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PDF file - 316K, Supplementary Fig. S1: Effective depletion of PDCA-1+ cells in mouse splenocytes. Supplementary Fig. S2: Validation of gene expression by quantitative real-time PCR. Supplementary Fig. S3: Effects of TLR ligands on body weight changes in mice treated with subcutaneous injections of LLC/SeV/GM cells. Supplementary Fig. S4: Increased number of TVS-infiltrating PDCA-1+ cells in mice treated with combined irLLC/SeV/GM cells and imiquimod. Supplementary Fig. S5: Increased serum IFN-a level in mice treated with combined irLLC/SeV/GM cells and imiquimod. Supplementary Fig. S6: Prophylactic tumor vaccination using irLLC/SeV/GM cells induced significant antitumor effects in a syngeneic mouse model. Supplementary Fig. S7: Enhanced expression levels of maturation and costimulatory molecules including CCR7, CD40, MHC class I, and MHC class II in tumor cells-derived GM-CSF-sensitized DCs in TDLNs.

Vaccination with irradiated granulocyte macrophage colony-stimulating factor (GM-CSF)–transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity. However, its effectiveness is limited. We therefore attempted to improve the antitumor effect by identifying little-known key pathways in GM-CSF–sensitized dendritic cells (GM-DC) in tumor-draining lymph nodes (TDLN). We initially confirmed that syngeneic mice subcutaneously injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) remarkably rejected the tumor growth. Using cDNA microarrays, we found that expression levels of type I interferon (IFN)–related genes, predominantly expressed in plasmacytoid DCs (pDC), were significantly upregulated in TDLN-derived GM-DCs and focused on pDCs. Indeed, mouse experiments demonstrated that the effective induction of GM-CSF–induced antitumor immunity observed in immunocompetent mice treated with LLC/SeV/GM cells was significantly attenuated when pDC-depleted or IFNα receptor knockout (IFNAR−/−) mice were used. Importantly, in both LLC and CT26 colon cancer–bearing mice, the combinational use of imiquimod with autologous GVAX therapy overcame the refractoriness to GVAX monotherapy accompanied by tolerability. Mechanistically, mice treated with the combined vaccination displayed increased expression levels of CD86, CD9, and Siglec-H, which correlate with an antitumor phenotype, in pDCs, but decreased the ratio of CD4+CD25+FoxP3+ regulatory T cells in TDLNs. Collectively, these findings indicate that the additional use of imiquimod to activate pDCs with type I IFN production, as a positive regulator of T-cell priming, could enhance the immunologic antitumor effects of GVAX therapy, shedding promising light on the understanding and treatment of GM-CSF–based cancer immunotherapy. Cancer Immunol Res; 2(6); 568–80. ©2014 AACR.

BackgroundMatricellular glycoprotein, SPARC is a secreted molecule, that mediates the interaction between cells and extracellular matrix. SPARC functions as a regulator of matrix organization and modulates cell behavior. In various kinds of cancer, strong SPARC expression was observed in stromal tissues as well as in cancer epithelial cells. The function of SPARC in cancer cells is somewhat controversial and its impact on peritumoral stromal cells remains to be resolved.MethodsWe investigated the effects of SPARC expression in endometrial cancer cells on the surrounding stromal fibroblasts using in vitro co-culture system. Changes in characteristics of fibroblasts were examined by analysis of fibroblast-specific markers and in vitro contraction assay.ResultsSPARC induced AKT phosphorylation and epithelial-to-mesenchymal transition, consistent with previous reports. Cancer-associated fibroblasts of endometrial cancer expressed higher levels of mesenchymal- and fibroblast-associated factors and had a stronger contraction ability. Unexpectedly, cancer-associated fibroblasts expressed comparable levels of SPARC compared with fibroblasts from normal endometrium. However, co-culture of normal fibroblasts with SPARC-expressing Ishikawa cells resulted in activation of the fibroblasts. Immunodepletion of SPARC did not affect the activation of fibroblasts.ConclusionsOur data indicated that SPARC activated fibroblasts only in the presence of fibronectin, which was abundantly secreted from SPARC-expressing endometrial cancer cells. These results suggested that a SPARC-fibronectin-mediated activation of fibroblasts might be involved in enhanced mobility and invasion of cancer cells.

The prognosis of patients with high‐grade or advanced‐stage endometrial cancer remains poor. As cancer stem‐like cells (CSCs) are thought to be associated with endometrial cancers, it is essential to investigate the molecular mechanisms that regulate endometrial CSCs. Dual‐specificity phosphatase 6 (DUSP6) functions as a negative‐feedback regulator of MAPK–ERK1/2 signaling, but its role in endometrial cancer remains unknown. We investigated whether DUSP6 is involved in cancer cell stemness using endometrial cancer cell lines and specimens from endometrial cancer patients. DUSP6 induced the expression of CSC‐related genes including ALDH1, Nanog, SOX2 and Oct4A, increased the population of cells in the G0/G1 phase, and promoted sphere formation ability. DUSP6 knockdown resulted in reduced cell invasion and metastasis, whereas DUSP6 overexpression inhibited apoptosis under serum‐free conditions. Moreover, DUSP6 decreased phosphorylated ERK1/2 and increased phosphorylated Akt levels, which potentially induces CSC features. In patients with endometrial cancers, DUSP6 expression was determined using immunohistochemistry, and based on the results, the patients were dichotomized into high‐ and low‐DUSP6‐expression groups. Progression‐free survival and overall survival were significantly shorter in the high‐DUSP6‐expression group. These results suggest that DUSP6 has potential value as a biomarker of CSCs and as a target of therapies designed to eliminate CSCs in endometrial cancer., What's new? Although cancer stem‐like cells (CSCs) are involved in human endometrial cancers, the underlying molecular mechanisms and biomarkers for CSCs in endometrial cancers remain elusive. Here, the authors found that DUSP6 plays an important role in regulating endometrial CSC phenotypes by increasing self‐renewal ability and starvation resistance. DUSP6 expression was required for inducing invasion and metastasis and resulted in ERK1/2 dephosphorylation and Akt phosphorylation, which potentially contribute to the promotion of CSC phenotypes. As DUSP6 expression was also positively associated with worse progression‐free and overall survival, DUSP6 represents a potential biomarker for endometrial CSCs and a therapeutic target in endometrial cancers.

