Contributions of the N-terminal flanking residues of an antigenic peptide from the Japanese cedar pollen allergen Cry j 1 to the T-cell activation by HLA-DP5.

Cry j 1 is a major allergen present in Japanese cedar (Cryptomeria japonica) pollens. Peptides with the core sequence of KVTVAFNQF from Cry j 1 ('pCj1') bind to HLA-DP5 and activate Th2 cells. In this study, we noticed that Ser and Lys at positions -2 and -3, respectively, in the N-terminal flanking (NF) region to pCj1 are conserved well in HLA-DP5-binding allergen peptides. A competitive binding assay showed that the double mutation of Ser(-2) and Lys(-3) to Glu [S(P-2)E/K(P-3)E] in a 13-residue Cry j 1 peptide (NF-pCj1) decreased its affinity for HLA-DP5 by about 2-fold. Similarly, this double mutation reduced, by about 2-fold, the amount of NF-pCj1 presented on the surface of mouse antigen-presenting dendritic cell line 1 (mDC1) cells stably expressing HLA-DP5. We established NF-pCj1-specific and HLA-DP5-restricted CD4+ T-cell clones from HLA-DP5 positive cedar pollinosis (CP) patients, and analyzed their IL-2 production due to the activation of mouse TG40 cells expressing the cloned T-cell receptor by the NF-pCj1-presenting mDC1 cells. The T-cell activation was actually decreased by the S(P-2)E/K(P-3)E mutation, corresponding to the reduction in the peptide presentation by this mutation. In contrast, the affinity of NF-pCj1·HLA-DP5 for the T-cell receptor was not affected by the S(P-2)E/K(P-3)E mutation, as analyzed by surface plasmon resonance. Considering the positional and side-chain differences of these NF residues from previously reported T-cell activating sequences, the mechanisms of enhanced T-cell activation by Ser(-2) and Lys(-3) of NF-pCj1 may be novel.

Cancer therapy with major histocompatibility complex-deficient and interferon ß-producing myeloid cells derived from allogeneic embryonic stem cells.

We previously established a method to generate myeloid cells with a proliferative capability from pluripotent stem cells and designated them iPS-ML. Human iPS-ML cells share features with physiological macrophages including the capability to infiltrate into cancer tissues. We observed therapeutic effects of human iPS-ML cells expressing interferon ß (iPS-ML/interferon (IFN)-ß) in xenograft cancer models. However, assessment of host immune system-mediated therapeutic and adverse effects of this therapy is impossible by xenograft models. We currently evaluated the therapeutic effects of a mouse equivalent of human iPS-ML/IFN, a mouse embryonic stem (ES) cell-derived myeloid cell line producing IFN (ES-ML/IFN). The ES-MLs producing IFN-ß (ß-ML) and IFN-γ (γ-ML) and originating from E14 ES cells derived from the 129 mouse strain (H-2b ) were generated, and the MHC (H-2Kb , Db , and I-Ab ) genes of the ES-ML/IFN were disrupted using the clustered regularly interspaced short palindromic repeats (CRISPR)/CAS9 method. We used the ES-ML/IFN to treat allogeneic BALB/c mice (H-2d ) transplanted with Colon26 cancer cells. Treatment with ß-ML but not with γ-ML cells repressed the growth of colon cancer in the peritoneal cavity and liver. The transferred ES-ML/IFN infiltrated into cancer tissues and enhanced infiltration of T cells into cancer tissues. ES-ML/IFN therapy increased the number of immune cells in the lymphoid organs. Sensitization of both cancer antigen-specific CD8+ T cells and natural killer (NK) cells were enhanced by the therapy, and CD8+ T cells were essential for the therapeutic effect, implying that donor MHC-deficient ß-ML exhibited a therapeutic effect through the activation of host immune cells derived from allogeneic recipient mice. The results suggested the usefulness of HLA-deficient human iPS-ML/IFN-ß cells for therapy of HLA-mismatched allogeneic cancer patients.

Immune-suppressive effects of interleukin-6 on T-cell-mediated anti-tumor immunity.

