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PLENARY PAPER
From the Departments of Life Sciences and Chemistry and
Physics, Nottingham Trent University, Nottingham, United
Kingdom; Departments of Haematology and Immunology, University
of Liverpool, Liverpool, United Kingdom; and The Anthony
Nolan Research Institute, The Royal Free and University College Medical
School, London, United Kingdom.
The BCR-ABL oncogene is central in the pathogenesis of
chronic myeloid leukemia (CML). Here, tandem nanospray mass
spectrometry was used to demonstrate cell surface HLA-associated
expression of the BCR-ABL peptide KQSSKALQR on class I-negative CML
cells transfected with HLA-A*0301, and on primary CML cells from
HLA-A3-positive patients. These patients mounted a cytotoxic
T-lymphocyte response to KQSSKALQR that also killed autologous CML
cells, and tetramer staining demonstrated the presence of circulating
KQSSKALQR-specific T cells. The findings are the first demonstration
that CML cells express HLA-associated leukemia-specific immunogenic
peptides and provide a sound basis for immunization studies against
BCR-ABL.
(Blood. 2001;98:2887-2893) Chronic myeloid leukemia (CML) is a serious
neoplastic disorder of the hematopoietic stem cell, characterized by an
initial chronic phase of variable length, which commonly transforms to an incurable acute leukemia. Although an uncommon disease, CML has
attracted a disproportionate amount of research attention, because the
molecular genetics of the chronic phase are well understood and may
represent a model for many other malignant conditions. The Philadelphia
(Ph) translocation that typifies almost all cases juxtaposes the
ABL gene on chromosome 9q34 to BCR on chromosome 22q11. Although the genomic breakpoints on both chromosomes 9 and 22 may vary from patient to patient over several kilobases, they occur
only in introns.1 The resulting BCR-ABL
messenger RNA (mRNA) transcript comprises a proximal
BCR-derived portion ending with exon 2 or 3 (denoted by b2
or b3, respectively), fused to ABL exons 2 to 11 (denoted by
a2).2 Almost all Ph-positive CML patients express either
b3a2 or b2a2 fusion transcripts, depending on whether exon b3 is
included. When translated, b2a2 and b3a2 mRNA each generate a 210-kDa
BCR-ABL protein (p210), which is necessary and sufficient for leukemic
transformation.3 The BCR-ABL junction in b3a2
mRNA disrupts a triplet codon, producing a novel lysine (K) at the
junction in the b3a2 BCR-ABL protein product. A codon disruption also
occurs at the b2a2 fusion junction (Asp altered to Glu), but in this
case the novel amino acid may not be recognized as it is also present
at the normal a1a2 junction.
Endogenous antigen is presented to the immune system by initial
degradation to oligopeptides typically of 8 to 11 amino acids, which
are assembled in the endoplasmic reticulum lumen into a stable complex
with newly synthesized class I HLA BCR-ABL junctional peptides can induce in vitro CTL activity or
CD4+ cell proliferative responses in healthy
subjects.5,6,8-16 Antigen presenting cells (APCs) loaded
with BCR-ABL junctional 9-mers will elicit specific class I-restricted
CTL activity from autologous fresh peripheral blood mononuclear cells
(PBMC)s.5,6 By using T-cell blasts pulsed with high
concentrations of BCR-ABL peptide as APCs, we have recently reported
that BCR-ABL-specific CTLs elicited from healthy subjects will
kill CML cells in an HLA-restricted fashion,16 providing
indirect evidence that CML cells may naturally express BCR-ABL
junctional peptides. However, no evidence of BCR-ABL junctional
peptides was seen among peptides eluted from HLA molecules on CML
cells,17 although only HLA-A*0201 was studied. It is
possible that the technique used in that work (high-performance liquid
chromatography [HPLC]) may have been too insensitive to distinguish
the low level of BCR-ABL peptides from among the "noise" of other
extracted material. Furthermore, although tumor cell lysates have been
shown to contain HLA-bound peptides that are recognized by
antigen-specific CTLs,18-21 these experiments do not
discriminate between intracellular and cell surface expressed
HLA-peptide complexes. It is therefore not known whether CML cells can
express BCR-ABL-derived and thus leukemia-specific peptides on the
cell surface in the context of HLA. Direct proof of this would provide
powerful scientific support for the development of immunization
strategies against BCR-ABL.
Mass spectrometry with nanospray ionization (NSI/MS) is a sensitive
technique available for the identification of small quantities of
biological molecules. Skipper et al22 used mass
spectrometry to demonstrate that Melan A/MART 1 and gp100
HLA-A*0201-restricted epitopes can be eluted from surface class I HLA
molecules on cultured melanoma cell lines. However, the application of
mass spectrometry to study HLA-associated peptides on the surface of
tumor cells has been limited. In adapting mass spectrometry to study
primary tumor cells, a potential problem is that the signatures of
low-frequency tumor-specific peptides may be masked by those of other
cell surface peptides, expressed on other HLA alleles. Furthermore,
analysis is complicated by potential contamination by peptides/proteins from the intracellular compartments. To address this problem and using
CML as a model, we have conceived a strategy that allows for the
presentation and subsequent elution of peptides presented through a
single HLA allele. In this instance, this is achieved by transfecting
HLA-A*0301 into the HLA class I-negative CML cell line K562, which
expresses b3a2 BCR-ABL mRNA.
