novel diagnostic and potential therapeutic target for the clinical management of prostate cancer and, perhaps, other malignancies as well.

Expression of B7-H1 in Inflammatory Renal Tubular Epithelial Cells

Background: Renal tubular epithelial cells (TECs) function as antigen-presenting cells (APCs) as they constitutively express MHC-II molecules and have the capacity to present peptide antigen to T cells. Nevertheless, co-stimulatory signals provided by TECs for regulating T cell activation have not been fully characterized. We therefore investigated the expression of B7-H1, a member of the B7 superfamily, on TECs under normal or pathologic conditions in vivo and analyzed the regulation and functional role of it after proinflammatory factors treatment in vitro. Methods: Immunohistological staining for B7-H1 on cryostat sections of core needle biopsies from patients with different renal diseases was examined. Furthermore, we also detected B7-H1 protein expression on cultured human TECs stimulated by various inflammatory factors and performed TEC/T-cell co-cultured experiment to determine TEC-associated B7-H1 in regulating CD4⁺ T cell activation as well as antigen presentation. Results: Significant B7-H1 protein was detected in TECs of diseased renal samples. Although the presence of B7-H1 does not have any correlation with clinicopathological variables, marked B7-H1 expression on sections without interstitial inflammation revealed that B7-H1 has some protective function. In vitro, the expression of B7-H1 on TECs was increased after TECs were stimulated with IL-1α, LPS, TNF-α, IFN-γ or anti-CD40. Co-cultured experiments revealed that TEC-related B7-H1 was identified as a strong inhibitor of CD4⁺ T-cell activation as assessed by increased cytokine production (interleukin-2 and interferon-γ) and expression levels of the T cell activation marker (CD69) in the presence of a neutralizing antibody against B7-H1 (clone MIH1). Interestingly, IL-2 production by C10 T cells after antigen presentation by murine TECs was also enhanced when the B7-H1/PD-1 pathway was interrupted. Conclusion: This study clearly shows that B7-H1 is an inducible renal tubular epithelial antigen that inhibits T cell activation. It is speculated that B7-H1/PD-1 pathway might play a role in protecting tubular epithelium from immune-mediated damage and active delivery of the B7-H1 inhibitory signal represents a novel therapeutic strategy in autoimmune renal diseases.

B7-H4 overexpression in ovarian tumors

Objectives. Despite great advances in therapeutic management, the mortality rate for ovarian cancer has remained relatively stable over the past 50 years. This study was designed to evaluate the expression of B7-H4 protein, recently identified as a potential molecular marker of breast and ovarian cancer by quantitative PCR analysis, in benign tumors, tumors of low malignant potential and malignant tumors of the ovary. Methods. Archival formalin-fixed tissue blocks from serous, mucinous, endometrioid and clear cell ovarian tumors were evaluated by immunohistochemistry for the distribution of B7-H4 expression, and staining intensity was measured by automated image analysis. Univariate analyses were used to test for statistically significant relationships. Results. B7-H4 cytoplasmic and membranous expression was detected in all primary serous ( n = 32), endometrioid ( n = 12), and clear cell carcinomas ( n = 15), and in all metastatic serous ( n = 23) and endometrioid ( n = 7) ovarian carcinomas. By contrast, focal B7-H4 expression was detected in only 1/11 mucinous carcinomas. The proportion of positive cells and median staining intensity was greater in serous carcinomas than in serous cystadenomas or serous tumors of low malignant potential, and the differences were statistically significant ( P < 0.0001 and P = 0.034, respectively). The median staining intensity was also significantly greater in endometrioid carcinomas than in endometriosis ( P = 0.005). Conclusions. The consistent overexpression of B7-H4 in serous, endometrioid and clear cell ovarian carcinomas and the relative absence of expression in most normal somatic tissues indicates that B7-H4 should be further investigated as a potential diagnostic marker or therapeutic target for ovarian cancer.

