Hidetake Matsuyoshi

Prevention of Experimental Autoimmune Encephalomyelitis by Transfer of Embryonic Stem Cell-Derived Dendritic Cells Expressing Myelin Oligodendrocyte Glycoprotein Peptide along with TRAIL or Programmed Death-1 Ligand

Experimental autoimmune encephalomyelitis (EAE) is caused by activation of myelin Ag-reactive CD4+ T cells. In the current study, we tested a strategy to prevent EAE by pretreatment of mice with genetically modified dendritic cells (DC) presenting myelin oligodendrocyte glycoprotein (MOG) peptide in the context of MHC class II molecules and simultaneously expressing TRAIL or Programmed Death-1 ligand (PD-L1). For genetic modification of DC, we used a recently established method to generate DC from mouse embryonic stem cells (ES cells) in vitro (ES-DC). ES cells were sequentially transfected with an expression vector for TRAIL or PD-L1 and an MHC class II-associated invariant chain-based MOG epitope-presenting vector. Subsequently, double-transfectant ES cell clones were induced to differentiate to ES-DC, which expressed the products of introduced genes. Treatment of mice with either of the double-transfectant ES-DC significantly reduced T cell response to MOG, cell infiltration into spinal cord, and the severity of MOG peptide-induced EAE. In contrast, treatment with ES-DC expressing MOG alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus PD-L1, or coinjection with ES-DC expressing MOG plus ES-DC-expressing TRAIL or PD-L1 had no effect in reducing the disease severity. In contrast, immune response to irrelevant exogenous Ag (keyhole limpet hemocyanin) was not impaired by treatment with any of the genetically modified ES-DC. The double-transfectant ES-DC presenting Ag and simultaneously expressing immune-suppressive molecules may well prove to be an effective therapy for autoimmune diseases without inhibition of the immune response to irrelevant Ag.

Enhanced Priming of Antigen-Specific CTLs In Vivo by Embryonic Stem Cell-Derived Dendritic Cells Expressing Chemokine Along with Antigenic Protein: Application to Antitumor Vaccination

Dendritic cell (DC)-based immunotherapy is regarded as a promising means for anti-cancer therapy. The efficiency of T cell-priming in vivo by transferred DCs should depend on their encounter with T cells. In the present study, we attempted to improve the capacity of DCs to prime T cells in vivo by genetic modification to express chemokine with a T cell-attracting property. For genetic modification of DCs, we used a recently established method to generate DCs from mouse embryonic stem cells. We generated double-transfectant DCs expressing a chemokine along with a model Ag (OVA) by sequential transfection of embryonic stem cells, and then induced differentiation to DCs. We comparatively evaluated the effect of three kinds of chemokines; secondary lymphoid tissue chemokine (SLC), monokine induced by IFN-γ (Mig), and lymphotactin (Lptn). All three types of double transfectant DCs primed OVA-specific CTLs in vivo more efficiently than did DCs expressing only OVA, and the coexpression of SLC or Lptn was more effective than that of Mig. Immunization with DCs expressing OVA plus SLC or Mig provided protection from OVA-expressing tumor cells more potently than did immunization with OVA alone, and SLC was more effective than Mig. In contrast, coexpression of Lptn gave no additive effect on protection from the tumor. Collectively, among the three chemokines, expression of SLC was the most effective in enhancing antitumor immunity by transferred DCs in vivo. The findings provide useful information for the development of a potent DC-based cellular immunotherapy.

