Mitigation of regulatory T cell-mediated immunosuppression is crucial for optimal in vivo anti-tumor immune responses. In this study, we examined the anti-tumor effect induced by oral ingestion of an immunomodulating diet comprised of Lentinula edodes mycelia (L.E.M.) extract. C57BL ⁄ 6 mice were inoculated subcutaneously in the footpad with B16 melanoma and fed L.E.M. extract. Ingestion of L.E.M. extract significantly inhibited tumor growth, and this in vivo anti-tumor effect was not observed in nude mice, suggesting a T cell-dependent mechanism. In addition, ingestion of L.E.M. extract led to significant restoration of H-2K b -restricted and melanoma-reactive T cells in the spleen and draining lymph nodes of melanoma-bearing mice. Flow cytometry analysis revealed that the percentage of Foxp3 + CD4 + T cells increased in spleen and draining lymph nodes in melanoma-bearing mice, but decreased significantly with ingestion of L.E.M. extract. Importantly, selective depletion of CD8 + T cells abolished the L.E.M.-induced anti-tumor effect, whereas that of CD4 + T cells or CD25 + cells showed no additive influence on the effect. Real-time PCR analysis revealed that ingestion of L.E.M. extract by melanoma-bearing mice decreased the level of Foxp3 mRNA within the tumor tissues, and lowered plasma transforming growth factor (TGF)-b. Furthermore, an in vitro assay revealed that an immunosuppressive activity of CD4 + T cells from melanoma-bearing mice was canceled by ingestion of L.E.M. extract. Our results indicate that oral ingestion of L.E.M. extract restores immune responses of class I-restricted and melanoma-reactive CD8 + T cells in melanoma-bearing mice, presumably by a mitigation of regulatory T cells-mediated immunosuppression. (Cancer Sci 2011; 102: 516-521) A nticancer immunotherapy has received considerable attention recently as a new treatment modality. The immune system is capable of recognizing tumor antigens, and cancerreactive cytotoxic T lymphocytes are the most effective cancer destroying cells.(1-3) Although anticancer immunotherapies, including vaccines and adoptive immunotherapy, have been applied clinically, to date their efficacy has been unsatisfactory.(4) One explanation for the insufficient outcome of anticancer immunotherapy trials is the emergence of immunosuppressive cells, including CD4 + CD25 + regulatory T cells (Tregs) and ⁄ or myeloid-derived suppressor cells, in tumor-bearing states.(5,6) The presence of Tregs correlates strongly with unfavorable prognosis. (7,8) Treatment with antibodies or drugs has been proposed as a means of relieving Treg-mediated immunosuppression.(9-14) Nevertheless, safe and widely applicable treatment modalities to mitigate Treg-mediated immunosuppression in cancer patients must be therapeutically beneficial.A dried powder extracted from Lentinula edodes mycelia (L.E.M.) with hot water before germination and after culturing in a medium composed of bagasse and rice bran, (15) has been reported to exhibit anti-tumor activity and immunomodulatory effects in vit...

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Because cancer is associated with aging, immunological features in the aged should be considered in anticancer immunotherapy. In this study, we investigated antitumor immunity in aged mice using a CT26 colon carcinoma model. The tumor growth of CT26 was accelerated in aged mice compared with that in young mice, but this difference was not observed in nude mice. The serum levels of IL-6 and TNF-α were higher in aged mice than those in young mice, irrespective of the CT26-bearing state. The in vitro induction of CT26-specific CTLs from aged mice that were vaccinated with doxorubicin (DTX)-treated CT26 cells was impaired. In vivo neutralization of IL-6, but not TNF-α, showed a tendency to restore the in vitro induction of CT26-specific CTLs from vaccinated aged mice. Analyses on tumor-infiltrating immune cells as early as day 5 after CT26 inoculation revealed that monocytic and granulocytic MDSCs preferentially infiltrated into tumor sites in aged mice compared with young mice. Alternatively, oral administration of Lentinula edodes mycelia (L.E.M.) extract, which has the potential to suppress inflammation in tumor-bearing hosts, decreased the serum levels of IL-6 in aged mice. When administration of L.E.M. extract was started 1 week earlier, CT26 growth was retarded in aged mice and the in vivo priming of tumor-specific CTLs was improved in CT26-vaccinated aged mice. These results indicate early infiltration of MDSCs is related to impaired immunity of aged hosts and that oral administration of L.E.M. extract can mitigate the impairment.

