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Blood, Vol. 96 No. 1 (July 1), 2000:
pp. 297-306
NEOPLASIA
Inhibition of in vitro spontaneous apoptosis by IL-7 correlates
with Bcl-2 up-regulation, cortical/mature immunophenotype, and better
early cytoreduction of childhood T-cell acute lymphoblastic
leukemia
Leonid Karawajew,
Velia Ruppert,
Christian Wuchter,
Annett Kösser,
Martin Schrappe,
Bernd Dörken, and
Wolf-Dieter Ludwig
From the Department of Hematology, Oncology, and Tumor Immunology,
Robert-Rössle Clinic, Charité, Humboldt University of
Berlin, Berlin; and the Department of Pediatrics, Medical School of
Hannover, Hannover, Germany.
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Abstract |
In normal T-cell development, IL-7 plays a nonredundant role as an
antiapoptic factor by regulating Bcl-2 expression in pro-T cells. In
the current study, we addressed the roles of IL-7 and related cytokines
as apoptosis-modulating factors in precursor T-cell acute lymphoblastic
leukemia (T-ALL). To this end, leukemic blasts from pediatric patients
with T-ALL were prospectively investigated as to their responsiveness
to IL-7, IL-4, and IL-2 (in terms of modulation of spontaneous
apoptosis, assessed by flow cytometry), cytokine receptor expression
profiles, and expression levels of Bcl-2 and Bax proteins. IL-7, in
contrast to IL-4 and IL-2, was highly efficient in apoptosis inhibition
, and this effect correlated with the expression levels of IL-7R
chain and with the up-regulation of Bcl-2 protein expression (P
< .0001). Subclassification of T-ALL samples (n = 130)
according to their in vitro IL-7 responses revealed that IL-7
refractory samples were more frequently positive for CD34 (P
< .0001) and the myeloid-associated antigen CD33 (P = .01), whereas IL-7 responsiveness was associated with an
expression of more mature differentiation-associated T-cell antigens
(CD1a, surface CD3, CD4/8; P < .05). Furthermore, the extent
of apoptosis inhibition by IL-7 in vitro quantitatively correlated with
early cytoreduction as determined by the prednisone peripheral blood response on day 8 and cytoreduction in the marrow on day 15 (n = 87;
P < .05). Multivariate analysis of the apoptosis-related parameters investigated, including spontaneous apoptosis, its inhibition by IL-7, and expression levels of Bcl-2 and Bax, showed that
only IL-7 responsiveness has an independent impact on early cytoreduction (P < .05), thus indicating a potential
prognostic relevance of IL-7 sensitivity in T-ALL.
(Blood. 2000;96:297-306)
© 2000 by The American Society of Hematology.
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Introduction |
In normal hematopoiesis, responsiveness of progenitor
cells to cytokine signaling for proliferation, death, and survival
strongly depends on their lineage commitment and differentiation
state.1-3 Normal T-lineage development has recently been
shown to depend crucially on the so-called common  chain of the IL-2
receptor (IL-2R) shared by the receptors for cytokines IL-2, IL-4,
IL-7, IL-9, and IL-15.4 Early T-cell development was found
to be severely impaired in IL-2R chain knock-out mice,4
and the absence or markedly reduced number of T cells in patients with X-linked severe combined immunodeficiency (X-SCID) has been associated with -chain mutations.5 However, most of these cytokines
seem to be functionally redundant because mice deficient in single cytokines revealed unimpaired lymphopoiesis.6,7 The only exception is IL-7, which, when deficient in mice, has been shown to
result in a drastically reduced number of thymocytes, though with a
normal subset distribution.8,9 The critical role of IL-7
for early T-cell development in humans has also been demonstrated in
chimeric human-mouse thymus organ culture models in which antibodies blocking IL-7 and IL-7R have been used.3,10
The analysis of thymocytes from IL-7-deficient mice disclosed that
their developmental arrest occurs at the triple-negative (CD3 ,
CD4, CD8) stages.9 These cells revealed
increased rates of spontaneous apoptosis associated with decreased
levels of the anti-apoptotic protein Bcl-2 and increased levels of the proapoptotic Bcl-2 homolog Bax.11,12 Treatment of
triple-negative thymocytes with IL-7 resulted in the up-regulation of
Bcl-2 expression and the inhibition of apoptosis.12
Moreover, the overexpression of Bcl-2 in IL-7-deficient mice largely
restored normal T-cell development, suggesting that IL-7 may exert its
survival function by the up-regulation of Bcl-2.11
Acute leukemia results from a clonal expansion of hematopoietic
progenitor cells that have undergone malignant transformation at
distinct stages of differentiation.13 Because leukemia
cells may retain certain features of their normal counterparts, their characterization with respect to cytokine responsiveness can provide valuable information about the differentiation state of malignant cells
and their dependence on the microenvironment. Previous
studies14-16 investigated the effects of IL-7, IL-4, and
IL-2 regarding the induction of proliferation in childhood T-lineage
acute lymphoblastic leukemia (T-ALL), and their potential as growth
factors has been pointed out. However, the roles of these
cytokines as apoptosis-modulating factors in T-ALL have not yet been
studied in detail.