In this study, we present for the first time evidence for surface
HLA-A*0301-restricted b3a2 peptide expression, both on
HLA-A*0301-transfected K562 cells and on CML cells from
HLA- A3+/b3a2+ patients. Sequencing results
obtained by using tandem mass spectrometry confirmed the presence of
the HLA-A*0301-restricted peptide KQSSKALQR, with an identical
signature on both transfected K562 and primary CML cells. We also show
that these patients can mount a CTL response against the BCR-ABL
junctional peptide expressed on their CML cells, and we report on the
specificity of these CTLs. Furthermore, using tetramer staining, we
demonstrate that HLA-A3-restricted peptide-specific T cells are
detectable in the peripheral circulation of CML patients and that these
T cells can be expanded specifically in vitro to provide increased
levels of BCR-ABL-specific T cells.
Patient population
Cell lines and gene transfection
Synthetic peptide The BCR-ABL fusion peptide sequence KQSSKALQR has been previously described as binding to HLA-A*0301.4 This peptide was made by using an ABI synthesizer with F-moc chemistry and purified by HPLC to more than 90% purity, then confirmed by mass spectrometry. It was dissolved in 50% dimethyl sulfoxide before use in the refolding of the HLA-A*0301 molecule for tetramer preparation (see below).HLA-A*0301-associated peptide elution and purification The acid elution technique was adapted from Storkus et al.23 The essential modifications were that prior to use, all plastic and glassware were treated in Sigmacote (Sigma, Poole, United Kingdom) to minimize peptide losses by surface adsorption. Citrate phosphate buffer (0.131 M citric acid and 0.066 M sodium phosphate) was adjusted to pH 3.3 by using 10 M NaOH. A minimum of 109 freshly leukapheresed CML cells or 5 × 108 K562 transfectants/controls were washed in serum-free RPMI medium and centrifuged at 1500 rpm at 4°C for 5 minutes. Pelleted cells were resuspended in 20 mL citrate phosphate buffer for 5 minutes at room temperature and again centrifuged. The eluate-containing supernatant was collected and filtered through a 0.22-µm micropore filter and stored at 80°C. Cellular integrity
was assessed by using trypan blue, and the cells were found to be
intact after citrate phosphate buffer elution.
Synthetic hepatitis B MHC class II-restricted peptide (TPPAYRPPNAPIL128-140 900 fmol) was added to the cell eluate before biochemical purification and separation, as an internal recovery standard. Trichloroacetic acid (72% wt/vol, 2 mL) was added, and the sample was placed in an ultrasonic bath for 10 minutes, centrifuged for 10 minutes at 4°C at 15 000g, and followed by a further round of ultrasonication for 10 minutes. The supernatant was removed and applied to a cation exchange column (1 mL Econo S cartridge; BioRad, Hemel Hempstead, United Kingdom). The column was washed with 0.1 M HCl (30 mL), and the bound peptides were eluted with 0.1 M NaOH (25 mL). The eluted peptide sample from the cation exchange column was brought to neutral pH with 2.8% vol/vol trifluoroacetic acid (TFA, 70 µL) and loaded via an injection loop onto an RP-HPLC precolumn (1 × 30 mm ODS; Jupiter, Phenomonex, Macclesfield, United Kingdom). The column was washed in solvent A (water containing 0.1% vol/vol TFA, 200 µL/min) to remove any residual buffer or cation exchange salts. The solvent flow was then redirected via a 6-port switching valve (Rheodyne 7030) with 20% solvent B (acetonitrile containing 5% vol/vol glacial acetic acid and 0.01% vol/vol TFA) and 80% solvent C (water containing 5% glacial acetic acid vol/vol and 0.01% TFA vol/vol) flowing through both the precolumn and the online analytical column used for peptide separation (1 × 150 mm ODS; Jupiter, Phenomonex). The columns were then subjected to an elution gradient of 20:80% to 90:10% solvent B:C (45 minutes, 20 µL/min), and 3 to 5 µL fractions were collected in silanized 1.1-mL tapered glass vials. To achieve low flow rates, a flow restrictor (microflow splitter; SGE, Milton Keynes, United Kingdom) was used with the HPLC pumps (ConstaMetric; Milton Roy, Wokingham, United Kingdom). Mass spectrometric analysis and peptide identification RP-HPLC fractions were analyzed by using an in-house constructed nano-electrospray ionization source interfaced to a quadruple ion trap mass spectrometry (Finnigan, Hemel Hempstead, United Kingdom). An Au/Pd-coated nano-electrospray tip (Nano-ES tip; Protana, Denmark) supported in a PTFE holder was attached to an adjustable platform, allowing movement along all 3 axes for the precise positioning of the tip near the heated capillary inlet of the mass spectrometer. The tip was gently broken against the heated capillary to initiate spraying and was maintained at a voltage of 1.1 kV by connection to the instrumental high-voltage power supply.Mass spectrometric conditions were as follows: heated capillary 100°C, maximum injection time 400 ms, total microscans 3. The tube lens offset and heated capillary voltages were optimized for the doubly and triply protonated HLA-A*0301-associated peptide KQSSKALQR by running the synthetic peptide. The hepatitis B internal recovery standard peptide was isolated and analyzed to ensure that the level of recovery through the biochemical clean up was sufficient to allow mass spectrometric detection of natural peptides. Those peptides detected above the background noise were subjected to a zoom scan to ascertain their charge status. Ions found to be multiply charged in the mass range 300 to800 Da were subjected to collisionally activated dissociation with an isolation width of 3 arbitrary units, a relative collision energy of 30%, and an automatic gain control at 1 × 109 to generate peptide sequence data. CTL assay Autologous T-cell blasts were used as APCs, prepared as previously described,16 using KQSSKALQR as the pulsing peptide. CTLs were generated by using a modification of the method of Plebanski et al24 and Norbury et al.16 On day 0, PBMCs were plated out in 96-well U-bottomed plates at 37°C at 5% CO2 at 105 CD3+ cells per well with autologous APCs (105 per well) pulsed with peptide and 2-microglobulin in Iscoves modified Dulbecco media (IMDM;
Bio-Whittaker) with 10% heat-inactivated AB serum. The medium
contained 10 ng/mL interleukin-7 (IL-7; R&D systems, Abingdon, United
Kingdom). After 3 days an additional 25 µL IMDM with AB serum and
interleukin-2 (IL-2; First Link, West Midlands, United Kingdom) was
added, to give a final IL-2 concentration of 10 IU/mL.
