Cancer Immunology, Immunotherapy

, Volume 68, Issue 12, pp 1935–1947 | Cite as

Regulatory T cells induce CD4 NKT cell anergy and suppress NKT cell cytotoxic function

  • Fumie Ihara
  • Daiju Sakurai
  • Mariko Takami
  • Toshiko Kamata
  • Naoki Kunii
  • Kazuki Yamasaki
  • Tomohisa Iinuma
  • Toshinori Nakayama
  • Shinichiro Motohashi
  • Yoshitaka OkamotoEmail author
Original Article



Due to the strong tumoricidal activities of activated natural killer T (NKT) cells, invariant NKT cell-based immunotherapy has shown promising clinical efficacy. However, suppressive factors, such as regulatory T cells (Tregs), may be obstacles in the use of NKT cell-based cancer immunotherapy for advanced cancer patients. Here, we investigated the suppressive effects of Tregs on NKT cells and the underlying mechanisms with the aim to improve the antitumor activities of NKT cells.


Peripheral blood samples were obtained from healthy donors, patients with benign tumors, and patients with head and neck squamous cell carcinoma (HNSCC). NKT cells, induced with α-galactosylceramide (α-GalCer), and monocyte-derived dendritic cells (DCs) were co-cultured with naïve CD4+ T cell-derived Tregs to investigate the mechanism of the Treg suppressive effect on NKT cell cytotoxic function. The functions and phenotypes of NKT cells were evaluated with flow cytometry and cytometric bead array.


Treg suppression on NKT cell function required cell-to-cell contact and was mediated via impaired DC maturation. NKT cells cultured under Treg-enriched conditions showed a decrease in CD4 NKT cell frequency, which exert strong tumoricidal responsiveness upon α-GalCer stimulation. The same results were observed in HNSCC patients with significantly increased effector Tregs.


Tregs exert suppressive effects on NKT cell tumoricidal function by inducing more CD4 NKT cell anergy and less CD4+ NKT cell anergy. Both Treg depletion and NKT cell recovery from the anergy state may be important for improving the clinical efficacy of NKT cell-based immunotherapy in patients with advanced cancers.


Head and neck cancer Regulatory T cell Immune suppression NKT cell Anergy 





Antigen-presenting cells


Carboxyfluorescein succinimidyl ester


Cytotoxic T-lymphocyte-associated antigen


Dendritic cells




Head and neck squamous cell carcinoma



NKT cells

Invariant natural killer T cells


Mean fluorescence intensity


Non-small cell lung cancer


Peripheral blood mononuclear cells


Programmed cell death ligand 1


Regulatory T cells



We thank Saori Tagi for her excellent technical assistance and thank Katie Oakley, PhD, from Edanz Group ( for editing a draft of this paper.

Author contributions

FI, DS, SM and YO designed the study. FI, DS, MT, TK performed experiments. FI, DS, MK, TK, NK, KY, TI, TN, SM and YO discussed the experimental design. MT and TK contributed to analysis and interpretation. NK, KY and TI contributed to collecting the samples and analyzing the data. FI and DS analyzed the data and wrote the manuscript. TN, SM and YO critically revised the manuscript. All authors reviewed the manuscript.


This study was funded by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) KAKENHI (Grant numbers 15K10799 and 17K16892).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval and ethical standards

Blood samples from all healthy donors and patients were collected only after obtaining written informed consent. All procedures involving human participants were approved on August 2, 2013 by the institutional review board of Chiba University Hospital and were conducted in accordance with the Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants included in the study. All participants consented to providing their peripheral blood and the use of their specimens and data for research and for publication.

Supplementary material

262_2019_2417_MOESM1_ESM.pdf (589 kb)
Supplementary material 1 (PDF 589 kb)


