Published papers

Use of adequate adjuvant is necessary for induction of effective antitumor immune responses. To develop an effective adjuvant for cancer immunotherapy, we selected formalin-inactivated (f)-HSV as an adjuvant component, and analyzed the mechanisms underlying its adjuvant effects. First, we found that f-HSV can induce the tumor antigen-specific CTLs by enhancing antigen cross-presentation by dendritic cells (DCs), mainly through TLR2, but not TLR9. Next, f-HSV was also found to prevent the accumulation of myeloid-derived suppressor cells (MDSCs). We demonstrated that the expansion of MDSCs in the blood and spleen during tumor progression required B cells producing the inflammatory angiogenesis factors, vascular endothelial growth factor (VEGF)-A and neuropilin-1 (NRP-1), a co-receptor for VEGF receptor-2 (VEGFR-2). Interestingly, the transmembrane-type NRP-1 on B cells changed to soluble-type NRP-1 (sNRP-1) by f-HSV treatment. We further showed that the sNRP-1 and VEGF-A secreted from B cells by f-HSV treatment could abrogate the immunosuppressive ability of MDSCs. These results suggest that f-HSV can enhance antitumor immune responses as an adjuvant, not only through activation of DCs, but also inactivation of MDSCs via B cells.

Since the discovery of RNA interference (RNAi) in Caenorhabditis elegans in 1998, this mechanism has been found to be conserved in a wide variety of species, including insects, plants, and mammals. In mammals, small (or short) interfering RNA (siRNA) or short hairpin RNA (shRNA) can be expressed by using several expression vectors including lentiviral vectors. The lentiviral vector has several useful characteristics for RNAi experiments including broad host tropism and stable gene transduction to both dividing and nondividing cells, which permits stable depletion of target genes. This technology can be useful for several applications, including basic cancer research.

Background: The tumor associated antigen (TAA) gp100 was one of the first identified and has been used in clinical trials to treat melanoma patients. However, the gp100 epitope peptide restricted to HLA-A*2402 has not been extensively examined clinically due to the ethnic variations. Since it is the most common HLA Class I allele in the Japanese population, we performed a phase I clinical trial of cancer vaccination using the HLA-A*2402 gp100 peptide to treat patients with metastatic melanoma.Methods: The phase I clinical protocol to test a HLA-A*2402 gp100 peptide-based cancer vaccine was designed to evaluate safety as the primary endpoint and was approved by The University of Tokyo Institutional Review Board. Information related to the immunologic and antitumor responses were also collected as secondary endpoints. Patients that were HLA-A*2402 positive with stage IV melanoma were enrolled according to the criteria set by the protocol and immunized with a vaccine consisting of epitope peptide (VYFFLPDHL, gp100-in4) emulsified with incomplete Freund's adjuvant (IFA) for the total of 4 times with two week intervals. Prior to each vaccination, peripheral blood mononuclear cells (PBMCs) were separated from the blood and stored at -80°C. The stored PBMCs were thawed and examined for the frequency of the peptide specific T lymphocytes by IFN-γ- ELISPOT and MHC-Dextramer assays.Results: No related adverse events greater than grade I were observed in the six patients enrolled in this study. No clinical responses were observed in the enrolled patients although vitiligo was observed after the vaccination in two patients. Promotion of peptide specific immune responses was observed in four patients with ELISPOT assay. Furthermore, a significant increase of CD8+ gp100-in4+ CTLs was observed in all patients using the MHC-Dextramer assay. Cytotoxic T lymphocytes (CTLs) clones specific to gp100-in4 were successfully established from the PBMC of some patients and these CTL clones were capable of lysing the melanoma cell line, 888 mel, which endogenously expresses HLA-restricted gp100-in4.Conclusion: Our results suggest this HLA-restricted gp100-in4 peptide vaccination protocol was well-tolerated and can induce antigen-specific T-cell responses in multiple patients. Although no objective anti-tumor effects were observed, the effectiveness of this approach can be enhanced with the appropriate modifications.

Anti-tumor effects of current cancer vaccines are limited. However, detailed analyses of clinical trials revealed several important points to be improved towards immunological tumor rejection. These are, (1) identification of tumor antigens involved in tumor cell proliferation/survival and expressed in cancer stem cells, (2) development of in situ tumor destruction methods which release tumor antigens in an immunogenic manner to induce immune responses to multiple endogenous tumor antigens, (3) development of methods to augment antigen processing and presentation by dendritic cells, including effective adjuvants, (4) development of methods to expand CTL and helper T cells in vivo, (5) understanding of mechanisms underlying cancer induced immunosuppression and development of methods to overcome them. Comprehensive interventions on these points in the antitumor immune network will lead to effective cancer immunotherapy.