Embryonic Stem Cell–Derived Dendritic Cells Expressing Glypican-3, a Recently Identified Oncofetal Antigen, Induce Protective Immunity against Highly Metastatic Mouse Melanoma, B16-F10

We have recently established a method to generate dendritic cells from mouse embryonic stem cells. By introducing exogenous genes into embryonic stem cells and subsequently inducing differentiation to dendritic cells (ES-DC), we can now readily generate transfectant ES-DC expressing the transgenes. A previous study revealed that the transfer of genetically modified ES-DC expressing a model antigen, ovalbumin, protected the recipient mice from a challenge with an ovalbumin-expressing tumor. In the present study, we examined the capacity of ES-DC expressing mouse homologue of human glypican-3, a recently identified oncofetal antigen expressed in human melanoma and hepatocellular carcinoma, to elicit protective immunity against glypican-3-expressing mouse tumors. CTLs specific to multiple glypican-3 epitopes were primed by the in vivo transfer of glypican-3-transfectant ES-DC (ES-DC-GPC3). The transfer of ES-DC-GPC3 protected the recipient mice from subsequent challenge with B16-F10 melanoma, naturally expressing glypican-3, and with glypican-3-transfectant MCA205 sarcoma. The treatment with ES-DC-GPC3 was also highly effective against i.v. injected B16-F10. No harmful side effects, such as autoimmunity, were observed for these treatments. The depletion experiments and immunohistochemical analyses suggest that both CD8+ and CD4+ T cells contributed to the observed antitumor effect. In conclusion, the usefulness of glypican-3 as a target antigen for antimelanoma immunotherapy was thus shown in the mouse model using the ES-DC system. Human dendritic cells expressing glypican-3 would be a promising means for therapy of melanoma and hepatocellular carcinoma.

Proliferation Potential-Related Protein, an Ideal Esophageal Cancer Antigen for Immunotherapy, Identified Using Complementary DNA Microarray Analysis

Purpose: To establish effective antitumor immunotherapy for esophageal cancer, we tried to identify an useful target antigen of esophageal cancer. Experimental Design: We did cDNA microarray analysis to find a novel candidate antigen, proliferation potential-related protein (PP-RP). We examined cytotoxicity against tumor cells in vitro and in vivo of CTLs specific to PP-RP established from esophageal cancer patients. Results: In 26 esophageal cancer tissues, an average of relative ratio of the expression of the PP-RP mRNA in cancer cells versus adjacent normal esophageal tissues was 396.2. Immunohistochemical analysis revealed that, in 20 of the 22 esophageal cancer tissues, PP-RP protein was strongly expressed only in the cancer cells and not so in normal esophageal epithelial cells. PP-RP protein contains 10 epitopes recognized by HLA-A24–restricted CTLs. These CTLs, generated from HLA-A24–positive esophageal cancer patients, had cytotoxic activity against cancer cell lines positive for both PP-RP and HLA-A24. Furthermore, adoptive transfer of the PP-RP–specific CTL line inhibited the growth of a human esophageal cancer cell line engrafted in nude mice. Conclusions: The expression of PP-RP in esophageal cancer cells was significantly higher than in normal cells, and the CTLs recognizing PP-RP killed tumor cells in vitro and also showed tumor rejection effects in a xenograft model. Therefore, PP-RP may prove to be an ideal tumor antigen useful for diagnosis and immunotherapy for patients with esophageal cancer. cDNA microarray analysis is a useful method to identify ideal tumor-associated antigens.

Genetically Manipulated Human Embryonic Stem Cell‐Derived Dendritic Cells with Immune Regulatory Function

Genetically manipulated dendritic cells (DC) are considered to be a promising means for antigen‐specific immune therapy. This study reports the generation, characterization, and genetic modification of DC derived from human embryonic stem (ES) cells. The human ES cell‐derived DC (ES‐DC) expressed surface molecules typically expressed by DC and had the capacities to stimulate allogeneic T lymphocytes and to process and present protein antigen in the context of histocompatibility leukocyte antigen (HLA) class II molecule. Genetic modification of human ES‐DC can be accomplished without the use of viral vectors, by the introduction of expression vector plasmids into undifferentiated ES cells by electroporation and subsequent induction of differentiation of the transfectant ES cell clones to ES‐DC. ES‐DC introduced with invariant chain‐based antigen‐presenting vectors by this procedure stimulated HLA‐DR‐restricted antigen‐specific T cells in the absence of exogenous antigen. Forced expression of programmed death‐1‐ligand‐1 in ES‐DC resulted in the reduction of the proliferative response of allogeneic T cells cocultured with the ES‐DC. Generation and genetic modification of ES‐DC from nonhuman primate (cynomolgus monkey) ES cells was also achieved by the currently established method. ES‐DC technology is therefore considered to be a novel means for immune therapy.

