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VSIG4, a newly identified co-inhibitory molecule belonging to the B7-related family, is exclusively expressed on tissue-resident macrophages and is involved in the suppression of T cell proliferation and cytokine production. We sought to characterize the role of VSIG4 in anti-tumor immunity in the tumor microenvironment, focusing on VSIG4-expressing tumor-associated macrophages (TAMs). We found that VSIG4-expressing TAMs negatively regulated antigen-specific T cell proliferation and cytokine production through direct inhibition via cell cycle arrest, but not apoptosis, as well as through their arginase 1 activity. Furthermore, VSIG4-expressing TAMs suppress tumor-specific CD8

B7 homolog 1 (B7-H1)–expressing myeloma cells not only inhibit myeloma-specific cytotoxic T lymphocytes (CTL), but also confer a proliferative advantage: resistance to antimyeloma chemotherapy. However, it remains unknown whether B7-H1 expressed on myeloma cells induces cellular responses associated with aggressive myeloma behaviors. To address this question, we analyzed the proliferation and drug sensitivity of B7-H1–expressing myeloma cells transfected with B7-H1–specific short-hairpin RNA or treated with programmed cell death (PD)-1-Fc–coupled beads. Knockdown of B7-H1 expression in myeloma cells significantly inhibited cell proliferation and increased apoptosis induced by the chemotherapeutic alkylating agent melphalan, with downregulation of the expression of cell cycle–related genes (CCND3 and CDK6) and antiapoptotic genes (BCL2 and MCL1). B7-H1 molecules thus contributed to myeloma cell-cycle progression and suppression of drug-induced apoptosis. B7-H1–expressing myeloma cells had a higher affinity for PD-1 than for CD80. PD-1-Fc bead–treated myeloma cells also became resistant to apoptosis that was induced by melphalan and the proteasome inhibitor bortezomib. Apoptosis resistance was associated with the PI3K/AKT pathway. Both myeloma cell drug resistance and antiapoptotic responses occurred through the PI3K/AKT signaling pathway, initiated from “reverse” stimulation of B7-H1 by PD-1. Therefore, B7-H1 itself may function as an oncogenic protein in myeloma cells. The interaction between B7-H1 on myeloma cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces drug resistance in myeloma cells through the PI3K/AKT signaling pathway. These observations provide mechanistic insights into potential immunotherapeutic benefits of blocking the B7-H1–PD-1 pathway. Cancer Immunol Res; 4(9); 779–88. ©2016 AACR.

Supplementary Table S1 Primer Sequence. Supplementary Figure S1. (A) Surface antigen expression and (B) Cell morphology of MOSTI-1 cells, and (C) drug sensitivity to melphalan and bortezomib of MOSTI-1, U266, and RPMI8226 cells. Supplementary Figure S2. Expression of cell cycle-related and antiapoptotic genes in B7-H1.KMS28-PE (B7-H1) and mock.KMS28-PE (mock) cells. Supplementary Figure S3. No effect of cell aggregation on drug sensitivity in MOSTI-1.

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Purpose:Immune-checkpoint inhibitors have shown therapeutic efficacy in various malignant diseases. However, anti-programmed death (PD)-1 therapy has not shown clinical efficacy in multiple myeloma.Experimental Design:Bone marrow (BM) mononuclear cells were obtained from 77 newly diagnosed multiple myeloma patients. We examined the expression of immune-checkpoint receptors in BM CD8+ T cells and their functional restoration by ex vivo treatment with anti–PD-1 and TGFβ inhibitors.Results:We confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of multiple myeloma patients. PD-1–expressing CD8+ T cells from the BM of multiple myeloma patients coexpressed other checkpoint inhibitory receptors and exhibited a terminally differentiated phenotype. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. BM CD8+ T cells from multiple myeloma patients exhibited reduced proliferation and cytokine production upon T-cell receptor stimulation. However, anti–PD-1 did not increase the proliferation of BM CD8+ T cells from multiple myeloma patients, indicating that T-cell exhaustion in multiple myeloma is hardly reversed by PD-1 blockade alone. Intriguingly, anti–PD-1 significantly increased the proliferation of BM CD8+ T cells from multiple myeloma patients in the presence of inhibitors of TGFβ, which was overexpressed by myeloma cells.Conclusions:Our findings indicate that combined blockade of PD-1 and TGFβ may be useful for the treatment of multiple myeloma.

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The V-set and transmembrane domain-containing protein (VSTM) family is a newly discovered immunoglobulin (Ig) superfamily that shares structural similarities with the B7-like transmembrane proteins. Although most VSTM5 members have been reported to exert immune-related functions, VSTM5 has been described as a regulator of neuronal morphogenesis and migration in the brain. Based on its close phylogenetic relationship with two immune checkpoints, VISTA and TIGIT, we investigated the potential role of VSTM5 in T-cell immune responses. VSTM5.Ig inhibits T-cell proliferation and cytokine production, induces T-cell apoptosis, and promotes the generation of regulatory T cells (Tregs) in in vitro T-cell assays. VSTM5 also contributes to the maintenance of T-cell anergy in vitro. Similarly, serum VSTM5.Ig produced using a recombinant plasmid in ovalbumin (OVA)-immunized mice inhibits both naive and effector T-cell immune responses. In addition, VSTM5.Ig enhances oral tolerance of cell-mediated and antibody responses in OVA-fed mice by inducing Tregs and T-cell clonal deletion. Consequently, our findings suggest that VSTM5 is a novel immune checkpoint that could be used to improve the therapeutic efficacy of tolerance-based therapies for autoimmune diseases.

Objectives Lymphocyte-activation gene 3 (LAG-3) represents a potential immune checkpoint target for cancer treatment. We investigated LAG-3 expression and its prognostic value in patients with surgically treated clear cell renal cell carcinoma (RCC) and correlated LAG-3 expression with programmed cell death ligand 1(PD-L1). Methods We evaluated LAG-3 and PD-L1 expression using immunohistochemistry on tissue microarrays incorporating 134 primary excision specimens of clear cell RCC (ccRCC). The patients were analyzed as two groups: the whole cohort and those with metastatic RCC (mRCC). The cancer genome atlas (TCGA) data analysis of LAG-3 was done through UALCAN web servers. Results Using the UALCAN cancer transcriptional data analysis, we found that LAG-3 was overexpressed in ccRCC. LAG-3 expression was significantly correlated with PD-L1 expression in the whole cohort and in the mRCC group (all, p < 0.05). Both LAG-3⁺ RCC and PD-L1⁺ RCC presented with a higher TNM stage and higher Fuhrman nuclear grade (all, p < 0.05). PD-L1⁺/LAG-3⁺ RCC and PD-L1⁻/LAG-3⁺ RCC showed poorer cancer-specific survival (CSS) than PD-L1⁻/LAG-3⁻ RCC (all, p = 0.01). Similarly, PD-L1⁺/LAG-3⁺ mRCC and PD-L1⁻/LAG-3⁺ mRCC showed poorer CSS than PD-L1⁻/LAG-3⁻ mRCC (all, p < 0.05). Multivariate analysis showed that PD-L1⁺/LAG-3⁺ mRCC (hazard ratio: 3.19; 95% CI: 0.77–13.67; p = 0.033) was a predictor of poor CSS. Conclusion Both LAG-3⁺ and PD-L1⁺ RCC have adverse pathological features, and their coexpression predicts worse clinical outcomes. Our findings suggest LAG-3 blockade in combination with programmed cell death 1/PD-L1 blockade as a potential therapeutic approach for RCC.