A second and third stimulation with 105 peptide-pulsed autologous APCs was performed on days 7 and 14 in the presence of 10 ng/mL IL-7. IL-2 was again added after 3 days to a final concentration of 20 IU/mL. After 21 days of culture, individual wells containing the peptide-stimulated cells were assayed for peptide-specific CTL effector activity by using a standard 51Cr release assay.16 Wherever possible, CTLs were also tested against autologous CML cells. Data on 84 wells were available for all cytotoxicity assays. Results are expressed as the percentage of wells with 51Cr release more than 3 SDs above the mean background 51Cr release.16 The significance of differences in the percentages of positive wells between pairs of target cells (pulsed versus unpulsed) were tested by using Fisher exact test. Tetrameric MHC class I/peptide complexes The HLA-A*0301/KQSSKALQR tetramers were made as described previously.25 Briefly, the extracellular portion of the HLA-A*0301 coding DNA was amplified by polymerase chain reaction (PCR) and cloned into the pET 3d vector (Novagen) modified by adding a C-terminal sequence coding for a biotinylation target. This system allows the HLA-A*0301 heavy chain to be expressed in bacteria and harvested as insoluble inclusion bodies. The2-microglobulin chain
is produced in a similar fashion. Both molecules were solubilized in 8 M urea before being added to the KQSSKALQR peptide in a dilution
refolding under denaturing conditions. The refolded molecule was then
purified by FPLC on a Superdex 75 column (Pharmacia, United Kingdom).
Biotinylation was performed by using bacterially expressed Bir A
enzyme, with comparable efficiency to commercially available Bir A
(Avidity, Denver, CO). Monomeric biotinylated complexes were
further purified by gel filtration followed by anion exchange
chromatography. Tetrameric molecules were then formed by the addition
of phycoerythrin-labeled streptavidin at a 4:1 molar ratio. The
HLA-A2/NLVPMVATV tetramer used for the controls shown in the central
panels of Figure 2 was made in an analogous way.
Tetramer analysis Freshly isolated PBMCs or cultured effector CTLs were resuspended in RPMI 1640 medium supplemented with 10% FCS and 5 mM NaN3 to prevent internalization of the tetramers. Cells (106) were stained with the HLA-A*0301/KQSSKALQR tetramer for 30 minutes at 37°C. This staining was followed by a washing step and subsequent staining with fluorescein isothiocyanate-labeled anti-CD8 and peridinin chlorophyll protein-labeled anti-CD3 antibodies (Becton Dickinson, United Kingdom) for 30 minutes at 4°C. Cells were washed and fixed in RPMI with 1% paraformaldehyde and were analyzed within 24 hours. Data were acquired by using a FACScan flow cytometer using the CELLQuest software (Becton Dickinson). Identical methodology was used for the control HLA-A2/NLVPMVATV tetramer.mRNA transcript type The BCR-ABL mRNA transcript type was determined by using minor modifications of our previously reported technique.26 Briefly, total RNA from PBMCs was extracted by using RNeasy Mini Kit (QIAGEN, Crawley, United Kingdom). After synthesis of complementary DNA (cDNA), 2 µL was amplified by PCR using primers EK158 and EK159 as described previously.26
Identification of HLA-A*0301-associated peptide KQSSKALQR For each sample, the HPLC fractions found by NSI/MS to contain a triply charged ion corresponding to the HLA-A*0301-associated peptide KQSSKALQR (m/z 349.50) were subjected to tandem mass spectrometry (MS/MS). Figure 1A shows the resulting MS/MS product ion spectrum for a control cell eluate to which synthetic KQSSKALQR was added at a concentration of 2.3 fmol/µL. The ions resulting from the fragmentation of the parent ion of KQSSKALQR had been predicted, using MS Product within the Protein Prospector database (University of California at San Francisco). The observed fragment ions (as labeled in Figure 1) matched with the database prediction. The presence of KQSSKALQR peptide was also confirmed by inputting the observed fragment ion masses into MS-Tag tool, with the finding that the observed fragment ion masses were unique to KQSSKALQR.
