Yukiko Sagawa

Antitumour activity mediated by CD4+ cytotoxic T lymphocytes against MHC class II‐negative mouse hepatocellular carcinoma induced by dendritic cell vaccine and interleukin‐12

When BALA/c mice with BNL hepatocellular carcinoma (HCC) were treated with dendritic cells fused with BNL cells (DC/BNL) and recombinant murine interleukin (IL)‐12, tumour development was significantly suppressed, whereas treatment with either DC/BNL or IL‐12 alone did not show a tumour‐suppressive effect. Antitumour activity induced by DC/BNL + IL‐12 was abrogated by depletion of CD4+ T cells, but not by depletion of CD8+ T cells or natural killer cells. Splenic CD4+ T cells and CD8+ T cells from DC/BNL‐treated mice showed cytotoxic activity against BNL cells after 3 days of incubation with DC/BNL, although BNL cells do not express major histocompatibility complex (MHC) class II molecules even after treatment with interferon (INF)‐γ. Furthermore, CD4+ T cells killed syngeneic‐irrelevant CT26 cells and even allogeneic Hepa1‐6 cells. This cytotoxicity was blocked by concanamycin A, but not by an anti‐Fas ligand (FasL) monoclonal antibody, indicating that cytotoxic activity was mediated by perforin. Immunofluorescence microscopy demonstrated that abundant CD4+ T cells and MHC class II‐positive macrophages, but not CD8+ T cells, had infiltrated tumour tissue in mice treated with DC/BNL + IL‐12. Flow cytometric analysis of tumour‐infiltrating cells in mice treated with DC/BNL + IL‐12 showed increases in CD4+ T cells and MHC class II+ CD11b+ cells but not in CD8+ T cells or MHC class I+ CD11b+ cells. Our results suggest that, in BNL‐bearing mice treated with DC/BNL + IL‐12, tumour macrophages activated by INF‐γ produced by IL‐12‐stimulated T cells might present BNL tumour antigens and activate DC/BNL‐primed CD4+ cytotoxic T lymphocytes (CTLs) in a MHC class II‐dependent manner, leading to perforin‐mediated bystander killing of neighbouring MHC class II‐negative tumour cells.

Cancer immunotherapy using dendritic/tumour-fusion vaccine induces elevation of serum anti-nuclear antibody with better clinical responses

Dendritic cell (DC) vaccines might induce both anti‐tumour immunity and autoimmunity. In this report, we demonstrate elevated levels of anti‐nuclear antibody (ANA) in the sera of patients with cancer who had received immunotherapy with a dendritic/tumour‐fusion vaccine. Twenty‐two patients were treated with DC vaccine of fusion cells composed of autologous DCs and tumour cells (DC/tumour‐fusion vaccine), which was generated by treating each cell type with polyethylene glycol. Nine of the 22 patients were treated with both the DC/tumour‐fusion vaccine and systemic administration of recombinant human interleukin (rhIL)‐12. Serum levels of ANA were examined with an enzyme‐linked immunosorbent assay kit. One patient with gastric carcinoma (patient 1, DC/tumour‐fusion vaccine alone), one patient with breast cancer (patient 2, DC/tumour‐fusion vaccine alone) and one patient with ovarian cancer (patient 3, DC/tumour‐fusion vaccine + rhIL‐12) showed significant elevations of serum ANA levels during treatment. In patient 1 malignant ascitic effusion resolved and serum levels of tumour markers decreased. Patients 2 and 3 remained in good physical condition during treatment for 24 and 9 months, respectively. Immunoblot analysis indicated antibody responses to autologous tumour cells after vaccination in patient 2. None of the treated patients showed clinical symptoms suggesting autoimmune disease. Patients with elevated serum levels of ANA had significantly longer treatment periods than those without it. Elevated serum levels of ANA after DC/tumour‐fusion cell vaccine might be associated with anti‐tumour immune response induced by the vaccination.

