Cellular and Molecular Immunology (2018) 14, 1–10

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REVIEW

Functions of NKG2D in CD8+ T cells: an opportunity for

immunotherapy
Kushal Prajapati, Cynthia Perez, Lourdes Beatriz Plaza Rojas, Brianna Burke and
Jose A Guevara-Patino
Natural killer group 2 member D (NKG2D) is a type II transmembrane receptor. NKG2D is present on NK cells in
both mice and humans, whereas it is constitutively expressed on CD8+ T cells in humans but only expressed upon
T-cell activation in mice. NKG2D is a promiscuous receptor that recognizes stress-induced surface ligands. In NK
cells, NKG2D signaling is sufficient to unleash the killing response; in CD8+ T cells, this requires concurrent
activation of the T-cell receptor (TCR). In this case, the function of NKG2D is to authenticate the recognition of a
stressed target and enhance TCR signaling. CD28 has been established as an archetype provider of costimulation
during T-cell priming. It has become apparent, however, that signals from other costimulatory receptors, such as
NKG2D, are required for optimal T-cell function outside the priming phase. This review will focus on the
similarities and differences between NKG2D and CD28; less well-described characteristics of NKG2D, such as the
potential role of NKG2D in CD8+ T-cell memory formation, cancer immunity and autoimmunity; and the
opportunities for targeting NKG2D in immunotherapy.
Cellular and Molecular Immunology advance online publication, 5 February 2018; doi:10.1038/cmi.2017.161

Keywords: NKG2D; CD28; CD8+ T Cell; Cancer; Immunity; Auto-immunity; Memory; Immunotherapy

INTRODUCTION binding proteins 1–6 in humans.8 The expression of NKG2D

Natural killer group 2 member D (NKG2D) is a type II
transmembrane receptor encoded by the killer cell lectin-like
receptor subfamily K member 1 (Klrk1) gene.1,2 NKG2D is
present on all murine and human NK cells. It is constitutively
expressed on human CD8+ T cells, whereas its expression on
CD8+ T cells occurs after activation in mice.3 In addition to
NK and CD8+ T cells, NKG2D can also be expressed by natural
killer T (NKT) cells, γδ T cells, activated murine macrophages
and a small subgroup of CD4+ T cells.4 The functions of
NKG2D, however, differ between NK cells and CD8+ T cells.
In NK cells, NKG2D signaling alone is sufficient to mediate
direct killing of target cells,4 whereas in CD8+ T cells,
simultaneous activation of the T-cell receptor (TCR) is
required for NKG2D to be functional. In these cells, NKG2D
acts as a costimulatory receptor, and its signaling leads to
enhanced TCR activation and T-cell function.3–5
NKG2D is a promiscuous receptor, binding to a variety of
stress ligands. These ligands include members of the Rae-1ε
and H60a-c families6,7 in mice and major histocompatibility
complex (MHC) class-I-related chain (MIC) A/B and UL16
ligands on the cell surface is strictly regulated in the body.
Under healthy conditions, few or no NKG2D ligands are
expressed.6,7 However, expression of these ligands is
induced by transcriptional upregulation under stress-related
conditions, such as infection and transformation.6,7 Notably,
other forms of cellular insult (e.g., exposure to DNA-damaging
agents, TLR signaling, cell proliferation or exposure to certain
cytokines) can also result in surface expression of these
ligands.5,8,9
NKG2D has a short intracellular domain that lacks any
signaling motifs. In CD8+ T cells, NKG2D signals through the
adaptor protein DNAX-activating protein 10 (DAP10), whereas
in NK cells, it can pair with either DAP10 or its homolog
DAP12.3,10,11 DAP10 contains a YINM motif, which recruits
p85 to induce PI3K signaling and Grb2 to activate Vav-SOS
signaling.12,13 Interestingly, the same YxNM signaling motif
exists within the CD28 costimulatory receptor,14–16 suggesting
that these motifs are the result of coevolutionary adaptation, as
both NKG2D and CD28 function as costimulatory receptors in
CD8+ T cells. However, it is important to note that CD28 is

Loyola University Chicago, Oncology Institute, Maywood, IL 60153, USA

Correspondence: Dr JA Guevara, Loyola University Chicago, Oncology Institute, 2160 South First Avenue, Maywood, IL 60153, USA.
E-mail: jaguevara@luc.edu
Received: 30 September 2017; Revised: 30 November 2017; Accepted: 30 November 2017
 Functions of NKG2D in CD8+ T cells
JA Guevara-Patino et al
primarily required during the priming of naïve T cells, while
NKG2D is used by effector and memory CD8+ T cells.
In addition to its costimulatory activity, NKG2D appears to
be involved in non-canonical functions, such as memory
formation, although the few studies that exist concerning this
subject are conflicting. For instance, memory CD8+ T cells can
be activated by cytokines only under specific conditions, which
transform these cells into NK-like killer cells. In such cases,
they eliminate target cells through an NKG2D-mediated
mechanism without TCR engagement. Importantly, this
TCR-independent killing has been linked to the development
of several autoimmune disorders, highlighting the importance
of NKG2D under pathophysiological conditions. This review
will focus on the less well-described characteristics of NKG2D.
