Shigeaki Ishii

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.

Differential effect of cytotoxic T lymphocyte variant epitopes on generation and cytotoxicity in chronic hepatitis C virus infection.

Human leukocyte antigen (HLA) B44-restricted, hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) recognize HCV nucleoprotein amino acid residues 88-96 as an epitope. We previously reported the existence of variant peptide sequences at the epitope locus in three of 27 patients with HCV infection and HLA B44. Here we studied the effects of the variant peptide sequences on generation and cytotoxicity of CTLs. Two of the three variant peptides generated CTLs poorly although they activated well the cytotoxicity of CTLs. Such a differential activation of proliferation and cytotoxicity may contribute to the emergence of HCV with variant epitopes for CTLs.

Dendritic cells stimulated with cytidine‐phosphate‐guanosine oligodeoxynucleotides and interferon‐α‐expressing tumor cells effectively reduce outgrowth of established tumors in vivo

Dendritic cells (DC) are potent antigen‐presenting cells that elicit immune responses to foreign antigens. We have previously demonstrated the synergistic effects of cytidine‐phosphate‐guanosine (CpG) oligodeoxynucleotides (ODN) and interferon (IFN)‐α on DC maturation in vitro. In the present study, the antitumor effects of DC preincubated with IFN‐α gene‐overexpressing murine colorectal cancer MC38 cells (MC38‐IFN‐α) and CpG ODN were evaluated in a poorly immunogenic murine cancer system. When we injected DC preincubated with MC38‐IFNα and CpG ODN subcutaneously to mice bearing MC38 wild‐type tumors, the outgrowth of the established parental tumors was suppressed significantly compared with that following administration of DC with MC38‐IFN‐α (P = 0.008). All mice injected with DC preincubated with MC38‐IFN‐α and CpG ODN rejected a subsequent parental tumor challenge. Immunohistochemical and flow cytometric analyses showed that CD4+, CD8+, and NK1.1+ cells markedly infiltrated the established tumors of mice treated with DC preincubated with MC38‐IFN‐α and CpG ODN. From the results in immune cell‐depleted mice, CD4+ and asialo‐GM‐1+ cells seemed to contribute to the antitumor effects induced by the combination DC therapy. Furthermore, non‐specific cytolysis was detected when splenocytes of mice inoculated with DC preincubated with MC38‐IFNα and CpG ODN were used as effector cells. Using an interleukin (IL)‐12‐neutralizing antibody it was suggested that IL‐12 stimulates natural killer cells and contributes in part to the antitumor effects induced by DC incubated with CpG ODN and IFN‐α. As DC‐based immunotherapy with CpG ODN and IFN‐α‐expressing tumor cells induces a potent antitumor immune response, it should be considered for clinical application. (Cancer Sci 2008; 99: 1663–1669)

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

Abstract 4386: Effects of interferon-α-transduced tumor cell vaccines and blockade of programmed cell death 1 on the growth of established tumors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Progress in treatments for cancer has improved the prognosis of patients with colorectal cancer. However, 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 gene therapy using tumor cells that are genetically modified to produce cytokines has been studied in several therapeutic models. We have previously reported that the interferon (IFN)-α gene-transduced tumor-based vaccination therapy suppresses the outgrowth of established tumors. Although the suppressive effects on established tumors were observed, we did not see reductions in the size of all of the parental tumors. Therefore, further improvements in the treatment are needed before clinical application, we focused on programmed cell death 1 (PD-1), which has been identified as a marker of exhausted T cells. In this study, we evaluated the efficacy of the combination of IFN-γ-transduced tumor cell vaccines and PD-1 blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. A poorly immunogenic murine colorectal cancer cell line, MC38, was transduced to overexpress IFN-γ (MC38-IFNα). In a therapeutic model, parental tumor-bearing mice were inoculated with MC38-IFNα cells and an anti-PD-1 antagonistic antibody. Significant suppression of outgrowth of the established tumors was observed in the IFN-γ and anti-PD-1 combination treatment group (IFN+ anti-PD-1, 174.17 ± 35.54 mm2 vs control, 328.67 ± 26.36 mm2 on day 28, P = 0.0114 vs controls). Immunohistochemical analyses showed marked infiltration of CD4+ cells as well as CD8+ cells in the established tumors of mice treated with both IFN-γ and anti-PD-1. To investigate induction of tumor-specific immune responses, we stimulated splenocytes of IFN-γ or/and anti-PD-1 treated mice twice weekly by DCs in vitro. Significant tumor-specific cytolysis was detected when splenocytes of mice treated with both IFN-γ and anti-PD-1 were used as effector cells (58.1% ± 6.7% for MC38 and 14.1% ± 1.7% for YAC-1, effector:target = 20, P < 0.001). Our findings suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by IFN-γ. The combination of IFN-γ gene-transduced tumor cell vaccines and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials. 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 4386. doi:1538-7445.AM2012-4386