Accompanied by the growing clinical applications of immunotherapy in the treatment of cancer patients, development of novel therapeutic approaches to reverse the immune-suppressive environment in cancer patients is eagerly anticipated, because the success of cancer immunotherapy is currently limited by immune-suppressive effects in tumor-bearing hosts. Interleukin (IL)-6, a pleotropic proinflammatory cytokine, participates in tumor cell-autonomous processes that are required for their survival and growth, and is therefore known as a poor prognostic factor in cancer patients. In addition, an emerging role of IL-6 in modulating multiple functions of immune cells including T cells, dendritic cells, and macrophages is responsible for the dysfunction of innate and adaptive immunity against tumors. Therefore, the IL-6-targeting approach is of value as a promising strategy for desensitization and prevention of immune-suppressive effects, and should be an effective treatment when combined with current immunotherapies. The aim of the present review is to discuss the immune-suppressive aspects of IL-6, notably with modification of T-cell functions in cancer patients, and their relationship to anti-tumor immune responses and cancer immunotherapy.

Novel therapeutic strategies for advanced ovarian cancer by using induced pluripotent stem cell-derived myelomonocytic cells producing interferon beta.

Although first-line chemotherapy has a high rate of complete responses in ovarian cancer patients, the vast majority of patients present with recurrent disease that has become refractory to conventional chemotherapy. Peritoneal dissemination and malignant ascites are the hallmarks of recurrent or advanced ovarian cancer and severely reduce quality of life. Development of therapeutic measures to treat such patients is eagerly anticipated. Macrophage infiltration is observed in various types of cancer including epithelial ovarian cancer. In addition, macrophages are involved in the formation of spheroids in the malignant ascites of ovarian cancer and promote cancer growth. iPS-ML, macrophage-like myelomonocytic cells generated from human induced pluripotent stem (iPS) cells, made close contacts with ovarian cancer cells in vitro. We hypothesized that, if we inoculate iPS-ML-producing IFN-ß (iPS-ML/IFN-ß) into the peritoneal cavity of patients with ovarian cancer, IFN-ß produced by the iPS-ML/IFN-ß would efficiently act on the cancer cells to suppress cancer growth. To evaluate this hypothesis, we injected iPS-ML/IFN-ß into SCID mice bearing peritoneally disseminated human ovarian cancer cells, SKOV3. Immunohistochemical analysis of the intraperitoneal tumors detected iPS-ML/IFN-ß infiltrating into the cancer tissues. Therapy with iPS-ML/IFN-ß significantly suppressed tumor progression. In addition, dramatic reduction of cancer-related ascites was observed. Collectively, it is suggested that iPS-ML/IFN-ß therapy offers a new approach for the treatment of patients with advanced ovarian cancer.

iPS cell serves as a source of dendritic cells for in vitro dengue virus infection model.

The lack of an appropriate model has been a serious concern in dengue research pertinent to immune response and vaccine development. It remains a matter of impediment in dengue virus (DENV) studies when it comes to an in vitro model, which requires adequate quantity of dendritic cells (DC) with uniform characters. Other sources of DC, mostly monocyte derived DC (moDC), have been used despite their limitations such as quantity, proliferation, and donor dependent characters. Recent development of human iPS cells with consistent proliferation for long, stable, functional characteristics and desired HLA background has certainly offered added advantages. Therefore, we hypothesised that iPS derived cells would be a reliable alternative to the traditional DCs to be used with an in vitro DENV system. To develop a DENV infection and T cell activation model, we utilised iPS cells (HLA-A*24) as the source of DC. iPS-ML-DC was prepared and DENV infectivity was assessed apart from the major surface markers expression and cytokine production potential. Our iPS-ML-DC had major DC markers expression, DENV infection efficiency and cytokine production properties similar to that of moDC. Moreover, DENV infected iPS-ML-DC demonstrated the ability to activate HLA-matched T cell (but not mismatched) in vitro as evidenced by significantly higher proportion of IFN-γ+ CD69+ T cells compared to non-infected iPS-ML-DC. This affirmed the antigen-specific T cell activation by iPS-ML-DC as a function of antigen presenting cells. To conclude, maturation potential, DENV infection efficiency and T cell activation ability collectively suggest that iPS-ML-DC serves as an attractive option of DC for use in DENV studies in vitro.