The similarities and differences between NKG2D and CD28
will be discussed, as well as the potential role of NKG2D in
CD8+ T-cell memory formation. Last, the involvement of
NKG2D signaling under various pathological conditions, such
as autoimmune diseases and cancer, will also be discussed
(Figure 1).
NKG2D AND CD28 COSTIMULATORY FUNCTIONS IN
CD8+ T CELLS
In CD8+ T cells, engagement of the NK receptor, NKG2D,
results in enhanced TCR activation and function,1,3–5,9 which
facilitates the recognition and destruction of target cells.3 The
NKG2D receptor has been defined as a costimulatory molecule
in CD8+ T cells, and thus, it is often compared with CD28, an
extensively studied costimulatory receptor in CD4+ and CD8+
T cells. Despite sharing many similarities, both the NKG2D
and CD28 receptors possess distinct expression patterns,
structures, signaling pathways and functional outcomes.
In humans, both CD28 and NKG2D are expressed in CD8+
T cells; however, the proportions of these cells expressing these
receptors differ. NKG2D is expressed by all CD8+ T cells,3
whereas CD28 is expressed by ~ 50% of mature human CD8+
T cells, in addition to the vast majority (~95%) of human
CD4+ T cells.17 The NKG2D receptor has gained increasing
research interest based on the widespread expression of its
ligands compared to those of CD28 (CD80/CD86), which are
expressed only in antigen-presenting cells (APCs). NKG2D
ligands can be expressed in any cell type upon cell stress,
including viral infection and DNA damage. This is also true in
cancer cells, underscoring the relevance of this receptor under
pathophysiological conditions. While NKG2D ligands do not
have any known association with inhibitory receptors, the
CD28 ligands B7.1 and B7.2 are shared by the inhibitory
receptor cytotoxic T-lymphocyte-associated protein-4 (CTLA-
-4), which blocks CD28-dependent T-cell responses.18–20
Both NKG2D and CD28 receptors use a similar YxNM
motif to transduce signaling pathways. However, they differ
remarkably in their structural properties and assembly, which
determine the unique functional outcomes of their signals.
CD28 is expressed as a homodimer on the cell surface with an
extracellular ligand-binding domain, a transmembrane domain
and an intracellular domain containing the YMNM signaling
Cellular and Molecular Immunology
motif, among other motifs (Figure 2).14–16 NKG2D, lacking
any signaling motif, relies on the YINM motif-containing
adaptor protein DAP10 to initiate its signaling pathways.21,22
The NKG2D-DAP10 complex is expressed as a hexamer
consisting of a homodimer of two NKG2D molecules, each
bound to two DAP10 molecules (Figure 2).23 Thus, each CD28
and NKG2D-DAP10 complex consists of two and four YxNM
signaling motifs, respectively. In addition to these structural
and stoichiometric differences, CD28 expresses additional non-
YxNM signaling domains. These include the membrane
proximal proline-rich PRRP domain, which can recruit
inducible-2 (IL-2) T-cell kinase, and the distal PYAP domain,
which is capable of binding to Lck, Filamin A and Grb2.16
For both CD28 and NKG2D-DAP10 receptors, activation
leads to phosphorylation of tyrosine within the YxNM motif by
Src family kinase, which is followed by the recruitment of Grb2
and p85 subunits to asparagine and methionine residues,
respectively. This in turn results in the activation of a wide
array of well-characterized Grb2 → Vav and p85 → PDK1 → Akt
signaling pathways, ultimately resulting in AP-1, NFAT and
NF-κB nuclear translocation and subsequent increases in cell
survival, proliferation and expression of effector molecules and
cytokines, as well as the release of cytolytic granules.14–16,24,25
In NK cells, it has been shown that NKG2D-DAP10
specifically stimulates phospholipase C-γ2 (PLC-γ2) down-
stream of the Grb2-Vav1 pathway.26 For CD28, no such
preference for PLC-γ1 has been described. It is thought that
PLC-γ1 and PLC-γ2 have redundant functions,27,28 and thus
the functional relevance of this preferential signaling of
NKG2D is not clear. It is important to note that although
both of these isoforms are equally expressed in NK cells, PLC-
γ1 is preferentially expressed over PLC-γ2 in T cells.29 Based on
previous studies that have established PLC-γ as a key signaling
molecule for costimulation signaling, it is plausible that
NKG2D’s costimulatory potential is reduced compared with
CD28 in CD8+ T cells. The same study demonstrated that
overexpression of PLC-γ2, but not PLC-γ1, led to increased
NKG2D-mediated cytotoxicity in the majority of human CTL
clones.26 However, the differences between NKG2D and CD28
signaling in the context of differential PLC-γ isoform expres-
sion were not addressed in this work.