In the last decade, we have witnessed an unprecedented clinical success in cancer immunotherapies targeting the programmed cell-death ligand 1 (PD-L1) and programmed cell-death 1 (PD-1) pathway. Besides the fact that PD-L1 plays a key role in immune regulation in tumor microenvironment, recently a plethora of reports has suggested a new perspective of non-immunological functions of PD-L1 in the regulation of cancer intrinsic activities including mesenchymal transition, glucose and lipid metabolism, stemness, and autophagy. Here we review the current understanding on the regulation of expression and intrinsic protumoral activity of cancer-intrinsic PD-L1. [BMB Reports 2021; 54(1): 12-20].

Purpose: Immune-checkpoint inhibitors have shown therapeutic efficacy in various malignant diseases. However, anti-programmed death (PD)-1 therapy has not shown clinical efficacy in multiple myeloma. Experimental Design: Bone marrow (BM) mononuclear cells were obtained from 77 newly diagnosed multiple myeloma patients. We examined the expression of immune-checkpoint receptors in BM CD8+ T cells and their functional restoration by ex vivo treatment with anti–PD-1 and TGFβ inhibitors. Results: We confirmed the upregulation of PD-1 and PD-L1 expression in CD8+ T cells and myeloma cells, respectively, from the BM of multiple myeloma patients. PD-1–expressing CD8+ T cells from the BM of multiple myeloma patients coexpressed other checkpoint inhibitory receptors and exhibited a terminally differentiated phenotype. These results were also observed in BM CD8+ T cells specific to myeloma antigens NY-ESO-1 and HM1.24. BM CD8+ T cells from multiple myeloma patients exhibited reduced proliferation and cytokine production upon T-cell receptor stimulation. However, anti–PD-1 did not increase the proliferation of BM CD8+ T cells from multiple myeloma patients, indicating that T-cell exhaustion in multiple myeloma is hardly reversed by PD-1 blockade alone. Intriguingly, anti–PD-1 significantly increased the proliferation of BM CD8+ T cells from multiple myeloma patients in the presence of inhibitors of TGFβ, which was overexpressed by myeloma cells. Conclusions: Our findings indicate that combined blockade of PD-1 and TGFβ may be useful for the treatment of multiple myeloma.

Lymphocyte-activation gene 3 (LAG-3) protein is a potential immune checkpoint target for cancer treatment. Thus, we investigated LAG-3 expression and its prognostic value in clear cell renal cell carcinoma (ccRCC) and correlated LAG-3 expression with programmed cell death ligand 1 (PD-L1) using immunohistochemistry on tissue microarrays incorporating 134 ccRCC primary excision specimens. The study patients were analyzed as two groups: the whole cohort and those with metastatic renal cell carcinoma (mRCC). LAG-3 expression was significantly correlated with PD-L1 expression in the whole cohort and in the mRCC group (all, p p < 0.05). According to the International Metastatic Renal Cell Carcinoma Database Consortium risk classification, there were more intermediate- and poor-risk patients in both LAG-3⁺ mRCC and PD-L1⁺ mRCC than LAG-3⁻ mRCC and PD-L1⁻ mRCC (all, p < 0.05). PD-L1⁺/LAG-3⁺ RCC and PD-L1⁻/LAG-3⁺ RCC in the whole cohort showed poorer cancer-specific survival (CSS) than PD-L1⁻/LAG-3⁻ RCC (all, p = 0.01). Similarly, PD-L1⁺/LAG-3⁺ mRCC and PD-L1⁻/LAG-3⁺ mRCC showed poorer CSS than PD-L1⁻/LAG-3⁻ mRCC (all, p < 0.05). Multivariate analysis showed that PD-L1⁺/LAG-3⁺ mRCC (hazard ratio: 3.19; p = 0.033) was a predictor of poor CSS. Our findings provided a scientific rationale for LAG-3 blockade in combination with programmed cell death 1/PD-L1 blockade as a potential therapeutic approach for RCC.

Oral tolerance can prevent unnecessary immune responses against dietary antigens. Members of the B7 protein family play critical roles in the positive and/or negative regulation of T cell responses to interactions between APCs and T cells. V-set and Ig domain-containing 4 (VSIG4), a B7-related co-signaling molecule, has been known to act as a co-inhibitory ligand and may be critical in establishing immune tolerance. Therefore, we investigated the regulation of VSIG4 signaling in a food allergy and experimental oral tolerance murine models. We analyzed the contributions of the two main sites involved in oral tolerance, the mesenteric lymph node (MLN) and the liver, in VSIG4-mediated oral tolerance induction. Through the comparative analysis of major APCs, dendritic cells (DCs) and macrophages, we found that Kupffer cells play a critical role in inducing regulatory T cells (Tregs) and establishing immune tolerance against oral antigens via VSIG4 signaling. Taken together, these results suggest the possibility of VSIG4 signaling-based regulation of orally administered antigens.

The goal of the present study focused on the adverse reaction of contrast medium (CM) via the induction of inflammatory molecules in human umbilical vein endothelial cells (HUVECs). Ultravist-induced monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) gene expression was markedly increased in interleukin-4 (IL-4)-pretreated HUVECs in a time- and dose-dependent manner and was paralleled by concomitant production of MCP-1 and VCAM-1 proteins. MCP-1 and VCAM-1 gene expression by Ultravist in combination with IL-4 was mediated by the c-Jun N-terminal kinases (JNK1/2) signaling pathway. IL-4-pretreated Ultravist-stimulated HUVECs showed greatly increased migration and adhesion of THP-1 cells. Cell migration was decreased by treatment of CCR2 antagonist, and cell adhesion was also decreased by VCAM-1 blocking antibody. Furthermore, when tested in vivo under similar conditions, MCP-1 protein was significantly increased in Ultravist combined with IL-4-injected mice. Taken together, our findings suggest that MCP-1 blocking may be crucial in preventing the endothelial dysfunction induced by contrast medium in patients with inflammatory disease and atherosclerosis.