Y-box binding protein 2 (YBX2) has been associated with the properties of both germ cells and cancer cells. We hypothesized that YBX2 might contribute to the characteristics of cancer stem cells (CSCs). In this study, we clarified the function of YBX2 in endometrial cancer stem cells. We established a human YBX2-expressing Ishikawa (IK) cell line (IK-YBX2 cells). We analyzed gene expression associated with stemness and isolated SP cells from IK-YBX2 cells. The SP population of IK-YBX2 cells, the expression of ALDH1 and serial sphere-forming capacity were associated with levels of YBX2 expression. IK-YBX2 cells were resistant to anti-cancer drugs. In gene expression analysis, a gene for cancer testis antigen, CT45, was generally overexpressed in IK-YBX2 cells. YBX2-mediated CT45 expression was associated with increased levels of self-renewal capacity and paclitaxel resistance. The level of CT45 expression was enhanced in high-grade and/or advanced stages of human endometrial cancer tissues. We conclude that expression of YBX2 is essential for the stem cell-like phenotype. CT45 contributes to stemness associated with YBX2 and might be related to the progression of endometrial cancer.

Among cancer immunotherapies, granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-transduced tumor cell vaccine (GVAX) therapies appear promising and have been shown to be safe and effective in multiple clinical trials. However, the antitumor efficacies of GVAX therapy alone are in some cases limited. Here we showed that GVAX therapy targeting cancer stem cells (CSCs) substantially suppressed tumor development in syngeneic immunocompetent mice recapitulating normal immune systems. CSCs were isolated as side population (SP) cells from 4T1 murine breast carcinoma cell line and transduced with GM-CSF gene delivered by non-transmissible Sendai virus (4T1-SP/GM). Impaired tumorigenicity of subcutaneously injected 4T1-SP/GM depended on CD8+ T cells in concert with CD4+ T cells and natural killer cells. Mice therapeutically vaccinated with irradiated 4T1-SP/GM cells had markedly suppressed tumor development of subcutaneously transplanted 4T1-SP cells compared with those treated with irradiated cells of non-transduced 4T1-SP cells or non-SP (4T1-NSP/GM) cells. Tumor suppression was accompanied by the robust accumulation of mature dendritic cells at vaccination sites and T-helper type 1-skewed systemic cellular immunity. Our results suggested that CSC cell-based GVAX immunotherapy might be clinically useful for inducing potent tumor-specific antitumor immunity.

Innate γδ T cells expressing Vγ6 produce IL-17A at an early stage following infection with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In this study, we used IL-21 receptor knockout (IL-21R KO) mice and IL-21-producing recombinant BCG mice (rBCG-Ag85B-IL-21) to examine the role of IL-21 in the regulation of IL-17A-producing innate γδ T-cell response following BCG infection. IL-17A-producing Vγ6+ γδ T cells increased in the peritoneal cavity of IL-21R KO mice more than in wild type mice after BCG infection. In contrast, the number of IL-17A-producing Vγ6+ γδ T cells was significantly lower after inoculation with rBCG-Ag85B-IL-21 compared with control rBCG-Ag85B. Notably, exogenous IL-21 selectively induced apoptosis of IL-17A-producing Vγ6+ γδ T cells via Bim. Thus, these results suggest that IL-21 acts as a potent inhibitor of a IL-17A-producing γδ T-cell subset during BCG infection.

Around the world, cervical cancer is one of the most common neoplastic diseases among women, and the prognosis of patients in an advanced stage remains poor. To reduce the mortality rate of cervical cancer, early diagnosis and treatment are essential. DNA methylation is an important aspect of gene regulation, and aberrant DNA methylation contributes to carcinogenesis and cancer progression in various cancers. Although 5-methylcytosine (5mC) has been analyzed intensively, the function of 5-hydroxymethylcytosine (5hmC) has not been clarified. The purpose of our study was to identify the molecular biomarkers for early diagnosis of cervical tumors due to epigenetic alterations. To assess the clinical relevance of DNA methylation, we used immunohistochemistry (IHC) to characterize the level of 5hmC in 102 archived human cervical intraepithelial neoplasia (CIN) samples and cervical cancer specimens. The level of 5hmC was significantly decreased between CIN2 and CIN3. The progression of cervical tumors is caused by a reduction of TP53 and RB1 because of HPV infection. We observed that Tp53 and Rb1 were knocked down in mouse embryonic fibroblasts (MEF), a model of normal cells. The level of 5hmC was reduced in Tp53-knockdown cells, and the expression levels of DNA methyltransferase 1 (DNMT1) and ten-eleven translocation methylcytosine dioxygenase 1 (TET1) were induced. In contrast, there was no significant change in Rb1-knockdown cells. Mechanistically, we focused on apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) 3B (A3B) as a cause of 5hmC reduction after TP53 knockdown. In the human cell line HHUA with a wild-type TP53 gene, A3B was induced in TP53-knockdown cells, and A3B knockdown recovered 5hmC levels in TP53-knockdown cells. These data indicate that TP53 suppression leads to 5hmC reduction in part through A3B induction. Moreover, IHC showed that expression levels of A3B in CIN3 were significantly higher than those in both normal epithelium and in CIN2. In conclusion, 5hmC levels are decreased between CIN2 and CIN3 through the TP53-A3B pathway. Since A3B could impair genome stability, 5hmC loss might increase the chances of accumulating mutations and of progressing from CIN3 to cervical cancer. Thus, these epigenetic changes could predict whether CINs are progressing to cancer or disappearing.

In connection with our studies on antibacterial active compounds in the class of new oxazolidinones against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) strains, some molecular modifications were attempted. In this study, molecular modifications of 4-aminomethyloxazolidin-2-ones (3a) to the corresponding 4-acylaminomethyloxazolidin-2-one derivatives (3c–d) and preparations of the represented twin-drug type molecules (10–14) were investigated. Some additional 4-dialkylaminomethyloxazolidin-2-ones (2) were also synthesized. The synthesized compounds were evaluated for antibacterial activity with Gram-positive (S. aureus) and Gram-negative (E. coli) strains.

