REVIEWS

Perforin and granzymes: function,

dysfunction and human pathology
Ilia Voskoboinik1,2, James C. Whisstock3,4 and Joseph A. Trapani1,2
Abstract | A defining property of cytotoxic lymphocytes is their expression and regulated
secretion of potent toxins, including the pore-forming protein perforin and serine protease
granzymes. Until recently, mechanisms of pore formation and granzyme transfer into the
target cell were poorly understood, but advances in structural and cellular biology have
now begun to unravel how synergy between perforin and granzymes brings about
target cell death. These and other advances are demonstrating the surprisingly broad
pathophysiological roles of the perforin–granzyme pathway, and this has important
implications for understanding immune homeostasis and for developing immunotherapies
for cancer and other diseases. In particular, we are beginning to define and understand a
range of human diseases that are associated with a failure to deliver active perforin to
target cells. In this Review, we discuss the current understanding of the structural, cellular
and clinical aspects of perforin and granzyme biology.

Perforinopathies
A range of human
immune-mediated disorders
that are caused by impaired
perforin delivery or function.
1
Cancer Immunology
Program, Peter MacCallum
Cancer Centre, East
Melbourne, Victoria 3002,
Australia.
2
Sir Peter MacCallum
Department of Oncology,
University of Melbourne,
Parkville, Victoria 3010,
Australia.
3
Department of Biochemistry
and Molecular Biology,
Monash University, Clayton,
Victoria 3800, Australia.
4
Australian Research Council
Centre of Excellence in
Advanced Molecular Imaging,
Monash University, Clayton,
Victoria 3800, Australia.
Correspondence to I.V. and
J.A.T.
e‑mails:
ilia.voskoboinik@petermac.org;
joe.trapani@petermac.org
doi:10.1038/nri3839
Published online 22 May 2015
Central to both the innate and adaptive arms of the
immune system is the ability of cytotoxic lymphocytes
to recognize virus-infected or transformed cells and
kill them by apoptosis. The role of cytotoxic lympho-
cytes in defending against pathogens has long been
appreciated, but the more controversial notion of
immune surveillance of cancer — which was proposed
by Macfarlane Burnet in the 1950s — only recently
gained broad acceptance. Subsequently, the develop­
ment of immune-based therapies that exploit the
activity of cytotoxic lymphocytes has begun to revo-
lutionize cancer therapy. Thus, new cancer therapies
that promote antitumour cytotoxicity, such as gene-
engineered chimeric antigen receptor-expressing
T cells and recombinant antibodies, take advantage of
the perforin–granzyme effector pathway. Advancing
our understanding of cytotoxic lymphocyte biology
should support the further development of immune-
based therapies for cancer, as well as for infectious
diseases and autoimmunity.
Cytotoxic lymphocytes include cytotoxic T lympho­
cytes (CTLs) and natural killer (NK) cells. Despite
substantial differences in how these two cell types are
activated and how they recognize their targets, the key
pathways that mediate target cell death are conserved.
Once conjugated to a target cell, the cytotoxic secre-
tory granules traffic to the immunological synapse and
release a cargo of deadly proteins — including perforin,
granzymes (FIG. 1) and granulysin1 — into the synaptic
cleft. Defects of this cytotoxic pathway — particularly
a failure to secrete functional perforin — are associated
with various human disorders (which we have termed
‘perforinopathies’ (REF. 2)), including familial haemo­
phagocytic lymphohistiocytosis (FHL), protracted
viral infections and susceptibility to haemato­logical
malignancies3–5. Although the mutations that disrupt
granule trafficking or exocytosis are mostly rare, recent
attention has turned to a common polymorphism in
the perforin (PRF1) gene that encodes moderate-to-
severe dysfunction of the perforin protein2. In this
Review, we discuss the mechanisms by which perforin
and granzymes kill target cells, and the consequences
of defects in this essential facet of immune function.
The cytotoxic cargo of cytotoxic lymphocytes
The discovery of perforin and granzymes. The first
report that lymphocytes kill specific target cells was
more than 50 years ago6. Subsequently, the Golgi and
electron-dense secretory granules were shown to re‑­
orient (or become ‘polarized’) towards the target cell7,8,
and the dynamics of CTL secretory granule movement
towards the immunological synapse were determined9.
In 1980, pore-like membrane lesions were observed on
cells that were targeted by antibody-dependent degranu-
lation of NK cells10. The groups of Henkart, Okumura
and Podack11–16 then isolated a monomeric 67 kDa

