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Blood, Vol. 95 No. 7 (April 1), 2000:
pp. 2198-2203
PLENARY PAPER
Selective elimination of leukemic CD34+ progenitor
cells by cytotoxic T lymphocytes specific for WT1
Liquan Gao,
Ilaria Bellantuono,
Annika Elsässer,
Stephen B. Marley,
Myrtle Y. Gordon,
John M. Goldman, and
Hans J. Stauss
From the Departments of Immunology and Hematology, Imperial School
of Medicine, Hammersmith Hospital, London, UK.
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Abstract |
Hematologic malignancies such as acute and chronic myeloid leukemia
are characterized by the malignant transformation of immature CD34+ progenitor cells. Transformation is associated with
elevated expression of the Wilm's tumor gene encoded transcription
factor (WT1). Here we demonstrate that WT1 can serve as a target for cytotoxic T lymphocytes (CTL) with exquisite specificity for
leukemic progenitor cells. HLA-A0201- restricted CTL specific
for WT1 kill leukemia cell lines and inhibit colony formation by
transformed CD34+ progenitor cells isolated from patients
with chronic myeloid leukemia (CML), whereas colony formation by normal
CD34+ progenitor cells is unaffected. Thus, the
tissue-specific transcription factor WT1 is an ideal target for
CTL-mediated purging of leukemic progenitor cells in vitro and for
antigen-specific therapy of leukemia and other WT1-expressing
malignancies in vivo.
(Blood. 2000;95:2198-2203)
© 2000 by The American Society of Hematology.
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Introduction |
Cells of the hematopoietic system are derived from
hematopoietic stem cells (HSC) capable of self-renewal and
differentiation. Transplantation experiments in humans and mice
have shown that CD34+ cell populations contain HSC
capable of reconstituting the erythroid, myeloid, and lymphoid
lineages in myeloablated recipients.1 In addition,
HSC capable of reconstituting murine hosts were recently demonstrated in a rare population of
CD34 /lin bone marrow
cells.2
There is strong evidence that the critical transformation events in CML
and acute myeloid leukemia (AML) affect immature CD34+
progenitor cells. Because the majority of leukemic blast cells have
limited proliferative capacity, the malignant disease must be
maintained by a subpopulation of leukemic progenitor cells with
extensive proliferative and self-renewal capacities.3,4 Transplantation studies with purified cells from patients with AML
showed that only immature CD34+ cells were capable of
initiating leukemia in immunocompromised murine
recipients.5 Similarly, purified CD34+ cells
from patients with CML efficiently initiated leukemia in murine
recipients.6,7
The molecular events leading to uncontrolled progenitor cell
proliferation are not fully understood. Although BCR/ABL
fusion proteins associated with the t(9;22) chromosomal translocation is the hallmark of CML, BCR/ABL transcripts can also be found in
healthy persons, indicating that additional factors are required for
leukemia to develop.8 The Wilm's tumor gene (WT1)
transcription factor is a candidate protein contributing to
leukemogenesis. This transcription factor is normally expressed in
immature CD34+ progenitor cells, and differentiation is
associated with WT1 down-regulation.9,10 Elevated levels of
WT1 expression have been observed in unseparated mononuclear cells and
in purified CD34+ cells from patients with AML and
CML.11,12 In vitro studies showing that increased WT1
expression can block normal differentiation and enhance proliferation
of hematopoietic progenitor cells provide an explanation for the
potential of WT1 to contribute to leukemogenesis.13,14
The results of a recent study suggested that T lymphocytes specific for
CD34+ progenitor cells are critically important in
mediating antileukemic effects in patients with CML.15
However, there is no information concerning the nature of T-cell
recognized target antigens expressed by CD34+ cells. In
this study we explored the possibility of exploiting WT1 as a target
molecule to direct cytotoxic T lymphocytes against leukemic progenitor
cells. We tested the hypothesis that CML, but not normal
CD34+ progenitor cells, express sufficient WT1 protein to
trigger a cytotoxic T lymphocyte (CTL) attack.