The present status and future prospects of peptide-based cancer vaccines.

Tumor cells commonly express several antigens, such as tumor-associated antigens (TAAs) or mutation-derived antigens (neoantigens), that can be regarded as foreign antigens and elicit anti-tumor immune responses in cancer patients. Various TAAs or neoantigens expressed in cancer cells have been identified and utilized as targets for cancer vaccines. One approach to elicit tumor-specific immune responses is termed peptide-based cancer vaccination; it involves administrating TAAs or neoantigen-derived peptide for treatment of cancers. There have been several forms of peptide-based cancer vaccines depending on which effector cells, such as CTLs or CD4(+) T-helper cells, are targeted to be activated. Many phase I and II clinical trials of peptide-based cancer vaccines using TAA-derived CTL epitopes, T-helper cell epitopes or dendritic cells loaded with TAA-derived peptides for various malignant tumors have been conducted and provide clinical benefits in a small fraction of patients. Nowadays, to improve the efficiency of peptide-based cancer vaccines, combination immunotherapy of peptide-based cancer vaccines with the immune-checkpoint blockade therapies using mAbs specific for CTLA-4, programmed cell death 1 (PD-1), or PD-1 ligand 1 (PD-L1) have been developed for clinical application. Furthermore, along with the recent technological progress in genetic and bioinformatic analysis, it has become easier to identify neoantigens from individual cancer patients. It is expected that peptide-based cancer vaccines targeting neoantigens as a personalized cancer immunotherapy will be developed.

Therapy of peritoneally disseminated colon cancer by TAP-deficient embryonic stem cell-derived macrophages in allogeneic recipients.

We established a method to generate a large quantity of myeloid lineage cells from mouse embryonic stem (ES) cells, termed ES cell-derived proliferating myeloid cell lines (ES-ML). ES-ML continuously proliferated in the presence of M-CSF and GM-CSF. ES-ML genetically modified to express an anti-HER2 (neu) mAb single-chain V region fragment reduced the number of cocultured mouse Colon-26 cancer cells expressing HER2. Stimulation of ES-ML with IFN-γ plus LPS or TNF resulted in almost complete killing of the Colon-26 cells by the ES-ML, and the cytotoxicity was mediated, in part, by NO produced by ES-ML. When ES-ML were injected into mice with i.p. established Colon-26 tumors, they efficiently infiltrated the tumor tissues. Injection of ES-ML with rIFN-γ and LPS inhibited cancer progression in the mouse peritoneal cavity. Coinjection of TNF-transfected or untransfected ES-ML with rIFN-γ inhibited cancer growth and resulted in prolonged survival of the treated mice. In this experiment, transporter associated with Ag processing (TAP)1-deficient ES-ML exhibited therapeutic activity in MHC-mismatched allogeneic recipient mice. Despite the proliferative capacity of ES-ML, malignancy never developed from the transferred ES-ML in the recipient mice. In summary, TAP-deficient ES-ML with anticancer properties exhibited a therapeutic effect in allogeneic recipients, suggesting the possible use of TAP-deficient human-induced pluripotent stem cell-derived proliferating myeloid cell lines in cancer therapy.

Cancer immunotherapy using novel tumor-associated antigenic peptides identified by genome-wide cDNA microarray analyses.

Recent genome-wide cDNA microarray analysis of gene expression profiles in comprehensive tumor types coupled with isolation of cancer tissues by laser-microbeam microdissection have revealed ideal tumor-associated antigens (TAAs) that are frequently overexpressed in various cancers including head and neck squamous cell cancer (HNSCC) and lung cancer, but not in most normal tissues except for testis, placenta, and fetal organs. Preclinical studies using HLA-transgenic mice and human T cells in vitro showed that TAA-derived CTL-epitope short peptides (SPs) are highly immunogenic and induce HLA-A2 or -A24-restricted CTLs. Based on the accumulated evidence, we carried out a phase II clinical trial of the TAA-SP vaccine in advanced 37 HNSCC patients. This study showed a significant induction of TAA-specific CTLs in the majority of patients without serious adverse effects. Importantly, clinical responses including a complete response were observed in this study. Another phase II clinical trial of therapeutic TAA-SP vaccine, designed to evaluate the ability of prevention of recurrence, is ongoing in HNSCC patients who have received curative operations. Further studies in human preclinical studies and in vivo studies using HLA class I transgenic mice showed TAA-derived long peptides (TAA-LPs) have the capacity to induce not only promiscuous HLA class II-restricted CD4(+) T helper type 1 cells but also tumor-specific CTLs through a cross-presentation mechanism. Moreover, we observed an augmentation of TAA-LP-specific T helper type 1 cell responses and tumor antigen-spreading in HNSCC patients vaccinated with TAA-SPs. This accumulated evidence suggests that therapeutic TAA-SPs and LPs vaccines may provide a promising cancer immunotherapy.

