Table 1
NK cells are large granular lymphocytes with a diameter ranging from 7
to 12 µm, depending on the activation state. Upon staining, these cells
are easily recognised among other circulating lymphocytes by their
reniform nucleus and by the presence of azurophilic granules in the
cytoplasm (Whiteside & Herberman, 1994). These characteristics are not
exclusive of NK cells, as activated cytotoxic T cells may also present
such phenotype. Therefore, for the correct identification of the NK
phenotype, CD3 must be absent, and CD56 (neural cell adhesion molecule,
NCAM) and CD16 (FcγRIII) must be present. Moreover, NK cells also lack
surface immunoglobulins (Ig) and constitutively express IL-2 receptors
(Nagler, Lanier & Phillips, 1990; Whiteside & Herberman, 1994). Many
other surface markers are present in NK cells, as well in other
lymphocytes. However, the unique combination of the
CD3- CD56+ CD16+phenotype defines human NK cells (Abakushina, 2015). Additionally, two
subsets of NK cells may be defined, depending on the relative expression
of CD16 and CD56. These are called
CD56brightCD16dim/- and
CD56dimCD16+, where bright and dim
are associated with high and low levels of expression, respectively. The
first subset is outnumbered by the second in circulation but constitutes
the majority of NK cells in lymphoid tissues. These subsets present
different expression levels of other markers and receptors and different
cytotoxic activities, with CD56bright being
significantly less cytotoxic than CD56dim cells (Poli,
Michel, Theresine, Andres, Hentges & Zimmer, 2009). It has been
hypothesized that CD56bright cells are an immature
form of NK cells that differentiate into CD56dim NK
cells, which participate in natural and antibody-mediated cell
cytotoxicity (Chan et al., 2007). On the other hand,
CD56bright NK cells express higher levels of cytokine
receptors, such as the IL-2 receptors αβγ and βγ (IL-2Rαβγ and IL-2Rβγ,
respectively), higher levels of some activating and inhibiting
receptors, and chemokine receptors. These cells produce higher levels of
cytokines upon stimulation, namely of IL-10, an immunosuppressive
cytokine, and therefore are thought to have immunoregulatory properties
(Cooper et al., 2001).
NK cells also display a broad range of cell adhesion molecules (CAMs)
essential for their cytotoxic activities, as these participate in
interactions between NK cells, target cells, and accessory cells to
generate effective immune responses. Some of these CAMs are upregulated
upon activation, including lymphocyte function-associated antigen 1
(LFA-1; CD11a/CD18), LFA-3 (CD58) and intracellular adhesion molecule-1
(CD54). Interestingly, upon in vitro stimulation with IL-2, the
cell surface levels of all these CAMs increase, as do the levels of
CD56. Even more interesting is the fact that, upon this increase in CAM
expression, the cytotoxic activity of NK cells towards NK-sensitive
target cells also increases, and cells that were previously
non-susceptible to NK-killing become targets (Robertson, Caligiuri,
Manley, Levine & Ritz, 1990).
The cytotoxic response of NK
cells
The cytotoxic response of NK cells upon activation is divided into 4
steps – formation of the immunological synapse, microtubule
re-organization, lysosome docking to the membrane, and lysosome fusion
with the membrane (Paul & Lal, 2017).
In the first, an interface between the NK cell and the target cell is
formed, deemed immunological synapse. This occurs when the NK cell
approaches the target, either accidentally or “intentionally” due to
chemotactic signalling.
Upon contact, CD2 molecules present on the surface of NK cells recognise
the presence of stage-specific embryogenic antigen 1 (SSEA-1, also known
as CD15 or Sialyl-LewisX). At this point, if the
target cell presents markers for NK cell inhibition, the formation of
the immunological synapse is halted. Conversely, if activating receptors
are present, tight adhesion between the two cells is promoted by
receptor-ligand interactions of high affinity. LFA-1 and MHC-1 molecules
present on NK cells bind ligands present on the target cell surface. The
formation of these receptor-ligand complexes is enough to activate the
cytolytic response, to some extent. However, these processes are
considered more relevant in maintaining the immunological synapse than
in triggering NK cell cytotoxicity. Full activation of the cytotoxic
response depends on the engagement of specific receptors, such as the
natural cytotoxicity receptors, present on the surface of NK cells. The
immunological synapse is shaped in a way that both cells form a
ring-shaped interface. Within this ring, cytotoxic granules and other
cytotoxicity mediators are released directly and in a controlled fashion
towards the target-cell surface (Orange, 2008; Stinchcombe & Griffiths,
2007). The secretory lysosome exocytosis requires reorganization of the
cell cytoskeleton. In this step, the microtubule organizing centre
(MTOC) becomes polarized and the secretory lysosomes are transported
along the microtubules towards the synapse. Upon reaching the cell
membrane, the granules dock and fuse, releasing the contents towards the
target. This formally constitutes degranulation (Paul & Lal, 2017;
Topham & Hewitt, 2009).
The molecular mechanisms of target-cell
killing
As referred before, contrarily to T cells, NK cells do not rely on the
somatic rearrangement of receptor genes to accommodate the expression of
a variety of receptors. Instead, NK cell receptors are germ-line-encoded
and consistently expressed (Biassoni, 2008; Whiteside & Herberman,
1994). The homeostasis of NK cell activity is ensured by a specific set
of receptors with activating and inhibitory activities, some of which
overlap. Inhibitory receptors contribute to self-tolerance of NK cells,
preventing the lysis of normal healthy cells. On the other hand,
activating receptors trigger the lytic activity of these cells,
prompting the destruction of cells that present activating ligands.
