NK cell receptors and their ligands in leukemia

Article (PDF Available)inLeukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 22(2):249-57 · March 2008with34 Reads
DOI: 10.1038/sj.leu.2405040 · Source: PubMed
Abstract
Human natural killer (NK) cells are built to kill abnormal cells but to preserve autologous normal cells. To accomplish this task, they are equipped with a large number of inhibiting and activating receptors. Ligation with corresponding ligands will determine whether the NK cell becomes activated to destroy the abnormal cell. This review will focus on the abnormalities of NK cell receptors and their putative ligands found in patients with leukemia, which can lead to an inadequate function of NK cells allowing these malignant cells to escape from NK cell destruction. In recent years it has become clear that NK cells in the haploidentical hematopoietic stem cell transplantation (HSCT) setting are very effective in eliminating residual acute myeloid, but not acute lymphoid, leukemic cells. In this regard, we also reviewed published studies of retrospective cohorts of HSCT investigating the potential beneficial effect of killer-cell immunoglobulin-like receptors (KIRs) and human leukocyte antigen (HLA) ligands on NK alloreactivity. Manipulating NK cell inhibition or activation could lead to new forms of immunotherapy, ultimately leading to the elimination of resistant leukemic cells.
REVIEW
NK cell receptors and their ligands in leukemia
S Verheyden and C Demanet
Department of Hematology, HLA and Molecular Hematology Laboratory, Universitair Ziekenhuis Brussel (UZ Brussel), Brussels,
Belgium
Human natural killer (NK) cells are built to kill abnormal cells
but to preserve autologous normal cells. To accomplish this
task, they are equipped with a large number of inhibiting and
activating receptors. Ligation with corresponding ligands will
determine whether the NK cell becomes activated to destroy the
abnormal cell. This review will focus on the abnormalities of NK
cell receptors and their putative ligands found in patients with
leukemia, which can lead to an inadequate function of NK cells
allowing these malignant cells to escape from NK cell destruc-
tion. In recent years it has become clear that NK cells in the
haploidentical hematopoietic stem cell transplantation (HSCT)
setting are very effective in eliminating residual acute myeloid,
but not acute lymphoid, leukemic cells. In this regard, we also
reviewed published studies of retrospective cohorts of HSCT
investigating the potential beneficial effect of killer-cell
immunoglobulin-like receptors (KIRs) and human leukocyte
antigen (HLA) ligands on NK alloreactivity. Manipulating NK cell
inhibition or activation could lead to new forms of immunotherapy,
ultimately leading to the elimination of resistant leukemic cells.
Leukemia (2008) 22, 249–257; doi:10.1038/sj.leu.2405040;
published online 29 November 2007
Keywords: NK cells; NK cell receptors; hematopoietic stem cell
transplantation; Killer cell immunoglobulin-like receptors; HLA class I
ligands
Introduction
The ability of natural killer (NK) cells to destroy leukemic cells
as demonstrated by allogeneic transplantation of hematopoietic
stem cells indicates that this cell type is implicated in the control
and clearance of leukemia.
1,2
The number or activity of
autologous NK cells against leukemic cells is frequently reduced
and abnormalities at the level of receptors and/or ligands
determining NK cell function may be responsible for the
resistance of leukemic cells to NK cells.
3–9
There is also still a
high occurrence of leukemic relapse after high dose chemother-
apy and/or allogeneic hematopoietic stem cell transplantation
(HSCT), suggesting that leukemic cells can escape from the
immune system. A variety of studies are now ongoing to unravel
the mechanisms of NK cell killing of leukemic targets and to
explain how leukemia can escape from the innate immune
surveillance. This overview summarizes the most important
findings available from the literature about the role of NK cell
receptors and their ligands in the immunity against different
types of leukemia. Further understanding of the NK receptor
biology is necessary to develop therapeutic strategies that may
create an efficient NK cell immune response against leukemia.
Role of inhibitory KIRs and HLA class I interactions
The activity of NK cells is controlled by the expression of
inhibitory receptors that interact with human leukocyte antigen
(HLA) class I molecules. Prominent among these HLA class I-
specific receptors are the killer cell immunoglobulin-like
receptors (KIRs), which are encoded by a diverse and rapidly
evolving family of different polymorphic genes. Within the
human population, the variation of KIR haplotypes depends on
the number and type of KIR genes that they inherited and on
the allelic polymorphism. Consequently, diverse KIR repertoires
are generated across individuals with differences in the balance
between activating and inhibitory receptors, which may
influence the NK and/or T-cell activity to lyse tumor targets.
Only scant information is available from the literature
concerning the role of KIR and HLA genes in the pathogenesis
of leukemia. Here our study found an increased frequency of
specific inhibitory KIR–HLA class I interactions in different forms
of leukemia compared to the healthy volunteers.
10,11
This data
suggests that one escape mechanism from the innate immune
surveillance might be due to a dominance of inhibitory over
activating signals in leukemic patients. Along this line, a recent
study demonstrated that B lineage chronic lymphoid leukemia
(B-CLL) patients with advanced disease had significantly higher
percentages of cytotoxic T cells expressing inhibitory KIRs and
CD94 compared to B-CLL patients with nonprogressive dis-
ease.
12
In patients with chronic myeloid leukemia (CML) the
number of NK cells progressively decrease during disease
progression.
4,5
Substantial numbers of NK cells in an advanced
phase of the disease were found to be positive for t(9;22)
translocation-related breakpoint cluster region-abelson (BCR/
ABL) in contrast to those in the chronic phase.