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Materials and methods |
Cell lines
The K562 cell line was established from the pleural effusion of a
female patient with CML in blast crisis.16 The BV173 cell line was established from the peripheral blood of a male patient with
CML in blast crisis.17 The cell line 697 was established from the bone marrow of a 12-year-old boy with acute lymphoblastic leukemia.18 The C1R cell line is a HLA-A0201-negative
Epstein-Barr virus (EBV) transformed lymphoblastoid cell
line.19 The T2 cell line has been selected for loss of the
genes encoding TAP (transporter associated with antigen processing),
resulting in inefficient loading of human leukocyte antigen
(HLA) class I molecules with endogenous
peptides.20 As a consequence, the HLA-A0201 molecules of T2
cells can be efficiently loaded with exogenous peptides. Drosophila cells transfected with HLA-A0201, human  -2
microglobulin, B7.1, and ICAM-1 were a kind gift from Dr M. Jackson.
Synthetic peptides.
A peptide derived from human Wilms tumor antigen 1 P126 (RMFPNAPYL) and
a control HLA-A02 01-binding peptide derived from the E7 protein of
human papillomavirus type 16 were synthesized by the central peptide
synthesis laboratory of the Imperial College Medical School using
fluorenylmethoxycarbonyl chemistry. The quality of the peptides was
assessed by high-performance liquid chromatography analysis, and the
expected molecular weight was observed using matrix-assisted laser
desorption mass spectrometry. The peptides were dissolved in
phosphate-buffered saline (PBS; pH 7.4) to give a concentration of 2 mmol/L and were stored at  20°C.
Generation of allo-HLA-restricted CTL lines.
Peripheral blood mononuclear cells (PBMC) were separated from buffy
coat packs using Ficoll gradient centrifugation and were stained with
monoclonal antibodies HB54 (anti-HLA-A2, B17) and HB117 (anti-HLA-A2,
A28). A2-negative PBMCs were used as responders. Peptide-coated
Drosophila cells transfected with HLA-A0201, human 2-microglobulin, B7.1, and ICAM-1 were used as initial stimulators. Drosophila cells were induced in 100 mmol/L CuSO4
for 48 hours, washed 3 times with medium, and loaded with peptide at a
concentration of 100 µmol/L for 4 hours. Each well of a 24-well plate
received 2 × 106 responder PBMC and
2 × 105 stimulator cells in 2 mL T-cell medium. On
day 5, T cells were harvested and plated in fresh T-cell medium at a
density of 5 × 105 per well, with the addition of
2 × 106 autologous irradiated PBMC as feeders,
2 × 105 irradiated peptide-coated T2 cells, 10%
QS4120 culture supernatant (containing anti-CD4 antibodies), and 10 U/ml hu-rIL-2 (Roche Diagnostics, Lewes, UK). The cultures
were restimulated weekly using T2 cells coated with the immunizing
peptide as stimulators. After 2 to 3 cycles of stimulation, the bulk
cultures were cloned in 96-well plates at densities of 1, 10, and 30 cells per well; 104 peptide-coated T2 cells,
2 × 105 HLA-A2 negative PBMC feeders, and 2 U/mL
IL-2 were added to each well. The cytotoxicity of each well was tested
against T2 target cells coated with the immunizing peptide or a control
HLA-A0201-binding peptide. Peptide-specific microcultures were
expanded and restimulated weekly in 24-well plates by adding
2 × 106 feeders, 2 × 105
stimulator cells, and 10 U/mL IL-2. The T-cell line 77 (Figure 1B) was maintained for more than 1 year in
culture and served as a source of CTL for most experiments in
this study. Because this line consisted of CD4+
and CD8+ T cells, CD8+ subclones were used to
show that the specific killing activity was mediated by
CD8+ CTL. Unlike the parental 77 line, the in vitro
lifespan of CD8+ subclones was limited to a few months.
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| Fig 1.
Specificity of allo-restricted CTL generated against the
WT1-derived peptide P126.
CTL were isolated by limiting dilution cloning of T-lymphocyte bulk
cultures from HLA-A0201 donors stimulated with
HLA-A0201+ stimulator cells coated with P126 peptide. (A)
Isolated CTL lines killed the TAP-deficient T2 target cells coated with
the immunizing P126 peptide but not T2 cells coated with the
HLA-A0201-binding E7 control peptide. (B) Peptide titration
experiments showing that 3 anti-P126 CTL lines were of high avidity
recognizing low picomolar concentrations of P126, and that 3 CTL lines
were of low avidity because nanomolar P126 concentrations were required
for target cell recognition. T2 cells coated with the indicated
concentrations of P126 were used as CTL targets. High-avidity CTL were
used for all subsequent experiments because low-avidity CTL did not
recognize target cells expressing WT1 endogenously. (C) High-avidity
CTL killed the HLA-A0201+ leukemic cell lines BV173, 697 but not the HLA-A0201+, EBV-transformed B-lymphoid cells
C1R-A2. Coating of C1R-A2 with P126 resulted in efficient CTL killing.