Accumulating evidence suggests that in several types of brain tumors, including glioma, only a phenotypic subset of tumor cells called brain cancer stem cells (BCSCs) may be capable of initiating tumor growth. Recently, the isolation of side population (SP) cells using Hoechst dye has become a useful method for obtaining cancer stem cells in various tumors. In this study, we isolated cancer stem-like cells from human glioma cell lines using the SP technique. Flow cytometry analysis revealed that SK-MG-1, a human glioblastoma cell line, contained the largest number of SP cells among the five glioma cell lines that were analyzed. The SP cells had a self-renewal ability and were capable of forming spheres in a neurosphere culture medium containing EGF and FGF2. Spheres derived from the SP cells differentiated into three different lineage cells: neurons, astrocytes and oligodendrocytes. RT-PCR analysis revealed that the SP cells expressed a neural stem cell marker, Nestin. The SP cells generated tumors in the brains of NOD/SCID mice at 8 weeks after implantation, whereas the non-SP cells did not generate any tumors in the brain. These results indicate that SP cells isolated from SK-MG-1 possess the properties of cancer stem cells, including their self-renewal ability, multi-lineage differentiation, and tumorigenicity. Therefore, the SP cells from SK-MG-1 may be useful for analyzing BCSCs because of the ease with which they can be handled and their yield.

Cancer-induced immunotolerance mediated by inducible Treg (iTreg) is a major obstacle to cancer immunotherapy. In a basic study of immunotolerance, injection of an endogenous superantigen, i.e. the minor lymphocyte stimulatory (Mls)-1a, into specific TCR Vβ8.1-Tg mice enabled generation of anergic CD25- iTreg, the immunosuppressive function of which was maintained by IL-10 production via p38-MAPK activation. Interestingly, although p38-chemical inhibitor (p38-inhibitor) is capable of breaking CD25- iTreg-induced immunotolerance, the p38-inhibitor had hardly any immunotolerance breaking effect when CD25+ Treg were present, suggesting that depletion of CD25+ Treg is necessary for p38-inhibitor to be effective. Peptide OVA323-339 iv.-injection into its specific TCR-Tg (OT-II) mice also induced adaptive tolerance by iTreg. Peptide immunotherapy with p38-inhibitor after CD25+ Treg-depletion was performed in an OVA-expressing lymphoma E.G7-bearing tolerant model established by adoptive transfer of OT-II CD25- iTreg, which resulted in suppression of tumor growth. Similarly, the antitumor immunity induced by peptide immunotherapy in colon carcinoma CT26-bearingmice, in which the number of IL-10-secreting iTreg is increased, was augmented by treatment with p38-inhibitor after CD25 + Treg-depletion and resulted in inhibition of tumor progression. These results suggest that simultaneous inhibition of two distinct Treg-functions may be important to the success of cancer immunotherapy.

Malignant gliomas are the most aggressive human primary brain tumors and are currently incurable. Immunotherapies have the potential to target glioma and glioma stem cells (GSCs) that are resistant to conventional therapies. We previously identified SOX6 as a human glioma antigen and demonstrated that vaccination with SOX6 DNA induced cytotoxic T lymphocytes (CTLs) specific for glioma, thereby exerting therapeutic antitumor responses in glioma-bearing mice. In this study, we attempted to identify SOX6-derived peptides as specific targets for effective and safe T-cell-mediated immunotherapy targeting SOX6-positive glioma and GSCs. In vitro stimulation with human leukocyte antigen (HLA)-A*2402 (A24)-restricted peptides, RFENLGPQL (SOX6504) and PYYEEQARL (SOX6628) or the HLA-A*0201 (A2)-restricted peptide, ALFGDQDTV (SOX6447) was capable of inducing SOX6 peptide-specific CTLs in peripheral blood mononuclear cells derived from healthy donors and glioma patients. These CTLs were able to lyse a majority of glioma cell lines and a GSC line derived from human glioblastoma in an HLA Class I-restricted and an antigen-dependent manner. Furthermore, peptide vaccines of SOX6628, which was conserved in the murine SOX6 protein and expected to bind to major histocompatibility complex (MHC) H-2d, induced CTLs specific for SOX6628 in H-2d mice. Normal autologous cells from mice, in which SOX6-specific immune responses were generated, were not destroyed. These results suggest that these SOX6 peptides are potnetially immunogenic in HLA-A24 or -A2 positive glioma patients and should be considered as a promising strategy for safe and effective T-cell-based immunotherapy of patients with gliomas.