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a b
MTOC Secretory granule
Granzyme
Perforin
Cytotoxic
lymphocyte Target cell
c d
e microtubule-organizing centre (MTOC) of the cytotoxic
Dying cell
granule protein that oligomerized (at neutral pH and
in the presence of Ca2+) into membrane-spanning pores
and named it perforin. Crucially, these authors also
noted that perforin pores and the complement mem-
brane attack complex had marked similarity by electron
microscopy. Around the same time, several granzymes
were identified as abundant granule constituents17–20, and
they were later shown to induce target cell apoptosis in
synergy with otherwise non-lethal amounts of perforin
in vitro 21–28. The lipid-disrupting toxin granulysin —
which is secreted by human T cells — was discovered
soon after 29, but its orthologues exist only in ruminants
(BOX 1). Detailed biochemical studies characterized these
perforin-enriched and granzyme-enriched granules as
a subset of ‘secretory lysosomes’ with an electron-dense
core surrounded by small vesicles17–19,30.
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Microtubule
Secretory granule
trafficking
Release of perforin
and granzymes
Fusion of granules
with the presynaptic
membrane
Perforin
pore
↑ Ca2+
Ca2+
Figure 1 | Interaction of a cytotoxic lymphocyte with a
target cell. The cytotoxic lymphocyte recognizes its target
cell and forms an immunological synapse (part a). The
lymphocyte polarizes and secretory granules traffic towards
the presynaptic membrane (part b). The secretory granules
then fuse with the presynaptic membrane and release perforin
and granzymes into the synaptic cleft (parts c and d). At the
postsynaptic membrane, perforin forms large transmembrane
pores that enable the diffusion of granzymes into the target
cell cytosol. Granzymes then initiate apoptosis of the
target cell, and the cytotoxic lymphocyte detaches from
the dying cell (indicated by the arrow; part e) and can interact
with another target cell to carry out serial killing (not shown).
Knockout studies in mice showed that loss of per-
forin abolished granule-dependent target cell death;
Nature Reviews | Immunology
these data supported a role for perforin as the key
enabler of granzyme-induced apoptosis (reviewed in
REFS 31,32). Conversely, disrupting single granzyme
genes produced far subtler phenotypes, which high-
lighted some redundancy in the functions of gran-
zymes33–35. Great effort has been made to understand
how granzymes induce specific death signalling path-
ways and to understand their role in infectious dis-
eases, inflammation and cancer. More recently, the
discovery that congenital perforin deficiency causes
the fatal human hyperinflammatory disease FHL36
has reignited interest in perforin and demonstrated its
involvement in a far broader range of pathologies than
previously suspected.
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REVIEWS
Box 1 | Granulysin
Granulysin is a small, saposin-like, antibacterial toxin that is expressed by natural
killer (NK) cells and CD8+ T cells of primates and ruminants, but not rodents166.
Unlike co‑secreted perforin and granzymes, it kills intracellular bacteria — such as
Listeria monocytogenes and Mycobacterium tuberculosis — by rupturing their
membranes167. Similar to granzymes, the delivery of granulysin into infected cells
is dependent on perforin; granulysin diffuses through perforin pores and kills
pathogenic bacteria that are escaping from the endolysosomal compartment of
dying target cells168. To investigate granulysin delivery into a target cell and the
possible role of perforin and granzymes in its antibacterial activity, a recent study
used mice that were transgenic for granulysin. The results of this study confirmed
earlier reports that intracellular antibacterial activity of granulysin in vivo depends
on perforin; however, wild-type mice that were transgenic for granulysin cleared
L. monocytogenes as efficiently as granzyme-deficient animals that were also
transgenic for granulysin169. In vitro experiments have shown that granulysin can
deliver lethal levels of granzymes into bacteria, suggesting that perhaps under
certain conditions these effector proteins may synergize in vivo. Despite showing
little cytotoxicity towards mammalian cells under controlled conditions, high
levels of extracellular granulysin might contribute to Stevens–Johnson syndrome
and toxic epidermal necrolysis, both of which result from immune responses to
certain drugs and allergens170. In these conditions, the recruitment and activation
of NK cells and CD8+ T cells within epidermal blisters leads to the secretion of
granulysin, which causes potentially life-threatening skin necrosis170.
Regulation of perforin expression in cytotoxic lympho­
cytes. Stimulation with cytokines and/or specific
antigen causes naive CD8+ CTLs and NK cells to dif-
ferentiate into efficient killers, which require the rapid
synthesis, safe trafficking and storage of large amounts
of perforin and granzymes. A major advance in under-
standing PRF1 transcription in T cells came from the dis-
covery of ten DNase I hypersensitivity sites in its 150‑kb
long locus control region, which signifies an open chro-
matin configuration that enables gene transcription37,38.
Subsequently, some of the sites were shown to bind to
runt-related transcription factor 3 (RUNX3) — which
is necessary for PRF1 expression — and to an addi-
tional PRF1 transcriptional regulator, eomesodermin
(which is encoded by EOMES)38–40. More recent stud-
ies have shown that PRF1 is also post-transcriptionally
regulated. MicroRNAs (mi­RNAs) bound to the 3ʹ untrans-
lated region of Prf1 and PRF1 mRNAs directly down-
regulated their translation in mouse and human CTLs
and NK cells, whereas other mi­RNAs indirectly down­
regulated perforin expression by reducing the expression
of eomesodermin41–44. Depending on the mode of acti-
vation, several mi­RNAs can suppress PRF1 translation
including: miR‑27a* (REF. 42), miR‑30e and miR‑378 in
human NK cells44; miR‑150 in mouse NK cells41; and
miR‑139 and miR‑342 in human and mouse CTLs43.
Although modulation of miRNA expression and activ-
ity in cytotoxic lymphocytes in vitro markedly reduced
the production of perforin, the overall reduction in
cytotoxicity was relatively mild (approximately two-
Toxic epidermal necrolysis
A rare life-threatening skin fold)41,42, implying that residual perforin levels were
disease in which the dermis sufficient to support granzyme delivery. Despite this,
detaches from the epidermis. adoptively transferred miR‑150‑deficient NK cells
This disease often leads to with enhanced perforin expression rejected trans-
sepsis and death. It is also
known as Lyell syndrome;
planted tumours better than wild-type NK cells 41,
one of its forms is called and miR‑27a*-­overexpressing NK cells (with reduced
Stevens–Johnson syndrome. perforin levels) were less effective than control cells42.
390 | JUNE 2015 | VOLUME 15
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As discussed below, this and other lines of evidence sug-
gest that even mild changes in perforin activity, as occurs
in carriers of hypomorphic PRF1 mutations, might
contribute to immune‑based pathology over time.
Safe trafficking and storage of perforin in granules. How
do killer cells safely express and store large amounts of
highly toxic proteins in their granules? Granzyme traf-
ficking and activation are well understood45–47. By con-
trast, the trafficking of perforin and management of its
inherent toxicity are less well characterized. The activity
of perforin is highly dependent on pH and Ca2+; perforin
is active at the neutral pH of the immunological synapse,
but it is inactive under acidic conditions (that is, less than
pH 6)48–50. Therefore, the acidic secretory granule (which
is at pH 5) is a safe storage compartment for perforin,
which only becomes active upon secretion. However,
until recently, it was unclear how perforin is safely pro-
cessed and trafficked from the Ca2+-rich and pH-neutral
environment of the endoplasmic reticulum (ER) to the
granules. One hypothesis is that perforin is synthesized
as an inactive precursor, with an inhibitory carboxy‑
terminal dodecapeptide that blocks Ca2+ binding to the
C2 domain51. However, this is inconsistent with the find-
ing that recombinant perforin with an intact C terminus
has the same activity and stability as perforin that lacks
the C terminus52. Furthermore, the X‑ray crystal struc-
ture of the perforin monomer showed that the C termi-
nus is located far from the Ca2+-binding region of the
C2 domain, making the proposed mechanism unlikely53,54.
Instead, it has been suggested that the distal end of the
C‑terminal region encodes a highly efficient ER export
motif that is required for protein delivery from the ER to
the granules52. Deletion of the last 12 amino acids or even
a mutation of the last C-terminal residue, tryptophan,
results in the retention of functional and stable perforin
in the ER and the death of cytotoxic lymphocytes owing
to perforin pore-forming activity52. This is further sup-
ported by the observation that cytotoxic cells that express
an inactive perforin variant — which cannot properly
bind to Ca2+ — are protected from the toxic effects of
mutations of the C‑terminal region52. Accordingly, an
explanation for cell survival during perforin synthesis is
that it is rapidly exported from the ER, thus minimizing
the concentration of active perforin in the ER at any one
time. Furthermore, it is notable that N‑linked glycans are
essential to enable the delivery of perforin from the Golgi
to secretory granules. The exact carrier (or carriers) of
nascent perforin are unknown52; however, this process
may involve constitutive protein trafficking machinery
(such as lysosome-­associated membrane glycoprotein 1
(LAMP1)55 or the ‘leaky’ mannose 6‑phosphate receptor).
Finally, it is important to note that the perforin C‑terminal
peptide is removed; however, the precise purpose of this
processing (if any) remains unclear.
Granule exocytosis and the immunological synapse.
CTLs respond to T cell receptor-mediated recognition
of peptide–MHC complexes, and NK cells respond to
the balance of stimulatory versus inhibitory signals
that are expressed by target cells. In both cell types,
www.nature.com/reviews/immunol