The HLA-A0201 leukemia cell line K562 was not killed
by the CTL unless transfected with the HLA-A0201 gene.
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CTL assays.
Cytotoxic T lymphocyte assays were performed as described. Briefly,
106 T2 cells were incubated for 1 hour in 200 µL assay
medium (RPMI 1640 with 5% heat inactivated fetal calf serum) with 100 µmol/L synthetic peptide at 37°C. Peptide-coated T2 cells or
tumor cells were labeled with chromium 51 for l hour, washed, and added
to serial 2-fold dilutions of effector cells in round-bottom, 96-well plates to obtain a total volume of 200 µL/well. Assay plates were incubated for 4 hours at 37°C, 5% CO2, and 100 µL
supernatant was harvested and counted using a Wallac Gamma Counter,
Wallac, Milton Keynes, UK. The specific lysis was
calculated by the equation (experimental release spontaneous
release)/maximum release spontaneous release) × 100%.
Purification of hematopoietic CD34+
cells.
As a source of normal CD34+ cells, we used human bone
marrow from adult healthy donors (n = 5), leukapheresis products of
stem cells mobilized from solid-tumor patients in disease remission (n = 2), and cord blood (n = 1). Samples of cord blood were
obtained from discarded placental and umbilical tissues by drainage of the blood into sterile collection tubes. Informed consent for use of
these cells was obtained from donors or parents as appropriate. As a
source of leukemic CD34+ cells, peripheral blood was
obtained from patients who had CML in chronic phase and who had not
been treated with interferon in at least 3 months.
Samples were diluted 1:2 in Hanks balanced salt solution and enriched
for mononuclear cells by density-gradient centrifugation (Lymphoprep
1.077 g/mL; Nycromed Pharma AS, Oslo, Norway), and the
recovered mononuclear fraction was subject to magnetic microbead selection for the isolation of CD34+ fraction using the
Minimacs system and following the manufacturer's instructions
(Miltenyi Biotec, Bisley, UK). The purity of the cell
population ranged from 80% to 95% as estimated by FACS analysis using
an antihuman CD34 phycoerythrin (PE) mouse monoclonal antibody (Becton
Dickinson, Cowley, UK).
RNA extraction and reverse transcription-polymerase chain reaction
(RT-PCR).
Total RNA of 106 cells was isolated according to RNAzol B
protocol (AMS Bio, Witney, UK). cDNA synthesis of whole
RNA pellet was performed in a 40µL reaction. The dissolved RNA pellet
was first incubated with 2 µg oligo-dT 12-18 primer (Life
Technologies, Paisley, UK) at 65°C for 10 minutes,
followed by a 1-hour incubation at 42°C with a mixture of 50 U
murine leukemia virus reverse transcriptase, 10 mmol/L dithiothreitol,
1 mmol/L dNTP (Roche Diagnostics), and 40 U RNase
inhibitor (Promega, Southampton, UK). Five microliters cDNA preparation was used for polymerase chain reaction (PCR) amplification in a 50-µL volume of final reaction mixture containing 2.5 U Taq DNA polymerase (Qiagen, Crawley, UK), 1 mmol/L dNTP, and 20 OD/mL primer.
Amplification of the human WT1 coding region was achieved using sense
primers located in exon 7 (21 mer 5'-ggc atc tga gac cag tga
gaa-3') and antisense primers in exon 10 (22 mer 5'-gag agt
cag act tga aag cag t-3'). The expected size for the WT1 PCR product was 482 bp. RNA integrity was verified by amplifying the human
c-ABL gene in every sample using intron-spanning primers: 22 mer sense 5'-ccc aac ctt ttc gtt gca ctg t-3'; 22 mer
antisense 5'-cgg ctc tcg gag gag acg atg a-3'. The expected
size of the c-ABL PCR product was 385 bp. Hot-start PCR was performed
for 35 cycles with a thermal cycler (Techne Genius, Cambridge, UK) under the following conditions (same for ABL and WT1 amplification): denaturing at 95°C for 1 minute, primer annealing at 56°C for 1 minute, and chain elongation at 72°C for 2 minutes. The cycling was
initiated by a 5-minute denaturation step at 95°C to heat inactivate the reverse transcriptase, and it was terminated by a
10-minute final extension at 72°C. All reverse transcription (RT)-PCRs were performed at least twice, and negative control (no cDNA)
and positive control (cDNA from the WT1-expressing leukemic cell
line BV173) were included in every experiment. PCR products were electrophoresed through 1.5% agarose gels.