Fatty acid-binding protein-7 (FABP7) has been shown to be expressed in cutaneous melanoma; however, its role in tumor progression is unclear. Expression of FABP7 was assessed during melanoma progression through assessment of various clinicopathology stages of primary tumor progression and metastasis. FABP7 mRNA was highly expressed in 60 of 87 (69%) primary melanomas, compared with significant (P0.0001) reduction in 13 of 68 (19%) metastatic melanomas. Analysis of 37 paired primary and metastatic melanomas by immunohistochemistry with anti-FABP7 Ab showed 73 and 27% positivity, respectively (P0.001). FABP7 detection of metastatic tissues was inversely correlated with relapse-free (P0.0001) and overall (P0.0001) survival. To examine FABP7 expression loss in advanced melanomas, loss of heterozygosity (LOH) was assessed using microsatellite markers encompassing the FABP7 gene. LOH was identified in 10 of 20 (50%) metastatic melanomas at 6q22.31, compared with 0 of 14 primary melanomas (P0.0017). FABP7 as a surrogate biomarker for circulating tumor cells (CTCs) in the blood was assessed by quantitative real-time (qRT)-PCR from melanoma patients blood (n134). Assessment of patients blood showed that FABP7() CTC decreased with disease progression. FABP7 may function as a tumor progression gene and can be used as a potential diagnostic biomarker of early-stage melanoma systemic spreading in blood.

Chronic graft-versus-host disease (cGVHD) of the ocular surface and lacrimal gland is a vision-threatening condition that occurs after allogeneic bone marrow transplantation. In this study, we used immunohistochemistry and electron microscopy to investigate whether epithelial mesenchymal transition (EMT) contributed to the pathogenesis of ocular cGVHD. We detected down-regulation of E-cadherin and translocation of β-catenin from the intercellular junction to the cytoplasm and nucleus of cGVHD conjunctival basal epithelia and lacrimal gland myoepithelia. Notable findings included expression of Snail, an inducer of EMT, in the nucleus of ocular cGVHD epithelia. The fibrosis markers heat shock protein 47, α-smooth muscle actin, and fibroblast specific protein-1 were overexpressed in ocular cGVHD epithelia. In addition, p63, a marker of conjunctival basal epithelia, was observed in the nuclei of subconjunctival cells beneath disrupted basal lamina. Disrupted basal lamina and the presence of altered collagen bundles were observed in the cytoplasm and beneath cGVHD epithelia. In contrast, these observations were rarely observed in the normal conjunctiva and in Sjögren's syndrome lacrimal gland epithelia. These findings together indicate that ocular cGVHD epithelia gain the mesenchymal phenotype and the capacity to migrate into the subepithelial stroma. Our findings suggest that EMT may be partially responsible for the conjunctival and lacrimal gland fibrosis found in patients with cGVHD.

PURPOSE. CD44 is a cell-surface adhesion molecule and receptor for hyaluronan (HA), one of the major extracellular matrix components. The purpose of the present study was to clarify a role of HA and CD44 in the development of choroidal neovascularization (CNV). METHODS. Laser photocoagulation was used to induce CNV in C57BL/6 mice or CD44-deficient mice. The mRNA expression of CD44 and HA synthase (HAS)-2 in the retinal pigment epithelium (RPE)-choroid complex was evaluated by DNA microarray and real-time RT-PCR analyses 3 days after laser treatment. HA synthesis and CD44 expression were examined by immunohistochemistry 1 week after photocoagulation. Mice with laser-induced CNV were systemically administered the HA synthesis inhibitor 4-methylumbelliferone (MU) or an anti- CD44-neutralizing antibody. The response of CNV was analyzed by volumetric measurements 1 week after photocoagulation. Macrophage infiltration into CNV lesions was evaluated by real-time RT-PCR for F4/80 3 days after laser-induced injury. RESULTS. The induction of CNV led to a significant increase in expression of CD44 and HAS2 mRNA. HA and CD44 were immunopositive in the CNV lesions. Compared with vehicle treatment, the systemic application of MU significantly attenuated CNV volume in a dose-dependent fashion, together with macrophage infiltration into the lesions. Consistently, antibody- based blockade of CD44 resulted in a significant reduction of CNV volume, compared with the isotype control. In contrast, genetic ablation of CD44 significantly augmented CNV formation together with HA accumulation and macrophage infiltration, compared with wild-type mice. CONCLUSIONS. These results indicate a significant role of HA and its receptor CD44 in the development of CNV. (Invest Ophthalmol Vis Sci.