5,13
It has been
demonstrated that BCR/ABL can increase KIR expression and so
influence the activity of NK cells.
13
The importance of inhibitory
KIR–HLA class I interactions in the innate immune response
against leukemic cells has been further underscored in allo-
geneic HLA-mismatched HSCT. In the setting of KIR ligand-
mismatched HSCT, NK cell alloreactions are directed toward
allogeneic cells that lack inhibitory HLA class I ligands. A
fraction of the donor NK cells may only express inhibitory KIRs
that are not engaged by the HLA class I alleles of the recipient,
and thus have the potential to kill the residual leukemic cells of
the patient. In this context, a study published by Koh et al.
14
demonstrated that antibody blockade of murine NK inhibitory
receptors enhanced the lysis of leukemic cells both in vivo and
in vitro, supporting a pivotal role of NK inhibitory receptors in
decreasing the anti-leukemic NK cell response.
Cell surface expression of HLA class I molecules
Normal cells express a threshold of HLA class I molecules that
protect them from autologous NK cell killing, whereas
Received 5 July 2007; revised 22 October 2007; accepted 29 October
2007; published online 29 November 2007
Correspondence: Dr C Demanet, Department of Hematology, HLA
and Molecular Hematology Laboratory, Universitair Ziekenhuis
Brussel, Laarbeeklaan 105, 1090 Brussels, Belgium.
E-mail: christian.demanet@uzbrussel.be
Leukemia (2008) 22, 249–257
& 2008 Nature Publishing Group All rights reserved 0887-6924/08 $30.00
www.nature.com/leu
malignant cells often have a reduced expression or loss of
HLA class I alleles and therefore become susceptible to NK
cells. Most types of solid tumors display defects in HLA
class I expression, but the incidence of HLA class I down-
regulation and complete HLA class I allele loss varies among
different types of tumors.
15
In contrast to solid tumors, HLA class
I expression in leukemia has not been extensively studied,
except in acute leukemia
16–20
and B-CLL.
21
In most of these
studies, complete loss of HLA class I allele(s) in leukemic cells
was infrequent. A possible explanation for this low frequency is
that the time interval between the onset of leukemia and the
diagnosis is too short to permit leukemic cells to acquire
mutations in the genes involved in HLA class I expression. On
the other hand, our group has reported frequent partial
downregulation of certain HLA class I alleles on leukemic cells
in comparison with their normal autologous cells.
19
At variance
with complete loss, downregulation of HLA class I alleles is
rather caused by defects in regulatory mechanisms and is
generally restored by cytokines.
17
In the other studies, it is likely
that the frequency of HLA class I allelic downregulation was
underestimated
16,18,21
since labeling of leukemic cells was done
with a few anti-allelic mAbs or recognizing monomorphic
determinants of HLA class I antigens that do not detect
downregulation of HLA class I expression at the allelic level.
Our study on a heterogeneous group of leukemia further
showed that HLA class I molecules belonging to the Bw6
group were more frequently downregulated than those belong-
ing to the HLA-Bw4 group.
19
Since HLA-B molecules
containing the serologically defined Bw6 epitope cannot be
sensed by NK cells, downregulation of HLA-Bw6 may
provide leukemic cells with an escape mechanism only from
cytotoxic T cells.
19
Leukemic cells with a downregulated
HLA-Bw6 expression and a preserved HLA-Bw4 expression
inhibiting NK cells, can therefore escape from both cytotoxic
T cells and NK cells.
Until now, the expression of HLA-C on leukemic cells, which
interacts with the majority of inhibitory KIRs, has not been
studied. We recently analyzed the expression of certain HLA-C
alleles (Cw1, Cw3, Cw4, Cw0801, Cw1202 and Cw1402) on
acute myeloid leukemia (AML), B lineage acute lymphoblastic
leukemia (B-ALL) and B-CLL cells. Our study demonstrates that
HLA-C expression was significantly downregulated in all three
types of leukemic patients and was most pronounced in the AML
patients (unpublished data).
The aberrant expression of nonclassical HLA class I antigens
on tumor cells may provide a further escape mechanism from
the immune response by interacting with inhibitory receptors on
NK cells and certain T-cell subsets. HLA-G is a nonclassical
HLA class I molecule recognizing KIR2DL4, which is not
expressed by normal cells with the exception of trophoblast
tissue. In addition to its inhibitory function, KIR2DL4 can also
have an activating function that may depend on the type of
cell.
22
Despite the presence of HLA-G transcription in mature
lymphoid cells, HLA-G antigens are not expressed at the cell
surface of normal lymphocytes or leukemic cells in the absence
of IFN-g.
23–25
This finding is at variance with a study published
by Nuckel et al.
26
showing that a variable percentage of CLL-B
cells aberrantly expressed the nonclassical HLA-G molecule.
The surface expression of HLA-E depends on the binding of
peptides derived from HLA class I molecules. In general, HLA-E
surface expression correlates with the overall expression of HLA
class I molecules and can be recognized by the inhibitory
NKG2A receptor. In contrast to cells from healthy volunteers,
HLA-E is not transcribed and hence not expressed in different
types of leukemia.
27
Immunotherapy based on preventing NK cell inhibition
Hematopoietic stem cell transplantation
HSCT has been used for several years to cure leukemic patients.
However graft failure, graft-versus-host disease (GvHD), leuke-
mic relapse and susceptibility to infections remain significant
complications that limit the efficacy of HSCT.