Western blot analysis.
Separated CD34+ cells (2 × 105 cells)
were washed in PBS and lysed in Laemmli buffer. The cell lysate was
fractionated by a 12% sodium dodecyl sulphate-polyacrylamide gel
electrophoresis and transferred to a nitrocellulose membrane
(Amersham, Little Chalfont, UK) by wet transfer. The
membrane was then blocked in PBS containing 0.01% Tween 20 and 5%
nonfat dry milk for 1 hour at room temperature and incubated first with
rabbit antihuman WT-1 C19 polyclonal antibody (1:200 in blocker; Santa
Cruz Biotech, Santa Cruz, CA) overnight at 4°C and
then with rabbit anti-actin polyclonal serum (1:500 in blocker;
Sigma, Gillingham, UK) for 30 minutes at room temperature.
The signal was revealed by incubating the membrane with horseradish
peroxidase-conjugated swine antirabbit antibody (1:1000;
DAKO, Cambridge, UK) and ECL reaction (Amersham) according to the manufacturer's instructions.
Progenitor (CFU) assays.
Colony-forming unit (CFU) assays were performed by plating 1000 to 3000 CD34+ cells in methylcellulose medium supplemented with the
following recombinant human growth factors (Stem Cell
Technologies, Northampton, UK): stem cell factor (50 ng/mL), IL-3 (20 ng/mL), IL-6 (20 ng/mL), granulocyte macrophage
colony-stimulating factor (20 ng/mL), and granulocyte
colony-stimulating factor (20 ng/mL). The cultures were
incubated for 14 days at 37°C in humidified atmosphere at 5%
CO2 to allow the development of granulocyte macrophage
colony-forming units.
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Results |
Generation of WT1-specific CTL
Expression of the WT1 transcription factor in adults is detectable
in renal podocytes, testicular Sertoli cells, ovarian granulosa cells,
and CD34+ bone marrow cells.21 To avoid
possible immunologic tolerance to WT1, we used a previously described
approach of generating peptide-specific CTL from major
histocompatibility complex-mismatched donors. This approach is suitable
for generating CTL against any protein overexpressed in tumor cells,
independent of immunologic tolerance.22,23 A 9-amino
acid-long WT1-derived peptide epitope, P126 (RMFPNAPYL), was selected
as the CTL target because it bound to HLA-A0201 class I molecules, the
most frequent class I allele found in white populations
(not shown). Responder lymphocytes from
HLA-A0201 donors were cultured
in vitro with HLA-A0201+ stimulator cells presenting the
P126 peptide, and limiting dilution cultures were used to isolate
peptide-specific CTL lines. Experiments with peptide-coated T2 target
cells showed that the CTL were highly specific for the P126 peptide
(Figure 1A). Peptide titration indicated that the CTL could be divided
into high-avidity lines capable of recognizing low picomolar peptide
concentrations and low-avidity lines recognizing low nanomolar peptide
concentrations (Figure 1B). High-avidity CTL lines were selected for
further experiments.
WT1-specific CTL kill leukemia cell lines
Analysis of a panel of leukemia cell lines revealed that
P126-specific CTL killed the HLA-A0201+ cells BV173 and 697 (Figure 1C). The HLA-A0201 leukemia cell line K562
was only killed after transfection with HLA-A0201. In contrast, the
HLA-A0201+ EBV-transformed B cell line C1R-A2 was not
killed unless cells were coated with P126 peptides (Figure 1C; similar
results were seen with other EBV-transformed cells). The expression of
WT1 in the CTL target cells was analyzed at the RNA and protein levels. RT-PCR demonstrated that the leukemia cell lines, but not the EBV-transformed C1R-A2 cells, expressed WT1 RNA (Figure
2A). Similar results were obtained by
Western blot analysis showing that WT1 protein was only expressed in
leukemia cells but not in C1R-A2 cells (Figure 2C). Together the data
indicated that the CTL recognized A0201+ leukemia cell
lines and that CTL killing correlated with WT1 expression.
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| Fig 2.