Figure 1C shows the MS/MS spectrum for an eluate from K562 cells transfected with HLA-A*0301. A close match was observed with the fragment ion profile for the synthetic peptide. In contrast, no fragment ions characteristic of KQSSKALQR (as labeled in Figure 1) were seen above the limit of detection in eluates from nontransfected K562 cells. The data therefore demonstrate that K562 cells expressing HLA-A*0301 as the only class I MHC molecule are able to present KQSSKALQR. Mass spectrometric analysis was performed on 3 b3a2 BCR-ABL/HLA-A*0301-positive patients (cases 1-3) and one b3a2 BCR-ABL-positive/HLA-A*0301-negative patient (case 6). MS/MS product ion spectra characteristic of the KQSSKALQR peptide were found in eluates from the HLA-A*0301-positive cases expressing the b3a2 transcript. Figure 1B shows the MS/MS spectrum obtained from an eluate from the HLA-A*0301-positive primary CML cells (patient 1) in which the fragment ion pattern matches that observed for the synthetic peptide, confirming the presence of KQSSKALQR. In contrast, no evidence of the KQSSKALQR peptide was found above the mass spectrometric limit of detection in the HLA-A*0301-negative patient (case 6). Tables of BCR-ABL-derived peptides able to bind to HLA-A*0301 were also predicted, using the computer algorithms BIMAS (http://www.bimas.dcrt.gov) and Syfpeithi (http://www.uni-tuebingen.de/kxi). These data allowed us to confirm that our defined peptide would also be selected as a potential binding candidate by the 2 commonly used peptide prediction algorithms. CTL directed against BCR-ABL junctional peptides Seven CML patients (5 HLA-A*0301 positive and 2 HLA-A*0301 negative) and 2 HLA-A*0301-positive healthy subjects were studied for evidence of CTL against BCR-ABL junctional peptides, using autologous T-cell blasts pulsed with BCR-ABL peptides as APCs. All 7 CML patients had well-controlled chronic phase CML at the time of sampling. The results are shown in Table 2. Effector CTLs were grown from all 7 HLA-A*0301-positive subjects (5 CML patients and 2 healthy subjects), which were cytotoxic against autologous T-cell blasts expressing the HLA-A*0301-associated peptide KQSSKALQR. In all 7 HLA-A*0301-positive patients/controls, sufficient effectors were available to show that these CTLs were also cytotoxic against allogeneic HLA-A*0301-positive T-cell blasts pulsed with this peptide. Similarly, where sufficient CTL were available for study, these CTLs were also cytotoxic against unpulsed autologous and/or allogeneic HLA-A*0301-positive CML cells, although not against HLA-mismatched CML cells. Effector CTLs were not grown from the 2 HLA-A*0301-negative CML patients (cases 7 and 8 in Table 2).
Detection and expansion of peptide-specific peripheral blood T cells with HLA-A*0301 tetramers The results of the studies using the HLA-A*0301/KQSSKALQR peptide tetramer are given in Figure 2 and summarized in Table 3. Of the 6 patients studied, 2 patients (1 and 2) who were positive for b3a2 and HLA-A*0301 demonstrated circulating T cells reactive with the HLA-A*0301/KQSSKALQR peptide tetramer (Figure 2, panels 1A and 2A). Figure 2, panel 1C, demonstrates that stimulation with KQSSKALQR peptide (3 rounds) augmented tetramer-positive cells from 0.3% to 2.5%. The remaining b3a2 and HLA-A*0301-positive patient (patient 3), showed negligible levels of tetramer-positive cells (Figure 2, panel 3A). However, after 1 round of stimulation with KQSSKALQR, tetramer-positive cells were detectable (Figure 2, panel 3C). This finding illustrates that there is potential to expand leukemia-reactive T cells in vitro and that tetramer staining may be helpful in monitoring their expansion. This correlates with our finding that on in vitro stimulation with peptide it was possible to raise CTLs with a similar reactivity pattern in each of patients 1, 2, and 3 (Table 2). Expanded CTLs were not screened with tetramers in patients 2 and 5, as there were insufficient numbers of effector cells remaining after their use for specificity assays.
Patients 6 (b3a2-positive but HLA-A*0301-negative CML) and 9 (HLA-A*0301-negative AML) both showed no staining with the tetramer (Figure 2, panel 3A, 5A, and 6A). These cases serve as negative controls for tetramer staining. As further controls, each patient was tested with tetramers of HLA-A2 with the HLA-A2-associated CMV peptide NLVPMVATV (central panels of Figure 2). Both patients who were HLA-A2 negative (patients 2 and 3) are negative for this tetramer (Figure 2, panels 2B and 3B), even though patient 2 is CMV positive. Similarly, patient 3 is HLA-A2 positive and CMV positive and shows tetramer-positive cells (Figure 2, panel 3B).
