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Blood, Vol. 94 No. 3 (August 1), 1999:
pp. 1038-1045
By
From the Department of Immunohematology and Bloodbank, Leiden
University Medical Center, Leiden, The Netherlands.
Peptides corresponding to the fusion site in 210 kD BCR-ABL protein
b3a2 (p210b3a2) were previously shown to bind to several HLA class I
and II alleles. We have found that b3a2 peptide-specific CD4-positive
T-helper cells were able to recognize p210b3a2-positive chronic
myelogenous leukemia (CML) blasts in a DR4 restricted manner. Until
now, there were no reports of b2a2 breakpoint-specific human T-cell
responses. Here we show that repetitive stimulation of T lymphocytes
with a 17mer peptide covering the fusion region in p210b2a2 also leads
to specific T-cell responses. CD4 and CD4/CD8 double-positive clones
obtained from a b2a2 peptide-specific cell line were cytotoxic and
proliferative in an HLA-DR2a (DRB5*0101) restricted fashion. Autologous
Epstein-Barr virus (EBV) transformed cells, expressing
BCR-ABLb2a2 on transfection, and allogeneic HLA-DR matched
p210b2a2-positive cells from CML patients were, however, not lysed.
BCR-ABL peptide-specific T-cell clones did respond to autologous EBV
cells transfected with invariant chain (li) cDNA in which the HLA class
II-associated invariant chain peptide (CLIP) was replaced by a BCR-ABL
b2a2 fusion oligonucleotide sequence, illustrating the potential of these T cells to recognize an endogenous BCR-ABLb2a2 ligand.
THE CLASSICAL t(9;22) translocation in
leukemic cells from patients with chronic myelogenous leukemia (CML)
results in a fusion gene known as BCR-ABL. The breakpoint on chromosome 9 is nearly always located upstream of abl exon a2,1
whereas the breakpoint on chromosome 22 is usually located either
between bcr exons b2 and b3 or between exons b3 and b4.2,3
The differently fused genes encode principally two variant 210 kD
proteins: p210b2a2 or p210b3a2.4 Alternative splicing of
BCR-ABL mRNA can result in coexpression of oncoproteins p210b3a2 and/or
p210b2a24,5 with p190e1a2.6,7 BCR-ABL
oncoprotein-positive cells, which express a novel protein harboring
nonself and possibly antigenic peptides at the fusion site, may be
targets for appropriate cells of the immune system. The junction region
aa sequences in p210BCR-ABL, being well-characterized
antigens expressed by leukemic cells, can be considered of potential
importance in an immunotherapeutic approach of CML. In approximately
two thirds of adult CML patients, the b2a2 protein (in combination with
p210b3a2 and/or p190e1a2) is detected.6-13 Thus far,
however, only peptides derived from the p210b3a2 protein have been
shown to bind to several major histocompatibility complex (MHC) class
I14-16 and class II molecules17,18 and
peptide-specific human T lymphocytes have been generated only against
b3a2 sequences.15-23 Although a 23mer b2a2 peptide appeared to bind with intermediate affinity to HLA DR3,17 no T-cell
responses to the b2a2 breakpoint sequence have been described. In this
report, we show that in vitro immunization with a 17 aa peptide
representing the p210b2a2 fusion region resulted in HLA-DRB5*0101
restricted peptide-specific cytotoxic and/or proliferative
CD4+ T-cell clones. In view of the predominant DR
positivity of BCR-ABL-positive CML progenitors24,25 and
the observation of very low numbers of BCR-ABL transcripts in healthy
individuals,26,27 BCR-ABL breakpoint-specific DR restricted
T cells may play an important role in CML remission-induction after
transplantation and in immunosurveillance. Such T cells can be directed
toward the b3a2 and, as shown by our data, the b2a2 breakpoint sequence.
This study was approved by a local ethical committee. Blood samples
were collected from healthy donors after informed consent only.
Preparation of BCR-ABLb2a2-specific T-cell line, P-b2,
and clones.