WT1 RNA and protein expression in leukemic cell lines and
in CD34+ and CD34 cell populations freshly
isolated from patients with leukemia and normal donors.
(A) RT-PCR to measure WT1 RNA in leukemic cell lines and in the
B-lymphoid cell line C1R-A2. The same cell lines were used as CTL
targets in Figure 1C. The amplified WT1 product is 482 bp long. The RNA
of the housekeeping ABL gene was amplified to indicate the amount of
RNA in each sample. The ABL product is 385 bp long. (B) RT-PCR to
measure WT1 RNA expression in purified CD34+ and
CD34 cell populations from 4 patients with CML and 3 normal donors. The leukemic cell line BV173 served as a positive
control for WT1 expression. Similar results were obtained with samples
from 6 additional patients with CML. (C) Western blotting to measure
WT1 protein expression in leukemia cell lines and in purified
CD34+ and CD34 cell populations from 2 patients with CML and 2 normal donors. The expression of the
housekeeping actin protein was used as an indicator to control for the
amount of protein in each sample. The WT1 protein measures
approximately 54 kd and the actin protein approximately 42 kd.
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WT1-specific CTL kill fresh leukemic CD34+ cells
PBMCs from patients with chronic-phase CML were separated into
immature CD34+ and mature CD34
populations. As expected, cells isolated from
HLA-A0201 patients were not recognized by
P126-specific CTL (Figure 3A). When cells
from HLA-A0201+ patients with CML were analyzed, the CTL
selectively recognized the CD34+ cell population, whereas
no killing of the more mature CD34 population was
observed (Figure 3A). Cold target competition experiments showed that
the killing of CD34+ CML cells was inhibited by the
leukemia cell line BV173 but not by EBV-transformed C1R-A2 cells
(Figure 3B). This indicated that CD34+ cells from patients
with CML and BV173 cells share the CTL-recognized target antigen and
that this antigen is absent in C1R-A2 cells.
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| Fig 3.
Analysis of CTL-mediated killing of CD34+
cell populations purified from patients with leukemia and normal
donors.
(A) Representative experiment showing the level of killing by anti-P126
CTL against purified CD34+ and CD34 cell
populations isolated from patients with CML who were either
HLA-A0201+ or A0201.
CD34/A0201+ cells were not recognized by
the CTL unless coated with P126 peptide. The leukemic cell line BV173
and the TAP-deficient T2 cells coated with P126 or the control E7
peptide were used as positive and negative controls in all experiments.
(B) Cold target competition experiment. Shown is the killing by
anti-P126 CTL against chromium-labeled CD34+ targets from
an A0201+ patient with CML in the absence or presence of a
30-fold excess of cold BV173 and C1R-A2 targets. The killing of
chromium-labeled BV173 and C1R-A2 is shown for comparison. (C) Average
of the level of specific CTL killing of purified CD34+
cells from 11 different HLA-A0201+ patients with CML and
from 6 normal donors. The level of killing of CD34
cells purified from patients with CML and against the positive control
cells BV173 is also shown. The figure shows the mean level and standard
deviation of specific CTL killing. (D) Representative experiment
showing the level of killing by anti-P126 CTL of purified
CD34+ and CD34 cell populations isolated
from HLA-A0201+ normal donors. No CTL killing was
detectable unless target cells were coated with P126 peptide.
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Lack of killing of CD34 cells most likely resulted
from the insufficient expression of the WT1-derived target peptide
because the coating of these cells with exogenous P126 peptide resulted in CTL killing (Figure 3A). RT-PCR analysis revealed strong WT1 RNA
expression in CD34+ cells and low expression in
CD34 cells (Figure 2B). WT1 protein expression
detectable by Western blotting was restricted to CD34+, and
no protein was detectable in CD34 cells (Figure 2C).