Microtubule-organizing
centre
(MTOC). An intracellular
structure from which
microtubules originate.
In cytotoxic lymphocytes,
the MTOC moves towards the
immunological synapse.
Wiskott–Aldrich syndrome
A life-threatening X‑linked
immunodeficiency that is
caused by mutations in the
gene encoding the Wiskott–
Aldrich syndrome protein. The
condition is characterized by
thrombocytopaenia with small
platelets, eczema, recurrent
infections caused by
immunodeficiency and an
increased incidence of
autoimmune manifestations
and malignancies.
NATURE REVIEWS | IMMUNOLOGY
recognition of the target gives rise to signalling events
that result in a pronounced increase in cytosolic Ca2+
concentration. The microtubule-organizing centre (MTOC)
rapidly moves towards the target cell, and then cyto-
toxic granules migrate along the MTOC, fuse with the
presynaptic membrane and release their cargo into the
synaptic cleft 1,4,56–58 (FIG. 1). It was recently proposed
that granule clustering at the presynaptic membrane
might precede MTOC polarization and granule dock-
ing, which suggests some plasticity or redundancy in
mechanisms of cytotoxic immunological synapse forma-
tion59. Consistent with this notion, human disorders that
are caused by cytoskeletal abnormalities show varying
cytotoxic impairment — for example, individuals with
Wiskott–Aldrich syndrome (which is caused by disordered
F‑actin remodelling) show only mildly defective NK cell
cytotoxicity189. However, disorders that are associated
with mutations in adhesion molecules — such as leuko­
cyte adhesion deficiency 1 (LAD1), LAD3, hyper-IgE
recurrent infection syndrome (which is due to a defi-
ciency in the gene encoding dedicator of cytokinesis 8
(DOCK8)) and a deficiency in the gene encoding the
guanine nucleotide exchange factor VAV1 — all cause
an incomplete loss of lymphocyte cytotoxicity (for exam-
ple, see REFS 60,61). By contrast, failure to deliver per-
forin to the immunological synapse owing to biallelic
mutations in UNC13D (which encodes MUNC13‑4),
STX11 (which encodes syntaxin 11) and STXBP2 (which
encodes syntaxin-binding protein 2; also known as
MUNC18‑2) results in the development of FHL type 3,
FHL type 4 and FHL type 5, respectively 4,5. These pro-
teins regulate cytotoxic granule docking, priming or
fusion to the presynaptic membrane; consistent with
these loss‑of‑function consequences, data indicate that
their functions are non-redundant (discussed below).
Protection of cytotoxic cells following perforin secre­
tion. The notion that killer cells are resistant to the toxic
effects of perforin and granzymes following granule
exocytosis is remarkable, and how they do so remains
unclear 62–65. Without this resistance, it would not be pos-
sible for a single cytotoxic lymphocyte to kill multiple
target cells, and an infected host would need to gener-
ate at least as many cytotoxic cells as infected cells54,66.
However, cytotoxic lymphocytes are not intrinsically
resistant to granule exocytosis: CTLs were readily killed
when they presented cognate peptide–MHC class I to
identical, non-peptide-pulsed clonogenic CTLs, which
went unscathed90. Recently, it was proposed that, upon
degranulation, the NK cell surface becomes ‘coated’
with LAMP1, which protects it against newly released
perforin67. However, LAMP1‑deficient and wild-type
NK cells were equally cytotoxic in long-term in vitro
assays, which suggests that serial cytotoxicity is not
reduced in the absence of LAMP1 (REF. 67). It is still
possible that several such factors may contribute to
cytotoxic lymphocyte protection; a role has been pro-
posed for cathepsin B63, but the survival of cathepsin
B‑deficient cytotoxic cells following encounters with tar-
get cells is no different from wild-type cytotoxic cells64.
Finally, it has been noted that CTLs contain high levels
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of the granzyme B inhibitor serine protease inhibitor B9
(serpin B9)68. Knockout studies show that mice lack-
ing serpin B9 have much more CTL death during the
response to infection than wild-type mice69. Together,
these data suggest that although the reservoir of ser-
pin B9 in CTLs may be exhausted during a prolonged
immune attack, the presence of this inhibitor may
provide cytotoxic cells with limited protection from
perforin and granzyme toxicity during killing.
Death of the target cell
Structural basis of perforin oligomerization and pore
formation. Until recently, the molecular basis for
perforin pore formation remained unclear. The X‑ray
crystal structures of several perforin-related proteins70–72
showed that these proteins are homologous in their
pore-forming domain (known as the membrane attack
complex and perforin (MACPF) domain) to the bac-
terial cholesterol-dependent cytolysins (CDCs). This
unexpected relationship provided several mechanistic
clues about perforin function. CDCs initially bind to and
oligomerize on the membrane surface into a pre-pore of
20–50 monomers (FIG. 2a–c). Following pre-pore assem-
bly, two small clusters of α‑helices (transmembrane
helical region 1 (TMH1) and TMH2) in each mono-
mer unwind and insert into the membrane as a pair
of amphipathic β‑hairpins. Therefore, a full pore com-
prises a large β‑barrel of 80–200 strands73–75. Through an
alternative but similar mechanism, several studies have
shown that CDCs, and most likely perforin itself, form
incomplete pores (arc-like structures) that can penetrate
and damage the membrane76–78.
On the basis of these structural data, it was suggested
that perforin and other MACPF domain-containing
proteins form pores in a CDC-like manner 70,71. This pre-
diction supports the finding that mutations in TMH1 or
TMH2 — which are essential for membrane piercing —
frequently cause perforin dysfunction and disease53,79.
Furthermore, the crystal structure of perforin itself,
together with the 28 Å resolution cryo-electron micro­
scopy structure of the pore, provided broad support for
the idea that perforin forms pores through a CDC-like
mechanism53 (FIG. 2d–f).
Access of granzymes to the target cell cytosol. Early elec-
tron microscopy 10 and recent X‑ray and cryo-electron
microscopy studies have shown that perforin pores (with
an internal diameter of 16–22 nm) should easily enable
granzymes (with a diameter of approximately 4 nm) to
diffuse across the plasma membrane into the cytosol of
the target cell53,80. However, several studies using puri-
fied perforin could demonstrate only small solutes such
as Ca2+ or Na+ passing through the pores, whereas the
larger fluorescent molecule propidium iodide seemed to
be excluded from cells that were simultaneously under-
going perforin-dependent apoptosis81,82. This paradox
provided support to the ‘endosomolysis’ hypothesis,
which proposed that small perforin pores or rents in the
membrane are internalized together with granzyme mol-
ecules into clathrin-dependent and dynamin-dependent
endosomes by the target cell. Approximately 15 minutes
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later, perforin pores are thought to assemble within the
endosome (or a larger endosomal structure known as
the ‘gigantosome’) to form channels that are large
enough for granzymes to exit into the cytosol81–86.
Structural studies with perforin in liposomes found
no evidence of pores with a ‘small’ lumen, and full pore
structures comprising fewer than 16 monomers were
extremely rare. However, the finding by independ-
ent groups that recombinant perforin and structurally
related toxins can also form curved membrane disrup-
tions (‘arcs’) re‑enlivened the debate77,78. This situation
raised problems for both major hypotheses. On the one
hand, how did granzymes enter an intact target cell when
entry of propidium iodide could not be demonstrated
through transmembrane pores? On the other hand, how
could endosomolysis occur when the endosomal envi-
ronment (with an acidic pH and extremely low Ca2+
concentration87) seems unsuitable for perforin action49,65?

