DOI: 10.1002/JLB.

MR0718-269R

REVIEW

Mechanisms of natural killer cell-mediated cellular cytotoxicity

Isabel Prager Carsten Watzl

Department for Immunology, Leibniz Research

Centre for Working Environment and Human
Factors at TU Dortmund (IfADo),
Dortmund, Germany
Correspondence
Carsten Watzl, Leibniz Research Centre for
Working Environment and Human Factors
(IfADo) at TU Dortmund, Ardeystrasse 67, 44139
Dortmund, Germany.
Email: watzl@ifado.de
Abstract
Cellular cytotoxicity, the ability to kill other cells, is an important effector mechanism of the
immune system to combat viral infections and cancer. Cytotoxic T cells and natural killer (NK)
cells are the major mediators of this activity. Here, we summarize the cytotoxic mechanisms of
NK cells. NK cells can kill virally infected of transformed cells via the directed release of lytic
granules or by inducing death receptor-mediated apoptosis via the expression of Fas ligand or
TRAIL. The biogenesis of perforin and granzymes, the major components of lytic granules, is a
highly regulated process to prevent damage during the synthesis of these cytotoxic molecules.
Additionally, NK cells have developed several strategies to protect themselves from the cytotoxic
activity of granular content upon degranulation. While granule-mediated apoptosis is a fast pro-
cess, death receptor-mediated cytotoxicity requires more time. Current data suggest that these
2 cytotoxic mechanisms are regulated during the serial killing activity of NK cells. As many modern
approaches of cancer immunotherapy rely on cellular cytotoxicity for their effectiveness, unravel-
ing these pathways will be important to further progress these therapeutic strategies.

KEYWORDS
apoptosis, death receptors, degranulation, granzyme, perforin

1 INTRODUCTION
Natural killer (NK) cells are cytotoxic innate lymphocytes that can
lyse cancerous or virally infected cells.1 Their activity is regulated
by a variety of germline-encoded receptors that can mediate either
activating or inhibitory signals and thereby elicit NK cell responses
while also ensuring self-tolerance. NK cells can mediate their cyto-
toxic activity via 2 distinct pathways. They can release cytotoxic gran-
ules containing perforin and granzymes, or they can induce death
receptor-mediated apoptosis by expressing TRAIL and/or Fas ligand
(FasL) to engage TRAIL-R1/-R2 or CD95/Fas, respectively, on the sur-
face of diseased cells. NK cell or T cell-mediated cellular cytotoxicity
are essential for many approaches of immunotherapy against cancer.2
Antibody-based therapies such as the anti-CD20 antibody rituximab
against non-Hodgkin’s lymphoma depend on antibody-dependent
cellular cytotoxicity (ADCC) mediated by NK cells. Chimeric antigen
receptor T and NK cell therapies aim to directly target the activity of
Abbreviations: ADCC, antibody dependent cellular cytotoxicity; AIF, apoptosis-inducing
factor; DISC, death-inducing signaling complex; EGF, epidermal growth factor; ER,
endoplasmic reticulum; FasL, Fas ligand; M6P, mannose-6-phosphate; MACPF, membrane
attack complex/perforin; MTOC, microtubule organizing center; NK, natural killer; ROS,
reactive oxygen species; SB9, serpin B9.
these cytotoxic cells against tumors and checkpoint inhibitor therapy
enhances the activity of cytotoxic T cells. These examples highlight the
importance of understanding the mechanisms of cellular cytotoxicity.
In the following, we will summarize the current knowledge about the
cytotoxic mechanisms of NK cells.
2 PRODUCTION AND STORAGE
OF GRANZYME AND PERFORIN
IN LYTIC GRANULES
One mechanism of cellular cytotoxicity is the targeted release of
lytic granules that contain granzymes and perforin. The synthesis of
these cytotoxic proteins is tightly regulated. Granzymes are a family
of closely related serine proteases that are expressed in cytotoxic
T cells and NK cells.3 Most studies have investigated the biosynthesis
of granzyme B (Figure 1), which is expressed as a pre-pro-protein
(zymogen) containing an inhibitory dipeptide and an N-terminal
signal peptide that directs it to the endoplasmic reticulum (ER).4
After synthesis into the ER the signal peptide is cleaved off, resulting
in an inactive proenzyme. This proenzyme is then modified with a
mannose-6 phosphate (M6P) moiety by the N-acetylglucosamine-1-
phosphodiester 𝛼-N-acetyl-glucosaminidase in the cis compartment of