Therefore, in the current study we investigated cytokine
responsiveness, receptor expression profiles, and the expression of
apoptosis-regulating Bcl-2 and Bax proteins in leukemic blasts from
pediatric patients with T-ALL. IL-7, in contrast to IL-4 and IL-2, was
highly efficient in apoptosis inhibition, and this effect correlated
with the expression levels of the IL-7R chain and the up-regulation
of Bcl-2 protein expression. Given these findings, a large series of
T-ALL samples was classified according to in vitro IL-7 responses and
examined for immunophenotypic features (n = 130) and early therapy
response in vivo (n = 87). Our data demonstrating significant
correlations of IL-7 responsiveness in vitro with maturational stage
and early response to initial therapy in vivo point to a potential
prognostic relevance for the determination of IL-7 sensitivity in
T-ALL.
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Patients, materials, and methods |
Patient population
Leukemic samples from 130 children with T-lineage ALL who were
enrolled in the ALL-Berlin-Frankfurt-Münster (BFM)
95 trial were prospectively investigated for the current study.
Diagnosis of ALL was based on morphology and on cytochemical and
immunophenotypic features.17,18
All leukemic samples were characterized by immunophenotype.
Immunophenotyping was carried out on leukemic blasts isolated by
standard Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient
centrifugation, and cell-surface as well as intracytoplasmic antigens
were detected by a panel of monoclonal antibodies either by direct or
indirect immunofluorescence techniques as previously described.19,20 The criteria for marker positivity and for the subclassification of T-lineage ALL (pro-, pre-, cortical, and
mature T-ALL) were adopted from the guidelines proposed by the European
Group for the Immunological Characterization of
Leukemias.18 In the current study, 28 samples were
subclassified as pro-T-/pre-T-ALL, 79 samples as cortical T-ALL, and
23 samples as mature T-ALL.
Data on early responses to initial therapy, as measured by the in vivo
corticosteroid response in peripheral blood on day 8 and the response
in the bone marrow on day 15, were available for 87 and 62 patients,
respectively. Therapy in the first 14 days consisted of the
administration of intrathecal methotrexate (on day 1), prednisone
(daily, in rapidly increasing dosages from day 1), vincristine and
daunorubicin (on day 8), and asparaginase (on day 12).21 If
a patient had more than 1000 blood blasts/µL on day 8, it was defined
as a prednisone-poor response.
Cell culture
Freshly obtained leukemic blasts were purified either from bone
marrow (n = 113) or peripheral blood (n = 17) patient samples by
density gradient centrifugation using Ficoll-Hypaque separation (Pharmacia). Cell viability was always more than 90%, as determined by
trypan blue or propidium iodide (PI) exclusion (Sigma, Deisenhofen, Germany). All samples contained more than 90% leukemic cells based on
morphologic and immunophenotypic criteria.
Leukemic cells were maintained in RPMI 1640 (Biochrom, Berlin, Germany)
standard medium containing 2 mmol/L
L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin
and supplemented with 10% heat-inactivated fetal calf serum (FCS;
Gibco BRL, Paisley, UK). Functional assays (spontaneous apoptosis and
its modulation by cytokines) performed with samples from several
patients in the presence and absence of FCS showed similar results.
To assess spontaneous apoptosis, 0.5 × 106 leukemic
cells/well were cultured in 96-well microtiter plates (Nunc, Roskilde, Denmark) in standard medium at 37°C in a humidified atmosphere of
5% CO2 in air. To investigate cytokine-mediated effects,
leukemic cells were cultured in the presence of recombinant human
cytokines IL-7 (25 ng/mL), IL-4 (50 ng/mL), or IL-2 (25 ng/mL) (Pharma
Biotechnologie Hannover, Hannover, Germany) at concentrations found to
be optimal in different T-cell systems22,23 and in our own
titration experiments (not shown). In several experiments, blocking
anti-IL-7 rabbit polyclonal antibodies (10 µg/mL; Pharma
Biotechnologie Hannover) were used.
Assessment of apoptosis and cell cycle distribution
To determine the extent of apoptosis, cells were stained with
fluorescein isothiocyanate (FITC)-conjugated annexin V and PI using the
annexin V kit (Immunotech, Marseille, France) as recommended by the
manufacturer. Thereafter, samples were analyzed by flow cytometry
(FACScan; Becton Dickinson, San Jose, CA) for the presence of viable
(annexin V- and PI-negative), early apoptotic (annexin V-positive,
PI-negative), and late apoptotic (annexin V- and PI-positive) cells.
The extent of apoptosis (N%) was quantified as the percentage of
annexin V-positive cells. The extent of spontaneous apoptosis was
calculated as the difference of N% values in leukemic samples before
and after culture in medium,  Nspont. Leukemic cell
responsiveness to cytokines was evaluated in terms of cytokine-specific
apoptosis modulation, which was calculated as the difference between
the extent of apoptosis after culturing in the absence and presence of
the respective cytokine (NIL). In a number of
experiments (n = 38), apoptotic cells were identified, in addition
to the annexin/PI method, by their subdiploid DNA content.24
To study cell cycle distributions, cells were fixed and stained as
described elsewhere.24 Briefly, a pellet of 5 × 105 cells was fixed by adding 2 mL ice-cold 70% ethanol
for at least 1 hour at 4°C. After washing, the cells were
resuspended in 0.5 mL phosphate-buffered saline containing 50 µg/mL
PI, pH 7.5. After treatment with 10 µL of 10 mg/mL RNase (type I-A;
Boehringer Mannheim, Mannheim, Germany) for 30 minutes at room
temperature in the dark, the cells were stored at 4°C until flow
cytometric analysis. Cell cycle analysis was carried out using either
CellQuest (Becton Dickinson) or ModFit LT (Verity, Topsham, ME) software.