Before the infusion of hematopoietic stem cells (HSC),
recipients undergo a myeloablative conditioning regimen to
destroy the host immunohematopoietic system, suppressing its
immune system, making space for the donor HSC and reducing
the tumor burden in the patient. Afterwards, the infused HSC
will differentiate and proliferate to reconstitute a new immuno-
hematopoietic system of donor type. The lymphocytes, particu-
larly T cells, in the graft further eliminate host T cells, preventing
rejection and destroying residual leukemic cells that survive the
conditioning regimen, thereby decreasing the rate of relapse.
The risks of graft rejection, caused by host T-cell recognition of
donor HLA, and that of GvHD, caused by donor recognition
of host HLA antigens, are augmented with increasing numbers of
HLA mismatches.
28
Thus the success of HSCT depends on the
degree of HLA matching between the donor and recipient and
the best clinical outcome occurs when patients are transplanted
with a graft from an HLA-identical sibling. However only a
minority of patients will find a fully HLA-matched donor,
because there is only a 25% chance that siblings are HLA
identical. So leukemic patients who failed to find a related or
unrelated HLA-matched donor can be given a transplant from an
HLA-haploidentical family member. But HLA-haploidentical
transplants, in which the donor and recipient are incompatible
at the HLA loci of the unshared haplotype, are at high risk for
T-cell-mediated alloreactions. Therefore T-cell-mediated graft
rejection can only be prevented by a high intensity conditioning
regimen destroying the host T cells, followed by the infusion of
equal to or more than 10 10
6
CD34 þ cells per kg of HSC. In
addition, extensive T-cell depletion of the graft is required to
control GVHD. In contrast to the HLA-matched transplants, the
GvL effect in these haploidentical transplants relies on
alloreactivity of NK cells triggered by a mismatch between the
inhibitory KIRs on the donor NK cells and the HLA class I
molecules on the recipient cells. Because most individuals
express all inhibitory KIR genes, a potential for NK cell
alloreactivity is present when the recipient does not express
the donor’s KIR ligand(s) in the haploidentical HSCT setting. For
example, when the donor is heterozygous for HLA-C group 1
(HLA-C1) and group 2 (HLA-C2), this donor will have usually
the inhibitory KIR2DL2/3 and KIR2DL1 for these HLA-C ligands.
Transplanting a graft from this donor into a patient who is
homozygous for HLA-C1 or HLA-C2 will result in alloreactive
donor NK clones with inhibitory KIRs that cannot bind the HLA-
C ligand on the recipient cells. However this cannot be applied
to one-third of the patients who express all three KIR ligands
(HLA-C1, HLA-C2 and HLA-Bw4) and consequently inhibit NK
cells from every donor. A recent study published by Kim et al.
29
proposed that only immature NK cells expressing an inhibitory
receptor that can engage self HLA class I molecules, become
fully functional. Thus donor NK cells expressing solely
inhibitory KIRs for self HLA class I molecules that are present
in the donor and not in the recipient sense the missing self-
expression of HLA and can therefore mediate alloreactions. The
first study that demonstrated that KIR ligand incompatibility
could reduce the risk of relapse, promote engraftment and
protect against GvHD in AML patients transplanted with a graft
from related haploidentical donors was published by Ruggeri
et al.,
2
which is confirmed in their later study.
30
After this initial
NK cells and leukemia
S Verheyden and C Demanet
250
Leukemia
study, additional studies retrospectively analyzed cohorts of
related
31
and unrelated
32–39
HLA-mismatched HSCT to address
the impact of NK alloreactivity on different transplant outcome
variables (an overview is given in Table 1). A second study of
related haploidentical transplants published by Bishara et al.
31
failed to confirm any beneficial effect of KIR ligand incompati-
bility. The impact of KIR ligand incompatibility has also been
controversial for unrelated HSCT. Only two studies in unrelated
HSCT observed a decreased leukemic relapse rate in patients
with myeloid leukemia receiving a graft from KIR ligand
incompatible donors.
33,36
In the study published by Giebel
et al.,
33
all patients received anti-thymocyte globulin (ATG)
during the conditioning regimen resulting in vivo depletion of
patient and donor T cells. The results showed that the use of
ATG favored the beneficial effects of alloreactive NK cells.
However several other retrospective HSCT studies could not
confirm these beneficial effects of KIR ligand incompatibility
despite the use of ATG.
34,35,37,38
In contrast, some reports found
that KIR ligand incompatibility was even associated with
an increased rate of treatment-related mortality (TRM) caused
by infection.
35,37,38
A possible mechanism underlying the
increased infection-related mortality in these patients is that
donor alloreactive NK cells may kill the patient’s dendritic cells
and in this way interfere with immunity against opportunistic
infections.
Because KIR and HLA segregate independently from each
other, an individual can also express an inhibitory KIR but no
HLA ligand for that KIR. In this way the missing KIR ligand can
also be applied in HLA-matched HSCT, where the donors
express inhibitory KIRs for which neither donor nor recipient has
HLA class I ligand(s). Normally, NK clones that express
inhibitory KIRs in the absence of self-HLA class I ligands are
self-tolerant. However, one study showed that these NK clones
are still active but because this NK cell population persists at a
low frequency in a person’s blood, these cells cannot cause
autoreactions.
39
It has been postulated that these NK clones after
transplantation, which, on expansion in an altered hematopoie-
tic environment, may become alloreactive. Only a few studies
took into account the interactions between KIRs on donor NK
cells and recipient HLA ligands to study NK alloreactivity on
transplant outcome.
30,38,41,42,44
In related HLA-matched HSCT,
Hsu et al.