Identification of CDCA1-derived long peptides bearing both CD4+ and CD8+ T-cell epitopes: CDCA1-specific CD4+ T-cell immunity in cancer patients.

We recently identified a novel cancer-testis antigen, cell division cycle associated 1 (CDCA1) using genome-wide cDNA microarray analysis, and CDCA1-derived cytotoxic T lymphocyte (CTL)-epitopes. In this study, we attempted to identify CDCA1-derived long peptides (LPs) that induce both CD4+ helper T (Th) cells and CTLs. We combined information from a recently developed computer algorithm predicting HLA class II-binding peptides with CDCA1-derived CTL-epitope sequences presented by HLA-A2 (A*02:01) or HLA-A24 (A*24:02) to select candidate CDCA1-LPs encompassing both Th cell epitopes and CTL-epitopes. We studied the immunogenicity of CDCA1-LPs and the cross-priming potential of LPs bearing CTL-epitopes in both human in vitro and HLA-class I transgenic mice in vivo. Then we analyzed the Th cell response to CDCA1 in head-and-neck cancer (HNC) patients before and after vaccination with a CDCA1-derived CTL-epitope peptide using IFN-γ enzyme-linked immunospot assays. We identified two CDCA1-LPs, CDCA1(39­64)-LP and CDCA1(55­78)-LP, which encompass naturally processed epitopes recognized by Th cells and CTLs. CDCA1-specific CTLs were induced through cross-presentation of CDCA1-LPs in vitro and in vivo. In addition, CDCA1-specific Th cells enhanced induction of CDCA1-specific CTLs. Furthermore, significant frequencies of CDCA1-specific Th cell responses were detected after short-term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with CDCA1-LPs in HNC patients (CDCA1(39­64)-LP, 74%; CDCA1(55­78)-LP, 68%), but not in healthy donors. These are the first results demonstrating the presence of CDCA1-specific Th cell responses in HNC patients and underline the possible utility of CDCA1-LPs for propagation of both CDCA1-specific Th cells and CTLs.

A novel coculture system for assessing respiratory sensitizing potential by IL-4 in T cells.

Although several in vitro assays that predict the sensitizing potential of chemicals have been developed, none can distinguish between chemical respiratory and skin sensitizers. Recently, we established a new three-dimensional dendritic cell (DC) coculture system consisting of a human airway epithelial cell line, immature DCs derived from human peripheral monocytes, and a human lung fibroblast cell line. In this coculture system, compared to skin sensitizers, respiratory sensitizers showed enhanced mRNA expression in DCs of the key costimulatory molecule OX40 ligand (OX40L), which is important for T helper 2 (Th2) cell differentiation. Herein, we established a new two-step DC/T cell coculture system by adding peripheral allogeneic naïve CD4+ T cells to the DCs stimulated in the DC coculture system. In this DC/T cell coculture system, model respiratory sensitizers, but not skin sensitizers, enhanced mRNA expression of the predominant Th2 marker interleukin-4 (IL-4). To improve the versatility, in place of peripheral monocytes, monocyte-derived proliferating cells called CD14-ML were used in the DC coculture system. As in peripheral monocytes, enhanced mRNA expression of OX40L was induced in CD14-ML by respiratory sensitizers compared to skin sensitizers. When these cell lines were applied to the DC/T cell coculture system with peripheral allogeneic naïve CD4+ T cells, respiratory sensitizers but not skin sensitizers enhanced the mRNA expression of IL-4. Thus, this DC/T cell coculture system may be useful for discriminating between respiratory and skin sensitizers by differential mRNA upregulation of IL-4 in T cells.