a CDC monomer b CDC pre-pore c CDC pore

Lipid
bilayer

d Perforin domain structure e Perforin monomer f Perforin pore

MACPF domain (pore forming) EGF-like domain C-terminal domain
21 112–165 240–296 371 377 397 397 412 526 542

TMH1 TMH2 413 525

C2 domain
(Ca2+ and
membrane
binding)

Figure 2 | Structural transitions of cholesterol-dependent cytolysins
and perforin. a | The structure of a cholesterol-dependent cytolysin (CDC)
in its monomeric state, with the pore-forming ‘warhead’ (blue), the central
sheet (red) and the transmembrane helical (TMH) regions (orange). b | CDCs
initially interact with the lipid bilayer of the cell membrane through the
carboxy-terminal immunoglobulin-like domain (yellow; lower panel). Here,
monomers oligomerize to form a distinct pre-pore intermediate. A surface-
contoured view (upper panel) shows the proposed structure of the pre-pore.
c | CDC pre-pores undergo a marked collapse, which results in insertion of
the two pore-forming regions (orange) into the membrane as a large
β‑barrel (lower panel). Modelling studies suggest that the linker domain
(green) rotates during pre-pore collapse175. The β‑strands in the final pore
are thought to adopt a slightly tilted (off-vertical) architecture (upper
panel)75. d | The domain structure of perforin. The pore-forming membrane
attack complex and perforin (MACPF) domain contains two membrane-
penetrating regions (TMH1 and TMH2) and is followed
Nature by |an
Reviews epidermal
Immunology
growth factor (EGF)-like domain. The C‑terminal domain (comprising a
short disulfide-bonded region) contains the Ca 2+ -binding and
membrane-binding C2 domain. e | The structure of mouse perforin. The
pore-forming warhead is homologous to that of CDCs; the central, highly
twisted sheet is shown in red, the TMH regions are shown in orange and the
remainder of the MACPF domain is shown in blue. It is not known whether
perforin forms a pre-pore before membrane insertion. f | A cryo-electron
microscopy reconstruction of a perforin pore. The diameter of the pore is
calculated to be approximately 150 Å and therefore would easily enable the
passage of granzyme B, which is approximately 50 Å in diameter (not
shown). All of the images in this figure were prepared specifically for this
Review based on data from REFS 53,74,75,175.

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To address some of these issues, we recently re‑­
investigated whether propidium iodide could pass
through perforin pores. In previous studies, propidium
iodide had arbitrarily been used at 0.5–1.5 μM82,85,86 and
occasionally at 15 μM78,88. As the diffusion of a solute
through a pore is proportional to its concentration but
inversely proportional to its radius89, these conditions
favoured the diffusion of Ca2+ (which is typically used at a
concentration of 0.5–2 mM) into cells over that of propid-
ium iodide by several orders of magnitude. We found that
simply increasing the concentration of propidium iodide
to 100 μM was sufficient to demonstrate obvious free dif-
fusion of propidium iodide across the plasma membrane
in the presence of recombinant perforin65. Consistent with
these findings, we repeated these experiments in the con-
text of physiologically relevant immunological synapses
formed by human or mouse cytotoxic cells; these con-
ditions led to a pronounced entry of propidium iodide
into the target cell near to the immunological synapse,
which indicates the formation of large perforin pores
(FIG. 3). This influx of propidium iodide occurred within
60–80 seconds of Ca2+ flux into the cytotoxic cell cytosol,
which is the stimulus that signals degranulation65,90. The
potentially lytic effect of perforin on the target cell was
rapidly (within approximately 30 seconds) countered by
efficient exocytic membrane repair mechanisms91,93 that
prevented osmotic lysis, but still enabled the movement of
granzymes into the target cell cytosol65.
The authenticity of these kinetics was evident from
the direct cleavage of BH3‑interacting domain death
agonist (BID) — which is the cytosolic substrate of gran-
zyme B — within 2 minutes of adding it to cells in the
presence of recombinant perforin65. In the context of the
immunological synapse, clear morphological changes
(such as cell rounding and membrane blebbing) within
10 minutes of the propidium iodide influx indicated
granzyme-induced caspase activation; this occurred
much faster than predicted by endosomolysis. Human
and mouse cytotoxic cells all produced similar results
against diverse target cells90, indicating that the function
of perforin is conserved across species.
Taken together, we argue that these data do not sup-
port the endosomolysis hypothesis. However, these
findings do not preclude a role for arcs in permitting
rapid granzyme delivery in the context of the immuno-
logical synapse. Indeed, it has been proposed that arcs
may transition into pore-like structures92, and recent
studies on the CDC suilysin indicate that arcs that form
in close proximity may develop even larger membrane
ruptures77. Therefore, it is possible that perforin — the
secretion of which is concentrated onto a confined area
of the target cell membrane — may form a mixture of
arcs, pores and coalesced arcs that increase the diffusion
of granzymes into the target cell. At the same time, exo-
cytic membrane repair of the target cell at the immuno­
logical synapse65 seems to be sufficient to prevent lysis
while enabling enough time for granzymes to enter the
target cell and initiate apoptosis65,90.
Finally, it is important to note that many of the
studies that address perforin pore formation in the con-
text of the target cell rely (by technical necessity) on
applying recombinant perforin to cells78, liposomes, lipid
monolayers or lipid-coated chips53. Therefore, although
these in vitro studies are informative, it is important