The present study addresses 2 issues of importance to our understanding of immune mechanisms in cancer, namely the presentation of HLA-restricted tumor-specific peptides and the host T-cell response to such peptides. We focus on the CML-specific BCR-ABL protein. First, using the strategy of a class I-negative CML cell line transfected with a single HLA allele together with NSI/MS, we show that the HLA-A*0301-associated peptide KQSSKALQR is presented by transfected but not by nontransfected K562 cells. This finding demonstrates that a CML cell line can process the BCR-ABL fusion protein and can process and present peptides derived from the BCR-ABL junction on the cell surface in association with HLA class I antigen. The finding is in agreement with previous indirect evidence of HLA-associated BCR-ABL expression.16,27,28 We then extend this NSI/MS methodology to show that primary CML cells from b3a2/HLA-A*0301-positive patients also express KQSSKALQR on the CML cell surface. To our knowledge this represents the first direct evidence that a hematologic malignancy can express leukemia-specific peptides on the cell surface, in association with HLA class I antigens, which has until now remained unconfirmed. Second, we show that these patients can mount an in vitro CTL response against the BCR-ABL junctional peptide expressed on their CML cells and that these CTLs are capable of killing autologous CML cells. We also demonstrate by tetramer staining that KQSSKALQR-specific T cells are detectable in the peripheral circulation of these patients and that these T cells can be expanded specifically in vitro. The demonstration that BCR-ABL junctional peptides can be eluted directly from CML cells is consistent with observations that appropriate synthetic 9-mer b3a2 peptides can bind HLA-A*0301.4,7,8 None of the 9-mer b3a2 peptides "shifted" by a single amino acid in either N- or C-terminal directions from KQSSKALQR were detected in the eluates. This finding is strong evidence that the defined HLA-binding peptide was not derived from nonspecific proteolysis but was processed and presented in association with class I HLA at the surface of CML cells. Neither of these shifted 9-mers were predicted to bind with high affinity to either HLA-A*0301 or to other class I HLA molecules expressed by the patients. An eluate from a b3a2-positive HLA-A*0301-negative patient lacked the KQSSKALQR peptide, and this finding further supports the view that this peptide was derived from endogenously processed BCR-ABL protein presented in the HLA peptide binding groove in HLA-A*0301-positive CML cells. Although we demonstrate that both BCR-ABL antigen presentation and CTL response are intact in CML, these mechanisms cannot operate optimally in vivo, otherwise the disease would never come to clinical attention. The Ph-positive-initiating cell may not express BCR-ABL and may therefore escape immunologic detection. However, BCR-ABL transcripts have been detected in healthy subjects, using a sensitive reverse transcriptase-PCR assay,29 and both CTL and CD4+ proliferative responses against BCR-ABL can be elicited in healthy subjects.5,6,8,9-16 CML may rarely spontaneously regress.30-32 It is therefore interesting to speculate that certain CML clones may present suitably immunogenic peptides, which become targets for CTL. CML may be less common in HLA-A3-positive or B8-positive individuals,33 suggesting that peptide immunogenicity may differ according to HLA type. It is plausible that the BCR-ABL rearrangement may be more common than the prevalence of CML, but that a T-cell response may control or eliminate a BCR-ABL-positive clone. Recent data suggest that a T-cell response against proteinase 3, a granule protein overexpressed in CML cells, may be important in clearing leukemic cells after therapy.34 Thus, CML may come to clinical attention as a result of an immune tolerance or anergy to BCR-ABL, as well as the occurrence of the BCR-ABL rearrangement per se. CTL precursors capable of being activated in vitro to kill autologous tumor cells have been described in malignant melanoma.35 However, melanoma-associated antigens (MAGEs) are mostly unmutated self-antigens, such as MAGE1-3 or tyrosinase.36 A completely novel tumor-specific antigen such as BCR-ABL would be less likely to induce tolerance or anergy than a normal self-antigen. However, there are a number of possible explanations for the failure of BCR-ABL to induce effective CTL in vivo in CML patients. These explanations include low T-cell receptor avidity or weak antigen expression or T-cell tolerance in patients with a massive leukemic burden.37 Dendritic cells from CML patients are part of the leukemic clone,28 and, hence, there is the potential for induction of T-cell tolerance to BCR-ABL via endogenous expression by thymic dendritic cells. Specific CD4+ T cells, which are thought to be essential for most CD8+ T-cell responses,38 may be absent or anergic. It is notable that CD4+ T cells specific for b3a2 peptide could be detected in healthy donors but not in CML patients.13 The requirement for specific CD4+ T cells in generating b3a2-specific CTL may be circumvented during in vitro CTL assays by the use of IL-2 and IL-7. The potential promise of peptide vaccination for the therapy of malignant disease has yet to be realized. This may, in part, be due to difficulties in accurately defining tumor-specific peptides. The present strategy of using a single HLA-transfected cell line together with mass spectrometry demonstrates a novel approach for precise definition of HLA-associated peptides. This strategy may be widely applicable in many other malignant and nonmalignant disease states. The data support the view that immunization strategies against BCR-ABL may be helpful in boosting T-cell responses in CML. A recent preliminary report suggests that some CML patients may generate specific proliferative immune responses following vaccination with BCR-ABL junctional peptides,37 although CTL responses were not observed in that study. The present findings give encouragement to further studies to define the optimal composition, route, and timing of administration of immunization with BCR-ABL junctional peptides. In summary, the present data provide the first direct evidence that CML cells express surface BCR-ABL junctional peptides in association with HLA class I, and that antigen-specific T cells can target these peptides. CML can, therefore, be added to the list of examples of human tumors known to express a tumor-restricted peptide in the context of HLA.
Submitted June 22, 2001; accepted July 16, 2001.
Supported by the Nottingham Trent University Research award (J.R.L.), the John and Lucille van Geest Foundation (S.C.H.), and the North West Cancer Research Fund.
R.E.C., I.A.D., S.C.H., and J.R.L. contributed equally to the work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Robert C. Rees, Department of Life Sciences, Nottingham Trent University, Clifton Lane., Nottingham NG11 8NS, United Kingdom; e-mail: robert.rees{at}ntu.ac.uk.
