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Various proteins are highly expressed in cancer (e.g., epidermal growth factor receptor); however, the majority are also expressed in normal cells, although they may differ in expression intensity. Recently, we reported that CD271 (nerve growth factor receptor), a glycosylated protein, increases malignant behavior of cancer, particularly stemlike phenotypes in squamous cell carcinoma (SCC). CD271 is expressed in SCC and in normal epithelial basal cells. Glycosylation alterations generally occur in cancer cells; therefore, we attempted to establish a cancer-specific anti-glycosylated CD271 antibody. We purified recombinant glycosylated CD271 protein, immunized mice with the protein, and screened hybridomas using an ELISA assay with cancer cell lines. We established a clone G4B1 against CD271 which is glycosylated with O-glycan and sialic acid. The G4B1 antibody reacted with the CD271 protein expressed in esophageal cancer, but not in normal esophageal basal cells. This specificity was confirmed in hypopharyngeal and cervical cancers. G4B1 antibody recognized the fetal esophageal epithelium and Barrett's esophagus, which possess stem cell-like characteristics. In conclusion, G4B1 antibody could be useful for precise identification of dysplasia and cancer cells in SCC.

Cancer stem cells (CSCs) are responsible for therapy resistance and share several properties with normal stem cells. Here, we show that brain‐expressed X‐linked gene 2 (BEX2), which is essential for dormant CSCs in cholangiocarcinoma, is highly expressed in human hepatocellular carcinoma (HCC) lesions compared with the adjacent normal lesions and that in 41 HCC cases the BEX2high expression group is correlated with a poor prognosis. BEX2 localizes to Ki67‐negative (nonproliferative) cancer cells in HCC tissues and is highly expressed in the dormant fraction of HCC cell lines. Knockdown of BEX2 attenuates CSC phenotypes, including sphere formation ability and aldefluor activity, and BEX2 overexpression enhances these phenotypes. Moreover, BEX2 knockdown increases cisplatin sensitivity, and BEX2 expression is induced by cisplatin treatment. Taken together, these data suggest that BEX2 induces dormant CSC properties and affects the prognosis of patients with HCC., BEX2 is essential for dormant cancer stem cell and affects patients’ prognosis in hepatocellular carcinoma. BEX2 could be a promising therapeutic target.

Cancer stem cells (CSCs) are believed to cause cancer metastasis and recurrence. BEX2 (brain expressed X-linked gene 2) is a CSC-related gene that is expressed in dormant CSCs in cholangiocarcinoma and induces resistance against chemotherapy. The aim of the present study was to identify small compounds that have activity to inhibit BEX2 expression and result in the attenuation of CSC-related phenotypes. We screened 9600 small chemical compounds in high-throughput screening using cholangiocarcinoma cell line HuCCT1 expressing BEX2 protein fused with NanoLuc, and identified a compound, BMPP (1, 3-Benzenediol, [4-(4-methoxyphenyl)-1H-pyrazol-3-yl]). BMPP was found to exert decreasing effects on BEX2 protein expression and G0 phase population of the tumor cells, and increasing effects on ATP levels and chemotherapeutic sensitivity of the cells. These findings indicate that BMPP is a valuable chemical compound for reducing dormant CSC-related phenotypes. Thus, the identification of BMPP as a potential CSC suppressor provides scope for the development of novel therapeutic modalities for the treatment of cancers with BEX2 overexpressing CSCs.

Background Accumulating evidence indicates that CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in human epithelial carcinomas of multiple organs including the pancreas, but its functional role in carcinoma development has not yet been fully clarified. The aim of this study was to investigate the role of CD109 in the malignancy of pancreatic ductal adenocarcinoma (PDAC). Methods PDAC specimens of 145 cases were immunostained for CD109, and correlations between CD109 expression and clinicopathological conditions were analyzed. CD109 expression in PANC-1 cells, a PDAC-derived cell line, was decreased by siRNA or shRNA and its effect on the malignancy of PANC-1 cells was examined. Results Suppression of CD109 expression in PANC-1 cells resulted in reduction of in vitro cell motility and tumorigenicity in xenografts. Based on these results, we investigated the relationship between CD109 expression and metastasis of PDAC using tumor tissue specimens. Among 106 recurrent cases of 145 PDAC, there was a tendency for CD109-positive cases to be accompanied by distant metastasis. Conclusions CD109 plays a critical role in the promotion of tumorigenic ability and cellular motility relating to metastasis of PDAC cells.

CD271 is a common receptor for all neurotrophins that is localized to neurons, endothelial cells, and the basal layer of the epithelium in normal tissue. Recently, we and others reported that CD271 plays essential roles in the development of squamous cell carcinoma, especially in tumor-initiating cells. Since little is known about how CD271 regulates cancer cell initiation and proliferation, antibodies that recognize different domains of CD271 are needed to enable investigation. Therefore, this study aimed to develop an antihuman CD271 antibody by immunizing mice with a CD271 antigen produced by a baculovirus. The antibody was named hCD271mAb#13, and it recognized cysteine-rich domain 1 with a higher affinity than the commercially available antibody ME20.4. We determined that hCD271mAb#13 is suitable for flow cytometry, Western blotting, immunocytochemistry, and immunohistochemistry of formalin-fixed paraffin-embedded tissue. Use of hCD271mAb#13 for CD271 labeling could enable detailed analyses of cancer cell regulation and other biological processes.

Squamous cell carcinoma is a major type of cancer in the lung. While several therapeutic target molecules for lung adenocarcinoma have been identified, little is known about lung squamous cell carcinoma (LSCC). We recently reported that CD271 (p75 neurotrophin receptor) serves as a marker for tumor initiation and is a key regulator of cell proliferation in hypopharyngeal squamous cell carcinoma. In this study, we found that CD271 was also expressed in squamous cell carcinoma, but not in adenocarcinoma, of several tissues, including the lung, and the expression of CD271 was associated with a poor prognosis in LSCC. To examine CD271's role in LSCC, we established xenograft cell lines from LSCC patients. Within the sorted live LSCC cell population, the CD271high cells were primarily cycling through the G2/M phase, while the CD271low cells were mostly in the G0 phase. CD271 knockdown in the LSCC cells completely suppressed their proliferation and tumor-formation capability, and increased their cell-cycle arrest in the G0 phase. In the CD271-knockdown cells, ERK-phosphorylation was decreased, while no change was observed in the IκBα-phosphorylation, p65-phosphorylation, or Akt-phosphorylation. Treatment with the MEK inhibitor U0126 decreased the LSCC cells' proliferation capability. Microarray analysis revealed that CD271 knockdown attenuated the RAS-related pathways. The knockdown of TrkB, which forms a heterodimer with CD271 and accelerates its downstream signaling, partially inhibited the LSCC cell proliferation. These results indicated that LSCC exclusively depends on CD271 for cell proliferation, in part through ERK-signaling activation, and CD271 is a promising target for LSCC therapy.

