Nature Reviews Immunology | AOP, published online 6 March 2015; doi:10.

1038/nri3799 REVIEWS

Balancing natural killer cell activation

through paired receptors
Ludovic Martinet1,2 and Mark J. Smyth1,3
Abstract | Natural killer (NK) cells are innate lymphocytes that are crucial for the control of
infections and malignancies. NK cells express a variety of inhibitory and activating receptors
that facilitate fine discrimination between damaged and healthy cells. Among them, a family
of molecules that bind nectin and nectin-like proteins has recently emerged and has been
shown to function as an important regulator of NK cell functions. These molecules include
CD226, T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), CD96, and
cytotoxic and regulatory T cell molecule (CRTAM). In this Review, we focus on the recent
advances in our understanding of how these receptors regulate NK cell biology and of their
roles in pathologies such as cancer, infection and autoimmunity.

1
Immunology in Cancer
and Infection Laboratory,
QIMR Berghofer Medical
Research Institute, Herston,
Queensland 4006, Australia.
2
Institut National de la Santé
et de la Recherche Médicale
Unité Mixte de
Recherche 1037, Cancer
Research Center of Toulouse,
Toulouse F-31000, France.
3
School of Medicine,
University of Queensland,
Herston, Queensland 4006,
Australia.
Correspondence to M.J.S.
e‑mail: mark.smyth@
qimrberghofer.edu.au
doi:10.1038/nri3799
Published online 6 March 2015
Natural killer (NK) cells are innate lymphocytes that
are crucial for the early control of some pathogen infec-
tions and malignancies1. NK cells kill infected or trans-
formed cells through the release of cytolytic granules
that contain perforin and granzymes or through the
ligation of death-inducing receptors. NK cells also con-
tribute to the shaping of adaptive immune responses
through the production of a wide range of chemokines
and pro-­inflammatory cytokines1. These mainly include
interferon‑γ (IFNγ), tumour necrosis factor (TNF),
granulocyte–macrophage colony-stimulating factor
(GM‑CSF), interleukin‑6 (IL‑6), CC‑chemokine ligand 3
(CCL3), CCL4 and CCL5, and they can be released in
response to exogenous cytokine stimulation or following
ligation of NK cell-activating receptors.
NK cell activation is controlled by a wide range of
germline-encoded receptors that allow these cells to sense
and to rapidly respond to changes in their environment2.
Depending on the signal they transmit, these receptors are
usually classified in two categories: inhibitory and activat-
ing receptors. NK cells use their inhibitory receptors to
detect the absence of self molecules on potential target
cells — a recognition strategy that is known as the detec-
tion of ‘missing self’ (REF. 3). Inhibitory receptors — most of
which bind to MHC class I molecules — include the killer
cell immunoglobulin-like receptors (KIRs) and the leuko-
cyte immunoglobulin-like receptors (LIRs) in humans,
the LY49 family in mice and the CD94–NKG2A hetero­
dimers in both species. NK cell-activating receptors mainly
recognize pathogen or cell stress-induced ligands and
therefore allow further discrimination between healthy
cells and potential target cells (for a full review, see REF. 4).
An important family of adhesion molecules that bind
nectins and nectin-like family proteins has recently been
identified as a crucial regulator of NK cell functions5–7
(BOX 1; FIG. 1). CD226 (also known as DNAM1, PTA1
and TLISA1) is the most extensively studied member of
this family and was originally described as an adhesion
molecule that controls NK cell-mediated cyto­toxicity 8. Its
ligands, CD112 (also known as nectin 2 and PRR2) and
CD155 (also known as PVR and NECL5), are regulated
by cellular stress and are involved in different patho-
logical conditions ranging from cancer to infectious dis-
ease9–11. Interestingly, two other molecules that contain
inhibitory motifs — CD96 (also known as TACTILE) and
T cell immunoreceptor with immunoglobulin and ITIM
domains (TIGIT) — also bind to CD226 ligands and were
recently found to counterbalance the CD226‑mediated
activation of NK cells5,6,12–14. In this Review, we discuss the
most recent discoveries concerning the roles of CD226,
CD96 and TIGIT in NK cell biology, the pathological
implications of these receptors and their potential thera-
peutic applications compared with other well-known
paired NK cell receptor families.
Biochemistry and ligand specificity
CD226. CD226 is a transmembrane glycoprotein of the
immunoglobulin superfamily that is composed of an
extracellular domain containing two immuno­globulin-
like domains8. CD226 is expressed by most resting
human NK cells and by a large proportion of mouse
NK cells8,15. CD226 interacts with the nectin and nectin-
like family members CD112 and CD155 (REFS 9,16). Of
note, the recombinant CD226–Fc fusion protein bound

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Box 1 | Nectin and nectin-like family proteins

Nectins and nectin-like proteins are members of the large immunoglobulin superfamily that has central roles in cell
adhesion, cell movement, proliferation and survival, and that contributes to the morphogenesis of numerous tissue types
(for a review, see REF. 132). These receptors mediate cell adherens junctions by forming homodimers or heterodimers in
trans and lateral homodimers in cis to create a tight network of nectin ‘zippers’ between juxtaposed cells132. CD155
has a unique expression pattern among nectin and nectin-like proteins; it is expressed at very low levels in most adult
organs but is abundantly expressed in regenerating liver tissues133. CD155 expression is also frequently upregulated in
transformed cells and contributes to their loss of contact inhibition, which markedly enhances cell movement and
proliferation132,134. As a consequence, CD155 overexpression is associated with cancer invasiveness and metastasis135–138.
Several pathways have been involved in the regulation of CD155 expression, including the mitogen-activated protein
kinase and nuclear factor‑κB pathways47,134. In addition, several other studies have defined that DNA-damaging
conditions (such as genotoxic drugs or chemotherapy, dysregulated proliferation and oxidative stress) can promote the
expression of CD155 through the DNA damage sensors ataxia telangiectasia mutated (ATM) and ATM- and RAD3‑related
(ATR)53,66,74. The role of the DNA damage response in controlling CD155 expression has also been shown in the context
of viral infections, during which the DNA damage response pathway functions as an intrinsic defence response to the
incoming foreign genetic material139,140. The important immune-stimulatory functions of CD155 suggest that this
receptor provides a crucial direct link between intrinsic responses to cellular stress and surveillance by the immune
system. This hypothesis is supported by recent findings in a spontaneous B cell lymphoma model showing that
upregulation of CD155 expression induced by the DNA damage response in early-stage transformed B cells activates
natural killer (NK) cells and T cells, and leads to spontaneous tumour regression74.