Selective inhibition of nuclear factor-κB by nuclear factor-κB essential modulator-binding domain peptide suppresses the metastasis of highly metastatic oral squamous cell carcinoma.

Nuclear factor-κB (NF-κB) activation contributes to the development of metastasis, thus leading to a poor prognosis in many cancers, including OSCC. However, little in vivo experimental data are available about the effects of NF-κB inhibition on OSCC metastasis. OSCC sublines were established from a GFP-expressing parental cell line, GSAS, and designated GSAS/N3 and N5 according to the in vivo passage number after cervical lymph node metastasis by a serial orthotopic transplantation model. In vitro migration and invasion were assessed in these cells, and the NF-κB activities and expression of NF-κB-regulated metastasis-related molecules were also examined. In in vivo experiments, the metastasis and survival of tumor-engrafted mice were monitored. Furthermore, the effects of a selective NF-κB inhibitor, NEMO-binding domain (NBD) peptide, on metastasis in GSAS/N5-engrafted mice were assessed, and engrafted tongue tumors were immunohistochemically examined. Highly metastatic GSAS/N3 and N5 cells showed an enhanced NF-κB activity, thus contributing to increased migration, invasion, and a poor prognosis compared with the parent cells. Furthermore, the expression levels of NF-κB-regulated metastasis-related molecules, such as fibronectin, ß1 integrin, MMP-1, -2, -9, and -14, and VEGF-C, were upregulated in the highly metastatic cells. The NBD peptide suppressed metastasis and tongue tumor growth in GSAS/N5-inoculated mice, and was accompanied by the downregulation of the NF-κB-regulated metastasis-related molecules in engrafted tongue tumors. Our results suggest that the selective inhibition of NF-κB activation by NBD peptide may provide an effective approach for the treatment of highly metastatic OSCC.

Modulation of exosome-mediated mRNA turnover by interaction of GTP-binding protein 1 (GTPBP1) with its target mRNAs.

Eukaryotic mRNA turnover is among most critical mechanisms that affect mRNA abundance and are regulated by mRNA-binding proteins and the cytoplasmic exosome. A functional protein, guanosine-triphosphate-binding protein 1 (GTPBP1), which associates with both the exosome and target mRNAs, was identified. The overexpression of GTPBP1 accelerated the target mRNA decay, whereas the reduction of the GTPBP1 expression with RNA interference stabilized the target mRNA. GTPBP1 has a putative guanosine-triphosphate (GTP)-binding domain, which is found in members of the G-protein family and Ski7p, a well-known core factor of the exosome-mediated mRNA turnover pathway in yeast. Analyses of protein interactions and mRNA decay demonstrated that GTPBP1 modulates mRNA degradation via GTP-binding-dependent target loading. Moreover, GTPBP1-knockout models displayed multiple mRNA decay defects, including elevated nocturnal levels of Aanat mRNA in pineal glands, and retarded degradation of TNF-α mRNA in lipopolysaccharide-treated splenocytes. The results of this study suggest that GTPBP1 is a regulator and adaptor of the exosome-mediated mRNA turnover pathway.

Dual effects of TRAIL in suppression of autoimmunity: the inhibition of Th1 cells and the promotion of regulatory T cells.