Using flow cytometry, 10 000 and 20 000 cells were characterized
for apoptosis and cell cycle distribution analysis,
respectively. All tests were performed in duplicate. In
addition to flow cytometric analysis, the total number of viable cells
in a series of samples was estimated by trypan blue exclusion.
Assessment of surface expression of interleukin receptors
IL-2R (CD25), IL-7R (CDw127), and IL-2R (CD132) were
detected by direct immunofluorescence using mouse monoclonal antibodies conjugated either with FITC (CD25, clone 3G-10; Medac, Hamburg, Germany) or phycoerythrin (CDw127, clone R34.34; Immunotech; CD132, clone AG184; Pharmingen, Hamburg, Germany). IL-4R was detected by
indirect immunofluorescence using a specific monoclonal antibody (CDw124, clone S4-56C9; Immunotech) and FITC-conjugated goat antimouse polyclonal antibodies (Medac). Isotype-matched irrelevant mouse antibodies (Immunotech) were used as negative controls to determine background fluorescence. Antigen expression was quantified by flow
cytometry in units of molecules of equivalent soluble fluorochrome (MESF) using calibration beads as fluorescence standards (DAKO FluoroSpheres; DAKO, Glostrup, Denmark).
Assessment of Bcl-2 and Bax expression
To evaluate the expression of the intracellular proteins Bcl-2 and
Bax, leukemic cells were fixed and permeabilized using the Fix & Perm
kit (An-der Grub, Kaumberg, Austria) according to the manufacturer's
instructions. Bcl-2 antigen was detected by the FITC-conjugated
anti-Bcl-2 antibody clone 124 (DAKO). FITC-conjugated irrelevant mouse
antibodies of the appropriate immunoglobulin isotype (Immunotech) were
used as negative controls.
To detect Bax, Bax-specific rabbit polyclonal antibodies raised against
synthetic peptide sequences (I-19; Santa Cruz Biotechnology, Santa
Cruz, CA) and FITC-conjugated goat antirabbit serum (Medac) as a
secondary staining reagent were used. For negative controls, cells were
stained with Bax antibody in the presence of the blocking synthetic
peptide (provided by the manufacturer, Santa Cruz Biotechnology) at
saturation conditions (10 µg peptide vs 1 µg antibody), as determined by peptide titration experiments (data not shown).
Immunofluorescence analysis was performed on a FACScan (Becton
Dickinson). In cell samples with ongoing apoptosis, only antigen expression in viable and early apoptotic cells has been considered; late apoptotic cells might have had reduced protein content because of
cell disintegration. Viable and early apoptotic cells were differentiated from late apoptotic cells by the higher forward-scatter and lower side-scatter intensities.25,26
Antigen expression was quantified by flow cytometry in MESF units using
calibration beads as fluorescence standards (DAKO). Relative change of
expression (RCE) of Bcl-2 and Bax resulting from culturing in standard
medium was quantified as the ratio RCEspont =
MESFmedium/MESF0h. IL-7-specific RCE of Bcl-2
and Bax was calculated from the ratio of antigen expression levels in
IL-7-treated and untreated cells, so that RCEIL-7 = MESFIL-7/MESFmedium.
Statistical analysis
Mean values are given as mean ± SEM. Differences (P
values) were evaluated using the 2-tailed, nonparametric Mann-Whitney U test for continuous variables and the Fisher exact test for categorical variables. Associations (correlation coefficient
[r] and probability [P]) of the extent of
spontaneous or IL-7-modulated apoptosis with protein expression levels
in leukemic cells and blast counts in T-ALL patients were evaluated
using Spearman correlation statistics. Trend lines were calculated
using linear regression statistics. Differences were considered
significant for P < .05. Statistical analysis was performed
using SPSS software.
| |
Results |
IL-7, but not IL-4 or IL-2, is a highly potent inhibitor of
spontaneous apoptosis in T-ALL
Leukemic samples were investigated for their responsiveness to
cytokines according to the inhibition or enhancement of spontaneous apoptosis (Figure 1). To determine the
optimal incubation time periods, leukemic cell samples (n = 16) were
initially examined by flow cytometry after incubation periods of 24 and
72 hours. In all patients, cytokine responses in terms of apoptosis
modulation and cell cycle distribution changes (see below) measured
after 24 and 72 hours were similar. However, the extent of spontaneous cell death, measured after 24 hours in individual samples, was highly
variable (range, 6%-75%; mean, 33% ± 3%). Therefore, samples with high rates of spontaneous apoptosis could not be evaluated after
72 hours (data not shown). In addition, the total number of viable
cells was estimated after 3 and 7 days of culture. At day 7, leukemic
samples contained 50% to 100% of dead cells, and the total number of
cells did not exceed 20% of the initial number of 5 × 105 cells/well. Consequently, 24-hour incubation periods
were found to be optimal for standardizing cytokine response studies in
a larger series of T-ALL samples.
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| Fig 1.
Flow cytometric analysis of spontaneous apoptosis and
cytokine effects in leukemic cells from a patient with cortical T-ALL.
Blast cells were either cultured in medium alone or treated with 25 ng/mL rIL-7 for 24 hours. Freshly isolated (day 0) and cultured (24 hours; 24 hours +IL-7) cells were stained with annexin V-FITC and PI to
differentiate between viable (lower-left quadrant) and apoptotic
(lower- and upper-right quadrants) cells. Data shown are representative
of T-ALL responsive to IL-7.