Gemcitabine enhances Wilms’ tumor gene WT1 expression and sensitizes human pancreatic cancer cells with WT1-specific T-cell-mediated antitumor immune response

Wilms’ tumor gene (WT1), which is expressed in human pancreatic cancer (PC), is a unique tumor antigen recognized by T-cell-mediated antitumor immune response. Gemcitabine (GEM), a standard therapeutic drug for PC, was examined for the regulation of WT1 expression and the sensitizing effect on PC cells with WT1-specific antitumor immune response. Expression of WT1 was examined by quantitative PCR, immunoblot analysis, and confocal microscopy. Antigenic peptide of WT1 presented on HLA class I molecules was detected by mass spectrometry. WT1-specific T-cell receptor gene–transduced human T cells were used as effecter T cells for the analysis of cytotoxic activity. GEM treatment of human MIAPaCa2 PC cells enhanced WT1 mRNA levels, and this increase is associated with nuclear factor kappa B activation. Tumor tissue from GEM-treated MIAPaCa2-bearing SCID mice also showed an increase in WT1 mRNA. Some human PC cell lines other than MIAPaCa2 showed up-regulation of WT1 mRNA levels following GEM treatment. GEM treatment shifted WT1 protein from the nucleus to the cytoplasm, which may promote proteasomal processing of WT1 protein and generation of antigenic peptide. In fact, presentation of HLA-A*2402-restricted antigenic peptide of WT1 (CMTWNQMNL) increased in GEM-treated MIAPaCa2 cells relative to untreated cells. WT1-specific cytotoxic T cells killed MIAPaCa2 cells treated with an optimal dose of GEM more efficiently than untreated MIAPaCa2 cells. GEM enhanced WT1 expression in human PC cells and sensitized PC cells with WT1-specific T-cell-mediated antitumor immune response.

Dendritic/pancreatic carcinoma fusions for clinical use: Comparative functional analysis of healthy- versus patient-derived fusions.

Fetal calf serum (FCS)-independent pancreatic cancer cells were established in plasma protein fraction (PPF)-supplemented medium that is an agent of good manufacturing practice (GMP) grade. Dendritic cells (DCs) were activated with the Toll-like receptor agonist, penicillin-inactivated Streptococcus pyogenes (OK-432) that is also a GMP grade agent. Therefore, sufficient amounts of FCS-independent fusions were successfully generated with decreased potential hazards of FCS. The FCS-independent fusions expressed tumor-associated antigens, HLA-DR, costimulatory molecules, IL-12, and IL-10. Stimulation of T cells with fusions from healthy donors resulted in proliferation of T cells with high expression levels of perforin/granzyme B and IFN-gamma and efficient induction of antigen-specific cytotoxic T lymphocytes (CTLs). Selection and expansion of T-cell clones were confirmed by TCR Vbeta analysis. However, fusions from patients with metastatic pancreatic cancer induced increased expression levels of TGF-beta1 in CD4+ CD25high T cells and low levels of CTLs with decreased IFN-gamma production.

In vitro generation of cytotoxic and regulatory T cells by fusions of human dendritic cells and hepatocellular carcinoma cells

BackgroundHuman hepatocellular carcinoma (HCC) cells express WT1 and/or carcinoembryonic antigen (CEA) as potential targets for the induction of antitumor immunity. In this study, generation of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) by fusions of dendritic cells (DCs) and HCC cells was examined.MethodsHCC cells were fused to DCs either from healthy donors or the HCC patient and investigated whether supernatants derived from the HCC cell culture (HCCsp) influenced on the function of DCs/HCC fusion cells (FCs) and generation of CTL and Treg.ResultsFCs coexpressed the HCC cells-derived WT1 and CEA antigens and DCs-derived MHC class II and costimulatory molecules. In addition, FCs were effective in activating CD4+ and CD8+ T cells able to produce IFN-γ and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism. However, HCCsp induced functional impairment of DCs as demonstrated by the down-regulation of MHC class I and II, CD80, CD86, and CD83 molecules. Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated Streptococcus pyogenes. Interestingly, fusions of immature DCs generated in the presence of HCCsp and allogeneic HCC cells promoted the generation of CD4+ CD25high Foxp3+ Treg and inhibited CTL induction in the presence of HCCsp. Importantly, up-regulation of MHC class II, CD80, and CD83 on DCs was observed in the patient with advanced HCC after vaccination with autologous FCs. In addition, the FCs induced WT1- and CEA-specific CTL that were able to produce high levels of IFN-γ.ConclusionThe current study is one of the first demonstrating the induction of antigen-specific CTL and the generation of Treg by fusions of DCs and HCC cells. The local tumor-related factors may favor the generation of Treg through the inhibition of DCs maturation; however, fusion cell vaccination results in recovery of the DCs function and induction of antigen-specific CTL responses in vitro. The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.