Cancer stem cells (CSCs) define a subpopulation of cancer cells that are resistant to therapy. However, little is known of how CSC characteristics are regulated. We previously showed that dormant cancer stem cells are enriched with a CD274low fraction of cholangiocarcinoma cells. Here we found that BEX2 was highly expressed in CD274low cells, and that BEX2 knockdown decreased the tumorigenicity and G0 phase of cholangiocarcinoma cells. BEX2 was found to be expressed predominantly in G0 phase and starvation induced the USF2 transcriptional factor, which induced BEX2 transcription. Comprehensive screening of BEX2 binding proteins identified E3 ubiquitin ligase complex proteins, FEM1B and CUL2, and a mitochondrial protein TUFM, and further demonstrated that knockdown of BEX2 or TUFM increased mitochondria-related oxygen consumption and decreased tumorigenicity in cholangiocarcinoma cells. These results suggest that BEX2 is essential for maintaining dormant cancer stem cells through the suppression of mitochondrial activity in cholangiocarcinoma. Funding Statement: This research was supported in part by the Biomedical Research Core of Tohoku University Graduate School of Medicine. This research was supported in part by JSPS KAKENHI grant numbers JP: 19K08430, 17K10716, and 16K07132, and Practical Research for Innovative Cancer Control from AMED by grant number JP17ck0106197, The Cooperative Research Project Program of Joint Usage/Research Center at The Institute of Development, Aging and Cancer, Tohoku University, and Takeda Medical Foundation. Declaration of Interests: There are no financial conflicts of interest. Ethics Approval Statement: This study was conducted according to the principles expressed in the Declaration of Helsinki and was approved by the Ethics Committees at Miyagi Cancer Center (Natori, Japan) and Tohoku University Graduate School of Medicine (Sendai, Japan). The protocol of animal experiments was approved by the Miyagi Cancer Center Animal Care and Use Committee (permit number: MCC-AE-2019-8).

CD271, known as a neurotrophin receptor, is expressed in various cancers such as hypopharyngeal cancer (HPC) and melanoma. We recently reported that CD271 is a cancer-stem-cell biomarker of HPC, and that its expression is essential for cancer-cell proliferation and is correlated with a poor prognosis in this disease. Here, to develop a therapeutic antibody to CD271, we established a humanized anti-CD271 monoclonal antibody (hCD271 mA b). hCD271 mA b bound to the cysteine-rich domain 1 (CRD1) of human CD271 with high affinity (KD = 1.697 × 10−9 M). In vitro, hCD271 mA b exerted antibody-dependent cell-mediated cytotoxicity (ADCC) activity against SP2/0-CD271 (human CD271-transduced mouse cell line). Treatment with hCD271 mA b also exerted anti-tumor activity in graft models of three cell lines (HPCM2 (patient-derived xenograft cell line of hypopharyngeal cancer), MeWo-Luc (melanoma cell line), and SP2/0-CD271) in mice, resulting in smaller tumors compared to controls and reduced numbers of CD271-positive cells. Collectively, these data suggest that an antibody targeting CD271 is a promising therapeutic strategy.

Squamous cell carcinoma is a major type of cancer in the lung. While several therapeutic target molecules for lung adenocarcinoma have been identified, little is known about lung squamous cell carcinoma (LSCC). We recently reported that CD271 (p75 neurotrophin receptor) serves as a marker for tumor initiation and is a key regulator of cell proliferation in hypopharyngeal squamous cell carcinoma. In this study, we found that CD271 was also expressed in squamous cell carcinoma, but not in adenocarcinoma, of several tissues, including the lung, and the expression of CD271 was associated with a poor prognosis in LSCC. To examine CD271's role in LSCC, we established xenograft cell lines from LSCC patients. Within the sorted live LSCC cell population, the CD271

Several recent studies have revealed that microRNAs (miRNAs) have a role in carcinogenesis and cancer development, and that it is stably detectable in plasma/serum. The aim of this study was to test whether miR-483-3p as well as miR-21 could be plasma biomarkers for PDAC. The plasma samples were obtained from three groups including 32 pancreatic ductal adenocarcinoma (PDAC) patients, 12 patients with intraductal papillary mucinous neoplasm (IPMN) patients and 30 healthy controls (HC). We evaluated the plasma miR-483-3p and miR-21 expression level by quantitative RT-PCR. We compared the differences in the plasma level of these miRNAs among the three groups, and investigated the relevance of their plasma expression level to the clinical factors in PDAC. The expressions of miR-483-3p and miR-21 were detected in all examined plasma samples. The plasma expression levels of these miRNAs were significantly higher in PDAC compared to HC (P

The ESCRT-0 complex, consisting of the hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and the signal-transducing adaptor molecule (STAM) proteins, recognizes ubiquitylated cargo during the initial step of endosomal sorting. The endosomal accumulation of overexpressed Hrs has been reported previously to be associated with endosome enlargement. In this study, we have found that co-expressing exogenous STAM1 in Hrs-overexpressing cells leads to a diffuse localization for a large part of the Hrs accumulated on endosomes and a recovery of the impaired cargo protein degradation process, thus suggesting that exogenous STAM abrogates the abnormalities of the Hrs-positive endosomes. A fluorescently labeled Hrs, introduced into the cells by membrane permeabilization, exhibited endosomal localization in the absence of STAM1 and gradually dissociated from the endosomes upon the sequential addition of recombinant STAM1. Furthermore, when microinjected into cells, the fluorescently labeled Hrs also showed endosomal accumulation; however, ESCRT-0 complexes formed prior to the microinjection did not. Analysis of the state of the complex in HeLa cells using blue-native PAGE revealed that the membrane-associated Hrs exists partly as a monomer and not only in the STAM1-bound form. Thus, our data suggest that the membrane binding and dissociation cycle of the ESCRT-0 proteins on the endosomal membrane is a critical step during the cargo sorting process.