Genetic engineering of tumor cells to express immune-stimulatory molecules, including cytokines and co-stimulatory ligands, is a promising approach to generate highly efficient cancer vaccines. The co-signaling molecule, LIGHT, is particularly well suited for use in vaccine development as it delivers a potent co-stimulatory signal through the Herpes virus entry mediator (HVEM) receptor on T cells and facilitates tumor-specific T cell immunity. However, because LIGHT binds two additional receptors, lymphotoxin β receptor and Decoy receptor 3, there are significant concerns that tumor-associated LIGHT results in both unexpected adverse events and interference with the ability of the vaccine to enhance antitumor immunity. In order to overcome these problems, we generated tumor cells expressing the single-chain variable fragment (scFv) of anti-HVEM agonistic mAb on the cell surface. Tumor cells expressing anti-HVEM scFv induce a potent proliferation and cytokine production of co-cultured T cells. Inoculation of anti-HVEM scFv-expressing tumor results in a spontaneous tumor regression in CD4+ and CD8+ T cell-dependent fashion, associated with the induction of tumor-specific long-term memory. Stimulation of HVEM and 4-1BB co-stimulatory signals by anti-HVEM scFv-expressing tumor vaccine combined with anti-4-1BB mAb shows synergistic effects which achieve regression of pre-established tumor and T cell memory specific to parental tumor. Taken in concert, our data suggest that genetic engineering of tumor cells to selectively potentiate the HVEM signaling pathway is a promising antitumor vaccine therapy.

T-cell tolerance is the central program that prevents harmful immune responses against self-antigens, in which inhibitory PD-1 signal given by B7-H1 interaction plays an important role. Recent studies demonstrated that B7-H1 binds CD80 besides PD-1, and B7-H1/CD80 interaction also delivers inhibitory signals in T cells. However, a role of B7-H1/CD80 signals in regulation of T-cell tolerance has yet to be explored. We report here that attenuation of B7-H1/CD80 signals by treatment with anti–B7-H1 monoclonal antibody, which specifically blocks B7-H1/CD80 but not B7-H1/PD-1, enhanced T-cell expansion and prevented T-cell anergy induction. In addition, B7-H1/CD80 blockade restored Ag responsiveness in the previously anergized T cells. Experiments using B7-H1 or CD80-deficient T cells indicated that an inhibitory signal through CD80, but not B7-H1, on T cells is responsible in part for these effects. Consistently, CD80 expression was detected on anergic T cells and further up-regulated when they were re-exposed to the antigen (Ag). Finally, blockade of B7-H1/CD80 interaction prevented oral tolerance induction and restored T-cell responsiveness to Ag previously tolerized by oral administration. Taken together, our findings demonstrate that the B7-H1/CD80 pathway is a crucial regulator in the induction and maintenance of T-cell tolerance.

The membrane microdomains known as lipid rafts have been shown to act as platforms for the initiation of various receptor signals. Through proteomic analysis, we have identified a novel protein termed Raftlin (raft-linking protein) as a major protein in lipid rafts. To determine the physiological and immunological functions of Raftlin in mammals, we generated Raftlin-deficient mice, as well as Raftlin-transgenic (Tg) mice. Although Raftlin was originally identified in B cells, we observe no severe abnormalities in the B cells of these mice, presumably due to a high expression of Raftlin-homologue (Raftlin-2). T cells, in contrast, expressed a substantial amount of Raftlin but no Raftlin-2. In Raftlin-deficient mice, T cell-dependent Ab production was reduced, and experimental autoimmune encephalomyelitis, a Th17-dependent autoimmune disease model, was ameliorated. In Raftlin-Tg mice, in contrast, Ab production was enhanced and experimental autoimmune encephalomyelitis was more severe. Cytokine production, especially that of IL-17, was reduced in Raftlin-deficient T cells, while it was enhanced in Raftlin-Tg T cells. We found that these changes were associated with the strength of the TCR-mediated signals. Importantly, localization of Lck protein in the lipid rafts was enhanced by Raftlin overexpression and reduced by Raftlin deficiency. These data indicate that Raftlin modulates TCR signals and is necessary for the fine-tuning of T cell-mediated immune responses.

We studied the development and structure of the unusual trichotomous branching of Edgeworthia chrysantha. Three "branch primordia" are formed sequentially on the shoot apex of a main axis and develop into trichotomous branching. The branch primordia are clearly distinguishable from the typical axillary buds of other angiosperms; they develop much more rapidly than axillary buds, and the borders between the branch primordia and shoot apex of the main axis are anatomically unclear. Furthermore, at a later stage, leaves subtending the branch primordia produce typical axillary buds. These results suggest that the trichotomous branching in this species involves the division of the shoot apical meristem. Expression analysis of genes involved in branching or maintenance of the shoot apical meristem in this species should clarify the control mechanism of this novel branching pattern in angiosperms. We also observed the phyllotactic patterns in trichotomous branching and have related these patterns to the shoot system as a whole.

Suppressor of cytokine signaling (SOCS1/JAB) has been shown to play an important role in regulating dendritic cell (DC) function and suppressing inflammatory diseases and systemic autoimmunity. However, role of SOCS1 in DCs for the initiation of Th cell response has not been clarified. Here we demonstrate that SOCS1-deficient DCs induce stronger Th1-type responses both in vitro and in vivo. SOCS1-deficient DCs induced higher IFN-γ production from naive T cells than wild-type (WT) DCs in vitro. Lymph node T cells also produced a higher amount of IFN-γ when SOCS1-deficient bone marrow-derived DCs (BMDCs) were transferred in vivo. Moreover, SOCS1−/− BMDCs raised more effective anti-tumor immunity than WT BMDCs. Microarray analysis revealed that IFN-inducible genes were highly expressed in SOCS1-deficient DCs without IFN stimulation, suggesting hyper STAT1 activation in SOCS1−/− DCs. These phenotypes of SOCS1-deficient DCs were similar to those of CD8α+ DCs, and in the WT spleen, SOCS1 is expressed at higher levels in the Th2-inducing CD4+ DC subset, relative to the Th1-inducing CD8α+ DC subset. We propose that reduction of the SOCS1 gene expression in DCs leads to CD8α+ DC-like phenotype which promotes Th1-type hyperresponses.