1. Groffen J, Stephenson JR, Heisterkamp N, de Klein A, Bartram CR, Grosveld G. Philadelphia chromosome breakpoints are clustered within a limited region, bcr, on chromosome 22. Cell. 1984;36:93-99[CrossRef][Medline] [Order article via Infotrieve]. 2. Kurzrock R, Guttermann JU, Talpaz M. The molecular genetics of Philadelphia chromosome-positive leukaemias. N Engl J Med. 1988;319:990-998[Medline] [Order article via Infotrieve].
3.
McLaughlin J, Chianese E, Witte ON.
In vitro transformation of immature hematopoietic cells by the p210 bcr/abl oncogene product of the Philadelphia chromosome.
Proc Natl Acad Sci U S A.
1987;84:6558-6562
4.
Bocchia M, Wentworth PA, Southwood S, et al.
Specific binding of leukemia oncogene fusion protein peptides to HLA Class I molecules.
Blood.
1995;85:2680-2684
5.
Bocchia M, Korontsvit T, Xu Q, et al.
Specific human cellular immunity to bcr-abl oncogene-derived peptides.
Blood.
1996;87:3587-3592 6. Yotnda P, Firat P, Garcia-Pons F, et al. Cytotoxic T-cell response against the chimeric p210 BCR-ABL protein in patients with chronic myelogenous leukemia. J Clin Invest. 1998;101:2290-2296[Medline] [Order article via Infotrieve]. 7. Buzyn A, Ostankovitch M, Zerbib A, et al. Peptides derived from the whole sequence of BCR-ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes. Eur J Immunol. 1997;27:2066-2072[Medline] [Order article via Infotrieve]. 8. Berke Z, Andersen MH, Pedersen M, Fugger L, Zeuthen J, Haurum JS. Peptides spanning the junctional region of both the abl/bcr and the bcr/abl fusion proteins bind common HLA class I molecules. Leukemia. 2000;14:419-426[CrossRef][Medline] [Order article via Infotrieve]. 9. Barrett J, Guimaraes A, Cullis J, Goldman JM. Immunological characterisation of the tumour specific bcr/abl junction of Philadelphia chromosome positive chronic myeloid leukaemia. Stem Cells. 1993;11(suppl 3):104-108. 10. ten Bosch GJA, Toornvliet AC, Friede T, Melief CJM, Leeksma OC. Recognition of peptides corresponding to the joining region of p210BCR-ABL protein by human T cells. Leukemia. 1995;9:1344-1348[Medline] [Order article via Infotrieve].
11.
ten Bosch GJA, Joosten AM, Kessler JH, Melief CJM, Leeksma OC.
Recognition of BCR-ABL positive leukemic blasts by human CD4+ T cells elicited by primary in vitro immunization with a BCR-ABL breakpoint peptide.
Blood.
1996;88:3522-3527 12. Macintyre AR, Christmas SE, Clark RE. The influence of class II HLA type on the lymphoproliferative response of normal donors to a bcr-abl fusion peptide. Exp Hematol. 1996;24:1307-1311[Medline] [Order article via Infotrieve].
13.
Pawelec G, Max H, Halder T, et al.
BCR/ABL leukaemia oncogene fusion peptides selectively bind to certain HLA-DR alleles and can be recognised by T cells found at low frequency in the repertoire of normal donors.
Blood.
1996;88:2118-2124
14.
Mannering SI, McKenzie JL, Fearnley DB, Hart DNJ.
HLA-DR1 restricted bcr-abl (b3a2) specific CD4+ T lymphocytes respond to dendritic cells pulsed with b3a2 peptide and antigen-presenting cells exposed to b3a2 containing cell lysates.
Blood.
1997;90:290-297
15.
Yasukawa M, Ohminami H, Kaneko S, et al.
CD4+ cytotoxic T-cell clones specific for bcr-abl b3a2 fusion peptide augment colony formation by chronic myelogenous leukemia cells in a b3a2-specific and HLA-DR restricted manner.
Blood.
1998;92:3355-3361 16. Norbury LC, Clark RE, Christmas SE. b3a2 BCR-ABL fusion peptides as targets for cytotoxic T cells in chronic myeloid leukaemia. Br J Haematol. 2000;109:616-621[CrossRef][Medline] [Order article via Infotrieve].
17.
Papadopoulos KP, Suciu-Foca N, Hesdorffer CS, Tugulea S, Maffei A, Harris PE.
Naturally processed tissue and differentiation stage-specific autologous peptides bound by HLA Class I and II molecules of chronic myeloid leukaemia blasts.
Blood.
1997;90:4938-4946 18. Bellone M, Iezzi G, Manfredi AA, et al. In vitro priming of cytotoxic T lymphocytes against poorly immunogenic epitopes by engineered antigen presenting cells. Eur J Immunol. 1994;24:2691-2698[Medline] [Order article via Infotrieve].
19.
Celluzzi C.
Peptide pulsed dendritic cells induce antigen specific CTL mediated protective tumour immunity.
J Exp Med.
1996;183:283-287
20.
Protti MP, Imro MA, Manfredi AA, Consogno G, Bellone M, Rugarli C.
Particulate naturally processed peptides prime a cytotoxic response against human melanoma in vitro.
Cancer Res.