less efficiently to CD112 than CD155‑transfected cells16. TIGIT. TIGIT is an immune cell-specific immuno-
The first amino-terminal extracellular IgV-like domain globulin superfamily receptor of the CD28 family that
was shown to be crucial for CD226 interactions with its was discovered by three independent groups using
ligands15,17,18. A second splice variant that lacks this domain genome-wide strategies14,21,22. TIGIT is present at the
and the ability to interact with CD155 has been described cell surface of most human NK cells6,14 and is hardly
in mice, although the functional role of this variant detectable on naive mouse NK cells, but its expression
remains to be determined15. CD226 ligands are thought is upregulated following NK cell activation5,20,23. TIGIT
to be conserved between humans and mice19, but a recent consists of a short extracellular domain containing one
report suggests that mouse CD226 only interacts with extracellular IgV domain and a type 1 transmembrane
mouse CD155 (REF. 20). There is another activating recep- region14. A search for homology to the TIGIT sequence
tor that is similar to CD226, which is known as cytotoxic revealed that the immunoglobulin domain of TIGIT
and regulatory T cell molecule (CRTAM; also known as was similar to the amino-terminal domains of CD96,
CD355), but this receptor has been less well studied (BOX 2). CD155 and CD226 (REF.  14) . Similarly to CD226,
TIGIT binds to CD112 and CD155 (REFS 14,21,22).
Interestingly, its affinity for CD112 and CD155 is
CD44 αVβ3 CD226 LFA1 higher than that of CD226, and competition assays
153 μM
119 nM showed that TIGIT inhibited the interaction between
8.97 μM 0.4 μM CD155 and CD226 in a dose-dependent manner 14,20. It
Nectin 4 CD96
37.6 nM
CD155 CD112 was recently shown that TIGIT interacts with CD226
in cis and prevents CD226 homodimerization 24. In
360 nM
17.5 μM 228 μM 70.8 nM addition, TIGIT was shown to bind to the nectin
3.15 nM
family member CD113 (also known as nectin 3 and
NECL4 CD111
2.3 nM
CD113
38.9 nM
TIGIT PVRL3)14.

CD96. CD96 is a transmembrane glycoprotein

that has three extracellular immunoglobulin-like
NECL3 NECL1 NECL2 CRTAM
12.5 μM domains and is expressed by all resting human and
Figure 1 | Mode of interactions of nectin and nectin-like molecules.  Nectins (red mouse NK cells5,25,26. CD96 has approximately 20%
boxes) and nectin-like proteins (NECLs; blue boxes) form homophilic (looped
Nature Reviews arrows)
| Immunology homology with CD226 and its main ligand is also
and heterophilic (double-headed arrows) interactions in trans (thin arrows) and in cis CD155 (REFS 14,27). In addition, mouse CD96, but
(thick arrows) that may influence their relative binding affinity for CD226, T cell not human CD96, has been shown to bind to CD111
immunoreceptor with immunoglobulin and ITIM domains (TIGIT), CD96, cytotoxic and (also known as nectin 1 and PVRL1)27. The affinity
regulatory T cell molecule (CRTAM) and other immunoglobulin-like molecules (green of human CD96 for CD155 was found to be inter-
boxes). For example, CD112 forms cis and trans homodimers and it was suggested that mediate between TIGIT and CD226 (REF. 14), and a
CD112 homophilic interactions mask the CD226 binding site17. CD155 also associates in
recent study indicates that CD96 actively competes
cis with integrin αVβ3 (REF. 146) or CD44 (REF. 147) and in trans with CD113 (REF. 148).
Similarly, CD226 is physically associated with lymphocyte function-associated antigen 1 with CD226 for CD155 binding on naive mouse
(LFA1) on natural killer (NK) cells and this association is essential for CD226 signalling39. NK cells5. In addition, human, but not mouse, CD96 is
All of these interactions may greatly influence the accessibility and the binding of these expressed as two splice variants that differ in their sec-
receptors to their respective ligands. Known dissociation constants (Kd) are shown to ond immunoglobulin-like domain and their binding
indicate variations in affinity. affinity for CD155 (REF. 26).