  1. 1.
    Vicari AP, Zlotnik A (1996) Mouse NK1.1+ T cells: a new family of T cells. Immunol Today 17:71–76CrossRefGoogle Scholar
  2. 2.
    Taniguchi M, Koseki H, Tokuhisa T, Masuda K, Sato H, Kondo E, Kawano T, Cui J, Perkes A, Koyasu S, Makino Y (1996) Essential requirement of an invariant Vα 14 T cell antigen receptor expression in the development of natural killer T cells. Proc Natl Acad Sci USA 93:11025–11028CrossRefGoogle Scholar
  3. 3.
    Kawano T, Cui J, Koezuka Y, Toura I, Kaneko Y, Motoki K, Ueno H, Nakagawa R, Sato H, Kondo E, Koseki H, Taniguchi M (1997) CD1d-restricted and TCR-mediated activation of Vα 14 NKT cells by glycosylceramides. Science 278:1626–1629CrossRefGoogle Scholar
  4. 4.
    Kawano T, Nakayama T, Kamada N, Kaneko Y, Harada M, Ogura N, Akutsu Y, Motohashi S, Iizasa T, Endo H, Fujisawa T, Shinkai H, Taniguchi M (1999) Antitumor cytotoxicity mediated by ligand-activated human Vα 24 NKT cells. Cancer Res 59:5102–5105PubMedGoogle Scholar
  5. 5.
    Motohashi S, Nakayama T (2008) Clinical applications of natural killer T cell-based immunotherapy for cancer. Cancer Sci 99:638–645CrossRefGoogle Scholar
  6. 6.
    Nieda M, Nicol A, Koezuka Y, Kikuchi A, Lapteva N, Tanaka Y, Tokunaga K, Suzuki K, Kayagaki N, Yagita H, Hirai H, Juji T (2001) TRAIL expression by activated human CD4+Vα 24NKT cells induces in vitro and in vivo apoptosis of human acute myeloid leukemia cells. Blood 97:2067–2074CrossRefGoogle Scholar
  7. 7.
    Toura I, Kawano T, Akutsu Y, Nakayama T, Ochiai T, Taniguchi M (1999) Cutting edge: inhibition of experimental tumor metastasis by dendritic cells pulsed with α-galactosylceramide. J Immunol 163:2387–2391PubMedGoogle Scholar
  8. 8.
    Uchida T, Horiguchi S, Tanaka Y, Yamamoto H, Kunii N, Motohashi S, Taniguchi M, Nakayama T, Okamoto Y (2008) Phase I study of α-galactosylceramide-pulsed antigen presenting cells administration to the nasal submucosa in unresectable or recurrent head and neck cancer. Cancer Immunol Immunother 57:337–345CrossRefGoogle Scholar
  9. 9.
    Motohashi S, Ishikawa A, Ishikawa E, Otsuji M, Iizasa T, Hanaoka H, Shimizu N, Horiguchi S, Okamoto Y, Fujii S, Taniguchi M, Fujisawa T, Nakayama T (2006) A phase I study of in vitro expanded natural killer T cells in patients with advanced and recurrent non-small cell lung cancer. Clin Cancer Res 12:6079–6086CrossRefGoogle Scholar
  10. 10.
    Kunii N, Horiguchi S, Motohashi S, Yamamoto H, Ueno N, Yamamoto S, Sakurai D, Taniguchi M, Nakayama T, Okamoto Y (2009) Combination therapy of in vitro-expanded natural killer T cells and α-galactosylceramide-pulsed antigen-presenting cells in patients with recurrent head and neck carcinoma. Cancer Sci 100:1092–1098CrossRefGoogle Scholar
  11. 11.
    Yamasaki K, Horiguchi S, Kurosaki M, Kunii N, Nagato K, Hanaoka H, Shimizu N, Ueno N, Yamamoto S, Taniguchi M, Motohashi S, Nakayama T, Okamoto Y (2011) Induction of NKT cell-specific immune responses in cancer tissues after NKT cell-targeted adoptive immunotherapy. Clin Immunol 138:255–265CrossRefGoogle Scholar
  12. 12.
    Frydrychowicz M, Boruczkowski M, Kolecka-Bednarczyk A, Dworacki G (2017) The dual role of Treg in cancer. Scand J Immunol 86:436–443CrossRefGoogle Scholar
  13. 13.
    Wing JB, Sakaguchi S (2014) Foxp3+ Treg cells in humoral immunity. Int Immunol 26:61–69CrossRefGoogle Scholar
  14. 14.
    Lindau D, Gielen P, Kroesen M, Wesseling P, Adema GJ (2013) The immunosuppressive tumour network: myeloid-derived suppressor cells, regulatory T cells and natural killer T cells. Immunology 138:105–115CrossRefGoogle Scholar
  15. 15.
    Alizadeh D, Larmonier N (2014) Chemotherapeutic targeting of cancer-induced immunosuppressive cells. Cancer Res 74:2663–2668CrossRefGoogle Scholar
  16. 16.
    Ihara F, Sakurai D, Horinaka A, Makita Y, Fujikawa A, Sakurai T, Yamasaki K, Kunii N, Motohashi S, Nakayama T, Okamoto Y (2017) CD45RAFoxp3high regulatory T cells have a negative impact on the clinical outcome of head and neck squamous cell carcinoma. Cancer Immunol Immunother 66:1275–1285CrossRefGoogle Scholar
  17. 17.
    Feng C, Cao LJ, Song HF, Xu P, Chen H, Xu JC, Zhu XY, Zhang XG, Wang XF (2015) Expression of PD-L1 on CD4+CD25+Foxp3+ regulatory T cells of patients with chronic HBV infection and its correlation with clinical parameters. Viral Immunol 28:418–424CrossRefGoogle Scholar
  18. 18.
    Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, Parizot C, Taflin C, Heike T, Valeyre D, Mathian A, Nakahata T, Yamaguchi T, Nomura T, Ono M, Amoura Z, Gorochov G, Sakaguchi S (2009) Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity 30:899–911CrossRefGoogle Scholar