1996;56:1210-1213 21. Nair SK, Boczkowski D, Snyder D, Gilboa E. Antigen presenting cells pulsed with unfractionated tumour-derived peptides are potent tumour vaccines. Eur J Immunol. 1997;27:589-597[Medline] [Order article via Infotrieve]. 22. Skipper JCA, Gulden PH, Hendrickson RC, et al. Mass spectrometric evaluation of HLA-A0201-associated peptides identifies dominant naturally processed forms of CTL epitopes from MART-1 and gp100. Int J Cancer. 1999;82:669-677[CrossRef][Medline] [Order article via Infotrieve]. 23. Storkus WJ, Zeh HJ, Salter RD, Lotze MT. Identification of T cell epitopes: rapid isolation of class I-presented peptides from viable cells by mild acid elution. J Immunother. 1993;14:94-103. 24. Plebanski M, Allsopp CE, Aidoo M, Reyburn H, Hill AV. Induction of peptide-specific primary cytotoxic T lymphocyte responses from human peripheral blood. Eur J Immunol. 1995;25:1783-1787[Medline] [Order article via Infotrieve].
25.
Altman JD, Moss PAH, Goulder PJR, et al.
Phenotypic analysis of antigen-specific T lymphocytes.
Science.
1996;274:94-96 26. Broughton CM, Sherrington P, Pender N, Tidd DM, Clark RE. Molecular status of individual CFU-GM colonies derived from chemotherapy-mobilised peripheral blood stem cells in chronic myeloid leukaemia. Genes Chromosomes Cancer. 1997;18:292-298[CrossRef][Medline] [Order article via Infotrieve].
27.
Nieda M, Nicol A, Kikuchi A, et al.
Dendritic cells stimulate the expansion of bcr-abl specific CD8+ T cells with cytotoxic activity against leukaemic cells from patients with chronic myeloid leukaemia.
Blood.
1998;91:977-983
28.
Choudhury A, Gajewski JL, Liang JC, et al.
Use of leukaemic dendritic cells for the generation of antileukemic cellular cytotoxicity against Philadelphia chromosome-positive chronic myelogenous leukaemia.
Blood.
1997;89:1133-1142
29.
Biernaux C, Loos M, Sels A, Huez G, Stryckmans P.
Detection of major bcr-abl gene expression at a very low level in blood cells of some healthy individuals.
Blood.
1995;86:3118-3122 30. Fegan C, Morgan G, Whittaker JA. Spontaneous remission in a patient with chronic myeloid leukaemia. Br J Haematol. 1989;72:594-595[Medline] [Order article via Infotrieve]. 31. Provan AB, Majer RV, Herbert A, Smith AG. Spontaneous remission of chronic myeloid leukaemia with loss of the Philadelphia chromosome. Br J Haematol. 1991;78:578-579[Medline] [Order article via Infotrieve].
32.
Musashi M, Abe S, Yamada T, et al.
Spontaneous remission in a patient with chronic myelogenous leukemia.
N Engl J Med.
1997;336:337-339
33.
Posthuma EFM, Falkenburg JHF, Apperley JF, et al.
HLA-B8 and HLA-A3 coexpressed with HLA-B8 are associated with a reduced risk of the development of chronic myeloid leukaemia.
Blood.
1999;93:3863-3865 34. Molldrem JJ, Lee PP, Wang C, et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukaemia. Nat Med. 2000;6:1018-1023[CrossRef][Medline] [Order article via Infotrieve]. 35. van Pel A, van der Bruggen P, Coulie PG, et al. Genes coding for tumor antigens recognized by cytolytic T lymphocytes. Immunol Rev. 1995;145:229-250[CrossRef][Medline] [Order article via Infotrieve]. 36. Marchand M, van Baren N, Weynants P, et al. Tumor regressions observed in patients with metastatic melanoma treated with an antigenic peptide encoded by gene MAGE-3 and presented by HLA-A1. Int J Cancer. 1999;80:219-230[CrossRef][Medline] [Order article via Infotrieve].
37.
Pinilla-Ibarz J, Cathcart K, Korontsvit T, et al.
Vaccination of patients with chronic myelogenous leukaemia with bcr-abl oncogene breakpoint fusion peptides generates specific immune responses.
Blood.
2000;95:1781-1787 38. Clarke SR. The critical role of CD40/CD40L in the CD4-dependent generation of CD8+ T cell immunity. J Leuk Biol. 2000;67:607-614[Abstract].
© 2001 by The American Society of Hematology.
