[show abstract][hide abstract] ABSTRACT: Resistance to 5-fluorouracil (5-FU) is a major obstacle in treating oral squamous cell carcinoma (OSCC). However, little is known about apoptosis resistance, which contributes to 5-FU resistance in OSCC.
We focussed on the cellular inhibitor of apoptosis protein 2 (cIAP2) on the basis of a DNA microarray data using parental and 5-FU-resistant OSCC cell lines. The effects of cIAP2 downregulation on 5-FU sensitivity and apoptosis were evaluated. An immunohistochemical analysis of cIAP2 and related proteins, cIAP1 and X-linked IAP, was performed in 54 OSCC patients who were treated with 5-FU-based chemoradiotherapy and surgery.
The downregulation of cIAP2 significantly enhanced the sensitivity of the 5-FU-resistant cells to 5-FU, with a significant increase in apoptosis. The immunohistochemical analysis demonstrated a high cIAP2 tumour expression to significantly correlate with the pathological response to chemoradiotherapy. Furthermore, a Cox regression analysis revealed the cIAP2 expression status (hazard ratio, 4.91; P=0.037) and the pathological response to chemoradiotherapy (hazard ratio, 0.418; P=0.016) to be significant prognostic factors for OSCC patients.
These novel findings demonstrate that cIAP2 may represent a potentially useful therapeutic target for improving the treatment and survival of OSCC patients, particularly in the setting of 5-FU resistance.
British Journal of Cancer 09/2011; 105(9):1322-30. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Seminars in Immunopathology 04/2011; 33(6):603-12. · 5.38 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
International journal of hematology 02/2010; 91(3):392-400. · 1.17 Impact Factor
[show abstract][hide abstract] ABSTRACT: Serum amyloid A (SAA) is an acute-phase reactant, the blood level of which is often elevated in response to some types of neoplasia. Here, we investigated expression of the gene SAA1 and the protein SAA in head and neck squamous cell carcinoma (HNSCC) and normal oral mucosal tissues as well as blood SAA levels in HNSCC patients. Also, we investigated the effects of inhibiting signal transducer and activator of transcription 3 (STAT3) signaling on SAA1 expression in the HNSCC cell line SAS. Serum SAA levels in HNSCC patients were significantly higher than those in healthy volunteers. In addition, real-time quantitative reverse transcription-polymerase chain reaction analysis revealed a significantly higher SAA1 expression level in HNSCC than in normal mucosa (P < 0.0001). Furthermore, Western blot and immunohistochemical analyzes revealed that high expression of SAA in carcinomas was detected predominantly in tumor cells, but not in normal mucosal tissues. An inhibitor of STAT3 activation, AG490, significantly reduced SAA1 expression in SAS cells. These data demonstrated that SAA was up-regulated in HNSCC through the Janus kinase-STAT3 pathway.
Journal of Oral Pathology and Medicine 05/2009; 39(1):41-7. · 2.06 Impact Factor
[show abstract][hide abstract] ABSTRACT: Numerous tumor-associated antigens (TAA) have been identified and their use in immunotherapy is considered to be promising. For TAA-based immunotherapy to be broadly applied as standard anticancer medicine, methods for active immunization should be improved. In the present study, we demonstrated the efficacy of multiple TAA-targeted dendritic cell (DC) vaccines and also the additive effects of loading alpha-galactosylceramide to DC using mouse melanoma models. On the basis of previously established methods to generate DC from mouse embryonic stem cells (ES-DC), 4 kinds of genetically modified ES-DC, which expressed the melanoma-associated antigens, glypican-3, secreted protein acidic and rich in cysteine, tyrosinase-related protein-2, or gp100 were generated. Anticancer effects elicited by immunization with the ES-DC were assessed in preventive and also therapeutic settings in the models of peritoneal dissemination and spontaneous metastasis to lymph node and lung. The in vivo transfer of a mixture of 3 kinds of TAA-expressing ES-DC protected the recipient mice from melanoma cells more effectively than the transfer of ES-DC expressing single TAA, thus demonstrating the advantage of multiple as compared with single TAA-targeted immunotherapy. Loading ES-DC with alpha-galactosylceramide further enhanced the anticancer effects, suggesting that excellent synergic effects of TAA-specific cytotoxic T lymphocytes and natural killer T cells against metastatic melanoma can be achieved by using genetically modified ES-DC. With the aid of advancing technologies related to pluripotent stem cells, induced pluripotent stem cells, and ES cells, clinical application of DC highly potent in eliciting anticancer immunity will be realized in the near future.