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Box 2 | Role of CRTAM in the regulation of NK cell functions
Cytotoxic and regulatory T cell molecule (CRTAM) was discovered in 2000 using a cDNA
library generated from natural killer (NK) and T cells141. The structure of CRTAM has
some similarity to CD226 as it contains two extracellular immunoglobulin-like domains.
Only one CRTAM ligand has so far been described, the nectin-like family member 2
(NECL2; also known as CADM1 and TSLC1). Unlike CD226 and CD96, CRTAM is not
expressed by resting NK cells but expression of this receptor is rapidly and transiently
upregulated following NK cell activation142,143. CRTAM has been shown to enhance
NK cell adhesion and cytotoxicity, and to trigger the rejection of NECL2‑expressing
targets in vivo143. The generation of Crtam– /– mice revealed that CRTAM has a pivotal
role in CD8+ T cell retention within peripheral lymph nodes by favouring interactions
with NECL2‑expressing CD8α+ dendritic cells (DCs)144. CRTAM is also involved in
stabilizing the conjugation between CD4+ T cells and DCs145. A recent study revealed
that CRTAM interacts with the cell polarity regulator SCRIB to maintain T cell polarity
after the early phase of T cell activation145. CRTAM expression is rapidly upregulated
following activating receptor triggering on NK cells and future experiments will have to
determine whether, similarly to T cells, this receptor regulates NK cell interactions with
antigen-presenting cells.
Functions in NK cells
Regulation of NK cell adhesion and cytotoxicity.
Consistent with the crucial role of nectin and nectin-
like proteins in cell adhesion (BOX 1), CD226 was initially
defined for its ability to control NK cell adhesion and
cytotoxicity 8. Since then, the importance of CD226 in
NK cell-mediated killing has been supported by numer-
ous studies using a wide range of target cells, showing
that CD226–ligand interactions trigger a canonical acti-
vating pathway that enables the recognition of poten-
tial targets by NK cells28. Similarly to CD226, human
CD96 interactions with CD155 were originally shown to
enhance cell adhesion29. CD96 was considered to be a
potential co‑stimulatory receptor on the basis of its abil-
ity to increase the cytotoxicity of the human NK cell line
NK92 in redirected killing assays29. However, a recent
study using Cd96−/− mice failed to observe any effect of
CD96 loss on NK cell killing, and the role of CD96 on
NK cell-mediated cytotoxicity might differ between mice
and humans5.
Although TIGIT–CD155 interactions can also
enhance cell cluster formation30, there is now increasing
evidence that, unlike CD226, TIGIT inhibits NK cell-
mediated killing 6,20,31. For example, the transfection of
TIGIT limited cytotoxicity by the NK cell line YTS against
a Cd155‑transfected 721.221 B cell line and the cross­
linking of TIGIT with a specific antibody inhibited human
NK cell-mediated redirected killing 6,31. In addition,
TIGIT was also shown to limit mouse NK cell-mediated
cytotoxicity against CD155‑expressing B12 cells20.
Regulation of immune synapse formation. NK cell inter-
actions with target cells occur through a specialized
signalling domain known as the immunological syn-
apse (or lytic synapse)32. This dynamic cellular interface
involves numerous receptors, signalling molecules and
organelles that segregate in distinct regions. CD226 was
shown to polarize to the immunological synapse follow-
ing stimulation and recent reports indicate that CD226
deficiency or blockade impairs immunological synapse
formation18,33. The formation of the immunological syn-
apse is a multi­step process that begins with adhesion
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between the NK cell and its target cell34. The initial
adhesion is mainly mediated by the integrin lympho­
cyte function-associated antigen 1 (LFA1; also known as
integrin αL) that forms adhesion points in the periphery
of the synapse and is linked to the cytoskeleton to sta-
bilize conjugates35,36. CD226 also tightly associates with
the cytoskeleton by binding to the membrane-­associated
guanylate kinase (MAGUK) homologue DLG1 (also
known as SAP97)37,38, and several pieces of evidence sug-
gest that CD226 works together with LFA1 during this
initial process of actin cytoskeleton reorganization37,39,40.
Indeed, CD226 strongly associates with LFA1 following
activation of the cell and this interaction is required for
CD226 signalling and functions39. In addition, CD226 is
involved in LFA1‑mediated co‑stimulatory signals and its
signalling induces an LFA1 conformational change that
increases its affinity for its ligand intercellular adhesion
molecule 1 (ICAM1) on interacting cells37,40.
Interestingly, following target cell recognition,
TIGIT also clusters at the immunological synapse
and its engagement by CD155 has been shown to dis-
rupt polarization of the microtubule organizing centre
(MTOC) and cytotoxic granules31. Of note, the absence
of functional interactions between CD155 and CD226
in this study is a potential limitation and future experi-
ments will need to address whether TIGIT binding to
CD155 can antagonize CD226- and LFA1‑mediated
signalling during immunological synapse formation.
The role of CD96–CD155 interactions in immuno-
logical synapse formation is also worth considering, as
CD96 was originally shown to enhance human NK cell
adhesion to CD155‑expressing targets29.
Regulation of cytokine secretion. Recent evidence indi-
cates that CD226 and other nectin and nectin-like family
members also influence NK cell cytokine secretion. Indeed,
CD96 and CD226 were recently shown to oppose each other
in the regulation of NK cell production of IFNγ5,12,13. In
response to lipopolysaccharide (LPS), the absence of CD96
in Cd96−/− mice resulted in a marked increase in the propor-
tion of IFNγ-producing NK cells, whereas the frequency of
IFNγ-producing NK cells was reduced in Cd226−/− mice
compared with wild-type mice (FIG. 2a–c). CD96 was
shown to directly inhibit NK cell cytokine production,
but experiments using mice deficient in both CD226 and
CD96 (Cd226−/−Cd96−/− mice) revealed that CD96 also
functions independently of CD226 in limiting NK cell
IFNγ production5. CD226 seems to have a pivotal role in
promoting NK cell cytokine secretion during inflamma-
tion, and CD96 limits NK cell cytokine secretion at least
partially through a competition with CD226 for CD155
binding. These results are consistent with recent findings
showing that CD226 signalling is involved in the regula-
tion of T cell cytokine secretion41,42. Although TIGIT was
shown not to be involved in NK cell cytokine secretion
during LPS-driven inflammation5, a recent report indi-
cates that TIGIT may limit IFNγ production by hepatic
NK cells during liver regeneration43. Indeed, CD155 was
strongly expressed by hepatocytes and the absence of
TIGIT was associated with impaired liver regeneration
that depended on IFNγ production by hepatic NK cells.
REVIEWS

In addition, TIGIT binding to CD155 may limit the
NK cell IFNγ production elicited by target cell recogni-
tion20,44. In a recent study, NK cells from Tigit-transgenic
mice produced less IFNγ than wild-type mice, whereas
NK cells from Tigit−/− mice had enhanced IFNγ production
when co‑cultured with the YAC‑1 target T cell line44.
Crosstalk with DCs. CD112 and CD155 are highly
expressed by dendritic cells (DCs) and recent evidence
indicates that these receptors crucially influence NK cell
and DC interactions5,45–47. For example, it has been
reported that TIGIT interaction with CD155 affects DC
immunogenicity 14. Ligation of CD155 with TIGIT–Fc

a Perforin and b IL-12 and

granzymes IL-18
CD226 CD112 Lysis IL-12R
IL-18R LPS
+ + CD155
_ + TLR4

NK cell TIGIT CD155 CD226

Transformed or Cd96–/–
infected cell NK cell APC
IFNγ IFNγ hi

c IL-12 and
IL-18
IL-12R
TCR IL-18R LPS
MHC + CD155
TLR4
T cell
_
TIGIT
T cell CD96
proliferation Cd226–/–
NK cell APC
IL-10 IFNγ low
CD155
NK cell
Dendritic
cell
IL-12

d Killing e NK cell
LY49H
CD112 m157
CD226 CD155
Lysis

+ CD226
+ CD155 Proliferation
_
MCMV- Virus
Inhibitory MHC Immature infected cell
receptor class Ilow dendritic cell
Tolerance

CD226
LY49H+ activated
NK cells

+ Mature
_
_ dendritic
Differentiation
_ cell
NK cell LY49H+ long-lived
Inhibitory MHC
receptor class Ihi NK cells

Figure 2 | Regulation of NK cell functions by CD226, CD96 and
TIGIT.  a | CD226 binding to CD155 or CD112 at the cell surface of
transformed or infected cells triggers cytotoxic granule exocytosis and
target cell lysis by natural killer (NK) cells. Conversely, engagement of
T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT)
limits NK cell-mediated cytotoxicity and interferon‑γ (IFNγ) secretion.
b | CD96 and CD226 provide opposing signals in the regulation of NK cell
production of IFNγ. CD155 expression is upregulated at the cell surface
of antigen-presenting cells (APCs) after activation of Toll-like receptors
(TLRs), and the interaction of CD155 with CD226 or CD96 either
co‑stimulates or limits the NK cell production of IFNγ that is induced by
APC-derived cytokines. c | The interaction of TIGIT with CD155 affects
dendritic cell (DC) immunogenicity. TIGIT-mediated ligation of CD155
limits the production of interleukin‑12Nature Reviews
(IL‑12) | Immunology
and enhances the
production of IL‑10 by DCs. TIGIT-modified DCs are less potent inducers
of T cell proliferation. d | CD226 has a pivotal role in the NK cell-mediated
killing of immature DCs. Unlike immature DCs, mature DCs are protected
from CD226‑mediated killing as a result of their high expression of
MHC class I that engages NK cell-inhibitory receptors. e | The cooperative
signalling between CD226 and LY49H is required for the proliferation of
LY49H+ NK cells and for the differentiation of memory NK cells upon
recognition of their respective ligands (CD155 and m157, respectively) on
DCs that are infected with mouse cytomegalovirus (MCMV). IL‑12R, IL‑12
receptor; LPS, lipopolysaccharide; TCR, T cell receptor.