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  H.-J. Kolb Graft-versus-leukemia effects of transplantation and donor lymphocytes Blood, December 1, 2008; 112(12): 4371 - 4383. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Goldman How I treat chronic myeloid leukemia in the imatinib era Blood, October 15, 2007; 110(8): 2828 - 2837. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Scheich, J. Duyster, C. Peschel, and H. Bernhard The immunogenicity of Bcr-Abl expressing dendritic cells is dependent on the Bcr-Abl kinase activity and dominated by Bcr-Abl regulated antigens Blood, October 1, 2007; 110(7): 2556 - 2560. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Volpe, A. Cignetti, C. Panuzzo, M. Kuka, K. Vitaggio, M. Brancaccio, G. Perrone, M. Rinaldi, G. Prato, M. Fava, et al. Alternative BCR/ABL Splice Variants in Philadelphia Chromosome-Positive Leukemias Result in Novel Tumor-Specific Fusion Proteins that May Represent Potential Targets for Immunotherapy Approaches Cancer Res., June 1, 2007; 67(11): 5300 - 5307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Sinai, R. E. Berg, J. M. Haynie, M. J. Egorin, R. L. Ilaria Jr, and J. Forman Imatinib Mesylate Inhibits Antigen-Specific Memory CD8 T Cell Responses In Vivo J. Immunol., February 15, 2007; 178(4): 2028 - 2037. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
 M. J. Mauro and R. T. Maziarz Stem Cell Transplantation in Patients With Chronic Myelogenous Leukemia: When Should It Be Used? Mayo Clin. Proc., March 1, 2006; 81(3): 404 - 416. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Gannage, M. Abel, A.-S. Michallet, S. Delluc, M. Lambert, S. Giraudier, R. Kratzer, G. Niedermann, L. Saveanu, F. Guilhot, et al. Ex Vivo Characterization of Multiepitopic Tumor-Specific CD8 T Cells in Patients with Chronic Myeloid Leukemia: Implications for Vaccine Development and Adoptive Cellular Immunotherapy J. Immunol., June 15, 2005; 174(12): 8210 - 8218. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zeng, M. W. Graner, S. Thompson, M. Marron, and E. Katsanis Induction of BCR-ABL-specific immunity following vaccination with chaperone-rich cell lysates derived from BCR-ABL+ tumor cells Blood, March 1, 2005; 105(5): 2016 - 2022. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Azuma, T. Otsuki, K. Kuzushima, C. J. Froelich, S. Fujita, and M. Yasukawa Myeloma Cells Are Highly Sensitive to the Granule Exocytosis Pathway Mediated by WT1-Specific Cytotoxic T Lymphocytes Clin. Cancer Res., November 1, 2004; 10(21): 7402 - 7412. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lee, S. N. Sait, and M. Wetzler Characterization of dendritic-like cells derived from t(9;22) acute lymphoblastic leukemia blasts Int. Immunol., October 1, 2004; 16(10): 1377 - 1389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. M. Cristea, S. J. Gaskell, and A. D. Whetton Proteomics techniques and their application to hematology Blood, May 15, 2004; 103(10): 3624 - 3634. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. M. Butt, L. Wang, H. M. Abu-Eisha, S. E. Christmas, and R. E. Clark BCR-ABL-specific T cells can be detected in healthy donors and in chronic myeloid leukemia patients following allogeneic stem cell transplantation Blood, April 15, 2004; 103(8): 3245 - 3245. [Full Text] [PDF]
|
|
|
|
|
|
K. Cathcart, J. Pinilla-Ibarz, T. Korontsvit, J. Schwartz, V. Zakhaleva, E. B. Papadopoulos, and D. A. Scheinberg A multivalent bcr-abl fusion peptide vaccination trial in patients with chronic myeloid leukemia Blood, February 1, 2004; 103(3): 1037 - 1042. [Abstract] [Full Text] [PDF]
|
|
|
|
|
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T. Tamura, H. J. Kong, C. Tunyaplin, H. Tsujimura, K. Calame, and K. Ozato ICSBP/IRF-8 inhibits mitogenic activity of p210 Bcr/Abl in differentiating myeloid progenitor cells Blood, December 15, 2003; 102(13): 4547 - 4554. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Rezvani, M. Grube, J. M. Brenchley, G. Sconocchia, H. Fujiwara, D. A. Price, E. Gostick, K. Yamada, J. Melenhorst, R. Childs, et al. Functional leukemia-associated antigen-specific memory CD8+ T cells exist in healthy individuals and in patients with chronic myelogenous leukemia before and after stem cell transplantation Blood, October 15, 2003; 102(8): 2892 - 2900. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Goldman and J. V. Melo Chronic Myeloid Leukemia -- Advances in Biology and New Approaches to Treatment N. Engl. J. Med., October 9, 2003; 349(15): 1451 - 1464. [Full Text] [PDF]
|
|
|
|
 |
|
 A. Admon, E. Barnea, and T. Ziv Tumor Antigens and Proteomics from the Point of View of the Major Histocompatibility Complex Peptides Mol. Cell. Proteomics, June 1, 2003; 2(6): 388 - 398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
 |
|
 W. A. E. Marijt, M. H. M. Heemskerk, F. M. Kloosterboer, E. Goulmy, M. G. D. Kester, M. A. W. G. van der Hoorn, S. A. P. van Luxemburg-Heys, M. Hoogeboom, T. Mutis, J. W. Drijfhout, et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia PNAS, March 4, 2003; 100(5): 2742 - 2747. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. V. Melo, T. P. Hughes, and J. F. Apperley Chronic Myeloid Leukemia Hematology, January 1, 2003; 2003(1): 132 - 152. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. E. Heslop, F. K. Stevenson, and J. J. Molldrem Immunotherapy of Hematologic Malignancy Hematology, January 1, 2003; 2003(1): 331 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
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|
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A. Burchert, S. Wolfl, M. Schmidt, C. Brendel, B. Denecke, D. Cai, L. Odyvanova, T. Lahaye, M. C. Muller, T. Berg, et al. Interferon-alpha , but not the ABL-kinase inhibitor imatinib (STI571), induces expression of myeloblastin and a specific T-cell response in chronic myeloid leukemia Blood, January 1, 2003; 101(1): 259 - 264. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
chain and 
