B7-H1 expression on non-small cell lung cancer cells and its relationship with tumor-infiltrating lymphocytes and their PD-1 expression.

PURPOSE: B7-H1/PD-L1 (B7-H1) and B7-DC/PD-L2 (B7-DC) are ligands for the receptor PD-1, which is known to negatively regulate T-cell activation. In the present study, we investigated the expression of B7-H1 and B7-DC in tumor specimens of non-small cell lung cancer and their relationships with clinicopathological variables and postoperative survival. Furthermore, we examined the correlation between B7-H1 expression on tumor cells and the number of tumor-infiltrating lymphocytes (TILs) or PD-1 expression on TILs. EXPERIMENTAL DESIGN: The expression of B7-H1 and B7-DC in 52 surgically resected specimens of non-small cell lung cancer was evaluated immunohistochemically. RESULTS: Expression of B7-H1 and B7-DC was focally observed in all non-small cell lung cancer tumor specimens. No relationship was found between the expression of B7-H1 or B7-DC and clinicopathological variables or postoperative survival. However, in the same sections evaluated, significantly fewer TILs were identified in B7-H1-positive tumor regions than in B7-H1-negative tumor regions in a subset of five patients (P = 0.01). Moreover, the percentage of TILs expressing PD-1 was significantly lower in B7-H1-positive tumor regions than in B7-H1-negative tumor regions (P = 0.02). CONCLUSIONS: The expression of B7-H1 on tumor cells in local areas reciprocally correlated with the number of TILs, and this may contribute to negative regulation in antitumor immune responses in non-small cell lung cancer.     

Oral Squamous Carcinoma Cells Express B7-H1 and B7-DC Receptors in Vivo

B7-H1 and B7-DC ligands are members of the B7 family with important regulatory functions in cell-mediated immune response. Both receptors are ligands of the programmed death receptor PD-1. B7-H1 expression has been detected in the majority of human carcinomas in vivo. B7-H1 mediated signals are able to negatively regulate activated T cell functions and survival, and enable tumor cells to overcome host response. The aim of this study was to investigate the expression of B7-H1 and B7-DC proteins in oral squamous cell carcinomas (OSCC) in vivo. Tissues from 15 samples were cryo-sected and following histological routine staining (HE), incubated with antibodies against human B7-H1 and B7-DC. Immuno-staining of pan-cytokeratin was performed to ascertain the epithelial origin of the tissue and CK 19 to demonstrate the proliferating stage. Confocal laser scanning microscopy confirmed the presence of both B7-H1 and B7-DC in all 15 OSCC. The B7-H1 and B7-DC staining was located in areas of the tissue that were identified as cancerous lesions in the previously stained HE sections before. Staining with Pan-CK and CK19 provided evidence for the epithelial origin and the proliferating stage of the tissue. The in vivo expression of the B7-H1 and B7-DC receptors in oral squamous cell carcinomas suggest that general mechanisms for immune evasion of tumors are also found in OSCC.     

Expression of B7-H3 in hypopharyngeal squamous cell carcinoma as a predictive indicator for tumor metastasis and prognosis

B7-H3 is a member of the B7 family thought to be a co-regulatory factor of antigen-specific T-cell immune response via co-stimulatory and co-inhibitory receptors. We evaluated its potential expression in head and squamous cell carcinoma (SCC) cell lines, and in clinical tissue samples obtained from 37 patients with human hypopharyngeal SCC. All head and neck SCC cell lines tested expressed both the B7-H3 gene and cell surface protein. The staining intensity of immunoreactivity by tumor cells was blindly evaluated by two head and neck surgeons and the results were categorized into 4 grades according to staining intensity. Eighty-seven percent of patients expressed B7-H3. B7-H3 expression was inversely correlated with the number of tumor infiltrating CD8+ T-cells (r=-0.4339, p=0.023). Patients who developed distant metastasis after tumor-free periods showed significantly higher B7-H3 expression scores compared to patients who did not develop distant metastasis during follow-up periods (p=0.048). Distant metastasis control ratio in patients with strong B7-H3 expression was significantly lower compared to that in patients with no to intermediate B7-H3 expression (p=0.040). Cause-specific survival ratio in patients with strong B7-H3 expression was significantly lower compared to that in patients with no to intermediate B7-H3 expression (p=0.028). Moreover, multivariate analysis revealed that strong B7-H3 expression was an independent prognostic factor in tumor-specific death in hypopharyngeal SCC (hazard ratio: 9.803, confidence interval: 0.018-0.539, p=0.0110).     

