Isolation and Characterization of Innate Lymphoid Cells within the Murine Tumor Microenvironment

Part of the Methods in Molecular Biology book series (MIMB, volume 2121)


Innate lymphoid cells (ILCs) are important for both tissue immunity and tissue homeostasis. They are classified into three groups: Group 1 ILCs include NK cells, which are important in eliciting immunity against intracellular pathogens; group 2 ILCs protect against parasitic helminths; and group 3 ILCs protect against extracellular pathogens. The role of ILCs in cancer immunity remains unclear. In this chapter, we discuss methods for isolating and characterizing tumor-infiltrating ILC subsets within the tumor microenvironment in an experimental murine model of B16 melanoma. The chapter also highlights the expression of PD-1 on the various ILC subsets within the tumor microenvironment.

Key words

B16 Melanoma ILCs PD-1 



We would like to thank the Academy of Medical Sciences, Springboard Award [SBF003\1129] supported by Wellcome Trust and Newcastle University Research Fellowship for supporting this work. G.M. is supported by a Medical Research Council [MRC] Discovery Medicine North Ph.D. studentship. M.P. is supported by a Wellcome Trust Vacation Scholarship [216503/Z/19/Z].


  1. 1.
    Salimi M, Barlow JL, Saunders SP et al (2013) A role for IL-25 and IL-33-driven type-2 innate lymphoid cells in atopic dermatitis. J Exp Med 210(13):2939–2950CrossRefGoogle Scholar
  2. 2.
    Geremia A, Arancibia-Carcamo CV, Fleming MP et al (2011) IL-23-responsive innate lymphoid cells are increased in inflammatory bowel disease. J Exp Med 208(6):1127–1133CrossRefGoogle Scholar
  3. 3.
    Spits H, Artis D, Colonna M et al (2013) Innate lymphoid cells—a proposal for uniform nomenclature. Nat Rev Immunol 13(2):145–149CrossRefGoogle Scholar
  4. 4.
    Crellin NK, Trifari S, Kaplan CD et al (2010) Regulation of cytokine secretion in human CD127(+) LTi-like innate lymphoid cells by Toll-like receptor 2. Immunity 33(5):752–764CrossRefGoogle Scholar
  5. 5.
    Herberman RB, Nunn ME, Holden HT et al (1975) Natural cytotoxic reactivity of mouse lymphoid cells against syngeneic and allogeneic tumors. II. Characterization of effector cells. Int J Cancer 16(2):230–239CrossRefGoogle Scholar
  6. 6.
    Weizman OE, Adams NM, Schuster IS et al (2017) ILC1 confer early host protection at initial sites of viral infection. Cell 171(4):795–808.e12CrossRefGoogle Scholar
  7. 7.
    Klose CSN, Kiss EA, Schwierzeck V et al (2014) Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages. Cell 157(2):340–356CrossRefGoogle Scholar
  8. 8.
    Neill DR, Wong SH, Bellosi A et al (2010) Nuocytes represent a new innate effector leukocyte that mediates type-2 immunity. Nature 464(7293):1367–1370CrossRefGoogle Scholar
  9. 9.
    Huang Y, Guo L, Qiu J et al (2015) IL-25-responsive, lineage-negative KLRG1(hi) cells are multipotential ‘inflammatory’ type 2 innate lymphoid cells. Nat Immunol 16(2):161–169CrossRefGoogle Scholar
  10. 10.
    Sanos SL, Bui VL, Mortha A et al (2009) RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells. Nat Immunol 10(1):83–91CrossRefGoogle Scholar
  11. 11.
    Cella M, Fuchs A, Vermi W et al (2009) A human natural killer cell subset provides an innate source of IL-22 for mucosal immunity. Nature 457(7230):722–725CrossRefGoogle Scholar
  12. 12.
    Buonocore S, Ahern PP, Uhlig HH et al (2010) Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology. Nature 464(7293):1371–1375CrossRefGoogle Scholar
  13. 13.
    Sonnenberg GF, Monticelli LA, Ellosi MM et al (2011) CD4(+) lymphoid tissue-inducer cells promote innate immunity in the gut. Immunity 34(1):122–134CrossRefGoogle Scholar
  14. 14.
    Takatori H, Kanno Y, Watford WT et al (2009) Lymphoid tissue inducer-like cells are an innate source of IL-17 and IL-22. J Exp Med 206(1):35–41CrossRefGoogle Scholar
  15. 15.
    Morvan MG, Lanier LL (2016) NK cells and cancer: you can teach innate cells new tricks. Nat Rev Cancer 16(1):7–19CrossRefGoogle Scholar
  16. 16.
    Martini M, Testi MG, Passetto M et al (2010) IFN-gamma-mediated upmodulation of MHC class I expression activates tumor-specific immune response in a mouse model of prostate cancer. Vaccine 28(20):3548–3557CrossRefGoogle Scholar
  17. 17.
    Nakajima C, Uekusa Y, Iwasaki M et al (2001) A role of interferon-gamma (IFN-gamma) in tumor immunity: T cells with the capacity to reject tumor cells are generated but fail to migrate to tumor sites in IFN-gamma-deficient mice. Cancer Res 61(8):3399–3405PubMedGoogle Scholar
  18. 18.
    Beatty G, Paterson Y (2001) IFN-gamma-dependent inhibition of tumor angiogenesis by tumor-infiltrating CD4+ T cells requires tumor responsiveness to IFN-gamma. J Immunol 166(4):2276–2282CrossRefGoogle Scholar
  19. 19.
    Larmonier N, Cathelin D, Larmonier C et al (2007) The inhibition of TNF-alpha anti-tumoral properties by blocking antibodies promotes tumor growth in a rat model. Exp Cell Res 313(11):2345–2355CrossRefGoogle Scholar
  20. 20.
    Jovanovic IP, Pejnovic N, Radosavljevic GD et al (2014) Interleukin-33/ST2 axis promotes breast cancer growth and metastases by facilitating intratumoral accumulation of immunosuppressive and innate lymphoid cells. Int J Cancer 134(7):1669–1682CrossRefGoogle Scholar
  21. 21.
    Molofsky AB, Nussbaum JC, Liang HE et al (2013) Innate lymphoid type 2 cells sustain visceral adipose tissue eosinophils and alternatively activated macrophages. J Exp Med 210(3):535–549CrossRefGoogle Scholar
  22. 22.
    Huber S, Gagliani N, Zenewicz LA et al (2012) IL-22BP is regulated by the inflammasome and modulates tumorigenesis in the intestine. Nature 491(7423):259–263CrossRefGoogle Scholar
  23. 23.
    Chan IH, Jain R, Tessmer MS et al (2014) Interleukin-23 is sufficient to induce rapid de novo gut tumorigenesis, independent of carcinogens, through activation of innate lymphoid cells. Mucosal Immunol 7(4):842–856CrossRefGoogle Scholar
  24. 24.
    Schatton T, Murphy GF, Frank NY et al (2008) Identification of cells initiating human melanomas. Nature 451(7176):345–349CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Laboratory of T cell Regulation, Institute of Cellular MedicineNewcastle UniversityNewcastle upon TyneUK

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