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fusion protein diminished the production of IL‑12p40
and enhanced the production of IL‑10 by human DCs14
(FIG. 2c). Although the intracellular domain of CD155
contains an immunoreceptor tyrosine-based inhibitory
motif (ITIM) that can be phosphorylated upon TIGIT
ligation14,48,49, it is not clear how CD155 signalling affects
DC functions and NK cell–DC crosstalk. Recent analysis
of TIGIT–CD155 interactions revealed that TIGIT and
CD155 form a heterotetrameric complex that is com-
prised of two TIGIT and CD155 dimers. Interestingly,
TIGIT and CD155 homodimerization was shown to be
fundamental for the ITIM-mediated signalling of CD155
(REF. 30). Whether CD96 and CD226 can induce proper
CD155 dimerization and, similarly to TIGIT, alter DC
functions, is under debate12. The recently shown ability of
human TIGIT to interfere with CD226 homodimerization
in T cells in cis questions the relative importance of inter-
actions with DCs in trans 24. It is likely that both cis and
trans interference between different family members have
a role in regulating the outcome of immune responses.
Interactions between immature DCs and activated
NK cells can also result in the killing of DCs50 (FIG. 2d) —
a process that is known as DC editing. This process was
recently shown to occur in a cancer model as a mecha-
nism of DC selection that favours the development of
efficient antitumour adaptive immune responses51.
Interestingly, several reports in both humans and mice
indicate that CD226, together with other activating
receptors, has a pivotal role in DC editing by NK cells
in vitro15,45. Recent evidence indicates that NK cells have
similar regulatory functions directed at T cell responses
through the killing of activated T cells52. CD155 expres-
sion is upregulated by T cells following T cell receptor
activation and CD226 was shown to be crucial for the
NK cell-mediated elimination of activated T cells53.
Further work is now required to understand the rele­
vance of CD226 in DC and T cell killing in vivo, and
how CD96 and TIGIT may counterbalance this process.
NK cell memory. NK cells were long considered to be
short-lived cells that were unable to mount long-term
memory immune responses. However, accumulat-
ing evidence indicates that long-lived populations of
NK cells can differentiate after exposure to pathogens,
providing enhanced effector responses upon second-
ary challenge with similar antigens54–56. For example
in humans, NKG2C+ NK cells persist for months after
infection with human cytomegalovirus (HCMV)57,58.
In mice, a subpopulation of NK cells bearing the
receptor LY49H, which binds to mouse cytomegalo­
virus (MCMV) glycoprotein m157, undergoes a
rapid expansion and differentiates into long-lived
memory NK cells that persist for months in lymphoid
and non-lymphoid organs after MCMV infection55.
Interestingly, a recent study indicates that CD226 is
required for the proliferation of LY49H+ NK cells and
for the differentiation of memory NK cells following
MCMV infection 59. The expression of CD112 and
CD155 was found to be upregulated on DCs following
MCMV infection and it was hypothesized that cooper-
ative signalling between CD226 and LY49H is required
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for NK cell memory generation (FIG.  2e) . Memory
NK cell generation is not fully understood and future
studies will be needed to clarify how CD155–CD226
interactions regulate NK cell fate and whether CD96
or TIGIT are involved in this process.
Signalling
CD226. Unlike most classical NK cell-activating recep-
tors, CD226 does not associate with any immuno­receptor
tyrosine-based activation motif (ITAM)-bearing mole­
cules but it contains an intracellular domain that has
at least two phosphorylation sites — tyrosine residue
322 and serine residue 329 (REF. 8) (FIG. 3). The phos-
phorylation of Ser329 by protein kinase C was shown
to be crucial for ligand binding and for the association
between CD226 and LFA1 at the surface of NK cells39,60.
This physical association with LFA1 facilitates the phos-
phorylation of CD226 at Tyr322 by the SRC kinase FYN
and the propagation of CD226-induced downstream
signalling 39. CD226 signalling involves the sequential
phosphorylation of SH2 domain-containing leukocyte
protein of 76 kDa (SLP76; also known as LCP1) and
VAV1, which subsequently leads to phospholipase Cγ2
(PLCγ2) activation, Ca2+ mobilization and NK cell acti-
vation2. Interestingly, phosphorylation of VAV1 that is
induced by CD226 signalling alone is not sufficient to
trigger Ca2+ intracellular flux and combined engagement
with 2B4 is required to overcome the inhibition of VAV1
that is mediated by the E3 ubiquitin ligase Casitas B line-
age lymphoma (CBL)61,62. The synergistic phosphoryla-
tion (of Tyr113 and Tyr128) of SLP76 by CD226 and 2B4
is necessary for the binding of SLP76 to VAV1 and for
the subsequent activation of PLCγ2, Ca2+ mobilization
and NK cell degranulation63.
TIGIT. The cytoplasmic tail of TIGIT has an immuno-
globulin tail tyrosine (ITT)-like motif and an ITIM. The
ITT-like motif has an important role in TIGIT inhibi-
tory signalling, whereas the role of the ITIM remains
unclear 31,44. Indeed, recent reports indicate that TIGIT
binding to CD155 induces phosphorylation at Tyr225
in the ITT-like motif and the recruitment of SH2
domain-containing inositol‑5‑phosphatase 1 (SHIP1;
also known as phosphatidylinositol 3,4,5‑trisphosphate
5‑­phosphatase 1) through the cytosolic adaptor growth
factor receptor-bound protein 2 (GRB2)31,44. TIGIT-
mediated recruitment of SHIP1 is responsible for NK cell
inhibition by blocking phosphoinositide 3‑kinase (PI3K)
and mitogen-activated protein kinase (MAPK) signal-
ling pathways. In support of this, mutation of Tyr225 or
SHIP1 silencing abrogates TIGIT-mediated NK cell inhi-
bition. In addition, upon phosphorylation, the ITT-like
motif of TIGIT binds β‑arrestin 2 and recruits SHIP1 to
limit nuclear factor‑κB (NF‑κB) signalling 44.
CD96. Although a role for CD96 in NK cell biology
is emerging, little is known about the signalling of
this molecule5,25–27,29. Both mouse and human CD96
contain an ITIM-like motif (IXYXXI) in the intra-
cellular domain that potentially delivers inhibitory
signals26. In addition, a cysteine residue located in
REVIEWS