The present study was designed to investigate the rewarding effect, G-protein activation and dopamine (DA) release following partial sciatic nerve ligation in the rat. Here we show for the first time that morphine failed to produce a place preference in rats with nerve injury. Various studies provide arguments to support that the mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc), is critical of the motivational effects of opioids. In the present study, there were no significant differences between sham-operated and sciatic nerve-ligated rats in the increases in guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes of the N.Acc stimulated by either DA, the D1 receptor agonist SKF81297, the D2 receptor agonist N-propylnoraporphine or the D3 receptor agonist 7-hydroxy-2-dipropylaminotetralin (7-OH DPAT). In contrast, the increases in [35S]GTPgammaS binding to membranes of the VTA induced by either morphine or a selective micro -opioid receptor agonist [d-Ala2, NMePhe4, Gly(ol)5]enkephalin were significantly attenuated in nerve-ligated rats as compared with sham- operated rats. Furthermore, the enhancement of DA release in the N.Acc stimulated by morphine was significantly suppressed by sciatic nerve ligation. These findings suggest that attenuation of the morphine-induced place preference under neuropathic pain may result from a decrease in the morphine-induced DA release in the N.Acc with reduction in the mu-opioid receptor-mediated G-protein activation in the VTA.

The suppressor of cytokine signaling-3 (SOCS3/CIS3) has been shown to be an important negative regulator of cytokines, especially cytokines that activate STAT3. To examine the role of SOCS3 in neutrophils and the granulocyte colony-stimulating factor (G-CSF) signaling in vivo, we compared neutrophils from two types of conditional knockout mice, LysM-Cre:SOCS3(fl/fl) mice and Tie2-Cre:SOCS3(fl/fl) mice, in which the Socs3 gene had been deleted in mature neutrophils and hematopoietic stem cells, respectively. The size of the G-CSF-dependent colonies from Tie2-Cre:SOCS3(fl/fl) mouse bone marrow was much larger than that of colonies from control wild-type mice, while the size of interleukin-3-dependent colonies was similar. Moreover, LysM-Cre:SOCS3(fl/fl) mice had more neutrophils than SOCS3(fl/fl) mice, suggesting that SOCS3 is a negative regulator of G-CSF signaling in neutrophils. Consistent with this notion, G-CSF-induced STAT3 as well as mitogen-activated protein kinase activation was much stronger and prolonged in SOCS3-deficient mature neutrophils than in wild-type neutrophils. The preventive effect of G-CSF on apoptosis was more prominent in SOCS3-deficient mature neutrophils than in control neutrophils. These data indicate that SOCS3 negatively regulates granulopoiesis and G-CSF signaling in neutrophils and may contribute to neutrophilia or neutropenia.

Vaccination with irradiated granulocyte macrophage colony-stimulating factor (GM-CSF)–transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity. However, its effectiveness is limited. We therefore attempted to improve the antitumor effect by identifying little-known key pathways in GM-CSF–sensitized dendritic cells (GM-DC) in tumor-draining lymph nodes (TDLN). We initially confirmed that syngeneic mice subcutaneously injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) remarkably rejected the tumor growth. Using cDNA microarrays, we found that expression levels of type I interferon (IFN)–related genes, predominantly expressed in plasmacytoid DCs (pDC), were significantly upregulated in TDLN-derived GM-DCs and focused on pDCs. Indeed, mouse experiments demonstrated that the effective induction of GM-CSF–induced antitumor immunity observed in immunocompetent mice treated with LLC/SeV/GM cells was significantly attenuated when pDC-depleted or IFNα receptor knockout (IFNAR−/−) mice were used. Importantly, in both LLC and CT26 colon cancer–bearing mice, the combinational use of imiquimod with autologous GVAX therapy overcame the refractoriness to GVAX monotherapy accompanied by tolerability. Mechanistically, mice treated with the combined vaccination displayed increased expression levels of CD86, CD9, and Siglec-H, which correlate with an antitumor phenotype, in pDCs, but decreased the ratio of CD4+CD25+FoxP3+ regulatory T cells in TDLNs. Collectively, these findings indicate that the additional use of imiquimod to activate pDCs with type I IFN production, as a positive regulator of T-cell priming, could enhance the immunologic antitumor effects of GVAX therapy, shedding promising light on the understanding and treatment of GM-CSF–based cancer immunotherapy. Cancer Immunol Res; 2(6); 568–80. ©2014 AACR.

In connection with our studies on antibacterial active compounds in the class of new oxazolidinones against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) strains, some molecular modifications were attempted. In this study, molecular modifications of 4-aminomethyloxazolidin-2-ones (3a) to the corresponding 4-acylaminomethyloxazolidin-2-one derivatives (3c–d) and preparations of the represented twin-drug type molecules (10–14) were investigated. Some additional 4-dialkylaminomethyloxazolidin-2-ones (2) were also synthesized. The synthesized compounds were evaluated for antibacterial activity with Gram-positive (S. aureus) and Gram-negative (E. coli) strains.

A 57-year-old female was seen at another clinic because of diarrhea and abdominal pain in July, 1994. Since abnormal findings were pointed out in the blood examination, she was referred to the hospital and was diagnosed as acute promyelocytic leukemia (APL). After one course of BHAC-DMP therapy, fever raised up to 38_??_39°C and CRP also raised up. With abdominal US and CT, she was diagnosed as multiple splenic abscesses and cholecystolithiasis. Splenectomy and cholecystectomy were performed on November 25, 1994. Liver biopsy was also performed during the operation, since a lot of small indurations similar to those of the spleen were recognized in the liver. The weight of the spleen was 260 g and multiple small abscesses which sized 5_??_10 mm were also recognized in the section. They were pathologically diagnosed as fungal splenic and hepatic abscesses. After the operation, antifungal agent was also administrated systemically for the rest abscesses in the liver and the patient was discharged from the hospital on the postoperation day 10. We successfully treated a case of fungal hepatic and splenic abscesses occurred during the chemotherapy for leukemia and systemic administration of antifungal agent, for which there has been no established definitive therapy as yet.