In 2003, it was reported that NKG2D-DAP10 does not
activate LAT, an important adaptor molecule that recruits key
proteins in T-cell signaling, in NK cells.30 Notably, CD28 has
been shown to induce phosphorylation of LAT independent of
Syk or ZAP70 kinases.31 One study reported that although
CD28 is necessary to phosphorylate LAT, its signaling motif-
containing cytoplasmic tail is not involved in the initiation or
persistence of TCR-induced LAT phosphorylation.32 NKG2D’s
effects on LAT in the context of TCR stimulation remain to be
determined. As mentioned above, CD28 has two additional
signaling domains that are absent in DAP10, namely, PRRP
and PYAP. Itk binds to the PRRP domain, the importance of
which is not yet clear in CD28 signaling. PYAP recruits Lck,
Filamin A and Grb2, and activates signaling pathways that lead
to cytoskeletal rearrangement and greater cytokine mRNA

Figure 1 NKG2D’s role in physiology, diseases and its potential use
in medical interventions: In CD8+ T cells, NKG2D signaling is
thought to occur exclusively via DAP10 and requires simultaneous
TCR engagement, and thus acting as an enhancer rather than an
inducer of activation. DAP10 uses a YINM motif to recruit PI3K
and Grb2, and activate the associated pathways. Major outcomes of
the signaling is enhanced TCR signaling, cytotoxicity and enhanced
T-cell survival. However, alterations in the NKG2D signaling and
expression levels can lead to autoimmune diseases that are either
TCR dependent: vitiligo, type I diabetes, and RA or TCR-
independent: celiac disease. Based on the contribution of NKG2D
signaling to CD8+ T-cell function, approaches are being
investigated for the treatment of cancer, autoimmunity and some
infections diseases. DAP10, DNAX-activating protein 10; NKG2D,
natural killer group 2 member D; RA, rheumatoid arthritis; TCR,
T-cell receptor.
stability.16 Although it apparently lacks this signaling potential,
NKG2D-DAP10 has been linked to other non-YINM signaling
pathways. In experiments performed with human NK cells,
Sutherland et al.33 demonstrated that NKG2D stimulation leads
to specific phosphorylation of JAK2 and STAT5. However, the
molecular basis and functional relevance of this pathway is not
yet completely understood.
Both CD28 and NKG2D stimulation have been shown to
have canonical costimulatory effects on T cells, including
increases in cell survival, proliferation, expression of effector
molecules and cytokines, and release of cytolytic
granules.14–16,24,25 Many studies have reported that NKG2D
is capable of costimulating human and mouse T cells to an
extent similar to that of CD28 at the level of proliferation,
cytolytic function and interferon-γ (IFN-γ) production.4,34–36 It
is important to note that factors such as cell activation status
and cytokine treatment can alter the responsiveness of CD8+
T cells to NKG2D. For instance, Lanier and co-workers37 were
Functions of NKG2D in CD8+ T cells
JA Guevara-Patino et al
unable to costimulate human and murine CD8+ T cells by
engaging the NKG2D receptor (unlike CD28), presumably due
to different culture conditions and/or cytokine milieu. Owing
to the unique characteristics of the signaling pathways dis-
cussed above, it is not altogether surprising that NKG2D and
CD28 would have different functional effects on T cells. In
experiments performed on DUC18 transgenic CTLs, only
CD28 could promote survival upon antigen encounter, whereas
only NKG2D could form immunological synapses in the
absence of antigen.34 Interestingly, in another study, Barber
and Sentman38 demonstrated that in either non-transduced or
NKG2D-transduced human CD8+ T cells, NKG2D engage-
ment specifically activated β-catenin, increased the expression
of β-catenin-induced genes and led to reduced production of
the anti-inflammatory cytokines IL-9, IL-10, IL-13 and vascular
endothelial growth factor-α in a peroxisome proliferator-
activated receptor-γ-dependent manner. CD28 stimulation
either failed to exert these effects or caused effects opposite
to that of NKG2D in this study. It was also found that both
CD28 and NKG2D stimulation decreased the migration of
human CD8+ T cells; however, only NKG2D activated Cdc42, a
Rho GTPase involved in T-cell motility, and only NKG2D’s
effects on T-cell migration were dependent on N-WASP.39
These data suggest that CD28 and NKG2D may also use
different signaling pathways to produce similar functional
outcomes in T cells. There is also evidence that both receptors
can synergize with each other or modulate the other’s
function,36,38 suggesting possible crosstalk between the two
receptors at certain signaling notches. Hu et al.40 studied the
effects of CD28 signaling on the expression of NKG2D and
found that CD28 costimulation resulted in sustained NKG2D
expression in murine and human CD8+ T cells via Lck/JAK/
STAT3 signaling.
Collectively, both NKG2D and CD28, despite sharing a
similar YxNM motif and associated upstream signaling events,
lead to unique signaling cascades, most likely due to different
structures, recruitment of adaptor proteins, downstream effec-
tors and possible crosstalk with other immune receptors. This
results in NKG2D- or CD28-mediated costimulation of CD8+
T cells with unique features/functional outcomes. In addition
to the above factors, differential expression levels of these
receptors on T cells as well as those of the activating ligands in
different tissues and cytokine environments may dictate the
relevance of NKG2D and CD28 at the physiological level.