Microphthalmia-associated transcription factor (MITF) is a master gene regulating differentiation of melanocytes, and a lineage survival oncogene mediating pro-proliferative function in malignant melanoma. However, high expression of MITF also has an anti-proliferative effect. To clarify the therapeutic implication of MITF as a molecular target for human melanoma, we evaluated the role of MITF in cell proliferation in a panel of human melanoma cell lines which express different levels of MITF. We found that both MITF depletion and forced expression of MITF significantly inhibited proliferation, suggesting that endogenous MITF is regulated at an appropriate level for melanoma cell proliferation, and could be a molecular target for melanoma. However, half of the melanoma cell lines in this study were relatively resistant to MITF depletion, indicating other treatment strategies are required for therapy. Our microarray analysis indicated that regulation of several cell growth-associated molecules may be independent of MITF and dependent on BRAFV600E. Thus to enhance the anti-proliferative effect of MITF down-regulation, we combined shRNA-mediated MITF depletion with BRAFV600E inactivation, another known molecular target for melanoma. Indeed, simultaneous depletion of both MITF and BRAFV600E significantly inhibited melanoma growth even for the melanoma cell lines resistant to MITF depletion. These results suggest MITF may be an important molecular target for human melanoma and simultaneous inhibition of MITF and MAPK signaling may be an attractive strategy for melanoma treatment. (Cancer Sci 2009; 100: 1863-1869).

Purpose: Plasmacytoid dendritic cells (pDC) are specialized cells to produce type I IFN. Infiltration of pDCs in cancer tissues that have impaired ability to produce IFN-α has been suggested to play immunosuppressive roles in tumor immunity. To identify potential mechanisms causing pDC impairment in the cancer microenvironment, expression of immunoglobulin-like transcript 7 ligands (ILT7L), which inhibits pDC production of type I IFNs on the surface of various human cancer and noncancer cells, was examined. Experimental Design: To detect unidentified ILT7L, reporter cells, which express green fluorescent protein on interaction with ILT7L, were constructed. ILT7L expression on various human cancer cell lines as well as various noncancerous stromal cells and immune cells was examined. Cytokines and signals involved in the ILT7L expression were also investigated. Results: ILT7L was detected on all of the various types of human cancer cell lines tested. IFN-α, IFN-β, IFN-γ, tumor necrosis factor-α, interleukin-1β, transforming growth factor-β, lipopolysaccharide, and imiquimod induced ILT7L expression on cancer and noncancer cells. High ILT7L-expressing cancer cells inhibited production of IFN-α and tumor necrosis factor-α by pDC stimulated with CpG. ILT7L does not appear to be a member of classic or nonclassic HLAs. Additionally, NF-κB and mammalian target of rapamycin are involved in regulating ILT7L expression. Conclusions: ILT7L expression on cancer cells may be one of the mechanisms for impairment of pDCs in the cancer microenvironment. ILT7/ILT7L signaling may normally enable a negative immune response feedback following viral infection. Intervention of the ILT7L/ILT7 system may be useful for enhancing antitumor immunity as well as antiviral immunity.

Objective. To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods. An anti-CADM-140 antibody-positive prototype serum sample was used to screen a HeLa cell-derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro-transcribed and in vitro-translated products using anti-CADM-140 antibody-positive and anti-CADM-140 antibody-negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti-CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue Results. By cDNA library screening and immunoprecipitation of in vitro-transcribed and in vitro-translated products, we obtained a cDNA clone encoding melanoma differentiation-associated gene 5 (MDA-5). The anti-CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the "gold standard" immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion. Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti-CADM-140 antibody routinely available.

