Cancer Immunology, Immunotherapy

, Volume 61, Issue 11, pp 2143–2152 | Cite as

Combining a peptide vaccine with oral ingestion of Lentinula edodes mycelia extract enhances anti-tumor activity in B16 melanoma-bearing mice

  • Kousuke Tanaka
  • Satoru Ishikawa
  • Yasunori Matsui
  • Takashi Kawanishi
  • Makoto Tamesada
  • Nanae Harashima
  • Mamoru Harada
Original Article


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 2180–188 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-2Kb-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.


Regulatory T cells Cytotoxic T-lymphocyte Immunotherapy Biological response modifier Melanoma Tumor peptide vaccine 


Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

262_2012_1275_MOESM1_ESM.ppt (58 kb)
Supplementary material 1 (PPT 58 kb)


  1. 1.
    Rosenberg SA, Yang JC, Schwartzentruber DJ et al (1998) Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med 4:321–327PubMedCrossRefGoogle Scholar
  2. 2.
    Nestle FO, Alijagic S, Gilliet M et al (1998) Vaccination of melanoma patients with peptide-or tumor lysatepulsed dendritic cells. Nat Med 4:328–332PubMedCrossRefGoogle Scholar
  3. 3.
    Jager E, Gnjatic S, Nagata Y et al (2000) Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers. Proc Natl Acad Sci USA 97:12198–12203PubMedCrossRefGoogle Scholar
  4. 4.
    Oka Y, Tsuboi A, Fujiki F et al (2009) WT1 peptide vaccine as a paradigm for “cancer antigen-derived peptide”-based immunotherapy for malignancies: successful induction of anti-cancer effect by vaccination with a single kind of WT1 peptide. Anticancer Aqents Med Chem 9:787–797Google Scholar
  5. 5.
    Mine T, Sato Y, Noguchi M et al (2004) Humoral responses to peptides correlate with overall survival in advanced cancer patients vaccinated with peptides based on pre-existing, peptide-specific cellular responses. Clin Cancer Res 10:929–937PubMedCrossRefGoogle Scholar
  6. 6.
    Schwartzentruber DJ, Lawson DH, Richards JM et al (2011) gp100 peptide vaccine and interleukin-2 in patients with advanced melanoma. N Engl J Med 364:2119–2127PubMedCrossRefGoogle Scholar
  7. 7.
    Rosenberg SA, Yang JC, Restifo NP (2004) Cancer immunotherapy: moving beyond current vaccines. Nat Med 10:909–915PubMedCrossRefGoogle Scholar
  8. 8.
    Yamaguchi T, Sakaguchi S (2006) Regulatory T cells in immune surveillance and treatment of cancer. Semin Cancer Biol 16:115–123PubMedCrossRefGoogle Scholar
  9. 9.
    Ostrand-Rosenberg S, Sinha P (2009) Myeloid-derived suppressor cells: linking inflammation and cancer. J Immunol 182:4499–4506PubMedCrossRefGoogle Scholar
  10. 10.
    Curiel TJ, Coukos G, Zou L et al (2004) Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 10:942–949PubMedCrossRefGoogle Scholar
  11. 11.
    Sato E, Olson SH, Ahn J et al (2005) Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci USA 102:18538–18543PubMedCrossRefGoogle Scholar
  12. 12.
    Loeffler M, Kruger JA, Reisfeld RA (2005) Immunostimulatory effects of low-dose cyclophosphamide are controlled by inducible nitric oxide synthase. Cancer Res 65:5027–5030PubMedCrossRefGoogle Scholar
  13. 13.
    Wada S, Yoshimura K, Hipkiss EL et al (2009) Cyclophosphamide augments antitumor immunity: studies in an autochthonous prostate cancer model. Cancer Res 69:4309–4318PubMedCrossRefGoogle Scholar
  14. 14.
    Ghiringhelli F, Larmonier N, Schmitt E et al (2004) CD4+ CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol 34:336–344PubMedCrossRefGoogle Scholar
  15. 15.
    Onizuka S, Tawara I, Shimizu J, Sakaguchi S, Fujita T, Nakayama E (1999) Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res 59:3128–3133PubMedGoogle Scholar
  16. 16.
