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Prepublished online as a Blood First Edition Paper on April 30, 2002; DOI 10.1182/blood-2002-02-0350.
REVIEW ARTICLE
From the Department of Internal Medicine, Division of
Hematology/Oncology, and the Comprehensive Cancer Center, The Ohio
State University, Columbus; and The Division of Hematology and Clinical
Immunology, The Department of Clinical and Experimental Medicine,
University of Perugia, Perugia, Italy.
Natural killer (NK) cells have held great promise for the
immunotherapy of cancer for more than 3 decades. However, to date only
modest clinical success has been achieved manipulating the NK cell
compartment in patients with malignant disease. Progress in the field
of NK cell receptors has revolutionized our concept of how NK cells
selectively recognize and lyse tumor and virally infected cells while
sparing normal cells. Major families of cell surface receptors that
inhibit and activate NK cells to lyse target cells have been
characterized, including killer cell immunoglobulinlike receptors
(KIRs), C-type lectins, and natural cytotoxicity receptors (NCRs).
Further, identification of NK receptor ligands and their expression on
normal and transformed cells completes the information needed to begin
development of rational clinical approaches to manipulating
receptor/ligand interactions for clinical benefit. Indeed, clinical
data suggest that mismatch of NK receptors and ligands during
allogeneic bone marrow transplantation may be used to prevent leukemia
relapse. Here, we review how NK cell receptors control natural
cytotoxicity and novel approaches to manipulating NK receptor-ligand
interactions for the potential benefit of patients with cancer.
(Blood. 2002;100:1935-1947) Over the past 2 decades, a number of different
immune-based strategies aimed at eradication or suppression of residual
malignant disease have been proposed for the clearance of leukemic
cells. T-cell-mediated graft-versus-leukemia (GVL) has been
shown to be the most efficacious following allogeneic hematopoietic
stem cell transplantation and donor lymphocyte
infusions.1,2 Although specific antileukemic responses
have been documented, most T-cell-mediated alloreactions are thought
to be directed against minor or major histocompatibility antigens
shared by both leukemic and normal cells with potential for widespread
host tissue damage. Successful T-cell-based immunotherapy will,
therefore, ultimately require better definition of tumor-specific
antigens that will allow the direction of the immune response to the
tumor cells.3 Unfortunately, tumor-specific antigens have
only been identified on a minority of cancers.4
In contrast to antigen-specific T cells, effector cells of the innate
immune system lack the ability to rearrange the genes in the germ line
that encode receptor components, and, hence, they cannot recognize a
multitude of antigens in the context of classical major
histocompatibility complex (MHC) molecules. Natural killer (NK) cells
are innate immune lymphocytes that held early clinical promise because
of their ability to lyse tumor cells without specific antigen
recognition.5,6 Clinical trials attempting to harness the
antitumor effect of NK cells, either through in vivo or in vitro
activation, have met with only modest success to date.7-9
However, over the past decade our knowledge of how NK cells recognize
target cells using an integration of activating and inhibitory
receptors now points toward potential clinical utility for the
treatment of leukemia and other malignancies. In this review we
summarize our current understanding of the receptors involved in NK
cell recognition of tumor targets and discuss their potential clinical
role in immunotherapy of leukemia with and without hematopoietic stem
cell transplantation.