Supported by the Office of International Affairs, National Cancer Institute (NCI), the "US-Japan Workshop on Immunological Biomarkers in Oncology" was held in March 2009. The workshop was related to a task force launched by the International Society for the Biological Therapy of Cancer (iSBTc) and the United States Food and Drug Administration (FDA) to identify strategies for biomarker discovery and validation in the field of biotherapy. The effort will culminate on October 28th 2009 in the "iSBTc-FDA-NCI Workshop on Prognostic and Predictive Immunologic Biomarkers in Cancer", which will be held in Washington DC in association with the Annual Meeting. The purposes of the US-Japan workshop were a) to discuss novel approaches to enhance the discovery of predictive and/or prognostic markers in cancer immunotherapy; b) to define the state of the science in biomarker discovery and validation. The participation of Japanese and US scientists provided the opportunity to identify shared or discordant themes across the distinct immune genetic background and the diverse prevalence of disease between the two Nations. Converging concepts were identified: enhanced knowledge of interferon-related pathways was found to be central to the understanding of immune-mediated tissue-specific destruction (TSD) of which tumor rejection is a representative facet. Although the expression of interferon-stimulated genes (ISGs) likely mediates the inflammatory process leading to tumor rejection, it is insufficient by itself and the associated mechanisms need to be identified. It is likely that adaptive immune responses play a broader role in tumor rejection than those strictly related to their antigen-specificity; likely, their primary role is to trigger an acute and tissue-specific inflammatory response at the tumor site that leads to rejection upon recruitment of additional innate and adaptive immune mechanisms. Other candidate systemic and/or tissue-specific biomarkers were recognized that might be added to the list of known entities applicable in immunotherapy trials. The need for a systematic approach to biomarker discovery that takes advantage of powerful high-throughput technologies was recognized; it was clear from the current state of the science that immunotherapy is still in a discovery phase and only a few of the current biomarkers warrant extensive validation. It was, finally, clear that, while current technologies have almost limitless potential, inadequate study design, limited standardization and cross-validation among laboratories and suboptimal comparability of data remain major road blocks. The institution of an interactive consortium for high throughput molecular monitoring of clinical trials with voluntary participation might provide cost-effective solutions.

Previously, we demonstrated that implanted dendritic cells (DCs) in the injured spinal cord of adult mice exert a neurotrophic effect, resulting in the activation of endogenous neural stem/progenitor cells (NSPCs) and neurogenesis. Granulocyte-macrophage colony stimulating factor (GM-CSF), which is an essential cytokine for the generation of DCs from haematopoietic progenitor cells, has been shown to be beneficial for the treatment of spinal cord injury (SCI). In the present study, to evaluate the mechanisms underlying this therapeutic efficacy of GM-CSF, we investigated the effects of GM-CSF on the DC-like cells and NSPCs in the injured spinal cord. When GM-CSF was injected into the injured spinal cords of mice, the numbers of DC-like cells and activated microglia/macrophages around the lesion site increased, accompanied by an increase in BDNF expression. A significant increase in endogenous NSPCs was observed around the lesion site in the GM-CSF-treated mice compared with that in the controls. A neurosphere forming assay revealed that GM-CSF also induced the proliferation of NSPCs in vitro. Moreover, injection of GM-CSF into the lesion immediately after the SCI resulted in early recovery of the locomotor function of the injured mice. In conclusion, GM-CSF activated DC-like cells and NSPCs in the injured spinal cord, which was probably involved in its beneficial effects in cases of spinal cord injury.

To examine the role of genetic factors in development of immune thrombocytopenic purpura (ITP) in association with Helicobacter pylori infection, gene polymorphisms within the loci for human leukocyte antigen class II, interleukin (IL)-1β (-511), tumor necrosis factor-β (+252), immunoglobulin (Ig)G1 heavy chain (+643), and Igκ light chain (+573) were determined in 164 adults with ITP and 75 healthy controls. Of these gene polymorphisms, the IL-1β (-511) T allele was less frequently detected in H. pylori-infected than in H. pylori-uninfected (58% vs 81%, P = 0.01, odds ratio = 0.31) ITP patients diagnosed before age 50. These findings suggest that a single nucleotide polymorphism within the IL-1β (-511) may affect susceptibility to early-onset ITP associated with H. pylori infection.