Received: 7 February 2019 Revised: 22 March 2019 Accepted: 14 April 2019

J Leukoc Biol. 2019;105:1319–1329. www.jleukbio.org 2019
c Society for Leukocyte Biology 1319
1320
cis
Golgi
ER
pro-
granzyme
Nucleus
pre-pro-
granzyme
F I G U R E 1 Biogenesis of granzymes. Most of the events described in this figure stem from studies investigating granzyme B. Granzymes are
synthesized into the ER as a pre-pro-protein. In the ER, the signal peptide is cleaved off, resulting in inactive progranzyme. In the cis-Golgi,
mannose-6-phosphate is added, providing the necessary signal for sorting into lytic granules. In the granules, mannose-6-phosphate may be
removed due to the acidic pH and the inhibitory dipeptide is cleaved by cathepsin C or cathepsin H. Active granzymes are stored in complex
with serglycin
the Golgi apparatus, which provides a sorting signal that is necessary
to translocate the proenzyme to the secretory granules via the M6P-
receptor.5 Inside the secretory granules, M6P may be removed due to
the acidic environment of pH 5.56 ; however, it was also suggested that
granzyme B may be able to retain its M6P modification.7 The inhibitory
dipeptide is then removed by the cysteine proteases cathepsin C or
cathepsin H, which transform the granzyme into its active form.5,8,9
Granzymes are then stored in their active form inside the lytic gran-
ules. However, their activity inside the granules is inhibited by the
low pH and by associating with the chondroitin sulfate proteoglycan
serglycin,10 thereby preventing the cleavage of host cell proteins. As
an alternative to this secretory pathway, some granzymes may also be
secreted via a constitutive nongranule biosynthesis pathway.11
Perforin consists of 3 domains: The N-terminal membrane attack
complex perforin like (MACPF)-dependent cytolysin pore-forming
domain, a central epidermal growth factor (EGF) domain, and a C-
terminal membrane and Ca2+ -binding C2 domain.12 The pore-forming
activity of perforin is dependent on its polymerization and it is pH
and Ca2+ -dependent. Therefore, perforin is not active at the acidic pH
of the lytic granules, even if Ca2+ is present.13,14 However, it is still
unclear how cytotoxic lymphocytes evade the toxicity of perforin while
processing and trafficking it from the ER, containing a neutral environ-
ment and high Ca2+ -levels, to the lytic granules. One hypothesis is that
perforin is expressed as a zymogen similar to granzymes, carrying a
C-terminal dodecapeptide to block Ca2+ -binding to its C2 domain.15
However, recombinant perforin with and without the dodecapeptide
shows the same pore-forming activity16 and the X-ray structure of per-
forin indicates that its C-terminus is diverted away from Ca2+ -binding
region of the C2 domain,17 thereby challenging this hypothesis. Addi-
tionally, Ca2+ binding to the C2 domain actually seems to be neces-
PRAGER AND WATZL
Granzyme B
trans
Mannose-6-Phosphate
Golgi
Inhibitory dipeptide
Signal peptide
Serglycin
lytic granule
Active
granzyme
?
pH 5.5 cathepsin
sary for perforin protein stability and it regulates the export of perforin
from the ER to the lytic granules.18 However, studies using endogenous
instead of recombinant perforin showed that N-linked glycosylation of
the C-terminal dodecapeptide inhibits its oligomerization and thereby
its pore-forming activity.19 This suggests a role of the glycosylated C-
terminal dodecapeptide in preventing premature pore formation dur-
ing the biosynthesis of perforin. Additionally, fast and highly efficient
trafficking of perforin monomers from the ER to the Ca2+ -poor Golgi
apparatus may be key to protect cytotoxic lymphocytes from perforin
toxicity.16 This may be supported by calreticulin, a chaperone in the
