B7-H1 Pathway Research Articles

The PD-1/B7-H1 pathway modulates the natural killer cells versus mouse glioma stem cells.

PurposeGlioblastoma multiforme (GBM) is the most malignant primary type of brain tumor in adults. There has been increased focus on the immunotherapies to treat GBM patients, the therapeutic value of natural killer (NK) cells is still unknown. Programmed death-1 (PD-1) is a major immunological checkpoint that can negatively regulate the T-cell-mediated immune response. We tested the combination of the inhibiting the PD-1/B7H1 pathway with a NK-cell mediated immune response in an orthotopic mouse model of GBM.Methods and MaterialsMouse glioma stem cells (GL261GSCs) and mouse NK cells were isolated and identified. A lactate dehydrogenase (LDH) assay was perfomed to detect the cytotoxicity of NK cells against GL261GSCs. GL261GSCs were intracranially implanted into mice, and the mice were stratified into 3 treatment groups: 1) control, 2) NK cells treatment, and 3) PD-1 inhibited NK cells treatment group. Overall survival was quantified, and animal magnetic resonance imaging (MRI) was performed to determine tumor growth. The brains were harvested after the mice were euthanized, and immunohistochemistry against CD45 and PCNA was performed.ResultsThe mouse NK cells were identified as 90% CD3- NK1.1+CD335+ by flow cytometric analysis. In the LDH assay, the ratios of the damaged GL261GSCs, with the E:T ratios of 2.5:1, 5:1, and 10:1, were as follows: 1) non-inhibited group: 7.42%, 11.31%, and 15.1%, 2) B7H1 inhibited group: 14.75%, 18.25% and 29.1%, 3) PD-1 inhibited group: 15.53%, 19.21% and 29.93%, 4) double inhibited group: 33.24%, 42.86% and 54.91%. In the in vivo experiments, the mice in the PD-1 inhibited NK cells treatment group and IL-2-stimulated-NK cells treatment group displayed a slowest tumor growth (F = 308.5, P<0.01) and a slower tumor growth compared with control group (F = 118.9, P<0.01), respectively. The median survival of the mice in the three groups were as follows: 1) conrol group: 29 days, 2) NK cells treatment group: 35 days (P = 0.0012), 3) PD-1 inhibited NK cells treatment group: 44 days (P = 0.0024). Immunologic data of PCNA-positive cell ratios and CD45-positive cell ratios of the tumor specimens in the three groups were as follows: 1) control group: 65.72% (PCNA) and 0.92% (CD45), 2) NK treatment group: 27.66% (PCNA) and 13.46% (CD45), and 3) PD-1 inhibited NK cells treatment group: 13.66% (PCNA) and 23.66% (CD45) (P<0.001).ConclusionThe results demonstrated that blockade of PD-1/B7H1 pathway could promote mouse NK cells to kill the GL261GSCs, and the PD-1-inhibited NK cells could be a feasible immune therapeutic approach against GBM.

Mesenchymal stem cells suppress T cells by inducing apoptosis and through PD-1/B7-H1 interactions

Mesenchymal stem cells (MSCs) exert a suppressive role toward T cells which has been widely studied in recent decades. However, the underlying mechanisms utilized by MSCs are still not fully elucidated. Herein, we performed traditional suppressive assays using co-cultured MSCs and conventional CD4+CD25− T cells (Tconv) with and without transwell systems. We showed that the expression of programmed cell death-1 receptor (PD-1) on activated Tconv was significantly elevated after they were exposed to MSCs. And we demonstrated that PD-1/B7-H1 pathway was involved in the suppression of MSCs on activated Tconv. Further analysis revealed that the up-regulation of PD-1 was related to an increasing apoptosis of activated Tconv. Finally, we demonstrated that the PD-1/B7-H1 pathway was not related to the elevated immunosuppressive cytokines including IL-10 and TGF-β1 which in turn played dispensable roles in the up-regulation of PD-1 on activated Tconv in MSC-Tconv co-culture systems.

Antagonist Antibodies to PD-1 and B7-H1 (PD-L1) in the Treatment of Advanced Human Cancer—Response

The immune suppressive molecule programmed death-1 (PD-1) is upregulated in activated T lymphocytes and inhibits T-cell function upon binding to its ligands B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273). Substantial experimental data from in vitro cell culture systems and animal models, and more recently from clinical trials, indicate that PD-1/PD-1-ligand interactions are a major mechanism of immune suppression within the tumor microenvironment. Initial clinical studies of antibodies directed against PD-1 and B7-H1 showed both an encouraging safety profile and remarkable antitumor activity in subsets of patients with metastatic disease, including malignancies--such as lung cancer--which were previously thought to be unresponsive to immunotherapy. Preliminary data have suggested a correlation between tumor membrane B7-H1 expression and clinical response to anti-PD-1 antibodies. Several key challenges remain to optimize development of PD-1/B7-H1 pathway blockade, including defining the biologic significance of all potential ligand-receptor interactions in the tumor microenvironment, developing more accurate predictive biomarkers of response, determining the breadth of activity in human malignancies, and developing rational combinations of therapy that address key mechanisms involved in positive and negative regulation of antitumor immune responses.