42
demonstrated that AML and myelodysplastic
syndrome (MDS) patients who lacked the HLA ligand for the
donor KIR genotype had significantly increased disease-free and
overall survival. The same beneficial effect was recently
observed in early stage myeloid leukemia patients who lacked
one of the KIR ligands.
43
In contrast, an inferior survival was
observed in patients who were homozygous for HLA-C2, but
only when donors were positive for the activating KIR2DS2
receptor.
41
Although the importance of inhibitory KIRs has been
extensively investigated in HLA-mismatched HSCT, the role of
activating KIRs has only been studied in a few other HSCT
studies.
38,41,44–46
Our study in related HLA-identical HSCT
observed that the incidence of relapse decreased when patients
received a graft from activating KIR2DS1 and KIR2DS2 positive
donors.
44
Furthermore, Giebel et al.
46
found that the presence of
KIR2DS2 alone in the donor was associated with a high risk of
mortality following unrelated HSCT, presumably caused by a
higher incidence of GvHD. Finally, a study published by Gagne
et al.
45
showed that the risk for GvHD was very high when the
KIR genotype of an unrelated donor included activating KIRs
that were absent in the recipient KIR genotype.
Taken together, these studies show that not only KIR ligand
incompatibility but also the number of activating KIRs
38
and
presence of particular activating KIRs in the donor might
influence transplant outcome (Table 1). In contrast to haploi-
dentical HSCT, KIR ligand incompatibility is mostly not
associated with a decreased relapse in unrelated HSCT (Table 1).
Heterogeneity in the treatment procedures (conditioning regi-
men, graft composition and post-transplant immune suppres-
sion) and in the patient population (disease, risk category) might
explain the conflicting results concerning KIR ligand incompati-
bility (Table 2). More specifically, unrelated grafts contain more
T cells than haploidentical grafts and this has been shown to be
associated with poor reconstitution of potentially alloreactive
NK cells. Prospective studies in the unrelated setting should
therefore include functional assessment of donor NK alloreac-
tivity toward leukemic targets besides a more extensive T-cell
depletion of the graft.
Immunotherapy using the NK-92 cell line
A novel attractive immunotherapy for the treatment of leukemia
is the use of the NK-92 cell line because it has a superior
antitumor effect against a broad range of human leukemic cell
lines in vitro, albeit with a variable activity against B-ALL
cells.
47–49
The NK-92 cell line is an IL-2 dependent cell line
isolated from a patient with non-Hodgkin’s lymphoma at
diagnosis. The high cytotoxicity of the NK-92 cell line is thought
to be the result of an almost complete lack of inhibitory KIRs
with a preserved perforin- and granzyme-mediated cytotoxicity.
Consequently, in vivo administration of this NK cell line in
allogeneic leukemic patients could create a KIR ligand
mismatch situation similar to that encountered in an allogeneic
HSCT. Moreover, studies showed that leukemic blasts from
B-ALL patients were more resistant to killing by the NK-92 cell
line than the other types of leukemia.
47–49
It has also been
demonstrated that the NK-92 cell line does not induce
cytotoxicity toward normal bone marrow cells, suggesting that
malignant hematopoietic cells express additional activating ligands
and that resistance of certain leukemic cells could be caused by the
absence or downregulated expression of these NK activating
ligands.
47,50
In this regard, a study established that the NK-92 cell
line could be used as a purging agent to decrease or eliminate the
malignant contamination of autologous stem cell grafts in CML
patients.
51
Other clinical trials are currently underway using NK-92
for adoptive immunotherapy of malignancies.
52
Role of activating NK cell receptors and their ligands
Although the interaction of inhibitory NK cell receptors with HLA
class I molecules controls the NK cell activation, it is apparent
that an activating signal is needed when NK cells interact with
potential leukemic cells. Resistance of leukemic cells against NK
cells mediated by inhibitory KIR–HLA class I interaction may
therefore be further increased by additional mechanisms based
on defective activating NK receptor–ligand interactions.
An important tumor escape mechanism from immune
surveillance is the absence or downregulation of specific
activating antigens. Certain types of leukemia are resistant to
NK cells of an allogeneic KIR ligand-mismatched donor. In this
regard, a significant graft-versus-leukemia effect was observed in
AML patients but not in B-ALL patients following haploidentical
HSCT.
2
It has also been shown that B-ALL cells are negative for
the MHC class I-related protein (MICA/B) ligands known to
trigger the NKG2D receptor and loose or express low levels of
several other NK activating ligands such as PVR, Nectin-2,
CD48 and NK-T-B antigen (NTBA) (Table 3).
49,53
Presumably,
downregulated expression of other unidentified ligands for NK
NK cells and leukemia
S Verheyden and C Demanet
251
Leukemia
Table 1 Clinical outcomes of KIR and HLA in HSCT
References KIR typing Association Disease Relapse GvHD TRM Overall survival
Ruggeri et al.
2
No KIR ligand incompatibility
a
AML Decreased Decreased nt Increased
Bishara et al.
31
Yes KIR ligand incompatibility
44 donor activating KIRs
Mixed
b
NS
NS
NS
Increased
nt
nt
Decreased
NS
Cook et al.
41
Yes HLA-C2 homozygous and donor KIR2DS2 positive Myeloid leukemia NS NS NS Decreased
Hsu et al.
42
Yes KIR ligand absent
c
AML/MDS Decreased NS nt Increased
Verheyden et al.
44
Yes Presence of KIR2DS1 and KIR2DS2 in donor Leukemia Decreased NS NS Increased
Ruggeri et al.