Involvement of regulatory T cells in the experimental autoimmune encephalomyelitis-preventive effect of dendritic cells expressing myelin oligodendrocyte glycoprotein plus TRAIL.

We previously reported the protection from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by the adoptive transfer of genetically modified embryonic stem cell-derived dendritic cells (ES-DC) presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing TRAIL (ES-DC-TRAIL/MOG). In the present study, we found the severity of EAE induced by another myelin autoantigen, myelin basic protein, was also decreased after treatment with ES-DC-TRAIL/MOG. This preventive effect diminished, if the function of CD4+CD25+ regulatory T cells (Treg) was abrogated by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. The adoptive transfer of CD4+CD25+ T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient mice from MOG- or myelin basic protein-induced EAE. The number of Foxp3+ cells increased in the spinal cords of mice treated with ES-DC-TRAIL/MOG. In vitro experiments showed that TRAIL expressed in genetically modified ES-DC and also in LPS-stimulated splenic macrophages had a capacity to augment the proliferation of CD4+CD25+ T cells. These results suggest that the prevention of EAE by treatment with ES-DC-TRAIL/MOG is mediated, at least in part, by MOG-reactive CD4+CD25+ Treg propagated by ES-DC-TRAIL/MOG. For the treatment of organ-specific autoimmune diseases, induction of Treg reactive to the organ-specific autoantigens by the transfer of DC-presenting Ags and simultaneously overexpressing TRAIL therefore appears to be a promising strategy.

Pluripotent stem cells as source of dendritic cells for immune therapy

Dendritic cells (DC) are the most potent antigen-presenting cells. In vivo transfer of antigen-bearing DC has proven efficient in priming T cell responses specific to the antigen. DC-based cellular vaccination is now regarded as a powerful means for immunotherapy, especially for anti-cancer immunotherapy. Clinical trials of therapy with DC pulsed with peptide antigens or genetically modified to present antigens are currently carried out in many institutions. In addition, antigen-specific negative regulation of immune response by DC is considered to be a promising approach for treatments of autoimmune diseases and also for regulation of allo-reactive immune response causing graft rejection and GVHD in transplantation medicine. DC for transfer therapy are now generated by in vitro differentiation of peripheral blood monocytes of the patients. However, there is a limitation in the number of available monocytes, and the DC-differentiation potential of monocytes varies depending on the blood donor. Embryonic stem (ES) cells possess both pluripotency and infinite propagation capacity. We consider ES cells to be an ideal source for DC to be used in immunotherapy. Several groups, including us, have developed methods to generate DC from ES cells. This review introduces the studies on generation, characterization, and genetic modification of DC derived from ES cells or induced pluripotent stem (iPS) cells. The issues to be resolved before clinical application of pluripotent stem cell-derived DC will also be discussed.

Therapeutic effect of α‐galactosylceramide‐loaded dendritic cells genetically engineered to express SLC/CCL21 along with tumor antigen against peritoneally disseminated tumor cells