ER, which was also identified in lytic granules.20,21 Perforin contains
the binding motif sequence KVFF (single amino acid code), which could
mediate interaction with calreticulin and thereby limit its pore-forming
activity while in transit from the ER to the lytic granules.21–23 Also,
LAMP-1 (CD107a), which is widely used as a degranulation marker,
has a critical role in trafficking of perforin to the lytic granules.24 Once
inside the granules, the C-terminus of perforin is cleaved by cathep-
sin L25 and other proteases,19 enabling the activity of perforin once
it is secreted from the inhibitory acidic environment of the lytic gran-
ules. Additionally, there seems to be a second mechanism to transfer
newly synthesized perforin to the immunologic synapse independently
of secretory granules.26
3 THE ABC OF GRANZYMES
The family of human granzymes consists of 5 members: granzyme A,
granzyme B, granzyme H, granzyme K, and granzyme M.27 Together
with perforin and granulysin, they represent the major cytotoxic com-
ponents of secretory granules of NK cells and cytotoxic T cells. While
PRAGER AND WATZL
much is known about the functions of granzyme A and B (reviewed in
Refs. 27–31), the other granzymes have received little attention until
a few years ago, although they were also discovered in the late 1980s
and early 1990s. In the following section, we summarize the effect of
each granzyme individually. However, it should be noted that NK cells
release several different granzymes during their degranulation, so that
the killing of a target cell is likely a result of a combined action of
different granzymes.
3.1 Granzyme B
Granzyme B was initially named “cytotoxic T lymphocyte-associated
serine esterase 1” or granzyme 2. This serine protease has a wide
substrate specificity, but it preferentially cleaves after Asp residues.
One prominent substrate of granzyme B is caspase-3. Cleavage
of caspase-3 and of caspase-7 results in their activation and sub-
sequently induces caspase-dependent cell death by cleavage of
different cellular substrates, including a number of structural and regu-
latory proteins in the nucleus, in the cytosol and at the cytoskeleton.32
In addition to this, granzyme B can also induce cell death by cleav-
age of the BH3-only protein Bid.33 The resulting truncated form, tBid,
then relocates to the mitochondria where it interacts with the pro-
apoptotic proteins Bax and/or Bak.34 This results in a disruption of
the mitochondrial membrane integrity,35 the release of apoptotic fac-
tors such as smac/DIABLO, cytochrome c, apoptosis-inducing factor
(AIF), and Omi/HtrA2,33 and subsequently to apoptosis of the cell.
This granzyme B-induced apoptosis can be inhibited by overexpres-
sion of the antiapoptotic protein Bcl-2.35,36 Interestingly, human and
mouse granzyme B show differences in their substrate specificity.37–39
While granzyme B from both species can cleave and activate caspase-
3 and caspase-7, only human granzyme B is very efficient in cleaving
Bid. Therefore, caspase inhibitors, Bid deletion or Bcl-2 overexpression
can have different effects on granzyme B-mediated cell death depend-
ing on the species. This has created some controversies in the field in
the past and it is important to note that other granzymes also show
species-specific differences.39–41
3.2 Granzyme A
Granzyme A has a trypsin-like activity and cleaves substrates after
basic residues Arg or Lys. It can induce a fast form of cell death
independently of caspases and without the release of apoptotic fac-
tors from mitochondria, creating large DNA fragments, which are not
detectable by usual apoptosis assays.42–45 Within minutes, it activates
the production of reactive oxygen species (ROS) from the mitochon-
