Details of a Researcher - Kawakami, Yutaka

Papers - Kawakami, Yutaka

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Emerging concepts in biomarker discovery; The US-Japan workshop on immunological molecular markers in oncology

Tahara Hideaki, Sato Marimo, Thurin Magdalena, Wang Ena, Butterfield Lisa H., Disis Mary L., Fox Bernard A., Lee Peter P., Khleif Samir N., Wigginton Jon M., Ambs Stefan, Akutsu Yasunori, Chaussabel Damien, Doki Yuichiro, Eremin Oleg, Fridman Wolf Herevé, Hirohashi Yoshihiko, Imai Kohzoh, Jacobson James, Jinushi Masahisa, Kanamoto Akira, Kashani-Sabet Mohammed, Kato Kazunori, Kawakami Yutaka, Kirkwood John M., Kleen Thomas O., Lehmann Paul V., Liotta Lance, Lotze Michael T., Maio Michele, Malyguine Anatoli, Masucci Giuseppe, Matsubara Hisahiro, Mayrand-Chung Shawmarie, Nakamura Kiminori, Nishikawa Hiroyoshi, Karolina A. Karolina, Petricoin Emanuel F., Pos Zoltan, Ribas Antoni, Rivoltini Licia, Sato Noriyuki, Shiku Hiroshi, Slingluff Craig L., Streicher Howard, Stroncek David F., Takeuchi Hiroya, Toyota Minoru, Wada Hisashi, Wu Xifeng, Wulfkuhle Julia, Yaguchi Tomonori, Zeskind Benjamin, Zhao Yingdong, Zocca Mai Britt, Marincola Francesco M.

Journal of Translational Medicine 7 2009.06

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.

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Activation of dendritic-like cells and neural stem/progenitor cells in injured spinal cord by GM-CSF

Hayashi Kaori, Ohta Shigeki, Kawakami Yutaka, Toda Masahiro

Neuroscience Research 64 ( 1 ) 96 - 103 2009.05

ISSN 0168-0102

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.

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Single nucleotide polymorphism of interleukin-1β associated with Helicobacter pylori infection in immune thrombocytopenic purpura

Satoh T., Pandey J. P., Okazaki Y., Asahi A., Kawakami Y., Ikeda Y., Kuwana M.

Tissue Antigens 73 ( 4 ) 353 - 357 2009.04

ISSN 0001-2815

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.

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Cancer Metastasis Is Accelerated through Immunosuppression during Snail-Induced EMT of Cancer Cells

Kudo-Saito Chie, Shirako Hiromi, Takeuchi Tadashi, Kawakami Yutaka

Cancer Cell 15 ( 3 ) 195 - 206 2009.03

ISSN 1535-6108

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.

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Epithelial mesenchymal transition in human ocular chronic graft-versus-host disease

Ogawa Yoko, Shimmura Shigeto, Kawakita Tetsuya, Yoshida Satoru, Kawakami Yutaka, Tsubota Kazuo

American Journal of Pathology 175 ( 6 ) 2372 - 2381 2009

ISSN 0002-9440

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.

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Involvement of hyaluronan and its receptor CD44 with choroidal neovascularization

Mochimaru Hiroshi, Takahashi Eri, Tsukamoto Nobuo, Miyazaki Junichiro, Yaguchi Tomonori, Koto Takashi, Kurihara Toshihide, Noda Kousuke, Ozawa Yoko, Ishimoto Takatsugu, Kawakami Yutaka, Tanihara Hidenobu, Saya Hideyuki, Ishida Susumu, Tsubota Kazuo

Investigative Ophthalmology and Visual Science 50 ( 9 ) 4410 - 4415 2009

ISSN 0146-0404

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.

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Identification of a WT1 protein-derived peptide, WT1₁₈₇, as a HLA-A*0206-restricted, WT1-specific CTL epitope

Li Zheyu, Oka Yoshihiro, Tsuboi Akihiro, Fujiki Fumihiro, Harada Yukie, Nakajima Hiroko, Masuda Tomoki, Fukuda Yoko, Kawakatsu Mai, Morimoto Soyoko, Katagiri Takamasa, Tatsumi Naoya, Hosen Naoki, Shirakata Toshiaki, Nishida Sumiyuki, Kawakami Yutaka, Udaka Keiko, Kawase Ichiro, Oji Yusuke, Sugiyama Haruo

