Advertisement

A Flow Cytometric NK Cell-Mediated Cytotoxicity Assay to Evaluate Anticancer Immune Responses In Vitro

  • Seila Lorenzo-Herrero
  • Christian Sordo-Bahamonde
  • Segundo González
  • Alejandro López-SotoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1884)

Abstract

The immune system is able to detect and eliminate nascent and developing tumors. Thus, T lymphocytes of the adaptive immunity recognize cancer cells by detecting tumor-associated antigens, whereas certain innate immune cells scan for molecules that are mainly overexpressed on malignant and infected cells. The best example of the latter is natural killer (NK) cells. The activity of these immune cells is strictly governed by a balance between positive and negative signals provided by stress-regulated molecules that bind NK cell membrane receptors, such as the activatory receptor NKG2D. Given the key role of NK cells in eradicating cancer cells, the development of assays to study the cellular and molecular determinants of NK cell antitumor activity are of great relevance. In this chapter, we describe a flow cytometric in vitro assay to evaluate the cytotoxic activity of NK cells against a given target cell, including tumor cells. Moreover, this method is highly versatile, as it can be complemented with the use of antibodies blocking cell surface proteins (receptors or ligands) or a variety of chemical modulators, which allows the elucidation of molecules and signaling pathways that regulate NK cell anticancer function.

Key words

NK cells Cancer immunosurveillance NKG2D Blocking antibodies 

Notes

Acknowledgments

This work was supported by a Spanish grant from Instituto de Salud Carlos III (PI16/01485). S.L-H. holds a Severo Ochoa Fellowship by FICYT from Principado de Asturias, Spain (BP14–150).

References

  1. 1.
    Vivier E, Tomasello E, Baratin M, Walzer T, Ugolini S (2008) Functions of natural killer cells. Nat Immunol 9(5):503–510.  https://doi.org/10.1038/ni1582CrossRefPubMedGoogle Scholar
  2. 2.
    Caligiuri MA (2008) Human natural killer cells. Blood 112(3):461–469.  https://doi.org/10.1182/blood-2007-09-077438CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Waldhauer I, Steinle A (2008) NK cells and cancer immunosurveillance. Oncogene 27(45):5932–5943.  https://doi.org/10.1038/onc.2008.267CrossRefPubMedGoogle Scholar
  4. 4.
    Lopez-Soto A, Huergo-Zapico L, Acebes-Huerta A, Villa-Alvarez M, Gonzalez S (2015) NKG2D signaling in cancer immunosurveillance. Int J Cancer 136(8):1741–1750.  https://doi.org/10.1002/ijc.28775CrossRefPubMedGoogle Scholar
  5. 5.
    Gonzalez S, Lopez-Soto A, Suarez-Alvarez B, Lopez-Vazquez A, Lopez-Larrea C (2008) NKG2D ligands: key targets of the immune response. Trends Immunol 29(8):397–403.  https://doi.org/10.1016/j.it.2008.04.007CrossRefPubMedGoogle Scholar
  6. 6.
    Huergo-Zapico L, Acebes-Huerta A, Lopez-Soto A, Villa-Alvarez M, Gonzalez-Rodriguez AP, Gonzalez S (2014) Molecular bases for the regulation of NKG2D ligands in cancer. Front Immunol 5:106.  https://doi.org/10.3389/fimmu.2014.00106CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Orange JS, Ballas ZK (2006) Natural killer cells in human health and disease. Clin Immunol 118(1):1–10.  https://doi.org/10.1016/j.clim.2005.10.011CrossRefPubMedGoogle Scholar
  8. 8.
    Voskoboinik I, Whisstock JC, Trapani JA (2015) Perforin and granzymes: function, dysfunction and human pathology. Nat Rev Immunol 15(6):388–400.  https://doi.org/10.1038/nri3839CrossRefPubMedGoogle Scholar
  9. 9.
    Lopez-Soto A, Gonzalez S, Smyth MJ, Galluzzi L (2017) Control of metastasis by NK cells. Cancer Cell 32(2):135–154.  https://doi.org/10.1016/j.ccell.2017.06.009CrossRefPubMedGoogle Scholar
  10. 10.
    Lopez-Soto A, Folgueras AR, Seto E, Gonzalez S (2009) HDAC3 represses the expression of NKG2D ligands ULBPs in epithelial tumour cells: potential implications for the immunosurveillance of cancer. Oncogene 28(25):2370–2382.  https://doi.org/10.1038/onc.2009.117CrossRefPubMedGoogle Scholar
  11. 11.
    Guillerey C, Huntington ND, Smyth MJ (2016) Targeting natural killer cells in cancer immunotherapy. Nat Immunol 17(9):1025–1036.  https://doi.org/10.1038/ni.3518CrossRefPubMedGoogle Scholar
  12. 12.
    Muntasell A, Magri G, Pende D, Angulo A, Lopez-Botet M (2010) Inhibition of NKG2D expression in NK cells by cytokines secreted in response to human cytomegalovirus infection. Blood 115(25):5170–5179.  https://doi.org/10.1182/blood-2009-11-256479CrossRefPubMedGoogle Scholar
  13. 13.
    Lopez-Soto A, Huergo-Zapico L, Galvan JA, Rodrigo L, de Herreros AG, Astudillo A, Gonzalez S (2013) Epithelial-mesenchymal transition induces an antitumor immune response mediated by NKG2D receptor. J Immunol 190(8):4408–4419.  https://doi.org/10.4049/jimmunol.1202950CrossRefPubMedGoogle Scholar
  14. 14.
    Acebes-Huerta A, Lorenzo-Herrero S, Folgueras AR, Huergo-Zapico L, Lopez-Larrea C, Lopez-Soto A, Gonzalez S (2016) Drug-induced hyperploidy stimulates an antitumor NK cell response mediated by NKG2D and DNAM-1 receptors. Oncoimmunology 5(2):e1074378.  https://doi.org/10.1080/2162402X.2015.1074378CrossRefPubMedGoogle Scholar
  15. 15.
    Gasser S, Orsulic S, Brown EJ, Raulet DH (2005) The DNA damage pathway regulates innate immune system ligands of the NKG2D receptor. Nature 436(7054):1186–1190.  https://doi.org/10.1038/nature03884CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Aktas E, Kucuksezer UC, Bilgic S, Erten G, Deniz G (2009) Relationship between CD107a expression and cytotoxic activity. Cell Immunol 254(2):149–154.  https://doi.org/10.1016/j.cellimm.2008.08.007CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Seila Lorenzo-Herrero
    • 1
    • 2
  • Christian Sordo-Bahamonde
    • 1
    • 2
  • Segundo González
    • 1
    • 2
  • Alejandro López-Soto
    • 3
    Email author
  1. 1.Departamento de Biología Funcional, InmunologíaUniversidad of OviedoOviedoSpain
  2. 2.Instituto Universitario de Oncología del Principado de Asturias (IUOPA), Instituto de Investigación Sanitaria del Principado de Asturias (IISPA)OviedoSpain
  3. 3.Departamento de Biología Funcional, InmunologíaUniversidad de Oviedo, Instituto Universitario de Oncología del Principado de Asturias (IUOPA)OviedoSpain

Personalised recommendations