Abstract. We previously reported that oral ingestion of Lentinula edodes mycelia (L.E.M.) extract can inhibit the growth of a subcutaneously established melanoma in a T cell-dependent manner via mitigation of regulatory T cell (Treg)-mediated immunosuppression. In this study, we tested the antitumor effect and mechanism of oral ingestion of L.E.M. extract following inoculation of murine colon carcinoma colon-26 (C26) cells into the subserosal space of the cecum (i.c.) of syngeneic mice. In this model, the primary site of the immune response was gut-associated lymphoid tissue (GALT), which is known to be an immunological toleranceinducing site for numerous dietary antigens. Oral ingestion of the L.E.M. extract suppressed the growth of i.c.-inoculated C26 cells in a T cell-dependent manner and restored the T cell response of the mesenteric lymph nodes and the spleen, not only to a tumor antigen-derived peptide, presented on H-2L d molecules, but also to C26 cells. I.c. inoculation of C26 cells increased the potential of CD4 + T cells of the mesenteric lymph nodes to produce transforming growth factor (TGF)-β, but ingestion of the L.E.M. extract decreased the ability of both CD4 + and CD8 + T cells in the mesenteric lymph nodes to produce this immunosuppressive cytokine. Although ingestion of L.E.M. showed only a marginal effect on Tregs in this model, this treatment significantly reduced the plasma levels of TGF-β and IL-6, both of which were increased in the i.c. C26-inoculated mice. In summary, our results indicate that oral ingestion of L.E.M. extract can restore antitumor T cell responses of mice even when the primary antitumor immune response is elicited in GALT, and provide important implications for anticancer immunotherapy of human colon cancer. IntroductionStudies on tumor-reactive cytotoxic T lymphocytes (CTLs) and tumor-related antigens have enabled the design of specific anticancer immunotherapies (1,2). Anticancer vaccines and adoptive immunotherapy have been applied clinically, although their efficacy has been unsatisfactory (3). Although several explanations of their unsatisfactory performance could be proposed, the major reason that these therapies fail is believed to be due to the emergence of immunosuppressive cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) (4,5). In addition, the tumor-bearing state is accompanied by chronic inflammation, and it is thought that the inflammatory state of cancer patients inhibits the efficacy of anticancer immunotherapy (6,7).Lentinula edodes mycelia extract (L.E.M.) is a dried powder of a hot water extract of the mycelia of L. edodes before germination, which were cultured in a medium composed of bagasse and rice bran (8). This L.E.M. extract has been reported to exhibit antitumor activity and immunomodulatory effects both in vitro and in vivo (9,10). L.E.M. can mitigate inflammation in the liver of mice (11), suggesting that it also has an anti-inflammatory effect. In addition, we recently reported that oral ingestion of ...

New anticancer vaccines must overcome regulatory T cell (Treg)-mediated immunosuppression. We previously reported that oral ingestion of Lentinula edodes mycelia (L.E.M.) extract restores melanoma-reactive T cells in melanoma-bearing mice via a mitigation of Treg-mediated immunosuppression. In this study, we investigated the effect of oral ingestion of the extract on peptide vaccine-induced anti-tumor activity. The day after subcutaneous inoculation in the footpad with B16 melanoma, mice were freely fed the extract and were vaccinated with a tyrosinase-related protein 2(180-188) peptide. The peptide vaccine was repeated thrice weekly. Melanoma growth was significantly suppressed in mice treated with both the peptide vaccine and L.E.M. extract compared with mice treated with vaccine or extract alone, and the effect was CD8(+) T cell-dependent. The combination therapy increased H-2K(b)-restricted and B16 melanoma-reactive T cells in the draining lymph nodes and spleen. Flow cytometric and immunohistological analyses revealed that the combination therapy significantly decreased the percentage of Tregs in the draining lymph nodes and spleen of melanoma-bearing mice compared to treatment with vaccine or extract alone. Kinetic analyses of peptide-specific T cells and Tregs revealed that induction of peptide-specific T cells by the peptide vaccine alone was transient, but when combined with L.E.M. extract, it efficiently prolonged the duration of peptide-specific T cell induction without increasing the percentage of Tregs. These results indicate that combination therapy enhances peptide vaccine-induced anti-tumor activity due to attenuation of the increase in the percentage of Tregs in tumor-bearing hosts.