High plasma levels of soluble programmed cell death ligand 1 are prognostic for reduced survival in advanced lung cancer

OBJECTIVES Programmed cell death-ligand 1 (PD-L1) expressed in tumor tissues is a key molecule for immune suppression, given its role in immune checkpoints. The significance and implication of soluble PD-L1 (sPD-L1) in the blood of lung cancer patients remain unknown. PATIENTS AND METHODS Blood samples were prospectively collected from patients with advanced lung cancer, and the plasma sPD-L1 concentrations were measured by enzyme-linked immunosorbent assay. The correlations of the plasma sPD-L1 levels with clinico-pathological status, laboratory data, and survival of the patients were analyzed. RESULTS Ninety-six patients with advanced lung cancer were analyzed, including 73 with adenocarcinoma, 12 with squamous cell carcinoma, and seven with small-cell lung cancer. Sixty-five were naïve to chemotherapy, and 20 had received two or more lines of chemotherapy. The mean plasma sPD-L1 concentration of all the patients was 6.95±2.90ng/ml (range 2.30-20.0ng/ml), and this value is significantly increased compared with that previously reported for normal subjects. No correlation of the plasma sPD-L1 level with histological subtypes, adenocarcinoma genetic status, smoking history, clinical stage or laboratory data was found. However, overall survival was significantly reduced in patients with high (≥7.32ng/ml) compared with low (<7.32ng/ml) plasma sPD-L1 levels (13.0 vs. 20.4 months, p=0.037). Multivariate analysis revealed that high sPD-L1 levels were significantly related to poor prognosis (hazard ratio 1.99, p=0.041). CONCLUSION High plasma sPD-L1 levels were associated with poor prognosis in patients with advanced lung cancer, possibly associated with suppression of anti-tumor immunity. Clinical trial register and their clinical registration number: UMIN%000014760.

Combined treatment with dendritic cells and 5-fluorouracil elicits augmented NK cell-mediated antitumor activity through the tumor necrosis factor-alpha pathway.

Antitumor effects and mechanism of combined therapy with a dendritic cell (DC) vaccine and fluorouracil (5-FU) were investigated. Cytotoxic activity against MC38 cells, untreated or pretreated with 5-FU, was examined in splenocytes from mice inoculated with DCs: DCs pulsed with MC38 lysate or treated with LPS or both and untreated DCs. Inoculation with all types of DCs induced the significant cytotoxic activity of splenocytes, and pretreatment of MC38 cells with 5-FU significantly enhanced the cytotoxic activity of splenocytes. Depletion of natural killer (NK) cells, but not of CD8+ or CD4+ T cells, in the splenocytes from DC (without MC38 lysate-pulse or LPS treatment thereafter)-inoculated mice decreased the cytotoxic activity. The cytotoxic effect was eliminated by treatment with a monoclonal antibody (mAb) against tumor necrosis factor (TNF)-α and was partially inhibited by concanamycin A. Inoculation of mice with DCs upregulated TNFα expression on NK cells. MC38 cells pretreated with 5-FU exhibited enhanced expression of procaspase 8 and efficiently underwent apoptosis by TNF-α with activation of caspase 8. Although treatment with 5-FU upregulated Rae-1 expression on MC38 cells, the NK-cell–mediated cytotoxic activity was not suppressed by treatment with an anti–Rae-1 mAb or an antinatural killer group 2D mAb or both. These results indicate that combined therapy with a DC vaccine and 5-FU is a promising strategy for cancer treatment mediated by the tumoricidal activity of NK cells through the TNF-α pathway.

Mechanism of antitumor effect on mouse hepatocellular carcinoma by intratumoral injection of OK-432, a streptococcal preparation