B7 homolog 1 (B7-H1)–expressing myeloma cells not only inhibit myeloma-specific cytotoxic T lymphocytes (CTL), but also confer a proliferative advantage: resistance to antimyeloma chemotherapy. However, it remains unknown whether B7-H1 expressed on myeloma cells induces cellular responses associated with aggressive myeloma behaviors. To address this question, we analyzed the proliferation and drug sensitivity of B7-H1–expressing myeloma cells transfected with B7-H1–specific short-hairpin RNA or treated with programmed cell death (PD)-1-Fc–coupled beads. Knockdown of B7-H1 expression in myeloma cells significantly inhibited cell proliferation and increased apoptosis induced by the chemotherapeutic alkylating agent melphalan, with downregulation of the expression of cell cycle–related genes (CCND3 and CDK6) and antiapoptotic genes (BCL2 and MCL1). B7-H1 molecules thus contributed to myeloma cell-cycle progression and suppression of drug-induced apoptosis. B7-H1–expressing myeloma cells had a higher affinity for PD-1 than for CD80. PD-1-Fc bead–treated myeloma cells also became resistant to apoptosis that was induced by melphalan and the proteasome inhibitor bortezomib. Apoptosis resistance was associated with the PI3K/AKT pathway. Both myeloma cell drug resistance and antiapoptotic responses occurred through the PI3K/AKT signaling pathway, initiated from “reverse” stimulation of B7-H1 by PD-1. Therefore, B7-H1 itself may function as an oncogenic protein in myeloma cells. The interaction between B7-H1 on myeloma cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces drug resistance in myeloma cells through the PI3K/AKT signaling pathway. These observations provide mechanistic insights into potential immunotherapeutic benefits of blocking the B7-H1–PD-1 pathway. Cancer Immunol Res; 4(9); 779–88. ©2016 AACR.

Immune-checkpoint inhibitors have shown therapeutic efficacy in various malignant diseases. However, anti-programmed death (PD)-1 therapy has not shown clinical efficacy in multiple myeloma.Bone marrow (BM) mononuclear cells were obtained from 77 newly diagnosed multiple myeloma patients. We examined the expression of immune-checkpoint receptors in BM CD8We confirmed the upregulation of PD-1 and PD-L1 expression in CD8Our findings indicate that combined blockade of PD-1 and TGFβ may be useful for the treatment of multiple myeloma.

Our previous study demonstrated that G-CSF treatment increased the expression of TLR2 in donor grafts; this contributed to rapid engraftment after allogeneic hematopoietic stem cell transplantation (HSCT) in mice. In the current study, we investigated the effects of upregulated TLR2 expression in G-CSF-mobilized donor grafts on acute graft-versus-host disease (GVHD). We found that TLR2 was highly expressed on myeloid cell populations but not T and B cells from the spleens of G-CSF-treated donor mice. After transplantation, the mortality and disease severity in recipients were not significantly different between G-CSF-treated TLR2-/- and wt donor grafts. Although endogenous TLR2 ligand was detected in the serum of both recipients, T cells from TLR2-/- and wt donors have the same ability regarding alloreactivity. Moreover, the blockade of TLR2 signaling in recipients by administering anti-TLR2 blocking antibody after BMT did not lead to a significant difference in acute GVHD compared with control IgG treatment. However, the hematopoietic ability of G-CSF-mobilized lin−c-kit+ HSCs from TLR2-/- donor grafts was lower than that from wt donor grafts. Our results demonstrate that upregulated TLR2 expression in G-CSF-mobilized donor grafts has no effect on acute GVHD, suggesting that TLR2 is a valuable target for increasing HSCT efficiency in order to enhance engraftment without exacerbating acute GVHD.

Hepatitis C virus (HCV) E2 protein binds to CD81, which is a component of the B cell co-stimulatory complex. The E2-CD81 interaction leads to B cell proliferation, protein tyrosine phosphorylation and to the hypermutation of immunoglobulin genes. Epidemiological studies have reported a high prevalence of B cell non-Hodgkin lymphoma (NHL) in HCV-positive patients, suggesting a potential association between HCV and Epstein-Barr virus (EBV) in the genesis of B lymphocyte proliferative disorders. In the present study, in order to investigate the association between EBV and HCV in B cells, we created an in vitro EBV-induced B cell transformation model. CD81 was gradually overexpressed during transformation by EBV. B cells isolated from HCV-positive patients grew more rapidly and clumped together earlier than B cells isolated from healthy donors following EBV infection. Pre-stimulation of CD81 expressed by resting B cells with anti-CD81 monoclonal antibody (mAb) or HCV E2 accelerated the generation of lymphoblastoid cell lines (LCLs) by EBV infection. These cells proliferated prominently through the early expression of interleukin-10 and intracellular latent membrane protein (LMP)-l. By contrast, the overexpression of CD81 on EBV-transformed B cells by anti-CD81 mAb or HCV E2 protein induced apoptosis through reactive oxygen species (ROS)-mediated mitochondrial dysfunction. These results suggest that the engagement of CD81 expressed by B cells has differential effects on B cell fate (proliferation or apoptosis) according to EBV infection and the expression level of CD81.

Regulatory T (Treg) cells act as terminators of T cell immuniy during acute phase of viral infection; however, their role and suppressive mechanism in chronic viral infection are not completely understood. In this study, we compared the phenotype and function of Treg cells during acute or chronic infection with lymphocytic choriomeningitis virus. Chronic infection, unlike acute infection, led to a large expansion of Treg cells and their upregulation of programmed death-1 (PD-1). Treg cells from chronically infected mice (chronic Treg cells) displayed greater suppressive capacity for inhibiting both CD8+ and CD4+ T cell proliferation and subsequent cytokine production than those from naive or acutely infected mice. A contact between Treg and CD8+ T cells was necessary for the potent suppression of CD8+ T cell immune response. More importantly, the suppression required cell-specific expression and interaction of PD-1 on chronic Treg cells and PD-1 ligand on CD8+ T cells. Our study defines PD-1 upregulated on Treg cells and its interaction with PD-1 ligand on effector T cells as one cause for the potent T cell suppression and proposes the role of PD-1 on Treg cells, in addition to that on exhausted T cells, during chronic viral infection.

