Abstract
Natural killer (NK) cells provide a first line of defense against viral infections and prepare the ground for subsequent action of virus-specific T cells in a concerted way. Human NK cells use a sophisticated system of inhibitory and stimulatory receptors of the killer cell immunoglobulin-like receptor (KIR) gene family, which are expressed in a clonally distributed manner. Several studies suggest that KIR play a critical role in NK cell-mediated protection against HCV and HIV infection. As each NK cell expresses an individual set of KIR receptors that enables them to sense differences in HLA class I expression, classical measurement of NK cell function by analysis of target cell killing does not enable one to define and isolate the clinically relevant NK cell effector subsets. Here, we have developed a flow cytometry-based protocol to measure cytolytic activity together with KIR expression at a clonal level. Combined analysis of KIR expression in conjunction with cell surface mobilization of CD107 enables precise enumeration of cytolytic NK cells with defined specificity for HLA class I. Moreover, via inclusion of intracellular perforin or alternatively granzyme B, NK cells with deficient loading of cytotoxic granula can be identified. The present protocol enables identification and isolation of cytotoxic NK cells on a clonal level and enables reliable measurement in healthy as well as in pathological settings such as virus infection and hematological disease.
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References
Orange JS (2002) Human natural killer cell deficiencies and susceptibility to infection. Microbes Infect 4:1545–1558
Tiemessen CT, Shalekoff S, Meddows-Taylor S et al (2009) Cutting edge: unusual NK cell responses to HIV-1 peptides are associated with protection against maternal-infant transmission of HIV-1. J Immunol 182:5914–5918
Martin MP, Gao X, Lee JH et al (2002) Epistatic interaction between KIR3DS1 and HLA-B delays the progression to AIDS. Nat Genet 31:429–434
Jennes W, Verheyden S, Demanet C et al (2006) Cutting edge: resistance to HIV-1 infection among African female sex workers is associated with inhibitory KIR in the absence of their HLA ligands. J Immunol 177:6588–6592
Khakoo SI, Thio CL, Martin MP et al (2004) HLA and NK cell inhibitory receptor genes in resolving hepatitis C virus infection. Science 305:872–874
Trowsdale J, Barten R, Haude A, Stewart CA, Beck S, Wilson MJ (2001) The genomic context of natural killer receptor extended gene families. Immunol Rev 181:20–38
Valiante NM, Uhrberg M, Shilling HG et al (1997) Functionally and structurally distinct NK cell receptor repertoires in the peripheral blood of two human donors. Immunity 7:739–751
Schonberg K, Sribar M, Enczmann J, Fischer JC, Uhrberg M (2011) Analyses of HLA-C-specific KIR repertoires in donors with group A and B haplotypes suggest a ligand-instructed model of NK cell receptor aquisition. Blood 117:98–107
Yawata M, Yawata N, Draghi M, Little AM, Partheniou F, Parham P (2006) Roles for HLA and KIR polymorphisms in natural killer cell repertoire selection and modulation of effector function. J Exp Med 203:633–645
Anfossi N, Andre P, Guia S et al (2006) Human NK cell education by inhibitory receptors for MHC class I. Immunity 25:331–342
Yokoyama WM, Kim S (2006) Licensing of natural killer cells by self-major histocompatibility complex class I. Immunol Rev 214:143–154
Brunner KT, Mauel J, Cerottini JC, Chapuis B (1968) Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14:181–196
Jedema I, van der Werff NM, Barge RM, Willemze R, Falkenburg JH (2004) New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population. Blood 103:2677–2682
Betts MR, Brenchley JM, Price DA et al (2003) Sensitive and viable identification of antigen-specific CD8+ T cells by a flow cytometric assay for degranulation. J Immunol Methods 281:65–78
Penack O, Gentilini C, Fischer L et al (2005) CD56dimCD16neg cells are responsible for natural cytotoxicity against tumor targets. Leukemia 19(5):835–840
Peters PJ, Borst J, Oorschot V et al (1991) Cytotoxic T lymphocyte granules are secretory lysosomes, containing both perforin and granzymes. J Exp Med 173:1099–1109
Lieberman J (2003) The ABCs of granule-mediated cytotoxicity: new weapons in the arsenal. Nat Rev Immunol 3:361–370
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Schönberg, K., Hejazi, M., Uhrberg, M. (2012). Protocol for the Clonal Analysis of NK Cell Effector Functions by Multi-parameter Flow Cytometry. In: MacKenzie, C., Henrich, B. (eds) Diagnosis of Sexually Transmitted Diseases. Methods in Molecular Biology, vol 903. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-937-2_26
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DOI: https://doi.org/10.1007/978-1-61779-937-2_26
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Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-936-5
Online ISBN: 978-1-61779-937-2
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