A case of accidental ingestion of a hooked denture without symptoms for 6 years.

Six years prior, the patient accidentally swallowed a denture during a meal and immediately visited a nearby doctor. However, because spontaneous excretion was expected, regular imaging testing was used to monitor it. After 4 years, although the denture was still in the small bowel, as there were no symptoms, the regular follow-up was terminated. Because the patient's anxiety increased, he visited our hospital 2 years later. Surgery was performed, as it was determined that there was no possibility of spontaneous excretion. The denture was palpated in the jejunum. The small intestine was incised, and the denture was removed. As far as we know, there are no guidelines prescribing a clear follow-up period for accidental denture ingestion. In addition, there are no guidelines specifying surgical indications in asymptomatic cases. Nonetheless, there have been reports of gastrointestinal perforations with dentures, and we consider that earlier preventive surgical intervention is better.

Onodera's prognostic nutritional index correlates with tumor immune environment and survival in patients with oral squamous cell carcinoma undergoing chemoradiotherapy.

Pretreatment nutritional and immunological status is useful for predicting survival outcomes for various types of malignant tumors. Our objective was to determine the impact of the pretreatment Onodera's prognostic nutritional index (OPNI) on outcomes of patients who underwent definitive chemoradiotherapy for advanced oral squamous cell carcinoma (OSCC). We reviewed 47 patients treated for OSCC with definitive chemoradiotherapy (CRT) at our institution between January 2004 and December 2011. We determined the OPNI according to the following formula: 10 × serum albumin (g/dL) + 0.005 × total lymphocyte count (per µL). We determined the optimum OPNI cut-off through a receiver operating characteristic analysis. We analyzed the associations between OPNI status and various clinicopathological features and evaluated the effects of OPNI on the prognosis. We examined the relationships between OPNI and systemic inflammatory response parameters and analyzed intratumoral CD8+ T cells and their correlation with OPNI. The optimum OPNI cut-off was 42.7. A Kaplan-Meier curve analysis revealed that low OPNI was significantly associated with poor overall survival and cause-specific survival. The multivariate analysis revealed that low OPNI was independently correlated with poor 5 year overall survival and cause-specific survival. OPNI was significantly correlated with systemic inflammatory response parameters. Intratumoral CD8+ T cell counts in primary tumors were significantly lower for low OPNI than for high OPNI. The present data demonstrate that pretreatment OPNI is a valuable independent prognostic indicator of overall and cause-specific survival in advanced OSCC following definitive CRT. OPNI might reflect the tumor immune microenvironment characterization in OSCC.

Salivary duct carcinoma treated with cetuximab-based targeted therapy: A case report.

Salivary duct carcinoma is a highly aggressive disease with a poor prognosis. Surgical resection is currently the only curative treatment, as there is no effective systemic therapy for this malignancy. Recently, trastuzumab has been shown to exhibit therapeutic efficacy in the treatment of salivary duct carcinoma; similarly, molecularly targeted agents, such as cetuximab, are expected to be useful for salivary duct carcinoma treatment. We herein describe the case of a 56-year-old man diagnosed with salivary duct carcinoma in the left submandibular region, with ipsilateral multiple metastases to the neck lymph nodes. Radical resection of the tumor and submandibular gland with neck dissection were performed. One month after radical surgery, computed tomography (CT) scans indicated metastasis in the lower lobe of the left lung. CT-guided transthoracic fine-needle aspiration biopsy revealed a single metastasis and lung metastasectomy was immediately performed. The tumor cells of the primary lesion and those of the lung metastasis were immunohistochemically positive for epidermal growth factor receptor. One month later, multiple right lung metastases appeared, and the patient was treated with cisplatin/5-fluorouracil (5-FU) chemotherapy plus cetuximab, achieving a complete radiographic response. However, multiple lung metastases developed during adjuvant weekly cetuximab monotherapy. Subsequently, treatment with S-1 and weekly cetuximab was initiated, and the multiple lung metastases have been maintained as stable disease for 5 months. To the best of our knowledge, this is the first report of cetuximab use for the treatment of salivary duct carcinoma. Although cisplatin/5-FU chemotherapy plus cetuximab was efficacious in treating the lung metastasis, cetuximab monotherapy was insufficient for controlling tumor growth.

Soluble IL6R Expressed by Myeloid Cells Reduces Tumor-Specific Th1 Differentiation and Drives Tumor Progression.