Oral tolerance can prevent unnecessary immune responses against dietary antigens. Members of the B7 protein family play critical roles in the positive and/or negative regulation of T cell responses to interactions between APCs and T cells. V-set and Ig domain-containing 4 (VSIG4), a B7-related co-signaling molecule, has been known to act as a co-inhibitory ligand and may be critical in establishing immune tolerance. Therefore, we investigated the regulation of VSIG4 signaling in a food allergy and experimental oral tolerance murine models. We analyzed the contributions of the two main sites involved in oral tolerance, the mesenteric lymph node (MLN) and the liver, in VSIG4-mediated oral tolerance induction. Through the comparative analysis of major APCs, dendritic cells (DCs) and macrophages, we found that Kupffer cells play a critical role in inducing regulatory T cells (Tregs) and establishing immune tolerance against oral antigens via VSIG4 signaling. Taken together, these results suggest the possibility of VSIG4 signaling-based regulation of orally administered antigens.

The lung is a prototypic organ that was evolved to reduce immunopathology during the immune response to potentially hazardous endogenous and exogenous antigens. In this study, we show that donor CD4+ T cells transiently induced expression of indoleamine 2,3-dioxygenase (IDO) in lung parenchyma in an IFN-γ–dependent manner early after allogeneic hematopoietic stem cell transplantation (HSCT). Abrogation of host IDO expression by deletion of the IDO gene or the IFN-γ gene in donor T cells or by FK506 treatment resulted in acute lethal pulmonary inflammation known as idiopathic pneumonia syndrome (IPS). Interestingly, IL-6 strongly induced IDO expression in an IFN-γ–independent manner when deacetylation of STAT3 was inhibited. Accordingly, a histone deacetylase inhibitor (HDACi) could reduce IPS in the state where IFN-γ expression was suppressed by FK506. Finally, l-kynurenine produced by lung epithelial cells and alveolar macrophages during IPS progression suppresses the inflammatory activities of lung epithelial cells and CD4+ T cells through the aryl hydrocarbon receptor pathway. Taken together, our results reveal that IDO is a critical regulator of acute pulmonary inflammation and that regulation of IDO expression by HDACi may be a therapeutic approach for IPS after HSCT.

Aim B7-H1, which belongs to the B7 family of costimulatory molecules, is implicated in the ability of tumors to evade the host immune response. The development of evasion mechanisms within the tumor microenvironment may be responsible for poor therapeutic responses. In this manuscript, we report that the 15-deoxy-δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), peroxisome proliferator-activated receptor gamma (PPARγ) activator leads to the downregulation of the cancer-associated expression of B7-H1 in response to interferon-gamma (IFN-γ) and the associated signaling cascades. Main methods The expression of B7-H1 from IFN-γ-induced B16F10 melanoma cells was measured with flow cytometric analysis. The regulatory mechanisms of 15d-PGJ 2 on cellular signaling pathways were examined using Western blot and electrophoretic mobility shift assays. Key findings The flow cytometric analysis revealed that the B7-H1 costimulatory molecule is significantly upregulated in B16F10 melanoma cells by stimulation with IFN-γ. However, 15d-PGJ 2 strongly downregulates B7-H1 expression in IFN-γ-stimulated B16F10 melanoma cells. Furthermore, the significant damping effect of 15d-PGJ 2 on B7-H1 expression involves the inhibition of the tyrosine phosphorylation of Janus kinase (Jak) and signal transducer(s) and activator(s) of transcription (STAT) and, thereby, the interferon regulatory factor-1 (IRF-1) trans-activation of STAT. These effects of 15d-PGJ 2 were not abrogated by the PPARγ antagonist GW9662, indicating that they occur through a PPARγ-independent mechanism. Significance In this study, we demonstrate that 15d-PGJ 2 suppresses the IFN-γ-elicited expression of B7-H1 by the inhibition of IRF-1 transcription via the Jak/STAT signaling pathway through a PPARγ-independent mechanism in mouse melanoma cells.

Tumor cells have been used as the tumor antigen sources for developing cancer vaccines. Due to their low immunogenicity, tumor antigens are combined with various adjuvants to enhance immunogenicity of cancer vaccines. Among them, a natural killer T cell (NKT)-ligand, α-galactosylceramide (αGC) has been reported as a powerful adjuvant showing therapeutic effects in solid tumors as well as hematological malignancies including lymphoma. In this study, we applied αGC-based tumor cell vaccine in mouse multiple myeloma model. The αGC-loaded MOPC315BM myeloma cell vaccine efficiently retarded tumor growth, induced regression of established tumors, and protected surviving mice from tumor rechallenge. Therapeutic responses were associated with induction of strong humoral immune responses, including myeloma-specific antibodies, and cellular immune responses, including myeloma-specific CD8(+) cytotoxic T lymphocytes and memory T cells. In addition, regulatory T cells were significantly decreased in mice that received the αGC-loaded myeloma cell vaccine. Thus, our results demonstrated that αGC-loaded myeloma vaccine efficiently promoted NKT-dependent anti-tumor immunity in a mouse model. These findings are informative for improving the efficacy of tumor-cell-based immunotherapy for patients with MM and other CD1d-expressing tumors.

This paper presents a circulating tumor cell (CTC) microseparator for isolation of CTCs from human peripheral blood using immunomagnetic nanobeads with bound antiepithelial cell adhesive molecule (EpCAM) antibodies that specifically bind to epithelial cancer cells. The isolation is performed through lateral magnetophoresis, which is induced by high-gradient magnetic separation technology, involving a ferromagnetic wire array inlaid in the bottom substrate of a microchannel. Experimental results showed that the CTC microseparator isolates about 90% of spiked CTCs in human peripheral blood at a flow rate of up to 5 mL/h and purifies to approximately 97%. The overall isolation procedure was completed within 15 min for 200 μL of peripheral blood. CTCs from peripheral blood of patients with breast and lung cancers were isolated with the CTC microseparator, and the results were compared with those of healthy donors. Using a fluorescence-based viability assay, the viability of CTCs isolated from peripheral blood of patients with cancer was observed. In addition, the usefulness of the CTC microseparator for subsequent genetic assay was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of cancer-specific genes using CTCs isolated from patients with cancer.