Target cell
ITIM

CD113 CD112 CD155 CD111 NECL2

b CD226 c
CD96 d CRTAM
LFA1

a TIGIT

β-arrestin 2 ITT P ITIM P

PKC
P
GRB2 P ? SCRIB
ITIM FYN
DLG1 CDC42
SHIP1

PKCζ
Ub P
Ub SLP76
Ub
TRAF6 P VAV1 VAV1 P
Actin
cytoskeleton
PI3K
p50 p65 P ERK
• Cell adhesion
NF-κB • Cell polarity

Degranulation Cytokine secretion

NK cell

Figure 3 | TIGIT, CD226, CD96 and CRTAM ligand specificity and signalling.  a | Upon interaction with CD112, CD113
or CD155, the immunoglobulin tail tyrosine (ITT)-like motif of T cell immunoreceptor with immunoglobulin
Nature Reviewsand ITIM
| Immunology
domains (TIGIT) is phosphorylated on Tyr225 and binds the cytosolic adaptor growth factor receptor-bound protein 2
(GRB2), which can recruit SH2 domain-containing inositol‑5‑phosphatase 1 (SHIP1) to inhibit phosphoinositide 3‑kinase
(PI3K) and mitogen-activated protein kinase (MAPK) signalling. In addition, phosphorylated TIGIT recruits SHIP1 through
β‑arrestin 2 and impairs nuclear factor‑κB (NF‑κB) activation by blocking TNF receptor-associated factor 6 (TRAF6)
autoubiquitylation. b | The intracellular domain of CD226 gets phosphorylated on Ser329 and Tyr322 upon binding to
CD112 and CD155. Ser329 phosphorylation facilitates activation of protein kinase C (PKC) and the physical association of
CD226 with lymphocyte function-associated antigen 1 (LFA1). LFA1 is then required for FYN-mediated phosphorylation of
Tyr322 and CD226‑mediated downstream signalling. In addition, CD226 tightly associates with DLG1 and works together
with LFA1 during the initial process of actin cytoskeleton reorganization. c | CD96 binds to CD111 and to CD155. The
intracellular domain of CD96 contains an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like motif but whether
this motif is phosphorylated upon ligand binding and whether it accounts for CD96‑mediated inhibition of natural killer
(NK) cell interferon‑γ (IFNγ) secretion remain to be defined. Of note, the human cytoplasmic tail of CD96 also contains a
YXXM motif that may lead to PI3K activation. d | Binding of cytotoxic and regulatory T cell molecule (CRTAM) to nectin-like
family member 2 (NECL2) promotes cell adhesion and polarity through its intracellular association with SCRIB, PKCζ and
cell division control protein 42 (CDC42). ERK, extracellular signal-regulated kinase; SLP76, SH2 domain-containing
leukocyte protein of 76 kDa.

the transmembrane domain, together with positively Roles in disease

charged amino acids at the cytoplasmic–transmembrane
interface, could be a putative binding site for SRC-
related kinases26,64. Of note, human CD96 differs from
mouse CD96 by the presence of a YXXM motif in its
cyto­plasmic tail12,26 — a motif that is frequently found
in activating co‑receptors of the immunoglobulin super-
family, such as NKG2D and CD28 (REF. 65). This motif is
a potential SH2 domain-binding site for the p85 subunit
of PI3K and thus could function to initiate an activating
signal that might account for the reported co‑­stimulatory
functions of CD96 in humans65.
CD226, CD96 and TIGIT in cancer. The nectin CD112
and nectin-like protein CD155 are expressed by many
solid and haematological malignancies11,19,66–72 (BOX 1).
The recognition of these ligands, especially CD155, by
CD226 is crucial for an antitumour immune response,
as shown in several mouse tumour models7,19,69,73,74.
Enforced expression of CD155 was shown to mark-
edly increase NK cell-dependent tumour rejection19.
By contrast, NK cell control of experimental meta­
stasis was greatly impaired in the absence of CD226
(REFS 7,75), and Cd226−/− mice were more susceptible

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© 2015 Macmillan Publishers Limited. All rights reserved