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Apoptosis-modulating effects of IL-7, IL-4, and IL-2 were investigated
in leukemic samples from 38 patients. As Table
1 indicates, IL-2 was unable to inhibit
apoptosis in leukemic blasts, and IL-4 prevented spontaneous apoptosis
in only a few (7 of 37) patients. By contrast, IL-7 inhibited apoptosis
(NIL-7, >5%) in most T-ALL (24 of 38 patients; 63%)
patients. Apoptosis induction by IL-2 and IL-4 was observed in 1 and 2 patients, respectively (Table 1).
In individual samples, the inhibiting effect of IL-4 coincided with
that of IL-7 in almost all (6 of 7) patients. In 1 patient (patient
25), IL-4 and IL-7 demonstrated opposite (pro-apoptotic and
anti-apoptotic, respectively) effects on spontaneous apoptosis. Most
samples (17 of 24) were only responsive to IL-7.
To demonstrate the specificity of IL-7 effects, we used
cytokine-specific blocking antibodies against IL-7 during cell
culturing in the presence or absence of IL-7. These antibodies
successfully prevented the inhibition of spontaneous apoptosis by IL-7
but did not affect spontaneous apoptosis, indicating that IL-7 was not
produced by the blast cells themselves (data not shown).
IL-7, IL-4, and IL-2 do not affect cell cycle distribution
in T-ALL samples
In addition to their apoptosis-modulating
capacity, cytokines were tested for their ability to influence
proliferation kinetics. Evaluation of the cell cycle distribution
disclosed a low initial rate of proliferation that did not
significantly change after 24-hour incubation in the culture medium
(Table 2). No spontaneous proliferation
after in vitro was observed except in patient 38, in whom the
percentage of cells in S and G2/M phases increased from 12% to
22%.
The presence of cytokines in the culture medium did not affect cell
cycle distribution in most leukemic cell samples (Table 2). Marked
increases (>5%) of cells in the S and G2/M phases after 24-hour
incubation with cytokines were observed in a few patients (3 of 38 with
IL-7 and 2 of 37 with IL-4). The increased proliferation in these
patients, however, did not prevent a decrease in the overall number of
viable cells from spontaneous apoptosis, as assessed after 3 and 7 days
of incubation (data not shown).
Cell surface expression of IL-7R, IL-4R, IL-2R, and
IL-2R
Expression levels of IL-7R, IL-4R, IL-2R, and IL-2R were
examined, in addition to the functional studies, in a
subset of samples from T-ALL patients (n = 33, n = 21, n = 21, and n = 12, respectively). Expression values of IL-7R were highly variable and correlated with the extent of apoptosis inhibition by IL-7 (r = 0.63; P < .0001; Figure
2). Most (15 of 21) T-ALL samples were
negative for the expression of IL-4R, but IL-4 responding samples
revealed significantly higher expression levels than nonresponding samples (mean MESF × 10 3, 0.69 ± 0.25 vs
0.07 ± 0.07, respectively; P = .02). In contrast to IL-7R
and IL-4R, almost all (15 of 19) leukemic samples expressed, in spite
of their unresponsiveness to IL-2, low but distinct levels of IL-2R
(mean MESF × 103, 0.37 ± 0.1; range,
0-1.26). In addition, all samples studied were positive for IL-2R (n = 12; mean MESF × 103, 1.8 ± 0.2). Interestingly, in most samples, IL-2R expression levels were
relatively similar (MESF × 103; range,
1.5-3.0) and did not correlate with the responsiveness of leukemic
blasts to IL-7. In a T-ALL sample with relatively low IL-2R
expression (0.3 MESF × 103), neither
IL-7 response nor IL-7R expression was observed.
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| Fig 2.
Expression levels of IL-7R (CDw127) in T-ALL cells
correlate with the apoptosis-inhibiting effects of IL-7
( NIL-7).
Leukemic cells were incubated in the presence and absence of IL-7 for
24 hours at 37°C and assessed for apoptosis by flow cytometry, and
the cytokine-specific apoptosis modulation (N) was calculated as the
difference between the extent of apoptosis in the presence and absence
of the cytokine. CDw127 expression was quantified in MESF units using
calibration beads.
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Bcl-2 and Bax involvement in the IL-7 inhibition of spontaneous
apoptosis
Constitutive expression levels of Bcl-2 and Bax were determined by
flow cytometry as shown in Figure 3. All
samples (n = 45) expressed Bcl-2 and Bax with mean values of (4.9 ± 0.8) × 103 MESF and (186 ± 18) × 103 MESF, respectively. Expression levels in individual
samples were highly variable (0.5-25 × 103 MESF for
Bcl-2 and 40-553 × 103 MESF for Bax), which, however,
failed to correlate with the extent of spontaneous apoptosis or
apoptosis inhibition by IL-7 (data not shown).
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| Fig 3.
Flow-cytometric detection of Bcl-2 (A) and Bax (B)
antigens in untreated and IL-7-treated leukemic cells from a patient
with cortical T-ALL.
Blast cells were fixed, permeabilized, and stained by either direct or
indirect immunofluorescence for Bcl-2 (mouse monoclonal antibody) or
Bax (rabbit polyclonal antibodies against Bax peptide sequences),
respectively. Solid lines correspond to antigen staining in untreated
and IL-7-treated cells (bold and thin solid lines, respectively).
Dotted lines correspond to background fluorescence of cells stained
either with irrelevant antibodies (A and B) or with Bax antibody
blocked with an excess of the corresponding specific peptide (B).
Expression changes representative of T-ALL responsive to IL-7 are
shown.
