Tumor-infiltrating programmed death receptor-1+ dendritic cells mediate immune suppression in ovarian cancer

Within the ovarian cancer microenvironment, there are several mechanisms that suppress the actions of antitumor immune effectors. Delineating the complex immune microenvironment is an important goal toward developing effective immune-based therapies. A dominant pathway of immune suppression in ovarian cancer involves tumor-associated and dendritic cell (DC)-associated B7-H1. The interaction of B7-H1 with PD-1 on tumor-infiltrating T cells is a widely cited theory of immune suppression involving B7-H1 in ovarian cancer. Recent studies suggest that the B7-H1 ligand, programmed death receptor-1 (PD-1), is also expressed on myeloid cells, complicating interpretations of how B7-H1 regulates DC function in the tumor. In this study, we found that ovarian cancer-infiltrating DCs progressively expressed increased levels of PD-1 over time in addition to B7-H1. These dual-positive PD-1(+) B7-H1(+) DCs have a classical DC phenotype (i.e., CD11c(+)CD11b(+)CD8(-)), but are immature, suppressive, and respond poorly to danger signals. Accumulation of PD-1(+)B7-H1(+) DCs in the tumor was associated with suppression of T cell activity and decreased infiltrating T cells in advancing tumors. T cell suppressor function of these DCs appeared to be mediated by T cell-associated PD-1. In contrast, ligation of PD-1 expressed on the tumor-associated DCs suppressed NF-κB activation, release of immune regulatory cytokines, and upregulation of costimulatory molecules. PD-1 blockade in mice bearing ovarian cancer substantially reduced tumor burden and increased effector Ag-specific T cell responses. Our results reveal a novel role of tumor infiltrating PD-1(+)B7-H1(+) DCs in mediating immune suppression in ovarian cancer.     

B7-H3 drives immunosuppression and Co-targeting with CD47 is a new therapeutic strategy in β-catenin activated melanomas

In melanoma, immune cell infiltration into the tumor is associated with better patient outcomes and response to immunotherapy. T-cell non-inflamed tumors (cold tumors) are associated with tumor cell-intrinsic Wnt/β-catenin activation, and are typically resistant to anti-PD-1 alone or in combination with anti-CTLA-4 therapy. Reversal of the ‘cold tumor’ phenotype and identifying new effective immunotherapies are challenges. We sought to investigate the role of a newer immunotherapy agent, B7-H3, in this setting. RNA sequencing was used to identify co-targeting strategies upon B7-H3 inhibition in a well-defined preclinical melanoma model driven by β-catenin. We found that immune checkpoint molecule B7-H3 confers a suppressive tumor microenvironment by modulating antiviral signals and innate immunity. B7-H3 inhibition led to an inflamed microenvironment, up-regulation of CD47/SIRPa signaling, and together with blockade of the macrophage checkpoint CD47 resulted in additive antitumor responses. We found that the antitumor effects of the B7-H3/CD47 antibody combination were dependent on cytokine signaling pathways (CCR5/CCL5 and IL4).     

Immunohistochemical Staining of B7-H1 (PD-L1) on Paraffin-embedded Slides of Pancreatic Adenocarcinoma Tissue

B7-H1/PD-L1, a member of the B7 family of immune-regulatory cell-surface proteins, plays an important role in the negative regulation of cell-mediated immune responses through its interaction with its receptor, programmed death-1 (PD-1) ^1,2. Overexpression of B7-H1 by tumor cells has been noted in a number of human cancers, including melanoma, glioblastoma, and carcinomas of the lung, breast, colon, ovary, and renal cells, and has been shown to impair anti-tumor T-cell immunity^3-8.Recently, B7-H1 expression by pancreatic adenocarcinoma tissues has been identified as a potential prognostic marker^9,10. Additionally, blockade of B7-H1 in a mouse model of pancreatic cancer has been shown to produce an anti-tumor response¹¹. These data suggest the importance of B7-H1 as a potential therapeutic target. Anti-B7-H1 blockade antibodies are therefore being tested in clinical trials for multiple human solid tumors including melanoma and cancers of lung, colon, kidney, stomach and pancreas¹².In order to eventually be able to identify the patients who will benefit from B7-H1 targeting therapies, it is critical to investigate the correlation between expression and localization of B7-H1 and patient response to treatment with B7-H1 blockade antibodies. Examining the expression of B7-H1 in human pancreatic adenocarcinoma tissues through immunohistochemistry will give a better understanding of how this co-inhibitory signaling molecule contributes to the suppression of antitumor immunity in the tumor''s microenvironment. The anti-B7-H1 monoclonal antibody (clone 5H1) developed by Chen and coworkers has been shown to produce reliable staining results in cryosections of multiple types of human neoplastic tissues^4,8, but staining on paraffin-embedded slides had been a challenge until recently^13-18. We have developed the B7-H1 staining protocol for paraffin-embedded slides of pancreatic adenocarcinoma tissues. The B7-H1 staining protocol described here produces consistent membranous and cytoplasmic staining of B7-H1 with little background.     

