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IMMUNOBIOLOGY
From INSERM 487, Laboratoire de thérapie
cellulaire, Institut Gustave Roussy, Villejuif, France; INSERM 506 Hôpital Paul Brousse, Villejuif, France; and Laboratoire de
Chimie Médicale et d'Oncologie Médicale, Université
de Liège, Belgique.
Chronic myeloid leukemia is a clonal myeloproliferative expansion
of transformed primitive hematopoietic progenitor cells characterized
by high-level expression of BCR-ABL chimeric gene, which induces growth
factor independence. However, the influence of BCR-ABL expression on
cell-mediated cytotoxicity is poorly understood. In the present study,
we asked whether BCR-ABL expression interferes with leukemic target
sensitivity to natural killer (NK) cell cytolysis. Our approach was
based on the use of 2 BCR-ABL transfectants of the pluripotent
hematopoietic cell line UT-7 expressing low (UT-7/E8, UT-7/G6) and high
(UT-7/9) levels of BCR-ABL. As effector cells, we used
CD56bright, CD16 Natural killer (NK) cells play an important role in
the early defense against viral and malignant transformation. Their
activity is characterized as nonadaptive and major histocompatibility
complex (MHC)-unrestricted and is thought to play an important role in immune surveillance. Recent studies have shown that NK cell recognition is based on the expression of multiple cell surface receptors that bind
either HLA class I or non-HLA ligands and transduce either inhibitory
or activating signals.1 The balance between these signals
controls NK cell activation. In addition, adhesion molecules of the
Over 90% cases of CML are characterized by a reciprocal translocation
between chromosomes 9 and 22 leading to the formation of
BCR-ABL chimeric gene on the derivative Philadelphia (Ph)
chromosome.6 Depending of the breakpoint in the
BCR gene, 3 main types of BCR-ABL genes can be
formed.7,8 The predominant hybrid gene in CML encodes for a 210-kd fusion protein (p210 BCR-ABL), which exhibits deregulated protein tyrosine kinase activity compared to normal ABL.8 As a result, there is excessive tyrosine
phosphorylation of many intracellular proteins including the BCR-ABL
protein itself.9 Remarkably, BCR-ABL gene
transfer induces growth factor independence in growth factor-dependent
cell lines and antiapoptotic activity,10 which can be
reversed when the tyrosine kinase activity of BCR-ABL is inhibited by
the specific abl inhibitor STI571. It is also known that CML
progression from chronic to transformed phase is associated with an
increase of BCR-ABL messenger RNA,8,11 and results in cell
escape to antileukemic treatments. Interestingly, in the present study
we showed that leukemic cells expressing high levels of BCR-ABL were
efficiently recognized and killed by dNK cells suggesting that these
cells may constitute useful effectors for immunotherapeutic approaches
in the treatment of patients with CML.
Target cells, UT-7 cell line, and BCR-ABL transfectants UT-7/9
and UT-7/E8-1
In vitro differentiation of NK cells from cord blood
CD34+ cells
Phenotypic analysis Several fluorescein isothiocyanate (FITC)-, phycoerythrin (PE)-, or phycoerythrin-cyanin 5 (PE-Cy5)-coupled monoclonal antibodies (mAbs) were used, allowing double fluorescence analysis: CD34 (IgG1), CD16 (IgG1), CD56 (IgG1), CD94 (IgG1), CD158 (IgG1), and CD3 (IgG1) purchased from Immunotech (Marseille, France). Each mAb was used in a volume of 8 µL to label 105 cells. Cells were incubated for 20 minutes at 4°C, washed twice with phosphate-buffered saline (PBS), and fixed before analysis on a FACS-Sort (Becton Dickinson, Pont de Claix, France). For indirect fluorescence, 105 cells were incubated with the first mAb directed against p46 (IgG1), p30 (IgG1) (from A. Moretta, Genoa, Italy), ICAM-1 (IgG2a), LFA-1 (IgG1), LFA-3 (IgG1), HLA class I W6/32 (IgG2a), and Fas (ZB4, IgG1) purchased from Immunotech, followed by an FITC-conjugated goat antimouse immunoglobulin. Background levels were measured using isotypic controls. For 2-color labeling analysis, compensation was set up with single-stained samples. Low forward-scatter elements (red cells or debris) were excluded from the analysis and 10 000 events were collected and analyzed using the Cellquest software (Becton Dickinson).Cytotoxicity assay Susceptibility of K562, UT-7, UT-7/9, UT-7/E8.1, and UT-7/G6 to differentiated NK cytolysis was tested in a 4-hour 51Cr release assay. Effector-target ratios ranged from 10:1 to 1:1. Determinations were performed in triplicate or quadruplicate and lysis percentages were determined as previously described.4 SDs were less than 10%. In some experiments, cell lines were treated with 1 µM (1 nM for K562) STI571 for 48 hours before the test.Proliferation and viability assays Cell proliferation was assessed by the dimethyl-thiazolyl-diphenyl-tetrazolium bromide (MTT) assay, and 15 × 103 cells were plated in quadruplicate in 96-well plate in -MEM supplemented with 10% FBS, GM-CSF, and 0 or 1 µM STI. STI571 was kindly provided by Novartis (Novartis Pharma,
France). Wells were assayed for uptake of MTT at hour 0 and after a
48-hour culture. Absorbance (A), which was proportional to cell
viability, was measured at 570 nm. Cell viability was evaluated using
the following calculation: percent of viability = 100 × A1/A2
where A1 and A2 were the absorbances obtained after 48 hours culture
with and without STI, respectively.