// Jin Roh 1 , Youkyoung Jeon 6 , A-Neum Lee 4, 5 , Sang Min Lee 7 , YeonMee Kim 8 , Chang Ohk Sung 1 , Chan-Jeoung Park 3 , Jung Yong Hong 2 , Dok Hyun Yoon 2 , Cheolwon Suh 2 , Jooryung Huh 1 , Inhak Choi 6, * and Chan-Sik Park 1, 4, 5, * 1 Department of Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 2 Department of Oncology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 3 Department of Laboratory Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 4 Asan Institute for Life Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 5 Cell Dysfunction Research Center, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 6 Department of Microbiology and Immunology, Advanced Research Center for Multiple Myeloma, Inje University College of Medicine, Busan, Korea 7 Department of Hematology/Oncology, Busan Paik Hospital, Inje University College of Medicine, Busan, Korea 8 Department of Pathology, Haeundae Paik Hospital, Inje University College of Medicine, Busan, Korea * These authors have contributed equally to this work Correspondence to: Chan-Sik Park, email: csikpark@amc.seoul.kr Inhak Choi, email: miccih@inje.ac.kr Keywords: multiple myeloma, immune checkpoint, VSIG4, immunohistochemistry, prognosis Received: November 14, 2016 Accepted: June 20, 2017 Published: July 22, 2017 ABSTRACT Multiple myeloma (MM) remains as an incurable disease, despite recent substantial improvements in treatment. Therefore, development of novel biomarkers for risk stratification and new therapeutic targets are imperative. One of the emerging treatments for MM is the immune checkpoint blockades. V-set Ig domain-containing 4 (VSIG4) is a lately studied B7-related immune checkpoint modulator. We assessed the VSIG4 expression in patients with MM and its prognostic impact. We analyzed 81 bone marrow and 66 extramedullary biopsy samples of MM patients using immunohistochemistry. VSIG4 mRNA expression data from the Multiple Myeloma Genomics Portal (MMGP) were analyzed to validate our results. The overall survival (OS) of the high VSIG4 expression group was significantly poorer than that of the low VSIG4 expression group ( p = 0.046). VSIG4 expression was remained statistically significant after adjustment for revised international staging system (rISS) and Mayo stratification algorithm (mSMART) risk classification, respectively ( p = 0.019 and 0.017). Corroborating results were also observed on analyses of VSIG4 expression in patients with extramedullary MM and external data from the MMGP. Our results suggest that VSIG4 expression in MM is an independent indicator of poor prognosis, implying a possible therapeutic target for immunotherapy for MM.

V-set and Ig domain-containing 4 (VSIG4, CRIg, or Z39Ig), a newly identified B7-related cosignaling molecule, is a complement receptor and a coinhibitory ligand that negatively regulates T-cell immunity. Despite its exclusive expression on liver Kupffer cells (KCs) that play key roles in liver tolerance, the physiological role of VSIG4 in liver tolerance remains undefined. Mice lacking VSIG4 had poor survival rates and severe liver pathology in a concanavalin A (ConA)-induced hepatitis (CIH) model, which could be prevented by adoptive transfer of VSIG4+ KCs. The absence of VSIG4 rendered endogenous liver T- and natural killer T (NKT)-cells more responsive to antigen-specific stimulation and impaired tolerance induction in those cells against their cognate antigens. T-cell costimulation with VSIG4.Ig suppressed Th1-, Th2-, and Th17-type cytokine production and arrested the cell cycle at the G0/G1 phase but did not induce apoptosis in vitro. VSIG4-mediated tolerance induction and cell-cycle arrest were further supported by down-regulation of G1 phase-specific Cdk2, Cdk4, and Cdk6, and up-regulation of tolerance-inducing p27KIP-1 in VSIG4.Ig-stimulated T-cells. Administration of soluble VSIG4.Ig to wildtype mice prevented CIH development and prolonged the survival of mice with established CIH. Conclusion: Collectively, our results suggest that VSIG4+ KCs play a critical role in the induction and maintenance of liver T- and NKT-cell tolerance, and that modulation of the VSIG4 pathway using a VSIG4.Ig fusion protein may provide useful immunological therapies against immune-mediated liver injury including autoimmune hepatitis. (HEPATOLOGY 2012;56:1838–1848)

The immunoregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic peripheral blood cell transplantation (PBCT) have been demonstrated to reduce acute graft-versus-host disease (GVHD). However, the underlying mechanism is still not clear. In this study, we focused on the direct effects of G-CSF on donor CD4(+) T cell responses after transplantation. We observed that lethally irradiated B6D2F1 recipient mice that are transplanted with CD4(+) T cells from G-CSF-treated B6 donors showed mild attenuations in severity and mortality compared with recipients transplanted with PBS-treated CD4(+) T cells. Notably, skin GVHD was significantly reduced, but no such reduction was observed in other organs. Although there was no difference with respect to alloreactive expansion or Foxp3(+) Treg induction, the use of G-CSF-treated CD4(+) T cells significantly reduced the numbers of IL-17-producing and RORγt-expressing cells in the secondary lymphoid organs of allogeneic recipients after transplantation compared with the use of the control cells. Finally, we found that the suppressor of cytokine signaling-3 (SOCS3) expression in G-CSF-treated donor CD4(+) T cells was much higher than that in control CD4(+) T cells. Our results demonstrate that the inhibition of Th17 cell differentiation by SOCS3 induction is associated with the immunoregulatory role of G-CSF in CD4(+) T cell-mediated acute GVHD.

This report introduces an on-chip integrated reverse transcription (RT)-microchip, which includes two genetic functionalities of RNA extraction and cDNA synthesis. In the RNA extraction compartment, RNA is extracted from peripheral blood lysate within 1 min, by lateral magnetophoresis using magnetic oligo-dT beads. The extracted RNA is then collected and used directly to produce cDNA in the cDNA synthesis microchamber, which is monolithically integrated with the RNA extraction compartment. To verify the superiority of the proposed RT-microchip, RT-PCR amplification was performed using cDNA harvested from the RT-microchip, and the results were compared with those obtained using typical RNA extraction methods such as a silica matrix column and magnetic oligo-dT beads. The RT-PCR amplification results using 100 μl of blood showed that the intensity of the bands in gel electrophoresis of the RT-microchip was 2-fold stronger than that of the silica matrix column and 2.65-fold stronger than that of the magnetic oligo-dT beads. The results demonstrate that the RT-microchip technique is the most sensitive of the tested methods.

The immunological death induction by EY-6 on the human tumor cell lines was screened. Human colon carcinoma (HCT15, HCT116), gastric carcinoma (MKN74, SNU668), and myeloma (KMS20, KMS26, KMS34) cells were died by EY-6 treatment with dose-dependent manner. CRT expression, a typical marker for the immunological death, was increased on the EY-6-treated colorectal and gastric cancer cells. Interestingly, the effects on the myeloma cell lines were complicated showing cell line dependent differential modulation. Cytokine secretion from the EY-6 treated tumor cells were dose and cell-dependent. IFN-γ and IL-12 secretion was increased in the treated cells (200% to over 1000% of non-treated control), except HCT116, SNU668 and KMS26 cells which their secretion was declined by EY-6. Data suggest the potential of EY-6 as a new type of immuno-chemotherapeutics inducing tumor-specific cell death. Further studies are planned to confirm the efficacy of EY-6 including in vivo study.