NK cells are innate immune lymphocytes critical to host defense
against invading infectious pathogens and malignant transformation through elaboration of cytokines and cytolytic
activity.5,6,10 Human NK cells comprise approximately 10%
of all blood lymphocytes and are identified by the expression of the
CD56 surface antigen and the lack of CD3. Functionally, NK cells are an
important source of innate immunoregulatory cytokines (eg,
interferon- Two distinct subsets of human NK cells are identified according to
cell surface density of CD56 expression as recently reviewed elsewhere.10 The majority (90%) of human NK cells are
CD56dim and express high levels of CD16, whereas a minority
(10%) is CD56bright and CD16dim/neg. These NK
subsets are functionally distinct with the immunoregulatory CD56bright cells producing abundant cytokines and the
cytotoxic CD56dim cells likely functioning as efficient
effectors of natural and antibody-dependent target cell
lysis.15 CD56bright NK cells constitutively
express the high and intermediate affinity interleukin (IL)-2 receptors
and expand in vitro and in vivo in response to low (picomolar) doses of
IL-2.8,16 In contrast, resting CD56dim NK
cells express only the intermediate-affinity IL-2 receptor and
proliferate weakly in response to high doses of IL-2 (1-10 nM) in
vitro, even after induction of the high-affinity IL-2
receptor.16,17 Resting CD56dim NK cells are
more cytotoxic against NK-sensitive targets (K562 and COLO205 cell
lines) than CD56bright NK cells.18 However,
after activation with IL-2 or IL-12, CD56bright cells
exhibit similar or enhanced cytotoxicity against NK targets compared
with CD56dim cells.18-20 In addition, resting
CD56bright and CD56dim NK cell subsets show
differences in their NK receptor repertoires.21,22 Resting
CD56bright NK cells are large agranular cells and express
high levels of the C-type lectin CD94/NKG2 family with only very small
fractions expressing killer-cell immunoglobulin receptor (KIR)
family.18 Resting CD56dim NK cells, however,
express both KIR and C-type lectin NK receptors at relatively high
surface density along with an abundance of cytolytic granules packaged
in the cytoplasm.18
NK cells originate in the bone marrow from human CD34+
hematopoietic progenitor cells (HPCs) and require the bone marrow
microenvironment for complete maturation. Bone marrow stroma-derived
cytokines, including IL-15 in cooperation with c-kit ligand (KL) and
flt-3 ligand (FL), are critical physiologic factors for NK cell
development, as recently reviewed.23 Human NK cell
development can be divided into an early phase of development in which
a NK progenitor responds to early acting stromal cell growth factors,
KL and FL, and develops into an NK cell precursor intermediate with the
phenotype
CD34+IL-2/ IL-15R Unlike T and B lymphocytes, NK cells do not rearrange genes
encoding receptors for antigen recognition, but they have developed the
ability to recognize self-MHC class I or class I-like molecules through a unique class of receptors, NK cell receptors (NKRs), that can
inhibit or activate NK cell killing. Initially, Ljunggren and
Karre27 proposed the "missing-self" hypothesis,
wherein the function of NK cells is to recognize and destroy autologous cells that have lost or altered self-MHC class I molecules. Although tolerant to normal autologous cells, NK cells can recognize and attack
virus-infected and transformed cells that have down-regulated MHC class
I molecules. Human NK cells lyse class I-deficient Epstein-Barr virus
(EBV)-transformed B-lymphoblastoid cell lines, whereas transfection of
class I alleles into target cells inhibits NK lysis.28,29 Accordingly, over the past decade, a number of inhibitory NK receptors specific for classical (eg, HLA-A, -B, or -C) or nonclassical (eg,
HLA-E, -G) class I molecules have been recognized.
However, MHC class I is not always necessary for protection from lysis
by NK cells, and inhibition by MHC class I is not always sufficient to
prevent NK cytotoxicity. For example, NK cells are unable to reject MHC
class I-deficient nonhematopoietic tissues, such as skin grafts, and
in vitro they fail to lyse fibroblasts, even from
KIRs belong to the immunoglobulin superfamily and are
characterized structurally by 2 or 3 extracellular immunoglobulinlike domains. KIRs specifically recognize MHC class I alleles, including groups of HLA-A,36,37 HLA-B,38-40 and
HLA-C.38,41,42 There are 2 functionally distinct sets of
KIRs: inhibitory and activating. Each set has an identical
extracellular domain, and, consequently, each set binds to identical
ligands. However, because of differences in their transmembrane and
intracellular or cytoplasmic domains, one set of KIRs signals an
inhibitory response and one set signals an activating response
following their binding to identical MHC class I alleles (Figure
2).43,44
The KIR family of NKR, located on chromosome 19p13.4,45,46
includes 12 members and a number of allelic variants, of which 6 receptors are inhibitory and 6 are activating. These are monomeric (single chain) receptors with either 2 (KIR2D) or 3 immunoglobulinlike domains (KIR3D), which can be further subdivided into those with long
(L) cytoplasmic tails (KIR2DL and KIR3DL) and short (S) cytoplasmic tails (KIR2DS and KIR3DS) (Figure 2). The long tail KIRs generate an
inhibitory signal, whereas the short tail KIRs generate an activation
signal. The inhibitory signal results from the presence of
immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the
cytoplasmic domains of the long tail receptors. The short tail
receptors owe their activating signals to their association with
adaptor proteins bearing immunoreceptor tyrosine-based activating motifs (ITAMs) (Figure 2). Whereas KIRs are specific for a number of
MHC class I molecules, HLA-C is the predominant class I isotype involved in the inhibitory and activating regulation of human NK cells
to provide either protection from or induction of target cell lysis and
cytokine production. For the purposes of our discussion, we will focus
on KIR recognition of HLA-C class I ligands. A current listing of known
KIRs and their known ligands can be found in Table
1.