Abstract 1437: Prostate cancer cells up-regulate PD-L1 in response to pro-inflammatory cytokines.

Abstract In the last few years, the approval of two novel agents for advanced cancer, the anti-CTLA4 (cytotoxic T-lymphocyte antigen 4) antibody ipilimumab for patients with metastatic melanoma and the antigen presenting cell-based product sipuleucel-T for patients with castration-resistant prostate cancer, have transformed the field of immunotherapy. Knowing that cancer cells are continuously evolving due to genetic and epigenetic variability, it makes sense to attempt to eliminate the tumor cells using targeted antitumor immune responses, especially in prostate cancer where several studies have demonstrated infiltration of tumors with CD4 and CD8 T cells. Among the different mechanisms responsible for the failure of the immune system to develop a durable response towards tumors, the expression of immune checkpoint molecules on tumor infiltrating CD8 (killer) T cells has been shown to have a crucial role by suppressing CD8 T cell function. The PD-1 / B7-H1 pathway has recently emerged as a key player in this process, since single-agent blockade of PD-1 or PD-L1 has resulted in sustained responses in patients with renal cell carcinoma, melanoma and lung cancer. However, PD1 or PD-L1 blockade in prostate cancer have not had the same results and based on previously limited immunohistochemistry data, there is low expression of PD-L1 at prostate cancer samples. In our study,surprisingly, we were able to modify the immune checkpoint expression after treatment with cytokines known to be modulated by either vaccination or androgen deprivation. We tested a number of prostate cancer cell lines (LnCap, LnCap-C42B, LnCap-ABL, LAPC4, VCap, DU145, PC3, CWR22R and E006AA) for PD-L1, PD-L2, MHCII and B7-H3 expression by flow cytometry, either at baseline or after treatment for 48 hours with IFNγ, IL6, IL8, IL10 or TGFβ. At baseline, PD-L1, PD-L2, MHCII and B7-H3 was detected by flow cytometry in 4, 1, 2, and 6 of the cell lines respectively. When we treated the cell lines with IFNγ, 4 of them showed a robust up-regulation of PD-L1 while surprisingly several other cell lines up-regulated PD-L2, MHC-II and B7H3. Treatment with IL6, IL8 and IL10 and TGFβ was able to up-regulate PD-L1 in several cell lines. In this study we were able to show for the first time in prostate cancer regulation of immune checkpoints by IFNγ, IL6, IL8, IL10 and TGFβ. These data argue for a combinatorial approach, in which initially cytokines, regulated through an anti-tumor vaccine or androgen deprivation, sensitize the prostate cancer cells to immune response through alteration in expression of the immune checkpoints and subsequently this immune response is sustained by blocking the PD-1 / B7-H1 pathway. Citation Format: Sosipatros A. Boikos, Chris Nirschl, Allison Martin, Angela Alme, Timothy Harris, Charles G. Drake. Prostate cancer cells up-regulate PD-L1 in response to pro-inflammatory cytokines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1437. doi:10.1158/1538-7445.AM2013-1437

Antagonist antibodies to PD-1 and B7-H1 (PD-L1) in the treatment of advanced human cancer.

The immune suppressive molecule programmed death-1 (PD-1) is upregulated in activated T lymphocytes and inhibits T-cell function upon binding to its ligands B7-H1 (PD-L1, CD274) and B7-DC (PD-L2, CD273). Substantial experimental data from in vitro cell culture systems and animal models, and more recently from clinical trials, indicate that PD-1/PD-1-ligand interactions are a major mechanism of immune suppression within the tumor microenvironment. Initial clinical studies of antibodies directed against PD-1 and B7-H1 showed both an encouraging safety profile and remarkable antitumor activity in subsets of patients with metastatic disease, including malignancies--such as lung cancer--which were previously thought to be unresponsive to immunotherapy. Preliminary data have suggested a correlation between tumor membrane B7-H1 expression and clinical response to anti-PD-1 antibodies. Several key challenges remain to optimize development of PD-1/B7-H1 pathway blockade, including defining the biologic significance of all potential ligand-receptor interactions in the tumor microenvironment, developing more accurate predictive biomarkers of response, determining the breadth of activity in human malignancies, and developing rational combinations of therapy that address key mechanisms involved in positive and negative regulation of antitumor immune responses.