Tryptophan-derived metabolites, initiated by indoleamine 2,3-dioxygenase (IDO), preferentially induce activated T cell death, which is an important mechanism in IDO-mediated T cell suppression. However, the mechanism of this phenomenon remains unclear. We found that 3-hydroxyanthranilic acid (3-HAA), the most potent metabolite, selectively eliminated activated T cells, which were stimulated with the bacterial superantigen staphylococcal enterotoxin A (SEA), but not resting T cells, by inducing apoptosis. We observed 3-HAA-induced depletion of intracellular glutathione (GSH) in activated T cells. When GSH levels were maintained by addition of N-acetylcysteine (NAC) and GSH, 3-HAA-mediated T cell death was completely inhibited. This was associated with extrusion of GSH from activated T cells without increased reactive oxygen species (ROS) formation. Finally, we showed that administration of 3-HAA in mice after allogeneic bone marrow transplantation reduced acute graft-versus-host disease (GVHD) lethality by inhibition of alloreactive T cell expansion through intracellular GSH depletion. Our data suggest that direct depletion of intracellular GSH is the major mechanism of 3-HAA-mediated activated T cell death.

We identified a new immune checkpoint molecule, CPMA9, belonging to the immunoglobulin superfamily (IgSF). In TCGA (The Cancer Genome Atlas) data set, CPMA9 is highly expressed and associated with poor survival in human prostate, lung, liver, breast. Also, we showed that CPMA9 differentially expressed between responder and non-responder with Pembrolizumab, PD1 blockade, treated renal cell carcinoma patients (n=49). CPMA9 inhibits TCR mediated T cell activation through suppress of proliferation, various cytokine secretion. Furthermore, hydrodynamic injection of plasmid encoding CPMA9. Ig into mice that were immunized with ovalbumin inhibits T and B cell response to chicken OVA protein. These data suggest that CPMA9 directly inhibits T cell activation as new immune checkpoint.

Background and purpose: Cilostazol is a specific inhibitor of 3′-5′-cyclic adenosine monophosphate (cAMP) phosphodiesterase, which is widely used to treat ischemic symptoms of peripheral vascular disease. Although cilostazol has been shown to exhibit vasodilator properties as well as antiplatelet and anti-inflammatory effects, its cellular mechanism in microglia is unknown. In the present study, we assessed the anti-inflammatory effect of cilostazol on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-stimulated murine BV2 microglia. Experimental approach: We examined the effects of cilostazol on LPS-induced nuclear factor-kappaB (NF-κB) activation and phosphorylation of mitogen-activated protein kinases (MAPKs). Key results: Cilostazol suppressed production of nitric oxide (NO), prostaglandin E2 (PGE2) and the proinflammatory cytokines, interleukin-1 (IL-1), tumour necrosis factor-α, and monocyte chemoattractant protein-1 (MCP-1), in a concentration-dependent manner. Inhibitory effects of cilostazol were not affected by treatment with an adenylate cyclase inhibitor, SQ 22536, indicating that these actions of cilostazol were cAMP-independent. Cilostazol significantly inhibited the DNA binding and transcriptional activity of NF-κB. Moreover, cilostazol blocked signalling upstream of NF-κB activation by inhibiting extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK), but without affecting the activity of p38 MAPK. Conclusion and implications: Our results demonstrate that suppression of the NF-κB, ERK, JNK signalling pathways may inhibit LPS-induced NO and PGE2 production. Therefore, cilostazol may have therapeutic potential for neurodegenerative diseases by inhibiting pro-inflammatory mediators and cytokine production in activated microglia.

While programmed death-1 (PD-1), a co-inhibitory member of CD28 immunoglobulin superfamily plays negative roles in effector functions of T cells and B cells, little is known about the function of PD-1 expressed on innate immune cells. In this study, we demonstrate that IL-12 production was greatly suppressed in LPS-stimulated RAW264.7 cells upon PD-1 engagement with B7-H1.Fc fusion protein, and was restored in the presence of antagonistic anti-PD-1 mAb. PD-1-mediated suppression of IL-12 production in LPS-stimulated RAW264.7 cells was mediated by inhibition of Janus N-terminal-linked kinase (JNK) signaling pathway, and to a lesser extent, phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway through the recruitment of SHP-2 to PD-1 cytoplasmic tail. B7-H1.Fc-mediated PD-1 engagement also downregulates the expression of co-stimulatory molecules such as CD80, CD86, MHC class I and II proteins in LPS-stimulated RAW264.7 cells. Furthermore, the endocytic activity is enhanced but the allostimulatory capacity is suppressed in LPS-treated RAW264.7 cells upon PD-1 engagement. Taken together, our results reveal a novel function of macrophage PD-1 in the negative regulation of IL-12 synthesis and differentiation into dendritic cell-like cells.

Granulocyte colony-stimulating factor (G-CSF)-mobilized donor graft tissue used for peripheral blood stem cell transplantation contains a large number of immature myeloid cells that suppress alloreactive donor T cells, resulting in an inhibition of acute graft-versus-host disease (GVHD). However, the molecular mechanism underlying the suppressive function of immature myeloid cells is not fully understood. Here, we investigated whether indoleamine 2,3-dioxygenase (IDO) is related to the suppressive mechanism of G-CSF-induced immature myeloid cells (gMCs). We found that Gr-1(+) CD11b(+) cells were highly induced in G-CSF-injected donor graft tissue, which is a phenotype of immature myeloid cells, resulting in an inhibition of acute GVHD lethality by suppressing alloreactive donor T-cell expansion. IDO was not detected in primary isolated gMCs; however, this enzyme was markedly induced after treatment with interferon-gamma (IFN-gamma). This level was significantly higher in IFN-gamma-treated gMCs than in bone marrow myeloid cells, which promote alloreactive T-cell responses. We next investigated the functional role of IDO in gMC-mediated inhibition of acute GVHD lethality. We found no changes in gMC-mediated survival or alloreactive donor T-cell suppression when IDO activity was blocked using 1-methyl tryptophan. In addition, there was no difference in gMC-mediated survival rates between recipients transferred with either wild-type gMCs or IDO(-/-) gMCs. Taken together, our data suggest that gMC-mediated inhibition of lethal acute GVHD is through an IDO-independent mechanism.

The aim of the present work is focused on protective effects of an edible red alga, Laurencia undulata ethanolic (EtOH) extracts (LU) containing a large amount of polyphenols against OVA-induced murine allergic airway reactions using in vivo histological and cytokine assay. Mice sensitized and challenged with ovalbumin (OVA) showed typical asthmatic reactions as follows: an increase in the number of eosinophil in bronchoalveolar lavage fluid; a marked influx of inflammatory cells into the lung around blood vessels and airways, and airway luminal narrowing; the development of airway hyperresponsiveness; the detection of TNF-α and Th2 cytokines, such as IL-4 and IL-5 in the bronchoalveolar lavage (BAL) fluid; and detection of allergen-specific IgE in the serum. The successive intraperitoneal administration of LU before the last airway OVA-challenge resulted in a significant inhibition of all asthmatic reactions. These results suggest that L. undulata polyphenolic extracts possess therapeutic potential for combating bronchial asthma associated with allergic diseases.