than wild-type mice to spontaneous fibrosarcoma for-
mation induced by methylcholanthrene (MCA)73, a
model in which tumour initiation is NK cell depend-
ent 76. Although CD155 potentiates CD226‑dependent
tumour immuno­surveillance, the presence of CD96
has recently been shown to counterbalance CD226
functions and to limit tumour control by NK cells5.
Indeed, in a recent study using Cd96−/− mice, it was
shown that the absence of CD96 resulted in increased
resistance to MCA-induced fibrosarcomagenesis and
experimental lung metastases5. In these models, the
host-protective role of CD96 deficiency was depend-
ent on IFNγ production by NK cells. Importantly,
experiments carried out in Cd226−/−Cd96−/− mice also
revealed that the loss of CD226 greatly limited the
effect of CD96, which suggests that CD96 is a crucial
regulator of CD226‑dependent NK cell antitumour
functions in vivo.
On the basis of its ability to inhibit NK cell-­mediated
cytotoxicity, TIGIT may also be a potent barrier to effi-
cient NK cell-mediated antitumour immune responses.
However, a recent study using Tigit−/− mice failed to
see any effect of TIGIT loss on tumour metastasis, and
the functional role of TIGIT in antitumour NK cell-
mediated responses remains to be addressed5. By con-
trast, a role for TIGIT in mouse and human CD8+ T cell
exhaustion has recently been described in models of
chronic lymphocytic choriomeningitis virus infection
and tumours. In the tumour model, a strong synergy
between TIGIT blockade and programmed cell death
protein 1 (PD1)–PD1 ligand 1 (PDL1)-mediated inhi-
bition was detected24. This antitumour synergy of the
combined blockade was abrogated by CD226 block-
ade, which indicates that control of CD226 function is
important.
CD226, CD96 and TIGIT in viral infection. Recent
reports indicate that the absence of CD226 results
in higher viral titres and delayed virus clearance,
which suggests that CD226 may have an important
role in controlling viral infection in vivo 59,77. CD112
and CD155 are expressed by virus-infected cells and
CD226 may be involved in the recognition of these
cells by NK cells59. For example, the killing of HCMV-
infected myeloid DCs by NK cells was mainly depend-
ent on CD226 (REFS 78,79). As discussed in the previous
section, CD226 is also involved in the differentiation
and the expansion of memory NK cells that may be
required for optimal control of viral infections 59.
The importance of CD226 in NK cell control of viral
infections is also highlighted by the diverse evasion
strategies developed by virus to avoid CD226 recogni-
tion. Indeed, several viral proteins have been shown
to prevent the cell surface expression of both CD112
and CD155, thus affecting the cytotoxic responses of
NK cells79–81.
CD226, CD96 and TIGIT in autoimmunity. A single
nucleotide polymorphism in CD226, which leads to
one amino acid substitution (Gly307Ser) in CD226,
was recently associated with the development of several
NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 7
© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
autoimmune pathologies, including type 1 diabetes,
rheumatoid arthritis and systemic lupus erythema­
tosus82–86. Given the number of studies linking NK cells
to autoimmune pathologies (for a review, see REF. 87),
we can speculate that altered NK cell function resulting
from CD226 mutations may favour the development
of immune-related pathologies. However, the role of
CD226 in T cells is also potentially important and must
be considered. Future work will determine whether the
Gly307Ser substitution that occurs in the intra­cellular
domain of CD226 affects CD226 signalling, and the
functions of NK cells and T cells. In addition, it will
be important to understand the role of the CD226
antagonists CD96 and TIGIT in the development of
autoimmune disease. Indeed, a recent study reported
that Tigit−/− mice were more susceptible to experimen-
tal autoimmune encephalomyelitis than wild-type mice
and that TIGIT blockade accelerated collagen-induced
arthritis22.
Other paired receptors in NK cells
NK cells express a wide repertoire of activating and
inhibitory receptors. Akin to the CD226–CD96–TIGIT
family, many of these receptors are closely related
membrane proteins with similar binding specificities
but with either activating or inhibitory functions. In
this section, we briefly discuss the role of other well-
known paired NK cell receptor families and their rela-
tive importance in NK cell biology compared with the
receptors for nectin and nectin-like proteins.
KIR family molecules. KIRs are a prototypic group of
immunoglobulin superfamily receptors that interact
specifically with MHC class I molecules. Each KIR
recognizes a subgroup of HLA class  I allotypes 88.
Similarly to HLA molecules, KIRs are highly polymor-
phic and this high degree of polymorphism influences
KIR–HLA interactions89. Of note, KIRs do not exist in
mice but the C‑type lectin-like LY49 receptors have
similar functions as a result of their interaction with
MHC class I molecules90. KIR nomenclature is based
on the presence of a long or a short cytoplasmic tail
and the number of extracellular domains. KIRs with
long cytoplasmic tails (KIR‑L molecules) are charac-
terized by the presence of ITIMs that, upon ligand rec-
ognition, recruit the tyrosine phosphatase SHP1 (also
known as PTPN6) and inhibit NK cell activation 2.
Only one SHP1 substrate has so far been identified
in NK cells — the guanine nucleotide exchange factor
VAV1 (REFS 91,92) — and it is postulated that inhibi-
tory KIRs limit NK cell functions mainly by blocking
the most proximal signals that are required for the
recruitment and the phosphorylation of activating
receptors93,94. TIGIT differs from inhibitory KIRs as
a result of its preferential association with SHIP1 and
its more targeted inhibition of PI3K and NF‑κB signal-
ling pathways31,44. Consistent with the more restricted
distribution of its ligands CD112 and CD155 (BOX 1),
TIGIT signalling may have more specialized inhibitory
functions in NK cells than KIRs, although this requires
further investigation.
REVIEWS
Unlike KIR‑L molecules, KIRs with short cytoplas-
mic tails (KIR‑S molecules) lack ITIMs but associate
with DNAX activation protein 12 (DAP12; also known
as TYROBP), which is a protein adaptor that contains a
cytoplasmic region with an ITAM and that delivers acti-
vating signals89. The sequence homologies in the extra-
cellular portions of KIR‑L and KIR‑S molecules suggest
that activating KIRs should share the same ligand-­
binding specificity as their inhibitory counterparts, but
only the specificities of KIR2DS1 and KIR2DS4 have
been firmly established95–97. The functions of KIR‑S are
still mostly elusive. The presence of particular KIR‑S and
HLA class I combinations was shown to be associated
with several diseases in genetic studies, which suggests
that KIR‑S and HLA interactions affect NK cell activa-
tion88,98. For example, patients co‑expressing KIR3DS1
and its putative ligand HLA‑Bw4 with an isoleucine at
position 80 (HLA‑Bw4‑80Ile) were protected against
hepatitis C virus infection99 and had delayed progres-
sion to AIDS100,101. Consistent with the observation that
NK cell inhibition by inhibitory receptors dominates
over signals from activating receptors (as suspected
for CD96 and TIGIT, and CD226, respectively), it was
observed that the affinity of the activating KIRs for HLA
class I molecules is always lower than for their inhibi-
tory counterparts89. However, there is evidence that
inhibition resulting from interactions between KIRs and
MHC class I molecules is affected by the amino acid
sequence of the peptide bound to MHC class I102–105.
Although various MHC class I-binding peptides can
induce KIR-mediated inhibition, recognition by KIRs
can be impaired by certain peptides104,105. This suggests
that KIR-mediated inhibition can be released by MHC
class I-binding peptides that function as antagonists.
Conversely, the affinity of activating KIRs for HLA
molecules may be increased by the presentation of cer-
tain peptides and facilitate NK cell-mediated killing 106.
Thus, rapid changes in the peptide repertoire generated
during viral infections or cancer may alert NK cells to
alterations in their environment and may increase their
reactivity. Other non-HLA class I molecules that are
upregulated on target cells in pathogenic conditions
may also trigger signalling by activating KIRs, thereby
functioning as ligands or as co‑stimuli for these recep-
tors107. The upregulation of expression of CD155 follow-
ing cell stress and the subsequent interaction of CD155
with CD226, TIGIT or CD96 may affect the distribu-
tion of KIR‑L and KIR‑S within the cell membrane and
thereby their relative interactions with MHC class I at
the surface of contacting cells.
CD94–NKG2 receptor family molecules. The CD94–
NKG2 receptor family consists of a CD94 subunit that is
covalently associated with a member of the NKG2 fam-
ily (NKG2A, NKG2B, NKG2C, NKG2E or NKG2H)108.
The prototypic member of this family, CD94–NKG2A,
has an ITIM and, similarly to inhibitory KIRs, recruits
SHP1 to inhibit NK cell function109,110. By contrast,
CD94–NKG2C, CD94–NKG2E and CD94–NKG2H
associate with DAP12 and function as activating recep-
tors111. In humans, the ligand for the CD94–NKG2
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© 2015 Macmillan Publishers Limited. All rights reserved
receptor complex is the non-classical MHC class I
mole­cule HLA‑E112. Interestingly, HLA‑E presents pep-
tides that are derived from the conserved region of the
leader sequence of other MHC class I molecules includ-
ing HLA‑A, HLA‑B, HLA‑C and HLA‑G109. The inter­
action of HLA‑E with CD94–NKG2A enables NK cells
to monitor on target cells both the expression of MHC
class I molecules and the capacity to process and present
antigen112. HLA‑E has also been shown to bind viral pep-
tides in the absence of functional transporter associated
with antigen-­processing (TAP), and hijacking of HLA‑E
may be a common viral evasion mechanism to inhibit
NK cell activation113. As the main role of HLA‑E is to
prevent NK cell activation, this molecule may also protect
malignant cells from NK cell-mediated killing 114,115.
Unlike KIRs, the CD94–NKG2 family receptor–
ligand interactions are conserved between mice and
humans. In mice, CD94–NKG2A interacts with the
MHC class Ib molecule Qa1 in association with the Qa1
determinant modifier (Qdm), which is a leader
sequence derived from MHC class  I molecules 116.
Although CD94‑deficient mice lack expression of
NKG2A, NKG2C and NKG2E, these mice do not show
developmental or functional NK cell abnormalities117.
However, they are more susceptible to mouse poxvirus
infections and it was recently shown that the activa-
tion of NK cells by CD94–NKG2E has a crucial role
in the control of this virus118. Although the molecular
basis for the recognition of Qa1b by CD94–NKG2E
in poxvirus-infected mice is not clear, it suggests
that activating the CD94–NKG2 complex may have
a role in pathogen defence. The clonal expansion
of a population of NK cells that has a high density of
NKG2C in humans in response to HCMV infection is
consistent with this hypothesis119,120. Engagement of the
CD94–NKG2C complex specifically triggers NK cell
effector functions and the stimulation of peripheral
blood mononuclear cells with virus-infected fibroblasts
promotes the clonal expansion of NKG2C+ NK cells
in HCMV-infected donors120,121. This suggests that the
CD94–NKG2C complex itself might be involved in
driving NK cell clonal expansion in response to HCMV
infection122. Nevertheless, the nature of the putative viral
ligand for CD94–NKG2C remains elusive. The behav-
iour of human NKG2C+ NK cells in response to HCMV
is reminiscent of the behaviour of mouse LY49H +
NK cells that recognize the MCMV glycoprotein m157
(REF. 55). Interestingly, CD226 was shown to have a cru-
cial co‑stimulatory role in the expansion of the LY49H+
NK cell subset during MCMV infection, which raises the
question as to whether CD226 is also required for the
expansion of the NKG2C+ NK cell subset in humans59.
NK cell tolerance is thought to rely on signals
resulting from interactions between MHC class I and
cognate receptors, but no signs of NK cell-dependent
auto­immunity have been detected in mice or humans
with genetic defects that result in the lack of expression
of MHC class I molecules or in the absence of inhibi-
tory cell surface receptors for MHC class I molecules90.
Paradoxically, both mouse and human studies have
shown that in the absence of MHC class I-mediated
 REVIEWS