Triptolide inhibits B7-H1 expression on proinflammatory factor activated renal tubular epithelial cells by decreasing NF-κB transcription

Triptolide has been used extensively in China for the treatment of autoimmune diseases and tumor for many centuries. Nevertheless, little is known about its exact immunosuppressive and anti-inflammatory properties. Increasing recognition of the importance of renal tubular epithelial cells (TECs) in renal diseases raises the question whether triptolide can regulate TEC activity. In this study, various cultured human and murine TECs were exposed to tumor necrotic factor-α (TNF-α) and triptolide, followed to examine the expression of B7-H1 and B7-DC. Flow cytometric analysis revealed that B7-H1 but not B7-DC constitutively expresses on TECs, and the B7-H1 protein expression was profoundly up-regulated by the stimulation of TNF-α with a dose-dependent manner. However, triptolide under non-cytotoxic concentration could down-regulate B7-H1 expression on activated TECs at both mRNA and protein level. This effect was transcription factor NF-κB dependent. Interestingly, the significant damping effect of triptolide on B7-H1 signal could promote interleukin-2 production by T cell hybridoma (C10) after antigen presentation and enhance cytokine (IFN-γ and IL-2) secretion by anti-CD3 activated T cells. Our results indicated that triptolide could regulate TEC activity via B7-H1, in addition to previously reported it directly affects the production of some inflammatory factors by T cells, tumor cells and peripheral blood mononuclear cells.

B7-H4 Is Highly Expressed in Ductal and Lobular Breast Cancer

This study was designed to investigate the expression of B7-H4 protein, a member of the B7 family that is involved in the regulation of antigen-specific immune responses, in normal breast and in primary and metastatic breast carcinomas. Archival formalin-fixed tissue blocks from breast cancers and normal somatic tissues were evaluated for B7-H4 expression by immunohistochemistry with manual and automated image analysis. The proportion of B7-H4-positive cells and the intensity of B7-H4 staining were compared with histologic type, grade, stage, hormone receptor status, and HER-2/neu status. B7-H4 was detected in 165 of 173 (95.4%) primary breast cancers and in 240 of 246 (97.6%) metastatic breast cancers. B7-H4 staining intensity was greater in invasive ductal carcinomas [24.61 relative units (RU)] and in invasive lobular carcinomas (15.23 RU) than in normal breast epithelium (4.30 RU, P = 0.0003). Increased staining intensity was associated with negative progesterone receptor status (P = 0.014) and history of neoadjuvant chemotherapy (P = 0.004), and the proportion of B7-H4-positive cells was associated with negative progesterone receptor (P = 0.001) and negative HER-2/neu (P = 0.024) status. However, there was no statistically significant relationship between the proportion of B7-H4-positive cells or staining intensity and grade, stage, or other clinicopathologic variables. Low levels of B7-H4 expression were also detected in epithelial cells of the female genital tract, lung, pancreas, and kidney, but B7-H4 was generally absent in most other normal somatic tissues. The nearly ubiquitous expression of B7-H4 in breast cancer, independent of tumor grade or stage, suggests a critical role for this protein in breast cancer biology.

B7-H4, a Molecule of the B7 Family, Negatively Regulates T Cell Immunity

We identify a B7 family molecule, B7-H4, by protein sequence analysis and comparative molecular modeling. While B7-H4 mRNA is widely distributed in mouse and human peripheral tissues, cell surface expression of B7-H4 protein is limited and shows an inducible pattern on hematopoietic cells. Putative receptor of B7-H4 can be upregulated on activated T cells. By arresting cell cycle, B7-H4 ligation of T cells has a profound inhibitory effect on the growth, cytokine secretion, and development of cytotoxicity. Administration of B7-H4Ig into mice impairs antigen-specific T cell responses whereas blockade of endogenous B7-H4 by specific monoclonal antibody promotes T cell responses. B7-H4 thus may participate in negative regulation of cell-mediated immunity in peripheral tissues.

B7-H1 pathway and its role in the evasion of tumor immunity.

B7-H1 is a recently identified member of the B7 family molecules. Upon ligation to its receptors on T cells it regulates activation and differentiation of T cells. B7-H1 preferentially costimulates IL-10 production in resting T cells and further induces the apoptosis of activated T cells. PD-1 is a receptor of B7-H1 and is shown to mediate the inhibition of activated T cell response, presumably by inhibiting cell cycle progression. The expression of B7-H1 protein is limited to macrophage lineage of cells in normal tissues, although its mRNA transcription is found in a broad range of tissues. In contrast, B7-H1 is abundant in various human cancers. The tumor-associated B7-H1 increases apoptosis of antigen specific T cells, leading to growth of immunogenic tumor growth in vivo. Current data suggest that B7-H1 regulates the organ-specific tolerance in normal tissue and may contribute to immune evasion by cancers. Selective manipulation of B7-H1 pathway thus aids in the design of new regimens in the treatment of human autoimmune disease and the control of malignant cancers.