IFNγ Licensed Human Bone Marrow Derived Mesenchymal Stromal Cells Inhibit T Cell Cytokine Production by a Mechanism Independent of Indoleamine 2 3-Dioxygenase (IDO) Activity

Abstract 1255Human Bone marrow derived Mesenchymal Stromal Cells (MSCs) inhibit activation-mediated T cell proliferation. This functional interaction serves nearly universally as a surrogate assay for immune suppressive activity and potency of MSCs in vitro. However the role of MSCs in inhibiting T cell function, cytokine production in particular, has not been investigated thoroughly. In the present study we have compared resting MSCs with IFNγ licensed MSCs for their ability to inhibit activated T cell function such as cytokine secretion and degranulation through intracellular cytokine staining assays. PBMCs cocultured with resting or inflammatory cytokine licensed MSCs were stimulated with the super antigen Stephylococcal Enterotoxin B (SEB) for 12–14hours in the presence of Brefeldin A. Intracellular cytokine staining was performed on the T cells to detect the Interferon gamma (IFNγ), Tumor Necrosis Alpha (TNFα) and Interleukin-2 (IL-2). Our results demonstrate that resting MSCs do not inhibit T cell cytokine secreting function. However, IFNγ and IFNγ plus TNFα licensed MSCs dose dependently inhibit IFNγ secretion by T cells. Our results also show that IFNγ and IFNγ plusTNFα licensed MSCs not only inhibit cytokine secretion but also inhibit dregranulation of activated T cells. Boolean gating analysis demonstrates that IFNγ and IFNγ plusTNFα licensed MSCs inhibit triple cytokine producing highly inflammatory IFNγ +TNFα +IL2+T cells. Phenotypic analysis of MSCs demonstrates that IFNγ licensing upregulate indoleamine 2 3-dioxygenase (IDO) and B7-H1. Blocking of IDO activity with 1-Methyl D,L Tryptophan (1MT) will abolish MSC-mediated blockade of T cell proliferation. However, 1MT does not reverse the MSC blockade of T cell cytokine secretion. Phenotypical analysis of T cells demonstrate that PD1+ cells are inhibited while PD1- cells are not inhibited suggesting the role of B7-H1 and PD-1 inhibitory pathway is involved in the inhibitory potential of IFNγ licensed MSCs. Interestingly, the inhibitory potential of IFNγ licensed MSCs has been observed only with the SEB stimulation but not with anti-CD3, CD28 stimulation. This suggests that MHC-TCR interaction is necessary for the inhibitory effect of IFNγ licensed MSCs. Our results demonstrate the superiority of IFNγ licensed MSCs in inhibiting multiple cytokine producing T cell function independent of IDO activity. Our results also provide the rationale for use of IFNγ licensed MSCs in autologous immunosuppressive cell therapy. Disclosures:No relevant conflicts of interest to declare.

Marrow stromal cells induce B7-H1 expression on myeloma cells, generating aggressive characteristics in multiple myeloma

Tumor-associated B7-H1 molecules inhibit antitumor immunity in some malignancies. We found that B7-H1 expression on patient myeloma cells and human myeloma cell lines (HMCLs) was upregulated by cultivating the cells with autologous stromal cells and the human stromal cell line HS-5. Among major cytokines produced by HS-5 cells, interleukin (IL)-6-induced B7-H1 expression on HMCLs. Moreover, HS-5 cell-mediated B7-H1 expression was downregulated by inhibiting IL-6. B7-H1(+) HMCLs were more proliferative and less susceptible to antimyeloma chemotherapy compared with B7-H1(-) HMCLs. Moreover, the former cells showed higher levels of Bcl-2 and FasL expression than the latter. Finally, B7-H1 molecules on HMCLs induced T-cell apoptosis and anergy of tumor-specific T cells. Consistent with these in vitro observations, patients whose myeloma cells expressed high levels of B7-H1 had higher myeloma cell percentages in the bone marrow (BM) and higher serum lactate dehydrogenase levels compared with other myeloma patients. In addition, B7-H1 expression levels were often upregulated after myeloma patients relapsed or became refractory to therapy. Our data indicate that the BM microenvironment upregulates B7-H1 expression on myeloma cells, which links to the two biological actions of inducing T-cell downregulation and enhancing aggressive myeloma-cell characteristics. Modulating the B7-H1 pathway may be worthwhile in myeloma.

