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-␥ 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.
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)-kappaB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-kappaB 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.
PurposeWe investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC).Experimental DesignFifty 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.ResultsWe 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.ConclusionsThese 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.
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