B7-H1 is a newly identified member of the B7 family with important regulatory functions in cell-mediated immune responses, and it is expressed in human immune cells and several tumors. We first observed that expression of surface B7-H1 on B cells was increased during the immortalization process by EBV, which is strongly related to both inflammation and tumorigenesis. Cross-linking of B7-H1 on EBV-transformed B cells using anti-B7-H1 Ab (clone 130002) induced reactive oxygen species (ROS) generation, mitochondrial disruption, release of apoptotic proteins from mitochondria, and subsequent apoptosis. Inhibition of caspases and ROS generation recovered B7-H1-mediated apoptosis and proteolytic activities of caspase-8, -9, and -3. We observed that B7-H1 stimulation induced both transcription and translation of fasL. ZB4, an antagonistic anti-fas Ab, and NOK-1, an antagonistic anti-fasL Ab, effectively blocked apoptosis without exerting any influence on ROS generation. N-acetylcysteine (NAC) completely blocked the induction of fasL mRNA and protein. We found that B7-H1 stimulation activated the phosphorylation of JNK and c-jun and down-regulated ERK1/2 and p-Akt. NAC blocked the activation of JNK and down-regulation of ERK, but both z-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) and ZB4 did not inhibit JNK activation of B7-H1 stimulation. SP600125 blocked fasL induction and apoptosis but did not affect ROS generation after B7-H1 stimulation. Taken together, we concluded that B7-H1-mediated apoptosis on EBV-transformed B cells may be involved in the induction of fasL, which is evoked by ROS generation and JNK activation after cross-linking of B7-H1. These results provide a new concept for understanding reverse signaling through B7-H1 and another mechanism of tumor immunotherapy using anti-B7-H1.

A potent anti-inflammatory activity was detected from methanol extracts prepared from roots of Rhodiola sacra. In the preliminary test, methanol extracts from the roots of R. sacra could potently attenuate histological change in the lung tissue after lipopolysaccharide (LPS)-treated inflammatory stimulation. The active compound has been identified as 3-(3,4-dihydroxy-phenyl)-acrylic acid phenethyl ester (caffeic acid phenethyl ester, CAPE). In vivo study on protective effect of R. sacra CAPE against inflammatory response by endotoxin shock in the LPS-induced inflammatory mice showed significantly attenuated the intravenous LPS-induced increase in plasma TNF-α and IL-1β concentration. R. sacra CAPE significantly inhibited LPS-induced nuclear transcription factor-κB (NF-κB) activation. It also down-regulates enhanced matrix metalloproteinase-9 (MMP-9) activity triggered by LPS in mouse lung. In the present study, the results indicate that the intake of R. sacra CAPE could provide health benefits, reducing the magnitude of the inflammatory process triggered by endotoxin shock and the production of inflammatory mediators.

Ecklonia cava (EC) is a brown alga that evidences radical scavenging activity, bactericidal activity, tyrosinase inhibitory activity, and protease inhibitory activity. However, its anti-allergic effects remain poorly understood. In the current study, we attempted to determine whether pretreatment with EC induces a significant inhibition of asthmatic reactions in a mouse asthma model. Mice sensitized and challenged with ovalbumin (OVA) evidenced typical asthmatic reactions, as follows: an increase in the number of eosinophils in bronchoalveolar lavage fluid; a marked influx of inflammatory cells into the lung around blood vessels and airways, and airway luminal narrowing; the development of airway hyperresponsiveness; the detection of tumor necrosis factor-alpha (TNF-α) and Th2 cytokines, including IL-4 and IL-5 in the bronchoalveolar lavage (BAL) fluid; and the detection of allergen-specific immunoglobulin E (IgE) in the serum. However, the administration of EC extract prior to the final airway OVA challenge resulted in a significant inhibition of all asthmatic reactions. We also demonstrated that EC extracts treatment resulted in significant reductions on matrix metalloproteinase-9 (MMP-9) and Suppressor of cytokine signaling-3 (SOCS-3) expression and a reduction in the increased eosinophil peroxidase (EPO) activity. The treatment of animals with EC extracts resulted in a significant reduction in the concentrations of the Th2 cytokine (IL-4 and IL-5) in the airways, without any concomitant increase in the concentration of Th1 cytokines. These findings indicate that EC extracts may prove useful as an adjuvant therapy for allergic airway reactions via the inhibition of the Th2 response. Accordingly, this study may provide evidence that EC extract performs a critical function in the amelioration of the pathogenetic process of asthma in mice.

Caffeic acid phenethyl ester (CAPE) is a biologically active ingredient of propolis, which has several interesting biological properties, including antioxidant and anti-inflammatory; however, its anti-allergic effects are poorly understood. The objective of this study was to determine whether treatment with CAPE results in significant inhibition of asthmatic reactions in a mouse model. Mice sensitized and challenged with ovalbumin (OVA) had the following typical asthmatic reactions: an increase in the number of eosinophils in bronchoalveolar lavage (BAL) fluid; a marked influx of inflammatory cells into the lung around blood vessels and airways, and airway luminal narrowing; the development of airway hyperresponsiveness (AHR); the presence of tumor necrosis factor-alpha (TNF-alpha) and Th2 cytokines, including IL-4 and IL-5, in the BAL fluid; and the presence of allergen-specific IgE in the serum. Five successive intraperitoneal administrations of CAPE before the last airway OVA challenge resulted in significant inhibition of characteristic asthmatic reactions. We determined that increased generation of reactive oxygen species (ROS) by inhalation of OVA was diminished via the administration of CAPE in BAL fluid, as well as nuclear factor-kappaB (NF-kappaB) DNA binding activity. These findings indicate that oxidative stress may have a crucial function in the pathogenesis of bronchial asthma, and that CAPE may be useful as an adjuvant therapy for the treatment of bronchial asthma.