Blood was obtained at regular intervals from a healthy male donor P,
blood group A, Rhesus D-negative, with HLA type: A2; B7, B44; Cw5, Cw7;
DR2(DRB1*1501/DRB5*0101); DR4(DRB1*0401/DRB4*0101); DQ6, DQ8; DPB*0201,
DPB*0401. T-cell line, P-b2, was prepared from peripheral blood
mononuclear cells (PBMC) after incubation with 17mer p210b2a2
breakpoint peptide as described earlier19,21 with some
modifications. A total of 2 to 3 × 107 PBMC were
isolated from whole blood using Ficoll-amidotrizoate centrifugation
and, initially, cultured in 20 mL tissue culture flasks. Tissue culture
medium (Iscove's modified Dulbecco's modified Eagle's medium
[DMEM]; GIBCO-Life Technologies, Paisley, UK), supplemented with 10% autologous heat-inactivated serum, was partially renewed every 5 to 10 days. Final 17mer b2a2 peptide concentration was
maintained at 10 µg/mL (approximately 6 nmol/mL). A total of
107 autologous nonirradiated PBMC were added every 9 to 10 days until a proliferative response, as assessed by increasing T-cell
numbers, was seen. In case of a peptide-dependent T-cell
response, as evidenced in a proliferation assay using autologous
irradiated PBMC as antigen-presenting cells (APC),
restimulation with 30 Gy irradiated autologous PBMC was continued for
at least 40 days. Cultures were restimulated thereafter with allogeneic
pooled 30 Gy irradiated PBMC from five healthy donors mixed in a
4:1 ratio with 50 Gy irradiated B-lymphoblastoid cell lines
(B-LCL), prepared from Epstein-Barr virus (EBV)
transformed B cells from the T-cell donor, and peptide.
Peptide-specific T cells were cloned in round bottomed 96-well plates
(Costar Co, Cambridge, MA) from T-cell line, P-b2, by limiting dilution
using autologous irradiated B-LCL and allogeneic pooled irradiated PBMC as APC/feeder population. The culture medium was supplemented with 10%
heat inactivated pooled human serum (HPS) instead of autologous serum.
Ten percent T-cell growth factor (TCGF; Lymphocult; Biotest, Dreieich,
Germany) was added 48 hours after restimulation with APC and peptide.
Clones were maintained in 96-well plates and stimulated weekly with the
APC/feeder mixture, peptide, and TCGF as described.
Peptides.
Amino acid sequences of the p210b2a2 and p210b3a2 fusion region were
used to design BCR-ABL breakpoint representing 17mer length peptides
(b2a2: IPLTINKEEALQRPVAS; b3a2: ATGFKQSSKALQRPVAS),2,3 as
well as 15 b2a2 peptide analogs with single aa substitutions to
alanine. Peptides were synthesized on an Abimed 422 multiple peptide
synthesizer (Abimed, Langenfeld, Germany) at 10 µmol scale. TentagelS
AC resins28 were used in combination with Fmoc-protected amino acids applying a sixfold excess amino acid using
benzotriazole-1-yl-oxy-tris-(dimethylamino) phosphoniumhexafluorophosphate/N-methylmorpholine activation in N-methylpyrrolidone.29 After cleavage in trifluoroacetic
acid (TFA)/water, peptides were isolated and purified by repeated ether precipitations and analyzed by reversed phase high-performance liquid
chromatography (HPLC) on a 5C18 Lichrospher RP Select B column (Merck, Darmstadt, Germany), amino acid sequencing and/or mass
spectrometry. Peptides were stored at Proliferation assay.
Proliferation tests were performed using a standard
3H-thymidine incorporation assay as
described.19 Briefly, 105 30 Gy
irradiated APC were incubated with 2.5 to 5·104 responder
cells (in round-bottomed microtiter wells, Costar Co) in 150 µL
culture medium with 15% heat-inactivated serum autologous to the
responder cell donor. After 48 hours, 1 µCi 3H-thymidine
was added for another 15 hours before harvesting (Microcell harvester;
Skatron Co, Tranby, Norway) and counting (1205 Betaplate counter; Wallac Oy, Turku, Finland). All tests were repeated on separate occasions. Results are expressed as the mean (plus the standard deviation) of counts per minute (cpm) from triplicate experiments or as the proliferation index:
Cytotoxicity.
Target cells were labeled with 100 µCi
51chromium/106 cells for 1 hour at 37°C.