The close cooperation of both innate and acquired immunity is essential for the induction of truly effective antitumor immunity. We tested a strategy to enhance the cross‐talk between NKT cells and conventional antigen‐specific T cells with the use of αGalCer‐loaded dendritic cells genetically engineered to express antigen plus chemokine, attracting both conventional T cells and NKT cells. DC genetically engineered to express a model antigen, OVA, along with SLC/CCL21 or monokine induced by IFN‐γ/CXCL9, had been generated using a method based on in vitro differentiation of DC from mouse ES cells. The ES‐DC were loaded with α‐GalCer and transferred to mice bearing MO4, an OVA‐expressing melanoma, and their capacity to evoke antitumor immunity was evaluated. In vivo transfer of either OVA‐expressing ES‐DC, stimulating OVA‐reactive T cells, or α‐GalCer‐loaded non‐transfectant ES‐DC, stimulating NKT cells, elicited a significant but limited degree of protection against the i.p. disseminated MO4. A more potent antitumor effect was observed when α‐GalCer was loaded to ES‐DC expressing OVA before in vivo transfer, and the effect was abrogated by the administration of anti‐CD8, anti‐NK1.1 or anti‐asialo GM1 antibody. α‐GalCer‐loaded double transfectant ES‐DC expressing SLC along with OVA induced the most potent antitumor immunity. Thus, α‐GalCer‐loaded ES‐DC expressing tumor‐associated antigen along with SLC can stimulate multiple subsets of effector cells to induce a potent therapeutic effect against peritoneally disseminated tumor cells. The present study suggests a novel way to use α‐GalCer in immunotherapy for peritoneally disseminated cancer. (Cancer Sci 2005; 96: 889–896)

Immunotherapy with pluripotent stem cell-derived dendritic cells

In vivo transfer of dendritic cells (DC) has proven efficient in the priming of T cells and is regarded as a powerful means of providing anti-cancer immunotherapy. Clinical trials of anti-cancer therapy with DC pulsed with peptide antigens have been carried out in many institutions, although dramatic therapeutic effect has not been observed in most of the trials. Negative regulation of the immune response by DC might be applicable to treatment of autoimmune diseases and transplantation medicine. Currently, the DC used for anti-cancer vaccine therapy are generated from the peripheral blood monocytes of the patients. However, there is a limitation in the number of available monocytes and the potential of monocytes to differentiate into DC varies depending on the individual blood donors. To resolve the issue of the cell source for DC therapy, several groups have developed methods to generate DC from pluripotent stem cells. This review introduces methods to generate functional DC from pluripotent stem cells of mouse and human.

Examination of the effectiveness of the Active Head Shaking Nystagmus Test for the diagnosis of vestibular neuritis

実際の臨床の現場において,急性めまい症例に 対し初診時に温度刺激検査を施行することの有効 性をすでに我々は報告したが,時間的にもマン パワーの上でも検査が困難な場合もあると考えら れる。一方,頭振後検査(Head Shaking Nystagmus Test:HSNT)は簡便に一側性内耳障害を評 価する上で有効な検査である。しかし高齢化社 会において他動的に HSNTを行うことは,頸椎 損傷など予想外の医原性合併症や医事紛争を引き 起こす可能性が考えられる。このため,患者自身 によって頭振りを施行してもらう能動的 HNST (Active Head Shaking Nystagmus Test:AHSNT) を温度刺激検査施行時に併せて行った。温度刺激 検査の結果と,AHSNTの結果を前庭神経炎 (Vestibular Neuritis:VN)と後述する突発性め まいとで比較検討し,VNの診断における AHSN の有効性について評価した。 The Head Shaking Nystagmus Test (HSNT) is an effective test for simply evaluating unilateral inner ear disorders. However, performing the HSNT passively in elderly patients can cause unexpected iatrogenic complications. For this reason, the present study compared the Active Head Shaking Nystagmus Test (AHSNT), which was performed by the patients themselves, with the air caloric test. The two tests were performed at the same visit, and the results were compared between patients diagnosed as having vestibular neuritis (VN) and those diagnosed as having sudden vertigo. The effectiveness of AHSNT for the diagnosis of VN was then examined. The Active Head Shaking Nystagmus (AHSN) score was significantly higher in the VN group than in the sudden vertigo group among the acute vertigo cases. No complications arising from the AHSNT were seen. Consequently, the test was considered to be an effective and safe means of easily differentiating VN. 原 著