Microbiology and Immunology 52 ( 11 ) 551 - 558 2008.11

ISSN 0385-5600

The Wilms' tumor gene WT1 is overexpressed in various kinds of hematopoietic malignancies as well as solid cancers, and this protein has been demonstrated to be an attractive target antigen for cancer immunotherapy. WT1-specific CTL epitopeswith a restriction of HLA-A*2402 or HLA-A*0201 have been already identified. In the present study it has been demonstrated that a 9-mer WT1-derived WT1₁₈₇ peptide, which had already been shown to elicit a WT1-specific CTL response with a restriction of HLA-A*0201, can also elicit a CTL response with a restriction of HLA-A*0206. In all three different HLA-A*0206⁺ healthy donors examined, WT1187 peptide-specific CTL could be generated from peripheral blood mononuclear cells, and the CTL showed cytotoxic activity that depended on dual expression of WT1 and HLA-A*0206 molecules. The present study describes the first identification of a HLA-A*0206-restricted, WT1-specific CTL epitope. The present results should help to broaden the application of WT1 peptide-based immunotherapy fromonly HLA-A*0201- positive to HLA-A*0206-positive cancer patients as well.

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Helicobacter pylori eradication shifts monocyte Fcγ receptor balance toward inhibitory FcγRIIB in immune thrombocytopenic purpura patients

Asahi Atsuko, Nishimoto Tetsuya, Okazaki Yuka, Suzuki Hidekazu, Masaoka Tatsuhiro, Kawakami Yutaka, Ikeda Yasuo, Kuwana Masataka

Journal of Clinical Investigation 118 ( 8 ) 2939 - 2949 2008.08

ISSN 0021-9738

Immune thrombocytopenia purpura (ITP) is a bleeding disorder in which platelet-specific autoantibodies cause a loss of platelets. In a subset of patients with ITP and infected with Helicobacter pylori, the number of platelets recovers after eradication of H. pylori. To examine the role of H. pylori infection in the pathogenesis of ITP, the response of 34 ITP patients to treatment with a standard H. pylori eradication regimen, irrespective of whether they were infected with H. pylori, was evaluated. Eradication of H. pylori was achieved in all H. pylori-positive patients, and a significant increase in platelets was observed in 61% of these patients. By contrast, none of the H. pylori-negative patients showed increased platelets. At baseline, monocytes from the H. pylori-positive patients exhibited an enhanced phagocytic capacity and low levels of the inhibitory Fcγ receptor IIB (FcγRIIB). One week after starting the H. pylori eradication regimen, this activated monocyte phenotype was suppressed and improvements in autoimmune and platelet kinetic parameters followed. Modulation of monocyte FcγR balance was also found in association with H. pylori infection in individuals who did not have ITP and in mice. Our findings strongly suggest that the recovery in platelet numbers observed in ITP patients after H. pylori eradication is mediated through a change in FcγR balance toward the inhibitory FcγRIIB.

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Novel system evaluating in vivo pathogenicity of desmoglein 3-reactive T cell clones using murine pemphigus vulgaris

Takahashi Hayato, Amagai Masayuki, Nishikawa Takeji, Fujii Yoshiko, Kawakami Yutaka, Kuwana Masataka

Journal of Immunology 181 ( 2 ) 1526 - 1535 2008.07

ISSN 0022-1767

Autoreactive T cells are thought to be involved in the pathogenesis of autoimmune diseases, but evidence for their direct pathogenicity is almost lacking. Herein we established a unique system for evaluating the in vivo pathogenicity of desmoglein 3 (Dsg3)-reactive T cells at a clonal level in a mouse model for pemphigus vulgaris (PV), an autoimmune blistering disease induced by anti-Dsg3 autoantibodies. Dsg3-reactive CD4⁺ T cell lines generated in vitro were adoptively transferred into Rag-2^-/- mice with primed B cells derived from Dsg3-immunized Dsg3^-/- mice. Seven of 20 T cell lines induced IgG anti-Dsg3 Ab production and acantholytic blister, a typical disease phenotype, in recipient mice. Comparison of the characteristics between pathogenic and nonpathogenic Dsg3-reactive T cell lines led to the identification of IL-4 and IL-10 as potential factors associated with pathogenicity. Further in vitro analysis showed that IL-4, but not IL-10, promoted IgG anti-Dsg3 Ab production by primed B cells. Additionally, adenoviral expression of soluble IL-4Rα in vivo suppressed IgG anti-Dsg3 Ab production and the PV phenotype, indicating a pathogenic role of IL-4. This strategy is useful for evaluating the effector function of autoreactive T cells involved in the pathogenesis of various autoimmune diseases.