Abstract 524: Cytokine therapy by allogenic IFN-alpha-expressing murine colorectal cancer cells suppresses outgrowth of established tumors in a murine hepatocellular carcinoma model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Although several effective therapies for hepatocellular carcinoma (HCC) have been developed, many patients with both advanced cirrhosis and HCC are not able to receive those treatments because of their poor liver function. Thus, it is needed to establish a new effective therapy that has lesser side effects for those patients. We previously reported that immunotherapy by interferon-alpha (IFNa) and dendritic cells effectively suppressed outgrowth of established tumors and showed preventive effects in the murine colorectal cancer, MC38 cell model. We also revealed that tumor-specific cytotoxic T cell responses were important for these anti-tumor effects. In this study, we investigated the antitumor effect of allogenic cytokine (IFNa and IL-4)- expressing MC38 cells in the murine hepatocellular carcinoma, BNL model. There were no differences in cell growth between BNL cells and BNL cells co-incubated with cytokine-expressing MC38 cells in vitro. Balb/c mice were injected with 5x105 of BNL cells on their flank. Once BNL tumors were established, these mice received therapeutic injection of cytokine-expressing MC38 cells (2.5x105/mouse or 5x105/mouse) and tumor size was measured twice weekly. While tumor growth of the mice injected with MC38-IL4 was not suppressed (control; 421.0±146.9mm2, MC38-IL4 (5x105); 351.3±126.1mm2, p=0.33), injection of MC38-IFNa cells significantly suppressed the tumor growth by dose-dependent manner (MC38-IFNa: 5x105 cells; 183.5±46.2mm2, p=0.02). When we injected mice with both IFNa cells and IL-4 cells, any additive anti-tumor effects were not observed (mice injected both modified MC38: 5x105 cells each; 182±44.6 mm2, p=0.01). A BNL-specific cytolysis was detected when splenocytes of mice injected with MC38-IFNa were used as effector cells in a chromium-release assay. Furthermore, immunohistocemistrical analysis revealed that CD4+ T cells, CD8+ T cells and especially Gr-1+ cells infiltrated established BNL tumors of mice injected with MC38-IFNa. Our results suggest that the immunotherapy with allogenic IFNa-expressing cells has potent antitumor effects, and that it would be applicable for treatment to advanced HCC patients. 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 524. doi:1538-7445.AM2012-524

Two cases of tuberculous peritonitis with liver cirrhosis complicated by refractory ascites

We present two cases of tuberculous peritonitis with liver cirrhosis complicated by refractory ascites. Case 1 was a 59-year-old female with alcoholic liver cirrhosis. She was admitted to our hospital because of diarrhea, anorexia and inflammatory reactions on a blood test. She had a high fever of 38°C or more and refractory ascites. Tubercle bacilli infection was suspected based on increased levels of serum CA125 and adenosine deaminase (ADA) in ascites. Laparoscopic examination showed white nodules on the peritoneum, and histologic study confirmed tuberculous nodules. The same bacteria were isolated from culture of ascites. Case 2 was a 55-year-old female with hepatitis C virus-infected liver cirrhosis. She was admitted because of high fever and abdominal fullness due to ascites. High levels of serum CA125 and ADA in ascites and ineffectiveness of treatment with antibiotics plus diuretics led us to start anti-tuberculous therapy before definitive diagnosis. Tuberculus bacillus was later isolated from culture of ascites. It is difficult to make early diagnosis of tuberculous peritonitis in cirrhotic patients with ascites due to a lack of specific symptoms. However, determination of serum CA125 and ADA in ascites and the acid-fast bacterial culture of ascites are useful for early diagnosis.