T-cell homeostasis preserves the numbers, the diversity and functional competence of different T-cell subsets that are required for adaptive immunity. Naive CD4+ T (TN) cells are maintained in the periphery via the common γ-chain family cytokine IL-7 and weak antigenic signals. However, it is not clear how memory CD4+ T-cell subsets are maintained in the periphery and which factors are responsible for the maintenance. To examine the homeostatic mechanisms, CFSE-labeled CD4+CD44highCD62Llow effector memory T (TEM) cells were transferred into sublethally-irradiated syngeneic C57BL/6 mice, and the systemic cell proliferative responses, which can be divided distinctively into fast and slow proliferations, were assessed by CFSE dye dilution. We found that the fast homeostatic proliferation of TEM cells was strictly regulated by both antigen and OX40 costimulatory signals and that the slow proliferation was dependent on IL-7. The simultaneous blockade of both OX40 and IL-7 signaling completely inhibited the both fast and slow proliferation. The antigen- and OX40-dependent fast proliferation preferentially expanded IL-17-producing helper T cells (Th17 cells). Thus, OX40 and IL-7 play synergistic, but distinct roles in the homeostatic proliferation of CD4+ TEM cells.

Cholangiocarcinoma is an aggressive malignant tumor originating from intrahepatic or extrahepatic bile ducts. Its malignant phenotypes may be assumed by cancer stem cells (CSC). Here, we demonstrate that CD274 (PD-L1), known as an immunomodulatory ligand, has suppressive effects on CSC-related phenotypes of cholangiocarcinoma. Using two human cholangiocarcinoma cell lines, RBE and HuCCT1, we attempted to isolate the CD274(low) and CD274(high) cells from each cell line, and xenografted them into immunodeficient NOD⁄scid⁄γcnull (NOG) mice. We found that the CD274(low) cells isolated from both RBE and HuCCT1 are highly tumorigenic in NOG mice compared with CD274(high) cells. Furthermore, the CD274(low) cells possess several CSC-related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle. Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity. These findings are compatible with our observation that clinical cholangiocarcinoma specimens are classified into low and high groups for CD274 expression, and the CD274 low group shows poorer prognosis when compared with the CD274 high group. These results strongly suggest that CD274 has a novel function in the negative regulation of CSC-related phenotypes in human cholangiocarcinoma, which is distinct from its immunomodulatory actions.

The transplantation of human cancer cells into immunodeficient NOD/SCID/IL-2Rγc(null) (NOG) mice often causes highly malignant cell populations like cancer stem cells to emerge. Here, by serial transplantation in NOG mice, we established two highly tumorigenic adult T-cell leukemia-derived cell lines, ST1-N6 and TL-Om1-N8. When transplanted s.c., these cells formed tumors significantly earlier and from fewer initial cells than their parental lines ST1 and TL-Om1. We found that protein kinase B (AKT) signaling was upregulated in ST1-N6 and TL-Om1-N8 cells, and that this upregulation was due to the decreased expression of a negative regulator, INPP5D. Furthermore, the introduction of a constitutively active AKT mutant expression vector into ST1 cells augmented the tumorigenicity of the cells, whereas treatment with the AKT inhibitor MK-2206 attenuated the progression of tumors induced by ST1-N6 cells. Collectively, our results reveal that the AKT signaling pathway plays a critical role in the malignancy of adult T-cell leukemia-derived cells.

CD271 (p75 neurotrophin receptor) plays both positive and negative roles in cancer development, depending on the cell type. We previously reported that CD271 is a marker for tumor initiation and is correlated with a poor prognosis in human hypopharyngeal cancer (HPC). To clarify the role of CD271 in HPC, we established HPC cell lines and knocked down the CD271 expression using siRNA. We found that CD271-knockdown completely suppressed the cells’ tumor-forming capability both in vivo and in vitro. CD271-knockdown also induced cell-cycle arrest in G0 and suppressed ERK phosphorylation. While treatment with an ERK inhibitor only partially inhibited cell growth, CDKN1C, which is required for maintenance of quiescence, was strongly upregulated in CD271-depleted HPC cells, and the double knockdown of CD271 and CDKN1C partially rescued the cells from G0 arrest. In addition, either CD271 depletion or the inhibition of CD271-RhoA signaling by TAT-Pep5 diminished the in vitro migration capability of the HPC cells. Collectively, CD271 initiates tumor formation by increasing the cell proliferation capacity through CDKN1C suppression and ERK-signaling activation, and by accelerating the migration signaling pathway in HPC.

Hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is a component of the ESCRT-0 protein complex that captures ubiquitylated cargo proteins and sorts them to the lysosomal pathway. Although Hrs acts as a key transporter for ubiquitin-dependent endosomal sorting, we previously reported that Hrs is also involved in ubiquitin-independent endosomal sorting of interleukin-2 receptor beta (IL-2R beta). Here, we show direct interactions between bacterially expressed Hrs and interleukin-4 receptor alpha (IL-4R alpha), indicating that their binding is not required for ubiquitylation of the receptors, similar to the case for IL-2R beta. Examinations of the Hrs binding regions of the receptors reveal that a hydrophobic amino acid cluster in both IL-2R beta and IL-4R alpha is essential for the binding. Whereas the wild-type receptors are delivered to LAMP1-positive late endosomes, mutant receptors lacking the hydrophobic amino acid cluster are sorted to lysobisphosphatidic acid-positive late endosomes rather than LAMP1-positive late endosomes. We also show that the degradation of these mutant receptors is attenuated. Accordingly, Hrs functions during ubiquitin-independent endosomal sorting of the receptors by recognizing the hydrophobic amino acid cluster. These findings suggest the existence of a group of cargo proteins that have this hydrophobic amino acid cluster as a ubiquitin-independent sorting signal., Article, JOURNAL OF BIOLOGICAL CHEMISTRY. 286(17):15458-15472 (2011)