dria, cripples the mitochondrial electron transport and disrupts the
mitochondrial membrane potential.46 It targets the ER-associated SET
complex, which translocates to the nucleus where SET is cleaved by
granzyme A, releasing 2 nucleases, which mediate DNA damage.47
The exonuclease TREX1 is in the SET complex and acts in concert
with NM23–H1 to degrade DNA during granzyme A-mediated cell
death.48 Other granzyme A substrates include HMG-2,49 lamins,50 his-
tone H1,51 Ku70,50,52 and the DNA damage sensor poly(adenosine
5′ -diphosphate-ribose) polymerase-1.53 Conflicting reports describe
that cell death induced by mouse granzyme A does not require cas-
1321
pase activation, mitochondrial disruption, or the generation of ROS,
but instead induces a novel form of cell death termed “athetosis,”
which was dependent on the actin cytoskeleton.54 Therefore, several
aspects of granzyme-induced cell death are controversial.41 Granzyme
A also has species-specific effects. Human granzyme A was reported
to be poorly cytotoxic39 and may even be more involved in promoting
proinflammatory immune responses,55 thereby challenging the above
described cell death inducing activity.
3.3 Granzyme K
Similar to granzyme A, granzyme K has a trypsin-like activity and
cleaves substrates after basic residues Arg or Lys.56 It induces rapid
caspase-independent cell death with the production of ROS from mito-
chondria, chromatin condensation, and apoptotic nuclear morphol-
ogy analogous to granzyme A.57–59 It also cleaves the nucleosome
assembly protein SET to induce single-stranded DNA nicks.57,58 There-
fore, granzyme K seems to be a “backup” granzyme for the activ-
ity of granzyme A, which is supported by the fact that granzyme
A-deficient cytotoxic T cells can still mediate cell death via granzyme
K.60 Granzyme K also shows some unique function by cleaving the
pre-mRNA-binding protein heterogeneous ribonuclear protein K.61
However, granzyme K-deficient mice do not show impaired antiviral
responses, suggesting that granzyme K does not play an essential role
in this process.62
3.4 Granzyme M
Granzyme M has an unusual enzyme specificity, preferring cleavage
after methionine or leucine.63 Together with perforin it rapidly induces
cell death that is independent of caspases, fails to trigger hallmark
mitochondrial changes, occurs in the absence of discernible DNA frag-
mentation and cannot be inhibited by Bcl-2.64 One granzyme M sub-
strate is the nucleolar phosphoprotein nucleophosmin, which induces
cell death upon cleavage.65 Additionally, granzyme M can cleave and
inactivate Serpin B9 (SB9),66 an inhibitor of granzyme B, which likely
promotes granzyme B-induced apoptosis after degranulation. In con-
trast, Serpin B4 was found to be an inhibitor of granzyme M-induced
cell death.67 Granzyme M is specifically expressed in NK cells, CD56+
T cells and 𝛾𝛿-T cells with little to no detectable expression in CD4 or
CD8 T cells.68
3.5 Granzyme H
Not much is known about the function of granzyme H, which shows
homology to granzyme B, but which does not seem to have an ortholog
in rodents. Despite its homology, granzyme H has a chymotrypsin-
like activity with a cleavage preference for hydrophobic amino acid
residues (Phe or Tyr)69 and is predominantly expressed in NK cells.70
Granzyme H was shown to cleave the adenovirus DNA-binding pro-
tein, thereby interfering with viral replication in infected cells and to
inactivate the adenovirus 100K assembly protein, a major inhibitor
of granzyme B.71 Additionally, it can cleave the cellular phosphopro-
tein La, which is necessary for hepatitis C virus replication,72 demon-
strating interesting antiviral activities of this granzyme. Granzyme H
1322 PRAGER AND WATZL