T helper responses are maintained by basal-like breast cancer cells and confer to immune modulation via upregulation of PD-1 ligands

A conspicuous T cell infiltration is frequently observed in triple-negative and/or basal-like breast cancers. Since the immunological course of breast cancer is explicitly directed by helper T cells, this study aims to determine the influence of basal-like breast cancer (BLBC) cells on CD4⁺ T cell responses. Co-cultures were established with breast cancer cell lines and CD4⁺ T cells under stimulatory conditions. Helper T cell activation, proliferation, cytokine secretion, and differentiation were assessed. Protein and mRNA expression of PD-1 ligands were determined on breast cancer cell lines. Blockade assays were performed in order to determine the functional assets of PD-1 ligation. In contrast to luminal breast cancer cells, BLBC cells allowed CD4⁺ T cell activation, proliferation, and IFN-γ secretion, but only to a certain extent. A substantial population of CD25⁺CD127^low/? regulatory T (Treg) cells was also induced in BLBC co-cultures. In return, IFN-γ stimulated the upregulation of PD-L1 (B7-H1) and/or PD-L2 (B7-DC) inhibitory molecules on the basal-like cells. In prolonged periods of co-culturing, blockade of PD-1 ligands on BLBC cell lines impaired Treg differentiation, restored IL-2 secretion, and increased CD8⁺ T cell activation. In conclusion, T helper responses were maintained by BLBC cells. On the other hand, IFN-γ secreted from Th1 and other immune cells upregulated the expression of PD-1 ligands on BLBC cells and modulated the immune reactions. Our results indicate the capacity of BLBCs to adapt to IFN-γ-mediated anti-tumor immune responses and to evade immunity via upregulation of PD-1 ligands.     

T-cell coregulatory molecule expression in urothelial cell carcinoma: clinicopathologic correlations and association with survival

PURPOSE: Aberrant expression of T-cell coregulatory molecules has been investigated as a mechanism by which certain cancers may evade host immune surveillance. We evaluated expression of the T-cell coregulators B7-H1, B7-H3, and PD-1 in urothelial cell carcinoma (UCC) of the bladder. EXPERIMENTAL DESIGN: Immunohistochemistry for B7-H1, B7-H3, and PD-1 was done on paraffin-embedded sections from 318 consecutive patients with UCC who underwent radical cystectomy. Expression was correlated with clinicopathologic outcomes and postoperative survival. RESULTS: B7-H3 was widely expressed in UCC, as 222 of 314 (70.7%) tumors showed positive staining. Expression of B7-H3 in UCC was significantly increased compared with adjacent, nontumor urothelium, as a median of 70% of tumor cells expressed B7-H3, compared with 20% of cells in nontumor specimens (P < 0.001). The increase in B7-H3 expression was independent of tumor stage (P = 0.13). Expression of B7-H1 by UCC tumors (P < 0.001) and PD-1 by tumor-infiltrating lymphocytes (P = 0.012) were significantly associated with increased pathologic stage. Patients who had received intravesical bacillus Calmette-Guerin before cystectomy tended to show increased expression of B7-H3 (P = 0.023) and PD-1 (P = 0.071) but were less likely to express B7-H1 (P = 0.027). Moreover, for the subset of patients with organ-confined disease (n = 167), B7-H1 expression independently predicted all-cause mortality after cystectomy (hazard ratio, 3.18; 95% confidence interval, 1.74-5.79; P < 0.001). CONCLUSIONS: B7-H3 is highly expressed in UCC across tumor stages, whereas B7-H1 and PD-1 expression are associated with advanced disease. B7-H1 expression predicts mortality after cystectomy for patients with organ-confined tumors. These molecules may represent novel diagnostic or prognostic markers, as well as therapeutic targets, for patients with UCC.     