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In contrast to the constitutive expression levels, changes of the Bax
expression after in vitro culturing of T-ALL cells were associated with
the extent of spontaneous apoptosis (r = 0.51; P
= .02) (Figure 4). However, changes
in neither Bcl-2 levels (Figure 4) nor Bcl-2/Bax ratios (not shown)
correlated with spontaneous apoptosis.
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| Fig 4.
Relative changes of expression (RCEspont) of
Bcl-2 (A) and Bax (B) in T-ALL cells after culturing in standard medium
for 24 hours.
RCEspont values for Bax, but not Bcl-2, positively
correlated with the extent of spontaneous apoptosis
(Nspont). RCE values were calculated
as the ratio RCEspont =
MESFmedium/MESF0h. Bcl-2 and Bax expression
were assessed by flow cytometry as described in Figure 3 and were
quantified in MESF units using calibration beads as fluorescence
standards.
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To investigate the involvement of Bcl-2 and Bax in the IL-7-induced
inhibition of spontaneous apoptosis, we examined their expression
levels in IL-7-treated cells in relation to those in untreated cells
(Figure 5). Bcl-2 expression after IL-7
treatment increased significantly when in comparison with untreated
samples, and these changes highly correlated with the extent of
apoptosis reduction by IL-7 (r = 0.75; P < .0001)
(Figure 5A). In contrast to Bcl-2, IL-7-induced changes of Bax were
relatively low and did not correlate with the effect of IL-7, though a
tendency to a negative correlation was observed (Figure 5B). Moreover,
examination of the Bcl-2/Bax ratios did not reveal a better correlation
with IL-7 effect than the analysis of Bcl-2 levels alone (r =
0.55; P = .0002).
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| Fig 5.
Correlation between IL-7-induced changes of Bcl-2 (A)
and Bax (B) expression levels and extent of IL-7-specific apoptosis
inhibition (NIL-7).
Changes of Bcl-2 but not Bax positively correlated with the
anti-apoptotic effect of IL-7. IL-7-specific relative changes of
expression of Bcl-2 and Bax were calculated from the ratio of antigen
expression levels in IL-7-treated and untreated cells
(RCEIL-7 =
MESFIL-7/MESFmedium). Bcl-2 and Bax expression
were assessed by flow cytometry as described in Figure 3 and were
quantified in MESF units using calibration beads as fluorescence
standards.
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Unresponsiveness to IL-7 is associated with an immature
or aberrant immunophenotype and with lower spontaneous apoptosis
To investigate the diagnostic and clinical relevance of
IL-7 responsiveness in vitro, we examined prospectively a series of 130 patients with immunophenotypically characterized T-ALL. Only few (10 of
130) patients could not be evaluated because of the absence of
spontaneous apoptosis (Nspont, <5%). Of the 120 samples that revealed different extents of spontaneous apoptosis
(range, 7%-75%), 64 (53%) were sensitive to IL-7
(NIL-7, >5%), whereas 56 were IL-7 refractive
(NIL-7,  5%). Interestingly, IL-7-sensitive T-ALL
samples revealed a significantly higher extent of spontaneous apoptosis
when compared with IL-7-refractive samples (39% vs 28%; P =
.002).
There was no correlation between responsiveness to IL-7 and initial
laboratory and clinical parameters of the 130 patients with T-ALL,
including white blood cell count, hemoglobin level, platelet count,
age, organomegaly, and mediastinal mass. Furthermore, we studied
whether the sensitivity of T-ALL blasts to IL-7 in terms of spontaneous
apoptosis inhibition might be associated with a particular
immunophenotype pattern. A significant positive correlation with the
response to IL-7 was found for CD1a (P < .05; Figure
6), surface (s)CD3 (P < .05), CD8
(P < .01), and CD4/CD8 coexpression (P < .05). An
even stronger, but negative, correlation was observed between the
expression of CD34 and IL-7 responsiveness (P < .0001; Figure
6). Blast cells exhibiting aberrant expression of the myeloid antigen
CD33 were mainly refractory to IL-7 (P < .01; Figure 6).
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| Fig 6.
Correlation of sensitivity to IL-7 in terms of inhibition
of spontaneous apoptosis (NIL-7) with expression of
surface markers in T-ALL cells.
Antigen positivity was determined by a 20% cutoff criterion that is,
a sample was considered positive for the surface antigen if more than
20% of the leukemic cells expressed fluorescence intensity greater
than 98% of the negative control cells.
|
|
In addition, we investigated the correlation between IL-7R
expression levels and immunophenotypic features associated with more
mature differentiation stages (n = 33). We observed higher IL-7R
expression levels in the T-ALL samples positive for CD1a, CD4, CD8, and
CD3 and negative for CD34 and CD33, with cortical T-ALL revealing the
highest and pro-T-ALL and pre-T-ALL revealing the lowest
expression values (mean MESF × 103, 1.1 ± 0.3 vs 1.7 ± 0.2 vs 1.4 ± 0.6 for 8, 17, and 8 patients with
pro-/pre-, cortical, and mature T-ALL, respectively). However, the
observed differences were not statistically significant (P > .05). Leukemic cell samples with extremely low levels of spontaneous apoptosis (Nspont, <5%) did not reveal any particular
phenotype or subtype (4, 3, and 2 samples of pro-/pre-, cortical, and
mature T-ALL, respectively).