[show abstract][hide abstract] ABSTRACT: 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.
The Journal of Immunology 12/2008; 181(9):6635-43. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: 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.
The Journal of Immunology 02/2007; 178(2):918-25. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: We previously reported that glypican-3 (GPC3) was overexpressed, specifically in hepatocellular carcinoma (HCC) and melanoma in humans, and it was useful as a novel tumor marker. We also reported that the preimmunization of BALB/c mice with dendritic cells pulsed with the H-2K(d)-restricted mouse GPC3(298-306) (EYILSLEEL) peptide prevented the growth of tumor-expressing mouse GPC3. Because of similarities in the peptide binding motifs between H-2K(d) and HLA-A24 (A*2402), the GPC3(298-306) peptide therefore seemed to be useful for the immunotherapy of HLA-A24+ patients with HCC and melanoma. In this report, we investigated whether the GPC3(298-306) peptide could induce GPC3-reactive CTLs from the peripheral blood mononuclear cells (PBMC) of HLA-A24 (A*2402)+ HCC patients. In addition, we used HLA-A2.1 (HHD) transgenic mice to identify the HLA-A2 (A*0201)-restricted GPC3 epitopes to expand the applications of GPC3-based immunotherapy to the HLA-A2+ HCC patients.
We found that the GPC3(144-152) (FVGEFFTDV) peptide could induce peptide-reactive CTLs in HLA-A2.1 (HHD) transgenic mice without inducing autoimmunity. In five out of eight HLA-A2+ GPC3+ HCC patients, the GPC3(144-152) peptide-reactive CTLs were generated from PBMCs by in vitro stimulation with the peptide and the GPC3(298-306) peptide-reactive CTLs were also generated from PBMCs in four of six HLA-A24+ GPC3+ HCC patients. The inoculation of these CTLs reduced the human HCC tumor mass implanted into nonobese diabetic/severe combined immunodeficiency mice.
Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of HCC patients.
Clinical Cancer Research 06/2006; 12(9):2689-97. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
Cancer Research 03/2006; 66(4):2414-22. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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 alpha GalCer-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-gamma/CXCL9, had been generated using a method based on in vitro differentiation of DC from mouse ES cells. The ES-DC were loaded with alpha-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 alpha-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 alpha-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. alpha-GalCer-loaded double transfectant ES-DC expressing SLC along with OVA induced the most potent antitumor immunity. Thus, alpha-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 alpha-GalCer in immunotherapy for peritoneally
Cancer Science 01/2006; 96(12):889-96. · 3.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: Dendritic cells (DC) genetically modified to present tumor-associated antigen are a promising means for anti-cancer immunotherapy. By introducing expression vectors into ES cells and subsequently inducing differentiation to DC (ES-DC), we can generate transfectant DC expressing the transgenes. In the future clinical application of this technology, the unavailability of human ES cells genetically identical to the patients will be a problem. However, in most cases, semi-allogeneic ES cells sharing some of HLA alleles with recipients are expected to be available. In the present study, we observed that model tumor antigen (OVA)-expressing mouse ES-DC transferred into semi-allogeneic mice potently primed OVA-reactive CTL and elicited a significant protection against challenge with OVA-expressing tumor. Genetic modification of ES-DC to overexpress SPI-6, the specific inhibitor of granzyme B, further enhanced their capacity to prime antigen-specific CTL in semi-allogeneic recipient mice. These results suggest the potential of ES-DC as a novel means for anti-cancer immunotherapy.
Biochemical and Biophysical Research Communications 10/2005; 335(1):5-13. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
The Journal of Immunology 03/2005; 174(4):1888-97. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: To establish effective antitumor immunotherapy for esophageal cancer, we tried to identify an useful target antigen of esophageal cancer.
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.
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.
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.
Clinical Cancer Research 11/2004; 10(19):6437-48. · 7.84 Impact Factor