Masashi Sakaki

IFN-γ production by lung NK cells is critical for the natural resistance to pulmonary metastasis of B16 melanoma in mice

NK cells are effector lymphocytes playing a critical role in the natural resistance against tumors. However, the precise mechanisms underlying NK cell‐mediated natural resistance against tumor metastasis are still unrevealed. B16 cells, mouse melanoma cells, were resistant to freshly isolated NK cell‐mediated killing; nevertheless, NK cells were critical for natural resistance against experimental lung metastasis of B16 cells. We found that lung metastasis was increased significantly in IFN‐γ–/– mice but not pfp–/–, IFN‐αR–/–, or IL‐12/IL‐18–/– mice. Interestingly, freshly isolated lung NK cells, but not spleen or liver NK cells, displayed augmented IFN‐γ production after B16 inoculation. Adoptive transfer of pfp–/– NK cells, but not IFN‐γ–/– NK cells, significantly decreased B16 lung metastasis in IFN‐γ–/– and pfp/IFN‐γ–/–mice. Lung metastases of IFN‐γRDN B16 was also increased in NK cell‐depleted or IFN‐γ–/– mice, suggesting that the IFN‐γ response of host cells was required in the NK cell and IFN‐γ‐mediated antimetastatic effect. Our results demonstrate that IFN‐γ production from lung resident NK cells is a key response in the natural resistance to the experimental lung metastasis of NK cell‐resistant tumor cells.

Intrahepatic status of regulatory T cells in autoimmune liver diseases and chronic viral hepatitis

Aim:  Regulatory T cells (Tregs) maintain immunological tolerance and suppress autoreactive immune responses. We evaluated the intrahepatic status of Tregs in patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), chronic hepatitis C (CH‐C), or chronic hepatitis B (CH‐B).

Cyclooxygenase‐2 gene promoter polymorphisms affect susceptibility to hepatitis C virus infection and disease progression

Aim:  Because polymorphisms of cyclooxygenase‐2 (COX‐2) and osteopontin (OPN) promoter regions and a promoter/enhancer region of forkhead box protein 3 (FOXP3) gene are known to affect immune responses, we examined whether these polymorphisms can influence susceptibility to hepatitis C virus (HCV) infection and progression of liver disease.

Genetic polymorphism in cyclooxygenase‐2 promoter affects hepatic inflammation and fibrosis in patients with chronic hepatitis C

Summary.  Cyclooxygenase (COX)‐2 is involved in inflammation, anti‐apoptosis and carcinogenesis. The ‐1195GG genotype of single nucleotide polymorphism (SNP) in COX‐2 promoter was associated with low platelet counts in patients with chronic hepatitis C. Polymorphism of patatin‐like phospholipase domain‐containing protein 3 (PNPLA3) gene (rs738409 C>G) have been reported to be associated with cirrhosis, and the major genotype of SNPs near interleukin (IL)28B are related to viral clearance. The present study was designed to assess the contribution of these SNPs to disease progression in patients with chronic hepatitis C. The study enrolled 220 Japanese patients with chronic hepatitis C. Three SNPs, ‐1195 COX‐2, PNPLA3 and IL28B (rs8099917), were genotyped in order to analyze their association with hepatic fibrosis and inflammation. The ‐1195GG genotype in COX‐2 was associated with advanced fibrosis and higher levels of inflammation in the liver tissues. The major genotype of IL28B was also associated with advanced fibrosis, but the polymorphism of PNPLA3 was neither associated with fibrosis nor inflammation. Multivariate analysis showed that ‐1195GG in COX‐2 is an independent factor associated with advanced fibrosis, while the major genotype of IL28B and HCV genotype 2 were other independent factors. In conclusion, the ‐1195GG genotype in COX‐2 is a genetic marker for liver disease progression, while the PNPLA3 genotypes are not associated with disease progression in Japanese patients with chronic hepatitis C.

