Tomoyuki Tano

Expression of Toll-Like Receptor 4 on Dendritic Cells Is Significant for Anticancer Effect of Dendritic Cell-Based Immunotherapy in Combination with an Active Component of OK-432, a Streptococcal Preparation

A lipoteichoic acid-related molecule OK-PSA is an active component of OK-432, a Streptococcus-derived anticancer immunotherapeutic agent. In the present study, we first examined the effect of OK-PSA on the maturation of dendritic cells (DCs) in vitro by using the DCs derived from 5 healthy donors and 10 patients with head and neck cancer with or without expression of toll-like receptor 4 (TLR4) or MD-2 mRNA. OK-PSA treatment effectively increased the surface expression of MHC class II, CD80, CD83, and CD86. OK-PSA-stimulated DCs secreted the cytokines that can induce helper T-cell 1 (Th1)-type T-cell response, and stimulated allogeneic T cells to produce IFN-γ and to elicit an allogeneic antigen-specific cytotoxicity. These activities almost depended on expression of TLR4 and MD-2 genes. We next investigated the in vivo anticancer effect of intratumoral administration of syngeneic DCs followed by OK-PSA against established tumors in mice. C57BL/6 mice, which express wild-type TLR4, and C57BL/6-derived TLR4-knockout (TLR4−/−) mice were used. Although OK-PSA accelerated the antitumor effect of intratumoral DC administration in wild-type mice bearing syngeneic tumors, the antitumor effect of OK-PSA as well as of the combination therapy with DCs and OK-PSA was not significant in TLR4−/− mice. Interestingly, an administration of wild-type-mouse-derived DCs followed by OK-PSA exhibited a marked antitumor effect even in the TLR4−/− mice. These findings suggest that OK-PSA may be a potent adjuvant for local DC therapy, and that DC therapy followed by OK-PSA is able to elicit anticancer activity even in a TLR4-deficient host when TLR4 is expressed only in DCs injected intratumorally.

Mechanism of anticancer host response induced by OK-432, a streptococcal preparation, mediated by phagocytosis and Toll-like receptor 4 signaling

It has previously been reported by our group that Toll-like receptor (TLR) 4 is involved in anticancer immunity induced by OK-432, a Streptococcus-derived immunotherapeutic agent. However the detailed mechanism of the OK-432-induced immune response via TLR4 remained uncertain, because it may not be possible for OK-432, which consists of whole bacterial bodies, to bind directly to TLR4. In the current study, we conducted in vitro and in vivo experiments to investigate the hypothesis that OK-432 may first be captured and dissolved by phagocytes and that the active components released by the cells may then induce host responses via TLR4. TS-2 monoclonal antibody, which recognizes an active component of OK-432 designated OK-PSA was used in the current study. First, it was observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited in vitro by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining using TS-2 demonstrated that OK-432 was captured and dissolved by phagocytes. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by enzyme-linked immunosorbent assay using TS-2. Supernatants from OK-432-treated DC culture increased nuclear factor (NF)-κB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-κB in these cells. In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B. In this case, the amount of OK-PSA, an active component of OK-432, in the sera was also reduced by cytochalasin B. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the immune effect of OK-432.

Severe impairment of anti-cancer effect of lipoteichoic acid-related molecule isolated from a penicillin-killed Streptococcus pyogenes in toll-like receptor 4-deficient mice.

A lipoteichoic acid-related molecule (OK-PSA) isolated from OK-432, a penicillin-killed Streptococcus pyogenes, is a potent inducer of Th1 cytokines, and elicits anti-cancer effect in tumor-bearing mice. Toll-like receptor (TLR) 4 is a member of the recently identified toll-like receptor family of proteins that has been implicated in lipopolysaccharide-induced cell signaling. In the present study, we have examined the role of TLR4 for OK-PSA-induced Th1-cytokine production and anti-tumor effect by using C3H/HeJ mice in which TLR4 function is impaired. Although OK-PSA strikingly induced Th1 cytokines [interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-2, IL-12 and IL-18] in the splenocytes derived from control animals (C3H/HeN), OK-PSA did not induce the cytokines in the splenocytes from C3H/HeJ. Furthermore, C3H/HeJ-derived splenocytes acquired the responsiveness to OK-PSA stimulation by overexpression of TLR4 gene. Finally, OK-PSA administration significantly inhibited the tumor growth and lung metastasis of syngeneic squamous cell carcinoma cells in C3H/HeN; however, no effect of OK-PSA was observed in C3H/HeJ. These findings strongly suggest that TLR4 signaling is involved in regulating OK-PSA-induced anti-cancer immunity.