A NK cell B
Granule Vesicle

Granzyme

Perforin FasL
TRAIL
TRAIL R FAS
FADD

Caspase
Granzyme –8 or –10

BID Caspase-
cleavage
Caspase-
Target cell independent
Mitochondrium

Apoptosis

F I G U R E 2 Two ways to kill. (A) Granule-mediated cytotoxicity is initiated by the targeted release of lytic granules toward a locally attached
target cell. Granzymes can then enter the target cell by perforin-pores in the plasma membrane (left) or by endocytosis and perforin-aided escape
from endosomes (right). Granzymes can then induce caspase activation, mitochondrial dysfunction, or caspase-independent apoptosis. (B) Death
receptor-mediated cytotoxicity is induced by surface expression of FasL or TRAIL, which can engage and activate their respective receptor. This
results in caspase activation, mitochondrial dysfunction, and apoptosis

can induce cell death with mitochondrial depolarization, ROS, DNA
degradation, and chromatin condensation, which is independent of
caspase or Bid cleavage and does not involve cytochrome c release
from mitochondria.73
3.6 Granulysin
Granulysin was found as a gene to be induced 3–5 days after T cell
activation.74 It is expressed by cytotoxic T cells and NK cells and
belongs to the saposin-like protein family.75 Granulysin is synthesized
as a 15 kDa molecule, which is then processed to a 9 kDa form by
proteolytic cleavage within the lysosomal compartment.76 The pro-
cessed Granulysin is found in lytic granules, whereas the 15 kDa
form can be constitutively secreted.77 Granulysin has cytolytic activ-
ity against tumors and microbes, including Gram-positive and Gram-
negative bacteria, fungi/yeast and parasites.78–81 Like other members
of the saposin-like protein family, it has pore-forming activity, which
alters membrane permeability and results in osmotic lysis and mito-
chondrial damage.82,83
4 NK CELL DEGRANULATION AND ENTRY
OF GRANZYMES INTO THE TARGET CELL
The killing of virally infected or malignant transformed cells by NK
cells is a multistep process, which is initiated by integrin-mediated
adhesion of the NK cell to the target cell84,85 and the formation of
the immunologic synapse (reviewed in Ref. 86). Lytic granules are
transported along the microtubules in the direction of the microtubule
organizing center (MTOC) using dynein motor proteins and are then
polarized toward the synapse together with the MTOC.87,88 At the
synapse, the granules fuse with the plasma membrane with the help
of small GTPases of the Rab family, MUNC proteins and the formation
of a SNARE complex, resulting in the release of granular content into
the synaptic cleft (reviewed in Ref. 89). The synapse forms a gasket-
like structure with the help of LFA-1-mediated adhesion, which lim-
its the diffusion of granular content and promotes granule-mediated
cytotoxicity.90
In the neutral pH of the extracellular space perforin can bind to
the target cell membrane and in the presence of Ca2+ the monomers
aggregate and form pores in the membrane (Figure 2).91 These
ring-shaped pores have a structure equivalent to a transmembrane
channel with a pore lumen of 13–20 nm17 that may even coa-
lesce into larger pores by recruiting additional perforin oligomers.92
As granzyme B has a stokes radius of 5 nm, it should be able to
enter the target cell directly through the perforin pores,93 which
was demonstrated in recent studies.94,95 As an alternative mech-
anism of granzyme entry into target cells, it was proposed that
granzymes are taken up by the target cell via endocytosis.96 This may
be facilitated by binding of granzyme B to the mannose 6-phosphate
receptor7,97 or by membrane repair processes induced by perforin-
mediated membrane damage and Ca2+ influx.98 Granzymes would
then escape their endocytotic vesicles with the help of perforin.99
However, given the speed of perforin pore formation at the plasma
membrane that was shown to deliver granzymes within 80 s,95 it is
likely that the direct delivery of granzymes through perforin pores at
the plasma membrane is the dominant pathway for granzyme deliv-
ery. However, it cannot be excluded that a slower endocytic delivery
PRAGER AND WATZL
of granzymes operates in parallel as a potential backup mechanism
to guarantee efficient delivery of granzymes into the cytosol of the
target cell.
5 SELF-PROTECTION OF NK CELLS
NK cells and cytotoxic T cells have various mechanisms to prevent
cellular damage during the synthesis and the storage of perforin and
granzymes. But what protects these cells once the content of the lytic
granules is released into the synaptic cleft? In other words, why does
perforin not attack the NK cell membrane, delivering granzymes into
the cytosol and thereby killing the NK cell? The delivery of perforin
pores at the synapse appears to be unidirectional only toward the tar-