  19. 19.
    Schmidt A, Oberle N, Krammer PH (2012) Molecular mechanisms of treg-mediated T cell suppression. Front Immunol 3:51PubMedPubMedCentralGoogle Scholar
  20. 20.
    Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, Kuchroo VK, Strom TB, Robson SC (2007) Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 204:1257–1265CrossRefGoogle Scholar
  21. 21.
    Amodio G, Gregori S (2012) Human tolerogenic DC-10: perspectives for clinical applications. Transplant Res 1:14CrossRefGoogle Scholar
  22. 22.
    Wing K, Sakaguchi S (2010) Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nat Immunol 11:7–13CrossRefGoogle Scholar
  23. 23.
    Mahnke K, Ring S, Johnson TS, Schallenberg S, Schonfeld K, Storn V, Bedke T, Enk AH (2007) Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation. Eur J Immunol 37:2117–2126CrossRefGoogle Scholar
  24. 24.
    Raker VK, Domogalla MP, Steinbrink K (2015) Tolerogenic dendritic cells for regulatory T cell induction in man. Front Immunol 6:569CrossRefGoogle Scholar
  25. 25.
    Schwartz RH, Mueller DL, Jenkins MK, Quill H (1989) T-cell clonal anergy. Cold Spring Harb Symp Quant Biol 54(Pt 2):605–610CrossRefGoogle Scholar
  26. 26.
    Schwartz RH (2003) T cell anergy. Annu Rev Immunol 21:305–334CrossRefGoogle Scholar
  27. 27.
    van den Heuvel MJ, Garg N, Van Kaer L, Haeryfar SM (2011) NKT cell costimulation: experimental progress and therapeutic promise. Trends Mol Med 17:65–77CrossRefGoogle Scholar
  28. 28.
    Robertson FC, Berzofsky JA, Terabe M (2014) NKT cell networks in the regulation of tumor immunity. Front Immunol 5:543CrossRefGoogle Scholar
  29. 29.
    Gumperz JE, Miyake S, Yamamura T, Brenner MB (2002) Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J Exp Med 195:625–636CrossRefGoogle Scholar
  30. 30.
    Takahashi T, Chiba S, Nieda M, Azuma T, Ishihara S, Shibata Y, Juji T, Hirai H (2002) Cutting edge: analysis of human Vα 24+CD8+ NK T cells activated by α-galactosylceramide-pulsed monocyte-derived dendritic cells. J Immunol 168:3140–3144CrossRefGoogle Scholar
  31. 31.
    Terabe M, Matsui S, Noben-Trauth N, Chen H, Watson C, Donaldson DD, Carbone DP, Paul WE, Berzofsky JA (2000) NKT cell-mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nat Immunol 1:515–520CrossRefGoogle Scholar
  32. 32.
    Singh AK, Gaur P, Shukla NK, Das SN (2015) Differential dendritic cell-mediated activation and functions of invariant NKT-cell subsets in oral cancer. Oral Dis 21:e105–e113CrossRefGoogle Scholar
  33. 33.
    Krijgsman D, Hokland M, Kuppen PJK (2018) The role of natural killer T cells in cancer-a phenotypical and functional approach. Front Immunol 9:367CrossRefGoogle Scholar
  34. 34.
    Hua J, Liang S, Ma X, Webb TJ, Potter JP, Li Z (2011) The interaction between regulatory T cells and NKT cells in the liver: a CD1d bridge links innate and adaptive immunity. PLoS ONE 6:e27038CrossRefGoogle Scholar
  35. 35.
    Oh KH, Lee C, Lee SW, Jeon SH, Park SH, Seong RH, Hong S (2011) Activation of natural killer T cells inhibits the development of induced regulatory T cells via IFNγ. Biochem Biophys Res Commun 411:599–606CrossRefGoogle Scholar
  36. 36.
    Kamata T, Suzuki A, Mise N, Ihara F, Takami M, Makita Y, Horinaka A, Harada K, Kunii N, Yoshida S, Yoshino I, Nakayama T, Motohashi S (2016) Blockade of programmed death-1/programmed death ligand pathway enhances the antitumor immunity of human invariant natural killer T cells. Cancer Immunol Immunother 65:1477–1489CrossRefGoogle Scholar
  37. 37.
    Parekh VV, Wilson MT, Olivares-Villagomez D, Singh AK, Wu L, Wang CR, Joyce S, Van Kaer L (2005) Glycolipid antigen induces long-term natural killer T cell anergy in mice. J Clin Invest 115:2572–2583CrossRefGoogle Scholar
  38. 38.
    Okita R, Saeki T, Takashima S, Yamaguchi Y, Toge T (2005) CD4+CD25+ regulatory T cells in the peripheral blood of patients with breast cancer and non-small cell lung cancer. Oncol Rep 14:1269–1273PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Fumie Ihara
    • 1
    • 2
  • Daiju Sakurai
    • 1
  • Mariko Takami
    • 2
  • Toshiko Kamata
    • 2
  • Naoki Kunii
    • 1
  • Kazuki Yamasaki
    • 1
  • Tomohisa Iinuma
    • 1
  • Toshinori Nakayama
    • 3
  • Shinichiro Motohashi
    • 2
  • Yoshitaka Okamoto
    • 1
    Email author
  1. 1.Department of Otorhinolaryngology, Head and Neck Surgery, Graduate School of MedicineChiba UniversityChibaJapan
  2. 2.Department of Medical Immunology, Graduate School of MedicineChiba UniversityChibaJapan
  3. 3.Department of Immunology, Graduate School of MedicineChiba UniversityChibaJapan

Personalised recommendations