Tumor cell programmed death ligand 1-mediated T cell suppression is overcome by coexpression of CD80

Programmed death ligand 1 (PDL1, or B7-H1) is expressed constitutively or is induced by IFN-γ on the cell surface of most human cancer cells and acts as a "molecular shield" by protecting tumor cells from T cell-mediated destruction. Using seven cell lines representing four histologically distinct solid tumors (lung adenocarcinoma, mammary carcinoma, cutaneous melanoma, and uveal melanoma), we demonstrate that transfection of human tumor cells with the gene encoding the costimulatory molecule CD80 prevents PDL1-mediated immune suppression by tumor cells and restores T cell activation. Mechanistically, CD80 mediates its effects through its extracellular domain, which blocks the cell surface expression of PDL1 but does not prevent intracellular expression of PDL1 protein. These studies demonstrate a new role for CD80 in facilitating antitumor immunity and suggest new therapeutic avenues for preventing tumor cell PDL1-induced immune suppression.     

B7-H1和B7-H4在口腔鳞状细胞癌组织中的表达及临床意义

目的:探讨口腔鳞状细胞癌(Oral Squamous Cell Carcinoma,OSCC)中B7-H1和B7-H4的表达及其临床意义,并为OSCC的临床诊断、治疗、判断预后及预防等提供依据。方法:采用免疫组织化学S-P法检测B7-H1及B7-H4在60例OSCC及20例非肿瘤患者正常口腔黏膜组织(NOM)中的表达情况,分析两者与OSCC临床病理特征的相关性。结果:B7-H1在OSCC组织中表达显著高于在NOM组织中表达(29例,48.3%v4例,20%,x~2=4.969,P0.05);B7-H4在OSCC组织中表达亦显著高于在NOM组织中表达(31例,51.7%v5例,25%,x~2=4.310,P0.05)。B7-H1与B7-H4在OSCC组织的表达都与TNM分期、淋巴结转移和肿瘤分化程度显著相关(P0.05),而与年龄、性别及肿瘤直径大小等无关。OSCC组织中B7-H1和B7-H4的高表达呈显著性正相关性(x~2=5.613 P0.05),60例组织中B7-H1和B7-H4共表达现象有11例(18.3%),NOM中未发现两者共表达现象。结论:B7-H1和B7-H4过表达与OSCC发生、发展及预后有关,可以作为预后指标。     

miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究*

目的: 探讨miR-34a-5p在三阴性乳腺癌(triple negative breast cancer,TNBC)中的表达,分析miR-34a-5p对TNBC细胞增殖、凋亡、迁移的作用,对TNBC荷瘤小鼠肿瘤生长的影响以及在TNBC中对B7-H1表达的影响。方法: 利用RT-qPCR、Western blot分析TNBC细胞中miR-34a-5p、B7-H1的表达,并利用Kaplan-Meier分析二者的表达与TNBC患者的生存关系;将miR-34a-5p转染TNBC细胞,通过CCK-8、流式细胞术及划痕实验检测miR-34a-5p对TNBC细胞增殖、凋亡、迁移的影响;利用RT-qPCR、Western blot检测miR-34a-5p、B7-H1表达水平的变化,双荧光素酶基因报告验证miR-34a-5p与B7-H1的相互作用;利用RT-qPCR、Western blot、IHC检测miR-34a-5p对MDA-MB-231荷瘤小鼠miR-34a、B7-H1表达的影响。结果: TNBC细胞中miR-34a-5p呈低表达,B7-H1呈高表达,二者均与TNBC患者的不良预后有关,差距具有统计学意义(P<0.01);miR-34a-5p抑制TNBC细胞增殖、侵袭,促进细胞凋亡,并且在TNBC细胞中靶向抑制B7-H1;miR-34a-5p agomir在体内抑制MDA-MB-231成瘤裸鼠的肿瘤生长和B7-H1表达。结论: miR-34a-5p在TNBC发生、发展中发挥着重要作用,靶向miR-34a-5p/B7-H1可能成为TNBC患者新的分子治疗策略。     

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-H3 enhances tumor immunity in vivo by costimulating rapid clonal expansion of antigen-specific CD8+ cytolytic T cells

B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.     

Tumor cell-intrinsic CD274/PD-L1: A novel metabolic balancing act with clinical potential

Tumor expression of the immune co-signaling molecule CD274/PD-L1 was originally described as impeding antitumor immunity by direct engagement of its receptor, PDCD1/PD-1, on antitumor T cells. Melanoma-intrinsic PDCD1 was recently shown to promote tumor growth and MTOR signals in cooperation with tumor CD274, and sarcoma-intrinsic CD274 signaling promotes glucose metabolism to impede antitumor immunity. Our recent report shows that tumor cell-intrinsic CD274 promotes MTORC1 signaling in mouse melanoma and mouse and human ovarian cancer, inhibits autophagy and sensitizes some tumors to clinically available pharmacological autophagy inhibitors and confers resistance to MTOR inhibitors. Tumor CD274 could be a biomarker of autophagy or MTOR inhibitor response in selected tumors, and these inhibitors could improve anti-CD274 or anti-PDCD1 cancer immunotherapy. As we found that distinct tumor types exhibit this CD274-driven phenotype, it could be widely applicable.     

B7-H1 Expression Is Associated with Poor Prognosis in Colorectal Carcinoma and Regulates the Proliferation and Invasion of HCT116 Colorectal Cancer Cells

Background And Objective

The investigation concerning the B7-H1 expression in colorectal cancer cells is at an early stage. It is unclear whether B7-H1 expression may have diagnostic or prognostic value in colorectal carcinoma. Additionally, how B7-H1 is associated with the clinical features of colorectal carcinoma is not known. In order to investigate the relationship between B7-H1 and colorectal cancer, we analyzed B7-H1 expression and its effect in clinical specimens and HCT116 cells.

Methods

Paraffin-embedded specimens from 143 eligible patients were used to investigate the expression of CD274 by immunohistochemistry. We also examined whether B7-H1 itself may be related to cell proliferation, apoptosis, migration and invasion in colon cancer HCT116 cells.

Results

Our results show that B7-H1 was highly expressed in colorectal carcinoma and was significantly associated with cell differentiation status and TNM (Tumor Node Metastasis) stage. Patients with positive B7-H1 expression showed a trend of shorter survival time. Using multivariate analysis, we demonstrate that positive B7-H1 expression is an independent predictor of colorectal carcinoma prognosis. Our results indicate that B7-H1 silencing with siRNA inhibits cell proliferation, migration and invasion. Furthermore, cell apoptosis was also increased by B7-H1 inhibition.

Conclusions

Positive B7-H1 expression is an independent predictor for colorectal carcinoma prognosis. Moreover, knockdown of B7-H1 can inhibit cell proliferation, migration and invasion.     