Upon activation, NK cells release a series of lytic enzymes through the
degranulation process already described, namely perforins and granzymes.
Granzymes induce target-cell apoptosis, but their actions depend on
being appropriately delivered by perforins (Boivin, Cooper, Hiebert &
Granville, 2009). Together with these enzymes, a wide variety of
cytokines is also released, including interferon γ (IFN-γ),
tumour-necrosis factor α (TNF-α), granulocyte-macrophage
colony-stimulating factor (GM-CSF), IL-10, IL-5 and IL-13, chemokine
macrophage inflammatory proteins 1α and 1β (MIP-1α, MIP-1β), IL-8, and
chemokine (C-C motif) ligand 5 (CCL5, also known as RANTES) (Paul &
Lal, 2017).
Besides the direct effect of granzymes (and perforins), cytokines such
as IFN-γ also play an important role in inducing target-cell death.
IFN-γ, as seen before, is an important activator of macrophages, as well
of APCs. Stimulation of the latter, as shown before, upregulates the
expression of cytokine IL-12, as well as IL-18 and co-stimulatory
molecules CD86, that enhance Th1 differentiation and CTL
function. IFN-γ can also exert antiproliferative effects on tumour cells
by enhancing the expression of the cell cycle inhibitor proteins p27Kip,
p16 or p2 (Ni & Lu, 2018). TNF-α is also implicated in the collapse of
tumour vasculature, but can additionally induce tumour regression by
triggering apoptosis, T cell activation (by Tregs’ blockage), and
neutrophil and monocyte chemoattraction to the tumour region, and by
downregulating the immunosuppressive phenotypes of tumours (Josephs et
al., 2018).
GM-CSF is a part of the inflammatory cascade, recruiting monocytes and
inducing the differentiation, proliferation, and migration of
granulocytes (neutrophils, eosinophils and basophils) to the
inflammation site. It is also essential for the differentiation of
dendritic cells that, as seen before, are responsible for processing and
presenting of tumour antigens and for activating cytotoxic T lymphocytes
(Yan, Shen, Tien, Chen & Liu, 2017).
IL-5 and IL-13 are associated with B cell growth, eosinophil activation,
and regulation of inflammatory and immune responses (Minty et al.,
1993). IL-10, on the other hand, seems to have a pleiotropic effect on
the immune response. It downregulates Th1 responses,
cytokine secretion, such as TNF-α, IFN-γ and IL-12, and
CD4+ T cell activation (de Waal Malefyt, Abrams,
Bennett, Figdor & de Vries, 1991; de Waal Malefyt et al., 1991).
However, IL-10 can also induce the secretion of granzymes and perforins
by CD8+ T cells and potentiate TCR-dependent IFN-γ
secretion (Emmerich et al., 2012). Lastly, MIP-1α/1β, IL-8 and CCL-5
have chemotactic activities towards granulocytes, neutrophils and T
cells, eosinophils and basophils (Kohidai & Csaba, 1998; Wolpe et al.,
1988).
Through the secretion of these cytokines and chemokines, NK cells are
able to recruit other immune cells, namely dendritic cells that can
infiltrate the tumour tissue and trigger robust and sustained immune
responses (Bottcher et al., 2018).
Balance between activation and inactivation of NK
cells
NK cells express a wide variety of receptors with activating and
inhibiting functions that allow for the fine discrimination between
healthy and ailing cells in a matter of seconds; the activating and
inhibitory signalling pathways involved are depicted in Figure
1 . Most of the inhibitory receptors detect the absence of MHC I
molecules, while activation receptors probe for the presence of specific
ligands that flag the target cells as harmful (Leung, 2014). Because NK
cells are extremely cytotoxic when active and can very quickly deliver
their lytic response without prior sensitization, contrarily to other
lymphocytes, a complex activation/inactivation system tightly regulates
their responses. Activation depends on tipping the balance between
stimulatory and inhibitory signals, an equilibrium that is dictated by
the engagement of different receptors. After cell-to-cell contact, NK
cells integrate, within seconds, the signals from their activating and
inhibiting surface receptors. Normal cells, expressing normal MHC I
molecules, will engage the inhibitory receptors, rendering NK cells
unresponsive, without compromising their functionality
(licensing ). Cells lacking MHC I and presenting surface
activating ligands will trigger the cytolytic response. A dynamic
equilibrium is reached when the target cells present both MHC I
molecules and activating ligands. In this case, no response is triggered
as the positive and negative feedback loops cancel each other. If more
activating ligands are engaged, the activation signal dominates and
target-cell lysis occurs (Figure 2 ) (Vivier, Ugolini, Blaise,
Chabannon & Brossay, 2012).
The engagement of different receptors is translated into activating and
inactivating signals through ITIM and ITAM motifs, present in either the
receptors themselves or in adaptor proteins. The ITAM and ITIM
signalling cascades are combined intracellularly to determine the
response of NK cells. Upon binding of activating ligands, the ITAM
motifs are phosphorylated, triggering the recruitment of Syk-family
kinases and the subsequent activation of cytolytic responses. However,
engagement of inhibitory receptors also leads to the phosphorylation of
ITIM motifs on these receptors, a process that is promoted by the same
Src kinase responsible for ITAM phosphorylation. These now
phosphorylated ITIMs recruit the tyrosine phosphatases SHP1 or 2, which
can in turn terminate intracellular signals emanating from ITAM
signalling receptors via their phosphatase activity, rendering NK cells
inactive (Figure 2 )(Linnartz-Gerlach, Kopatz & Neumann, 2014).