To assess their susceptibility to Fas-mediated cell lysis, UT-7 and UT-7/9 cells (5 × 103) were incubated in quadruplicate with agonistic anti-Fas mAb (CH-11, IgM) or irrelevant IgM for 24 hours in a 96-well plate and wells were assayed for uptake of MTT as described above. Nuclear protein extraction and electrophoretic mobility shift assay Nuclear protein extraction for electrophoretic mobility shift assay (EMSA) was described previously.12 The palindromic B probe was 5'-TTGGCAACGGCAGGGGAATTCCCCTCTCCTTA-3', labeled with ( 32P) adenosine triphosphate (ATP) using T4 kinase.
Nuclear extracts of UT-7 and UT-7/9 cells were performed before and 1, 4, 12, and 48 hours after treatment with 1 µM STI571.
Transient transfection and luciferase assay To quantify NF-B activity, the 3 cells lines were transfected
with the NF-B LUC plasmid reporter as previously
described.13 Briefly, cells were transfected with 0.5 µg
pCDNA3 vector containing 3 times NF-kB(pIC)-tk-LUC using the Fugene
system (Roche, Meylan, France) and luciferase activity was
determined as recommended by the manufacturer (Boehringer Mannheim, Germany).
Confocal microscopy analysis For double staining of NF-B and nuclear compartment, cells
were permeabilized with ORTHOpermeafix (Ortho Diagnostic Systems, Raritan, NJ). Then, cells were incubated for 30 minutes with 1 µg/mL
rabbit polyclonal purified IgG anti-NF-B p65 (Santa Cruz Biotechnologies, Santa Cruz, CA) followed by incubation with Alexa Fluor 488 goat antirabbit conjugate (Molecular Probes, Eugene, OR). Nuclei were stained with propidium iodide (red staining). Confocal analyses were performed on UT-7 and UT-7/9 cells before and 1, 12, and 48 hours after treatment with 1 µM STI. Stained cells were
washed with PBS, cytocentrifuged in a cytospin 3 (Shandon, Pittsburgh,
PA), and analyzed by laser scanning confocal microscopy using a Leica
TCS Confocal System (Wetzler, Germany).
IL-15 dNK cells from cord blood CD34+ cells exhibit higher lytic efficiency against UT-7/9 BCR-ABL transfectants The present studies were based on the use of dNK cells from CD34+ cord blood cells. IL-15 dNK cells express high levels of CD56 antigen (mean fluorescence intensity [MFI] > 1000) and CD16 expression is restricted to about 30% of CD56+ cells. We further showed that natural cytotoxicity receptors (NCRs), namely, p30 and p46 were expressed on most dNK cells, whereas Fas-L protein expression was low. As depicted in Table 1, CD94 receptor associated with NKG2-A was strongly expressed on more than 80% of CD56+ cells, whereas less than 10% of dNK cells expressed the KIR p58, p70, and p140 receptors. Concerning coreceptors involved in lysis, 70% of dNK cells express LFA-1, whereas CD2 expression was low.
To examine the antileukemic effect of dNK cells and the influence of
BCR-ABL expression on target cell lysis, the killing of UT-7, and its
BCR-ABL transfectants UT-7/E8.1, UT-7/G6, and UT-7/9 by dNK cells were
determined using 51Cr release assay. Data shown in Figure
1A indicate that UT-7/9 cells are
constantly found more susceptible to NK cell-mediated killing than
UT-7 and UT-7/E8.1 cells, suggesting that the level of BCR-ABL
expression is involved in the modulation of target susceptibility to
NK-induced lysis. Levels of BCR/ABL expression in the 4 cell lines are
depicted in Figure 1B.
