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Chapter 32 : Measurement of NK Cell Phenotype and Activity in Humans

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Abstract:

Natural killer (NK) cells are a subset of innate lymphocytes initially identified for their ability to specifically kill virally infected and transformed cells without prior antigen sensitization. NK cells efficiently kill target cells through directed release of perforin-containing secretory lysosomes, a feature shared with cytotoxic T lymphocytes (CTLs) (1). Despite similarities in effector mechanisms, the strategies employed by NK cells and CTLs for target cell recognition are distinct yet complementary with respect to immune defense. Whereas CTLs express recombined, clonally distributed antigen receptors that dictate their activation and are selected for recognition of cells presenting nonself peptides in the context of major histocompatibility (MHC) class I molecules, NK cells rely on dynamic integration of signals from various germ line-encoded receptors for target cell discrimination. NK cells express numerous inhibitory receptors to detect normal expression of MHC class I and can selectively kill target cells that downregulate these molecules (2). NK cell activation by target cells with low MHC class I levels does not occur by default, but rather is mediated through engagement of different activating receptors (3). Representing an effector arm of humoral immunity, NK cells express the low-affinity Fc receptor CD16, which facilitates antibody-dependent cellular cytotoxicity (4). Moreover, supporting first-line defense against virally infected or stressed cells, a multiplicity of activating receptors that participate in natural cytotoxicity have been identified. In general, engagement of each such receptor alone is not sufficient to induce NK cell cytotoxicity. However, certain combinations of receptor signals can synergistically activate NK cell effector functions (5). Reflecting the expression of several activating receptors on NK cells that bind ligands exclusively expressed on hematopoietic cells, the ability of NK cells to kill autologous, activated immune cells is increasingly appreciated as an important immunoregulatory mechanism to control and shape adaptive immune responses (6). Upon activation, NK cells not only release granules but also abundantly produce chemokines and cytokines (7).

Citation: Chiang S, Bryceson Y. 2016. Measurement of NK Cell Phenotype and Activity in Humans, p 300-309. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch32
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Figures

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FIGURE 1

Gating strategy for flow cytometric analysis of NK cells. The strategy for consecutive gating of CD3CD56 NK cells is depicted. First, lymphocytes are gated on forward-scatter versus side-scatter characteristics. Second, single cells are gated on forward-scatter height versus forward-scatter area characteristics. Third, a histogram representing a dump channel with staining for dead cells, CD14 monocytes, and CD19 B cells. Viable, nonmonocyte, non-B cells are gated. Fourth, CD3CD56 NK cells are gated on a CD3 versus CD56 plot. Finally, phenotypic or functional readouts can be assessed in histograms.

Citation: Chiang S, Bryceson Y. 2016. Measurement of NK Cell Phenotype and Activity in Humans, p 300-309. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch32
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Image of FIGURE 2
FIGURE 2

Lytic granule expression in CD3CD56 NK cells from patients and healthy controls. PBMCs were stained and assessed according to protocol 1. Histograms depict expression of perforin (A, B) or granzyme B (C) in a patient with biallelic mutations (solid line) relative to a healthy control (dashed line) (A) or a patient with biallelic mutations (solid line) relative to a healthy control (dashed line) (B, C).

Citation: Chiang S, Bryceson Y. 2016. Measurement of NK Cell Phenotype and Activity in Humans, p 300-309. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch32
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Image of FIGURE 3
FIGURE 3

Functional responses by CD3CD56 NK cells from patients and healthy controls. PBMCs were mixed with target cells and assessed according to protocol 2. Histograms depict expression of CD107a, CD69, and TNF, as indicated, on NK cells from a healthy control or patients with biallelic mutations in , or . Expression in unstimulated cells (filled histograms), cells incubated with K562 cells (dashed lines), or P815 cells with added anti-CD16 mAb (solid lines) is depicted.

Citation: Chiang S, Bryceson Y. 2016. Measurement of NK Cell Phenotype and Activity in Humans, p 300-309. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch32
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Tables

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TABLE 1

Suggested antibody panels

Citation: Chiang S, Bryceson Y. 2016. Measurement of NK Cell Phenotype and Activity in Humans, p 300-309. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch32
Generic image for table
TABLE 2

Genes associated with strong defects in NK cell development or function

Citation: Chiang S, Bryceson Y. 2016. Measurement of NK Cell Phenotype and Activity in Humans, p 300-309. In Detrick B, Schmitz J, Hamilton R (ed), Manual of Molecular and Clinical Laboratory Immunology, Eighth Edition. ASM Press, Washington, DC. doi: 10.1128/9781555818722.ch32

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