Immune Response of Cytotoxic T Lymphocytes and Possibility of Vaccine Development for Hepatitis C Virus Infection

Immune responses of cytotoxic T lymphocytes (CTLs) are implicated in viral eradication and the pathogenesis of hepatitis C. Weak CTL response against hepatitis C virus (HCV) may lead to a persistent infection. HCV infection impairs the function of HCV-specific CTLs; HCV proteins are thought to actively suppress host immune responses, including CTLs. Induction of a strong HCV-specific CTL response in HCV-infected patients can facilitate complete HCV clearance. Thus, the development of a vaccine that can induce potent CTL response against HCV is strongly expected. We investigated HCV-specific CTL responses by enzyme-linked immuno-spot assay and/or synthetic peptides and identified over 40 novel CTL epitopes in the HCV protein. Our findings may contribute to the development of the HCV vaccine. In this paper, we describe the CTL responses in HCV infection and the attempts at vaccine development based on recent scientific articles.

Interleukin-4 and CpG oligonucleotide therapy suppresses the outgrowth of tumors by activating tumor-specific Th1-type immune responses.

Because IL-4 and CpG oligodeoxynucleotides (CpG-ODNs) are immune stimulants, we evaluated the antitumor effects of IL-4 gene therapy and CpG-ODN treatment in a poorly immunogenic murine cancer model. We used a murine colorectal cancer MC38 cell line overexpressing the IL-4 gene (MC38-IL4). Incubation with MC38-IL4 and CpG-ODN enhanced bone marrow-derived dendritic cell (DC) maturation in vitro. In addition, interferon (IFN)-γ production was significantly increased in naïve splenocytes after they were coincubated with MC38-IL4 and CpG-ODN. When mice bearing MC38 wild-type tumors were inoculated subcutaneously with MC38-IL4 cells and CpG-ODN, the outgrowth of established parental tumors was significantly suppressed compared to those in the MC38-IL4-treated group (IL-4 vs. IL-4 + CpG-ODN, p=0.015). A marked infiltration of CD8+ cells in the established parental tumors of mice treated with MC38-IL4 and CpG-ODN was confirmed by immunohistochemical analyses (MC38-IL4, 2.8 ± 1.9 cells/field vs. MC38-IL4 + CpG-ODN, 20.7 ± 15.3 cells/field, p=0.027). Significant tumor-specific cytolysis was detected when splenocytes of MC38-IL4 + CpG-ODN-treated mice were stimulated by γ-irradiated MC38-IL4 cells and CpG-ODN twice weekly in vitro and used as effector cells in a chromium-release assay (32.2 ± 3.5% for MC38 cells vs. 3.2 ± 1.1% for YAC-1 cells; at an effector to target ratio of 40). These results suggest that IL-4 and CpG-ODN treatment promotes potent Th1-type antitumor immune responses. Therefore, the combination of IL-4 gene therapy and CpG-ODN treatment for cancer should be evaluated in clinical trials.

Magnitude of CD8+ T‐cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection

Aim:  We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)‐specific CD8+ T‐cell responses and the clinical course of acute HCV infection.