Labeled cells were washed and, when appropriate, pulsed with relevant
peptide for 1 hour. The effector cells were mixed at various effector
to target ratios with target cells for 4 hours at 37°C, after which
cytoxicity was determined by calculating the percent specific lysis
from counts of 51Cr released:
MHC restriction testing. Functional HLA class I and HLA-DR blocking experiments were performed as described21 using murine monoclonal antibody (MoAb) W6/32 and murine MoAb B8.11.230 in proliferation or MoAb anti-HLA-DR B-D (Becton Dickinson Co, Mountain View, CA) in cytotoxicity assays, respectively. The DQ restricted anti M. Leprae CD4+ clone R3F1031 was used to exclude aspecific blocking by anti-DR. HLA-typed donor-derived fresh PBMC were used in a panel to test the MHC restriction of peptide sensitive T cells. Murine L-cell line LDR2a32,33 expressing DRB5*0101 (kindly provided by Dr J. Bodmer, Imperial Cancer Research Fund, London, UK), HLA-DR cDNA negative murine cells LTKcd323 (kindly provided by Dr K. Moore, DNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA), and T2 DRB5*0101 transfectants (kindly provided by Dr A. Vogt, Heidelberg, Germany) were used to confirm HLA-DR2a (DRB5*0101) allele restriction. Flow cytometry.
Double immunostaining of T-cell clones was performed with fluorescein
isothiocyanate (FITC)-labeled Moab anti-CD4 (Becton Dickinson) and
phycoerythrin (PE)-conjugated anti-CD8 (anti-CD8 HLA-DR peptide binding assay. T2-DR2a cells were cultured in RPMI 1640 (GIBCO), supplemented with 2 mmol/L L-glutamine (GIBCO-Life Technologies), 100 U penicillin/100 µg streptomycin/mL (GIBCO), and 10% heat-inactivated fetal calf serum (FCS) (GIBCO). Cells were lysed at a concentration of 108 cells/mL in 50 mmol/L Tris-HCl, pH 8.5, containing 2% Renex (Accurate Chemicals and Scientific Corp, Westbury, NY), 150 mmol/L NaCl, 5 mmol/L EDTA, and 2 mmol/L phenylmethyl sulfonyl fluoride (PMSF). The lysates were cleared of nuclear and other debris by centrifugation at 10,000g for 20 minutes. DR molecules were purified essentially as described.34,35 Purified DR molecules (60 to 600 nmol/L) were incubated at pH 4.5 for 48 hours with 100 fmol (6.7 nmol/L) fluorescent-labeled DR binding class II-associated invariant chain peptide (CLIP, p87-p101)36 in 15 µL (final volume) 100 mmol/L Na-phosphate buffer containing 75 mmol/L NaCl, 1 mmol/L CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-propanesulfonate, Merck, Darmstadt, Germany) and 15% (vol/vol) CH3CN in the presence of a mixture of protease inhibitors (final concentration [f.c.] 1 µmol/L chymostatin, 5 µmol/L leupeptin, 10 µmol/L pepstatin A, 1 mmol/L EDTA, 200 µmol/L pefabloc). Each DR preparation was titrated in the presence of 100 fmol of the reference peptide (CLIP p87-101) to determine the DR concentration necessary to bind 10% to 20% of the total fluorescent signal. All subsequent inhibition assays were then performed at this concentration. Peptides of which the DR binding capacity was to be determined were added to DR molecules simultaneously with the reference peptide. The DR peptide complexes were separated from free peptide by gel filtration on a Synchropak GPC 100 column (250 mm × 4.6 mm; Synchrom Inc, Lafayette, IN). Fluorescent emission was measured at 528 nm on a Jasco FP-920 fluorescence detector (B & L Systems, Zoetermeer, The Netherlands). The percentage of labeled peptide bound was calculated as the amount of fluorescence bound to MHC divided by total fluorescence. Peptides were tested at concentrations ranging from 70 µmol/L to 0.7 nmol/L.The concentration of unlabeled test peptide yielding 50% inhibition of binding (IC50) of FITC-labeled CLIPp 87-101 peptide was deduced from the dose-response curve. Each peptide was tested in three separate experiments. P210BCR-ABL transfected B-LCL. B lymphoblastoid cells were obtained by EBV transformation of PBMC. Full-length BCR-ABLb2a2 cDNA in plasmids pCDXX (kindly provided by Dr G. Grosveld, St Jude Children's Research Hospital, Memphis, TN) was recloned into vector PDR2 (Clontech Laboratories Inc, Palo Alto, CA). Unaltered PDR2 was used as negative control vector (empty