Increased B7-H1 Expression on Peripheral Blood T Cells in Oral Lichen Planus Correlated with Disease Severity

Oral lichen planus (OLP) is a chronic and T cell-mediated autoimmune disease whose immunopathogenesis may involve antigen-presentation, T cells activation and migration as well as keratinocytes apoptosis. PD-1/B7-H1 pathway may have a unique function in regulating self-reactive T cells associated with inflammatory response and maintaining tolerance in peripheral tissues. In this study, we aimed to explore the contribution of PD-1/B7-H1 pathway to OLP. We determined the expression of PD-1 and B7-H1 on peripheral blood T cells from OLP cases and analyzed their association with disease severity assessed by RAE (reticular, atrophic and erosive lesion) scoring system. In addition, interferon-γ, interleukin (IL)-2, IL-4, IL-10 and soluble PD-1 concentrations in serum were measured using ELISA. Then, we explored the regulation of PD-1/B7-H1 pathway on T cells immune response in OLP by blockade of PD-1 or B7-H1. We found that PD-1 and B7-H1 were up-regulated on peripheral blood T cells from OLP patients and B7-H1 expression positively correlated with disease severity of OLP. It is suggested that Th1 dominant inflammatory situation might contribute to the high expression of PD-1 and B7-H1 in OLP. Blockade of PD-1/B7-H1 pathway significantly increased the proliferation, and IFN-γ and IL-2 production of T cells. PD-1/B7-H1 pathway may play an important role in negatively modulating T cell-mediated immune response in OLP, and provide the rationale to employ B7-H1 expression on peripheral blood T cells as a marker of severity of OLP and to develop agonists targeting PD-1/B7-H1 pathway as a promising immunotherapeutic strategy for OLP.

In vitro-activated tumor-bearing host T cells and the effectiveness of tumor vaccine immunotherapy

BACKGROUND AND OBJECTIVEVaccination during periods of lymphopenia may facilitate immune responses to weak self-antigens and enhance antitumor immunity. The objective of this study was to determine the effectiveness of tumor vaccine immunotherapy combined with immune reconstruction using tumor-bearing host immune cells in lymphopenia, and to investigate the role of tumor-bearing host T cells activated in vitro during immunotherapy.DESIGN AND SETTINGAnimal study conducted in the First Affiliated Hospital of Xi’an Jiaotong University from January 2009 to January 2010.PATIENTS AND METHODSLymphopenia was induced by cyclophosphamide. A reconstituted immune system with different syngeneic lymphocytes was employed, including lymphocytes from naïve rats (unsensitized group), tumor-bearing rats (tumor-bearing group), and tumor-bearing rats activated in vitro (activated group). All rats were immunized with granulocyte-macrophage colony-stimulating factor (GM-CSF)-modified NuTu-19 ovarian cancer (GM-CSF/NuTu-19) cells. Tumor vaccine-draining lymph nodes (TVDLNs) were harvested, and then stimulated to induce effector T cells (TE). TE were then adoptively transferred to rats bearing a 3-day pre-established abdominal tumor (NuTu-19), and the survival rate was calculated.RESULTSCompared with the unsensitized group, the levels of interleukin-2 (IL-2) were significantly lower in the tumor-bearing group, whereas that of IL-4 were significantly higher (P<.05). The number of CD4+ T cells secreting interferon-γ and the specific cytotoxicity of CD8+ cytotoxic T lymphocytes were significantly lower (P<.05). The survival was significantly higher in the activated group compared with the other groups.CONCLUSIONSLymphocytes from tumor-bearing rats activated in vitro can effectively reverse the immunosuppressive effects of tumor-bearing hosts.

B7-H1 expression is associated with expansion of regulatory T cells in colorectal carcinoma

To investigate the expression of B7-H1 in human colorectal carcinoma (CRC) to define its regulating effects on T cells in tumor microenvironment. One hundred and two paraffin blocks and 33 fresh samples of CRC tissues were subject to this study. Immunohistochemistry was performed for B7-H1 and CD3 staining in CRC tissues. Ficoll-Hypaque density gradient centrifugation was used to isolate peripheral blood mononuclear cells of fresh CRC tissues; flow cytometry and immunofluorescence staining were used for detection of regulatory T cells. Data was analyzed with statistical software. Costimulatory molecule B7-H1 was found strongly expressed in CRC tissues, localized in tumor cell membrane and cytoplasm, while weak or none expression of B7-H1 was detected in pared normal colorectal tissues. Meanwhile, CD3 positive T cells were found congregated in CRC tumor nest and stroma. Statistic analysis showed that B7-H1 expression level was negatively correlated to the total T cell density in tumor nest (P < 0.0001) and tumor stroma (P = 0.0200) of 102 cases of CRC tissues. Among the total T cells, a variable amount of regulatory T cells with a clear Foxp3⁺ (forkhead box P3) staining could be detected in CRC tissues and patients' blood. Interestingly, in the 33 samples (15 cases of B7-H1(high) CRC tissues and 18 cases of B7-H1(low) CRC tissues) of freshly isolated mononuclear cells from CRC tissues, the percentages of CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ regulatory T cells were found remarkably higher in B7-H1(high) CRC tissues than in B7-H1(low) CRC tissues (P = 0.0024, P = 0.0182), indicating that B7-H1 expression was involved in proliferation of regulatory T cell. No significant difference was found in CRC peripheral blood (P = 0.0863, P = 0.0678). PD-1 is the specific ligand for B7-H1 pathway transferring inhibitory signal to T cell, which is expressed by activated T cell. Our further analysis of PD-1 expression on T cells in CRC tissues showed that conventional T cells (CD4⁺Foxp3⁻/CD8⁺Foxp3⁻), which was thought to contribute to the anti-tumor immune response, highly expressed PD-1; while regulatory T cells (CD4⁺Foxp3⁺/CD8⁺Foxp3⁻) almost failed to express PD-1. The average percentage of PD-1 expression on regulatory T cells was significantly higher than the percentage of PD-1 on conventional T cells (CD4⁺Foxp3⁻ T cell, P < 0.0001; CD8⁺Foxp3⁻ T cell, P < 0.0001). The diverse expression of PD-1 might lead to different fate of T cell subsets in B7-H1 over-expression CRC tumor microenvironment. B7-H1 expression in tumor cells can inhibit the conventional T cell proliferation in tumor microenvironment through the PD-1 expression on conventional T cells.