inhibitory receptor signalling, NK cells are hypo­

Genotoxic stress Oncogene activation responsive to various stimuli, which establishes that the
Drugs, oxygen free radicals or virus
engagement of inhibitory receptors is involved in the
responsiveness of NK cells123–125. It has been suggested
that the recognition of MHC class I molecules by inhibi-
ATM tory receptors ‘licenses’ NK cells to acquire their effector
or ATR
Dysregulated functions in a process referred to as ‘arming’ (REF. 126).
proliferation Alternatively, it has been proposed that NK cells integrate
Cd155
activating signals that desensitize them, leading to their
AP-1 hyporesponsiveness — a process referred to as ‘disarming’.
In accordance with this theory, the responsiveness of
NF-κB Loss of cell-contact
inhibition NK cells was shown to decrease upon chronic engage-
ment of activating receptors127. Thus, although NK cells
can respond to rapid modifications in their environment
Invasiveness (for example, downregulating MHC class I molecules or
TLR increasing their expression of stress-induced ligands),
when these alterations are long lasting, NK cells adapt
to them by ceasing to be responsive. These observations
CD155 led to a new immune paradigm that is referred to as ‘the
discontinuity theory’. This theory proposes that NK cells,
and immune cells in general, induce an effector response
when there is a discontinuity in the molecular motifs
with which their receptors interact, whereas they tend to
Inhibition of
angiogenesis TCR Tumour-specific become tolerant to continuously expressed motifs128. This
T cells theory may explain the presence of evolutionarily con-
Tumour Lysis served paired receptors that recognize similar ligands and
that transmit activating or inhibitory signals. With regard
CD226 to the biology of receptors for nectin and nectin-like
proteins, it was shown that the constitutive expression of
Tumour killing CD155 on antigen-presenting cells constantly limits the
CD155
expression of CD226, which suggests that a small propor-
CD96
IFNγ TIGIT tion of CD226 is constantly engaged at the cell surface of
CD96 Perforin
and TNF
DC maturation NK cells in steady-state conditions129. Therefore, the pres-
ence of the inhibitory receptors CD96 and TIGIT may
Therapeutic
antibody CD226 be required to avoid CD226‑mediated chronic activation
_
_ + and hyporesponsiveness of NK cells. Indeed, owing to
TIGIT
their increased affinity, these receptors are predominantly
Immune editing engaged upon interaction with resting antigen-presenting
Immune escape
cells that express low levels of their cognate ligand CD112
NK cell or CD155 (REFS 5,14). This dynamic inhibition by CD96
and TIGIT might be relieved in pathogenic conditions,
Figure 4 | Regulation of NK cell-mediated cancer immunosurveillance through during which cellular stress results in the upregulation
CD155 expression.  CD155 is frequently overexpressedNature Reviews
by cancer Immunology
cells.| Indeed, of these ligands and increases the engagement of CD226,
genotoxic stress that is associated with dysregulated proliferation, oncogene leading to full NK cell activation. Evaluation of mice
activation or viral transformation directly stimulates the transcription of Cd155 lacking both TIGIT and CD96 will be of interest in
through the DNA damage sensors ataxia telangiectasia mutated (ATM) and ATM-
investigating this idea in the future.
and RAD3‑related (ATR; top panel). In addition, activator protein 1 (AP‑1) activation
by oncogenic RAS or nuclear factor‑κB (NF‑κB) in response to Toll-like receptor (TLR)
ligation can also increase CD155 expression. CD155 has a dual role in the regulation Concluding remarks
of cancer development as it promotes cancer invasiveness and proliferation through Given the importance of host immune evasion in
the loss of contact inhibition but also provides a crucial direct link between intrinsic chronic infection and cancer and the promising clini-
responses to cellular stress and surveillance by the immune system. Indeed, the cal responses obtained by monoclonal antibodies
recognition of CD155 by CD226 is pivotal for natural killer (NK) cell-mediated cancer targeting lymphocyte-inhibitory pathways in cancer
immunosurveillance through perforin-mediated tumour cell destruction and the treatment 130, interest in the role of the receptor–ligand
release of anti-angiogenic cytokines such as tumour necrosis factor (TNF) and families that regulate lymphocyte effector functions is
interferon‑γ (IFNγ; lower panel). These cytokines and others that are released by growing. CD226, TIGIT and CD96 are crucial regula-
NK cells also help to shape the adaptive immune response by promoting dendritic
tors of lymphocyte-mediated effector functions against
cell (DC) maturation and tumour-specific T cell clonal expansion. Unfortunately,
the presence of CD96 and T cell immunoreceptor with immunoglobulin and ITIM tumours and may be promising new therapeutic tar-
domains (TIGIT) at the cell surface of NK cells limits CD226–CD155 interactions gets for the treatment of malignancies (FIG.  4). This
and inhibits NK cell activation, which subsequently favours immune escape. is particularly true for settings in which the tumour is
These two receptors represent promising targets for therapeutic antibodies to mainly non-immunogenic (that is, it does not express
increase or to restore tumour immunogenicity. TCR, T cell receptor. stress ligands or co‑stimulatory molecules), which is a