A single KIR recognizes determinants that are shared between members of
a group of HLA-C alleles. Two HLA-C allotype groups are identified
according to amino acid residues present at positions 77 and 80 in the
Importantly, the number and role of inhibitory KIRs in NK cell biology
are still evolving.48 A few additional inhibitory KIRs are
listed in Table 1 with their respective ligands, when known. It appears
clear that corresponding inhibitory KIRs to many HLA-A and HLA-B
alleles do not exist, indicating that the KIR repertoire is not all
inclusive for human classical class I allotypes.49 One
long cytoplasmic tail KIR, KIR2DL4, recognizes the nonclassical MHC
class I allele HLA-G.50,51 HLA-G is a molecule that
displays limited polymorphism, and its expression has a unique
restricted tissue distribution on fetal extravillous trophoblasts that
invade the maternal decidua during pregnancy.52 In
contrast to other KIRs that are clonally distributed, KIR2DL4 is
thought to be expressed by all NK cells,51,53 although one study found that only decidual NK cells (all of which are
CD56bright) expressed this receptor and that peripheral
blood NK cells did not express KIR2DL4.54 Although
initially classified as an inhibitory receptor because of the presence
of ITIM in its cytoplasmic domain, recent evidence indicates that
ligation of KIR2DL4 on resting NK cells results in activation with the
unique property of inducing IFN- Another group of inhibitory receptors belongs to the immunoglobulin
superfamily and is represented by the immunoglobulinlike transcripts
(ILTs), also referred to as leukocyte immunoglobulinlike receptors
(LIRs).57,58 These receptors are encoded by a series of
genes on chromosome 19, close to the region encoding KIR. ILT receptors
are expressed primarily on myeloid cells, dendritic cells, and B
cells.57 ILT-2 (LIR-1) is also expressed on NK cells and
interacts directly with a broad spectrum of HLA class I molecules,
including HLA-G.57-59
A second family of human NK receptors is structurally
characterized by C-type lectin extracellular domains and is expressed as heterodimers composed of a common subunit (CD94) covalently bonded
to a distinct chain encoded by a gene of the C-type lectin NKG2
family.49,60-63 CD94 is a product of a single
nonpolymorphic gene and essentially lacks a cytoplasmic domain for
intrinsic signal transduction capacity.64 The
extracellular and cytoplasmic domains of the NKG2 molecules are
structurally diverse, consistent with differences in ligand recognition
and signal transduction.65-67 Homodimers of CD94 exist and
are of uncertain physiologic function.68 Four closely
related transcripts of the NKG2 family, with corresponding genes, have
been identified: NKG2A (and its splice variant NKG2B), NKG2C, NKG2E
(and its splice variant NKG2H), and NKG2F.65-67,69 NKG2D
is a fifth distantly related member that displays only a low sequence
similarity with the other NKG2 members and does not interact with CD94
(discussed later). The CD94 and NKG2
genes are all closely linked on chromosome 12p12.3-p13.1 in the human NK gene complex.69,70
CD94/NKG2 heterodimers are selectively expressed by NK cells and
cytotoxic T lymphocytes.71 Of the C-type lectin NK
receptors, only CD94/NKG2A is inhibitory, whereas other heterodimers
are activating receptors (Tables 1 and
2). NK clones may selectively bear either
inhibitory or activating CD94/NKG2 receptors, yet NKG2A and NKG2C can
be detected by revere transcription polymerase chain reaction (RT-PCR)
in some NK clones, and indirect functional data suggest that a subset
of NK cells may coexpress both receptors.68 The inhibitory
receptor CD94/NKG2A complex binds the nonclassical class I molecule
HLA-E.72-74 Interestingly, HLA-E binds leader peptides
derived from HLA-A, -B, -C, and -G, and, therefore, CD94/NKG2A functionally senses overall expression of HLA class I molecules at the
cell surface. For the same peptide/HLA-E complex, binding to the