30
Yes KIR ligand incompatibility AML Decreased nt nt Increased
Davies et al.
32
No KIR ligand incompatibility Myeloid leukemia NS NS nt Iecreased
Giebel et al.
33
No KIR ligand incompatibility Myeloid leukemia Decreased NS Decreased Increased
Bornhauser et al.
34
No KIR ligand incompatibility Myeloid leukemia Increased NS NS NS
Schaffer et al.
35
No KIR ligand incompatibility Leukemia NS NS Increased Decreased
Giebel et al.
46
Yes
d
Presence of KIR2DS2 in donor Leukemia NS NS NS Decreased
Hsu et al.
40
No KIR ligand absent in HLA mm HSCT Leukemia Decreased nt nt NS
Farag et al.
37
No KIR ligand incompatibility Myeloid leukemia NS NS Increased Decreased
Kroger et al.
38
Yes KIR ligand incompatibility
High number of donor activating KIRs
Leukemia NS
Increased
NS
NS
Increased
NS
Decreased
Decreased
Miller et al.
43
No KIR ligand absent Early stage myeloid leukemia
CML41 year from diagnosis
Decreased
NS
NS
Increased
nt
nt
nt
nt
Gagne et al.
45
Yes Recipient KIR included in donor KIR genotype of unrelated HSCT
e
Mixed nt Increased nt nt
Beelen et al.
36
No KIR ligand incompatibility Myeloid leukemia Decreased NS NS NS
Abbreviations: GvHD, graft-versus-host disease; HLA mm, HLA mismatched pairs; KIR, killer cell immunoglobulin-like receptors; NS, not significant; nt, not tested; TRM, transplant-related mortality.
a
KIR ligand incompatibility was deduced from HLA typing of donors and patients and was assigned when the recipient lacks KIR ligands that are present in the donor.
b
Genetic diseases of blood cells and different hematological malignancies.
c
KIR ligand absence was defined when the recipient lacks HLA ligands for the identified donor inhibitory KIRs regardless of the donors HLA type.
d
Only two KIRs, 2DS1 and 2DS2, were genotyped.
e
The recipient KIR genotype is included in the donor KIR genotype when the donor has KIR genes supplementary to those of the recipient.
NK cells and leukemia
S Verheyden and C Demanet
252
Leukemia
activating receptors, including the natural cytotoxicity receptors
(NCRs) or the polymorphic KIRs, may be involved in the NK
resistance of B-ALL. This data suggests that resistance of most
B-ALL cells to NK cells in haploidentical HSCT maybe caused
by the lack of expression of crucial activating ligands.
It was recently shown that AML cells are negative for or have
low levels of the NKG2D ligands (MICA/B and unique long 16-
binding proteins (ULBP)) and the putative NCR ligands
53,54
and
that this could be caused by cell maturation arrest during
malignant transformation.
54
By contrast, the levels of ULBP and
NCR ligands on normal myeloid cells are higher because they
increase during hematopoietic cell differentiation.
54
These
normal myeloid cells would be protected against lysis by
autologous NK cells by the expression of HLA class I molecules
interacting with the inhibitory KIRs. However, antibody-
mediated blockade of activating receptor pathways revealed a
dominant role for DNAX accessory molecule-1 (DNAM-1) and
NCRs, but a complementary contribution of NKG2D signaling in
AML cell recognition.
6,53
The importance of the activating
DNAM-1 receptor in the lysis of AML blasts was also confirmed
by the consistent expression of PVR and nectin-2 ligands on their
cell membrane.
53
This was at variance with the study published
by Salih et al.
55
who found that a substantial number of AML
patients expressed NKG2D ligands that can render leukemic
cells susceptible to NK cells. Taken together, these studies
suggest that the leukemic blasts of most AML patients express
ligands able to trigger allogeneic NK cells. But there are still
conflicting results on the importance of the activating NKG2D in
the lysis of AML cells.
53,55
An upregulation of NKG2D ligands on
leukemic cells by growth factors could enhance NK cell-
mediated killing.
7
An overview of the expression of different
NK activating ligands on AML cells is given in Table 3.
Furthermore, Costello et al.
6,56
demonstrated that the majority
of NK cells in AML patients display a low NCR surface density
correlating with their weak cytolytic activity against autologous
leukemic cells. Downregulation of the activating receptors on
NK cells from AML patients may explain the observed reduced
NK killing of autologous AML cells and the HLA class I-deficient
K562 cell line.
6,7,9,56
Unfortunately, the expression of the
activating DNAM-1 receptor on NK cells from AML patients
has not yet been analyzed. It has also been suggested that direct
cell contact between leukemic blasts and NK cells rather than
secretion of TGF-b 1 may induce downregulation of the NCRs.
56
High levels of soluble MICA/B have been found in the serum of
patients with AML
55
and CML
57
and might cause downregula-
tion of the activating NKG2D receptor.
58,59
Nevertheless a
Table 2 Patient and graft characteristics in HSCT studies
References No. of patients HLA matching GvHD prophylaxis
T-cell depletion Immune suppression
In vitro In vivo
Related HSCT
Ruggeri et al.
2
92 Haploidentical 92 0 Unknown
Bishara et al.
31
62 Haploidentical 62 0 0
Cook et al.
41
220 220 HLA m Unknown Unknown 220 Cs
±
MTX
Hsu et al.
42
178 178 HLA m 178 0 178 Cs
Verheyden et al.
44
65 65 HLA m 34
0
0
0
0
31 Cs+MTX
Ruggeri et al.