Although various therapies are available to treat cancers, including surgery, chemotherapy, and radiotherapy, cancer has been the leading cause of death in Japan for the last 30 years, and new therapeutic modalities are urgently needed. As a new modality, there has recently been great interest in oncolytic virotherapy, with measles virus being a candidate virus expected to show strong antitumor effects. The efficacy of virotherapy, however, was strongly limited by the host immune response in previous clinical trials. To enhance and prolong the antitumor activity of virotherapy, we combined the use of two newly developed tools: the genetically engineered measles virus (MV-NPL) and the multilayer virus-coating method of layer-by-layer deposition of ionic polymers. We compared the oncolytic effects of this polymer-coated MV-NPL with the naked MV-NPL, both in vitro and in vivo. In the presence of anti-MV neutralizing antibodies, the polymer-coated virus showed more enhanced oncolytic activity than did the naked MV-NPL in vitro. We also examined antitumor activities in virus-treated mice. Complement-dependent cytotoxicity and antitumor activities were higher in mice treated with polymer-coated MV-NPL than in mice treated with the naked virus. This novel, polymer-coated MV-NPL is promising for clinical cancer therapy in the future.

BLT1 is a high-affinity receptor for leukotriene B4 (LTB4) that is a potent lipid chemoattractant for myeloid leukocytes. The role of LTB4/BLT1 axis in tumor immunology, including cytokine-based tumor vaccine, however, remains unknown. We here demonstrated that BLT1-deficient mice rejected subcutaneous tumor challenge of GM-CSF gene-transduced WEHI3B (WGM) leukemia cells (KO/WGM) and elicited robust antitumor responses against second tumor challenge with WEHI3B cells. During GM-CSF–induced tumor regression, the defective LTB4/BLT1 signaling significantly reduced tumor-infiltrating myeloid-derived suppressor cells, increased the maturation status of dendritic cells in tumor tissues, enhanced their CD4+ T-cell stimulation capacity and migration rate of dendritic cells that had phagocytosed tumor-associated antigens into tumor-draining lymph nodes, suggesting a positive impact on GM-CSF–sensitized innate immunity. Furthermore, KO/WGM mice displayed activated adaptive immunity by attenuating regulatory CD4+ T subsets and increasing numbers of Th17 and memory CD44hiCD4+ T subsets, both of which elicited superior antitumor effects as evidenced by adoptive cell transfer. In vivo depletion assays also revealed that CD4+ T cells were the main effectors of the persistent antitumor immunity. Our data collectively underscore a negative role of LTB4/BLT1 signaling in effective generation and maintenance of GM-CSF–induced antitumor memory CD4+ T cells.

Dendritic cells (DCs) induce immunity and immunological tolerance as APCs. It has been shown that DCs secreting IL-10 induce IL-10+ Tr1-type regulatory T (Treg) cells, whereas Foxp3-positive Treg cells are expanded from naive CD4+ T cells by coculturing with mature DCs. However, the regulatory mechanism of expansion of Foxp3+ Treg cells by DCs has not been clarified. In this study, we demonstrated that suppressors of cytokine signaling (SOCS)-3-deficient DCs have a strong potential as Foxp3+ T cell-inducing tolerogenic DCs. SOCS3−/− DCs expressed lower levels of class II MHC, CD40, CD86, and IL-12 than wild-type (WT)-DCs both in vitro and in vivo, and showed constitutive activation of STAT3. Foxp3− effector T cells were predominantly expanded by the priming with WT-DCs, whereas Foxp3+ Treg cells were selectively expanded by SOCS3−/− DCs. Adoptive transfer of SOCS3−/− DCs reduced the severity of experimental autoimmune encephalomyelitis. Foxp3+ T cell expansion was blocked by anti-TGF-β Ab, and SOCS3−/− DCs produced higher levels of TGF-β than WT-DCs, suggesting that TGF-β plays an essential role in the expansion of Foxp3+ Treg cells. These results indicate an important role of SOCS3 in determining on immunity or tolerance by DCs.

Background: Although there have recently been much progress in cancer treatment including surgery, chemotherapy and radiotherapy, cancers have long been the leading cause of death in Japan in the last 30 years and the development of new therapeutic modalities is imminently required. As a new modality, there has recently been great interest in oncolytic virotherapy. Particularly, measles virus is one of the candidate viruses expected for strong antitumor effects. The efficacy of virotherapy, however, was strongly limited by the immune response of the host in previous clinical trials.Methods: To enhance and prolong the antitumor activity of the virotherapy in vivo, we combined newly developed tools of the genetically engineered measles virus (MV-NPL) and multilayer virus coating method of layer-by-layer deposition of ionic polymers. We compared the oncolytic effects of this polymer-coated MV-NPL with naked MV-NPL in vitro and in vivo.Results: In the presence of anti-MV neutralizing antibodies, the polymer-coated virus showed more enhanced oncolytic activity than naked MV-NPL in vitro. We also examined the antitumor immunity in virus treated mice. Complement-dependent cytotoxicity activities in mice treated with polymer-coated MV-NPL were higher than those with naked virus and stronger antitumor activity was observed in these mice.Conclusion: This novel polymer-coated MV-NPL may be promising for future clinical cancer therapy.