TRAIL is known to play a pivotal role in the inhibition of autoimmune disease. We previously demonstrated that administration of dendritic cells engineered to express TRAIL and myelin-oligodendrocyte glycoprotein reduced the severity of experimental autoimmune encephalomyelitis and suggested that CD4(+)CD25(+) regulatory T cells (Tregs) were involved in mediating this preventive effect. In the current study, we investigated the effect of TRAIL on Tregs, as well as conventional T cells, using TRAIL-deficient mice. Upon induction of experimental autoimmune encephalomyelitis, TRAIL-deficient mice showed more severe clinical symptoms, a greater frequency of IFN-γ-producing CD4(+) T (Th1) cells, and a lower frequency of CD4(+)Foxp3(+) Tregs than did wild-type mice. In vitro, conventional T cells stimulated by bone marrow-derived dendritic cells (BM-DCs) from TRAIL-deficient mice showed a greater magnitude of proliferation than did those stimulated by BM-DCs from wild-type mice. In contrast, TRAIL expressed on the stimulator BM-DCs enhanced the proliferative response of CD4(+)CD25(+) Tregs in the culture. The functional TRAILR, mouse death receptor 5 (mDR5), was expressed in conventional T cells and Tregs upon stimulation. In contrast, the decoy receptor, mDc-TRAILR1, was slightly expressed only on CD4(+)CD25(+) Tregs. Therefore, the distinct effects of TRAIL may be due to differences in the mDc-TRAILR1 expression or the signaling pathways downstream of mouse death receptor 5 between the two T cell subsets. Our data suggest that TRAIL suppresses autoimmunity by two mechanisms: the inhibition of Th1 cells and the promotion of Tregs.

Peptides derived from human insulin-like growth factor-II mRNA binding protein 3 can induce human leukocyte antigen-A2-restricted cytotoxic T lymphocytes reactive to cancer cells.

Insulin-like growth factor-II mRNA binding protein 3 (IMP-3) is an oncofetal protein expressed in various malignancies including lung cancer. This study aimed to identify immunogenic peptides derived from IMP-3 that can induce tumor-reactive and human leukocyte antigen (HLA)-A2 (A*02:01)-restricted cytotoxic T lymphocytes (CTL) for lung cancer immunotherapy. Forty human IMP-3-derived peptides predicted to bind to HLA-A2 were analyzed to determine their capacity to induce HLA-A2-restricted T cells in HLA-A2.1 (HHD) transgenic mice (Tgm). We found that three IMP-3 peptides primed HLA-A2-restricted CTL in the HLA-A2.1 Tgm. Among them, human CTL lines reactive to IMP-3 (515) NLSSAEVVV(523) were reproducibly established from HLA-A2-positive healthy donors and lung cancer patients. On the other hand, IMP-3 (199) RLLVPTQFV(207) reproducibly induced IMP-3-specific and HLA-A2-restricted CTL from healthy donors, but did not sensitize CTL in the HLA-A2.1 Tgm. Importantly, these two IMP-3 peptide-specific CTL generated from healthy donors and cancer patients effectively killed the cancer cells naturally expressing both IMP-3 and HLA-A2. Cytotoxicity was significantly inhibited by anti-HLA class I and anti-HLA-A2 monoclonal antibodies, but not by the anti-HLA-class II monoclonal antibody. In addition, natural processing of these two epitopes derived from the IMP-3 protein was confirmed by specific killing of HLA-A2-positive IMP-3-transfectants but not the parental IMP-negative cell line by peptide-induced CTL. This suggests that these two IMP-3-derived peptides represent highly immunogenic CTL epitopes that may be attractive targets for lung cancer immunotherapy.

A novel tumor-associated antigen, cell division cycle 45-like can induce cytotoxic T-lymphocytes reactive to tumor cells.

The present study attempted to identify a useful tumor-associated antigen (TAA) for lung cancer immunotherapy and potential immunogenic peptides derived from the TAA. We focused on cell division cycle 45-like (CDC45L), which has a critical role in the initiation and elongation steps of DNA replication, as a novel candidate TAA for immunotherapy based on a genome-wide cDNA microarray analysis of lung cancer. The CDC45L was overexpressed in the majority of lung cancer tissues, but not in the adjacent non-cancerous tissues or in many normal adult tissues. We examined the in vitro and in vivo anti-tumor effects of cytotoxic T-lymphocytes (CTL) specific to CDC45L-derived peptides induced from HLA-A24 (A*24:02)-positive donors. We identified three CDC45L-derived peptides that could reproducibly induce CDC45L-specific and HLA-A24-restricted CTL from both healthy donors and lung cancer patients. The CTL could effectively lyse lung cancer cells that endogenously expressed both CDC45L and HLA-A24. In addition, we found that CDC45L (556) KFLDALISL(564) was eminent in that it induced not only HLA-A24 but also HLA-A2 (A*02:01)-restricted antigen specific CTL. Furthermore, the adoptive transfer of the CDC45L-specific CTL inhibited the growth of human cancer cells engrafted into immunocompromised mice. These results suggest that these three CDC45L-derived peptides are highly immunogenic epitopes and CDC45L is a novel TAA that might be a useful target for lung cancer immunotherapy.