NKG2D AND CD8+ T-CELL MEMORY FORMATION
During the normal course of an immune response, the
formation of memory CD8+ T cells is one of the fundamental
goals of the adaptive immune system. T-cell differentiation into
effector or memory cells is a tightly regulated process that is
still not fully understood. Current views suggest that T-cell
memory formation involves a passive transition from effector
to memory T cells and that memory is the result of initial
signaling cues promoting long-term survival.41 Among the
known cues, CD4+ T cells and cytokines, such as IL-15, have
been shown to play an essential role in not only activating
Cellular and Molecular Immunology
 Functions of NKG2D in CD8+ T cells
JA Guevara-Patino et al
Figure 2 Differences in the structure and signaling motifs of CD28
and NKG2D receptors: NKG2D-DAP10 complex is a hexameric
structure with two NKG2D molecules linked to total of four DAP10
adaptors, whereas CD28 is expressed as a homodimer on the cell
membrane. Both receptors contain YxNM signaling motif capable of
recruiting Grb2 and p85 subunit of PI3K. In addition, CD28
possesses PRRP, and PYAP domains capable of recruiting Itk, Lck,
Fil A and Grb2. DAP10, DNAX-activating protein 10; ltk, IL-2-
inducible T-cell kinase; NKG2D, natural killer group 2 member D;
PI3K, phosphatidylinositol-3 kinase.
CD8+ T cells but also programing them for memory
formation.42–47 However, recent work has increasingly estab-
lished that other factors, including NKG2D signaling, are
responsible for CD8+ T-cell memory formation.48–52 Mamma-
lian target of rapamycin complex 1 (mTORC1) has been
shown to play a pivotal role in deciding the fate of primed
CD8+ T cells. Low mTORC1 levels favor differentiation into
memory precursor cells, while high levels of mTORC1 activity
leads to the development of terminally differentiated CD8+ T
cells.49 Interestingly, McQueen et al.49 demonstrated that both
CD28 and NKG2D signaling activated the mTORC1 pathway
but to different extents. CD28 signaling induced strong
mTORC1 signaling, which led to the transcription of marker
genes of terminally differentiated effector CD8+ T cells, that is,
T-bet, BLIMP1 or IRF-4. In contrast, activation of NKG2D
weakly induced the mTORC1 signaling pathway,49 resulting in
the transcription of memory-associated genes, such as CD62L,
Eomes and CD127. Therefore, mTORC1 activation by CD28
and/or NKG2D mediates CD8+ T-cell differentiation into
terminally differentiated cells or memory cells.
CD8+ T-cell priming in the absence of CD4+ T-cell help
results in the formation of so-called ‘helpless’ or ‘unhelped’
CD8+ T cells.42–44 Notably, these helpless CD8+ T cells have
reduced effector functions and little to no memory
formation.42–44 Published work from our lab has demonstrated
that immunological help for CD8+ T cells in memory forma-
tion can also be provided via NKG2D signaling.52 We demon-
strated, using a murine vaccination model of helpless CD8+
T cells overexpressing NKG2D ligands during immunization of
mice depleted of CD4+ T cells, that these mice completely
restored the quantity and quality of the CD8+ T-cell memory
response (Figure 3). Importantly, this rescue was dependent on
Cellular and Molecular Immunology
Figure 3 NKG2D rescues the function of CD4+ T-cell-unhelped
CD8+ T cells. (a–c) depict three possible scenarios of CD8+ T cells
priming by an APC, which lead to three different outcomes for the
CD8+ T cells: classical CD8+ T-cell priming by p:MHC-I (peptide-
MHC-I complex) on APCs in the presence of CD4+ T-cell help,
which results in the optimal proliferation, CTL capacity and
formation of memory (a). These properties are defective when CD8+
T cell are activated in the absence of CD4+ T-cell help (b), which
can be rescued by engaging NKG2D on CD8+ T cells during their
priming (c). APC, antigen-presenting cell; CTL, cytotoxic
T-lymphocyte; NKG2D, natural killer group 2 member D; MHC,
major histocompatibility complex.
NKG2D expression on CD8+ T cells and was independent of
NK cells. This was shown in experiments using CD8+ T cells
from NKG2D-deficient mice and NK cell-depleted animals.
Further indicating the biological importance of these findings,
we also determined that NKG2D engagement by antibody
crosslinking was able to rescue the function of extremely
suppressed CD8+ T cells from HIV+ patients with low CD4+
T-cell counts. At the molecular level, we found that this rescue
was associated with the repression of T-bet, a transcription
factor involved in the terminal differentiation of effector cells.52
Paradoxically, while NKG2D signaling was able to rescue CD8+
T-cell memory formation, the initial effector response
remained compromised.52 These studies indicate that NKG2D
signaling on CD8+ T cells extends beyond known canonical
functions (e.g., facilitating target recognition and favored
killing), by providing a prosurvival signal. Moreover, these
data support the hypothesis that the effector and memory
functions of T cells can be disconnected, and CD8+ T-cell
memory formation can occur in the absence of an efficient
effector response.