Bone metabolism and the immune system have a correlative relationship, and both are controlled by various common cytokines, such as IFNs and ILs, produced in the bone microenvironments. The suppressor of cytokine signaling-1 (SOCS1) and SOCS3 are negative regulators of such cytokines. Although SOCSs are shown to be induced during osteoclast differentiation, their physiological roles in osteoclast differentiation and function have not been clarified. Thus, we examined the roles of SOCS1 and SOCS3 in osteoclastogenesis using SOCS1- and SOCS3-deficient mice. IFN-γ-mediated inhibition of osteoclast differentiation from bone marrow-derived monocytes (BMMs) was strongly enhanced in SOCS1-deficient BMMs, but was diminished in SOCS1-overexpressing BMMs. Moreover, LPS-induced osteoclastogenesis and bone destruction in vivo were suppressed in SOCS1+/− mice compared with those in wild-type mice, suggesting that SOCS1 antagonizes the inhibitory effect of IFN-γ on osteoclastogenesis. SOCS3 did not alter the inhibitory effect of IFNs in osteoclastogenesis in both gain and loss of functional assays; however, the suppressive effect of IL-6 on osteoclast differentiation was greater in SOCS3-deficient BMMs than in wild-type BMMs in vitro. In addition, IL-6 significantly prevented LPS-induced bone destruction in SOCS3-deficient mice, although it failed in wild-type mice in vivo. In SOCS3-deficient BMMs, expression levels of TNF-receptor-associated factor-6 and IκB were drastically reduced and receptor activator of the NF-κB ligand-induced IκB phosphorylation was severely impaired in the presence of IL-6. These data suggest that both SOCS1 and SOCS3 regulate osteoclastogenesis by blocking the inhibitory effect of inflammatory cytokines on receptor activator of the NF-κB ligand-mediated osteoclast differentiation signals. Selective suppression of SOCS1 and SOCS3 in osteoclast precursors may be a possible therapeutic strategy for inflammatory bone destruction.

We report a 72-year-old man with sporadic intra-abdominal desmoid tumor manifesting as acute abdomen. CT scan revealed an air-containing tumor 7 cm in diameter; three weeks later, the tumor had shrunk to 4 cm on antibiotics. At surgery, a tumor arising from the transverse colon mesentery and infiltrating the jejunum was resected. No recurrence occurred over a 1-year follow-up.

The present study was designed to investigate the rewarding effect, G-protein activation and dopamine (DA) release following partial sciatic nerve ligation in the rat. Here we show for the first time that morphine failed to produce a place preference in rats with nerve injury. Various studies provide arguments to support that the mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc), is critical of the motivational effects of opioids. In the present study, there were no significant differences between sham-operated and sciatic nerve-ligated rats in the increases in guanosine-5'-o-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to membranes of the N.Acc stimulated by either DA, the D1 receptor agonist SKF81297, the D2 receptor agonist N-propylnoraporphine or the D3 receptor agonist 7-hydroxy-2-dipropylaminotetralin (7-OH DPAT). In contrast, the increases in [35S]GTPgammaS binding to membranes of the VTA induced by either morphine or a selective micro -opioid receptor agonist [d-Ala2, NMePhe4, Gly(ol)5]enkephalin were significantly attenuated in nerve-ligated rats as compared with sham- operated rats. Furthermore, the enhancement of DA release in the N.Acc stimulated by morphine was significantly suppressed by sciatic nerve ligation. These findings suggest that attenuation of the morphine-induced place preference under neuropathic pain may result from a decrease in the morphine-induced DA release in the N.Acc with reduction in the mu-opioid receptor-mediated G-protein activation in the VTA.

Because blood cells can be obtained with relatively easy and safe procedure, they have been routinely used for transfusion and transplantation purposes. And they are now considered as attractive cell sources for developing new gene therapies including cancer therapy using various immune cells, and regenerative therapy using hematopoietic stem cells or induced pluripotent stem cells (iPS) cells. For example, chimeric antigen receptor modified autologous T cells have been considered as effective therapy for various cancers. And iPS cells have been easily established from peripheral T cells for the purpose of treating various diseases. However, in spite of these possibilities, the development of the safer and more efficient genetic modification methods of hematopoietic cells is imminent. In this study, we developed the novel measles viral (MV) vector which enables us to transduce multiple genes into immune cells. The wild type measles virus is one of the aerosol-transmitted viruses and has strong infectious capacity to immune cells, and epithelial cells via signaling lymphocyte activation molecule (SLAM) or nectin-4. First, we modified the wild type measles virus genome to non-transmissible and non-lytic, and equipped with the ability of transducing multiple genes, at most six genes, into target cells. Briefly, the intrinsically non-segmented wild type virus genome was divided into two segments and point mutations were introduced into the virus genes encoding hemagglutinin and the matrix protein. Moreover, as the fusion protein gene was removed from the virus genome, the virus could not replicate in neighbor cells. We examined the gene transduction efficiency of the gene modified measles virus (H8-Fd-MV vector) into hematopoietic cells. We first constructed the H8-Fd-MV vector with GFP gene and transduced into hematopoietic progenitor cells and immune cells from human cord blood and peripheral blood. We observed that almost all of HPCs from cord blood (99.7% in floating cells expressed CD34), T cells (99.9% in CD3+ cells), and B cells (98.2% in CD19+ cells) from peripheral blood expressed GFP at two days after the transduction. Especially, to express GFP gene in human peripheral T cells, it was not necessary to pre-stimulate them with CD3/CD28 beads (99.6% in stimulating T cells (72.9% in SLAM+ cells) v.s. 82.6 % in non-stimulating cells (37.4% in SLAM+ cells)). T cells from cord blood showed almost all naïve phenotype (CD4+ cells: 93.2±1.8% in CD45RA+CCR7+ cells, 1.7±1.1% in CD45RA+CCR7- cells; CD8+ cells: 41.6±5.7% in CD45RA+CCR7+ cells, and 41.9±11.0% in CD8+CD45RA+CCR7- cells) and T cells transduced by MV vector expressed GFP more (CD4+ cells: 80.3±13.7%, and CD8+ cells: 82.5±8.5%) than those transduced by Sendai viral vector (CD4+ cells: 15.5±0.7%, and CD8+ cells: 17.4±5.4%). These data suggested that H8-Fd-MV vector could transduce GFP gene efficiently into various T cell lineages including naïve T cells, which had been difficult to be transduced with classical gene transduction methods. We next generated H8-Fd-MV vector for expressing 6 genes (OCT4, SOX2, KLF4, L-MYC, PIN1, and GFP) and transduced into stimulated T cells. After 3 days, GFP+ T cells expressed all of these 6 genes. We also detected that more than 50% of the stimulated T cells with IL-2 expressed GFP at 14 days after the transduction. After 27 days from transfection, embryonic stem cell (ES cell)-like colonies were picked up and analyzed the character. These cells showed ES cell morphology over 20 times passages and expressed pluripotent marker (NANOG, OCT4, Tra-1-60, Tra-1-81). We also found T cell receptor rearrangements in these cells. Embryoid bodies from these cells expressed three germ line markers in vitro. We next examined the hematopoietic differentiation of these cells using coculture system with murine embryonic aorta-gonad-mesonephros region-derived stromal cell line (AGM-3 cells). The co-cultured cells harvested at day 12 expressed CD34 and CD45. These data suggested that we established iPS cells from terminal differentiated T cells using H8-Fd-MV vector for expressing reprogramming factor. These results indicated that multiple genes were expressed efficiently in immune cells using H8-Fd-MV vector. Highly efficient transduction ability of MV vector for T cells would enable us to develop new gene therapy targeting cancer using gene modified T cells as well as organ regeneration using iPS cells. Disclosures No relevant conflicts of interest to declare.