Response to IL-7 correlates with a good early response to initial
therapy
Within the subgroup of patients with T-ALL showing spontaneous
apoptosis in vitro (n = 120), analysis of the available data on the
rate of early cytoreduction revealed significant differences between
IL-7-responding and IL-7-nonresponding samples. Both
parametersblast counts in peripheral blood at day 8 (r = 0.28; P = .01; n = 82) and in bone marrow at day 15 (r = 0.27; P = .04; n = 59)were negatively associated with the extent of apoptosis inhibition by IL-7
(ie, leukemic cells with higher IL-7 sensitivity were more sensitive to
the initial therapy) (Figure 7). This
correlation was observed in T-ALL patients irrespective of their
immunophenotypic features. The only exception was the
TcR/ + T-ALL subgroup, who had highly variable but
IL-7-independent responses to initial therapy (r = 0.03; P
= .9; n = 13). Interestingly, the exclusion of
TcR/+ T-ALL from the analysis considerably improved
the correlation parameters (r = 0.38 and P =
.001; r = 0.37 and P = .009; day 8 and day 15, respectively). Similarly, a statistically significant difference was
observed between T-ALL patients classified as good and poor responders
to prednisone (cut-off criterion, >1000 leukemic blasts/µL or
1000 leukemic blasts/µL in peripheral blood at day 8) when patients
with TcR/+ T-ALL were not considered (Table
3).
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| Fig 7.
Correlation of IL-7-specific apoptosis inhibition
(NIL-7) with the percentage of leukemic blasts at day 8 in peripheral blood (A) and at day 15 in bone marrow (B).
Blast cell counts inversely correlated with the extent of apoptosis
inhibition.
|
|
Notably, the small group of patients with T-ALL who did not show
spontaneous apoptosis in vitro revealed a considerably higher, though
not a statistically significant, mean number of blast counts (%) on
day 8 (results available in 5 of 10 patients) than patients with T-ALL
who exhibited spontaneous apoptosis (33 ± 15 vs 22 ± 3, respectively). Within the latter group of patients with T-ALL, however,
no correlation was found between the extent of spontaneous apoptosis
and blast counts on day 8. With respect to blast counts on day 15, a
tendency to a negative correlation with the extent of spontaneous
apoptosis was observed (r = 0.24; P = .076; n = 59). Analysis of constitutive expression levels of Bcl-2 and Bax in
T-ALL patients tested (n = 45) also did not reveal a significant correlation with early cytoreduction parameters, though Bcl-2 levels
showed a tendency to a positive correlation with blast counts on days 8 and 15 (mean values, r = 0.20 and P = .17).
Interestingly, in the multivariate analysis of apoptosis-related
parameters of T-ALL patients tested in this study (ie, extent of
spontaneous apoptosis, the extent of apoptosis inhibition by IL-7, and
the expression levels of Bcl-2 and Bax), only IL-7 responsiveness revealed an independent impact on early cytoreduction and prednisone response parameters (P < .05).
| |
Discussion |
In the current study, T-ALL cells from pediatric patients were
characterized according to their cytokine responsiveness in terms of
apoptosis modulation in vitro. IL-7, but not IL-2 or IL-4, was found to
be a highly potent inhibitor of apoptosis in T-ALL blasts. The extent
of apoptosis inhibition by IL-7 correlated with the expression levels
of IL-7R and was strongly associated with an up-regulation of Bcl-2.
Most interesting, the classification of a large series of T-ALL cells
according to their in vitro IL-7 responsiveness revealed significant
differences between IL-7-sensitive and IL-7-refractive leukemic
samples with respect to the expression of maturation-associated and
myeloid markers and early responses to therapy.
In normal T-cell development, thymocyte responsiveness to cytokines has
been found to depend strongly on thymocyte maturational stage (reviewed
in Zlotnik and Moore3). Although thymocytes at the most
immature pro-T-cell stage of intrathymic development selectively
respond to IL-7 but not to IL-2 or IL-4,27 they lose the
ability to respond to cytokines after entering a cortical, double-positive (CD4+CD8+) differentiation
stage.28 After positive and negative selection, mature
single-positive (CD4+CD8 or
CD4 CD8+) thymocytes not only regain
their IL-7-responsiveness but acquire the ability to respond to IL-2
and IL-4.3 Therefore, our findings demonstrating selective
responsiveness to IL-7 in approximately two thirds of patients suggest
that the cytokine responsiveness profile in T-ALL largely corresponds
to that of pro-T thymocytes.
Cytokines have been shown able not only to inhibit apoptosis but also
to induce it.1 IL-2 and IL-4, but not IL-7, were reported
to induce apoptosis in thymocytes29,30 and activated T
cells.31 Highly cytotoxic effects caused by the induction of apoptosis by IL-4 were found in B-lineage ALL.32,33 In
our study, we observed, albeit rarely, an induction of apoptosis in T-ALL blasts by IL-2 and IL-4, but not by IL-7 (2 cases for IL-4 and 1 case for IL-2).