Both RT-PCR and Western analysis showed variation in the level of WT1
expression in leukemic CD34+ cell populations. Thus, we
explored whether the observed variation in the level of WT1 expression
resulted in a variation in the level of CTL killing. However, analysis
of 11 different patients with CML showed that the CTL consistently
lysed approximately 20% (SD, 5%) of the CD34+ population
(Figure 3C). This result raised the possibility that WT1 expression was
restricted to a subpopulation of approximately 20% of
CD34+ cells and that the expression level in all 11 patients with CML was sufficient to render most of these cells
susceptible to CTL killing. We explored whether the subpopulation
recognized by CTL included the clonogenic progenitor cells that can
give rise to colonies of the granulocyte, macrophage, and erythroid
lineages. When CD34+ populations isolated from 9 patients
with CML were treated with P126-specific CTL, this resulted in 80% to
100% inhibition of colony formation (Figure
4A). This indicated that the majority of
colony-forming progenitor cells were removed by P126-specific CTL. The
"escape" colonies seen in CTL-treated samples were small when
compared to the colonies in untreated samples. This is consistent with
the possibility that the escape colonies were derived from progenitor
cells that had already initiated differentiation toward the
granulocyte/myeloid lineage associated with the down-regulation of WT1
expression. Such partially differentiated progenitors might escape
recognition by WT1-specific CTL, and the small size of the colonies
observed in the CFU assay might reflect the reduced clonal burst size
of these progenitors.
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| Fig 4.
Analysis of CTL-mediated inhibition of colony formation
of CD34+ cell populations purified from patients with
leukemia and normal donors.
(A) CTL-mediated inhibition of colony formation by purified
CD34+ cells cocultured for 4 hours with CTL at an
effector/target cell ratio of 10:1. Untreated control CD34+
cells were cultured under the same conditions without CTL. CTL treated
and untreated control cells were then plated in methylcellulose, and
after 14 days the numbers of granulocyte macrophage colony-forming
units (GM-CFU) were counted. Shown is the percentage of GM-CFU after
CTL treatment using the GM-CFU observed in the untreated controls as
100% reference. This figure shows the mean and standard deviation of
independent experiments with CD34+ cells from 9 HLA-A0201+ patients with CML and 7 normal donors and with
CD34+ cells from 5 HLA-A0201 patients
with CML. (B) Colony formation by A0201+/CD34+
CML cells that were untreated or treated for 4 hours with high-avidity
P126-specific CTL (line 81) or with low-avidity CTL (line 85) before
plating. Shown are GM-CFU and BFU-E using the number of colonies
observed in the untreated controls as 100% reference.
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Importantly, colony formation by CD34+ cells from
HLA-A0201 patients with CML was unaffected,
indicating that the elimination of progenitors with colony-forming
activity was dependent on HLA-restricted antigen recognition by the
CTL. Furthermore, the elimination of colonies of the granulocyte,
macrophage, and erythroid lineages in CD34+ cells from
HLA-A0201+ patients with CML was
dependent on high-avidity recognition of the P126 peptide. Only
high-avidity CTL eliminated the progenitors of CFU-GM and BFU-E,
whereas low-avidity CTL had no effect (Figure 4B).
Finally, we explored whether high-avidity CTL discriminated between
leukemic and normal CD34+ progenitor cells. Normal
CD34+ cells were isolated from bone marrow, peripheral
blood, or umbilical cord blood of HLA-A0201+ donors and
were used as CTL targets. Independent of the source of
CD34+ cells, anti-P126 CTL did not inhibit colony formation
by these cells (Figure 4A). Furthermore, normal CD34+ cells
were not killed when used as targets in cytotoxicity assays (Figures 3C
and 3D). The selective CTL killing of leukemic versus normal
CD34+ cells can be explained by differences in WT1
expression. WT1 RNA expression was higher in leukemic than in normal
CD34+ cells (Figure 2B), and Western blot analysis detected
WT1 protein only in leukemic but not in normal CD34+ cell
populations (Figure 2C).
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Discussion |
The infusion of donor lymphocytes to patients in relapse after
previous allogeneic stem cell transplantation can engender strong
graft-versus-leukemia effects.24,25 A recent study showed that complete remission in patients with CML undergoing donor lymphocyte infusion was associated with an increased frequency of T
cells recognizing leukemic CD34+ progenitor
cells.15 In contrast, T-cell recognition of more mature
CD34 cells in patients with CML was not associated
with a favorable clinical response. This suggested that T lymphocytes
with specificity for CD34+ CML progenitor cells were
critically important in mediating antileukemic effects in vivo.
However, there is no information concerning the nature of target
antigens that can direct T-cell responses selectively against leukemic
CD34+ progenitor cells.