Anti-tumor effect of an intratumoral administration of dendritic cells in combination with TS-1, an oral fluoropyrimidine anti-cancer drug, and OK-432, a streptococcal immunopotentiator: Involvement of toll-like receptor 4

The authors investigated the in vivo anti-tumor effect of intratumoral administration of bone marrow-derived dendritic cells (DCs) after chemotherapy using an oral fluoropyrimidine anti-cancer drug TS-1, and followed by immunotherapeutic agent OK-432, in two syngeneic tumor-bearing mouse models. Both in Meth-A fibrosarcoma-bearing BALB/c mice and in SCCVII-bearing C3H/HeN mice, 1 week of oral administration of TS-1 effected partial eradication of established tumors. Intratumoral injection of DCs and OK-432 caused only slight inhibition of the tumor growth. However, TS-1 administration followed by DCs and OK-432 resulted in a marked inhibition in the tumor growth and also contributed to a greater prolongation of survival. By the injection of DCs and OK-432 after TS-1 administration, a significant infiltration of immune cells, especially CD8+ T cells, was observed. Furthermore, the cytotoxic activities of tumor-infiltrating lymphocytes and draining lymph node cells against inoculated tumor cells were significantly increased by the therapy, while activities against nonspecific target cells were not. Cytotoxic memory T cells were also induced; the main effectors were MHC class I-restricted, CD8+ T cells. The same therapy was also applied to SCCVII-bearing C3H/HeJ mice in which the Toll-like receptor (TLR) 4 is mutated and its function impaired; no immunotherapeutic effect was observed in the TLR4-deficient mouse model. These findings suggest that the local DC therapy in combination with TS-1 and OK-432 may be a useful strategy for the treatment of solid tumors, and that TLR4 signaling is involved in the success of this therapy.

Enhancement of anti-cancer immunity by a lipoteichoic-acid-related molecule isolated from a penicillin-killed group A Streptococcus

Abstract. We isolated the lipoteichoic-acid-related molecule (OK-PSA) from OK-432, a streptococcal preparation, by affinity chromatography on CNBr-activated Sepharose-4B-bound monoclonal antibody TS-2, which neutralizes the interferon (IFN)-γ-inducing activity of OK-432. We have previously reported that OK-PSA is a potent inducer of Th1-type cytokines in human peripheral blood mononuclear cells in vitro. In this study, we conducted an animal experiment to examine whether OK-PSA exhibits an anti-tumor effect in vivo by acting as a Th1 inducer in syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. It was found that OK-PSA induced Th1-type cytokines [IFN-γ, tumor necrosis factor-α, interleukin (IL)-2, IL-12 and IL-18] in BALB/c mice bearing Meth-A tumor and caused a marked anti-tumor effect. Although it was suggested by an in vitro study, using spleen cells derived from the animals, that IL-18 plays the greatest role in the induction of the Th1-dominant state and tumor cell killing induced by OK-PSA, the in vivo experiments demonstrated that both IL-12 and IL-18 are essential in the anti-tumor effect exhibited by OK-PSA. These findings strongly suggest that OK-PSA is a major effector molecule of OK-432 and may be a useful immunotherapeutic agent, as a potent Th1 inducer, for cancer patients with a Th2-dominant state.

Prognostic significance of interleukin-8 and CD163-positive cell-infiltration in tumor tissues in patients with oral squamous cell carcinoma

Purpose We investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC). Experimental Design Fifty OSCC patients who received radical resection of their tumor(s) were enrolled. Preoperative sera were measured for IL-8 by ELISA. Expression of IL-8 and the infiltration of immune cells in tumor tissues were analyzed by an immunohistochemical staining of surgical specimens. Results We found that disease-free survival (DFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p = 0.001). The tumor expression of IL-8, i.e., IL-8(T) and the density of CD163-positive cells in the tumor invasive front, i.e., CD163(IF) were correlated with the serum IL-8 level (p = 0.033 and p = 0.038, respectively), and they were associated with poor clinical outcome (p = 0.007 and p = 0.002, respectively, in DFS) in all patients. A multivariate analysis revealed that N status, IL-8(T) and CD163(IF) significantly affected the DFS of the patients. Further analysis suggested that combination of N status with serum IL-8, IL-8(T) or CD163(IF) may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Interestingly, the in vitro experiments demonstrated that IL-8 enhanced generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10. Conclusions These findings indicate that IL-8 may be involved in poor clinical outcomes via generation of CD163-positive M2 macrophages, and that these factors in addition to N status may have prognostic value in patients with resectable OSCSS.