Abstract LB-135: A pilot study of peptide-based vaccines in combination with poly ICLC in patients with WHO grade 2 low-grade glioma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Adult patients with WHO grade 2 low-grade glioma (LGG) have a significant risk of tumor progression despite treatment with surgery or surgery followed by radiation therapy (RT) and /or chemotherapy, and most patients eventually die of the disease. High-risk subsets of these LGG patients display astrocytoma or oligoastrocytoma histology plus any one of the following conditions: 1) age ≥40 with any extent resection; 2) age 18-39 with incomplete resection; or 3) age 18-39 with neurosurgeon-defined gross total resection with tumor size ≥ 4 cm in diameter. These patients have as high as an 89% risk of recurrence by 5 years following surgery. Methods: Based on encouraging data from a phase I vaccine study targeting multiple glioma-associated antigen (GAA) epitopes in adult high-grade glioma (HGG) patients, we initiated a bi-institutional pilot study of subcutaneous vaccinations of subcutaneous vaccinations with synthetic peptides for GAA epitopes emulsified in Montanide-ISA-51 every 3 weeks for 8 courses, and intramuscular administration of poly-ICLC in HLA-A2+ patients with: newly diagnosed high-risk LGG without prior RT (Cohort 1); newly diagnosed high-risk LGG with prior RT (Cohort 2), or recurrent LGG (Cohort 3). Primary endpoints were safety and CD8+ T-cell responses against vaccine-targeted GAAs, assessed by Enzyme-Linked Immuno-SPOT (ELISPOT) assays. Treatment response was evaluated clinically and by MR imaging. Targeted GAAs were EphA2, interleukin (IL)-13 receptor-α2, survivin, and WT1. As these GAAs are expressed not only in a subset of LGG cells but even at higher levels in HGG cells, our vaccine approach may offer both immunotherapeutic and immunoprophylactic potential to reduce the risk of tumor recurrence. Results: To date, 13, 1 and 10 patients have been enrolled in Cohorts 1, 2 and 3, respectively. No dose-limiting non-CNS toxicity has been encountered except for one case with Grade 3 fever (Cohort 1). ELISPOT assays, completed in 7 and 1 patients in Cohorts 1 and 2, respectively, demonstrated robust and sustained interferon (IFN)-γ (type-1) responses against at least 3 of the GAA epitopes in all cases, while IL-5 (type-2) responses were absent or transient in all cases. The magnitude of the IFN-γ ELISPOT responses in the current study was significantly higher than that observed in our previous phase I/II study in HGG patients (Okada H et al. 2011). Although evaluation of progression-free survival would require a longer observation period, among 9, 1 and 7 patients who completed the 8 vaccinations spanning 24 weeks, 6, 1 and 3 patients in Cohorts 1, 2 and 3, respectively, are currently with stable disease. Conclusion: Our preliminary results demonstrate that the regimen in these patients is well tolerated, and induces robust type-1 anti-GAA T-cell responses. These data suggest that patients with LGG are suitable for vaccine therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-135. doi:1538-7445.AM2012-LB-135

Increased expression of immuno-inhibitory molecules on peripheral blood lymphocytes may suppress disease progression in autoimmune hepatitis.

Accumulation of activated lymphocytes in the liver has been reported in patients with autoimmune liver diseases; the activity of these cells may be related to the disease immunopathogenesis. Although immuno-inhibitory molecules on lymphocytes are thought to play an important role in immunomodulation, there are only a few reports on the status of these molecules in autoimmune liver diseases. We focused on representative immunoinhibitory molecules, such as tumor necrosis factorrelated apoptosis-inducing ligand receptor 2/death receptor 5 (DR-5)/CD262, Fas/APO-1/CD95, programmed cell death-1 (PD-1)/CD279 and cytotoxic T-lymphocyte antigen-4 (CTLA-4)/CD152 on the surface of Tlymphocytes in patients with autoimmune liver disease, and investigated the relationship between inhibitory molecules on lymphocytes and the clinical features of patients with autoimmune liver diseases A limited number of patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and chronic hepatitis C (CHC) (n=10/group) and 10 healthy volunteers (control) were enrolled in this study. All patients and healthy volunteers gave written informed consent according to a protocol approved by the ethics committee of Showa University. Expression of inhibitory molecules DR5, Fas, PD-1 and CTLA-4 on the surface of peripheral CD4 and CD8 cells was assessed by staining withmonoclonal antibodies and by analyzing with flow cytometry. Differences between groups were assessed by ANOVA with Tukey’s honestly significant difference test and were considered statistically significant at P<0.05. Death receptor 5 expression was significantly upregulated on CD4 cells, but not on CD8 cells of patients with AIH compared with the controls (Fig. 1a). We found no differences in the expression of Fas molecules between patients and controls (Fig. 1b). PD-1 expression was more common on CD4 and CD8 cells of patients with AIH than on the control cells (Fig. 1c). The CD4 and CD8 cells of patients with AIH expressed CTLA-4 molecules more frequently than those of the controls, as shown in Figure 1(d). By comparison of the groups, significant difference could not be found between the control group and the disease groups other than AIH. In three AIH patients, the frequency of CTLA-4 positive cells was extremely high (Fig. 1d). The characteristics of these patients are presented in Table 1. They presented high frequencies of DR-5 and PD-1 expression as well. We noticed no signs of hepatitis activation such as elevated serum transaminase levels during the observation period. The other seven AIH patients had been treated with more than 5mg/day of prednisolone to control AIH. High expression of inhibitory molecules on CD4 and CD8 cells may suppress the disease activity of AIH. Upregulation of these immuno-inhibitory molecules may be a strategy for controlling AIH. We have reported that AIH patients have fewer intrahepatic regulatory T cells than do PBC patients. Immuno-inhibitory molecules also play important roles in immune regulation to prevent excessive inflammation. It has been reported that intrahepatic expression of PD-1 may play a crucial role in immunopathogenesis of AIH. Comprehensive study examining expression of inhibitory molecules on the surface of peripheral lymphocytes has not been reported, and it remains unclear whether overexpression of each molecule affects disease progression. Although each characteristic detail of inhibitory molecules has not been defined, several reports have compared CTLA-4 and PD-1. Each molecule inhibits T-cell activation by a different mechanism. PD-1 may be antigen-specific. Elucidation of the role of these molecules in immunerelated diseases would contribute to our understanding of the immunopathogenesis and the development of the treatment strategy. Further investigations including functional assay of inhibitory moleculeoverexpressing cells are needed.