vector). PDR2 vectors were transfected into EBV B-LCL by electroporation: cells were washed in serum free Iscove's medium containing penicillin. Approximately 17 × 106 cells in 0.7 mL medium were transferred to a gene pulser cuvet (Bio-Rad Laboratories, Hercules, CA) and 20 µg plasmid was added per cuvet. Electroporation was performed at 960 microfarad and 250 mV. Cells were kept in the cuvet for 10 minutes after electroporation and subsequently cultured at 106/mL in medium supplemented with 20% FCS (Sera Tech Zellbiologische Produkte, Skt Salvator, Germany). After 24 hours, dead cells were removed by ficoll centrifugation and viable cells were cultured in fresh medium under addition of 100 µg/mL hygromycine B (GIBCO Life Technologies Inc). The hygromycine concentration was increased with 100 µg/mL every 3 days to a final concentration of 500 µg/mL, at which the remaining B-LCL appeared hygromycine resistant. Invariant chain-b2a2 fusion insert-cDNA (Iib2a2) transfected B-LCL. A cassette vector was constructed from human cDNA coding for Ii, as described earlier,37 in which 33 CLIP-encoding nucleotides were replaced by a BCR-ABL p210b2a2 fusion cDNA sequence. The sequence of the b2a2-oligonucleotide was as follows: 5'-ACCATCAATAAGGAAGAAGCCCTTCAGCGGCCA-3' (replacing wt CLIP nt 5'-TTCTACGCGTACCGGTGGGGCGACGACTACGTC-3'). The vector-insert was checked by DNA sequencing. The construct Iib2a2 encodes Ii protein with TINKEEALQRP b2a2 fusion peptide (instead of wt CLIP aa KMRMATPLLMQ) within the CLIP aa sequence. Western blotting.
Laemmli sample buffer solution (60 mmol/L TRIS HCL pH6.8, 10%
glycerol, 10 mmol/L EDTA, 2% sodium dodecyl sulfate [SDS], 2% T-cell receptor (TCR) analysis.
RNA was extracted from P-b2-derived T-cell clones, Aa3 and Aa14, using
RNAzol B (Cinna/Biotecx Laboratories, Houston, TX). Oligo-dT-primed
first strand cDNA was reverse transcribed from 2 to 5 µg RNA using
the Riboclone cDNA synthesis system (Promega Corp, Madison, WI). cDNA
was subjected to PCR amplification with 29 TCRAV and 24 TCRBV
family-specific primers as described elsewhere.40 PCR
fragments were electrophoresed in agarose and subsequently purified
using Whizard columns (Promega Corp). The PCR products were used for
direct sequencing using the dideoxy-nucleotide chain termination
method41 with the T7 sequencing Kit (Pharmacia LKB, Uppsala, Sweden). DNA sequences were compared with TCR V/J
T-cell line, P-b2, is BCR-ABLb2a2 breakpoint
peptide-specific and HLA-DR2 restricted.
Primary in vitro stimulation of PBMC from a healthy donor with a 17mer
b2a2 fusion sequence peptide resulted in a peptide-specific T-cell line
designated P-b2. P-b2 proliferated in response to autologous irradiated
PBMC incubated with 5 µg/mL b2a2 peptide. P-b2 did not show
cross-reactivity to equal length BCR-ABLb3a2 fusion
peptide, which also contains the 8 carboxy terminal aa residues encoded
by ABL exon a2 (Fig 1). Proliferative
responses could be blocked by preincubation of peptide-loaded APC with
anti HLA-DR MoAbs (data not shown). A panel of partially
HLA-DR-matched APC indicated the HLA-DR2 restriction of the b2a2
peptide-specific T-cell response (Table 1).
Cloning of T-cell line, P-b2.
Forty-one peptide-specific T-cell clones were obtained from P-b2 by
limiting dilution. FACS analysis showed CD4 positivity in all cases,
but several clones expressed CD4/CD8 (Fig
2).
B2a2 peptide binding to HLA DR2a.
The relative importance of different aa residues within the b2a2 17 mer
for T-cell recognition or MHC interaction was evaluated by measuring
the effectiveness of the b2a2 peptide with its different alanine
substitutions to compete for binding to immunopurified DR2a molecules
with a fluorescence-labeled invariant chain control peptide. The
results (Table 3) allow, together with the
data from Table 2, a designation for the different T-cell clones of putative T-cell contact residues at positions 6, 7, 8, 9, 11, and 14 within the b2a2 peptide and strongly support a role for I5
and R13, as so called anchor residues necessary for MHC
binding.
B2a2 fusion peptide-specific T cells fail to lyse autologous B-LCL
and allogeneic CML cells expressing p210b2a2.