A new orthotopic esophageal cancer model was developed by implanting fragments of xenografts of T.T human esophageal squamous carcinoma cells into the cervical esophagus of athymic rats. The rats had symptoms analogous to the human clinical course such as respiratory distress, dysphagia, vomiting of blood, or Horner syndrome, followed by death resulting from suffocation. Microscopic metastases of lymph node were observed around the tumor in 3 of 18 rats. A new cell line (T.T-1) was established from these metastases. Flow cytometry showed that T.T-1 and T.T parental cells had nearly the same surface levels of beta1-integrin, alpha2-integrin, alpha3-integrin and E-cadherin, and no expression of CD44v3, CD44v6 and alpha5-integrin. T.T-1 cells had a higher level of CD44H, however, and a greater binding efficiency to the extracellular matrix components; laminin, type IV collagen, hyaluronic acid, and fibronectin than T.T cells. Anti-CD44H antibody significantly decreased the binding efficiency of T.T-1 cells. T.T-1 cells were also significantly more invasive than T.T cells through all the extracellular matrix components except hyaluronic acid. After orthotopic implantation histological examination showed that T.T-1 tumors invaded beyond the esophageal mucosa and tracheal muscle layer and obstructed the esophagus and trachea. No invasion was observed with T.T tumors. Rats with T.T-1 or T.T tumors survived an average of 32.0 and 50.7 days, respectively (p < 0.01). In addition T.T-1 tumors expressed higher levels of CD44H mRNA than T.T tumors. In summary, our newly developed orthotopic implantation model is a valid model of esophageal cancer because it followed the same clinical course experienced by humans. Moreover, using cells derived from this model, we were able to demonstrate that CD44H is involved in esophageal cancer cell invasion.

We report a case of hepatic abscess associated with the sump syndrome. The patient was a 66-year-old woman who had undergone cholecystectomy and side-to-side choledochoduodenostomy for a common bile duct (CBD) stone in 1983, and who presented with fever and right lower chest pain. A hepatic abscess was diagnosed; after it was drained, percutaneous transhepatic biliary drainage was performed. Bacteriological studies revealed the presence of Bacteroides fragilis and Streptococcus intermedius in the pus in the hepatic abscess cavity, and Klebsiella pneumoniae and Pseudomonas aeruginosa in the bile. The hepatic abscess and cholangitis rapidly resolved in response to two drainage procedures. At surgery, simple closure of the anastomosis was performed, because free drainage was observed from the distal CBD into the duodenum, despite the existence of a periampullary diverticulum.

Vaccination with irradiated granulocyte macrophage-colony stimulating factor (GM-CSF)-transduced autologous tumor cells (GVAX) has been shown to induce therapeutic antitumor immunity presumably through the activated maturation of myeloid dendritic cells (DCs). However, its effectiveness is limited, and little is known about the biological properties related to GM-CSF-sensitized DCs (GM-DCs) in tumor-draining lymph nodes (TDLNs). We therefore attempted to enhance the antitumor effect of GVAX therapy by identifying the key pathways in GM-DCs in TDLNs. We initially confirmed that syngeneic mice subcutaneously (s.c.) injected with poorly immunogenic Lewis lung carcinoma (LLC) cells transduced with Sendai virus encoding GM-CSF (LLC/SeV/GM) significantly rejected the tumor growth. Using cDNA microarrays, we obtained a large number of gene expression data from CD86+ GM-DCs and control DCs in TDLNs, and found that the expressions of type I interferons (IFNs)-related genes, including IRF7 and TLR7, known to be abundantly expressed in plasmacytoid DCs (pDCs), were upregulated in GM-DCs. Indeed, to further activate pDCs, mice s.c. challenged with LLC/SeV/GM cells in combination with a TLR7 ligand, imiquimod, but not a TLR4 ligand, LPS, significantly suppressed the tumor growth compared with mice treated with LLC/SeV/GM cells alone. In contrast, pDCs-depleted mice challenged with LLC/SeV/GM cells facilitated the tumor growth, strongly suggesting that pDCs are essential immune subpopulation in exerting GM-CSF-initiated antitumor effects. Furthermore, the additional use of imiquimod overcame the refractoriness of therapeutic vaccines with irradiated LLC/SeV/GM cells in mice with pre-established LLC tumors. Moreover, similar improvement of GVAX therapy was also observed in a mouse model of CT26 colorectal cancer. Mechanistically, mice treated with the combined vaccination displayed increased cell ratio of PDCA-1+ pDCs and expression levels of CD86, CD9, which correlate with an antitumor phonetype, and Siglec-H, which promote CD8+ T cell proliferation, in TDLNs. Indeed, allogeneic MLR test showed that bone marrow-derived pDCs matured by in vitro culture with GM-CSF plus imiquimod elicited a superior capacity of CD8+ T cell proliferation compared with those with GM-CSF only. On the other hand, the ratio of CD4+CD25+FoxP3+ regulatory T cells (Tregs) was decreased in TDLNs mice treated with the combined vaccination. These findings collectively elucidated that pDCs play positive roles in GM-CSF-initiated antitumor immunity and that further activation of pDC by imiquimod targeting TLR7-IRF7 dependent type I IFNs pathway enhance the antitumor effects of GM-CSF-based tumor vaccination. Disclosures: No relevant conflicts of interest to declare.