B7-H1 expression is regulated by MEK/ERK signaling pathway in anaplastic large cell lymphoma and Hodgkin lymphoma

B7-H1 is a member of the B7 family that inhibits the function of T-cells through its receptor programmed death-1 (PD-1). We examined B7-H1 expression in anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) and found that it was constitutively expressed in both clinical samples and cell lines. In anaplastic lymphoma kinase–positive (ALK⁺) ALCL cells, B7-H1 expression was suppressed by the blocking of extracellular signal-regulated kinase (ERK) signaling and upregulated by the augmentation of ERK activity by phorbol 13-myristate 12-acetate stimulation, suggesting that B7-H1 expression is regulated by ERK signaling pathway in ALCL. ERK is one of the downstream mediators of nucleophosmin (NPM)/ALK signaling in ALK⁺ALCL, and pharmacological inhibition of ALK was shown to dephosphorylate ERK and down-regulate B7-H1. The involvement of NPM/ALK in B7-H1 expression was also demonstrated by introducing the construct into human non-ALCL lymphoid cell lines, which resulted in B7-H1 expression. In the case of HL, B7-H1 expression was shown to be dependent on the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways. These results suggest that B7-H1 expression is controlled by common ERK signaling pathways in ALCL and HL cells. Our findings provide a potentially effective immunotherapeutic strategy for these B7-H1-expressing tumors. ( Cancer Sci 2009)     

Clinical significance and regulation of the costimulatory molecule B7-H1 in pancreatic cancer

We investigated the expression pattern and clinical significance of the costimulatory ligands B7-1, B7-2, B7-H1, and B7-DC, and their counter-receptors CTLA-4 and PD-1 in pancreatic cancer. Gene expression of all examined costimulatory molecules was significantly upregulated in pancreatic cancer tissues. B7-1, B7-2, B7-H1, and B7-DC protein was detectable in pancreatic cancer cells. Only the expression of B7-H1 significantly correlated with postoperative survival (p<0.0001). B7-H1 was inducible in cultured pancreatic cancer cells by IFN-gamma and significantly correlated with the level of IFN-gamma expression in human pancreatic cancer tissues (Spearman rho=0.4536,p=0.0029). B7-H1 positive tumors showed an increased prevalence of tumor-infiltrating regulatory T cells (T(regs)) compared to B7-H1 negative tumors. Among the investigated costimulatory molecules only tumor-associated B7-H1 seems to be of prognostic relevance in pancreatic cancer. B7-H1 might, therefore, be involved in the downregulation of antitumor responses through regulation of T(regs) in pancreatic cancer. Our findings also suggest a dual role of IFN-gamma in antitumor response. Through induction of B7-H1 in pancreatic cancer cells IFN-gamma might contribute to the evasion of antitumor immunity.     

Reconstructed Adeno-Associated Virus with the Extracellular Domain of Murine PD-1 Induces Antitumor Immunity

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmeddeath-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing totumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumortherapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain ofmurine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which theexpression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediateearly promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated withthe AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR,and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenatesrevealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with controllevels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, alsoenhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was alsofound expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression ofthe soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects onT cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapiesto augment T-cell responses in patients with malignancies.     

B7-H1及其受体PD-1在胃癌组织中的表达与意义

目的 探讨B7-H1和PD-1信号通路与胃癌发生的关系.方法 利用免疫组化技术和ANAE染色技术检测胃组织中B7-H1和PD-1的原位表达及肿瘤浸润淋巴细胞(TIL)浸润情况,利用逆转录聚合酶链反应(RT-PCR)技术检测胃癌组织中B7-H1和PD-1 mRNA的表达,利用Western blot技术检测B7-H1蛋白的表达.结果 B7-H1和PD-1在胃癌组织中表达升高,阳性率分别为64.4%和43.8%,而在正常胃组织中不表达.胃癌组织中,B7-H1表达与TIL浸润负相关.B7-H1表达与胃癌的浸润深度、周围淋巴结转移、远处转移及pTNM分期有关,而PD-1与其无关.结论 胃癌组织中B7-H1和PD-1表达升高,B7-H1/PD-1信号通路可能抑制抗肿瘤免疫.B7-H1的表达与胃癌的病程有关,有可能作为判断胃癌预后的指标和胃癌治疗的靶点.     