Degradation of fibrinogen and collagen by staphopains, cysteine proteases released from Staphylococcus aureus.

Staphylococcus aureus is the most frequently isolated pathogen in gram-positive sepsis often complicated by a blood clotting disorder, and is the leading cause of infective endocarditis induced by bacterial destruction of endocardial tissues. The bacterium secretes cysteine proteases referred to as staphopain A (ScpA) and staphopain B (SspB). To investigate virulence activities of staphopains pertinent to clotting disorders and tissue destruction, we examined their effects on collagen, one of the major tissue components, and on plasma clotting. Both staphopains prolonged the partial thromboplastin time of plasma in a dose- and activity-dependent manner, with SspB being threefold more potent than ScpA. Staphopains also prolonged the thrombin time of both plasma and fibrinogen, indicating that these enzymes can cause impaired plasma clotting through fibrinogen degradation. Whereas SspB cleaved the fibrinogen Aα-chain at the C-terminal region very efficiently, ScpA degraded it rather slowly. This explains the superior ability of the former enzyme to impair fibrinogen clottability. Enzymically active staphopains, at concentrations as low as 10 nM, degraded collagen with comparable efficiency. These results show novel virulence activities of staphopains in degrading fibrinogen and collagen, and suggest an involvement of staphopains in the clotting impairment and tissue destruction caused by staphylococcal infection.

The forkhead box M1 transcription factor as a candidate of target for anti-cancer immunotherapy.

The present study attempted to identify a target antigen for immunotherapy for cholangiocarcinoma. Forkhead box M1 (FOXM1) was selected as a candidate antigen based on the data of previous cDNA microarray analysis of clinical samples of cholangiocarcinoma. The level of FOXM1 mRNA was more than 4 times higher in cancer cells in comparison to adjacent normal epithelial cells, in all of 24 samples of cholangiocarcinoma tissues. An immunohistochemical analysis also detected FOXM1 protein in the cancer cells but not in the normal cells. Twenty-three human FOXM1-derived peptides predicted to bind to HLA-A2 were analyzed to determine their ability to induce HLA-A2-restricted T cells in HLA-A2 transgenic mice. FOXM1(362-370) (YLVPIQFPV), FOXM1(373-382) (SLVLQPSVKV), and FOXM1(640-649) (GLMDLSTTPL) peptides primed HLA-A2-restricted cytotoxic T lymphocytes (CTLs) in the HLA-A2 transgenic mice. Human CTL lines reactive to these 3 peptides could also be established from HLA-A2-positive healthy donors and cancer patients. Natural processing of the 3 epitopes from FOXM1 protein was confirmed by specific killing of HLA-A2-positive FOXM1-transfectants by peptide-induced CTLs. FOXM1 is expressed in various types of cancers and it is also functionally involved in oncogenic transformation and the survival of cancer cells. Therefore, FOXM1 may be a suitable target for immunotherapy against various cancers including cholangiocarcinoma.

Identification of SPARC as a candidate target antigen for immunotherapy of various cancers.

To establish efficient anticancer immunotherary, it is important to identify tumor-associated antigens (TAAs) directing the immune system to attack cancer. A genome-wide cDNA microarray analysis identified that secreted protein acidic and rich in cysteine (SPARC) gene is overexpressed in the gastric, pancreatic and colorectal cancer tissues but not in their noncancerous counterparts. This study attempted to identify HLA-A24 (A*2402)-restricted and SPARC-derived CTL epitopes. We previously identified H-2K(d)-restricted and SPARC-derived CTL epitope peptides in BALB/c mice, of which H-2K(d)-binding peptide motif is comparable with that of HLA-A24 binding peptides. By using these peptides, we tried to induce HLA-A24 (A*2402)-restricted and SPARC-reactive human CTLs and demonstrated an antitumor immune response. The SPARC-A24-1(143-151) (DYIGPCKYI) and SPARC-A24-4(225-234) (MYIFPVHWQF) peptides-reactive CTLs were successfully induced from peripheral blood mononuclear cells by in vitro stimulation with these two peptides in HLA-A24 (A*2402) positive healthy donors and cancer patients, and these CTLs exhibited cytotoxicity specific to cancer cells expressing both SPARC and HLA-A24 (A*2402). Furthermore, the adoptive transfer of the SPARC-specific CTLs could inhibit the tumor growth in nonobese diabetic/severe combined immunodeficient mice bearing human cancer cells expressing both HLA-A24 (A*2402) and SPARC. These findings suggest that SPARC is a potentially useful target candidate for cancer immunotherapy.