    Tawara I, Take Y, Uenaka A, Noguchi Y, Nakayama E (2002) Sequential involvement of two distinct CD4+regulatory T cells during the course of transplantable tumor growth and protection from 3-methylcholanthrene-induced tumorigenesis by CD25-depletion. Jpn J Cancer Res 93:911–916PubMedCrossRefGoogle Scholar
  17. 17.
    Yamaguchi T, Hirota K, Nagahama K et al (2007) Control of immune responses by antigen-specific regulatory T cells expressing the folate receptor. Immunity 27:145–159PubMedCrossRefGoogle Scholar
  18. 18.
    Tanaka K, Ishikawa S, Matsui Y, Tamesada M, Harashima N, Mamoru H (2011) Oral ingestion of Lentinula edodes mycelia extract inhibits B16 melanoma growth via mitigation of regulatory T cell-mediated immunosuppression. Cancer Sci 102:516–521PubMedCrossRefGoogle Scholar
  19. 19.
    Harada M, Tamada K, Abe K et al (1998) Characterization of B16 melanoma-specific cytotoxic T lymphocytes. Cancer Immunol Immunother 47:198–204PubMedCrossRefGoogle Scholar
  20. 20.
    Kojima H, Akaki J, Nakajima S, Kamei K, Tamesada M (2010) Structural analysis of glycogen-like polysaccharides having macrophage-activating activity in extracts of Lentinula edodes mycelia. J Nat Med 64:16–23PubMedCrossRefGoogle Scholar
  21. 21.
    Zeh HJ III, Perry-Lalley D, Dudley ME, Rosenberg SA, Yang JC (1999) High avidity CTLs for two self-antigens demonstrate superior in vitro and in vivo antitumor efficacy. J Immunol 162:989–994PubMedGoogle Scholar
  22. 22.
    Kato Y, Adachi Y, Ohno N (2008) Characterization of rat beta-glucan receptor dectin-1. Microbiol Immunol 52:418–428PubMedCrossRefGoogle Scholar
  23. 23.
    Manicassamy S, Ravindran R, Deng J et al (2009) Toll-like receptor 2-dependent induction of vitamin A-metabolizing enzymes in dendritic cells promotes T regulatory responses and inhibits autoimmunity. Nat Med 15:401–409PubMedCrossRefGoogle Scholar
  24. 24.
    Muraoka D, Kato T, Wang L et al (2010) Peptide vaccine induces enhanced tumor growth associated with apoptosis induction in CD8+ T cells. J Immunol 185:3768–3776PubMedCrossRefGoogle Scholar
  25. 25.
    Zhou LD, Zhang QH, Zhang Y, Liu J, Cao YM (2009) The shiitake mushroom-derived immuno-stimulant lentinan protects against murine malaria blood-stage infection by evoking adaptive immune-responses. Int Immunopharmacol 9:455–462PubMedCrossRefGoogle Scholar
  26. 26.
    Lu H, Yang Y, Gad E et al (2011) Polysaccharide krestin is a novel TLR2 agonist that mediates inhibition of tumor growth via stimulation of CD8T cells and NK cells. Clin Cancer Res 17:67–76PubMedCrossRefGoogle Scholar
  27. 27.
    Lee KS, Scanga CA, Bachelder EM, Chen Q, Snapper CM (2007) TLR2 synergizes with both TLR4 and TLR9 for induction of the MyD88-dependent splenic cytokine and chemokine response to Streptococcus pneumoniae. Cell Immunol 245:103–110PubMedCrossRefGoogle Scholar
  28. 28.
    Yoshioka Y, Tamesada M, Nagayama A (2009) The safety of excessive intake of the food containing extract of cultured Lentinula edodes mycelia (LEM) in healthy adult volunteers. JCAM 6:9–15 (in Japanese)Google Scholar
  29. 29.
    Yoshioka Y, Matsui Y, Kobayashi M et al (2010) Safety evaluation of extract from cultured Lentinula edodes mycelia; study of acute toxicity, genotoxicity and inhibiting effect of drug-metabolizing enzyme, cytochrome P-450 3A4. JCAM 7:51–57 (in Japanese)Google Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Kousuke Tanaka
    • 1
  • Satoru Ishikawa
    • 1
  • Yasunori Matsui
    • 1
  • Takashi Kawanishi
    • 1
  • Makoto Tamesada
    • 1
  • Nanae Harashima
    • 2
  • Mamoru Harada
    • 2
  1. 1.Central R & D Laboratory Kobayashi Pharmaceutical Co., Ltd.Ibaraki-city, OsakaJapan
  2. 2.Department of ImmunologyShimane University Faculty of MedicineIzumo, ShimaneJapan

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