To date, few studies have investigated the role of NKG2D
signaling in CD8+ T-cell memory formation. A study by
Wensveen et al.51 showed that NKG2D signaling enhanced
IL-15-mediated PI3K activity in activated CD8+ T cells, thus
assisting in memory commitment. In a series of adoptive
transfer experiments, the authors found that NKG2D

deficiency in OT-I cells resulted in impaired immunity against
challenge with LCMV expressing OVA. Moreover, in vitro-
activated T cells upregulated the antiapoptotic Mcl-1 protein,
which was found to be downregulated in NKG2D-KO CD8+ T
cells,51 suggesting that NKG2D induces memory formation by
increasing levels of survival proteins, such as Mcl-1. In contrast
to these findings, a study by Andre et al.48 suggested that
NKG2D had no major role in the generation and expansion of
memory CD8+ T cells, but instead substantially enhanced the
cytolytic effector responses of reactivated memory T cells,
thereby contributing to efficacious tumor rejection. In this
study, the authors used a transgenic mouse model in which the
human NKG2D ligand MHC class-I chain-related proteins A
(MICA) was ubiquitously and constitutively expressed, result-
ing in severe dysfunction of NKG2D signaling due to negative
feedback loops. These mice were used to assess the rejection of
OVA-expressing tumors (EG7) by tumor-specific memory
CD8+ T cells. The authors observed that the generation and
magnitude of memory CD8+ T-cell responses were indepen-
dent of NKG2D function, whereas in the effector phase,
efficient tumor rejection by CD8+ T cells was dependent on
NKG2D. However, there may be a possible relationship
between strong antigens and the need for NKG2D signaling.
In this case, OVA antigen could be considered a strong TCR
agonist, and OVA-reactive CD8+ T cells may therefore be
NKG2D-independent. Moreover, in these studies, NKG2D
signaling was lacking throughout the life of the CD8+ T cells
(e.g., CD8+ NKG2D-KO T cells or dysfunctional NKG2D),
which may have unexpected effects on their peripheral
behavior.
These studies indicate that CD8+ T-cell memory formation
is a dynamic process in which multiple factors have distinct
and redundant contributions. Based on these data, the role of
NKG2D signaling in memory formation may be to transcrip-
tionally certify CD8+ T cells that have actually recognized and
killed stressed cells. While this may not apply to CD8+ T cells
with high-affinity TCRs, this may play an important role in
CD8+ T cells that have weaker TCRs or are responding in a
subinflammatory environment. Moreover, these studies raise
important questions, such as whether the reduced quality of a
less-potent memory response is due only to the lack of NKG2D
signaling or whether it is a reflection of a poor effector phase.
Future studies will be required to fully delineate the role of
NKG2D in CD8+ T-cell memory formation.
NKG2D LIGANDS AND THEIR REGULATION
In humans, NKG2D ligands include two families: MICA and
MICB and HCMV UL16-binding proteins (ULBP1–6). While
these ligands are HLA class I homologs, they do not associate
with β-2m and play no known role in antigen presentation. In
mice, there are three families of ligands for NKG2D: the Rae-1
family, which includes five members (Rae-1α, Rae-1β, Rae-1γ,
Rae-1δ and Rae-1ε); the H60 family, which contains three
members (H60a, H60b, H60c); and the UL16-binding protein-
like transcript 1 family, consisting only of MULT1.
Functions of NKG2D in CD8+ T cells
JA Guevara-Patino et al
The expression of NKG2D ligands is strictly regulated in
normal cells and little or no NKG2DL is expressed in healthy
adult tissues.6 NKG2D ligands can be upregulated following
activation of the ATM/ATR (ataxia telangiectasia mutated/
ATM- and Rad3-related) DNA damage repair pathway. Studies
have shown that DNA-damaging agents (fluorouracil or
cisplatin) or local ionizing radiation can induce NKG2D ligand
surface expression in an ATM/ATR-dependent manner.53 In
addition, activation of other pathways, for example, by TLR4
and TLR7/8 agonists, also induces NKG2D ligand expression.54
Moreover, cytokines such as IFN-γ and tumor growth factor-β
can affect the expression of NKG2D ligands. IFN-γ down-
regulates the expression of NKG2D ligands in murine and
human melanoma cells,55,56 while transforming growth factor-
β has also been shown to decrease the transcription of several
NKG2D ligands in glioma cells.57 Interestingly, studies have
demonstrated that inhibition of the proteasomal pathway can
also induce the expression of NKG2D ligands.58 Previous
research has shown that heat shock protein (HSP70) and
several NKG2D ligands share the same promoter sequence,
which serves as a docking site for HSP 1 (HSF1).59 In response
to cellular stress, HSF1 is released in the cytoplasm. Following
nuclear translocation, HSF1 binds to the promoter regions of
both HSP70i and NKG2D ligands, initiating their
transcription.59 Expression of NKG2D ligands acts as a clear
indicator of cell damage, which can be interpreted as suicide by
proxy (in NKG2D-expressing cells). However, the spatiotem-
poral control of HSP70i (prosurvival) and NKG2D ligand
(suicide by proxy) expression in stressed cells remains to be
elucidated, representing an important area of translational
research.