Although pathways of cytokine-mediated inhibition of apoptosis are
poorly understood, there is abundant evidence that members of the Bcl-2
family are involved in the regulation of these processes. Overexpression of Bcl-2 delays cytokine deprivation-triggered apoptosis
in growth factor-dependent cell lines, whereas overexpression of Bax
induces or enhances spontaneous cell death without additional factors.34-36 Studies on the constitutive expression of
Bcl-2 and Bax in fresh leukemic cells reveal correlations between the
extent of spontaneous apoptosis and the expression levels of these
proteins in acute myeloid leukemia (AML), B-lineage ALL, and chronic
lymphocytic leukemia.25,32,37-39 However, we did not
observe clear-cut correlations between Bcl-2 expression levels and the
extent of spontaneous apoptosis in a relatively large series of ALL (n = 95)24 and AML (n = 100)40 patients. In the
current study, the constitutive expression of Bcl-2 and Bax did not
correlate with the extent of either spontaneous or IL-7 modulated
apoptosis in T-ALL. Recently, changes of expression levels after cell
treatment rather than constitutive expression levels of untreated cells
have been suggested to be associated with the apoptosis-regulative
function of Bcl-2 family members.41 A correlation between
increased levels of Bcl-2 and cytokine-induced inhibition of apoptosis
was observed in IL-7-treated mouse normal pro-T cells, malignant
T-lymphoma cells, and human activated T
lymphocytes.11,12,42-44 Apoptosis-associated up-regulation
of Bax and its prevention by IL-7 was reported for pro-T
cells.12 However, Bax expression levels did not change in
human activated T cells42 and in mouse malignant T-lymphoma cells.44 In the current study, we found that expression
changes of Bax, but not of Bcl-2, were positively correlated with the extent of spontaneous apoptosis in T-ALL. By contrast, IL-7-induced effects were strongly correlated with changes of Bcl-2 (P < .0001) but not Bax expression levels. Interestingly, consideration of the Bcl-2/Bax ratios, which have been shown to better predict apoptosis-related events in several but not all cell
systems,45-47 showed a weak influence on the correlation
parameters found for Bcl-2 alone. Therefore, our data suggest
differential involvement of Bcl-2 and Bax in the induction of
spontaneous apoptosis in vitro and its modulation by IL-7 in
T-ALL.
The specific Bcl-2-dependent role of IL-7 as an anti-apoptotic factor
in T-ALL in vitro resembles its crucial role in the normal early T-cell
development. Mice lacking IL-7 or IL-7R genes, in contrast to IL-2-
and IL-4-deficient mice, have been found to be severely deficient in
thymocyte development.8,9 Analysis of thymocytes from
IL-7-deficient mice disclosed their developmental arrest at the
triple-negative (CD3, CD4, and
CD8) stages.9 Freshly isolated
triple-negative cells from IL-7-deficient mice showed spontaneous
apoptosis in vitro, which could be inhibited by the addition of
IL-7.48 Because Bcl-2-deficient mice also revealed
impaired development of lymphoid lineages49 and Bcl-2 overexpression in IL-7-deficient mice largely restored the maturation of thymocytes,11 it has been suggested that IL-7 exerts its anti-apoptotic function by up-regulating or maintaining Bcl-2 levels in
pro-T cells.48 Interestingly, the IL-7-specific inhibition of spontaneous apoptosis in vitro has not been accompanied by changes
of cell cycle distribution in the subpopulation of isolated pro-T
cells, as demonstrated by flow cytometry.12 Based on these data, Kim et al12 suggested IL-7 to be a trophic rather
than a growth factor in early T-cell development. Similarly, our flow cytometric analysis, which showed that IL-7 was not able to affect cell
cycle distribution in T-ALL samples, points to the role of IL-7 as
survival or trophic, but not as proliferative, at least for the bulk of
T-ALL cells.
Comparison of the IL-7 response in vitro with the clinical response in
vivo revealed a better prednisone response and early cytoreduction of
IL-7 responding cells. Mechanisms underlying this correlation are
unclear. The extent of spontaneous apoptosis, though significantly
increased in IL-7-sensitive cells, did not reveal a statistically
significant impact on early cytoreduction in univariate analysis. The
latter observation is in line with studies in pediatric patients with
ALL that did not find a correlation between the extent of spontaneous
apoptosis and response to initial therapy.50 In contrast to
ALL, several studies in AML demonstrated the predictive value of the
extent of spontaneous apoptosis.40,51 We also did not
observe a predictive significance of Bcl-2 expression levels, though
the observed tendency to a positive correlation with blast counts is in
line with previous reports on Bcl-2 in T-ALL.52 Moreover, a
multivariate analysis of the investigated apoptosis-related parameters,
including spontaneous apoptosis, its inhibition by IL-7, and expression
levels of Bcl-2 and Bax, showed that only IL-7 responsiveness has an
independent impact on early cytoreduction (P < .05),
indicating a potential prognostic relevance of IL-7 sensitivity in
T-ALL.
Spontaneous death of leukemic cells observed in vitro might be in part
caused by a deprivation of humoral and cellular factors capable of
protecting these cells from apoptosis in vivo. Because glucocorticoids
are known to exert powerful anti-inflammatory effects from the
suppression of cytokine gene expression,53 prednisone
treatment in vivo might result in the overall down-regulation of
cytokine production, including leukemia survival factors. In survival
factor-dependent leukemic cells, this effect would result in the
induction of apoptosis by cytokine deprivation and might account for
the observed correlation between IL-7 effects in vitro and early
cytoreduction in vivo. This effect of apoptosis induction by the
down-regulation of survival factors would represent an antileukemic
mechanism that is in addition to the well-known effect of apoptosis
induction by glucocorticoids and other therapeutic drugs directly in
target cells.54,55
The nature of survival factors in T-ALL remains largely unknown, though
our data point to the putative role of IL-7 as a T-ALL survival factor.
Further studies, including the investigation of patients' IL-7 serum
levels and the application of cytokine blocking antibodies during in
vitro cultivation of T-ALL blasts with autologous serum or stroma
cells, are necessary to clarify the role of IL-7.