00:00 10 μm 03:25 04:55

05:25 06:15 06:45

Figure 3 | Perforin pore formation in action. The cytotoxic T lymphocyte (CTL) was labelled with a Ca2+ fluorophore
and added to adherent target cells in medium that was supplemented with 100 μM of propidium Nature Reviews
iodide . After
65,90 | Immunology

3–5 minutes, CTL degranulation was observed, as measured by an increase in green fluorescence (which reflected Ca2+
influx). Approximately 1 minute and 30 seconds later, the target cell membrane was punctured, as shown by a focal influx
of propidium iodide into the cytosol, where it fluoresced red following binding to RNA. The propidium iodide then spread
throughout the target cell, which started to acquire a round morphology (a sign of caspase activation). Finally, the CTL
detached from the rounded target. The whole process from degranulation to detachment was remarkably rapid and
took less than 5 minutes. These images have not been previously published but are analogous to those in REFS 65,90.

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Table 1 | Hierarchical granzyme-mediated cell death pathways

Cell type Cytotoxicity* Morphology Phosphatidylserine Propidium Initiation Refs
(functional exposure‡ iodide phase§
granzymes) uptake‡ (minutes)
Wild-type +++++ Classical apoptosis Early Late <7 108
NK cells (secondary
(predominantly necrosis)
granzyme B)
GzmA−/− +++++ Classical apoptosis Early Late <7 108
NK cells (secondary
(predominantly necrosis)
granzyme B)
GzmB−/− NK cells ++ Athetosis (worm-like Simultaneous Simultaneous ~15 108
(predominantly morphology, (secondary necrosis) (secondary
granzyme A) elongated cells and no necrosis)
membrane blebbing)
GzmA−/−GzmB−/− +/− Similar to apoptosis Late Late Typically 108,
CTLs (other but slower (secondary >20 176
granzymes) necrosis)
CTL, cytotoxic T lymphocyte; Gzm, granzyme; NK, natural killer. *The relative amount of cell death in a typical 4‑hour assay, in
which ‘+’ indicates the death of approximately 20% of target cells. +/– indicates the death of <20% of target cells. ‡Propidium
iodide used at 1 μM, as used in conventional cell death assays that measure annexin V binding and propidium iodide uptake.
Using propidium iodide at 100 μM demonstrates a much earlier breach of the target cell plasma membrane at the time of perforin
pore formation. §Defined as the time elapsed from conjugation to target cell rounding.

to note that they may not accurately reflect the in vivo
situation. For example, the concentration of perforin
and granzymes at the immunological synapse and the
structures that these molecules form within the synapse
remain to be understood. Recent advances in high-
resolution electron tomography may provide key
insights into these issues in the future.
Lethal and non-lethal granzyme function
Since the mid‑1990s, the increased interest in how gran-
zymes induce cell death led to diminished research on
the non-cytotoxic functions of granzymes. Granzymes
are characterized by their individual substrate speci-
ficity and the gene cluster by which they are encoded.
Despite being broadly labelled as trypsin-like or
chymotrypsin-like, granzymes induce tightly regulated
signalling (but not indiscriminate protein digestion),
as exemplified by their highly orchestrated activation
of caspase-driven cell death pathways21,94. Granzymes
also regulate inflammation95 and can directly subvert
intracellular infections by interfering with viral bio-
synthetic pathways96. However, as much of the work
on granzymes has been done in vitro, there are gaps in
our understanding of the potential pathophysiological
relevance of interesting laboratory findings. Whereas
congenital human perforin deficiency is well character-
ized, no known human disease arises from granzyme
deficiency. Papillon–Lefèvre syndrome (which is associ-
ated with cathepsin C deficiency) was proposed to cause
granzyme inactivity, as all pro-granzymes were thought
to require cathepsin C for amino-terminal process-
ing 46,97. Although this is the case for granzyme A and
the myeloid proteases cathepsin G and elastase, gran-
zyme B is also activated by cathepsin H45, which explains
why neutrophils of patients with Papillon–Lefèvre syn-
drome are poorly bactericidal, but NK cell cytotoxicity
remains intact.
Granzymes and cell death. Granzymes induce target
cell death through diverse, non-redundant pathways
that function in a complementary and hierarchical
manner (as recently reviewed in REF. 98; TABLE 1). In an
evolutionary context, this reflects a balance between
three mechanisms: first, the immune mechanisms that
induce suicide of infected cells; second, the opposing
pathways that are activated by intracellular patho-
gens to provide a safe haven (for example, protection
from neutralizing antibodies); and third, the cellular
machinery of replication. Granzyme B is the most pow-
erful pro-apoptotic granzyme, as its ability to cleave
target cell proteins at sites after selected aspartate
residues mimics the caspases. This ‘imposed’ death
is rapid and effective; target cells that are exposed to
nanomolar amounts of recombinant granzyme B and
an otherwise innocuous dose of recombinant perforin
(which provides access for granzyme B to the target
cell cytosol) die within 5–8 minutes by apop­tosis65,99–101.
Granzyme A cleaves proteins at sites after basic amino
acids and activates a slower form of cell death through
different substrates (see below). Although most other
granzymes also activate cell death in vitro, many of
the assays do not include granzyme delivery across a
bona fide immunological synapse, and thus it remains
unclear whether granzymes other than granzyme A and
granzyme B have generic cytotoxic activity (BOX 2).
There is still controversy about what the physio­logical
substrates of granzymes are, but recent findings have
resolved several contentious issues. The most impor-
tant finding was reported by several groups showing
that there are species-specific differences in the fine sub-
strate specificity of orthologous granzymes102–104. It took
a decade to reach a consensus on whether granzyme B
preferentially processes pro-caspases directly or activates
the mitochondrial cell death pathway by cleaving BID.
In fact, human granzyme B very efficiently activates the

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© 2015 Macmillan Publishers Limited. All rights reserved