NKG2D AND AUTOIMMUNITY
The prevention of autoimmunity is regulated by both central
and peripheral mechanisms. The central mechanisms involve
thymic education, in which early self-reactive T cells that
strongly react with peptide:MHC complexes undergo negative
selection in the thymus, during which cell death is induced.
Nevertheless, many T cells expressing low-affinity TCRs against
self-antigen are able to populate the periphery. In most cases,
these self-reactive T cells remain inactive. However, under
certain pathological conditions, they can become activated and
mediate host tissue destruction, causing autoimmunity. There
is emerging evidence that NKG2D signaling is involved in
certain forms of T-cell-mediated autoimmunity.52,60,61
Although interactions between autoreactive TCRs and pep-
tide:MHC complexes are normally of low affinity, the outcome
of these interactions can be influenced by factors outside the
TCR:peptide:MHC complex. NKG2D is an activating receptor
that enhances TCR signaling,3 potentially reducing the thresh-
old for T-cell activation via the TCR. In the great majority of
cases, autoimmunity does not occur during CD8+ T-cell
responses against intracellular infections. However, in certain
instances, TCR-independent cytotoxic functions of NKG2D
have been observed with higher expression levels on CD8+
T cells when NKG2D is engaged by its ligands.
Cellular and Molecular Immunology
 Functions of NKG2D in CD8+ T cells
JA Guevara-Patino et al
The involvement of NKG2D in potentiating CD8+ T-cell
responses against low-affinity peptides has been demonstrated
by our group and others in the context of antitumor immunity
and vitiligo.62 In the latter, there is an association between early
inflammatory events (e.g., exposure to UV light, a bleaching
agent, phenols or trauma) and the onset of disease.63 This is
significant, as the expression of NKG2D ligands is upregulated
in response to cell stress, rendering these cells visible to
NKG2D-expressing immune cells.1,3–5,9 Consistent with these
observations, we demonstrated that engagement of NKG2D,
but not 2B4 (another activating receptor), resulted in the
exacerbation of CD8+ T-cell-mediated vitiligo using vaccine
mouse models.62 These data indicate that autoimmune vitiligo
is limited by insufficient signaling, despite plentiful self-reactive
T cells in the peripheral immune system. Importantly, studies
have also shown a direct role for CD8+ T cells expressing
NKG2D in another skin-related autoimmune disease, alopecia
areata,64–66 indicating strong similarities between both diseases.
A similar situation has been reported for type I diabetes, in
which mouse studies from the Lanier group demonstrated that
NKG2D is involved during the onset of the disease. The
authors found that Rae-1 is present in prediabetic pancreas
islets of non-obese diabetic mice and that autoreactive CD8+
T cells infiltrating the pancreas express NKG2D, resulting in
the destruction of insulin-producing β-cells by T cells.67
Additionally, treatment with a non-depleting anti-NKG2D
monoclonal antibody (mAb) during the prediabetic stage
completely prevented disease by impairing the expansion and
function of autoreactive CD8+ T cells.
In chronic inflammatory rheumatoid arthritis (RA), data
from Andersson et al.60 work has demonstrated the expression
of NKG2D and its ligands on human RA synovial cells and in
the paws of arthritic mice. The authors demonstrated that, in
established collagen-induced arthritis, in vivo blockade of
NKG2D with an anti-NKG2D mAb ameliorated the disease
and protected against pathologic joint damage. In this study,
histologic analysis of arthritic joints from anti-NKG2D-treated
mice demonstrated significant joint protection compared with
that of control mice.60 These studies presented compelling data
demonstrating that self-reactive CD8+ T cells can be activated
by NKG2D-mediated mechanisms that can lead to the
destruction of host tissues. Considering that tumors are
stressed self-tissues, it would be expected that NKG2D signal-
ing plays a role in the recognition of NKG2D ligand-expressing
cancer cells.
While NKG2D is a potent costimulator of TCR-mediated
effector functions, its physiological behavior is tightly associated
with TCR function, and NKG2D is not expected to function
independently of TCR signaling.4,30,68 However, in celiac
disease, an autoimmune disease elicited by gluten intolerance
characterized by the destruction of the small intestine, studies
by Bana Jabri and co-workers61,69 have shown that IL-15 drives
the upregulation of NKG2D, ultimately enabling CD8+ T cells
to kill in a TCR-independent manner through NKG2D-
mediated mechanisms. The authors found that NK cells
deficient in NKG2D-DAP10 expression were unable to respond
Cellular and Molecular Immunology
to IL-15. Alternatively, the same study reported that IL-15-
activated Jak3 could phosphorylate DAP10 and thus prime the
NKG2D-DAP10 signaling pathway. In this way, IL-15 was
capable of converting effector T cells into NK-like ‘lympho-
kine-activated killers’ (LAK cells) both in vitro and in vivo in
celiac patients. Based on these data, it has been proposed that
DAP10 couples with the IL-15 receptor. Notably, in this study,
it was shown that the IL-15/NKG2D/DAP10 pathway conferred
TCR-independent cytolytic functions on T cells in the intestinal
epithelium.