inhibitory receptor CD94/NKG2A is stronger than binding to the
activating receptor CD94/NKG2C.75
Only when loaded with the appropriate nonamer peptides derived from the signal sequences of classic class I MHC molecules can the HLA-E molecule be transported to the cell surface.72-74 The detection of HLA-E by the CD94/NKG2 receptors may therefore be a sensitive mechanism for the immunosurveillance for normal biosynthesis of HLA class I molecules, a process that can be altered in virally infected or tumor cells. In addition, the ability of CD94/NKG2 receptors to discriminate among different peptide/HLA-E complexes might also influence reactivity against allogeneic cells. The spectrum of HLA molecules covered by KIRs and, indirectly, by CD94/NKG2 receptors, is only partially overlapping, suggesting that both systems play a complementary role for monitoring the biosynthesis/expression of most HLA class I molecules.76 The relative importance of each system and their interaction in modulating the reactivity of NK cells against autologous virus-infected and transformed cells, or allogeneic cells, remains to be elucidated. The biologic significance for the existence of paired inhibitory and activating receptors for MHC class I remains unclear. In both cases of KIRs and CD94/NKG2 receptors, the affinity of the activating receptor is lower than that of the corresponding inhibitory receptor,47,77 ensuring a predominance of the inhibitory signal when both activating and inhibitory receptors recognizing HLA molecules are expressed on the same NK cells. However, only a minority of NK cell clones express both activating and inhibitory isoforms that recognize the same HLA allotype.48,53,78 Much more commonly, NK cell clones expressing an activating receptor coexpress at least one inhibitory receptor specific for a different HLA class I allele that, when engaged, predominates. Therefore, the MHC class I-specific activating receptors may only signal when target cells have lost the expression of an HLA allele recognized by the inhibitory receptor, thus allowing NK cells' activating receptor to engage its ligand. In this way, NK cell surveillance may be important for removal of cells that have down-regulated or lost a single MHC class I allele while normal cells would be left unaffected.79 For example, virus-infected or transformed cells may selectively down-regulate HLA-A and -B allotypes while leaving HLA-C and -E unaffected.80,81 In this and other instances, positive target cell recognition by activating receptors is also essential to triggering NK cell cytotoxicity, and the balance between opposite signals delivered by inhibitory and/or activating receptors regulates NK cell functions. Activating receptors can be broadly grouped into those that are counterparts of the inhibitory receptors that recognize MHC class I molecules (discussed earlier) and those that do not have inhibitory counterparts and recognize inducible non-MHC molecules on target cells (Table 2).
Although the activating KIRs and CD94/NKG2 receptors may be
important in mediating NK cytotoxicity against MHC class I-bearing targets, other activating receptors are important in mediating cytotoxicity against MHC class I-deficient or negative targets. A
number of activating receptors with no apparent specificity for MHC
class I molecules have been reported, although many act as coactivators
rather than direct stimulators of NK cell function. In humans, a group
of receptors called natural cytotoxicity receptors (NCRs)82-86 and the NKG2D receptor have emerged as
activating receptors important in recognizing tumor cells in an
MHC-independent manner. Three NCRs (NKp46, NKp44, and NKp30) have been
identified. NKp46 and NKp30 are constitutively expressed by all
peripheral blood NK cells and are not found on other immune
cells.82,86 NKp44 is not expressed by resting NK cells but
is up-regulated on NK cells after IL-2 stimulation84 and
may be important for the cytotoxicity of IL-2-activated NK cells.