Characterization of dendritic cells and macrophages generated by directed differentiation from mouse induced pluripotent stem cells.

Methods have been established to generate dendritic cells (DCs) from mouse and human embryonic stem (ES) cells. We designated them as ES-DCs and mouse models have demonstrated the induction of anti-cancer immunity and prevention of autoimmune disease by in vivo administration of genetically engineered ES-DCs. For the future clinical application of ES-DCs, the histoincompatibility between patients to be treated and available human ES cells and the ethical concerns associated with human ES cells may be serious obstacles. However, recently developed induced pluripotent stem (iPS) cell technology is expected to resolve these issues. This report describes the generation and characterization of DCs derived from mouse iPS cells. The iPS cell-derived DCs (iPS-DCs) possessed the characteristics of DCs including the capacity of T-cell-stimulation, antigen-processing and presentation and cytokine production. DNA microarray analyses revealed the upregulation of genes related to antigen-presenting functions during differentiation into iPS-DCs and similarity in gene expression profile in iPS-DCs and bone marrow cell-derived DCs. Genetically modified iPS-DCs expressing antigenic protein primed T-cells specific to the antigen in vivo and elicited efficient antigen-specific anti-tumor immunity. In addition, macrophages were generated from iPS cells (iPS-MP). iPS-MP were comparable with bone marrow cell-derived macrophages in the cell surface phenotype, functions, and gene expression profiles.

Activation of antigen-specific cytotoxic T lymphocytes by beta 2-microglobulin or TAP1 gene disruption and the introduction of recipient-matched MHC class I gene in allogeneic embryonic stem cell-derived dendritic cells.

A method for the genetic modification of dendritic cells (DC) was previously established based on the in vitro differentiation of embryonic stem (ES) cells to DC (ES-DC). The unavailability of human ES cells genetically identical to the patients will be a problem in the future clinical application of this technology. This study attempted to establish a strategy to overcome this issue. The TAP1 or beta(2)-microglobulin (beta(2)m) gene was disrupted in 129 (H-2(b))-derived ES cells and then expression vectors for the H-2K(d) or beta(2)m-linked form of K(d) (beta2m-K(d)) were introduced, thus resulting in two types of genetically engineered ES-DC, TAP1(-/-)/K(d) ES-DC and beta(2)m(-/-)/beta(2)m-K(d) ES-DC. As intended, both of the transfectant ES-DC expressed K(d) but not the intrinsic H-2(b) haplotype-derived MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) and TAP1(-/-)/K(d) ES-DC were not recognized by pre-activated H-2(b)-reactive CTL and did not prime H-2(b) reactive CTL in vitro or in vivo. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC and TAP1(-/-)/K(d) ES-DC had a survival advantage in comparison to beta(2)m(+/-)/beta(2)m-K(d) ES-DC and TAP1(+/+)/K(d) ES-DC, when transferred into BALB/c mice. K(d)-restricted RSV-M2-derived peptide-loaded ES-DC could prime the epitope-specific CTL upon injection into the BALB/c mice, irrespective of the cell surface expression of intrinsic H-2(b) haplotype-encoded MHC class I. Beta(2)m(-/-)/beta(2)m-K(d) ES-DC were significantly more efficient in eliciting immunity against RSV M2 protein-expressing tumor cells than beta(2)m(+/-)/beta(2)m-K(d) ES-DC. The modification of the beta(2)m or TAP gene may therefore be an effective strategy to resolve the problem of HLA class I allele mismatch between human ES or induced pluripotent stem cells and the recipients to be treated.