In chronic inflammatory RA, data from Andersson and co-
workers’60 work has demonstrated the expression of NKG2D
and its ligands on human RA synovial cells and in the paws of
arthritic mice. The authors demonstrated that, in established
collagen-induced arthritis, in vivo blockade of NKG2D with an
anti-NKG2D mAb ameliorated the disease and protected
against pathologic joint damage. In this study, histologic
analysis of arthritic joints from anti-NKG2D-treated mice
demonstrated significant joint protection compared with that
of control mice. These studies presented compelling data
demonstrating that self-reactive CD8+ T cells can be activated
by NKG2D-mediated mechanisms that can lead to the
destruction of host tissues. Considering that tumors are
stressed self-tissues, it would be expected that NKG2D signal-
ing plays a role in the recognition of NKG2D ligand-expressing
cancer cells. Owing to its role in enhancing the cytotoxic
response of CD8+ T cells (in a TCR-dependent and TCR-
independent manner) and its pathophysiological role, NKG2D
may be able to be exploited as a target for autoimmune disease
treatment. Notably, since antitumor T-cell responses can be
viewed as a form of autoimmunity, the relationship between
these two different diseases may provide an opportunity for the
development of novel cancer therapies.
NKG2D AND CANCER IMMUNITY
Initial support for a role for NKG2D in tumor immunity was
provided by studies using tumor cell lines engineered to express
NKG2D ligands. In these studies, mice were able to reject
tumor cells expressing NKG2D ligands but not unmanipulated
counterparts.70 The mechanisms mediating rejection were
based on NK cells and CD8+ T cells. Subsequent studies by
Smyth et al.71 using methylcholanthrene-treated mice demon-
strated that in vivo NKG2D neutralization resulted in a higher
incidence of sarcoma. This observation was followed by studies
conducted by Guerra et al.70 using NKG2D-deficient mice
crossed with TRAMP mice (SV40 large T-driven prostate
cancer). In this case, the absence of NKG2D in TRAMP mice
resulted in early tumor development and growth compared
with TRAMP mice expressing NKG2D. These observations
demonstrated that tumor onset and progression were acceler-
ated in the absence of NKG2D or NKG2D ligands.
Importantly, studies by Liu et al.72 demonstrated dual and
opposing roles for NKG2D in cancer. The authors used mice
modified to express human MICB and a shedding-resistant
mutant MICB variant under the control of a prostate-specific
promoter. These mice were then crossed to TRAMP mice.

TRAMP/MICB mice (MICB is shed) developed prostate
tumors at a faster rate than TRAMP mice. However,
TRAMP/shedding-resistant-MICB mice remained tumor-free.
This interesting dichotomy was explained by elevated levels of
soluble MICB and the associated depletion of NK cells in
TRAMP/MICB mice.
Challenging several of the conclusions drawn from these
observations, studies using mice with severe dysfunction of
NKG2D signaling due to systemic expression of MICA revealed
that these mice were able to reject EG7 tumors with similar
efficacy as their wild-type counterparts.48 However, that EG7
cells are a thymoma cell line engineered to express the highly
immunogenic antigen chicken ovalbumin. Thus, it is possible
that CD8+ T-cell responses against OVA-derived peptide will
be mediated by high-affinity TCRs, which would be sufficient
to mediate tumor killing in an NKG2D-independent manner.
Despite their differing conclusions, these studies emphasize
the important role of NKG2D in immune responses against
tumors and suggest the possibility of exploiting this antitumor
role in the design of novel therapeutic approaches against
cancer.
FINAL REMARKS
Based on the physiological role of NKG2D in immune
responses against stressed cells (both infected and cancerous
cells) and the broad expression of its ligands on cancer cells,
NKG2D is an attractive target for clinical development of novel
therapeutic agents. Current strategies are focused on inducing
NKG2D signaling during CD8+ T-cell priming (i.e., vaccine
approaches), engaging NKG2D receptors on effector cells (i.e.,
CD8+ T cells), targeting NKG2D ligands on cancer cells (i.e.,
NKG2D-CAR T cells), inducing ligands for NKG2D in cancer
cells (i.e., radiotherapy) and preventing the suppressive effects
of shed-off NKG2D ligands on immune cells (i.e., blocking
antibodies).