12-O-tetradecanoyl phorbol 13-acetate induces the expression of B7-DC, -H1, -H2, and -H3 in K562 cells

Induction of the B7 family molecules by 12-O-tetradecanoyl phorbol 13-acetate (TPA) has been reported, however, the mechanism by which TPA up-regulates these molecules remains poorly understood. In this study, the expression of B7-DC, -H1, -H2, and -H3 in response to TPA was markedly induced in K562 cells. TPA also induced activation of ERK, p38 mitogen-activated protein kinase (MAPK), JNK, phosphatidylinositol-3-kinase (PI-3K), or nuclear factor (NF)-kappaB. Pre-treatments with protein kinase C (PKC) inhibitors significantly inhibited TPA-induced expression of B7-DC, -H1, -H2, and -H3 mRNA as well as TPA-induced phosphorylation of ERK, p38 MAPK, JNK, and PI-3K. TPA-induced expression of B7-DC, -H1, -H2, and -H3 mRNA was abrogated by pre-treatments with inhibitors of ERK and p38 MAPK. However, inhibition of PI-3K and JNK only caused decrease of TPA-induced B7-DC mRNA and B7-H3 mRNA, respectively. TPA-induced degradation of IkappaB-alpha was markedly abrogated by treatments with PKC inhibitors, but not by treatments with inhibitors of ERK, p38 MAPK, JNK, or PI-3K. NF-kappaB inhibitors significantly attenuated the expression of B7-DC, -H1, -H2, and -H3 mRNA in response to TPA. These results suggest that TPA induces the expression of B7-DC, -H1, -H2, and -H3 mRNA in K562 cells via activation of PKC, ERK, p38 MAPK, and NF-kappaB. Distinctly, the expression of B7-DC mRNA and -H3 mRNA in response to TPA is also PI-3K- and JNK-dependent, respectively.     

Blockade of B7-H1 and PD-1 by monoclonal antibodies potentiates cancer therapeutic immunity

Contemporary approaches for vaccination and immunotherapy are often capable of eliciting strong T-cell responses against tumor antigens. However, such responses are not parallel to clinical tumor regression. The development of evasion mechanisms within tumor microenvironment may be responsible for poor therapeutic responses. We report here that constitutive or inducible expression of B7-H1, a B7 family molecule widely expressed by cancers, confers resistance to therapeutic anti-CD137 antibody in mice with established tumors. The resistance is accompanied with failure of antigen-specific CD8+ CTLs to destroy tumor cells without impairment of CTL function. Blockade of B7-H1 or PD-1 by specific monoclonal antibodies could reverse this resistance and profoundly enhance therapeutic efficacy. Our findings support that B7-H1/PD-1 forms a molecular shield to prevent destruction by CTLs and implicate new approaches for immunotherapy of human cancers.     

Effect of TLR4 and B7-H1 on Immune Escape of Urothelial Bladder Cancer and its Clinical Significance

Background/Aim: Toll-like receptor 4 (TLR4) and B7-H1, both normally expressed restricted to immune cells,are found to be aberrantly expressed in a majority of human tumors and may play important roles in regulationof tumor immunity. It has been shown that urothelial bladder cancer (UBC) patients can manifest tumoralimmune escape which may be a potential critical factor in tumor pathogenesis and progression. However, so far,the mechanisms of UBC-related immune escape have not been clarified. The aim of this study was to investigatethe effect of TLR4 and B7-H1 on immune escape of UBC. Methods: Bladder cancer T24 cells were pre-incubatedwith LPS and co-cultured with tumor specific CTLs. CTL cytotoxicity and apoptosis rates were measured by MTTassay and flow cytometry, respectively. The effects of an ERK inhibitor on B7-H1 expression and CTL cytotoxicityagainst T24 cells were also evaluated. In addition, TLR4, B7-H1 and PD-1 protein expression was analyzed byimmunohistochemistry in 60 UBC specimens and 10 normal urothelia. Results: TLR4 activation protected T24cells from CTL killing via B7-H1 overexpression. However PD98059, an inhibitor of ERK, enhanced CTL killingof T24 cells by reducing B7-H1 expression. TLR4 expression was generally decreased in UBC specimens, whileB7-H1 and PD-1 were greatly overexpressed. Moreover, expression of both B7-H1 and PD-1 was significantlyassociated with UICC stage and WHO grade classification. Conclusions: TLR4 and B7-H1 may contribute toimmune escape of UBC. Targeting B7-H1 or the ERK pathway may offer new immunotherapy strategies forbladder cancer.     