Malignant B Cells Induce the Conversion of CD4+CD25− T Cells to Regulatory T Cells in B-Cell Non-Hodgkin Lymphoma

Recent evidence has demonstrated that regulatory T cells (Treg) were enriched in the tumor sites of patients with B-cell non-Hodgkin lymphoma (NHL). However, the causes of enrichment and suppressive mechanisms need to be further elucidated. Here we demonstrated that CD4+CD25+FoxP3+CD127lo Treg were markedly increased and their phenotypes were different in peripheral blood (PB) as well as bone marrow (BM) from newly diagnosed patients with B-cell NHL compared with those from healthy volunteers (HVs). Involved lymphatic tissues also showed higher frequencies of Treg than benign lymph nodes. Moreover, the frequencies of Treg were significantly higher in involved lymphatic tissues than those from PB as well as BM in the same patients. Suppression mediated by CD4+CD25+ Treg co-cultured with allogeneic CFSE-labeled CD4+CD25− responder cells was also higher in involved lymphatic tissues from B-cell NHL than that mediated by Treg from HVs. In addition, we found that malignant B cells significantly induced FoxP3 expression and regulatory function in CD4+CD25− T cells in vitro. In contrast, normal B cells could not induce the conversion of CD4+CD25− T cells to Treg. We also showed that the PD-1/B7-H1 pathway might play an important role in Treg induction. Taken together, our results suggest that malignant B cells induce the conversion of CD4+CD25− T cells to Treg, which may play a role in the pathogenesis of B-cell NHL and represent a promising therapeutic target.