Box 2 | Which granzymes are authentic inducers of cell death?
Many granzymes have been reported to cause cell death in vitro, but there are many
inconsistencies among the findings. For example, three groups (including ours) have
reported that granzyme M kills target cells in vitro but through very different and
incompatible mechanisms133,171–174. These studies used different methods to deliver
recombinant granzyme M to target cells (perforin, synthetic detergent or adenovirus).
The reports differed on whether activated caspases were required for cell death, and
they identified various substrates or ligands, including inhibitor of caspase-activated
DNase (ICAD; also known as DFFA), the cytoskeletal proteins ezrin and α‑tubulin,
FAS-associated death domain protein (FADD) and topoisomerase IIα. Granzyme
M‑deficient mice have no defect in generic cell death pathways114, even when
possible masking effects of granzyme A and granzyme B are absent108. Overall, the
notion that granzyme M has authentic cytotoxic activity remains unproven.
In addition to the possibility that some granzymes might have redundant functions,
there are many reasons why in vitro findings may not translate into the in vivo setting.
Granzyme substrate specificity can vary between species, but many studies combine
cells and reagents from disparate species. Moreover, in examinations of whether
a substrate is genuinely processed, robust kinetic analyses are rarely performed.
Commonly used commercially available reagents, such as monoclonal antibodies,
can give misleading results owing to a lack of specificity175. Granzyme concentrations
used in vitro vary from low nanomolar to high micromolar, but it is not known how
these levels relate to cell death induced by intact cytotoxic T lymphocytes (CTLs)
or natural killer (NK) cells. Finally, contaminants other than granzymes, such as
lipopolysaccharide or proteases, can markedly influence results. Indeed, we have
found that trypsin (which is commonly used to harvest adherent cells) caused
dose-dependent death of HeLa cells when delivered with recombinant perforin
(J.A.T. and K. A. Brown, unpublished observations).
Thus, caution is advised when extrapolating in vitro results to intact animals or
humans without corroborating genetic evidence. Perforin-deficient CTLs and NK cells
from both mice and humans have severely reduced cytotoxicity, but genetic evidence
for cytotoxic functions has only been provided for mouse granzyme A and granzyme B.
BID-dependent, B cell lymphoma 2 (BCL‑2)‑inhibitable
pathway, but mouse granzyme B preferentially activates
caspases directly 102. Both mouse and human gran-
zyme B are polymorphic, with variability of residues
in mouse granzyme B having an effect on substrate
choice102. Owing to strong founder effects, there is only
one granzyme B allele in all inbred laboratory mouse
strains (designated granzyme BP)105. However, C57BL/6
mice that are congenic for the most divergent ‘wild’
allele (designated granzyme BW) showed a profound
susceptibility to mouse cytomegalovirus (MCMV) due
to unrestrained viral replication in the liver 106. Failure to
control MCMV was due to BCL‑2‑like pro-survival viral
proteins that blocked killing by granzyme BW‑expressing
CTLs, but not by granzyme BP‑expressing CTLs. By con-
trast, granzyme genes that are closely linked to the gene
encoding granzyme B have minimal poly­morphism,
indicating that they are less crucial for defence against
specific viruses than granzyme B, and that they have
other roles105. The pro-apoptotic role of granzyme B
was also confirmed in granzyme B‑deficient mice that
have similar susceptibility to MCMV as granzyme
BW‑congenic mice35,106,107.
In granzyme B‑deficient mice, which lack the rapidly
acting granzyme B-mediated cell death pathway, a novel
granzyme A‑induced cell death pathway was revealed108.
Mouse granzyme A acts more slowly than granzyme
B through a distinct, caspase-independent mechanism;
recombinant mouse granzyme A that is delivered to tar-
get cells by purified perforin causes athetosis, which is
NATURE REVIEWS | IMMUNOLOGY
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REVIEWS
a form of cell death that is dependent on an intact actin
cytoskeleton and is marked by a writhing (‘twisting
and turning’) morphology of the dying cell108. Mouse
CTLs and NK cells deficient in granzyme B phenocopy
athetosis that is induced by recombinant granzyme A.
Athetosis does not occur when both granzyme A and
granzyme B are absent, confirming granzyme A as the
inducer of athetosis. However, human granzyme A has
minimal cytotoxic activity even at high concentrations
(in the micromolar range), which also suggests that
different species have been shaped by their microbial
environment and have evolved their own ‘customized’
range of granzyme-dependent cell death pathways109.
In the absence of both granzyme A and granzyme B,
mouse CTLs and NK cells induce an even slower form of
cell death than that activated by granzyme A alone. The
morphology of the double-deficient dying cells resem-
bles that of cells undergoing apoptosis, but phosphatidyl­
serine exposure on the dying cell is greatly delayed110.
Loss of this signal for phagocytosis reduces uptake of
the dying cells by professional antigen-presenting cells
and reduces their immunogenicity 110. It is likely that this
‘residual’ cell death is activated by mouse granzyme M
and by human granzyme M and granzyme H111–113, and
disrupting these minor pathways in otherwise normal
CTLs has little effect on cell death in vitro or in intact
mice114. Genetic evidence supporting a pro-apoptotic
role for granzyme M is still lacking 114, but several cell
death pathways can potentially be activated in vitro
(BOX 2). This lack of phenotype or specific function for
granzymes other than granzyme A and granzyme B has
led to them being referred to as ‘orphan’ granzymes,
although granzyme H is known to be crucial for defence
against human adenovirus V, as it disrupts capsid
synthesis and viral assembly 115.
Studies have catalogued many potential granzyme
substrates to decipher the ‘proteome’ by mixing cell
lysates with an arbitrary concentration of recombinant
granzyme116. One database currently lists 1,984 granzyme
B substrates, which is more than for all but three other
proteases117. Although unbiased, this approach does not
explain how granzyme B could ever encounter its sub-
strates physiologically, thus drawing some criticism116
(BOX 2). By contrast, functional genomics approaches
that are designed to identify whole pathways, and there-
fore some ‘indirect’ granzyme targets, are now providing
insight. This approach showed that granzyme B‑mediated
processing of BID is controlled epigenetically; loss of
the transcriptional cofactors histone acetyltransferase
KAT2B (also known as PCAF) or transcriptional adap-
tor 3 markedly reduced target cell death due to aberrant
trafficking of uncleaved BID118. This powerful approach
can identify key substrates in granzyme B signalling that
are not cleaved by granzyme B or any other protease118.
Granzymes in inflammation and infection. A long hiatus
separated the first two studies reporting that granzymes