High level of BCR-ABL expression on UT-7/9 transfectants correlates with overexpression of ICAM-1 and LFA-1 To evaluate the effect of BCR-ABL on the expression of molecules potentially involved in dNK/target cell interactions, surface expressions of HLA class I, ICAM-1, LFA-1, and LFA-3 on UT-7, UT-7/9, and UT7-E8.1 cells were assessed using FACS analysis. The expression of LFA-1 and LFA-3 was similar in UT-7 cells and in the 2 BCR-ABL transfected clones, whereas HLA-I was slightly increased in UT-7/9 cells. However, the major difference concerned the expression of ICAM-1 that was significantly higher on transfectant UT-7/9 cells (× 5), displaying the strongest BCR-ABL expression (Figure 2A). In addition, incubation of UT-7 and UT-7/9 cells with anti-ICAM-1 mAb before the cytotoxicity assay resulted in a significant decrease (< 50%) of the lysis of both cell lines (Figure 2B).
The increase of UT-7/9 transfectant susceptibility to dNK cells involves the Ca++-dependent killing pathway In addition to an increased expression of ICAM-1 and LFA-1 molecules, UT-7/9 transfectants that express a high level of BCR-ABL exhibit an increased membrane Fas expression (Figure 3A). To determine if the Fas/Fas-L pathway is involved in the increased cytotoxicity toward UT-7/9 cells, cell-mediated lysis was assessed in the presence of EGTA that inhibits the perforin-granzyme pathway. EGTA dramatically decreased the lytic activity of dNK cells against UT-7 and UT-7/9 transfectants, suggesting that dNK cells used the classical perforin-granzyme pathway to lyse BCR-ABL targets (Figure 3B). Furthermore, UT-7 and UT-7/9 cell lines treated by anti-Fas antagonist mAb (CH-11) proliferated similarly to control IgM-treated cells (Figure 3C), indicating that despite an increased Fas expression, UT-7/9 cells were resistant to Fas-mediated apoptosis and that the susceptibility of UT-7/9 transfectants to NK cell lysis did not involve Fas/Fas-L pathway.
Down-regulation of ICAM-1 expression in UT7/9 cells by STI571 results in a decrease of their sensitivity to dNK cell cytolysis Because oncogenic transformation by BCR-ABL is dependent on tyrosine kinase activity, selective inhibitors of the ABL tyrosine kinase were developed. Among these, STI571 is able to reduce the tyrosine phosphorylation of cellular proteins, largely affecting BCR-ABL protein level.l4 This compound was used to further investigate the involvement of BCR-ABL expression in the modulation of target susceptibility to NK cell cytolysis. Incubation of UT-7/9 cells with STI571 (1 µM) significantly inhibited their proliferation ( 30%) as determined by MTT assay, whereas it had no
effect on parental UT-7 cell proliferation (data not shown). As
depicted in Figure 4A, treatment by
STI571 for 48 hours dramatically decreased the expression of ICAM-1 by UT-7/9 cells, whereas it had no significant effect on parental UT-7
cells. It should be noted that a slight decrease in HLA class I
molecule expression by UT-7/9 cells was observed following STI571 treatment (Figure 4A). Interestingly, STI571 treatment of UT-7/9 and
K562 cells resulted also in a clear decrease in cell susceptibility to
dNK cytolysis, whereas the lysis of UT-7 and UT-7/G6 cells was not
significantly affected by STI571 treatment (data not shown and Figure
4B). These data suggested a prominent role of the interaction of ICAM-1
and LFA-1 in the modulation of UT-7/9 susceptibility to NK cell
lysis.
STI571 inhibits activation of NF- B in
UT-7/9 cells (Figure 5A) and experiments
using a NF-B-dependent reporter gene confirmed the increased
NF-B activity in the UT-7/9 cells (Figure 5B). Western blot analysis
evidenced an increased phosphorylation of IB in the UT-7/9 cells
(Figure 5C). Analysis by confocal microscopy of NF-B p65
localization confirmed the nuclear localization of NF-B, detected by
a yellow nuclear staining in most of the UT-7/9 cells, whereas the
parental UT-7 exhibited a green cytoplasmic staining (Figure
6). STI571 treatment of UT-7/9 cells
inhibited NF-B translocation as shown by the absence of nuclear
yellow staining of treated cells and the cytoplasmic expression of p65
NF-B subunit shown by the green staining. Treatment of UT-7/9 cells
by BAY 11-7082, a specific IB kinase inhibitor that prevents
degradation of IB and specifically abrogates NF-B DNA binding,
resulted in a clear decrease in nuclear NF-B staining (Figure
6).