B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium

Previous studies indicated that B7-H4, the youngest B7 family, negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors. Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy. However, the link between B7-H4 and tumor stem cells is unclear. In this study, we investigated B7-H4 expression in the medium of human glioma U251 cell cultures. Immunofluorescence results showed that U251 cells cultured in serum-free medium （supplemented with 2% B27, 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor） maintained stem-like cell characteristics, including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2. In contrast, U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein. Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serum- containing medium-cultured U251 cells （24%-35% vs. 8%-11%, P 〈 0.001）. Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of BT-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium. Moreover, conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4 expression compared with serum-containing conditioned medium （P 〈 0.01）. Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells, and conditioned medium from these cells more effectively induced monocytes to express BT-H4 than conditioned medium from U251 cells cultured in the presence of serum. Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.     

Expression of the B7-related molecule B7-H1 by glioma cells: a potential mechanism of immune paralysis

Human glioblastoma is a highly lethal tumor that is known for its immune inhibitory capabilities. B7-homologue 1 (B7-H1), a recently identified homologue of B7.1/2 (CD80/86), has been described to exert costimulatory and immune regulatory functions. We investigated the expression and the functional activity of B7-H1 in human glioma cells in vitro and in vivo. Although lacking B7.1/2 (CD80/86), all 12 glioma cell lines constitutively expressed B7-H1 mRNA and protein. Exposure to IFN-gamma strongly enhanced B7-H1 expression. Immunohistochemical analysis of malignant glioma specimens revealed strong B7-H1 expression in all 10 samples examined, whereas no B7-H1 expression could be detected on normal brain tissues. To elucidate the functional significance of glioma cell-related B7-H1 expression, we performed coculture experiments of glioma cells with alloreactive CD4+ and CD8+ T cells. Glioma-related B7-H1 was identified as a strong inhibitor of CD4+ as well as CD8+ T-cell activation as assessed by increased cytokine production (IFN-gamma, interleukin-2, and interleukin-10) and expression levels of the T-cell activation marker (CD69) in the presence of a neutralizing antibody against B7-H1 (mAb 5H1). B7-H1 expression may thus significantly influence the outcome of T-cell tumor cell interactions and represents a novel mechanism by which glioma cells evade immune recognition and destruction.     

B7家族负性共刺激分子在急性髓系白血病中的表达特征

目的:探讨B7家族4种负性共刺激分子B7-H1、B7-H3、B7-H4和B7-H6在人急性髓系白血病（AML）中的表达及分布特征。方法:采用流式细胞术检测8株AML细胞株和22例初治AML患者骨髓原始细胞中B7-H1、B7-H3、B7-H4和B7-H6膜蛋白的表达,设8例志愿者骨髓血细胞为对照,并应用阳性表达率、平均荧光强度（MFI）及原始细胞和淋巴细胞的MFI比值（MFI比）分析各B7分子的表达特征。结果:AML细胞株中,多数细胞膜表面表达B7-H3和B7-H6,B7-H3表达较强的细胞株有SKM-1和SHI-1,B7-H6表达较强的细胞株有K562和HL-60;B7-H1仅在HEL中表达较强,而其余AML细胞中基本不表达;B7-H4在AML细胞株中均不表达。初治AML患者的骨髓血细胞中,B7-H3和B7-H6在原始细胞膜表面的表达率及原始细胞和淋巴细胞的MFI比均显著高于对照组; B7-H3高表达组的外周血白细胞数显著高于低表达组,B7-H6高表达组的年龄也显著高于其低表达组;B7-H3和B7-H6在急性单核细胞白血病患者中表达显著升高。B7-H1和B7-H4在初治AML患者中的表达水平低下或不表达,且与对照组相比均无显著差异。结论:4种B7家族负性共刺激分子在AML中的表达和分布存在差异。B7-H3和B7-H6在初治AML患者的原始细胞中异常高表达,提示可能参与急性髓系白血病的发生发展。     

PD-L1在肿瘤中的表达及其相关机制研究

免疫逃逸作为恶性肿瘤的一个重要特征,更好地理解这种机制对于发展有效的抗肿瘤策略至关重要。程序性细胞死亡配体1（PD-L1,B7-H1）／程序性细胞死亡1（PD-1,CD279）信号通路已经被证实是免疫逃逸的主要机制之一。近年来,以PD-L1／PD-1为靶点的免疫靶向治疗取得令人鼓舞的进展。然而,PD-L1在肿瘤微环境中表达上调的相关机制十分复杂,理解这些错综复杂的关系及肿瘤如何利用这一途径有助于我们设计更有效的肿瘤治疗策略。本文就PD-L1在恶性肿瘤中表达的作用、意义及相关上调机制作一综述。