Our analysis of IL-7 response of T-ALL cells in the context of their
phenotypic features revealed an association between IL-7 unresponsiveness and an immature phenotype characterized by the expression of CD34 and CD33. Conversely, IL-7-sensitive cells were
found to express preferentially cortical and mature T-cell antigens.
These correlations point to IL-7 responsiveness as a factor that could
be involved in differentiation and maturation processes of leukemic T
cells. Because leukemic cells are thought to originate from either
transformed multipotential stem cells or from lineage-restricted
progenitor cells,13,56,57 IL-7-refractive T-ALL cells
could arise from relatively immature pluripotent progenitor cells,
whereas IL-7-sensitive blasts might originate from more differentiated
hematopoietic progenitors already committed to the lymphoid lineage.
IL-7R expression revealed only a tendency to correlate with
immunophenotype and, therefore, could not account for the observed correlation between IL-7 responsiveness and immunophenotype. Moreover, considerable variability in IL-7 responsiveness, observed in T-ALL samples with similar levels of receptor expression (Figure 2), indicates a complex regulation of signal transduction pathways in
leukemic blasts. Although molecular pathways of cytokine signaling and
their influence on spontaneous apoptosis are poorly understood, the
early signaling events triggered by engagement of the IL-7 receptor
have been shown to be intimately linked to a functional tyrosine-specific protein kinase pathway and to involve stimulation of
the inositol phospholipid turnover in human fetal thymocytes as well as
in T-lineage ALL blasts.14,58 Numerous transcription factors are subsequently activated, including Stat5 and
c-myc.59,60 It would be of interest to analyze
whether potential defects in these signal transduction pathways are
involved in the differential responsiveness of T-ALL cells to IL-7.
Normal TcR/+ cells represent a distinct T-cell
population with respect to requirement for IL-7.61
IL-7R-mediated signaling has been shown to be crucial for normal
TcR/+ cell development and to be responsible for the
rearrangement of TcR genes.62 However, because Bcl-2
overexpression has not been associated with the rescue of
TcR/+ T cells in IL-7R-deficient mice, it has been
suggested that IL-7 is involved in the generation rather than the
survival of TcR/+ cells.11 Interestingly,
our examination of IL-7 response in TcR/+ T-ALL, a
rare subgroup accounting for approximately 15% of T-ALL,63 did not reveal any correlation between IL-7 response and therapy response, indicating that IL-7 may be irrelevant in this subgroup as a
survival factor in vivo.
Maturational stage of leukemic T lymphoblasts has been suggested as a
means for subgrouping patients with T-lineage ALL (reviewed in Uckun et
al64). In the ALL-BFM 86 trial, children with
CD1a-positive cortical immunophenotypes disclosed significantly better
in vivo responses to prednisone and longer durations of event-free
survival than those with immature (pro-/pre-T) or mature
sCD3+ CD1a phenotypes.65 A
detailed update of 316 children enrolled in the ALL-BFM 86 and the
ALL-BFM 90 trials66 and recent data from the German COALL
multicenter studies67 confirmed the better in vivo
responses to prednisone and the more favorable outcomes of patients
with cortical T-ALL compared with other immunophenotypic subsets. This
indicates pharmacologic differences in responsiveness to cytotoxic
treatment among T-ALL maturational subgroups. In view of these
findings, our data on the correlation between IL-7 sensitivity and
immunophenotype suggest that the susceptibility of leukemic blasts to
apoptosis modulation by IL-7 may represent a valuable in vitro
parameter reflecting distinct developmental stages of T-ALL, with
differing accessibility to apoptotic programs. Furthermore, early
cytoreduction in vivo, which has been shown to be an independent
prognostic factor in pediatric patients with ALL,66,68,69
revealed a quantitative correlation with the extent of IL-7-mediated
apoptosis inhibition in vitro, thus suggesting that the classification
of T-ALL according to IL-7 sensitivity may have prognostic
significance. However, studies with larger numbers of
patients and longer follow-up times are obviously needed to elucidate
the predictive value of this parameter and its impact on the event-free
and overall survival in childhood T-ALL.
In conclusion, our results show that IL-7, but not IL-2 or IL-4, is a
potent inhibitor of spontaneous apoptosis in T-ALL. Similar to the
recently described effects of IL-7 in pro-T cells during normal T-cell
development,11,48 the IL-7-specific anti-apoptotic signaling in T-ALL blasts was associated with an up-regulation of Bcl-2
expression. From a clinical point of view, our findings demonstrating a
correlation between IL-7-induced apoptosis inhibition in vitro and
immunophenotype, and the early response to therapy in vivo, suggest
that IL-7 responsiveness may be a useful surrogate marker reflecting
differential survival factor dependence, apoptosis regulation, and
treatment response in T-ALL.
| |
Acknowledgments |
We thank Karin Ganzel, Mathilde Martin, Karin Liebezeit, and Grit
Czerwony for their valuable technical assistance, and we thank all the
clinicians who provided samples for our investigations.
| |
Footnotes |
Submitted July 27, 1999; accepted March 2, 2000.
Supported in part by the Deutsche
Leukämie-Forschungshilfe (grant no. DLFH-98.04) and by the
Deutsche Jose Carreras Leukämie-Stiftung (grant no.
DJCLS-98/NAT-3).
L.K. and V.R. contributed equally to this work.
Reprints: Leonid Karawajew, Robert-Rössle
Clinic, Humboldt University Berlin, Lindenberger Weg 80, D-13125
Berlin, Germany; e-mail: karawajew{at}rrk-berlin.de.
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.
| |
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Abstract
Introduction