directly influence inflammation109,119 by releasing pro-
inflammatory cytokines from monocytes, which focused
initially on granzyme A and granzyme B, but more
recently also on granzyme K and granzyme M120–124.
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REVIEWS
Emperitosis
A process by which a
granzyme B‑expressing
cytotoxic lymphocyte is
taken up by the target cell
and killed by apoptosis.
396 | JUNE 2015 | VOLUME 15
Granzyme M‑deficient mice resist lethal Toll-like recep-
tor 4 (TLR4)-mediated endotoxicosis owing to reduced
circulating levels of interleukin‑1α (IL‑1α), IL‑1β and
tumour necrosis factor 124. This study did not examine
whether granzyme M directly processes these cytokines,
but granzyme B can directly cleave pro‑IL‑1α close to
sites that are recognized by calpains and elastin125, and
direct processing of IL‑18 has also been proposed126,127.
Granzyme K might also function by promoting the bind-
ing of lipopolysaccharide to CD14; surprisingly, this
effect persisted with enzymatically inactive granzyme K,
raising questions about the mechanism underlying this
process128. Given their potential role in inflammation,
therapeutic targeting of granzymes has been considered.
For example, the concept of treating septic shock with
granzyme inhibitors was supported by a recent study
showing that granzyme A‑deficient mice survived lethal
infection and did not show compromised CTL-mediated
killing of infected cells129.
In addition to mediating cell death and depriving
viruses of the cellular processes that are necessary for
their survival and replication, granzymes can directly
limit the release of viable virus following the demise of
the cell96. Thus, mouse CTLs that are specific for influ-
enza virus upregulate the expression of granzyme K130,
which interferes with viral replication by cleaving
host importin‑α1 and importin‑β, and by preventing
the nuclear transport of viral nucleoprotein121. In an
analogous manner, heterogeneous nuclear ribonucleo­
protein K (hnRNPK) is cleaved by granzyme M to pre-
vent the replication of human CMV123, and eukaryotic
initiation factor 4γ3 (eIF4γ3) is cleaved by granzyme B
to stop viral protein translation in dying cells131.
Granzymes in tumour immunity. As described below,
perforin-deficient mice and humans are susceptible to
cancer. As perforin-dependent cell death is mediated by
granzymes, some granzyme substrates are likely to be
important for clearing transformed cells. Thus, cleav-
age of hnRNPK by several granzymes (including gran-
zyme H, granzyme K and granzyme M) reduces tumour
cell viability 123,132. Granzyme M also targets topoiso­
merase IIα113 and processes human (but not mouse)
FAS-associated death domain protein (FADD), caus-
ing FADD dimerization and increasing pro-caspase 8
processing 133,134. Some granzyme-mediated cell death
pathways are subverted by tumour cells. For example,
transformed cells may target granzyme B for degrada-
tion when undergoing autophagy 135. Granzyme B can
also contribute to the demise of CTLs that are internal-
ized by tumour cells through a process of engulfment
known as emperitosis136. Cancer cells often overexpress
BCL‑2 or related pro-survival molecules that protect
against myriad metabolic insults and block the mito-
chondrial death pathway that is activated by human
granzyme B137. Pertinent to tumour cell survival, pro-
cessing of BID by human granzyme B is long-lived (with
a half-life greater than 16 hours), which potentially offers
sufficient opportunity to administer pharmaco­logical
BCL‑2 antagonists, such as ABT‑737, to restore the
therapeutic benefit of granzyme B‑expressing CTLs138.
© 2015 Macmillan Publishers Limited. All rights reserved
Perforinopathies: defects of granule exocytosis
The term perforinopathy denotes the range of autosomal-­
recessive, immune-mediated diseases that are caused by
insufficient perforin delivery to the immunological syn-
apse, due either to PRF1 mutations or to impaired granule
exocytosis2. In approximately half of the cases in which
PRF1 is not mutated139, UNC13D, STX11 or STXBP2 are
the genes that are most commonly mutated139,141–143. The
corresponding proteins (MUNC13‑4, syntaxin 11 and
STXBP2, respectively) all take part in secretory granule
transport to the immunological synapse and/or mem-
brane fusion; therefore, deleterious mutations in these
genes impair perforin secretion and the killing activity
of cytotoxic cells139,141–142. Perforinopathies are classified
into three groups: acute (also known as early-onset ‘clas-
sic’ FHL, which invariably occurs before 2 years of age),
subacute (such as atypical FHL or another pathology,
which usually occurs after 5 years of age) and chronic (in
which heterozygosity can predispose to disease in adults);
the chronic disease grouping has not yet been well char-
acterized and requires further study. Complete loss of
perforin function or secretion almost invariably results in
FHL by 12 months of age (with a median of 3–9 months),
whereas hypomorphic PRF1 alleles result in delayed
FHL, or other inflammatory or neoplastic disorders32,144.
As classic FHL has been previously reviewed5, we
highlight one recent advance that causally links failed
target cell death with the fatal ‘cytokine storm’, which
is the hallmark of FHL. It was shown that detachment
of cytotoxic lymphocytes from their targets requires
a caspase-dependent signal from dying cells140. In the
absence of perforin, or if caspases are inhibited in the
target cell, the duration of engagement of cytotoxic
cells with the target cell is increased by approximately
fivefold. A failure to detach results in many successive
rounds of Ca2+ flux into the cytotoxic cell, which trig-
gers substantial secretion of pro-inflammatory cytokines
and chemokines. In turn, secreted interferon‑γ causes
hyperactivation of the myeloid cell compartment and
stimulates macrophages to secrete IL‑6, which is a major
instigator of the fatal systemic inflammation of FHL140.
Below, we focus on subacute and chronic perforin­
opathies caused by PRF1 mutations.
Partial perforin deficiency increases cancer risk. The role
of perforin in surveillance of carcinogen-induced cancer
or transplanted tumours in mice was demonstrated many
years ago145,146, but of greater physiological relevance was
the finding that approximately 60% of Prf1‑deficient
mice spontaneously developed B cell lymphomas with
age147. Broadly speaking, congenital or acquired immune
deficiency has long been associated with increased
cancer risk in humans, particularly those that are caused
by viruses148, but a direct link with impaired cytotoxic
lymphocyte function has been difficult to prove. We pre-
viously reported that carriers of biallelic PRF1 mutations
have a bimodal age distribution32, and patients presenting
in the later peak (that is, at 5–20 years of age) typically
have subacute perforinopathy 144. Approximately 40%
of patients present with various haematological cancers
(but generally not those with a known viral cause),
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Table 2 | Association of the Ala91Val polymorphism of perforin with human immunopathologies*