IL6 produced by tumor cells promotes their survival, conferring a poor prognosis in patients with cancer. IL6 also contributes to immunosuppression of CD4+ T cell-mediated antitumor effects. In this study, we focused on the impact of IL6 trans-signaling mediated by soluble IL6 receptors (sIL6R) expressed in tumor-bearing hosts. Higher levels of sIL6R circulating in blood were observed in tumor-bearing mice, whereas the systemic increase of sIL6R was not prominent in tumor-bearing mice with myeloid cell-specific conditional deletion of IL6R even when tumor cells produced sIL6R. Abundant sIL6R was released by CD11b+ cells from tumor-bearing mice but not tumor-free mice. Notably, IL6-mediated defects in Th1 differentiation, T-cell helper activity for tumor-specific CD8+ T cells, and downstream antitumor effects were rescued by myeloid-specific deletion of sIL6R. Expression of the T-cell transcription factor c-Maf was upregulated in CD4+ T cells primed in tumor-bearing mice in an IL6-dependent manner. Investigations with c-Maf loss-of-function T cells revealed that c-Maf activity was responsible for IL6/sIL6R-induced Th1 suppression and defective T-cell-mediated antitumor responses. In patients with cancer, myeloid cell-derived sIL6R was also possibly associated with Th1 suppression and c-Maf expression. Our results argued that increased expression of sIL6R from myeloid cells and subsequent c-Maf induction were adverse events for counteracting tumor-specific Th1 generation. Overall, this work provides a mechanistic rationale for sIL6R targeting to improve the efficacy of T-cell-mediated cancer immunotherapy. Cancer Res; 77(9); 2279-91. ©2017 AACR.

IL-6 controls resistance to radiation by suppressing oxidative stress via the Nrf2-antioxidant pathway in oral squamous cell carcinoma.

BACKGROUND: In promoting tumour malignancy IL-6 signalling is considered to have an important role. However, the biological roles of IL-6 on radiosensitivity in oral squamous cell carcinoma (OSCC) remain largely unclear. The objective of this study is to determine the effects and molecular mechanisms of IL-6 on radiosensitivity in OSCC. METHODS: Two OSCC cell lines, and OSCC tissue samples with radioresistant cells were used. We examined the effects of IL-6, or tocilizumab, a humanised anti-human IL-6 receptor antibody, or both on radiosensitivity and DNA damage after X-ray irradiation in vitro. In addition, we investigated the involvement of the Nrf2-antioxidant pathway in IL-6-mediated radioresistant mechanisms using OSCC cell lines and tissues. RESULTS: Increased levels of IL-6 suppressed radiation-induced cell death, and the blockade of IL-6 signalling by tocilizumab sensitised tumour cells to radiation. The radioresistant effect of IL-6 was associated with decreased DNA damage after radiation. We also found that IL-6 promotes the activation of not only the downstream molecule STAT3 but also the Nrf2-antioxidant pathway, leading to a significant decrease in oxidative stress by upregulating Mn-SOD. CONCLUSIONS: These results indicate that the blockade of IL-6 signalling combined with conventional radiotherapy could augment the treatment response and survival rate in patients with radioresistant OSCC.

Enhancement of active MMP release and invasive activity of lymph node metastatic tongue cancer cells by elevated signaling via the TNF-α-TNFR1-NF-κB pathway and a possible involvement of angiopoietin-like 4 in lung metastasis.

To study the role of TNF-α in tongue cancer metastasis, we made highly metastatic cells from a human oral squamous cell carcinoma cell line (SAS) by repeating the passage in which the cells were injected into a nude mouse tongue and harvested from metastasized cervical lymph nodes. Cancer cells after 5 passages (GSAS/N5) increased invasive activity 7-fold in a TNF-α receptor 1 (TNFR1)-dependent manner and enhanced mRNA expression of TNF-α and TNFR1. In the highly metastatic cells, NF-κB activation was upregulated via elevated phosphorylation of Akt and Ikkα/ß in the signaling pathway and secretion of TNF-α, active MMP-2 and MMP-9 increased. Suppression of increase of TNF-α mRNA expression and MMP secretion by NF-κB inhibitor NBD peptide suggested a positive feedback loop in GSAS/N5 cells; TNF-α activates NF-κB and activated NF-κB induces further TNF-α secretion, leading to increase of active MMP release and promotion of invasion and metastasis of the cells. GSAS/N5 cells that had been injected into the nude mouse tongue and harvested from metastasized lungs multiplied angiopoietin-like 4 (angptl4) expression with enhanced migration activity, which indicated a possible involvement of angptl4 in lung metastasis of the cells. These results suggest that TNF-α and angptl4 promote metastasis of the oral cancer cells, thus, these molecules may be therapeutic targets for patients with tongue cancer.

An oncofetal antigen, IMP-3-derived long peptides induce immune responses of both helper T cells and CTLs.