Preferential activation of circulating CD8⁺ and γδ T cells in patients with active Behçet's disease and HLA-B51

Yasuoka Hidekata, Yamaguchi Yukie, Mizuki Nobuhisa, Nishida Tomomi, Kawakami Yutaka, Kuwana Masataka

Clinical and Experimental Rheumatology 26 ( 4 SUPPL. 50 ) 2008.07

ISSN 0392-856X

Objective. To evaluate the activation status of circulating CD4⁺, CD8⁺, and γδ T cells in patients with active and inactive Behçet's disease (BD). Methods. We studied 11 subjects with active BD, 28 with inactive BD, and 13 healthy controls. The expression of CD4, CD8, pan-γd, Vδ1, and Vδ2 along with the early activation marker CD69 was analyzed by 3-color flow cytometry. Results. Proportions of activated CD8⁺ and γδ T cells were significantly greater in patients with active BD than in those with inactive BD or healthy control subjects, but the proportion of activated CD4⁺ T cells did not differ among these 3 groups. In addition, significantly greater proportions of the Vδ1⁺ and Vδ2⁺ γδ T-cell subsets were activated in patients with active BD than in those with inactive BD or healthy controls; in active BD, the balance of activation between these subsets favored the Vδ1⁺ T cells. No significant differences in these proportions were found between subjects with inactive BD and healthy controls. These findings were observed exclusively in patients with HLA-B51. A comparison of samples from 5 patients taken during active BD and after resolution of BD-related symptoms showed the proportions of activated CD8⁺ and γδ T cells dropped when the patients' BD became inactive. Conclusion. CD8⁺ and γδ T cells, rather than CD4⁺ T cells, were activated in vivo in patients with active BD and HLA-B51, but not in those with inactive BD, suggesting that these potentially cytotoxic T cells play a critical role in BD flares.

Functional recovery after spinal cord injury in mice through activation of microglia and dendritic cells after IL-12 administration

Yaguchi Masae, Ohta Shigeki, Toyama Yoshiaki, Kawakami Yutaka, Toda Masahiro

Journal of Neuroscience Research 86 ( 9 ) 1972 - 1980 2008.07

ISSN 0360-4012

We have previously reported that the transplantation of dendritic cells (DCs) brings about functional recovery after spinal cord injury in mice through the activation of endogenous microglia/macrophages and neural stem/progenitor cells. In this study, the effect of interleukin-12 (IL-12), which is secreted from DCs, was evaluated for the treatment of spinal cord injury in mice. Administration of IL-12 into the injured site significantly increased the number of activated microglia/macrophages and DCs as well as the expression of brain-derived neurotrophic factor surrounding the lesion site. Immunohistochemical analyses showed that de novo neurogenesis and remyelination were induced by IL-12 treatment. Furthermore, an open field test using Basso-Beattie-Brenham scoring revealed a significant improvement of locomotor function in mice treated with IL-12. These results suggest that IL-12 administration into the injured spinal cord results in a functional recovery through the activation of microglia/macrophages and DCs.

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Induction of protective and therapeutic antitumor immunity by a DNA vaccine with a glioma antigen, SOX6

Ueda Ryo, Kinoshita Eiko, Ito Rena, Kawase Takeshi, Kawakami Yutaka, Toda Masahiro

International Journal of Cancer 122 ( 10 ) 2274 - 2279 2008.05

ISSN 0020-7136

We previously reported identifying SOX6 as a glioma antigen by serological screening using a testis cDNA library. Its preferential expression and frequent IgG responses in glioma patients indicate that SOX6 may be a useful target for immunotherapy. To examine whether cytotoxic T-lymphocyte (CTL) responses specific for SOX6 to destroy glioma can be generated in vivo, we treated glioma-bearing mice by vaccination with a plasmid DNA encoding murine full-length SOX6 protein. Following SOX6-DNA vaccination, CTLs specific for SOX6-expressing glioma cells were induced, while normal autologous-cells that had restrictedly expressed SOX6 during embryogenesis were not destroyed. Furthermore, DNA vaccination with SOX6 exerted protective and therapeutic antitumor responses in the glioma-bearing mice. This antitumor activity was abrogated by the depletion of CD4 positive T cells and/or CD8 positive T cells. These results suggest that the SOX6 protein has multiple CTL and helper epitopes to induce antitumor activity and the effectiveness of SOX6-DNA vaccine for the prevention and treatment of glioma.