NKp44 is also found on a proportion of
The NKG2D is the best characterized activating receptor described
on NK cells. It is a C-type lectin surface receptor that is
misleadingly named as a member of the NKG2 family, encoded within the
NK gene complex on human chromosome 12.66,69,70 Unlike the
other NKG2 proteins presented earlier, NKG2D has little sequence
homology and does not associate with CD94 but is expressed as a
homodimer.88 The surface expression of NKG2D requires
association with a newly described adaptor subunit designated
DAP1088 or KAP10.89 The intracellular domain
of NKG2D does not have any signaling motif, and, therefore, signaling
is exclusively through its association with DAP10, which does not
contain a cytoplasmic ITAM but recruits phosphatidylinositol (PI)-3
kinase after phosphorylation that, in turn, induces cytotoxicity.
Importantly, because NKG2D has a signaling pathway that is distinct
from the activating KIR and C-type lectin NKR described earlier,
triggering via NKG2D is likely less susceptible to signals mediated by
inhibitory receptors. NKG2D is constitutively expressed by all NK cells
but is also expressed by almost all human
In contrast to the NCRs, target cell ligands for NKG2D have been identified and are induced by "stress" or neoplastic transformation, suggesting that NKG2D mediates the killing of cells that have been altered by these processes. The expression of these ligands may, therefore, be signals of "altered self" or "danger" to the innate immune system to promote NK and T-cell responses. In the human, these recently defined ligands belong to 2 distinct families, the MHC class I chain-related (MIC) antigens, and the UL16 binding proteins (ULBPs).91-94 The MIC antigens are encoded by a distinct family of genes stationed
along the entire MHC class I region on human chromosome 6. They have a
low degree of homology to other MHC-encoded class I genes, distinct
transcriptional control elements, and a peculiar pattern of
polymorphism.91 There are 7 MIC loci,
A to G, but only the MICA and
MICB genes are transcribed; MIC-C, -D, -E, -F and -G are pseudogenes.91-93 The MIC
glycoproteins contain 3 MHC-like Unlike MHC class I, MIC genes are not ubiquitously expressed. Bahram91 and Shiina et al92 have demonstrated that MIC transcripts are not expressed in the spleen and cells of the lympho-hematopoietic lineage, although they are expressed in fibroblast and epithelial cell lines as well as almost all tissues harboring these cell types. The expression of MICA and MICB are under the control of promoter elements similar to those of the heat shock protein gene PSP70.96 In this respect, exposure of MIC-expressing epithelial lines to heat shock was shown to increase expression of MIC transcripts and proteins.96 Furthermore, MIC expression on fibroblasts and epithelial cells was strongly up-regulated following infection with cytomegalovirus (CMV).97 Importantly, high MICA and MICB expression was detected on many human epithelial tumors98,99 and more recently on the JA3 and Raji leukemic cell lines,98 as well as on primary acute myeloid leukemia (AML) blasts (S.S.F., T.A.F., and M.A.C., unpublished observations, February 2002). Therefore, induction or up-regulation of MIC expression may occur with cellular stress, viral infection, or neoplastic transformation and may facilitate attack of these altered cells by NK cells and some T cells. The second family of NKG2D ligands reported are human cellular
proteins, initially identified by their ability to bind the human CMV
protein UL16, which is a type I transmembrane protein known to be
expressed by CMV-infected cells.100 UL16 binds to MICB and
2 proteins designated ULBP-1 and ULBP-2.94 By
expression-cloning a third member of this family of proteins has been
reported, ULBP-3, that, however, does not bind UL16.94 The
3 ULBPs possess The expression of ULBPs is more widespread than that of MIC proteins. With the use of RT-PCR, ULBP transcripts were detected in heart, lung, testis, brain, lymph nodes, thymus, tonsil, liver, and bone marrow.94 However, in some tissues where high ULBP mRNA expression was detected, no cell surface expression was found by monoclonal antibody,94 suggesting that surface expression of ULBPs may be regulated at a posttranscriptional level. This finding has important implications for the study of these ligands in tumor cell recognition by NK cells.
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Top
Abstract
Introduction
[IFN-
[TNF-
+CD56
. This
precursor is then responsive to IL-15 for maturation into a
functional CD56+ NK cell. The NK cells resulting from adult
CD34+ HPCs in stroma-free cultures following addition
of IL-15, however, resemble the CD56bright NK cell
population both functionally and phenotypically
(CD56brightCD16dim/neg),
including their NK receptor repertoire.
T cells.
and/or Fc
RI