Accumulating evidence suggests that cancer stem cells (CSC) play an important role in tumorigenicity. Epithelial cell adhesion molecule (EpCAM) is one of the markers that identifies tumor cells with high tumorigenicity. The expression of EpCAM in liver progenitor cells prompted us to investigate whether CSC could be identified in hepatocellular carcinoma (HCC) cell lines. The sorted EpCAM(+) subpopulation from HCC cell lines showed a greater colony formation rate than the sorted EpCAM(-) subpopulation from the same cell lines, although cell proliferation was comparable between the two subpopulations. The in vivo evaluation of tumorigenicity, using supra-immunodeficient NOD/scid/γc(null) (NOG) mice, revealed that a smaller number of EpCAM(+) cells (minimum 100) than EpCAM(-) cells was necessary for tumor formation. The bifurcated differentiation of EpCAM(+) cell clones into both EpCAM(+) and EpCAM(-) cells was obvious both in vitro and in vivo, but EpCAM(-) clones sustained their phenotype. These clonal analyses suggested that EpCAM(+) cells may contain a multipotent cell population. Interestingly, the introduction of exogenous EpCAM into EpCAM(+) clones, but not into EpCAM(-) clones, markedly enhanced their tumor-forming ability, even though both transfectants expressed a similar level of EpCAM. Therefore, the difference in the tumor-forming ability between EpCAM(+) and EpCAM(-) cells is probably due to the intrinsic biological differences between them. Collectively, our results suggest that the EpCAM(+) population is biologically quite different from the EpCAM(-) population in HCC cell lines, and preferentially contains a highly tumorigenic cell population with the characteristics of CSC.

Exosomes are nanovesicles derived from multivesicular bodies (MVBs) in antigen-presenting cells. The components of the ESCRT (endosomal sorting complex required for transport) pathway are critical for the formation of MVBs, however the relationship between the ESCRT pathway and the secretion of exosomes remains unclear. We here demonstrate that Hrs, an ESCRT-0 protein, is required for fascilitating the secretion of exosomes in dendritic cells (DCs). Ultrastructural analyses showed typical saucer-shaped exosomes in the culture supernatant from both the control and Hrs-depleted DCs. However, the amount of exosome secretion was significantly decreased in Hrs-depleted DCs following stimulations with ovalbumin (OVA) as well as calcium ionophore. Antigen-presentation activity was also suppressed in exsosomes purified from Hrs-depleted DCs, while no alteration in OVA degradation was seen in Hrs-depleted DCs. These data indicated that Hrs is involved in the regulation of antigen-presentation activity through the exosome secretion.

Maternal T cells acquire a transient state of tolerance specific for paternal alloantigens during pregnancy. CD4 + CD25 + regulatory T (Treg) cells play a central role in induction and maintenance of tolerance. We have studied the role of Treg cells for the maintenance of allogeneic pregnancy during the implantation period, early pregnancy period and late pregnancy period. We performed depletion of Treg cells using treatment with anti-CD25 monoclonal antibody (mAb) in allogeneic or syngeneic pregnant mice. BALB/c or C57BL/6 female mice were mated with BALB/c or C57BL/6 male mice, and anti-CD25 mAb was injected intraperitoneally on day 2.5 post-coitum (pc), or days 4.5 and 7.5 pc, or days 10.5 and 13.5 pc. Administration of 0.5mg of anti-CD25 mAb induced depletion of CD4 + CD25 + Foxp3 + Treg cells in both allogeneic and syngeneic pregnancy. The extent of depletion of CD4 + CD25 + Treg cells in spleen cells was 82.7%. This mAb treatment on day 2.5 pc of pregnancy induced implantation failure in allogeneic pregnant mice, but not in syngeneic pregnant mice. In addition, anti-CD25 mAb treatment on days 4.5 and 7.5 pc significantly increased resorption rates in allogeneic pregnant mice, but not in syngeneic pregnant mice. Interestingly, anti-CD25mAb treatment on days 10.5 and 13.5 pc reduced Treg cell numbers, but this treatment did not induce any abnormal pregnancy parameters such as intrauterine growth restriction, hypertension, or proteinuria. These findings suggest that CD4 + CD25 + Foxp3 + Treg cells are important to mediate maternal tolerance to the allogeneic fetus in the implantation phase and early stage of pregnancy, but Treg cells might not be necessary for maintenance of the late stage of allogeneic pregnancy.

OX40-OX40 ligand (OX40L) interactions have been proposed to support induction of allergic airway inflammation, which may be attributable to OX40 signaling in CD4(+) helper T cells for adaptive immune responses. However, a possible involvement of natural killer T (NKT) cells in the pathogenesis suggests that the underlying mechanisms are not yet fully elucidated.We aimed to characterize the OX40-modulated cellular contribution to allergic airway inflammation in a mouse model of house dust mite (HDM) allergen exposure.Mice were sensitized to HDM and, 3 weeks later, challenged with HDM on three consecutive days through the airways. Two days after the last exposure, bronchoalveolar lavage fluids and blood samples and lung tissues were evaluated for the airway inflammation.The development of HDM-induced eosinophilic airway inflammation was dependent on OX40 of both CD4(+) T cells and NKT cells; OX40 engagement on CD4(+) T cells in the sensitization led to pulmonary OX40L augmentation after the allergen challenge, which stimulated pulmonary NKT cells through OX40 to provide the pathogenic cytokine milieu. This was ablated by OX40L blockade by inhalation of the neutralizing antibody during the challenge, suggesting the therapeutic potential of targeting pulmonary OX40-OX40L interactions. Moreover, OX40 expression in CD4(+) T cells, but not in NKT cells, was reciprocally regulated by the helper T cell type 1-skewing transcription factor Runx3.OX40 on not only CD4(+) T cells but also NKT cells is involved in allergic airway inflammation. Notably, pulmonary blockade of OX40 ligation on NKT cells has therapeutic implications.