get cell95 and cytotoxic lymphocytes were shown to have some form of
resistance toward cell-mediated cytotoxicity.100 Several mechanisms
have been discussed to account for this resistance. First, cytotoxic
T cells and NK cells express the granzyme B inhibitor SB9 in their
cytosol, which may protect them from misdirected granzyme B that
escapes the exocytotic pathway.101 SB9 contains a C-terminal reac-
tive center loop that represents a substrate for granzyme B.102 Cleav-
age of this SB9 loop by granzyme B induces a rapid conformational
change in the serpin, resulting in the formation of a stable serpin–
proteinase complex, effectively inactivating granzyme B.103,104 As a
second mechanism, it was shown that CD107a/LAMP-1 protects the
NK cell from degranulation-associated self-injury.105 CD107a is exter-
nalized during the degranulation of lytic granules and thereby lines
the NK cell plasma membrane at the immunologic synapse. CD107a
can inhibit the binding of perforin to the NK cell plasma membrane,
thereby preventing damage to the NK cell. A similar potential regu-
lator of perforin-mediated lysis is the lipid packing. It was proposed
that certain phospholipids are more tightly packed in the membrane
of the effector cell, which can reduce the capacity of perforin to
bind to or to intercalate with the lipid bilayer.106 As a final mech-
anism, it was proposed that cathepsin B, which also reaches the
plasma membrane of cytotoxic lymphocytes during degranulation, can
cleave and inactivate perforin.107 Interestingly, tumor cells may use
the same mechanism of secreting cathepsin B at the synapse to escape
killing by cytotoxic T cells.108 However, in cathepsin B-deficient mice,
cytotoxic lymphocytes did not show enhanced apoptosis during the
killing of target cells,109 demonstrating that cathepsin B is not nec-
essary for the self-protection of cytotoxic cells. However, it is likely
that the protection of cytotoxic cells from their own lytic machinery
is such an important aspect that there are several redundant mecha-
nisms at play to guarantee the survival of the cell even if one of these
mechanisms should fail.
6 DEATH RECEPTOR-MEDIATED KILLING
In addition to the exocytosis of lytic granules, cytotoxic lymphocytes
can also kill target cells by engaging so-called death receptors. There
are 3 different receptor/ligand systems that can mediate apoptosis
upon activation: TNF, binding to either TNF receptor-1 or -2, FasL
engaging the CD95 (APO-1/Fas) receptor (reviewed in Ref. 110), and
1323
TRAIL, which can bind to different TRAIL receptors (reviewed in
Ref. 111). Cytotoxic lymphocytes can use all 3 receptor systems to kill
target cells. In the following, we will focus on the regulation of FasL and
TRAIL-mediated killing (Figure 2).
7 FasL-MEDIATED CYTOTOXICITY
The FasL is a 40-kDa type II transmembrane protein belonging to the
TNF superfamily.112 It can be expressed on the cell surface of acti-
vated T cells and NK cells.113 After synthesis in the ER, it is transported
via the Golgi apparatus to secretory lysosomes. For this trafficking,
a proline-rich domain in the cytoplasmic tail of FasL is important.114
Kinases associate with this domain, resulting in phosphorylation of
FasL, which is essential for its trafficking.115 In addition, ubiquitina-
tion of sequences adjacent to the proline-rich region is necessary for
the correct sorting of FasL to secretory granules. As FasL is stored
in these secretory granules, surface expression is dependent on the
degranulation of T cells and NK cells.116,117 However, there seem to
exist 2 distinct species of secretory granules in T cells and NK cells
that either contain perforin and granzymes or FasL118–120 indicating
that preformed FasL is stored in secretory granules that are distinct
from classical cytotoxic granules that contain perforin and granzymes.
Therefore, the signals necessary for the externalization and surface
expression of FasL may be different from the ones required for clas-
sical degranulation.121 FasL brought to the surface by degranulation
will quickly diffuse in the plasma membrane,122 but diffusion may be
constrained within the synapse by LFA-1-mediated adhesion.90 Alter-
natively, FasL may be transiently exposed to the surface by incom-
plete granule fusion.122 Upon surface expression of FasL it can engage
and multimerized the CD95 receptor on a locally attached target cell
and thereby activate its apoptotic signaling cascade. Membrane-bound
FasL can be cleaved by metalloproteinases123–125 and the resulting sol-
uble FasL does not exert cytotoxicity126 and may in some cases even
inhibit apoptosis induced by membrane-bound FasL.126–129 Stimula-
tion of the CD95 receptor activates an intracellular signaling program,
which starts with the assembly of the death-inducing signaling complex