Perforin mutations Total FHL cases Haematological Other pathology Healthy
(late onset)‡ malignancy
Ala91Val + null§ 11 (7) 3 1 1
Ala91Val + Ala91Val|| 7 (6) 6 – 5
FHL, familial haemophagocytic lymphohistiocytosis. *Data from REFS 163,165,177–188. The number of cases of late-onset FHL
‡

of the total number of FHL cases is shown in brackets. §Indicates co‑inheritance of Ala91Val with perforin-inactivating frameshift,
nonsense or missense mutations. ||Indicates homozygous Ala91Val polymorphisms.

approximately 20% of patients present with a life-
threatening viral infection (commonly Epstein–Barr
virus149) and the remainder of patients suffer with vari-
able manifestations of FHL or a relapsing, corticosteroid-
refractory, systemic inflammatory disorder 144 .
Surprisingly, most of the PRF1 missense mutations that
have been identified in these patients had no function
in vitro and the protein was retained in the ER, raising
the issue of why FHL did not develop during child-
hood144. Culturing the CTLs from these patients in con-
ditions that optimized protein folding also improved
perforin trafficking and partly rescued CTL function,
and thus it is likely that these mutations are ‘leaky’ in
intact organisms144, as previously proposed for a range
of protein-misfolding diseases, including cystic fibrosis
and α1‑antitrypsin deficiency. Consistent with this, PRF1
mutations with the greatest recoverable function pre-
sented later in life, and presented more commonly with
leukaemia or lymphoma than with inflammation144. We
concluded that subacute perforinopathy caused by PRF1
mutations represent a new protein-misfolding disease
that is similar to other misfolding disorders150,151.
These observations directly linked partial perforin
deficiency with predisposition to haematological cancer,
providing that sufficient residual perforin function
remained to avoid early-onset FHL (designated acute
perforinopathy)3. Although it has still not been demon­
strated, we hypothesize that hypomorphic defects of
MUNC13‑4, STX11 or STXBP2 might have similar
outcomes owing to inadequate perforin delivery 152–154.
Although broad immune suppression states (particularly
in HIV infection, AIDS and the post-transplant setting)
can greatly increase cancer risk148,155–157, the association of
hypomorphic PRF1 mutations with cancer links cancer
susceptibility with a protein for which the only known
function is to mediate target cell death. Impaired clear-
ance of transformed cells by cytotoxic lymphocytes
seems to be the most feasible way to link PRF1 mutations
with cancer predisposition, and it would be consistent
with the remarkable recent success of immune modula-
tors that are directed at programmed cell death protein 1
(PD1) and CTL antigen 4 (CTLA4) as cancer therapeu-
tics: these agents de‑repress cytotoxic lymphocyte killing
by inhibiting signalling by these proteins158–160.
The common human perforin polymorphism Ala91Val
and disease. The prediction that monoallelic mutations
in PRF1 can lead to pathology (mainly cancer) in later
life — tentatively designated chronic perforinopathy —
requires far more study, particularly at the population
level. The highly prevalent (8–9% among the Caucasian
population) perforin polymorphism C>T272 (which
causes the Ala91Val substitution) provides an oppor-
tunity to test this proposition. Although initially con-
sidered functionally neutral, Ala91Val impairs perforin
activity in several ways: it reduces protein stability and
slows intracellular trafficking, which reduces cyto­
toxicity by 50–90%, depending on the assay 161. Notably,
primary NK cells from healthy carriers of monoallelic
Ala91Val were less cytotoxic than wild-type cells162. So
far, of the 34 reported cases of Ala91Val homozygosity
or compound heterozygosity (that is, Ala91Val inher-
ited with a second PRF1 mutation), almost 80% pre-
sented either with atypical FHL or with haematological
cancer (TABLE 2).
Can monoallelic inheritance of Ala91Val be patho-
genic? Apart from its own reduced function, Ala91Val
perforin also has dominant-negative function, presum-
ably by disrupting pore formation when co‑packaged
and released with wild-type perforin161. Genotype analy-
sis of large cohorts of patients with cancer showed that
Ala91Val was not over-represented in more than 2,000
cases of paediatric leukaemia163; however, Ala91Val was
over-represented threefold in the subset of patients with
acute lymphocytic leukaemia with BCR−ABL trans­
locations (that is, fusion of ABL1 on chromosome 9
with BCR on chromosome 22, which is found in approx-
imately 10% of cases)163. In another study, 4 of 15 patients
with a lifetime history of both malignant melanoma
and lymphoma carried either the monoallelic Ala91Val
polymorphism or the Arg28Cys mutation, which closely
phenocopies Ala91Val164, suggesting that silencing one
PRF1 allele may predispose to dual cancers in humans.
Although significance was achieved in these studies,
the number of patients was small and the findings need
further testing in other cohorts. Intriguingly, individu-
als with a single mutation in UNC13D, STX11, STXBP2
or RAB27A, together with the Ala91Val perforin allele,
can present with late-onset FHL165, raising the possibil-
ity that mutations affecting different components of the
cytotoxic pathway may be complementary.
Concluding remarks
Recent advances in molecular, structural and cellular
biology have enhanced our understanding of how cyto-
toxic lymphocytes kill their targets, but many fascinat-
ing questions remain. The perforin–granzyme pathway
is increasingly being studied in contexts that are patho-
physiologically relevant, such as in viral immunity, and
for understanding susceptibility to various inflamma-
tory and neoplastic diseases. The importance of per-
forin and granzyme poly­morphisms is being explored,

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© 2015 Macmillan Publishers Limited. All rights reserved

REVIEWS
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mouse models will guide this work, but the focus must
shift to human subjects. At last, rational immune-based
therapies are showing promise in patients with cancer,
and more detailed knowledge of how tumour cells
are killed by the immune system, and of intratumoral
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Acknowledgements
I.V. and J.C.W. are supported by fellowships from the National
Health and Medical Research Council of Australia (NHMRC).
I.V., J.C.W. and J.A.T. are supported by project grants from the
NHMRC. J.C.W. is supported by the Australian Research
Council (ARC) and he acknowledges the support of an ARC
Federation fellowship. J.A.T. is supported by a programme
grant from the NHMRC. The authors thank the many members
of their laboratories, especially J. A. Lopez and A. J. Brennan,
and their collaborators, especially H. Saibil, M. A. Dunstone,
R. H. P. Law and N. Lukoyanova, for their contributions to
many of the findings discussed in this Review.
Competing interests statement
The authors declare no competing interests.
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