In order to facilitate the discovery and investigation of anti-cancer therapeutics under physiological conditions, we have engineered the ovarian cancer cell line, HM-1/luc, in mice. This cell stably expresses firefly luciferase and produces light that can be detected using an in vivo imaging system (IVIS). Parental HM-1 cells cause severe carcinomatous peritonitis to B6C3F1 mice, but not to C57BL6 mice. Established HM-1/luc cells showed pathologically similar findings to HM-1 cells. HM-1/luc cells were injected into the peritoneal cavity of B6C3F1 mice and IVIS 2000 was conducted weekly after inoculation to monitor intra-peritoneal tumor growth. The mice were divided into three groups: non-CDDP-treated (control) and CDDP-treated (0.2 and 0.4 mg). A disease-suppressive effect of the CDDP was reflected by the significantly prolonged survival of the CDDP-treated mice (control 23 +/- 1.9 days, CDDP 0.2 mg 29.6 +/- 2.9 days; p < 0.05); the total photon and area of flux were decreased. The optical imaging of intraperitoneal tumors via in vivo bioluminescence is effective for noninvasive monitoring and semi-quantitative analysis. Our syngeneic mouse model has the relevant clinical features of ovarian cancer, which makes it a useful model for developing new ovarian cancer therapies.

We found that the butanol fraction of Cinnamomi Cortex (CC/Fr.2) showed moderate inhibitory activity in wild-type severe acute respiratory syndrome coronavirus (wtSARS-CoV) and HIV/SARS-CoV S pseudovirus infections. The inhibition on pseudovirus was also seen in cells pretreated with the CC and CC/Fr.2 (IC(50S), 283.4+/-16.3 and 149.5+/-13.5 microg/ml, respectively), however the highest activities on wtSARS-CoV were observed when the viruses were treated by the extracts before challenging (IC(50S), 43.1+/-2.8 and 7.8+/-0.3 microg/ml; SIs, 8.4 and 23.1, respectively). Among the compounds fractionated from CC, procyanidin A2 and procyanidin B1 showed moderate anti-wtSARS-CoV activity (IC(50S), 29.9+/-3.3 and 41.3+/-3.4 microM; SIs, 37.35 and 15.69, respectively). We also sought to determine whether they could interfere with the clathrin-dependent endocytosis pathway using transferrin receptor (TfR) as an indicator. CC/Fr.2 inhibited the internalization of TfR but the procyanidins did not. Taken together, CC/Fr.2 contains unknown substances, that could inhibit the infection, probably by interfering with endocytosis, and it also contains procyanidins that did not inhibit the internalization but inhibited the infection. Therefore, CC extracts contain anti-virus activities that act through distinct mechanisms according to differences in the compounds or mixtures.

The endosomal sorting complex required for transport (ESCRT) proteins form multimolecular complexes that control multivesicular body formation, endosomal sorting, and transport ubiquitinated membrane proteins (including cell-surface receptors) to the endosomes for degradation. There is accumulating evidence that endosomal dysfunction is linked to neural cell degeneration in vitro, but little is known about the relationship between neural disorders and ESCRT proteins in vivo. Here we specifically deleted the hrs gene, ESCRT-0, in the neurons of mice by crossing loxP-flanked hrs mice with transgenic mice expressing the synapsin-I Cre protein (SynI-cre). Histological analyses revealed that both apoptosis and a loss of hippocampal CA3 pyramidal neurons occurred in the hrs(flox/flox);SynI-cre mice. Notably, the hrs(flox/flox);SynI-cre mice accumulated ubiquitinated proteins, such as glutamate receptors and an autophagy-regulating protein, p62. These molecules are particularly prominent in the hippocampal CA3 neurons and cerebral cortex with advancing age. Accordingly, we found that both locomotor activity and learning ability were severely reduced in the hrs(flox/flox);SynI-cre mice. These data suggest that Hrs plays an important role in neural cell survival in vivo and provide an animal model for neurodegenerative diseases that are known to be commonly affected by the generation of proteinaceous aggregates.

Aims Mobilization of stem cells/progenitors is regulated by the interaction between stromal cell-derived factor-1 (SDF-1) and its ligand, CXC chemokine receptor 4 (CXCR4). Statins have been suggested to ameliorate pulmonary arterial hypertension (PAH); however, the mechanisms involved, especially their effects on progenitors, are largely unknown. Therefore, we examined whether pravastatin ameliorates hypoxia-induced PAH in mice, and if so, which type of progenitors and what mechanism(s) are involved. Methods and results Chronic hypoxia (10% O2 for 5 weeks) increased the plasma levels of SDF-1 and mobilization of CXCR4+/vascular endothelial growth factor receptor (VEGFR)2+/c-kit+ cells from bone marrow (BM) to pulmonary artery adventitia in Balb/c mice in vivo , both of which were significantly suppressed by simultaneous oral treatment with pravastatin (2 mg/kg/day). Furthermore, in vitro experiments demonstrated that hypoxia enhances differentiation of VEGFR2+/c-kit+ cells into α-smooth muscle actin+ cells. Importantly, pravastatin ameliorated hypoxia-induced PAH associated with a decrease in the number of BM-derived progenitors accumulating in the pulmonary artery adventitia. The expression of intercellular adhesion molecule-1 (ICAM-1) and its ligand, CD18 (β2-integrin), were enhanced by hypoxia and were again suppressed by pravastatin. Conclusions These results suggest that pravastatin ameliorates hypoxia-induced PAH through suppression of SDF-1/CXCR4 and ICAM-1/CD18 pathways with a resultant reduction in the mobilization and homing of BM-derived progenitor cells.

Interleukin 6 (IL-6), a pleiotropic cytokine, functions in cells through its interaction with its receptor complex, which consists of two ligand-binding alpha subunits and two signal-transducing subunits known as gp130. There is a wealth of studies on signals mediated by gp130, but its downregulation is less well understood. Here we found that IL-6 stimulation induced lysosome-dependent degradation of gp130, which correlated with an increase in the K63-linked polyubiquitination of gp130. The stimulation-dependent ubiquitination of gp130 was mediated by c-Cbl, an E3 ligase, which was recruited to gp130 in a tyrosine-phosphorylated SHP2-dependent manner. We also found that IL-6 induced a rapid translocation of gp130 from the cell surface to endosomal compartments. Furthermore, the vesicular sorting molecule Hrs contributed to the lysosomal degradation of gp130 by directly recognizing its ubiquitinated form. Deficiency of either Hrs or c-Cbl suppressed gp130 degradation, which leads to a prolonged and amplified IL-6 signal. Thus, our present report provides the first evidence for involvement of a c-Cbl/SHP2 complex in ubiquitination and lysosomal degradation of gp130 upon IL-6 stimulation. The lysosomal degradation of gp130 is critical for cessation of IL-6-mediated signaling.