30
112 Haploidentical 112 112 ATG 0
Unrelated HSCT
Davies et al.
32
175 175 HLA mm 60
0
0
0
0
115 Cs
±
MTX
Giebel et al.
33
130 61 HLA m
69 HLA mm
Unknown 130 ATG 130 Cs+MTX
Bornhauser et al.
34
118 54 HLA m
64 HLA mm
Unknown 118 ATG Unknown
Schaffer et al.
35
190 94 HLA m
96 HLA mm
11
0
11 ATG
179 ATG
0
179 Cs
±
MTX
Giebel et al.
46
25 23 HLA m
2 HLA mm
Unknown 25 ATG 25 Cs+MTX
Hsu et al.
40
1202 580 HLA m
622 HLA mm
Unknown Unknown Unknown
Farag et al.
37
1571 1004 HLA m
567 HLA mm
310
0
0
199 ATG
Unknown
Unknown
Kroger et al.
38
142 103 HLA m
39 HLA mm
Unknown 142 ATG 142 Cs+MTX
Miller et al.
43
2062 831 HLA m
884 HLA mm
Unknown
a
Unknown
a
Unknown
Mixed (related and unrelated)
Gagne et al.
45
75 56 HLA m19 HLA mm 7 Unknown Unknown
Beelen et al.
36
374 237 HLA m137 HLA mm Unknown 0 374 Cs+MTX
Abbreviations: ATG, anti-thymocyte globulin; BM, bone marrow; Cs, cyclosporin; GvHD, graft-versus-host disease; HLA m, HLA matched pairs;
HLA mm, HLA mismatched pairs; MTX, methotrexate; PBSC, peripheral blood stem cells.
Most published HSCT studies included a mixed group of patients with hematological malignancies.
a
T-cell depletion was only mentioned for CML patients more than 1 year from diagnosis.
NK cells and leukemia
S Verheyden and C Demanet
253
Leukemia
recent study found no correlation between reduced NKG2D
surface expression on MDS-NK cells and serum levels of MICA/
B, suggesting that additional mechanisms such as the secretion of
immunomodulatory cytokines can downregulate NKG2D.
9
In contrast to AML patients, most CML patients express high
levels of MICA/B molecules.
57
Blocking experiments with anti-
NKG2D showed that the activating NKG2D receptor plays an
important role in the killing of CML cells and might be able to
override the inhibitory signals provided by the KIR–HLA
interactions.
57
Some of the CML patients expressed reduced
levels of the NKG2D receptor, which could explain why
autologous NK cells display a decreased killing of CML cells.
Finally, there are limited studies concerning the role of the NK
activating pathway in B-CLL patients. However, one study
reported that the inability of gd T cells to efficiently kill
autologous leukemic B cells might be attributable to the lack of
ligands for the activating NKG2D receptor.
60
Immunotherapy based on increasing NK cell activation
Cytokines
It has been demonstrated that the addition of cytokines,
particularly IL-2, has the capacity to increase NK cell prolifera-
tion and to kill NK cell-resistant cell lines and human tumor
cells of different types. The first clinical trials started in the
1980s, comparing IL-2 alone to IL-2 with autologous ex vivo IL-
2 expanded and activated lymphokine-activated killer (LAK)
cells in patients with advanced solid tumors.
61
Generally, no
significant beneficial effects were observed with the addition of
LAK cells and IL-2 based immunotherapies were mostly
associated with severe toxic effects.
Although it has been demonstrated that autologous LAK cells
have the capacity to kill leukemic cells in vitro,
62,63
only a few
clinical trials transplanting autologous LAK cells have been
reported for the treatment of leukemia.
64–68
However, trans-
plantation of autologous LAK cells showed a limited anti-
leukemic effect in the patients. No definitive conclusion can be
drawn because of the limited numbers of patients with poor
prognosis included in these studies. In contrast to IL-2, IL-15
upregulates, the anti-apoptotic protein Bcl-2 that gives IL-15-
stimulated NK cells a survival advantage.
69
Administration of
IL-15 can therefore increase immune reconstitution following
transplantation, because it plays a pivotal role in the survival of
NK cells, leading to increased NK cell numbers.
70
The cytokines
IL-12 and IL-18 induce IFN-g production in NK cells.
71
Another
cytokine that has the ability to activate NK cells is IL-21. This
cytokine plays an important role in the maturation of NK cells
and thus allows the induction of full NK cell maturation.
72
Finally, it has been shown that combinations of different
cytokines at low doses are associated with a synergistic
antitumor effect and lower toxicity and hence could be used
as immune therapy for treatment of leukemia.
73
So far only IL-2
and IL-12 have been administered to humans to investigate their
clinical use as immunotherapy. On the other hand, IL-15, IL-18
and IL-21 are still preclinical cytokines, but their beneficial
effects on NK cells shown in vitro and in vivo imply further
clinical investigation.
Upregulating the activating NK cell receptors or ligands
The recent understanding of the importance of activating NK
receptors in the recognition and killing of leukemia cells
suggests new therapeutic strategies. The functional activity of
NK cells is regulated by an array of distinct inhibitory and
activating receptors integrating a balance between negative and
positive signals that decides whether the NK cell will be
inhibited or activated. Modulating the balance toward activation
by upregulating NK activating receptors can induce NK cell
cytotoxicity. However there is limited knowledge about how we
Table 3 Expression of activating NK cell ligands and receptors in leukemia
Leukemia Activating NK cell ligands Expression References
B-ALL MICA/B Negative Romanski et al.,
49
Pende et al.