Intratumoral (i.t.) injection of OK-432, a streptococcal preparation, into implanted tumors of mouse hepatocellular carcinoma (MIH-2) showed antitumor effect including tumor eradication. Intraperitoneal administration of same dose OK-432 did not exhibit tumor suppressive effect. In vitro cytotoxic test suggested that direct cytotoxic effect of OK-432 was not associated with antitumor activity by i.t.-OK-432 treatment. It was also found that Toll-like receptor 4 signaling was not involved in i.t.-OK-432 treatment. Three mice out of five, which had shown tumor eradication by i.t.-OK-432 treatment did not reject re-challenge of MIH-2 cells. Splenocytes from i.t.-OK-432 treated mice did not produce IFN-gamma by stimulation with MIH-2 cells in vitro, but produced abundant IFN-gamma by stimulation with OK-432. Immunofluorescence microscopy demonstrated that CD4+T cells, but not CD8+T cells, infiltrated to i.t.-OK-432 treated tumor tissue produced IFN-gamma. Tumor-infiltrating CD4+T cells from i.t.-OK-432 treated tumor tissue produced IFN-gamma by in vitro stimulation with OK-432 higher than those from untreated tumor tissue. IFN-gamma directly induced apoptosis of MIH-2 cells in vitro. Collectively, i.t.-OK-432 treatment induced priming of CD4+T cells to antigenecity of OK-432, and repetitive i.t.-OK-432 treatment induced IFN-gamma production from OK-432-sensitized CD4+T cells in tumor site, leading to apoptosis of MIH-2 cells susceptible to IFN-gamma.

Antigenic stimulation with cytochrome P450 2J expressed in mouse hepatocellular carcinoma cells regulates host anti-tumour immunity

Cytochrome P450 2J subfamily (CYP2J) enzymes expressed in mouse hepatocellular carcinoma (HCC) cells were identified as an antigen recognized by specific CD4+ T cells and the structure of its T cell epitope was determined by proteomics‐based exploration. The major histocompatibility complex (MHC) class II binding peptides were isolated from I‐Ak/peptide complex of dendritic cells (DCs) loaded or unloaded with MIH‐2 mouse HCC cells. MHC class II‐binding peptides found in MIH‐2‐loaded DCs but not in unloaded DCs were determined by tandem mass spectrometric analysis. The peptide, consisting of amino acid 276–290 (DFIDAFLKEMTKYPE) of mouse CYP2J enzymes, was identified as an antigenic peptide presented in the context of MHC class II. Preventive treatment of mice with CYP2J peptide stimulated interferon (IFN)‐γ production of splenocytes and suppressed the growth of implanted CYP2J‐positive MIH‐2 cells but not CYP2J‐negative murine bladder tumour cells. However, continuous treatment of MIH‐2‐bearing mice with CYP2J peptide significantly suppressed IFN‐γ production of splenocytes and accelerated the growth of implanted MIH‐2 tumours in vivo. Increased frequencies of CD4+forkhead box P3 regulatory T cells and CD11b+Gr‐1+ myeloid suppressor cells were observed in splenocytes from the continuously immunized mice. These results indicate that antigenecity of CYP2J isoforms expressed in HCC cells activate host anti‐tumour immunity at an initial stage of HCC, but suppress host anti‐tumour immunity with excessive antigenic stimulation at an advanced stage.

Vaccination with vascular progenitor cells derived from induced pluripotent stem cells elicits antitumor immunity targeting vascular and tumor cells

Vaccination of BALB/c mice with dendritic cells (DCs) loaded with the lysate of induced vascular progenitor (iVP) cells derived from murine-induced pluripotent stem (iPS) cells significantly suppressed the tumor of CMS-4 fibrosarcomas and prolonged the survival of CMS-4-inoculated mice. This prophylactic antitumor activity was more potent than that of immunization with DCs loaded with iPS cells or CMS-4 tumor cells. Tumors developed slowly in mice vaccinated with DCs loaded with iVP cells (DC/iVP) and exhibited a limited vascular bed. Immunohistochemistry and a tomato-lectin perfusion study demonstrated that the tumors that developed in the iVP-immunized mice showed a marked decrease in tumor vasculature. Immunization with DC/iVP induced a potent suppressive effect on vascular-rich CMS-4 tumors, a weaker effect on BNL tumors with moderate vasculature, and nearly no effect on C26 tumors with poor vasculature. Treatment of DC/iVP-immunized mice with a monoclonal antibody against CD4 or CD8, but not anti-asialo GM1, inhibited the antitumor activity. CD8+ T cells from DC/iVP-vaccinated mice showed significant cytotoxic activity against murine endothelial cells and CMS-4 cells, whereas CD8+ T cells from DC/iPS-vaccinated mice did not. DNA microarray analysis showed that the products of 29 vasculature-associated genes shared between genes upregulated by differentiation from iPS cells into iVP cells and genes shared by iVP cells and isolated Flk-1+ vascular cells in CMS-4 tumor tissue might be possible targets in the immune response. These results suggest that iVP cells from iPS cells could be used as a cancer vaccine targeting tumor vascular cells and tumor cells.