Invariant natural killer T (iNKT) cells are a special subset of alphabeta T cells with invariant TCR, which recognize alpha-galactosylceramide (alpha-GalCer) presented by CD1d. In addition to signals through the invariant TCR upon stimulation with alpha-GalCer, costimulatory signals, such as signals through CD28 and OX40, are indispensable for full activation of iNKT cells. In this study, we investigated the functions of a well-known costimulatory molecule, glucocorticoid-induced TNF receptor (GITR), on Ag-induced iNKT cell activation. Unexpectedly, engagement of GITR by agonistic mAb DTA-1 suppressed proliferation and cytokine production of iNKT cells upon alpha-GalCer stimulation. In addition, GITR signals in iNKT cells during only the Ag-priming phase was sufficient to inhibit the iNKT cell activation. Consistent with these results, the GITR-deficient iNKT cells showed enhanced proliferation and increased cytokine production upon alpha-GalCer stimulation both in vitro and in vivo. Furthermore, the in vivo administration of alpha-GalCer suppressed tumor metastasis more efficiently in GITR-deficient mice than in wild-type mice. Collectively, GITR plays a co-inhibitory role in Ag-induced iNKT cell activation.

Several lines of evidence have revealed that ubiquitylation of membrane proteins serves as a signal for endosomal sorting into lysosomes or lytic vacuoles. The hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) interacts with ubiquitylated cargoes through its ubiquitin-interacting-motif domain (UIM domain), and plays an essential early role in endosomal sorting. Here, we show that the C-terminal region of Hrs, which does not contain the UIM domain, can bind to interleukin-2 receptor beta (IL-2Rbeta). We found a direct interaction between bacterially expressed IL-2Rbeta and Hrs in GST pull-down assays, indicating that their binding is independent of ubiquitin. Trafficking and degradation assays revealed that, similarly to wild-type IL-2Rbeta, an IL-2Rbeta mutant lacking all the cytoplasmic lysine residues is sorted from Hrs-positive early endosomes to LAMP1-positive late endosomes, resulting in degradation of the receptor. By contrast, an IL-2Rbeta mutant lacking the Hrs-binding region passes through early endosomes and is mis-sorted to compartments positive for the transferrin receptor. The latter mutant exhibits attenuated degradation. Taken together, these results indicate that precise sorting of IL-2Rbeta from early to late endosomes is mediated by Hrs, a known sorting component of the ubiquitin-dependent machinery, in a manner that is independent of UIM-ubiquitin binding.

The exceptional immunostimulatory capacity of DCs makes them potential targets for investigation of cancer immunotherapeutics. We show here in mice that TNF-alpha-stimulated DC maturation was accompanied by increased expression of OX40 ligand (OX40L), the lack of which resulted in an inability of mature DCs to generate cellular antitumor immunity. Furthermore, intratumoral administration of DCs modified to express OX40L suppressed tumor growth through the generation of tumor-specific cytolytic T cell responses, which were mediated by CD4+ T cells and NKT cells. In the tumors treated with OX40L-expressing DCs, the NKT cell population significantly increased and exhibited a substantial level of IFN-gamma production essential for antitumor immunity. Additional studies evaluating NKT cell activation status, in terms of IFN-gamma production and CD69 expression, indicated that NKT cell activation by DCs presenting alpha-galactosylceramide in the context of CD1d was potentiated by OX40 expression on NKT cells. These results show a critical role for OX40L on DCs, via binding to OX40 on NKT cells and CD4+ T cells, in the induction of antitumor immunity in tumor-bearing mice.

Autophagy is an evolutionarily conserved system responsible for the degradation of cellular components and contributes to the increasing of amino acid pool, organelle turnover, and elimination of intracellular bacteria. The molecular process of autophagy is still unclear. Here we demonstrate that Hrs, a master regulator in endosomal protein sorting, plays critical roles for the autophagic degradation of non-specific proteins and Streptococcus pyogenes. We found that Hrs containing FYVE domain is localized to autophagosomes. Hrs depletion resulted in a significant decrease in the number of mature autophagosomes (autophagolysosomes) detected by the co-localization of autophagosome marker LC3 and lysosome marker LAMP-1. In contrast, formation of the primary autophagosome, detected by LC3 immunoblotting and lysosomal degradation of non-specific proteins, were not significantly altered by Hrs depletion. Based on these results, we propose a novel function of Hrs, as a crucial player in the maturation of autophagosomes.

IL-21 regulates certain functions of T cells, B cells, NK cells and dendritic cells. Although activated CD4(+) T cells produce IL-21, data identifying the specific CD4(+) T cell subsets that produce IL-21 are conflicting. In a previous study, mouse IL-21 message was detected in T(H)2, whereas human IL-21 (hIL-21) message was found in both T(H)1 and follicular helper T cells. To identify the IL-21-secreting cell populations in human, we established a hybridoma cell line producing an anti-hIL-21 mAb. Intracellular hIL-21-staining experiments showed that hIL-21 was mainly expressed in activated CD4(+) central memory T cells and in activated CD4(+) effector memory T cells, but not in activated CD4(+) naive T cells. Moreover, IL-21 was produced upon activation by some IFN-gamma-producing T(H)1-polarized cells and some IL-17-producing T(H)17-polarized cells, but not by IL-4-producing T(H)2-polarized cells. These results suggest that specific CD4(+) T cell populations produce IL-21. In the functional analysis, we found that IL-21 significantly enhanced the cytokine-driven proliferation of CD4(+) helper T cells synergistically with IL-7 and IL-15 without T cell activation stimuli. Taken together, IL-21 produced from CD4(+) memory T cells may have a supportive role in the maintenance of CD4(+) T cell subsets.