Epithelial-mesenchymal transition (EMT) is a key step toward cancer metastasis, and Snail is a major transcription factor governing EMT. Here, we demonstrate that Snail-induced EMT accelerates cancer metastasis through not only enhanced invasion but also induction of immunosuppression. Murine and human melanoma cells with typical EMT features after snail transduction induced regulatory T cells and impaired dendritic cells in vitro and in vivo partly through TSP1 production. Although Snail+ melanoma did not respond to immunotherapy, intratumoral injection with snail-specific siRNA or anti-TSP1 monoclonal antibody significantly inhibited tumor growth and metastasis following increase of tumor-specific tumor-infiltrating lymphocytes and systemic immune responses. These results suggest that inhibition of Snail-induced EMT could simultaneously suppress both tumor metastasis and immunosuppression in cancer patients.

Human tumor antigens were identified using various immunological and genetic methods, and immune responses to the identified antigens were evaluated in cancer patients. Autologous tumor specific unique antigens derived from genetic alterations in cancer cells were isolated from patients with favorable prognosis after immunotherapy, indicating that they are attractive targets for immunotherapy. Immunogenicity of shared antigens was found to differ among patients due to antigen expression in cancer cells and patients' immunoreactivity. These observations suggest that personalization may be applied for cancer immunotherapy. We therefore developed intratumoral DC administration protocols that are able to induce immune responses to both unique and shared tumor antigens expressed in each individual cancer. By combining cryoablative tumor pretreatment and TLR stimulated DC, the anti-tumor effect of the intratumoral DC administration was significantly augmented in a murine tumor model. This improved protocol enhanced systemic induction of anti-tumor CD8 + CTL, and was able to regress relatively large remote untreated tumors. In clinical trials, systemic immune induction was observed by intraturmoral DC administration following cryoablative tumor treatment, although anti-tumor effects are relatively weak, indicating that additional interventions are required for more effective immunotherapy.

Background: Cationic liposomes containing the human interferon β (HuIFNβ) gene (IAB-1) was used for the clinical trial for glioma patients. HuIFNβ gene therapy showed much higher anti-tumor activity compared with the administration of HuIFNβ protein for melanoma. These results suggest that HuIFNβ gene therapy is an attractive strategy for the treatment of melanoma. Methods: Stage IV or III melanoma patients with cutaneous or subcutaneous metastatic lesions were enrolled in this pilot study. IAB-1 was dissolved by sterile PBS at a concentration of 30 μg DNA/ml and was injected into cutaneous or subcutaneous metastatic nodules three times a week for 2 weeks and the effect on the injected and non-injected metastatic lesions was evaluated. Results: Clinical responses were as follows (five patients): mixed response (MR) and no change in each one patient, and progressive disease in three patients. In the MR patient, the IAB-1 injected lesion disappeared clinically and histopathologically and one-half of IAB-1 non-injected skin metastases were transiently inflamed and mostly regressed. In the responded non-injected lesions of this patient, histopathologically, infiltration of CD4 positive T cells was observed around the melanoma cells in the dermis, which expressed the HLA-Class II antigen. Adverse events due to this gene therapy were not recognized in any of the patients. Conclusions: The efficacy of this gene therapy was generally insufficient; however, some immunological responses were recognized in one patient. No adverse events were observed. HuIFNβ gene therapy could be an attractive strategy for treatment of a variety of malignancies, including melanoma, though some modifications should be required.

The aim of this study was to evaluate the characteristics of autoreactive T cells to myeloperoxidase (MPO) in patients with MPO-antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Peripheral blood T cells from 15 patients with MPO-ANCA-associated vasculitis and 14 healthy individuals were cultured with three recombinant proteins that together comprised the entire MPO sequence (L, all 112 amino acids (AA) of the light chain; HI, AA 1-227 of the heavy chain; HII, AA 212-467 of the heavy chain), and the antigen-specific T-cell proliferative response was measured by 3H-thymidine incorporation. T-cell responses to MPO-L and HI were both detected in four patients and three healthy donors, and responses to MPO-HII were detected in four patients and seven healthy donors. These findings indicate that at least three independent T-cell epitopes exist on the MPO molecule. Interestingly, the patients whose T cells showed these MPO-induced responses were mainly in remission. Peripheral blood T cells reactive with MPO were primarily of the HLA-DR-restricted CD4+ phenotype. In summary, we successfully used recombinant MPO fragments to detect autoreactive CD4+ T cells to multiple MPO epitopes in blood samples from patients with MPO-ANCA-associated vasculitis and healthy individuals.