B7-H1 Molecules on Myeloma Cells Induce Aggressive Cell Behavior

Abstract 474 Introduction:B7-H1 (CD274), a B7 family molecule, is expressed on antigen-presenting cells and plays crucial roles in T-cell regulation in various immune responses. We found that expression of B7-H1 molecules is also detected on some tumor cells and inhibits tumor-specific cytotoxic T lymphocytes (CTLs). Consistent with these data, it was reported that B7-H1 expression on tumor cells was associated with poor prognosis in some cancers, i.e., renal cell carcinoma, breast and ovarian cancer, etc. The expression levels of B7-H1 on plasma cells from multiple myeloma (MM) patients were reported to be higher than on those cells from patients with monoclonal gammopathy of undetermined significance (MGUS) and normal controls, suggesting that B7-H1 expression may be involved in the pathophysiology of MM. In the current study, we investigated the mechanism by which B7-H1 expression is induced on MM cells in the bone marrow (BM) microenvironment and analyzed characteristics of B7-H1+ MM cells in comparison with B7-H1– MM cells, i.e., proliferative potential, drug resistance, and sensitivity to myeloma-specific CTLs and whether B7-H1+ MM cells are associated with patients’ disease status. Methods:We examined 14 human myeloma cell lines (HMCLs) and plasma cells from 40 MM patients, 10 MGUS patients, and 10 hematologically normal controls. B7-H1 expression levels were analyzed by flow cytometry (FCM) and real-time polymerase chain reaction (RT-PCR). Changes in B7-H1 expression levels on MM cells were analyzed after the cells were cultured in the presence of the human BM stromal cell line HS-5, its culture supernatant, various cytokines, or inhibitors of cytokines and transcription factors. Proliferative potential was compared between B7-H1+ and B7-H1– MM cells, including cell cycle status analyzed by propidium iodide (PI) staining, BrdU and Ki67 expression, and cell number in liquid culture. Finally, sensitivity to dexamethasone (DEX) and melphalan (MEL) and expression of apoptosis-related genes were compared between B7-H1+ and B7-H1– MM cells using FCM and RT-PCR, respectively. Results:1) Although B7-H1 mRNA was detected in 9 of 14 HMCLs, only 3 cell lines expressed B7-H1 on their surface in FCM. B7-H1 expression on MM cells was significantly upregulated by co-culture with HS-5 cells or their culture supernatant. 2) Among cytokines present in the HS-5 cell supernatants, i.e., interleukin (IL)-6, IL-8, granulocyte-colony stimulating factor, and macrophage inflammatory protein 1 alpha, IL-6 alone induced B7-H1 expression on MM cells. Moreover, IL-6-neutralizing antibody dose dependently inhibited B7-H1 expression in the HS-5 cell culture supernatant. B7-H1 expression was downregulated by inhibiting STAT3, a transcription factor mediating IL-6 signaling. 3) The proliferative potential was higher in B7-H1+ cells in all assays. 4) Although KMS-27 myeloma cells lacking B7-H1 expression were killed by specific CTLs, KMS-27 cells that had B7-H1 expression induced on their surface resisted CTLs. 5) Nearly 20% of RPMI8226 cells expressed B7-H1, and DEX- and MEL-induced apoptosis was observed in the B7-H1– cell fraction alone. B7-H1 gene transfection in KMS-28PE cells conferred apoptosis resistance. Compared with B7-H1– RPMI8226 cells, B7-H1+ RPMI8226 cells showed higher gene expression levels of Bcl-2 and FasL and lower gene expression levels of caspase-8, caspase-9, and Fas. 6) Plasma cells from MM patients expressed significantly higher levels of B7-H1 than those cells from MGUS patients. One of 10 MM patients with International Scoring System (ISS) stage I, and 8 of 30 MM patients with ISS stage II/III were B7-H1 positive. Moreover, patients with CD45– myeloma, who were reported to have shorter survival, had higher expression levels of B7-H1 compared with CD45+ myeloma patients. In 4 of 8 MM patients, B7-H1 expression levels were upregulated in relapse or refractory status compared with levels at initial diagnosis. Conclusions:Our study indicated that IL-6 derived from BM stromal cells upregulates B7-H1 expression on myeloma cells, giving the cells gain more aggressive intrinsic proliferative potential and resistance to chemotherapeutic drugs and CTLs. The modulating B7-H1 pathway may have therapeutic potential in myeloma. Disclosures:No relevant conflicts of interest to declare.

Programmed Death-1/B7-H1 Negative Costimulation Protects Mouse Liver Against Ischemia and Reperfusion Injury

Programmed death-1 (PD-1)/B7-H1 costimulation acts as a negative regulator of host alloimmune responses. Although CD4 T cells mediate innate immunity-dominated ischemia and reperfusion injury (IRI) in the liver, the underlying mechanisms remain to be elucidated. This study focused on the role of PD-1/B7-H1 negative signaling in liver IRI. We used an established mouse model of partial liver warm ischemia (90 minutes) followed by reperfusion (6 hours). Although disruption of PD-1 signaling after anti-B7-H1 monoclonal antibody treatment augmented hepatocellular damage, its stimulation following B7-H1 immunoglobulin (B7-H1Ig) fusion protected livers from IRI, as evidenced by low serum alanine aminotransferase levels and well-preserved liver architecture. The therapeutic potential of B7-H1 engagement was evident by diminished intrahepatic T lymphocyte, neutrophil, and macrophage infiltration/activation; reduced cell necrosis/apoptosis but enhanced anti-necrotic/apoptotic Bcl-2/Bcl-xl; and decreased proinflammatory chemokine/cytokine gene expression in parallel with selectively increased interleukin (IL)-10. Neutralization of IL-10 re-created liver IRI and rendered B7-H1Ig-treated hosts susceptible to IRI. These findings were confirmed in T cell-macrophage in vitro coculture in which B7-H1Ig diminished tumor necrosis factor-α/IL-6 levels in an IL-10-dependent manner. Our novel findings document the essential role of the PD-1/B7-H1 pathway in liver IRI. This study is the first to demonstrate that stimulating PD-1 signals ameliorated liver IRI by inhibiting T cell activation and Kupffer cell/macrophage function. Harnessing mechanisms of negative costimulation by PD-1 upon T cell-Kupffer cell cross-talk may be instrumental in the maintenance of hepatic homeostasis by minimizing organ damage and promoting IL-10-dependent cytoprotection.

PD‐1/B7‐H1 Interaction Contribute to the Spontaneous Acceptance of Mouse Liver Allograft

The programmed death-1 (PD-1)/B7-H1 pathway acts as an important negative regulator of immune responses. We herein investigated the role of the PD-1/B7-H1 pathway in establishing an immunological spontaneous tolerance status in mouse liver allografting. B7-H1 is highly expressed on the donor-derived tissue cells and it is also associated with the apoptosis of infiltrating T cells in the allografts. Strikingly, a blockade of the PD-1/B7-H1 pathway via anti-B7-H1mAb or using B7-H1 knockout mice as a donor led to severe cell infiltration as well as hemorrhaging and necrosis, thus resulting in mortality within 12 days. Furthermore, the expression of the FasL, perforin, granzyme B, iNOS and OPN mRNA in the liver allografts increased in the antibody-treated group in comparison to the controls. Taken together, these data revealed that the B7-H1 upregulation on the tissue cells of liver allografts thus plays an important role in the apoptosis of infiltrating cells, which might play a critical role of the induction of the spontaneous tolerance after hepatic transplantation in mice.