To identify such antigens, we used the allo-restricted CTL approach
that, independent of immunologic tolerance, is suitable for raising CTL
against any protein expressed at elevated levels in transformed
cells.22,23,26 Because the triggering of the cytotoxic
effector function is a threshold phenomenon, it is possible to select
CTL that are only triggered by elevated target protein levels in
transformed cells but not by physiological levels of protein in normal
cells.23
The transcription factor WT1 was chosen as a candidate target protein
for several reasons. There is evidence of overexpression in leukemic
CD34+ cells,12 elevated WT1 expression can
contribute to transformation,13,14 normal WT1 expression is
restricted to a small number of cells in postnatal life,21
and, in addition to leukemia, elevated WT1 expression has been observed
in renal cell carcinoma, ovarian cancer, advanced breast cancer, and
melanoma.27-30 Therefore, CTL selectively recognizing WT1
overexpressing malignant cells are invaluable reagents for
antigen-specific therapy of leukemia and other more common
malignancies. Furthermore, tumor escape by down-regulation of WT1
expression is unlikely to occur if overexpression is required to
maintain the transformed phenotype.31-33
The allo-restricted CTL described here were isolated from
HLA-A0201 donors, and they were specific for
leukemic progenitor cells presenting the WT1-derived P126 peptide in
the context of HLA-A0201 class I molecules. The P126 peptide was highly
immunogenic because in vitro stimulation of lymphocytes from different
HLA-A0201 donors consistently induced
peptide-specific, HLA-A0201-restricted CTL. Therefore, P126-specific
CTL are novel reagents for antigen-specific therapy of
HLA-A0201+ patients with leukemia undergoing stem cell
transplantation from donors displaying a 1-locus HLA-mismatch involving
the HLA-A0201 allele. A 1-locus HLA mismatch is clinically acceptable,
as demonstrated in a recent study showing comparable prognoses in
patients with leukemia receiving transplants from 1-locus-mismatched
and HLA-matched unrelated donors.34 Thus, a
1-locus-mismatch transplant provides an ideal setting for
antigen-specific therapy with allo-restricted CTL derived from the
donor. The in vitro stimulation protocol described here, in combination
with the selection of relevant CTL by staining with HLA-A0201 tetramers
containing P126 peptides, will allow rapid isolation of P126-specific
CTL for adoptive therapy.
In addition, it is possible that WT1 can be exploited for
antigen-specific therapy in the autologous setting. This is supported by our observation that P126-specific CTL can be isolated from HLA-A0201+ donors (unpublished data). Because WT1
expression in adults is restricted to a relatively small number of
cells (eg, CD34+ bone marrow cells, renal podocytes,
testicular Sertoli cells, and ovarian granulosa cells), tolerance of
autologous T lymphocytes to WT1 is probably incomplete. Therefore, it
may be possible to exploit the identified P126 epitope for the design
of anti-WT1 vaccine preparations aimed at stimulating CTL responses
against leukemia and other malignancies with elevated WT1 expression, such as renal cell carcinoma, ovarian cancer, melanoma, and
breast cancer.
In addition to in vivo therapy, the WT1-specific CTL provides a tool
for in vitro purging of autologous bone marrow cells harvested from
patients with leukemia. The CTL removed leukemic progenitors of the
granulocyte/macrophage lineage (Figure 4A) and also progenitors of the
erythroid lineage (Figure 4B). In contrast, the CTL did not recognize
normal progenitors of the 3 lineages. The selective removal of
transformed CD34+ progenitor cells should reduce the risk
for reinfusing leukemic progenitor cells, thus overcoming a
major limitation of autologous stem cell
transplantation.35
To date, tissue-specific minor histocompatibility antigens and
lineage-specific antigens, such as proteinase 3, have been studied as
potential targets for leukemia-reactive CTL.36-39 The WT-1
transcription factor is the first target antigen capable of directing
CTL responses selectively against leukemic progenitor cells.
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Acknowledgments |
We thank Drs E. Simpson and R. I. Lechler for critically reading the
manuscript and Dr F. Dazzi and F. Grant for useful discussions and
practical help. We also thank Prof Fisk and Dr C. Campagnoli for help
with cord blood samples and Robert J. Davidson for excellent technical support.
| |
Footnotes |
Submitted June 30, 1999; accepted November 30, 1999.
Supported by a project and program grant from the Leukemia Research Fund.
L.G. and I.B. contributed equally to this work.
Reprints: Hans J. Stauss, Department of Immunology, Imperial
College School of Medicine, Hammersmith Hospital, Du Cane Road, London
W12 0NN, UK; e-mail: h.stauss{at}ic.ac.uk.
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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Top
Abstract
Introduction