Increasing evidence suggests that only a small percentage of cancer cells in solid tumors have tumorigenic potential. These highly tumorigenic cells share with normal stem cells the ability to proliferate and give rise to diversified tumor cell types including those with the capacity for self-renewal. These cells are termed cancer stem cells (CSCs) and are believed to contribute to CSCs resistance to conventional therapies. Therefore, specific targeting of CSCs is an attractive and novel therapeutic approach. Measles virus Edmonston strain (MV-Edm) is thought to possess remarkable oncolytic activity that selectively destroys human tumor cells. Recently, we succeeded and reported the development of novel oncolytic measles virus (MV) by genetic engineering of MV-Edmonston strain (MV-Edm), that have demonstrated its remarkable oncolytic activity against wide ranges of human tumor cells, with the N, P, and L genes of wild-type MV (MV-NPL). The oncolytic activities of the MV-NPL on human renal cell carcinoma were superior to those of MV-Edm both in vitro and in vivo studies even in the presence of IFN[[Unsupported Character - ‐]]α[[Unsupported Character - ]](Xing, M. et. al. Mol Ther 2010). Here, we demonstrated that lung CSCs, side population fraction of human lung cancer cells of A549 (A549-SP), expressed high level of CD46 required for MV-NPL infection and MV-NPL had a superior oncolytic capacity against both A549-SP and A549-NSP (non SP) cells in a time and dose-dependent manner without damaging normal lung cells. Our results of pan-caspase inhibiton assays showed the partial contribution of caspases-dependent apoptosis in the oncolysis of A549-SP cells by MV-NPL, even though the expression levels of anti-apoptotic protein of both XIAP and Mcl-1 were higher in A549-SP cells than A549-NSP cells. Additionally, we demonstrated that an addition of either PI3K inhibitor or MEK inhibitor further enhanced the oncolytic effects of MV-NPL. Furthermore, our in vivo studies using nude mice bearing A549 xenografts revealed that serial intratumoral MV-NPL administrations exhibited significant antitumor effects without severe side effects. Collectively, these data suggested that genetically engineered MV-NPL could be a promising oncolytic agent to effectively eradicate lung CSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-370. doi:1538-7445.AM2012-LB-370

Abstract 3548 Poster Board III-485 CD26 is a 110-kDa cell surface glycoprotein with known dipeptidyl peptidase IV (DPPIV) activity in its extracellular domain, capable of cleaving amino-terminal dipeptides with either L-proline or L-alanine at the penultimate position. CD26 activity is dependent on cell type and the microenvironment, which influence its multiple biological roles. CD26 plays an important role in immunology, autoimmunity, diabetes and cancer. The role of CD26 in immune regulation has been extensively characterized, with our recent findings elucidating its linkage with signaling pathways and structures involved in T cells activation as well as antigen presenting cell (APC)-T-cell interaction. CD26 is preferentially expressed on human CD4+ memory T cells and CD4+CD45RO+CD26+ T cells exhibit response maximally to the recall antigen tetanus toxoid. Human T-helper 1 (Th1) cells display a higher expression of CD26 and are much more sensitive to CD26-mediated costimulation than human Th2 cells. In contrast, the role of CD26 in human CD8+ T cells still remains to be elucidated, while CD8+ T cells express CD26 as well as CD4+ T cells. In this study, we studied the effector function of CD26-mediated costimulation of CD8+ T cells. In comparison with CD28-medaited costimulation, which is one of representative T cell costimulatory pathway, CD26-mediated costimulation in CD8+ T cells showed delayed proliferation than that of CD28 stimuatlion, but finally expanded to a similar extent. The secretion of inflammatory cytokines, TNF-alpha and IFN-gamma, was strongly induced after CD26-mediated costimulation. In contrast, the secretion of IL-2 and IL-5 was significantly less as compared with CD28-mediated costimulation. Finally, we showed that the expression of Granzyme B, one of the major effector molecules in the cytotoxic activity of CD8+ T cells, was markedly enhanced by CD26-mediated costimulation in dose dependent manner of anti-CD26 monoclonal antibody stimulation. Moreover, with CD26-mediated costimulation, CD8+ T cells were observed to kill target cells in a Granzyme B-dependent manner. Next, we examined CD26-dependent organ injury by human T cells using xenogeneic chronic GVHD (x-cGVHD) mice model. About 6 weeks after transplantation of human peripheral blood mononuclear cells (PBMC) into NOG-Scid mice, mice develop loss pf hair, loss of weight, and human CD8+ lymphocytes infiltration into liver, lung, and salivary glands, which exhibit pathological changes of cGVHD. Administration of anti-human CD26 mAb decreased onset of x-cGVHD, while human lymphocytes were grafted successfully. Finally, we examined whether GVL effect is exerted via CD26-mediated costimulation, using x-cGVHD mice model in which P388 leukemic cells were grafted subcutaneously. In this tumor-graft model, P388 cells were not grafted in mice with onset of x-cGVHD, which indicated that GVL effect was exerted by human lymphocytes. On the other hand, P388 cells were also diminished in mice with decreased x-cGVHD by administration of anti-CD26 mAb. Taken together, our data strongly suggest that CD26-mediated costimulation in CD8+ T cells deeply involves in the pathology of cGVHD. The present study offers a novel notion that CD26 may become one of therapeutic targets of GVHD without impairment of GVL effect. Disclosures: Yamada: Y's Therapeutics: Membership on an entity's Board of Directors or advisory committees. Dang:Y's Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Morimoto:Y's Therapeutics: Patents & Royalties, a board member.

Immune and inflammatory systems are controlled by multiple cytokines, including interleukins and interferons. Many of these cytokines exert their biological functions through JAKs (Janus tyrosine kinases) and STAT (signal transduction and activators of transcription) transcription factors. CIS (cytokine-inducible SH2 (Src homology 2) protein) and SOCS (suppressor of cytokine signaling) are a family of intracellular proteins, several of which have emerged as key physiological regulators of cytokine-mediated homeostasis, including innate and adaptive immunity. In this review we focus on the molecular mechanism of the action of CIS/SOCS family proteins and their roles in immune regulation and inflammatory diseases including rheumatoid arthritis.