The penetration of various viruses into host cells is accomplished by hijacking the host endocytosis machinery. In the case of severe acute respiratory syndrome coronavirus (SARS-CoV) infection, viral entry is reported to require a low pH in intracytoplasmic vesicles; however, little is known about how SARS-CoV invades such compartments. Here we demonstrate that SARS-CoV mainly utilizes the clathrin-mediated endocytosis pathway for its entry to target cells by using infectious SARS-CoV, as well as a SARS-CoV pseudovirus packaged in the SARS-CoV envelope. The SARS-CoV entered caveolin-1-negative HepG2 cells, and the entry was significantly inhibited by treatment with chlorpromazine, an inhibitor for clathrin-dependent endocytosis, and by small interfering RNA-mediated gene silencing for the clathrin heavy chain. Furthermore, the SARS-CoV entered COS7 cells transfected with the mutant of ACE2 with the cytoplasmic tail deleted, SARS-CoV receptor, as well as the wild-type ACE2, and their entries were significantly inhibited by treatment with chlorpromazine. In addition, ACE2 translocated into EEA1-positive early endosomes immediately after the virus attachment to ACE2. These results suggest that when SARS-CoV binds ACE2 it is internalized and penetrates early endosomes in a clathrin-dependent manner and that the cytoplasmic tail of ACE2 is not required for the penetration of SARS-CoV.

We have recently demonstrated that endogenous erythropoietin (Epo)/Epo receptor (EpoR) system plays an important protective role in hypoxia-induced pulmonary hypertension. However, it remains to be examined whether vascular EpoR system contributes to angiogenesis in response to ischemia. We examined angiogenesis in EpoR −/− -rescued mice that lack EpoR in most organs including cardiovascular system except erythroid-lineage cells. Two weeks after femoral artery ligation, blood flow recovery, activation of VEGF/VEGF receptor system, and mobilization of endothelial progenitor cells were all impaired in EpoR −/− -rescued mice as compared with wild-type (WT) mice. Bone marrow (BM) transplantation with WT-BM cells in EpoR −/− -rescued mice partially but significantly improved blood flow recovery after hindlimb ischemia. The extent of VEGF upregulation and the number of BM-derived cells in ischemic tissue were significantly less in EpoR −/− -rescued mice compared with WT mice even after BM reconstitution with WT-BM cells. Similarly, the recovery of blood flow was significantly impaired in recipient EpoR −/− -rescued mice that had been transplanted with WT-BM or EpoR −/− -rescued-BM as compared with recipient WT mice. Furthermore, the Matrigel implantation assay and aortic ring assay showed that microvessel growth in vitro was significantly reduced in EpoR −/− -rescued mice as compared with WT mice. These results indicate that vascular EpoR system also plays an important role in angiogenesis in response to hindlimb ischemia through upregulation of VEGF/VEGF receptor system, both directly by enhancing neovascularization and indirectly by recruiting endothelial progenitor cells and BM-derived proangiogenic cells.

Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TL-Om1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR.

Signals through the OX40 costimulatory receptor on naive CD4 T cells are essential for full-fledged CD4 T cell activation and the generation of CD4 memory T cells. Because the ligand for OX40 is mainly expressed by APCs, including activated B cells, dendritic cells, and Langerhans cells, the OX40-OX40 ligand (OX40L) interaction has been thought to participate in T cell-APC interactions. Although several reports have revealed the expression of OX40L on T cells, the functional significance of its expression on them is still unclear. In this study, we demonstrate that Ag stimulation induced an increase in the surface expression and transcript levels of OX40L in CD4 T cells. Upon contact with OX40-expressing T cells, the cell surface expression of OX40L on CD4 T cells was markedly down-regulated, suggesting that OX40-OX40L binding occurs through a novel T cell-T cell interaction. To investigate the function of this phenomenon, we examined the proliferative response and survival of OX40L-deficient CD4 T cells when challenged with Ag. In vitro studies demonstrated markedly less CD3-induced proliferation of OX40L-deficient CD4 T cells compared with wild-type CD4 T cells. When using TCR transgenic CD4 T cells upon Ag stimulation, survival of OX40L-deficient T cells was impaired. Furthermore, we show that upon antigenic stimulation, fewer OX40L-deficient CD4 T cells than wild-type cells survived following transfer into wild-type and sublethally irradiated recipient mice. Taken together, our findings indicate that OX40L-expressing T cells have an autonomous machinery that provides OX40 signals through a T cell-T cell circuit, creating an additional mechanism for sustaining CD4 T cell longevity.

In this review Kazuo Sugamura and Yorio Hinuma summarize developments in studies on the human retrovirus associated with a unique human T-cell malignancy, adult T-cell leukemia; they also discuss the possible mechanisms of retrovirus-induced leukemogenesis.

Adult T cell leukemia (ATL) is an aggressive neoplastic disease, in which a quarter of the patients develop opportunistic infections due to cellular immunodeficiency. However, the underlying mechanism responsible for the immunosuppression has remained unclear. Recent studies have demonstrated that the leukemia cells from a subset of patients with ATL express Foxp3, a specific marker for CD25+CD4+ regulatory T (Treg) cells, which regulate the immune response by suppressing CD4+ T cell functions. However, whether there is a functional resemblance between ATL cells that have Foxp3 expression and Treg cells is still unknown. In this report, we confirmed the high expression of Foxp3 in leukemia cells from 5 of 12 ATL patients and demonstrated that ATL cells from 3 patients suppressed the proliferation of CD4+ T cells. Similarly, one of six HTLV-I-infected cell lines showed both high Foxp3 expression and suppressive activity. Like Treg cells, the suppression induced by the ATL cells from two patients and the HTLV-infected cell line appeared to be mediated by a cell-cell contact-dependent mechanism. Nevertheless, among the ATL cells that strongly expressed Foxp3, those from two of the five patients showed no apparent suppressive activity. Furthermore, retroviral transfection of Foxp3 did not confer any suppressive function on low Foxp3-expressing HTLV-I-infected cell lines. These results indicate that Foxp3 may be essential but is not sufficient for the Treg-cell-like suppressive activity of ATL cells and HTLV-I-infected cell lines.