Accelerated Course of Experimental Autoimmune Encephalomyelitis in PD-1-Deficient Central Nervous System Myelin Mutants

It is assumed that the onset and course of autoimmune inflammatory central nervous system (CNS) disorders (eg, multiple sclerosis) are influenced by factors that afflict immune regulation as well as CNS vulnerability. We challenged this concept experimentally by investigating how genetic alterations that affect myelin (primary oligodendrocyte damage in PLPtg mice) and/or T-cell regulation (deficiency of PD-1) influence both the onset and course of an experimental autoimmune CNS inflammatory disease [MOG(35-55)-induced experimental autoimmune encephalomyelitis (EAE)]. We observed that double pathology was associated with a significantly earlier onset of disease, a slight increase in the neurological score, an increase in the number of infiltrating cells, and enhanced axonal degeneration compared with wild-type mice and the respective, single mutant controls. Double-mutant PLPtg/PD-1(-/-) mice showed an increased production of interferon-gamma by CNS immune cells at the peak of disease. Neither PD-1 deficiency nor oligodendropathy led to detectable spread of antigenic MHC class I- or class II-restricted epitopes during EAE. However, absence of PD-1 clearly increased the propensity of T lymphocytes to expand, and the number of clonal expansions reliably reflected the severity of the EAE disease course. Our data show that the interplay between immune dysregulation and myelinopathy results in a stable exacerbation of actively induced autoimmune CNS inflammation, suggesting that the combination of several pathological issues contributes significantly to disease susceptibility or relapses in human disease.

Maternal PD-1 regulates accumulation of fetal antigen-specific CD8 + T cells in pregnancy

The failure to reject the semi-allogeneic fetus suggests that maternal T lymphocytes are regulated by potent mechanisms in pregnancy. The T cell immunoinhibitory receptor, Programmed Death-1 (PD-1), and its ligand, B7-H1, maintain peripheral tolerance by inhibiting activation of self-reactive lymphocytes. Here, we investigated the role of the PD-1/B7-H1 pathway in maternal tolerance of the fetus. Antigen-specific maternal T cells both proliferate and upregulate PD-1 in vivo at mid-gestation in response to paternally inherited fetal antigen. In addition, when these cells carry a null deletion of PD-1, they accumulate excessively in the uterus-draining lymph nodes ( P < 0.001) without a concomitant increase in proliferation. In vitro assays showed that apoptosis of antigen-specific CD8 + PD-1 −/− cells was reduced following peptide stimulation, suggesting that the accumulation of these cells in maternal lymph nodes is due to decreased cell death. However, the absence of neither maternal PD-1 nor B7-H1 had detectable effects on gestation length, litter size, or pup weight at birth in either syngeneic or allogeneic pregnancies. These results suggest that PD-1 plays a previously unrecognized role in maternal–fetal tolerance by inducing apoptosis of paternal antigen-specific T cells during pregnancy, thereby controlling their abundance.

Blockade of PD-1/B7-H1 Interaction Restores Effector CD8+ T Cell Responses in a Hepatitis C Virus Core Murine Model

The impaired function of CD8(+) T cells is characteristic of hepatitis C virus (HCV) persistent infection. HCV core protein has been reported to inhibit CD8(+) T cell responses. To determine the mechanism of the HCV core in suppressing Ag-specific CD8(+) T cell responses, we generated a transgenic mouse, core(+) mice, where the expression of core protein is directed to the liver using the albumin promoter. Using a recombinant adenovirus to deliver Ag, we demonstrated that core(+) mice failed to clear adenovirus-LacZ (Ad-LacZ) infection in the liver. The effector function of LacZ-specific CD8(+) T cells was particularly impaired in the livers of core(+) mice, with suppression of IFN-gamma, TNF-alpha, and granzyme B production by CD8(+) T cells. In addition, the impaired CD8(+) T cell responses in core(+) mice were accompanied by the enhanced expression of the inhibitory receptor programmed death-1 (PD-1) by LacZ-specific CD8(+) T cells and its ligand B7-H1 on liver dendritic cells following Ad-LacZ infection. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway (using a B7-H1 blocking antibody) in core(+) mice enhanced effector function of CD8(+) T cells and cleared Ad-LacZ-infection as compared with that in mice treated with control Ab. This suggests that the regulation of the PD-1/B7-H1 inhibitory pathway is crucial for HCV core-mediated impaired T cell responses and viral persistence in the liver. This also suggests that manipulation of the PD-1/B7-H1 pathway may be a potential immunotherapy to enhance effector T cell responses during persistent HCV infection.