53
ULBPs Mostly negative Pende et al.
53
PVR and Nectin-2 Negative or low Pende et al.
53
CD48 and NTBA Negative or low Pende et al.
53
MDS MICA/B Positive in 30% of patients Epling-Burnette et al.
9
AML MICA/B Negative Pende et al.
53
MICA/B Mostly positive Salih et al.
55
PVR and Nectin-2 Positive Pende et al.
53
ULBPs Negative or very low Pende et al.,
53
Nowbakht et al.,
54
Salih et al.
55
NCR ligands Negative or very low Nowbakht et al.
54
CML MICA/B Mostly positive Sconocchia et al.
57
B-CLL MICA Negative Poggi et al.
60
ULBP1, 2 and 4 Negative Poggi et al.
60
ULBP3 Low Poggi et al.
60
Leukemia Activating NK cell receptors Expression References
MDS NKp30 Downregulated Epling-Burnette et al.
9
NKp46, NKp44 Normal Epling-Burnette et al.
9
NKG2D Normal or downregulated Epling-Burnette et al.
9
AML NCRs Downregulated Costello et al.,
6
Fauriat et al.
56
NKp46 Normal Siegler et al.
7
NKG2D Downregulated Siegler et al.
7
NKG2D Normal Costello et al.
6
CML NKG2D Downregulated Sconocchia et al.
57
Abbreviations: MICA/B, major histocompatibility complex class I chain-related molecules A and B; NCR, natural cytotoxicity receptors; PVR,
poliovirus receptor; ULBP, unique long 16-binding proteins.
NK cells and leukemia
S Verheyden and C Demanet
254
Leukemia
can modulate the expression of NK cell activating receptors. It is
generally accepted that IL-2 induces the expression of the
activating NKp44 receptor on NK cells.
74
Different cytokines,
including IL-2, IL-15 and IL-18, have been shown to increase the
expression of activating NKG2D.
75,76
On the other hand, it is
possible that the administration of cytokines activates NK cells
but also increases the inhibitory signals that counterbalance any
NK cell activation. In this context it has been shown that IL-15
not only upregulates the activating NKG2D receptor but also
provides an appropriate stimulus for the expression of the
inhibitory CD94/NKG2A receptor.
77
IL-21 induces rapid and
full maturation by which NK cells express various NK receptors
such as the NKp46 and NKp30 triggering receptors, CD16, 2B4
and the inhibitory CD94/NKG2A and KIRs.
72
Conversely, IL-21
has the ability to downregulate NKG2D/DAP10 expression that
correlates with a downregulated NK cell function.
78
An alternative approach to augment NK lysis is the upregula-
tion of NK activating ligands on the tumor cell surface.
Leukemic blasts in AML patients express very low levels of
ULBP ligands. Treatment with hematopoietic differentiation-
promoting agents, myeloid growth factors, together with IFN-g,
upregulates the cell surface levels of ULBP1 on AML blasts,
increasing their susceptibility to NK cell-mediated lysis.
54,79
Antibodies
The majority of NK cells express the FcgRIII (CD16) receptor that
binds to the Fc component of the IgG antibody bound on the
target cell surface. Upon binding of the Fc receptor, NK cells are
activated, leading to degranulation and secretion of cytotoxic
granules that kill the target cell. In this regard, chimeric IgG anti-
bodies against specific antigens present on leukemic cells have
been used to induce ADCC against these leukemic cells.
80–82
Another therapeutic approach is the application of bispecific
antibodies to link NK cell activating receptors with tumor cells.
In this regard, an antibody against CD16 and CD19 has been
developed to target B non-Hodgkin’s lymphoma.
83
Conclusion
Further studies considering both inhibitory and activating NK
cell pathways are needed to identify the exact innate immune
escape mechanism used by each type of leukemia. In this way,
an efficient NK cell immune response, by either blocking
inhibitory KIR-HLA or by upregulating activating NK cell
receptor–ligand interactions, or by both, could be developed
against these different forms of leukemia. The importance of
inhibitory KIR and HLA class I genes in susceptibility to
leukemia has been underscored. So one approach to increase
NK cell killing of leukemic targets would be to block specific
inhibitory KIRs–HLA interactions so that the inhibitory signal
could be removed. On the other hand, we observed that partial
downregulation of HLA class I allele expression occurs
frequently in leukemic cells. This observation raises an
important question as to how much HLA class I is needed to
be expressed to inhibit NK cells. More functional tests with
leukemic cells expressing a decreased KIR ligand level have to
be performed in the future, to correlate HLA-C or/and -Bw4
expression with NK cell function. Moreover, leukemic cells
might also use other resistance mechanisms such as down-
regulation of ligands for activating receptors on NK cells and/or
T cells. In this regard, a positive outcome of inhibitory KIR–HLA
mismatch has been demonstrated in patients with myeloid
leukemia, while patients with B-ALL seem not to benefit from
this NK alloreactivity. The difference observed in susceptibility
of AML and ALL cells to NK alloreactivity can be explained by
insufficient levels of ligands on ALL cells that are necessary for
NK cell-mediated killing. Modulating the balance toward
activating signals through upregulation of the activating NK cell
ligands on the acute lymphoid blasts could therefore enhance
NK killing.
Acknowledgements
The authors’ work was supported by a grant from the ‘Fonds voor
Wetenschappelijk Onderzoek Vlaanderen’ (FWO-Vlaanderen;
no. G.0481.06) and a clinical research grant from the ‘Stichting
tegen Kanker’ (SCIE 2005-33).
