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Chapter 11.14 : Natural Killer Cell Assays

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Citation: Leber A. 2016. Natural Killer Cell Assays, p 11.14.1.1-11.14.6.7. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.14
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Figures

Image of Figure 11.14.2–1
Figure 11.14.2–1

Identification of NK cells and NK cell subsets. PBMC were stained using a dead cell discriminator stain followed by the NK cell anchor panel. NK cells were then identified by exclusion of doublets (A), gating on lymphocytes (B), exclusion of dead cells (C), exclusion of CD3 T cells, CD14 monocytes, and CD19 B cells (D), and gating on CD56 and/or CD16 cells (E). NK cell subsets were identified based on their expression level of CD56, taking the expression of CD16 into account (F).

Citation: Leber A. 2016. Natural Killer Cell Assays, p 11.14.1.1-11.14.6.7. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.14
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Image of Figure 11.14.2–2
Figure 11.14.2–2

Gating strategy for the KIR panel. PBMC were stained according to the strategy outlined for NK staining panel A. A gating strategy was optimized to identify certain KIR populations where specific antibodies were not available. After gating on NK cells as described in the legend to Fig. 11.14.2–1 , CD158b (which binds to both KIR2DL2 and KIR2DL3) was gated against KIR2DL3. Matched KIR genotyping data confirmed that individuals homozygous for KIR2DL2 displayed a CD158b KIR2DL3 phenotype (A), individuals homozygous for KIR2DL3 displayed a CD158b KIR2DL3 phenotype (B), and heterozygous individuals displayed populations of both CD158b KIR2DL3 and CD158b KIR2DL3 cells (C).

Citation: Leber A. 2016. Natural Killer Cell Assays, p 11.14.1.1-11.14.6.7. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.14
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Image of Figure 11.14.3–1
Figure 11.14.3–1

NK cell degranulation assay. Following stimulation for 5 h with medium, K562, 721.221, or antibody-coated p815 cells in the presence of anti-CD107a antibody, brefeldin A, and monensin, PBMC were stained according to the degranulation antibody panel in Table 11.14.5–1 . NK cells were identified as demonstrated in Fig. 11.14.2–1 , and the expression of CD107a and intracellular cytokines was assessed on this population. Robust but variable responses were observed in response to all cell lines, and some background CD107a and MIP1β expression was seen, as expected. Gates for CD107a and MIP1β were set using FMOs, whereas gates for tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ) were set based on the medium-only negative control.

Citation: Leber A. 2016. Natural Killer Cell Assays, p 11.14.1.1-11.14.6.7. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.14
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Image of Figure 11.14.4–1
Figure 11.14.4–1

NK cell fluorescence killing assay. K562 target cells were stained with the membrane dye PKH26 and the cytoplasmic dye CFSE before being incubated for 5 h with either medium or PBMC effector cells at an effector/target ratio of 10:1. A separate tube of effectors only was also made. After gating on the targets cells (A), the effector-only sample was used to exclude the effector cell population (B), and the target cell-only sample was used to set the gate for viable cells and to measure background (C). Killed cells were identified as PKH26 CFSE, and the killing ability was reported as the percentage of targets displaying this phenotype (D).

Citation: Leber A. 2016. Natural Killer Cell Assays, p 11.14.1.1-11.14.6.7. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.14
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References