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.

The degradation and sorting of cytoplasmic and cell-surface proteins are crucial steps in the control of cellular functions. We previously identified three mammalian Vps (vacuolar protein sorting) proteins, Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and signal transducing adaptor molecule (STAM) 1 and -2, which are tyrosine-phosphorylated upon cytokine/growth factor stimulation. Hrs and the STAMs each contain a ubiquitin-interacting motif and through formation of a complex are involved in the vesicle transport of early endosomes. To explore the mechanism and cellular function of this complex in mammalian cells, we established an Hrs-defective fibroblastoid cell line (hrs(-/-)); embryos with this genotype died in utero. In the hrs(-/-) cells only trace amounts of STAM1 and STAM2 were detected. Introduction of wild-type Hrs or an Hrs mutant with an intact STAM binding domain (Hrs-dFYVE) fully restored STAM1 and STAM2 expression, whereas mutants with no STAM binding ability (Hrs-dC2, Hrs-dM) failed to express the STAMs. This regulated control of STAM expression by Hrs was independent of transcription. Interestingly, STAM1 degradation was mediated by proteasomes and was partially dependent on the ubiquitin-interacting motif of STAM1. Revertant Hrs expression in hrs(-/-) cells not only led to the accumulation of ubiquitinated proteins, including intracytoplasmic vesicles, but also restored STAM1 levels in early endosomes and eliminated the enlarged endosome phenotype caused by the absence of Hrs. These results suggest that Hrs is a master molecule that controls in part the degradation of STAM1 and the accumulation of ubiquitinated proteins.

OX40/OX40 ligand (OX40L) interactions are implicated in costimulation for both CD4(+) T and B cells in a bidirectional manner. To determine the role of OX40/OX40L interactions in recipient antidonor responses after multiple allogeneic transfusions, we examined alloreactive cytotoxic T lymphocyte (allo-CTL) activity and alloantibody production in repeatedly alloimmunized OX40L-deficient mice. After the fifth alloimmunization, whereas OX40L-deficient mice showed allo-CTL activity with levels comparable to those of wild-type mice, alloantibody production in OX40L-deficient mice was significantly reduced, accompanied by fewer memory B and CD4(+) T cells with reduced function. Furthermore, nu/nu mice that received OX40L-deficient T cells still exhibited impaired alloantibody production with fewer memory CD4(+) T and B cells. In contrast, RAG-2-deficient mice that received both wild-type T cells and OX40L-deficient B cells produced scant alloantibodies with fewer memory B cells, but sufficient memory CD4(+) T cells. Thus, OX40L on B cells, rather than on T cells, is apparently required for adequate and persistent production of alloantibodies after repeated alloimmunizations.

Objectives . NS1 expression of human parvovirus B19 is a critical event in B19 pathogenesis. However, the mechanisms of NS1-induced cytotoxicity in B19 infection have yet to be clarified mainly because of the absence of multifunctional monoclonal antibodies (Mab) against NS1. The purpose of this study was to establish such Mab, which function in immunoprecipitation assays, immunoblotting, indirect immunofluorescence, and immunohistochemical staining. Methods . Balb/c mice were immunized with the recombinant NS1 protein and hybridoma cell lines producing Mab against the NS1 protein were screened by ELISA, immunoprecipitation, immunoblotting, and indirect immunofluorescence staining. The Mab were used for immunohistochemical staining of formalin-fixed tissues from an intrauterine B19 infected fetus. Results . ParC-NS1, the monoclonal antibody against the NS1 protein, worked in all tested screening methods. ParC-NS1 could also detect NS1 protein expressed in clinical tissue specimens. Conclusions . The ParC-Ns1 monoclonal antibody was specific against the NS1 protein. In addition, NS1 protein expression was successfully identified in human tissues. These data indicated that this monoclonal antibody should be a useful tool to study the kinetics and sites of NS1 expression and NS1-induced cytotoxicity in B19 infection.

Granulysin is a cytolytic granule protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTL) with a broad range of antimicrobial and tumoricidal activities. Two molecular forms of granulysin, the 15-kDa precursor and 9-kDa mature form, are produced in these cells. In this study, we developed monoclonal antibodies against granulysin and found that the 15-kDa granulysin is spontaneously secreted by peripheral blood NK and T cells via a non-granule exocytotic pathway. When NK cells killed the target cells, the released granulysin levels in culture supernatants significantly increased through the granule exocytosis. The granulysin protein was found in the sera of healthy individuals at an average concentration of 3.7 +/- 3.2 ng/ml (age 0-99 years, n=244). The serum levels of granulysin were transiently highly elevated among patients with acute viral infections. In addition, the serum granulysin levels in patients with severe immunodeficiency treated bycell therapy fluctuated proportionately to the improvement of other immunological parameters. Our results suggest that granulysin is well associated with diverse activities of NK cells and CTL in physiological and pathological settings and could be a useful novel serum marker to evaluate the overall status of host cellular immunity.

Bronchial asthma is characterized by massive infiltration of eosinophils and airway hyperreactivity (AHR), which are caused by overproduction of Th2 cytokines (IL-4, IL-5, and IL-13) by allergen-specific T cells. We recently demonstrated a critical contribution of OX40 ligand (OX40L) to the development of Th2-mediated experimental leishmaniasis. In this study, we have examined the role of OX40L in the development of Th2-mediated pulmonary inflammation by utilizing OX40L-deficient mice and a neutralizing anti-OX40L mAb in a murine model of asthma. Sensitization and airway challenge with ovalbumin in wild-type BALB/c mice induced a typical allergic asthma characterized by AHR, accumulation of eosinophils, increased mucus production, and high levels of Th2 cytokines in the lung. All these asthmatic responses were not induced in OX40L-deficient BALB/c mice. Administration of neutralizing anti-OX40L mAb in wild-type BALB/c mice during the sensitization period also abolished the induction of asthmatic responses. In contrast, administration of anti-OX40L mAb during the challenge period did not inhibit the asthmatic responses. These results indicate a critical role for OX40L in the induction phase, which leads to the development of pathogenic Th2 cells, but not in the effector phase, which includes migration and activation of pathogenic Th2 cells in the lung.