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NK cells and leukemia
S Verheyden and C Demanet
257
Leukemia
    • "DNAM-1 ligands were homogeneously expressed (Table S2) and thus were not suitable to subgroup the blasts and to explain the different lysis of the tested AML blasts, even though DNAM-1 triggering is important in the lysis of several tumor targets including AML [23, 32]. By subgrouping the blasts according to NKG2DL expression, a difference of cytolysis was revealed (Figure 5(a) andFigure S6), strongly indicating a major impact of NKG2D on NK cell mediated lysis of childhood AML blasts [24, 25, 30]. This hypothesis was further supported by a significant inverse correlation of HLA I expression and NK cell mediated cytolysis of NKG2DL low blasts (Figure 5(c)). "
    [Show abstract] [Hide abstract] ABSTRACT: Natural killer cells have been shown to be relevant in the recognition and lysis of acute myeloid leukemia. In childhood acute lymphoblastic leukemia, it was shown that HLA I expression and KIR receptor-ligand mismatch significantly impact ALL cytolysis. We characterized 14 different primary childhood AML blasts by flow cytometry including NKG2D ligands. Further HLA I typing of blasts was performed and HLA I on the AML blasts was quantified. In two healthy volunteer NK cell donors HLA I typing and KIR genotyping were done. Blasts with high NKG2D ligand expression had significantly higher lysis by isolated NK cells. Grouping the blasts by NKG2D ligand expression led to a significant inverse correlation of HLA I expression and cytolysis in NKG2D low blasts. Furthermore, a significant positive correlation of NKG2D ligand expression and blast cytolysis was shown. No impact of KIR ligand-ligand mismatch was found but a significantly increased lysis of homozygous C2 blasts by KIR2DL1 negative NK cells (donor B) was revealed. In conclusion, NKG2D signaling leads to NK cell mediated lysis of childhood AML despite high HLA I expression.
    Full-text · Article · Aug 2015
    • "The association between NK cell receptors and their ligands is very variable: in some cases, they increase the susceptibility to some cancers, while in others they might be protective from some cancers [22, 23]. Contradictory results about the interaction between KIR and its ligands were reported in various hematologic malignancies [19, 24, 25]. In two studies evaluating KIR and its ligands in NHL, no association between disease development and the interaction of KIR with its ligands was found [10, 16] . "
    [Show abstract] [Hide abstract] ABSTRACT: ABSTRACT NK cells' killer immunoglobulin-like receptors (KIR) contribute to the pathogenesis of many diseases. We determined the association between polymorphisms of KIR and its ligands with susceptibility to NHL, clinical features and prognosis. We included 90 NHL patients and 94 controls. In the NHL group, KIR2DS1, HLA-Bw4 (Thr80), and HLA-Bw4 (Thr80)+/Bw4 (Iso80)- ligands were significantly more frequent. Early-stage NHL patients had more frequent KIR2DL5 and KIR2DL5B than advanced-stage patients. During a median follow-up of 27 months, 26 NHL patients died. Poor prognostic factors in univariate analysis were KIR2DL5A, KIR2DS1 and KIR3DS1 genotypes. In multivariate Cox regression analysis, advanced age and early relapse stood out as poor prognostic factors; KIR genes and ligands had no significant effect on survival. The activating KIR2DS1 gene might have activated NK cells, contributing to production of more lymphoma cells. In addition to KIR2DS1, KIR2DL5A and KIR3DS1 might also be associated with poor prognosis in NHL.
    Full-text · Article · Feb 2015
    • "Acute myeloid leukemia (AML) is a frequent malignant hematological disease characterized by the initial accumulation of immature leukemia cells in the bone marrow and their subsequent migration into the blood circulation [8]. Natural Killer (NK) cells are key players in the immune surveillance of AML [9], and able to eradicate leukemic cells in an autologous or allogeneic setting [10], [11]. NK cell activity has been positively correlated with relapse-free survival after haematopoietic stem cell transplantation [12], [13]. "
    [Show abstract] [Hide abstract] ABSTRACT: Integrins are a large family of heterodimeric proteins that are involved in cell adhesion, migration, and proliferation. Integrin diversity and function is regulated by alternative splicing. Membrane-bound and truncated β3-integrins were shown to be key players in cancer metastasis. However, the immunomodulatory functions of the soluble (s) β3-integrin have not been investigated yet. In this study, we described a novel form of sβ3-integrin in acute myeloid leukaemia (AML) patients. Furthermore, we assessed the role of the sβ3-integrin in the modulation of natural killer (NK)-cell activity. Levels of sβ3-integrin were analysed in plasma samples of 23 AML patients and 26 healthy donors by ELISA. The capacity of sβ3-integrin to regulate NK cell activity was investigated using proliferation, cytokine secretion, and cytotoxicity assays. Circulating sβ3-integrin was detected in the plasma of 8 AML patients. NK cells showed significantly higher proliferation rates after stimulation with sβ3-integrin and IL-2, IL-15 (73%). Significant increases in the NK cells' secreted levels of TNF-α, IFN-γ were measured in presence of sβ3-integrin. In addition, sβ3-integrin caused the upregulation of Granzyme B transcripts levels as well as FasL expression levels in NK cells. Most importantly, significantly higher K562 or AML blast target cell lysis rates were observed when NK cells were exposed to sβ3-integrin. This study reports the identification of a novel sβ3-integrin in AML patients and provides novel insights into its role in the immunomodulation of NK cell activity.
    Full-text · Article · Jun 2014
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