We have shown using a DNA vaccination approach that
genes encoding NKG2D ligands can be incorporated in a
vaccine, along with the desired antigen. In this study, vaccina-
tion with genes encoding Rae-1ε or H60 and the melanocyte
shared antigen Trp1 resulted in numerically and functionally
enhanced CD8+ T-cell immune responses against Trp1-derived
peptides compared to vaccination with Trp1 alone.62 Other
approaches are being considered, such as in vitro activation of
NKG2D and TCR pathways in CD8+ T cells before adoptive
cell transfer. This approach would provide transferred CD8+
T cells with enhanced cytolytic and survival capacities optimal
for antitumor immune responses. One additional benefit may
be that increasing or maintaining the expression of NKG2D in
CD8+ T cells could result in resistance to immunosuppressive
factors in the tumor environment.
An alternative approach is the use of NKG2D-CAR T cells.
Here, the rationale is that these engineered T cells can eliminate
tumors that express ligands for NKG2D. Studies in mouse
models of lymphoma, ovarian cancer and multiple myeloma
have shown that targeting NKG2D ligands with NKG2D-CAR
T cells results in effective antitumor responses.73–75 However,
Functions of NKG2D in CD8+ T cells
JA Guevara-Patino et al
NKG2D ligand expression on non-tumor cells, as part of the
response to activation of DNA repair mechanisms involving the
ATM/ATR repair pathways, represents a safety concern. This
could result in ‘on-target, off-tumor’ responses, leading to
significant and potentially lethal toxicity. Indeed, murine
studies have demonstrated that adoptive transfer of NKG2D-
CAR T cells resulted in fatal responses.76,77 Importantly,
investigators have established that the severity of such toxicity
varied, depending on the mouse strain used as the recipient.
Specifically, BALB/c mice were more sensitive to toxicity than
C57BL/6 mice.77 These findings are of significance and must be
considered when attempting to treat human patients, who are
highly heterogeneous. Thus, approaches that target tumor cells
expressing NKG2D ligands must be considered with caution.
Notably, the use of NKG2D-CAR T cells is currently being
tested in a phase I clinical trial, and safety and toxicity results
are expected soon.
NKG2D ligands represent an attractive target for therapies,
and inducing their expression represents a viable approach for
the induction of antitumor immune responses. Irradiation
induces activation of the ATM/ATR repair pathways, resulting
in the expression of NKG2D ligands. To take advantage of this,
one could envision the combined use of local (tumor) radiation
and immune checkpoint therapies, namely, anti-PD-1 mAbs.
Moreover, several drugs can trigger the expression of NKG2D
ligands. Among these are DNA alkylating agents, proteasome
inhibitors58 and HDAC inhibitors.78–80 Again, however, a key
consideration must be that the expression of NKG2D ligands is
limited to cancer cells; otherwise, the development of auto-
immune responses would be a likely outcome.
In addition to cancer, NKG2D’s role has also been evaluated
in anti-viral immunity in various studies. In patients with
chronic hepatic viral infections, such as HBV and HCV,
NKG2D expression was found to be higher on intrahepatic
CD8+ T cells and was able to costimulate HBV-specific CD8+
T cells.81 Similar costimulatory roles of NKG2D have been
reported for virus-specific CD8+ T cells in cytomegalovirus.68
Furthermore, in a study focusing on virus-induced encephalitis
in mice, blocking NKG2D receptors resulted in a reduction of
the cytolytic capacity of the virus-specific CD8+ T cells and an
increase in viral titers, as well as an increase in associated
mortality.82 Along similar lines, blockade of NKG2D signaling
led to reduced clearance of Theiler's murine encephalomyelitis
virus in the murine CNS.83 Recently, Kavazović et al.84 showed
that a lack of NKG2D on virus-specific murine CD8+ T cells
attenuated their cytokine production and ability to clear
cytomegalovirus infection. Taken together, these data demon-
strate the important role of NKG2D in enhancing the effector
function of CD8+ T cells against viral infections. Based on these
and our findings, one can speculate that NKG2D-based
approaches may be used for the treatment of intracellular
pathogens. For example, in the case of HIV infection, which
results in low CD4+ T-cell levels, exogenous NKG2D activation
would be expected to provide a useful substitute for CD4+
T-cell assistance to CD8+ T cells. An additional benefit of
NKG2D signaling would be that the functionally exhausted
Cellular and Molecular Immunology
 Functions of NKG2D in CD8+ T cells
JA Guevara-Patino et al
phenotype that characterizes CD8+ T cells from HIV patients
may be reversed. Similar assumptions can also be made
regarding the use of NKG2D in other chronic infections, such
as HCV and HBV. Based on our current understanding of
NKG2D in autoimmunity, studies investigating how NKG2D
contributes to the onset of these diseases and its role in
autoimmune disease perpetuation are warranted.
Therapeutically, one can envision the development of
NKG2D blocking reagents that could prevent the progression
of disease by interfering with the recognition of NKG2D
ligands by CD8+ T cells. Based on the discussed studies, we
strongly believe that continued efforts towards enhancing
immunity in cancer treatment and controlling autoimmunity
will follow NKG2D-based approaches.
In summary, although much remains to be elucidated
concerning NKG2D’s diverse roles in the immune system,
significant evidence suggests that it may represent a novel
therapeutic target for multiple historically intransigent diseases.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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