Targeting molecular and cellular inhibitory mechanisms for improvement of antitumor memory responses reactivated by tumor cell vaccine

Development of effective vaccination approaches to treat established tumors represents a focus of intensive research because such approaches offer the promise of enhancing immune system priming against tumor Ags via restimulation of pre-existing (memory) antitumoral helper and effector immune cells. However, inhibitory mechanisms, which function to limit the recall responses of tumor-specific immunity, remain poorly understood and interfere with therapies anticipated to induce protective immunity. The mouse renal cell carcinoma (RENCA) tumor model was used to investigate variables affecting vaccination outcomes. We demonstrate that although a whole cell irradiated tumor cell vaccine can trigger a functional antitumor memory response in the bone marrows of mice with established tumors, these responses do not culminate in the regression of established tumors. In addition, a CD103+ regulatory T (Treg) cell subset accumulates within the draining lymph nodes of tumor-bearing mice. We also show that B7-H1 (CD274, PD-L1), a negative costimulatory ligand, and CD4+ Treg cells collaborate to impair the recall responses of tumor-specific memory T cells. Specifically, mice bearing large established RENCA tumors were treated with tumor cell vaccination in combination with B7-H1 blockade and CD4+ T cell depletion (triple therapy treatment) and monitored for tumor growth and survival. Triple treatment therapy induced complete regression of large established RENCA tumors and raised long-lasting protective immunity. These results have implications for developing clinical antitumoral vaccination regimens in the setting in which tumors express elevated levels of B7-H1 in the presence of abundant Treg cells.     

Tumor expression of B7-H4 predicts poor survival of patients suffering from gastric cancer

To establish the prognostic value of B7-H4 expression by tumor cells in gastric cancer patients, we evaluated the association of B7-H4 expression with clinicopathologic factors and overall survival of gastric cancer patients. A retrospective cohort study including 156 gastric cancer patients was performed in the present report. Immunohistochemical assay was used to evaluate the expression of B7-H4 in the surgical specimens of gastric cancer tissues. Multi-univariate COX model was then used to evaluate the association of B7-H4 expression with the patients’ survival and clinicopathological parameters. B7-H4 expression in the gastric cancer cells was observed in about 44.9% gastric cancer specimens. Univariate analysis demonstrated that there was no correlation between B7-H4 expression and sex, age, histological type, pathological grade or tumor size. In contrast, B7-H4 expression correlated positively with cancer invasiveness and lymph node metastasis. In addition, the median overall survival time of patients with lower B7-H4 expression was 13 months longer than that of patients with higher expression (χ² = 12.38, P < 0.0001), and the median disease-free survival time of patients with lower B7-H4 expression was significantly longer than that of patients with higher expression (33 vs. 16 months, χ² = 14.977, P < 0.0001). After adjustment for other confounding factors, the COX model analysis indicated that the death risk was significantly higher in patients with higher B7-H4 expression than those with lower expression (RR = 1.85, 95% CI = 1.15–2.96). The present study demonstrated that higher B7-H4 expression in cancer cells was associated with poor prognosis of gastric cancer patients. This is consistent with the idea that B7-H4 promotes cancer progression, likely via inhibition of anti-tumor immune responses.     

Generation of avian-derived anti-B7-H4 antibodies exerts a blockade effect on the immunosuppressive response

For highly conserved mammalian protein, chicken is a suitable immune host to generate antibodies. Monoclonal antibodies have been successfully targeted with immunity checkpoint proteins as a means of cancer treatment; this treatment enhances tumor-specific immunity responses through immunoregulation. Studies have identified the importance of B7-H4 in immunoregulation and its use as a potential target for cancer treatment. High levels of B7-H4 expression are found in tumor tissues and are associated with adverse clinical and pathological characteristics. Using the phage display technique, this study isolated specific single-chain antibody fragments (scFvs) against B7-H4 from chickens. Our experiment proved that B7-H4 clearly induced the inhibition of T-cell activation. Therefore, use of anti-B7-H4 scFvs can effectively block the exhaustion of immunity cells and also stimulate and activate T-cells in peripheral blood mononuclear cells. Sequence analysis revealed that two isolated scFv S2 and S4 have the same VH complementarity-determining regions (CDRs) sequence. Molecule docking was employed to simulate the complex structures of scFv with B7-H4 to analyze the interaction. Our findings revealed that both scFvs employed CDR-H1 and CDR-H3 as main driving forces and had strong binding effects with the B7-H4. The affinity of scFv S2 was better because the CDR-L2 loop of the scFv S2 had three more hydrogen bond interactions with B7-H4. The results of this experiment suggest the usefulness of B7-H4 as a target for immunity checkpoints; the isolated B7-H4-specific chicken antibodies have the potential for use in future cancer immunotherapy applications.     

