Eijiro Nagasaki

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.

Synergistic Induction of Antigen-Specific CTL by Fusions of TLR-Stimulated Dendritic Cells and Heat-Stressed Tumor Cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- γ at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+IFN-γ+CD8+ T cells and CD154+ IFN-γ+CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.

Regulation of Tumor Immunity by Tumor/Dendritic Cell Fusions

The goal of cancer vaccines is to induce antitumor immunity that ultimately will reduce tumor burden in tumor environment. Several strategies involving dendritic cells- (DCs)- based vaccine incorporating different tumor-associated antigens to induce antitumor immune responses against tumors have been tested in clinical trials worldwide. Although DCs-based vaccine such as fusions of whole tumor cells and DCs has been proven to be clinically safe and is efficient to enhance antitumor immune responses for inducing effective immune response and for breaking T-cell tolerance to tumor-associated antigens (TAAs), only a limited success has occurred in clinical trials. This paper reviews tumor immune escape and current strategies employed in the field of tumor/DC fusions vaccine aimed at enhancing activation of TAAs-specific cytotoxic T cells in tumor microenvironment.

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.

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.

Immunologic Monitoring of Cellular Responses by Dendritic/Tumor Cell Fusion Vaccines

Although dendritic cell (DC)- based cancer vaccines induce effective antitumor activities in murine models, only limited therapeutic results have been obtained in clinical trials. As cancer vaccines induce antitumor activities by eliciting or modifying immune responses in patients with cancer, the Response Evaluation Criteria in Solid Tumors (RECIST) and WHO criteria, designed to detect early effects of cytotoxic chemotherapy in solid tumors, may not provide a complete assessment of cancer vaccines. The problem may, in part, be resolved by carrying out immunologic cellular monitoring, which is one prerequisite for rational development of cancer vaccines. In this review, we will discuss immunologic monitoring of cellular responses for the evaluation of cancer vaccines including fusions of DC and whole tumor cell.

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.

Gemcitabine enhances rituximab‐mediated complement‐dependent cytotoxicity to B cell lymphoma by CD20 upregulation

Although rituximab, a chimeric monoclonal antibody that specifically binds to CD20, has significantly improved the prognosis for diffuse large B cell lymphoma (DLBCL), one‐third of DLBCL patients demonstrate resistance to rituximab or relapse after rituximab treatment. Thus, a novel approach to rituximab‐based treatment is likely to be required to improve the efficacy of DLBCL treatment. As complement dependent cytotoxicity (CDC) is a key mechanism mediating rituximabs tumoricidal activity, rituximab binding to CD20 on tumor cells is a critical factor for effective rituximab‐based treatments against DLBCL. We found that gemcitabine (GEM), but not lenalidomide (LEN) or azacitidine (AZA), can upregulate CD20 expression in TK and KML‐1 cells, two human DLBCL cell lines. Treatment of TK and KML‐1 cells with GEM enhanced CD20 expression at both the mRNA and protein levels. CD20 upregulation by GEM treatment was accompanied by increased rituximab binding to CD20. In TK cells, GEM treatment synergistically increased rituximab‐mediated CDC activity in a dose‐dependent manner. In KML cells, GEM treatment also induced upregulation of complement regulatory proteins, possibly leading to resistance to CDC. Treatment with LEN, a drug that did not upregulate CD20, did not enhance rituximab‐mediated CDC activity. GEM treatment activated nuclear factor‐kappa B (NF‐kB) signaling in these cells. Furthermore, a specific inhibitor to NF‐kB suppressed GEM‐induced CD20 upregulation, indicating that GEM‐induced NF‐kB activation is closely associated with CD20 upregulation. These results suggest that when used in combination, GEM might enhance the antitumor efficacy of rituximab against DLBCL due to its unique ability to upregulate CD20.

Up-regulation of HER2 by gemcitabine enhances the antitumor effect of combined gemcitabine and trastuzumab emtansine treatment on pancreatic ductal adenocarcinoma cells

BackgroundAlthough pancreatic ductal adenocarcinomas (PDAs) widely express HER2, the expression level is generally low. If HER2 expression in PDA cells could be enhanced by treatment with a given agent, then combination therapy with that agent and trastuzumab emtansine (T-DM1), a chemotherapeutic agent that is a conjugate of trastuzumab, might lead to significant antitumor effects against PDA.MethodsCell proliferation was examined by spectrophotometry. HER2 expression was examined by flow cytometry, immunoblot and quantitative reverse transcription polymerase chain reaction. T-DM1 binding to cells was examined by flow cytometry and enzyme-linked immunosorbent assay.ResultsOut of 5 tested human PDA cell lines, including MIA PaCa-2, three showed increases in HER2 expression after gemcitabine (GEM) treatment. The binding of T-DM1 to GEM-treated MIA PaCa-2 cells was higher than to untreated MIA PaCa-2 cells. Treatment with GEM and T-DM1 showed synergic cytotoxic effects on MIA PaCa-2 cells in vitro. Cells in the G2M phase of the cell cycle were retained after GEM treatment and showed higher levels of HER2 expression, possibly contributing to the synergic effect of GEM and T-DM1.ConclusionsCombined treatment with GEM and T-DM1 might confer a potent therapeutic modality against PDA as a result of GEM-mediated HER2 up-regulation.