NATURE REVIEWS | IMMUNOLOGY ADVANCE ONLINE PUBLICATION | 9

© 2015 Macmillan Publishers Limited. All rights reserved

REVIEWS

common situation for many epithelial cell malignan-
cies75. Co‑inhibitory receptors can suppress immune
responses by direct signalling in cis, by indirect com-
petition with complementary co‑stimulatory receptors
or by inducing ligand signalling in trans. The comple-
mentary co‑stimulatory and co‑inhibitory receptor pair
CD28 and cytotoxic T lymphocyte antigen 4 (CTLA4)
has proved to be a prototypic target for cancer immuno-
therapy. Therapeutic possibilities in cancer include the
co‑blockade of TIGIT and CD96 or agonism of CD226
signalling using novel monoclonal antibodies. Of course,
it is possible that the expression of CD226, TIGIT and/or
CD96 on non‑NK cell populations, such as effector
T cells and regulatory T cells, might be just as relevant
(or more so) in the function of these molecules in the
context of graft-versus-host disease131 or in immuno-
therapy of primary tumours7,73. The recent description
of the synergistic antitumour effects mediated by CD8+
T cells following blockade of both TIGIT and PD1
or PDL1 generates greater interest in the roles of the
TIGIT–CD96–CD226 family in cancer immunology24.
The ligand CD155 has many potential pro-­
tumorigenic effects on malignant cells and this biol-
ogy deserves greater attention given the widespread
expression of this molecule in cancers (FIG. 4). CD155
might also transmit an intrinsic signal following
binding to its receptors and thus may have func-
tional roles in antigen-presenting cells, such as DCs,
and in non-haematopoietic cells beyond its abil-
ity to regulate lympho­c yte activity. These roles need
far more exploration in mouse models of disease by
studying CD155‑deficient mice and by investigating
the potential CD155 signalling in antigen-presenting
cells. Further work is required to validate CD112 as
a ligand for TIGIT and CD226, particularly to deter-
mine the biological importance of CD112 in vivo. The
fact that CD96 does not seem to interact with CD112
calls into question the possible role of CD112 relative to
CD155 in vivo. Recent advances in the understanding
of the key cell signals that promote CD112 and CD155
expression provide new clues to therapeutic opportuni-
ties that involve increasing their expression by tumour
cells through genotoxic drugs66.
CD96 biology remains poorly studied and more
work is required to understand whether this receptor
improves or limits lymphocyte reactivity in humans. In
humans, CD96 has features, such as spliced forms and
an additional potential activation motif, that suggest
that it might be capable of cell inhibition or activation,
depending on the context. The signalling pathways by
which human and mouse CD96 functions in lympho-
cytes require molecular definition. Future work should
discriminate the relative roles of TIGIT and CD96 as
negative regulators of CD226 activation in human ver-
sus mouse immune responses. Inhibitory pathways are
required to regulate the activation of lymphocytes and
to limit the programme of intracellular events driven
by the activating receptors. In the case of inhibition of
CD226 by CD96 and TIGIT, at least one ligand, CD155,
is shared. The higher affinity of TIGIT, and to a lesser
extent CD96, than CD226 in binding to CD155 main-
tains a general state of CD226 inactivation. In naive
mouse NK cells, the relatively higher constitutive expres-
sion of CD96 than TIGIT may determine its dominant
role in regulating CD226 function. However, in NK cells
that are activated by various mechanisms, the upregula-
tion of TIGIT expression may enable TIGIT to have a
more important role than CD96 in regulating NK cell
function. Careful studies will be required to determine
the relative expression and function of CD96 and TIGIT
on various lymphocyte populations at different stages of
the immune response. It remains unclear whether these
inhibitory receptors regulate cytokine and/or cytotoxic
effector functions. In addition, structural studies to
determine the molecular details of CD226, TIGIT and
CD96 binding to CD155 in both mice and humans will
probably shed light on why two inhibitory receptors are
required to control one activating receptor. Higher order
structures involving TIGIT, CD96 and CD226 may also
contribute to their interaction with ligands. The role of
these receptor–ligand pairs in trans versus in cis remains
under debate and may further complicate their biologi-
cal roles. An intrinsic role of CD226, CD96 and TIGIT
interacting with CD155 on lymphocytes may represent
a very interesting mechanism of control to investigate
in the future.

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Acknowledgements
The authors thank members of their laboratory for contribu‑
tions in this area. L.M. and M.J.S. are supported by a
National Health and Medical Research Council of Australia
Fellowship and Project Grant. L.M. is supported by the
Association pour la Recherche Contre le Cancer.
Competing interests statement
The authors declare competing interests: see Web version for
details.