Insulin-like growth factor II mRNA-binding protein 3 (IMP-3), an oncofetal antigen identified using genome-wide cDNA microarray analyses, is overexpressed in several malignancies. IMP-3-derived cytotoxic T lymphocyte (CTL) epitopes have been used for peptide-based immunotherapies against various cancers. In addition to CTLs, induction of tumor-associated antigen (TAA)-specific helper T (Th) cells is crucial for establishment of effective antitumor immunity. In this study, we aimed to identify IMP-3-derived long peptides (IMP-3-LPs) carrying CTL and promiscuous Th-cell epitopes for use in cancer immunotherapy. IMP-3-derived Th-cell epitopes that bind to multiple HLA-class II molecules were predicted by in silico analysis, and their immunogenicity was determined by utilizing human T cells. We identified two highly immunogenic IMP-3-LPs presented by multiple HLA-class II molecules. One of the IMP-3-LPs encompassed two CTL epitopes that have been used for peptide-vaccine immunotherapy in ongoing clinical trials. IMP-3-LPs-specific Th cells responded to autologous dendritic cells (DCs) loaded with the recombinant IMP-3 proteins, suggesting that these s (LPs) can be naturally processed and presented. The IMP-3-LPs and specific Th cells augmented the expansion of IMP-3-specific CTLs, which was further enhanced by programmed cell death-1 (PD-1) blockade. In addition, IMP-3-LP encapsulated in liposomes was efficiently cross-presented in vitro, and this LP successfully cross-primed CTLs in HLA-A2 transgenic mice (Tgm) in vivo. Furthermore, one of the IMP-3-LPs induced IMP-3-specific Th cells from peripheral blood mononuclear cells (PBMCs) of head-and-neck malignant tumor (HNMT) patients. These findings suggest the potential usefulness of IMP-3-LPs in propagating both Th cells and CTLs and may have implications for IMP-3-LPs-based cancer immunotherapy.

Sentinel lymph node biopsy reduces the incidence of secondary neck metastasis in patients with oral squamous cell carcinoma.

It has recently been established that sentinel node biopsy (SNB) is an applicable and feasible procedure for the prediction of neck lymph node status in patients with early oral squamous cell carcinoma (OSCC) who are clinically negative for neck metastasis (cN0). The aim of this study was to retrospectively compare excision followed by watchful waiting with excision and SNB, in order to determine the effectiveness of SNB. A total of 125 patients with cN0 early OSCC were divided into two groups, namely the excision alone (n=78) and excision with SNB (n=47) groups. The clinical data of these two groups between 2006 and 2013 were analyzed. In the excision with SNB group, the negative predictive value and false-negative rate of SNB were 94% (30/32) and 18% (2/11), respectively. Secondary neck metastasis, also known as delayed neck metastasis, occurred in 24.2% of the patients in the excision alone group and 4.9% of the patients in the excision with SNB group. The 5-year overall survival (OS) rates were 84.0 and 97.5% in the excision alone and excision with SNB groups, respectively. Significant differences were found in the rate of secondary neck metastasis and OS between the two groups. SNB may be effective in the detection of occult neck lymph node metastasis, with a reduction in the incidence of secondary neck metastasis and improvements in the 5-year OS in patients with early-stage (stage I/II) oral cancer.

Phase II clinical trial of multiple peptide vaccination for advanced head and neck cancer patients revealed induction of immune responses and improved OS.

PURPOSE: The peptides derived from ideal cancer-testis antigens, including LY6K, CDCA1, and IMP3 (identified using genome-wide cDNA microarray analyses), were used in immunotherapy for head and neck squamous cell cancer (HNSCC). In this trial, we analyzed the immune response to and safety and efficacy of vaccine therapy. EXPERIMENTAL DESIGN: A total of 37 patients with advanced HNSCC were enrolled in this trial of peptide vaccine therapy, and the OS, PFS, and immunologic response were evaluated using enzyme-linked ImmunoSpot (ELISPOT) and pentamer assays. The peptides were subcutaneously administered weekly with IFA. The primary endpoints were evaluated on the basis of differences between HLA-A*2402-positive [A24(+)] patients treated with peptide vaccine therapy and -negative [A24(-)] patients treated without peptide vaccine therapy among those with advanced HNSCC. RESULTS: Our cancer vaccine therapy was well tolerated. The OS of the A24(+) vaccinated group (n = 37) was statistically significantly longer than that of the A24(-) group (n = 18) and median survival time (MST) was 4.9 versus 3.5 months, respectively; P < 0.05. One of the patients exhibited a complete response. In the A24(+) vaccinated group, the ELISPOT assay identified LY6K-, CDCA1-, and IMP3-specific CTL responses in 85.7%, 64.3%, and 42.9% of the patients, respectively. The patients showing LY6K- and CDCA1-specific CTL responses demonstrated a longer OS than those without CTL induction. Moreover, the patients exhibiting CTL induction for multiple peptides demonstrated better clinical responses. CONCLUSIONS: The immune response induced by this vaccine may improve the prognosis of patients with advanced HNSCC.

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.

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.

Aberrant expression of serum amyloid A in head and neck squamous cell carcinoma.

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.

Multiple antigen-targeted immunotherapy with alpha-galactosylceramide-loaded and genetically engineered dendritic cells derived from embryonic stem cells.

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.

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

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

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.

Identification of HLA-A2- or HLA-A24-restricted CTL epitopes possibly useful for glypican-3-specific immunotherapy of hepatocellular carcinoma.

PURPOSE AND EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSION: Our study raises the possibility that these GPC3 peptides may therefore be applicable to cancer immunotherapy for a large number of HCC patients.

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.

Therapeutic effect of alpha-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 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 prevention with semi-allogeneic ES cell-derived dendritic cells.

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.