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1. Moretta A, Bottino C, Mingari MC, Biassoni R, Moretta L. 2002. What is a natural killer cell? Nat Immunol 3:68.
2. Waggoner SN, Cornberg M, Selin LK, Welsh RM. 2012. Natural killer cells act as rheostats modulating antiviral T cells. Nature 481:394398.
3. Alter G, Altfeld M. 2011. Mutiny or scrutiny: NK cell modulation of DC function in HIV-1 infection. Trends Immunol 32:219224.
4. Paust S, Gill HS, Wang B-Z, Flynn MP, Moseman EA, Senman B, Szczepanik M, Telenti A, Askenase PW, Compans RW, von Andrian UH. 2010. Critical role for the chemokine receptor CXCR6 in NK cell-mediated antigen-specific memory of haptens and viruses. Nat Immunol 11:11271135.
5. Moffett-King A. 2002. Natural killer cells and pregnancy. Nat Rev Immunol 2:656663.
6. Lanier L. 2008. Up on the tightrope: natural killer cell activation and inhibition. Nat Immunol 9:495502.
1. Alter G, Malenfant JM, Altfeld M. 2004. CD107a as a functional marker for the identification of natural killer cell activity. J Immunol Methods 294:1522.
1. Sheehy ME, McDermott AB, Furlan SN, Klenerman P, Nixon DF. 2001. A novel technique for the fluorometric assessment of T lymphocyte antigen specific analysis. J Immunol Methods 249:99110.
2. Gomez-Roman VR, Florese RH, Patterson LJ, Peng B, Venzon D, Aldrich K, Robert-Guroff M. 2006. A simplified method for the rapid fluorometric assessment of antibody-dependent cell-mediated cytoxicity. J Immunol Methods 308:5367.
1. Bryant J, Day R, Whiteside TL, Herbeman RB. 1992. Calculation of lytic units for the expression of cell-mediated cytotoxicity. J Immunol Methods 146:91103.
11. Douglas SD, Durako S, Tustin NB, Houser J, Muenz L, Starr SE, Wilson C, Adolescent Medicine HIV/AIDS Research Network. 2001. Natural killer cell enumeration and function in HIV-infected and high-risk uninfected adolescents. AIDS Res Hum Retroviruses 17:543552.
12. Evans D, Lynch K, Benton T, Dube B, Gettes D, Tustin N, Lai J, Metzger D, Douglas S. 2008. Selective serotonin reuptake inhibitor and substance P antagonist enhancement of natural killer cell innate immunity in human immunodeficiency virus/acquired immunodeficiency syndrome. Biol Psychiatr 63:899905.
13. Hay R, Caputo J, Chen TR, Macy M, McClintock P, Reid Y (ed). 1994. American Type Culture Collection (ATCC) Catalogue of Cell Lines and Hybridomas, 8th ed. 1994 reference guide. American Type Culture Collection, Manassas, VA.
14. Lozzio CB, Lozzio BB. 1975. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45:321334.
15. Whiteside T, Rindalo CR, Herberman RB. 1992. Cytolytic cell functions, p 220–230. In Rose NR, Conway de Macario E, Fahey JL, Friedman H, Penn GM (ed), Manual of Clinical Laboratory Immunology, 4th ed. American Society for Microbiology, Washington, DC.
16. Whiteside TL. 2006. Measurement of NK cell activity in humans, p 296–300. In Detrick B, Hamilton RG, Folds JD (ed), Manual of Clinical Laboratory Immunology, 7th ed. ASM Press, Washington, DC.
1. Hay R, Caputo J, Chen TR, Macy M, McClintock P, Reid Y. 1994. American Type Culture Collection (ATCC) Catalogue of Cell Lines and Hybridomas, 8th ed. 1994 reference guide. American Type Culture Collection, Manassas, VA.
2. Lozzio CB, Lozzio BB. 1975. Human chronic myelogenous leukemia cell-line with positive Philadelphia chromosome. Blood 45:321334.

Tables

Generic image for table
Table 11.14.1–1

Overview of the main activating and inhibitory receptors on NK cells

Citation: Leber A. 2016. Natural Killer Cell Assays, p 11.14.1.1-11.14.6.7. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.14
Generic image for table
Table 11.14.2–1

Antibody combinations for composite analysis of NK cell phenotype

Citation: Leber A. 2016. Natural Killer Cell Assays, p 11.14.1.1-11.14.6.7. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.14
Generic image for table
Table 11.14.3–1

Antibody combinations for NK cell degranulation assay

Citation: Leber A. 2016. Natural Killer Cell Assays, p 11.14.1.1-11.14.6.7. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.14
Generic image for table
Table 11.14.5–1

Antibody combinations for redirected cytotoxicity assay

Citation: Leber A. 2016. Natural Killer Cell Assays, p 11.14.1.1-11.14.6.7. In Clinical Microbiology Procedures Handbook, Fourth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818814.ch11.14

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