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Isolation and characterization of dendritic cells from common marmosets for preclinical cell therapy studies

Ohta Shigeki, Ueda Yoko, Yaguchi Masae, Matsuzaki Yumi, Nakamura Masaya, Toyama Yoshiaki, Tanioka Yoshikuni, Tamaoki Norikazu, Nomura Tatsuji, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

Immunology 123 ( 4 ) 566 - 574 2008.04

ISSN 0019-2805

Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC-associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)-DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen-presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM-derived CD11c⁺ cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)-12, and to stimulate Xenogenic T cells. Moreover, BM-derived DCs produced the neurotrophic factor NT-3, which is also found in murine splenic DCs. These results suggest that BM-derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer.

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Suppression of alkali burn-induced corneal neovascularization by dendritic cell vaccination targeting VEGF receptor 2

Mochimaru Hiroshi, Usui Tomohiko, Yaguchi Tomonori, Nagahama Yasuharu, Hasegawa Go, Usui Yoshihiko, Shimmura Shigeto, Tsubota Kazuo, Amano Shiro, Kawakami Yutaka, Ishida Susumu

Investigative Ophthalmology and Visual Science 49 ( 5 ) 2172 - 2177 2008.04

ISSN 0146-0404

Purpose. To investigate whether the induction of cytotoxic T lymphocytes (CTLs) targeting VEGF receptor 2 inhibits corneal neovascularization caused by alkali injury. Methods. H-2Db-restricted peptide corresponding to amino acids 400 to 408 of VEGF receptor 2 (VEGFR2_400-408) was used as an epitope peptide. Dendritic cells (DCs) were harvested from bone marrow progenitors of C57BL/6 mice. Six- week-old C57BL/6 mice received subcutaneous injections of VEGFR2_400-408- or gp70-pulsed mature DCs three times at 6-day intervals. After the third immunization, corneal neovas- cularization was induced by alkali injury. Two weeks after the injury, the corneal vascularized area was evaluated by lectin angiography. To confirm the peptide-specific CTL activities in C57BL/6 mice, CD8⁺ T cells from immunized mice were subjected to ELISA for interferon (IFN)-γ and tumor necrosis factor (TNF)-α production and ⁵¹Cr-release cytotoxicity assay. To determine the in vivo effector T cells, the immunized mice were intraperitoneally injected with an anti-CD4 or -CD8 depletion antibody. Results. Corneal neovascularization was significantly attenuated in mice immunized with VEGFR2_400-408 compared with those not immunized or immunized with gp70. VEGFR2_400-408 or gp70, but not β-gal_96-103 application led to dose-dependent induction of IFN-γ and TNF-α in the CD8⁺ T cells cocultured with stimulator cells. Cytotoxicity assays showed the specific lysis of major histocompatibility complex-matched cells expressing VEGFR2, but not β-gal_96-103. In vivo depletion of CD8⁺, but not CD4⁺, T cells significantly reversed the suppressive effect of VEGFR2_400-408 immunization on corneal neovascularization to the level observed in nonimmunized or gp70-immunized animals. Conclusions. These results indicate the possibility of DC vaccination targeting VEGFR2 as a novel therapeutic strategy for corneal chemical injury.

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Dendritic cell based personalized immunotherapy based on cancer antigen research

Kawakami Yutaka, Fujita Tomonobu, Kudo Chie, Sakurai Toshiharu, Udagawa Masaru, Yaguchi Tomonori, Hasegawa Go, Hayashi Emiko, Ueda Yoko, Iwata Tomoko, Wang Qinfu, Okada Starlyn, Tsukamoto Nobuo, Matsuzaki Yuriko, Sumimoto Hidetoshi

Frontiers in Bioscience 13 ( 5 ) 1952 - 1958 2008

ISSN 1093-9946

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.

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Autoreactive T-cell responses to myeloperoxidase in patients with antineutrophil cytoplasmic antibody-associated vasculitis and in healthy individuals

Seta Noriyuki, Tajima Michiko, Kobayashi Shigeto, Kawakami Yutaka, Hashimoto Hiroshi, Kuwana Masataka

Modern Rheumatology 18 ( 6 ) 593 - 600 2008

ISSN 1439-7595

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.