Blocking of monocyte-associated B7-H1 (CD274) enhances HCV-specific T cell immunity in chronic hepatitis C infection

The establishment of a chronic hepatitis C (CHC) infection is associated with defective HCV-specific T cell responses. Recent studies suggest that negative T cell regulators such as programmed death 1 (PD-1) contribute to the impairment of virus-specific T cell functions in chronic viral infections. However, the implication of peripheral monocytes from CHC patients in the inhibition of HCV-specific T cell responses is only partially defined. In this study, we found that B7-H1, a ligand of PD-1, was significantly up-regulated on monocytes of CHC patients. Proliferation of T cells in response to anti-CD3 antibody was directly suppressed by B7-H1+CD14+ monocytes, and this suppression was reversed by addition of antagonistic B7-H1 mAb. Furthermore, blocking of monocyte-associated B7-H1 (moB7-H1) significantly enhanced the frequency of IFN-gamma-producing, HCV-specific CD4+ and CD8+ effector T cells and the production of Th1 cytokines, such as IL-2 but not Th2 cytokines, including IL-4 and IL-10. Upon B7-H1 blockade, production of perforin was also increased in CD8+ T cells stimulated with HCV peptides. Our findings suggest that moB7-H1 inhibits HCV-specific CD4+ and CD8+ T lymphocyte proliferation and suppresses Th1 cytokine production and perforin secretion. Blockade of the B7-H1 pathway thus represents an attractive approach in the treatment of chronic HCV infection.

Blockade of PD-1/B7-H1 interaction restores antigen-specific CD8+ T cell responses in HCV core transgenic mice (47.8)

Abstract Impaired CD8+ T cells have been implicated to play a role in the establishment of HCV persistent infection. HCV has evolved multiple strategies to avoid T cell responses. HCV core protein has been shown to contain immunomodulatory functions. To determine HCV core-mediated regulation of antigen-specific CD8+ T cell responses in vivo, a transgenic mouse where the expression of core protein is directed to the liver using the albumin (Alb) promoter was generated. Using this HCV core transgenic murine model system, we demonstrated that core-expressing mice failed to clear adenovirus-LacZ (Ad-LacZ) infection in the liver. Particularly, the generation of Ad-LacZ specific CD8+ T cells was impaired in the liver of HCV core transgenic mice having suppression of the production of IFN-gamma and TNF-alpha by CD8+ T cells and alteration of the production of the cytolytic protease, granzyme-B. Interestingly, HCV core transgenic mice exhibited enhanced expression of the inhibitory receptor PD-1 by Ad-LacZ specific CD8+ T cells and its ligand B7-H1 following Ad-LacZ infection. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway using a B7-H1 blocking antibody in core transgenic mice enhances effector function of CD8+ T cells and clearance of Ad-LacZ-infected cells. It suggests that the regulation of the PD-1/B7-H1 inhibitory pathway is crucial for HCV core-mediated impaired T cell responses and viral persistence/maintenance. Also, these findings implicate that manipulation of PD-1/B7-H1 pathway may be a potential immunotherapy to enhance effector T cell responses during persistent infection.

Overexpression of B7-H1 (PD-L1) significantly associates with tumor grade and postoperative prognosis in human urothelial cancers.

The programmed death-1 (PD-1)/B7-H1 (also called PD-L1) pathway negatively regulates T cell activation and has been suggested to play an important role in regulating antitumor host immunity. To investigate the clinical significance of B7-H1 expression to the tumor grade and postoperative prognosis of patients with urothelial cancer, we analyzed the relationship between B7-H1 expression and various clinicopathological features and postoperative prognosis. Sixty-five urothelial cancer cases were examined. B7-H1 expression in tumors and the numbers and phenotypes of tumor-infiltrating lymphocytes were evaluated by immunohistochemistry and flow cytometry. A substantial expression of B7-H1 was observed in all urothelial cancers investigated. Tumor specimens from patients with higher WHO grade or primary tumor classifications showed significantly higher percentages of tumor-associated B7-H1. Tumor-associated B7-H1 expression was significantly associated with a high frequency of postoperative recurrence and poor survival rate. Furthermore, multivariate analysis indicated that tumor-associated B7-H1 was more significant prognostic factor than WHO grade. Our results demonstrate that the aberrant expression of B7-H1 in urothelial cancer is associated with aggressive tumors, suggesting a regulatory role of tumor-associated B7-H1 in antitumor immunity. Therefore, the manipulation of tumor-associated B7-H1 may become a beneficial target for immunotherapy in human urothelial cancer.