(DISC), leading to the activation of caspases 8 and 10, subsequently
resulting in a caspase cascade, the depolarization of the mitochon-
drial membrane potential and ultimately in the induction of apoptosis
(reviewed in Ref. 110).
8 TRAIL-mediated cytotoxicity
TRAIL is a type II transmembrane protein with homology to FasL
and TNF.130 It is expressed by cytotoxic cells such as T cells131 and
NK cells,132 but it can also be found on other cells such as mono-
cytes, macrophages and dendritic cells.132,133 While freshly isolated
NK cells do not express detectable levels of TRAIL on their surface,
stimulation with IL-2, IL-15 or IL-12 can induce the surface expres-
sion of TRAIL.134,135 TRAIL-mediated cytotoxicity has been shown to
be important for NK cell-mediated control of viral infections and can-
cer, especially for liver NK cells.135–137 In Jurkat T cells TRAIL was
1324
shown to colocalize with CD107a/LAMP1 positive vesicles.138 How-
ever, the mechanism of how TRAIL traffics to the lysosomal compart-
ment and if it is stored in the same vesicles as FasL is unknown. It is also
unclear, how target cell exposure can induce the surface expression of
TRAIL within the synapse. In case of cytokine-induced expression, it is
unlikely that TRAIL is concentrated at any specific part of the NK cell
surface. Therefore, the question arises how this surface expression can
contribute to directed target cell killing. In addition to the membrane-
bound form, TRAIL can also be shed by the activity of an unknown cys-
teine protease.130 However, in contrast to soluble FasL, soluble TRAIL
can retain its apoptotic activity and potentially result in killing of cells
within a close proximity of TRAIL secreting NK cells.
Human TRAIL can bind to 4 distinct membrane receptors and 1
soluble receptor.111 TRAIL-R1 and TRAIL-R2 are the only receptors
that can induce apoptosis,139,140 whereas TRAIL-R3, TRAIL-R4 and
osteoprotegerin mostly induce NF-𝜅B signaling and may function as
decoy receptors to limit TRAIL binding to the pro-apoptotic TRAIL
receptors.139,141,142 Proapoptotic signaling of TRAIL-R1 and TRIAL-
R2 is very similar to CD95-mediated signaling, with the formation of
a DISC and the activation of caspase-8.111
9 KINETICS OF GRANULE- AND DEATH
RECEPTOR-MEDIATED CYTOTOXICITY
Cytotoxic T cells and NK cells can use the release of perforin and
granzymes from lytic granules and the surface expression of ligands
for death receptors to kill virally infected or transformed cells (Fig-
ure 2). When comparing the kinetics of these 2 cytotoxic mechanisms,
several studies documented that death receptor-mediated killing is
slower.143–145 For example, while lytic granule-mediated killing can
be observed within minutes after target cell contact, death receptor-
mediated killing by perforin-deficient cytotoxic cells can take 1–2 h.146
There are several possible explanations for this kinetic difference.
As perforin and granzymes were suggested to be in different gran-
ules compared to FasL or TRAIL, their timing of mobilization may
differ. CD107a externalization as a marker for the release of lytic
granules147 can be observed within minutes after NK cell activation.
We only observed clearly detectable accumulation of FasL on the sur-
face after about 30 min to 1 h after target cell contact (unpublished
data), whereas others observed low levels of FasL within 15–30 min.117
Another explanation could be that the dose necessary to induce tar-
get cell death may differ between perforin/granzyme and FasL-induced
killing. While the release of as little as 2–4 lytic granules was shown to
be sufficient to induce target cell death148 it may take more degranu-
lation events to accumulate the amount of surface FasL necessary to
induce apoptosis. And finally, the kinetics of the 2 cell death pathways
could differ inside the target cell. Engagement of CD95 induces recep-
tor micro-aggregates, followed by the assembly of the DISC and the
induction of larger receptor clusters.149 DISC formation can be seen
within just 1 min after receptor triggering, while the formation of larger
clusters takes about 15 min. In addition, 2 different CD95 apoptosis
pathways were described.150 In type I cells, caspase 8 is activated at
the DISC in quantities sufficient to directly activate caspase 3. How-
PRAGER AND WATZL
ever, in type II cells, formation of the DISC is inefficient and only little
caspase 8 is activated, which is insufficient to process caspase 3, but
sufficient to cleave the BH3-only protein Bid, resulting in the apopto-
genic activation of mitochondria and making CD95-induced apoptosis
in type II cells sensitive to Bcl-2 expression. Therefore, depending on
the cell type, FasL-induced apoptosis is a complex process that involves
many regulated steps. In contrast, granzyme B can directly cleave and