In situ expression of IFN-γ-inducible T cell α chemoattractant in breast cancer mounts an enhanced specific anti-tumor immunity which leads to tumor regression

Increased evidence indicates that chemokines are involved in tumor growth. ITAC, a key member of chemokines, possesses the ability to recruit T cells and enhance immune responses. Therefore, ITAC might contribute to antitumor immunity. In this study, we evaluated the relationship between the expression of ITAC and human breast cancer advancement. We further investigated whether forced expression of ITAC in tumor sites could mediate enhanced antitumor immunity in a murine breast cancer model. Results showed that ITAC expression level was down-regulated in 31 breast cancer specimens compared to normal mammary tissues, and associated negatively with the stages of breast cancer. Contrarily, forced expression of ITAC in murine 4T1 tumor cells resulted in tumor regression after initial growth upon injection into naïve Balb/c mice. More lymphocytes were recruited to the site of tumor inoculated by 4T1-ITAC and more than 80% of these T cells expressed the ITAC receptor, CXCR3. ITAC-recruited TILs exhibited 4T1-specific proliferation and cytotoxicity, and an increased IFN-γ but decreased IL-4 production. Importantly, forced expression of ITAC in 4T1 tumor nodules inhibited tumor growth. These findings demonstrated that the decreased expression of ITAC is associated with the advancement of breast cancer in patients. Forced expression of ITAC in tumor site not only induces increased T cell-recruitment and elicits a specific antitumor immunity, but also mediates regression of established 4T1 tumors, indicating the potential application of ITAC-expressing tumor cells in cancer immunotherapy and vaccine designing.     

Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion

B7-H1, a recently described member of the B7 family of costimulatory molecules, is thought to be involved in the regulation of cellular and humoral immune responses through the PD-1 receptor on activated T and B cells. We report here that, except for cells of the macrophage lineage, normal human tissues do not express B7-H1. In contrast, B7-H1 is abundant in human carcinomas of lung, ovary and colon and in melanomas. The pro-inflammatory cytokine interferon-gamma upregulates B7-H1 on the surface of tumor cell lines. Cancer cell-associated B7-H1 increases apoptosis of antigen-specific human T-cell clones in vitro, and the apoptotic effect of B7-H1 is mediated largely by one or more receptors other than PD-1. In addition, expression of B7-H1 on mouse P815 tumor increases apoptosis of activated tumor-reactive T cells and promotes the growth of highly immunogenic B7-1(+) tumors in vivo. These findings have implications for the design of T cell-based cancer immunotherapy.     

B7-h1 expressed by activated CD8 T cells is essential for their survival

An immunoinhibitory role of B7 homologue 1 (B7-H1) expressed by non-T cells has been established; however, the function of B7-H1 expressed by T cells is not clear. Peak expression of B7-H1 on Ag-primed CD8 T cells was observed during the contraction phase of an immune response. Unexpectedly, B7-H1 blockade at this stage reduced the numbers of effector CD8 T cells, suggesting B7-H1 blocking Ab may disturb an unknown function of B7-H1 expressed by CD8 T cells. To exclusively examine the role of B7-H1 expressed by T cells, we introduced B7-H1 deficiency into TCR transgenic (OT-1) mice. Naive B7-H1-deficient CD8 T cells proliferated normally following Ag stimulation; however, once activated, they underwent more robust contraction in vivo and more apoptosis in vitro. In addition, B7-H1-deficient CD8 T cells were more sensitive to Ca-dependent and Fas ligand-dependent killing by cytotoxic T lymphocytes. Activation-induced Bcl-x(L) expression was lower in activated B7-H1-deficient CD8 T cells, whereas Bcl-2 and Bim expression were comparable to the wild type. Transfer of effector B7-H1-deficient CD8 T cells failed to suppress tumor growth in vivo. Thus, upregulation of B7-H1 on primed T cells helps effector T cells survive the contraction phase and consequently generate optimal protective immunity.