Abstract 2810: Dendritic cell-based immunotherapy in combination with programmed cell death 1 blockade reduced established tumors by activating Th-1 type immune responses in a murine hepatocellular carcinoma model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Progress in treatments for hepatocellular carcinoma (HCC) has improved the prognosis of patients with HCC. However, HCC is usually associated with cirrhosis and often recurs even after complete treatment of the tumors in the remaining part of the cirrhotic liver. Thus, there is a strong need for the development of a new intervention therapy that suppresses the occurrence or recurrence effectively with fewer side effects. Immunotherapy may be such a treatment and several clinical trials have been performed for HCC treatment. Dendritic cells (DCs) are known as professional antigen-presenting cells characterized by their potent ability to elicit immune responses to tumor-specific T cells against tumor-associated antigens. Programmed cell death 1 (PD-1) has been identified as a marker of exhausted T cells in chronic disease states, and blockade of PD-1-PD-ligand interactions has been shown to partially restore T cell function. In this study, we evaluated the efficacy of the combination of DC-based vaccine and PD-1 blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. In protection model, mice were injected with DCs or/and PD-1-antibody before the murine HCC tumor cell (BNL cell) challenge. 40% of mice treated with both DCs and PD-1-antibody rejected tumor challenge, whereas other groups observed a palpable tumor in all mice tested. In therapeutic model, tumor-bearing mice were inoculated with DCs or/and PD-1-antibody. Significant suppression of outgrowth of the established tumors was observed in the DCs and anti-PD-1 combination treatment group (DCs + anti-PD-1, 138.00 ± 56.66 mm2 vs control, 402.33 ± 40.63 mm2 on day 42, P = 0.0073 vs controls). Immunohistochemical analyses of therapeutic model showed marked infiltration of CD4+ cells, CD8+ cells and CD11c+ cells in the established BNL tumors of mice treated with both DCs and PD-1-antibody. To investigate induction of tumor-specific immune responses, we stimulated splenocytes of DCs or/and PD-1-antibody treated mice twice weekly by DCs in vitro. Significant tumor-specific cytolysis was detected when splenocytes of mice treated with both DCs and anti-PD-1 were used as effector cells (30.0% ± 2.8% for BNL and 7.3% ± 0.1% for YAC-1, effector to target ratio; E/T=40). Our findings suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by DC vaccine. The combination of DC vaccine and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials. Citation Format: Eiichi Hayashi, Junichi Eguchi, Masashi Sakaki, Hiroyoshi Doi, Risa Oomori, Atsushi Kajiwara, Hitoshi Yoshida, Shigeaki Ishii, Kazumasa Hiroishi, Michio Imawari. Dendritic cell-based immunotherapy in combination with programmed cell death 1 blockade reduced established tumors by activating Th-1 type immune responses in a murine hepatocellular carcinoma model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2014-2810