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A pilot study of human interferon β gene therapy for patients with advanced melanoma by in vivo transduction using cationic liposomes

Matsumoto Kazuhiko, Kubo Hitomi, Murata Hiroshi, Uhara Hisashi, Takata Minoru, Shibata Shinichi, Yasue Satoshi, Sakakibara Akihiro, Tomita Yasushi, Kageshita Toshiro, Kawakami Yutaka, Mizuno Masaaki, Yoshida Jun, Saida Toshiaki

Japanese Journal of Clinical Oncology 38 ( 12 ) 849 - 856 2008

ISSN 0368-2811

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.

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Identification of a neuron-specific human gene, KIAA1110, that is a guanine nucleotide exchange factor for ARF1

Hattori Yujiro, Ohta Shigeki, Hamada Kenji, Yamada-Okabe Hisafumi, Kanemura Yonehiro, Matsuzaki Yumi, Okano Hideyuki, Kawakami Yutaka, Toda Masahiro

Biochemical and Biophysical Research Communications 364 ( 4 ) 737 - 742 2007.12

ISSN 0006-291X

To identify neuron-specific genes, we performed gene expression profiling, cDNA microarray and in silico ESTs (expressed sequence tags) analyses. We identified a human neuron-specific gene, KIAA1110 (homologue of rat synArfGEF (Po)), that is a member of the guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF). RT-PCR analysis showed that the KIAA1110 gene was expressed specifically in the brain among adult human tissues, whereas no apparent expression was observed in immature neural tissues/cells, such as fetal brain, glioma tissues/cells, and neural stem/precursor cells (NSPCs). The KIAA1110 protein was shown to be expressed in mature neurons but not in undifferentiated NSPCs. Immunohistochemical analysis also showed that KIAA1110 was expressed in neurons of the human adult cerebral cortex. Furthermore, the pull-down assay revealed that KIAA1110 has a GEF activity toward ARF1 that regulates transport along the secretion pathway. These results suggest that KIAA1110 is expressed specifically in mature neurons and may play an important role in the secretion pathway as a GEF for ARF1.

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Lentiviral vector-mediated RNAi and its use for cancer research

Sumimoto Hidetoshi, Kawakami Yutaka

Future Oncology 3 ( 6 ) 655 - 664 2007.12

ISSN 1479-6694

RNAi is a useful tool for functional analysis of genes and developing a potential therapeutic strategy for various diseases including cancers. RNAican be applied in various forms. HIV vectors are useful for the stable transduction of genes to both replicating and quiescent cells with a broad host tropism, and have been developed for basic and clinical research of gene therapy. HIV vectors can deliver shRNAs for post-transcriptional silencing of specific genes with high efficiency, and have been used to evaluate various genes far their potential involvement in cancer development and malignant features, and may be useful for future cancer gene therapy. Here we describe the development of shRNA-expressing HIV vectors and their use in cancer research, as well as perspectives for their future use in cancer gene therapy.

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Dendritic cells transduced with autoantigen FCRLA induce cytotoxic lymphocytes and vaccinate against murine B-cell lymphoma

Inozume Takashi, Mitsui Hiroshi, Okamoto Takashi, Matsuzaki Yuriko, Kawakami Yutaka, Shibagaki Naotaka, Shimada Shinji

Journal of Investigative Dermatology 127 ( 12 ) 2818 - 2822 2007.12

ISSN 0022-202X

We previously reported early evidence of the human Fc receptor-like A (hFCRLA), an antigen (Ag) that was specifically expressed in melanocytes, melanoma cells, and some B-cell states, and was recognized by IgG antibodies from melanoma patients. Recently, it has been demonstrated that hFCRLA is expressed in most human B-cell lymphoma tissues. In this report, we investigated the potential of FCRLA as a tumor-associated Ag of B-cell lymphoma for immunotherapy. We confirmed that murine FCRLA (mFCRLA) was expressed and distributed in murine tissues similar to hFCRLA. Recombinant mFCRLA fusion protein was constructed with a polyarginine (R9)-protein-transduction domain (PTD) (rR9-HA-mFCRL), and was transduced into bone marrow-derived dendritic cells (DC) ex vivo. Mice immunized with rR9-HA-mFCRL-treated DC primed cytotoxic T-lymphocyte (CTL) that killed the B-cell lymphoma cell line (A20), which express mFCRLA abundantly. In a tumor challenging study, A20 tumor growth inoculated in skin was significantly suppressed in mice vaccinated with rR9-HA-mFCRL-treated DC, compared with control mice. These results indicated that FCRLA is a potential target Ag in immunotherapy for B-cell lymphoma. In addition, our experimental system using R9-PTD-containing full-length proteins might be a useful method to analyze the immunogenicity of novel candidates of tumor-associated Ags in vivo.

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