Comparison of cytokine-inducing activity in a lipoteichoic acid-related molecule isolated from a penicillin-killed group A Streptococcus and from untreated bacteria.

We previously generated a monoclonal antibody, TS-2, that neutralizes the interferon (IFN)-gamma-inducing activity of OK-432, a penicillin-killed streptococcal preparation [J. Immunother. 13 (1993) 232]. Expression of the TS-2-binding antigen was markedly higher in the cell wall of the penicillin-treated Streptococcus pyogenes (OK-432) than in the untreated bacteria (Su-BBM). We here isolated the antigens from OK-432 and Su-BBM, designated OK-PSA and Su-PSA, respectively. OK-432 markedly induced IFN-gamma and interleukin (IL)-18 as compared with Su-BBM in human peripheral blood mononuclear cells (PBMC). Furthermore, all of the Thl-type and Th1-inducing cytokines tested [IFN-gamma, tumor necrosis factor (TNF)-alpha, IL-12 and IL-18] were secreted by OK-PSA-stimulated PBMC far better than by Su-PSA-treated PBMC. In addition, the cytolytic activities of the PBMC were accelerated by the stimulation with OK-432 or OK-PSA far better than by the stimulation with Su-BBM or Su-PSA. These findings strongly suggested that OK-PSA is a highly important molecule of OK-432 and may be a useful immunotherapeutic agent for the patients with malignant diseases as a potent Th inducer. It was also shown that penicillin treatment effectively enhances OK-PSA-induced anti-cancer immunity.

Antitumor effect of OK-432-derived DNA: one of the active constituents of OK-432, a streptococcal immunotherapeutic agent.

OK-432 is a Streptococcus-derived immunotherapeutic agent for malignancies. Our group has tried to identify the effective components of OK-432 and has succeeded in isolating a lipoteichoic acid–related preparation designated as OK-PSA, which is a strong inducer of T helper 1 (TH1) cells, and elicits an anticancer effect via Toll-like receptor (TLR) 4. Conversely, bacterial DNA with unmethylated CpG motifs can stimulate a TH1-type host response via TLR9. The unmethylated CpG DNA contained in OK-432 may play a role in its anticancer effect. In the current study, we investigated the effect of OK-432–derived DNA (OK-DNA) in augmenting the anticancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and MspI revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced TH1-type cytokines such as interferon-γ, tumor necrosis factor-α, interleukin (IL)-12, and IL-18 and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA–stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. The antitumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced compared with that in wild-type mice, whereas the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG DNA in OK-432 functions as an active component in OK-432–induced anticancer immunity via TLR9.

Toll-Like Receptor 4 Mediates the Antitumor Host Response Induced by a 55-Kilodalton Protein Isolated from Aeginetia indica L., a Parasitic Plant

ABSTRACT A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-κB-dependent reporter plasmid, AILb-A induced NF-κB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-κB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.

Th1-cytokine induction and anti-tumor effect of 55 kDa protein isolated from Aeginetia indica L., a parasitic plant

Abstract We have isolated a 55 kDa protein from the seed extract of Aeginetia indica L. (AIL), a parasitic plant, by affinity chromatography on an N-hydroxysuccinimide-activated Sepharose High Performance column bound with F3, a monoclonal antibody that neutralizes the cytokine-inducing and anti-tumor effect of AIL. In the present study, we examined this protein (AILb-A) for cytokine induction and anti-tumor effects by animal study, using syngeneic Meth-A tumor-bearing BALB/c mice, in which the Th2 response is genetically dominant. AILb-A administration resulted in markedly increased levels of Th1 cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, IL-12 and IL-18] in the sera derived from Meth-A-bearing mice. The in vitro re-stimulation with AILb-A of splenocytes derived from AILb-A-primed mice also selectively induced Th1-type cytokines and antigen-specific killer cell activity. The neutralizing test using cytokine-specific antibodies revealed that AILb-A-induced IL-18 plays a most significant role for IFN-γ- and killer cell-inducing activities. Furthermore, IL-12 and IL-18 induced by AILb-A inhibited specifically IL-10 and IL-4 production, respectively. Finally, we examined the anti-tumor effect of AILb-A in both Meth-A-bearing BALB/c mice and Meth-A-bearing nude mice with BALB/c background. AILb-A exhibited a striking anti-tumor effect in normal BALB/c mice inoculated with Meth-A cells. In athymic nude mice, the anti-tumor effect of AILb-A was relatively weak. These findings strongly suggested that AILb-A is a potent Th1 inducer and may be a useful immunotherapeutic agent for patients with malignant diseases.