The seven clones were not only proliferative, but displayed b2a2
peptide-specific cytolytic activity against peptide-loaded DR2-positive
target cells as well. Autologous B-LCL transfected with full-length
BCR-ABLb2a2 cDNA and allogeneic HLA-DR matched
BCR-ABLb2a2 RT-PCR-positive cells from CML patients
expressed BCR-ABL protein as shown by Western blotting
(Fig 3). However, B2a2 peptide-specific clones failed to show lytic or proliferative responses against these
cells, unless exogenous b2a2 peptide was added.
Iib2a2 cotransfection induces recognition of autologous
BCR-ABLb2a2 transfectants by b2a2 peptide-specific T-cell
clones.
Additional experiments were performed to investigate the possibility of
whether the immunogenic fusion region peptide from p210b2a2 could be
loaded endogenously and presented in MHC class II molecules of
BCR-ABL-transfected cells using a highly efficient endogenous class II
loading method. Autologous EBV cells were cotransfected with
full-length BCR-ABLb2a2 and invariant chain (Ii) cDNA, in
which a CLIP encoding sequence was replaced by a
BCR-ABLb2a2 oligonucleotide sequence (Iib2a2).
As control APC, identical cells were cotransfected with full-length BCR-ABLb2a2 and with invariant chain without such a CLIP
exchange (IiCLIP). As depicted in
Fig 4, clones Aa3 and clone Aa14 were both
able to kill autologous Iib2a2 transfectants, but not the
control Ii transfectants.
TCR analysis.
The TCR of clones Aa3 and Aa14 was analyzed by RT-PCR and direct
sequencing. The results were consistent with a clonal nature of both
T-cell populations (Table 4). Differences
were seen in their AJ gene usage, N(-D-N) regions and hence
complementary determining regions (CDR) 3
Although aberrant BCR-ABL fusion genes and BCR-ABL mRNA splicing can
occur,4 the majority of patients with CML express p210b3a2
and/or p210b2a2, often together with low levels of pl90e1a2 BCR-ABL
proteins.6,7 The immunogenicity of fusion region-derived peptides of p210b3a2 has been demonstrated by our and other
groups.15-23 Peptides corresponding to the b3a2 fusion
sequence were shown to bind to HLA A2,16
A3,14,15 A11 and B8,14 DRB1*0402, DRB1*0301,
and DR11.17 HLA A2,16 A3,15,16,20
A11,16,20 presumably A24,22 and
DRB1*090118 restricted b3a2 peptide-specific cytotoxic T
cells have been obtained, as well as HLA DR1,23 DRB1*1501,19 DRB1*0401,21
DR11,17,20 and DR17(DR317) (and own unpublished
observation, July 1996) restricted T-helper cells.
Processing of endogenous BCR-ABL proteins by CML cells has not been
proven biochemically, albeit that we have observed recognition of
HLA-DR4-positive b3a2 CML blasts by HLA-DR4-restricted b3a2 fusion
peptide-specific T-helper cells.21 Others have documented lysis of BCR-ABLb3a2 leukemic cells by CD8-positive b3a2
peptide-specific T cells in an MHC class
I-restricted16 and unrestricted22 manner. Indirect processing of p210b3a2 has been described in a murine system43 and with mitomycin C-treated CML patient-derived
PBMC as APC.23 No reports of BCR-ABL peptides in HLA
eluates have been published yet, but a peptide, apparently derived from
the BCR protein, was recently eluted from HLA A2.1
molecules.44
Submitted December 1, 1998; accepted April 6, 1999.
Supported by grants from the Dutch Cancer Society, Amsterdam, The
Netherlands, and the Fonds National de la Recherche Scientifique Fondation Bekales, Brussels, Belgium.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Onno C. Leeksma, MD, Department of
Immunohematology and Bloodbank, Leiden University Medical Center, PO
Box 9600, 2300 RC Leiden, The Netherlands.
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TOP
ABSTRACT
INTRODUCTION
70°C in aliquots of 1 mL in a concentration of 10 mg/mL in phosphate-buffered saline (PBS:
154 mmol/L NaCl, 1.4 mmol/L Na2HPO4, pH 7.5).
chain, Becton
Dickinson) and analyzed by FACscan (Becton Dickinson). CD8 expression
was not limited to CD8 
chain MoAb (kindly provided by Dr E. Reinhertz, Dana-Farber Cancer
Institute and Department of Medicine, Harvard Medical School, Boston, MA).