activate caspase 3 upon entry of the target cell and thereby induce
a very efficient and fast apoptotic program. The combination of dif-
ferences in mobilization, necessary dose and apoptotic signaling cas-
cades are probably responsible for the differences in kinetics between
granule- and death receptor-mediated cytotoxicity.
10 SERIAL KILLING
Soon after the discovery of cellular cytotoxicity, it was shown
that individual cytotoxic T cells can kill multiple targets in a serial
fashion.151–153 More recent studies have shown a similar serial killing
activity for NK cells.154–156 While the number of target cells killed by
an individual NK cell varies between studies, it became clear that only a
minority of NK cells is responsible for the majority of killing events.156
However, it is currently unclear how to identify and how to isolate this
subpopulation of serial killing NK cells. It will be important to charac-
terize these cells, as they would be ideal effector cells for therapeutic
applications of NK cells. The killing of multiple targets by a single
NK cell is a strictly serial process, as the NK cell polarizes its lytic gran-
ules with the help of the microtubules and the MTOC,157 facilitated
by signals from the integrin LFA-1.117 However, MTOC polarization
may not be strictly necessary for degranulation.158 This polarization is
needed for efficient cytotoxicity and for preventing bystander killing.
However, this also implies that the NK cell may only form 1 lytic
synapse with a target cell at a time. After a successful kill, the NK cell
needs to establish a new contact with another target cell. For this, the
detachment from the killed target cell is necessary. Interestingly, also
this detachment is a regulated process,159 and it may be aided by the
downregulation or shedding of activating receptors.160,161 Once a new
contact is established, the killing process can start all over again. How-
ever, there is a limit to how many targets an individual NK cell can
kill. The degranulation of as little as 2–4 granules was shown to be
sufficient to cause cell death.148 However, NK cells release about 10%
of their total granules in a single killing event.148 Therefore, during
the process of serial killing the NK cells lose the majority of their
preformed perforin and granzyme B.154 While this may be compen-
sated over time by de-novo synthesis, NK cells also lose activating
receptors from their surface159–161 and signaling pathways may be
attenuated. These events limit the serial killing activity of NK cells.
We recently developed fluorescence localization reporters to mea-
sure granule and death receptor-mediated killing of tumor cells by
NK cells.162 Using these reporters, we could show that NK cells use
granule-mediated killing for their first serial killing events, while
they switch to death receptor-mediated killing once their perforin
and granzymes have been depleted (unpublished results). This sug-
gests that NK cells alternate their cytotoxic mechanisms in a kinetic
PRAGER AND WATZL
fashion during serial killing. Cellular cytotoxicity and serial killing are
crucial for the effectiveness of monoclonal antibody-based anticancer
therapeutics154,160 and likely contribute to the effectiveness of many
cellular immunotherapies against cancer. Therefore, it will be impor-
tant for future studies to identify subpopulations of NK cells with the
best serial killing abilities and to find ways to enhance this activity.
11 CONCLUDING REMARKS
Cellular cytotoxicity is a very important immune-effector mechanism.
However, this deadly mechanism requires many regulatory steps to
ensure that cytotoxic cells do not damage themselves. Additionally,
cellular cytotoxicity has to be precisely targeted in order to prevent
damage to bystanders. This creates a mechanism with many differ-
ent layers of regulation and may explain why defects in the cyto-
toxic machinery not only cause defective immune responses against
pathogens but also result in immune-mediated pathologies themselves
(reviewed in Ref. 163). In the future, it will be important to investi-
gate which cytotoxic mechanism is used by specific subpopulations of
cytotoxic cells and under which circumstances. Additionally, species-
specific differences will have to be addressed in order to solve some
controversies in the field.
AUTHORSHIP
I.P. and C.W. jointly prepared and wrote the manuscript.
ACKNOWLEDGEMENTS
We thank all members of our laboratory for fruitful discussions and
input.
DISCLOSURES
The authors declare no conflicts of interest.
ORCID
Carsten Watzl https://orcid.org/0000-0001-5195-0995
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How to cite this article: Prager I, Watzl C. Mechanisms of
natural killer cell-mediated cellular cytotoxicity. J Leukoc
Biol. 2019;105:1319–1329. https://doi.org/10.1002/JLB.
MR0718-269R