To further confirm that NF-
We have previously shown that in vitro IL-15 dNK cells constituted a homogeneous NK cell population with high lytic activity.4 These cells differed from peripheral NK cells because they were CD56bright cells and did not express CD16 or CD2. CD94/NKG2-A, specific for HLA-E molecules, was expressed by more than 80% of dNK cells, whereas inhibitory receptors of the Ig (KIR) superfamily were expressed only by 5% to 10% of dNK cells. Such a phenotype was similar to the phenotype of NK cells isolated from peripheral blood of patients undergoing bone marrow transplantation from unrelated matched donors15 that were potentially implicated in the antitumoral graft-versus-leukemia effect.16 Recently we provided evidence for the alteration of in vitro NK cell differentiation from CML progenitors.5 We have demonstrated that this altered differentiation was related to an abnormal IL-15 transduction pathway that may account for the decreased peripheral NK activity in patients with advanced CML. The present studies were performed to gain more insights into the molecular basis of BCR-ABL-induced modulation of target susceptibility to NK lysis and to investigate the potential involvement of in vitro dNK cells from CD34+ progenitors in the lysis of leukemic cells expressing BCR-ABL oncoprotein. It has been reported that BCR-ABL-expressing leukemic cells were
highly resistant to apoptosis induced by chemotherapeutic drugs.17 One of the antiapoptotic pathways triggered by
BCR-ABL involved induction of bcl-XL through signal
transducers and activators of transcription (STAT5)
activation.18 In this regard, inhibition of BCR-ABL kinase
activity was effective in suppressing STAT5 interaction with the
bcl-XL promoter and subsequently leading to
bcl-XL down-regulation and increased
apoptosis.19 On the other hand, several reports evidenced
that human CML leukemic blasts may be killed by activated NK
cells20,21 and that activated NK cells specifically
suppress CML malignant hematopoïesis.22 This
suppression was not mediated by soluble factors but was dependent on
direct cell-to-cell contact and was reversed by
anti- It is clearly established that the regulation of NK cytolysis is
controlled by several cell-to-cell contacts including interactions of
KIR and NCRs with their ligands in combination with interactions of
Recently, evidence has been provided that NF- Our study evidenced that high-level BCR-ABL expression increased the
susceptibility to NK cytolysis by a mechanism involving at least in
part an increase of ICAM-1 expression favoring NK cell/target
attachment, through activation of NF- Although only a small percentage of patients benefit from this curative procedure,35 cellular therapy trials show that allogeneic marrow transplantation is firmly established as the treatment of choice for patients with CML. Current approaches to increase the graft-versus-leukemic effect is to isolate donor leukemia-specific T-cell clones using peptides spanning the BCR-ABL junction. These peptides induce cytotoxic T-cell response in vitro but it remains unclear whether the induced CTLs recognize and kill autologous CML cells. On the other hand, the opportuneness of using antineoplastic activity of allogeneic NK cells (selected for appropriate HLA-C and/or B mismatch) as an alternative to CTL, thus avoiding graft-versus-host disease, seems very promising. In addition, infusion of NK cells may lead to a rapid antileukemic activity appropriated to patients in blastic crisis. In that context, our results unexpectedly showing that oncogenic transformation by BCR-ABL improves susceptibility to non-MHC-restricted cytotoxicity by up-regulation of cellular ligands for NK cells further emphasize the interest of cellular therapy protocols using allogeneic NK cells in the treatment of patients with CML.
We would like to thank Yann Lécluse for immunofluorescence analyses. We thank Professor Alessandro Moretta for providing us with anti-NCR and anti-NKR mAbs and for helpful discussions.
J.G.-M. and A.G.T. contributed equally to the work.
Submitted January 29, 2001; accepted October 29, 2001.
Supported by grants awarded by INSERM, Institut Fédératif de Recherche IFR 54 and La Fondation de France (Nb 99003929). F.B. is postdoctoral researcher of the National Fund for Scientific Research (FNRS) Belgium.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Anne Caignard, Unité INSERM 487, Cytokines et Immunologie des Tumeurs Humaines, Institut Gustave Roussy, PR1, 39, rue Camille Desmoulins, F-94805 Villejuif, France; e-mail: caignard{at}igr.fr.
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Top
Abstract
Introduction
, CD2
2-integrin family, such as the intercellular adhesion
molecule 1 (ICAM-1) and its counterreceptor lymphocyte function-associated antigen 1 (LFA-1), also play a central role in the
regulation of NK cytolysis by modulating the positive
signal.