B7-H1 (also known as CD274 and PD-L1) is a cosignalling molecule regulating T-cell immunity positively or negatively in vivo. However, little is known about the role of endogenous B7-H1 in bacterial infection. We found that B7-H1 expression was up-regulated in various cell populations including CD4+ and CD8+ T cells, natural killer (NK) cells and macrophages following Listeria monocytogenes infection. Administration of the antagonistic B7-H1 monoclonal antibody resulted in a significant increase in mortality in mice infected with a lethal dose of L. monocytogenes compared with mice given the control immunoglobulin. In vivo blockade of B7-H1 greatly inhibited the production of tumour necrosis factor (TNF)-alpha and nitric oxide, key effector molecules responsible for intracellular killing by macrophages. B7-H1 blockade also suppressed the expression of granzyme B and interferon (IFN)-gamma by NK cells. Interestingly, blocking of endogenous B7-H1 selectively inhibited CD8+ T cells rather than CD4+ T cells in response to L. monocytogenes infection, as evidenced by the reduction of IFN-gamma production and the expression of effector surface markers including CD62L(int/low) and CD44(high) in CD8+ T cells from mice given anti-B7-H1 monoclonal antibody. In addition, we found that the proliferation of listeriolysin-O (LLO)-specific and IFN-gamma-producing L. monocytogenes-reactive CD8+ T cells was significantly decreased not only in the effector phase but also in the memory phase in the presence of anti-B7-H1 antibody. Our findings thus suggest that endogenous B7-H1 can provide positive costimulatory signals for innate and adaptive immunity leading to protection against intracellular bacterial infection.

Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts. It has several positive effects, including anti-inflammatory, anti-oxidation, anti-cancer, anti-bacterial, anti-viral, anti-fungal, and immunomodulatory effects. In particular, the suppressive effect of NF-kappaB may disrupt a component of allergic induction. The principal objective of this experimental study was to evaluate the effects of CAPE on the active systemic anaphylaxis induced by ovalbumin (OVA) challenge in mice. Mice were intraperitoneally sensitized and intravenously challenged with OVA. Histopathological analysis, nuclear factor (NF)-kappaB activation, and the plasma levels of histamine and total IgE after allergen challenge were evaluated. After challenges, all of the sham-treated mice developed anaphylactic symptoms, increased plasma levels of histamine and OVA-specific IgE, marked vascular leakage, NF-kappaB activation, platelet-activating factor (PAF) production, and histological changes including pulmonary edema and hemorrhage in the renal medullae within 20 min. By way of contrast, a reduction in the plasma levels of histamine and OVA-specific IgE and an inhibition of NF-kappaB activation and PAF release were observed in the CAPE-treated mice. In addition, a significant prevention of hemoconcentration and OVA-induced pathological changes were noted. These results indicate that CAPE demonstrates an anti-allergic effect, which may be the result of its protective effects against IgE-mediated allergy.

Phage display of single-chain variable fragment (scFv) antibodies is a powerful tool for selecting important, useful, and specific human antibodies. We constructed a library from three patients infected withPlasmodium vivax. Panning on recombinant PvRII enriched a population of scFvs that recognized region II of theP. vivaxDuffy binding protein (DBP). Three clones of scFvs that reacted with PvRII were selected, and their biological functions were analyzed. These scFvs inhibited erythrocyte binding to DBP. Clone SFDBII92 had the greatest affinity (dissociation constant = 3.62 × 10−8M) and the greatest inhibition activity (50% inhibitory concentration ≈ 2.9 μg/ml) to DBP. Thus, we demonstrated that human neutralizing antibody could be made from malaria patients using phage display and that these neutralizing scFvs should prove valuable for developing both passive and active immunization strategies based on DBP.

Silibinin is the primary active compound in silymarin. It has been demonstrated to exert anti-carcinogenic effects and hepato-protective effects. However, the effects of silibinin on the maturation and immunostimulatory activities exhibited by dendritic cells (DCs) remain, for the most part, unknown. In this study, we have attempted to determine whether silibinin can influence surface molecule expression, dextran uptake, cytokine production, capacity to induce T-cell differentiation, and the signaling pathways underlying these phenomena in murine bone marrow-derived DCs. Silibinin was shown to significantly suppress the expression of CD80, CD86, MHC class I, and MHC class II in the DCs, and was also associated with impairments of LPS-induced IL-12 expression in the DCs. Silibinin-treated DCs proved highly efficient with regard to Ag capture via mannose receptor-mediated endocytosis. Silibinin also inhibited the LPS-induced activation of MAPKs and the nuclear translocation of the NF-kappaB p65 subunit. Additionally, silibinin-treated DCs evidenced an impaired induction of Th1 response, and a normal cell-mediated immune response. These findings provide new insight into the immunopharmacological functions of silibinin, especially with regard to their impact on the DCs. These findings expand our current understanding of the immunopharmacological functions of silibinin, and may prove useful in the development of therapeutic adjuvants for acute and chronic DC-associated diseases.

Water-soluble proteoglycan (WSPG) of Agaricus blazei Murill has been known to stimulate the non-specific complements and humoral immune functions to act as polyclonal activators of B cells and to inhibit tumor growth and metastasis. However, little is known about its immunomodulating effects on murine bone marrow (BM)-derived dendritic cells (DC). In the present study, we examined the maturation process of murine BM-DC. BM cells were cultured in the presence of IL-4 and GM-CSF and the generated immature DC were stimulated with WSPG or LPS (WSPG-DC and LPS-DC, respectively) for 24 h. WSPG significantly enhanced the expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex (MHC) II, as did LPS. IL-12p70 production in WSPG-DC was also identical to that in LPS-DC. The antigen-uptake capacity of WSPG-DC was determined by FITC-labeled dextran uptake. WSPG-DC lost dextran uptake capacity comparable to LPS-DC. The antigen-presenting capacity of WSPG-DC as analyzed by allogeneic T cell proliferation was significantly increased in comparison with immature DC, was identical to LPS-DC, and induced higher levels of IL-2 in responding T cells. These results indicate the immunomodulatory properties of WSPG, which might be therapeutically useful in the control of cancers and immunodeficient diseases through the up-regulation of DC maturation.

This study investigated the therapeutic effects of combined local irradiation and anti-HER2/neu antibody in a mixed tumor mouse model comprised of a nonmetastatic neu-positive tumor and a metastatic neu-negative tumor. While local irradiation alone could control the primary tumor in a dose-dependent manner, it did not improve mouse survival. Combined treatment comprised of local irradiation and anti-neu antibody of tumor-bearing BALB/c mice significantly improved mouse survival (P < 0.5), even though the tumor growth was similar to that of the irradiated-alone group. The combined treatment significantly reduced metastatic tumor masses in the lung and increased immune cell infiltration in primary tumor tissues. However, immune deficient nude mice with tumors did not exhibit prolonged survival in response to the combined treatment. Collectively, these results show that combined local irradiation and anti-neu antibody can elicit an immune-mediated abscopal effect to extend survival. Although the mechanism for abscopal effects induced by the combined treatment of radiation and anti-HER2/neu antibody was not elucidated, to our knowledge this is the first published study to describe the abscopal effect induced by the combination of local irradiation and the anti-HER2/neu antibody.

Aberrant B7-H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7-H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7-H4 transcription in primary CD138(+) multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7-H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7-H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7-H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7-H4 expression. Furthermore, knockdown of cytoplasmic B7-H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7-H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7-H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment.