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Prepublished online as a Blood First Edition Paper on December 27, 2002; DOI 10.1182/blood-2002-06-1841.
IMMUNOBIOLOGY
From the Department of Immunology, Division of
Medicine, and Department of Haematology, Division of Investigative
Science, Faculty of Medicine, Imperial College at Hammersmith Hospital,
London; Transplantation Biology Group, Medical Research
Council (MRC) Clinical Sciences Centre (CSC), Imperial College at
Hammersmith Hospital, London; and The Ludwig Institute for
Cancer Research and Department of Biochemistry and Molecular Biology,
University College, London, United Kingdom.
Chronic myeloid leukemia (CML) is characterized by expression of
the BCR-ABL fusion gene that encodes a 210-kDa protein,
which is a constitutively active tyrosine kinase. At least 70% of the oncoprotein is localized to the cytoskeleton, and several of the most prominent tyrosine kinase substrates for p210BCR-ABL
are cytoskeletal proteins. Dendritic cells (DCs) are bone
marrow-derived antigen-presenting cells responsible for the initiation
of immune responses. In CML patients, up to 98% of myeloid DCs
generated from peripheral blood mononuclear cells are
BCR-ABL positive. In this study we have compared the
morphology and behavior of myeloid DCs derived from CML patients with
control DCs from healthy individuals. We show that the actin
cytoskeleton and shape of CML-DCs of myeloid origin adherent to
fibronectin differ significantly from those of normal DCs. CML-DCs are
also defective in processing and presentation of exogenous antigens
such as tetanous toxoid. The antigen-processing defect may be a
consequence of the reduced capacity of CML-DCs to capture antigen via
macropinocytosis or via mannose receptors when compared with DCs
generated from healthy individuals. Furthermore, chemokine-induced
migration of CML-DCs in vitro was significantly reduced. These
observations cannot be explained by a difference in the maturation
status of CML and normal DCs, because phenotypic analysis by flow
cytometry showed a similar surface expression of maturation makers.
Taken together, these results suggest that the defects in antigen
processing and migration we have observed in CML-DCs may be related to
underlying cytoskeletal changes induced by the p210BCR-ABL
fusion protein.
(Blood. 2003;101:3560-3567) Chronic myeloid leukemia (CML) is a
myeloproliferative disorder arising from the clonal expansion of an
altered pluripotent hematopoietic stem cell. The disease is
characterized by a t(9;22) chromosomal translocation that generates the
Philadelphia (Ph) chromosome. This contains a BCR-ABL fusion
gene, created by the juxtaposition of the ABL proto-oncogene
with the BCR gene, and encodes a 210-kDA
(p210BCR-ABL) fusion protein. p210BCR-ABL is a
constitutively active tyrosine kinase and probably initiates the
neoplastic process.1 As a result of the malignant
transformation, CML cells display abnormal adhesion properties to
stromal cells and extracellular matrix. In addition, cytoskeletal
abnormalities have been described in BCR-ABL-transformed
murine hematopoietic cells and CD34+ cells derived from the
peripheral blood of CML patients.2 These include an
increase in spontaneous cell motility, membrane ruffling, formation of
long actin extensions (filopodia), and an increase in the rate of
protrusion and retraction of pseudopodia on fibronectin-coated surfaces.
Dendritic cells (DCs) are bone marrow-derived antigen-presenting cells
(APCs) that play a central role in the development of both innate and
adaptive immune responses.3,4 DCs are heterogeneous and can be divided into 2 major populations based on
their origin, expression of surface markers, and function (myeloid and
plasmacytoid DCs).5 The state of activation of DCs is an important determinant of function, and this can be modified by local
cytokine production as well as by direct interaction with pathogens.
Immature DCs reside in peripheral tissues where they capture and
process antigens.6 Macropinocytosis has emerged as a key
mechanism of antigen capture by DCs, allowing substantial volumes of
the extracellular milieu to be engulfed and processed.7,8 Macropinosomes are believed to arise from deformations of the plasma
membrane, a process driven by constant remodeling of the actin cytoskeleton.
DC maturation is triggered by a number of signals, and this process is
characterized by a series of coordinated events. These include loss of
endocytic/phagocytic activity; up-regulation of costimulatory molecules
such as CD80, CD86, and CD40; expression of CD83; loss of adhesive
properties; changes in cytoskeletal organization that lead to
acquisition of high cellular motility; and changes in major
histocompatibility complex (MHC) class II-containing intracellular compartments. During maturation, DCs acquire the capacity
to migrate to the secondary lymphoid organs and prime naive T
cells.6
In CML patients, between 73% to 100% of monocyte-derived DCs are
positive for the chimeric BCR-ABL
gene.9-11 A number of groups have demonstrated that DCs
derived from both healthy subjects and patients with CML differentiated
and matured in culture in a similar way.11,12 However,
there are conflicting data regarding the ability of CML-DCs to
stimulate T cells compared with those from healthy
individuals.11,12
In this paper we describe differences in the organization of the actin
cytoskeleton in DCs generated from CML patients compared with normal
DCs. This observation led us to investigate whether these changes were
associated with functional abnormalities in CML-DCs in vitro. We
demonstrate that DCs generated from CML patients have a reduced
capacity to capture and process antigen when compared with DCs from
healthy controls. In addition, the capacity of CML-DCs to migrate is
also impaired.
The relevance of these findings to spontaneous and induced immunity to
CML antigens is discussed.
Patients
Generation of dendritic cells
Generation of DCs from CD34+ cells CD34+ cells derived from PBMCs of healthy individuals or CML patients were isolated by positive selection using an indirect immunomagnetic separation system (MACS CD34 Isolation Kit, Milteny Biotec, Sunnyvale, CA). The efficiency of the procedure was assessed by fluorescence-activated cell sorter (FACS) analysis, and the median purity of CD34+ cells after selection was higher than 90%, with cell viability more than 95% in all cases.CD34+ cells were cultured in RPMI 1640 supplemented with
10% fetal calf serum (FCS), 50 ng/mL GM-CSF, 30 ng/mL stem cell
factor (SCF) (First Link), 10 ng/mL TNF- Fluorescence in situ hybridization (FISH) The percentage of BCR-ABL fusion gene-positive DCs was determined by fluorescence in situ hybridization (FISH) using a dual-color DNA probe (Oncor, Gaithersburg, MD) containing a BCR-specific probe labeled with green fluorochrome and an ABL-specific probe labeled with red fluorochrome in accordance with the manufacturer's protocol. A total of 300 nuclei were counted for the reciprocal translocation between chromosome 9q34 and 22q11.Immunocytochemistry DCs were allowed to adhere to glass coverslips coated with 100 ng/mL fibronectin (Sigma, St Louis, MO) for 4 hours. The cells were then fixed with 4% buffered paraformaldehyde (Sigma) at 4°C. After 20 minutes, the slides were washed 3 times with PBS and the cells stained with 1 ng/mL tetramethyl rhodamine B isothiocyanate (TRITC)-conjugated phalloidin (Sigma) for 30 minutes at 37°C. The slides were washed 3 times with PBS, air dried, and mounted in mounting solution (DAKO, Cambridgeshire, United Kingdom).Wide field fluorescence microscopy Specimens were visualized with a Coolview 12 cooled CCD camera (Photonic Science, Newbury, United Kingdom) mounted over an Axiophot microscope (Zeiss, Welwyn Garden City, United Kingdom). A × 20 NA 0.6 objective and a standard epi-illuminating rhodamine fluorescence filter cube were used, and the 12-bit image data sets were generated as described elsewhere.13Confocal microscopy Specimens were visualized with an LSM 510 confocal microscope using a × 40 NA 1.3 Neofluar objective (Zeiss) aligned as described previously.14 The specimens were illuminated with nonsaturating levels of the 543 nm line from a HeNe laser. The fluorescent emissions were filtered out with a 560 nm long-pass filter where the 12-bit gray scale values in the final image data sets were proportional to the number of photons that emanated from the specimen.15 Each image data set was composed of a matrix of 1024 × 1024 pixels and represented the average of 16 passes of the illumination collected at 0.267 Hz.Phenotypic analysis of DCs by FACS analysis For cell surface marker analysis of in vitro-cultured DCs, the following monoclonal antibodies (mAbs), conjugated with either fluorescein isothiocyanate (FITC) or phycoerythrin (PE), were used: CD11 (DAKO), CD14 (Sigma), CD83 (Caltag, CA), CD86 (Caltag), HLA-DR (Sigma), and CCR1 (R&D Systems, Oxon, United Kingdom). Isotype controls were included. Cells were stained at a concentration of 1 × 105 in 100 µL. Samples were incubated for 30 minutes at 4°C with the conjugated antibody and then washed twice with PBS. The samples were analyzed using a FACSCalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA).Evaluation of mannose receptor-mediated endocytosis and macropinocytosis DCs (1 × 105 per tube) were suspended in serum-free RPMI 1640 media with either FITC-conjugated dextran (FITC-DX, Sigma) or Lucifer Yellow (LY, Sigma) at a final concentration of 1 mg/mL and incubated for 10 to 60 minutes. The cells were washed 3 times with cold PBS before FACS analysis. The background (cells pulsed at 0°C) was subtracted.8T-cell clones Tetanus toxoid (TT)-specific T-cell clones (TT1, CG1, and NIJ27) were derived from 3 individuals expressing DRB1*0701 by culturing PBMCs with 8 × 10 2 U/mL TT vaccine (EVANS
Vaccine Limited, Liverpool, United Kingdom) in RPMI/10%HS. TT-specific
T-cell lines were cloned by limiting dilution in U-bottomed 96-well
plates (Nunc) in the presence of irradiated PBMCs previously pulsed
with 10 µg/mL phytohemagglutinin (PHA) (Murex Biotech, Dartford,
United Kingdom) and recombinant IL-2 (rIL-2) (10 U/mL) (Roche,
Mannheim, Germany). HLA-DRB1*0701-restricted human T-cell clones (7P24
and 7P61) specific for influenza virus hemagglutinin peptide, residues
307-319 (HA 307-319) were generated as previously
described.16 T-cell clones were stimulated every 7 to 14 days with irradiated PBMCs previously pulsed with the relevant
cognate antigen in RPMI/10%HS and rIL-2. T-cell clones were used for
proliferative tests 1 or 2 weeks from their last stimulation.
T-cell proliferation assays Immature DCs were pulsed overnight with TT, HA307-319, or TT947-967 at the doses indicated in "Results." The following day, T-cell clones (1 × 104 per well) were cocultured with serial dilutions of antigen-prepulsed irradiated DCs in 96 U-bottomed well plates in a total volume per well of 200 µL. After 48 hours' incubation, tritiated thymidine (3HTdR) was added at 1 µCi (0.037 MBq) per well (Amersham Pharmacia, Bucks, United Kingdom). Thymidine incorporation was measured after a further 20 hours' incubation by a liquid-scintillation counter (Wallac).Mixed lymphocyte reactions (MLRs) were carried out in 96 U-bottomed well plates with a total volume per well of 200 µL. Sequential (1:3) dilutions of either irradiated iDCs or irradiated mDCs (1 × 103 to 1 × 105 per well) were performed in RPMI/10%HS. Purified CD4+CD45RA+ T cells (5 × 104 per well) were used as responders. After 5 days of culture, wells were pulsed with 1 µCi (0.037 MBq) per well 3HTdR. 3HTdR incorporation was measured after 20 hours by a liquid-scintillation counter. Assay for DC migration The migration of DCs was assessed in vitro in a 24-transwell cell culture plate (Costar, New York). DCs (2 × 105) were added to the 8-µm pore filter (Becton Dickinson). The lower chamber contained 1 mL RPMI/10%HS. MIP-1 (10 ng/mL) was added to
the lower chamber, and the number of DCs migrating to the lower chamber
after 2, 5, and 24 hours was assessed.17
CML-DCs show altered morphology and F-actin distribution It is well established that the p210BCR-ABL oncoprotein binds to actin, and a number of studies have demonstrated associated cytoskeletal changes.2 Several of the most prominent tyrosine kinase substrates for p210BCR-ABL are cytoskeletal proteins (tensin, talin, vinculin, paxillin, and p125FAK), and the colocalization of ABL with F-actin, paxillin, and vinculin has been described.18,19 In CML patients, between 73% to 100% of monocyte-derived DCs are positive for the chimeric BCR-ABL gene.9-11 To investigate the effects of the BCR-ABL gene on cytoskeletal organisation in DCs, we have compared the distribution of F-actin in myeloid DCs generated from CML patients and healthy volunteers. DCs were generated as described in "Patients, materials, and methods." The purity of monocyte (CD14+ cells) in the adherent populations was more than 98% in all DC preparations from healthy individuals and CML patients (data not shown). DCs were allowed to adhere to fibronectin-coated coverslips and incubated for 4 hours. The morphology of CML-DCs was very different from that of DCs generated from healthy volunteers (Figure 1). Most normal DCs assumed a polarized morphology (Figure 1A,D) and developed multiple small F-actin foci representing podosomes (Figure 1G,J). Podosomes are sites of adhesion to the extracellular matrix and have previously been described in osteoclasts, DCs, macrophages, and some oncogenically transformed fibroblasts.20-22 In contrast, CML-DCs remained more rounded, and the percentage of cells with a similar morphology to normal DCs was dramatically reduced (2% to 30%) (Figure 1B,C,E,F,H,I,K,L). Clusters of podosomes were localized primarily to one region in most (more than 90%) normal DCs, consistent with their role at the leading edge of migrating cells, whereas in CML-DCs this clustering was either absent or present in a reduced number of cells (less than 30%). In particular, DCs from 2 CML patients, patient 2 (Figure 1C,I) and patient 3 (Figure 1E,K), failed to spread significantly on fibronectin and lacked podosomes. Similar differences in morphology and F-actin distribution of normal DCs and CML-DCs were observed after adherence of DCs to gelatin-coated coverslips and lipopolysaccharide (LPS) stimulation (data not shown). We postulated that at least some of the cells with normal DC morphology in samples from patients were likely to be those that lack the BCR-ABL translocation. The same DC preparations were also analyzed for the presence of the BCR-ABL gene using FISH. The percentage of BCR-ABL-positive cells in each sample was 80% for patient 1 (Figure 1B,H), 92% for patient 2 (Figure 1C,I), 89% for patient 3 (Figure 1E,K), and 79% for patient 6 (Figure 1F,L).
DCs from CML patients are defective in antigen processing We hypothesized that BCR-ABL-induced alterations in DC morphology could interfere with the endocytic capacity of CML-DCs. Therefore, antigen processing was assessed.Immature DCs generated from DRB1*0701 CML patients and DRB1*0701
healthy volunteers were pulsed overnight with an optimal concentration
of intact TT (0.08 IU/mL) and their capacity to stimulate 3 different
DRB1*0701-restricted, TT-specific T-cell clones compared (Figure
2A-B, TT1; Figure 2B, CG2; Figure 2C, JNI27). Identical results were obtained at all doses of TT tested (data
not shown). CML-DCs were incompetent at inducing proliferation of
T-cell clones compared with DCs derived from healthy controls. These
data were confirmed on cells from 4 additional CML patients (data not
shown). The lack of antigen-processing capacity was independent of the
morphology of the CML-DC preparation. To determine whether these
results reflected a defect in antigen presentation or antigen
processing, T cells specific for a defined peptide of hemagglutinin
(HA) also restricted by DR7 were used. The same DC preparations were
pulsed with an optimal concentration (10 µg/mL) of either HA307-319
or TT947-967 peptide, and the response of DRB1*0701-restricted T-cell
clones was evaluated. In contrast to the T-cell responses to the intact
antigen TT, CML-DCs derived from patient 1 were as effective as normal
DCs in presenting an optimal concentration of peptide to
HA307-319-specific T-cell clones (Figure 2D). The comparable capacity
to present HA peptide was confirmed with DCs derived from 4 additional
CML patients (data not shown). Furthermore, the proliferation of the
T-cell clone JNI27, specific for peptide TT947-967, was similar in
response to peptide-pulsed DCs derived from healthy volunteers and from CML patient 7 (Figure 2F). However, a reduction in T-cell response was
seen when DCs derived from patient 2, for which the morphology was
grossly abnormal (Figure 1C), were compared with normal DCs (Figure 2E).
To further analyze this antigen-processing defect, DCs were generated
from CD34+ cells and compared with monocyte-derived DCs.
CD34+ cells from patients 1, 7, and 8 were positively
selected, and their capacity to present TT to T-cell clone TT1 was
evaluated (Figure 3). CML-DCs derived
from both the adherent population and CD34+ cells were
unable to present TT (Figure 3A,C). In contrast, DCs from either origin
from healthy individuals were very effective in processing and
presenting intact TT. As shown previously for monocyte-derived DCs, the
capacity to present the optimal concentration of peptide HA307-319 to
clone 7P24 was maintained in CD34+ CML-DCs and was similar
to the capacity of DCs derived from a healthy individual (Figure
3B,D).
DCs from CML patients are defective in presentation of limited amount of peptide The decreased ability of CML-DCs to present peptide was further investigated using DCs pulsed with varying doses of peptide. No significant difference in the capacity of DCs generated from patient 1 and normal healthy volunteers to present peptide was observed at the concentration range analyzed (1 to 0.01 µg/mL) (Figure 4A-C).Of the patients tested, DCs derived from patient 1 showed the most similar morphology to normal DCs (Figure 1B,H). In contrast, DCs generated from patient 2 were morphologically very different from normal DCs (Figure 1C), and a reduced capacity to present peptide was observed at concentration 1 µg/mL or less (Figure 4D-F). The heterogeneity of CML-DCs in their capacity to present peptide was
further investigated using DCs as stimulators and allogeneic naive T
cells as responders in a mixed lymphocyte reaction (MLR) (Figure
5). Mature CML-DCs derived from patients
5 and 3 and immature CML-DCs from patient 5 were compared with normal
DCs, and they were equally effective at inducing a T-cell response at
all DC numbers analyzed (1 × 103 to
3 × 104) (Figure 5A-B). In contrast, mature DCs derived
from patient 4, which showed a very similar morphology to those
from patient 2 (Figure 1C and data not shown), were less efficient
in stimulating allogeneic T cells compared with normal DCs (Figure
5C).
CML-DCs are defective in mannose receptor-mediated endocytosis and macropinocytosis To dissect the mechanisms involved in the antigen-processing defect, we compared the capacity of CML-DCs and DCs generated from healthy individuals to capture antigen via mannose receptors or macropinocytosis by coincubating DCs with FITC-DX or LY, respectively.8 At each time point analyzed the mean fluoresence intensity was lower than that observed with DCs generated from healthy individuals (Figure 6). Thus, the capacity of DCs generated from CML patients to capture antigen assessed by these methods was reduced compared with DCs from healthy controls, and this defect was independent of the phenotype of the CML-DCs described in Figure 1. However, DCs derived from 2 of the 15 CML patients analyzed were equally effective at capturing antigen when compared with normal DCs. In contrast, the capacity of DCs to process antigen was analyzed in 1 of these 2 patients (patient 7), and this was impaired (Figure 2C). This result suggests that a defect in antigen capture is not the only mechanism underlying the functional abnormalities observed.
DCs from CML patients and from healthy volunteers have comparable expression of maturation markers The capacity to capture antigen effectively is a characteristic of immature DCs, and thus we investigated the possibility that DCs from CML patients exhibited diverse differentiation characteristics when generated from monocyte precursors compared with DCs generated from healthy individuals. CML and normal DCs were incubated with different antibodies, and the expression of different markers was analyzed by flow cytometry. As expected, both DC preparations were CD11c+ and CD14. The 2 preparations of DCs
were similar in their maturation state as evaluated by the expression
of CD80, CD86, and HLA-DR (Figure 7).
All these markers were up-regulated following DC maturation (data not shown). The migratory ability of CML-DCs to a chemokine gradient is reduced compared with normal DCs The rounded morphology and altered actin distribution of CML-DCs suggested that they were less migratory than normal DCs (Figure 1). We therefore investigated the ability of CML-DCs to migrate to a chemokine gradient. The capacity of CML-DCs to migrate in the presence of MIP-1 was dramatically reduced when compared with normal DCs (Figure
8A-B). The difference in the
migratory capacity of CML-DCs compared with DCs from healthy
individuals was not due to a differential expression of CCR1, which
binds to MIP-1 (Figure 8C-D).
A significant statistical difference between the transmigration capacity of DCs derived from CML patients and healthy individuals was observed at each time point analyzed. Using multiple linear regression analysis, it was shown that significantly fewer CML-DCs transmigrated over time than DCs derived from healthy individuals (P < .001).
The results presented in this study show that DCs generated from CML patients have a decreased ability to spread and polarize and have fewer podosomes compared with normal DCs. This correlates with the impaired capacity of CML-DCs to migrate, capture, and process antigen. DCs derived from all the patients presented in Table 1 demonstrated similar behavior in the functional experiments. A summary of the experiments performed using DCs derived from each patient is shown in Table 1. Ph chromosome-positive CML originates in a single pluripotent hematopoietic stem cell, which serves as a precursor for granulocytic, erythroid, megakaryocytic, and monocyte/macrophage lineages.1 DCs are known to develop from CD34+ progenitors, monocytes, or lymphoid precursors. FISH analysis of monocyte-derived DCs generated from the peripheral blood of patients with CML has displayed the presence of the BCR-ABL fusion gene in 73% to 100% of DCs.9-11 This observation is consistent with the view that most myeloid DCs are part of the CML clone while a proportion are of normal origin. A broader range of BCR-ABL positivity (3% to 100%) has been reported in CML-DCs generated from bone marrow CD34+ cells.12,23 The morphology and distribution of F-actin in DCs generated from CML patients was very similar to that seen in DCs generated from patients lacking Wiskott-Aldrich syndrome protein (WASp), in which the formation of filopodia and podosomes is severely affected.20 The role of podosomes in cell spreading and migration and the mechanism of their formation or dissolution are not yet clearly understood, although they are generally localized to the front of migrating cells and may play a role in modifying the extracellular matrix to enhance migration. Altered cytoskeletal organization is one of the biologic consequences
of BCR-ABL transformation, and yet studies have yielded equivocal results. Many reports using BCR-ABL-transformed
cells or bone marrow from CML patients have documented a diminished capacity to adhere to stromal layers or to fibronectin and its proteolytic fragments.24-26 Futhermore, CML cells have
been reported to have increased adhesion to laminin and collagen type
IV.26 However, other studies have shown that the capacity
of CML cells to adhere to stromal surfaces was time
dependent.24 Short-term adhesion to fibronectin was
increased by BCR-ABL transformation, whereas longer-term
adhesion was decreased.24 More recently, Wertheim et al
have used a cell detachment device to measure adhesive changes and
reported that P210BCR-ABL expression increased adhesion to
fibronectin nearly 2-fold following transfection of the myeloblastic
cell line, 32D, compared with control vector.27 In
addition, Salgia et al described an increase in the level of
spontaneous motility in both BCR-ABL-transformed cells and
primary progenitor cells from patients with CML. Specifically, they
found an increased staining for F-actin and an enhanced rate of
formation and retraction of actin-containing protrusions such as
pseudopodia and filopodia.2 The active motility and
pseudopodia formation of BCR-ABL-transformed cells was
observed on fibronectin-coated as well as uncoated surfaces.
Altogether, these data suggest that the accumulation of the
p210BCR-ABL oncoprotein in the cytoskeleton may
affect a variety of important cell functions. This conclusion led us to
study 2 of the major functions of DCs DC maturation is intimately linked with their migration from the peripheral tissues to the draining lymphoid organs where DCs present antigen and initiate the adaptive phase of the immune response. In this study, the chemotactic migratory properties of CML-DCs and normal DCs were compared. The proportion of DCs that migrated was small, and a significant and consistent difference was observed between CML-DCs and normal DCs. In addition, the comparable levels of CCR1 expression measured in the 2 DC preparations suggests that the defect in the ability of CML-DCs to migrate was not due to a reduced sensitivity to chemokine. This finding appears to contradict observations of an increased level of spontaneous migration in BCR-ABL-transformed cell lines.2 However, Salgia et al2 did not investigate migration of CML-DCs, which may respond differently to BCR-ABL transformation compared with other cell types. In addition, we have measured migration through transwell filters, whereas Salgia et al2 used time-lapse videomicroscopy to monitor cell behavior. In agreement with our data are reports from a number of groups in which the function of polymorphonuclear leukocytes (PMNLs) from CML patients has been analyzed. Abnormalities in chemotaxis, fluid phase pinocytosis, phagocytosis, and degranulation in response to the chemotactic peptide formyl-methionyl-leucyl-phenylanine (fMLP) in PMNLs derived from CML patients have been described.28-30 One of the major findings of this study is that CML-DCs are defective in antigen processing. The use of 3 different T-cell clones specific for TT and derived from 3 individuals may suggest that the processing defect is general rather than specific for a particular T-cell epitope. This idea is further supported by the results demonstrating a decreased capacity to capture antigen via mannose receptors or macropinocytosis measured using FITC-dextran and Lucifer Yellow, respectively, in 13 of 15 patients tested. The results observed in one patient in whom the presentation of intact TT was impaired (patient 7) were not accompanied by a reduced ability to capture antigen, suggesting that the defect in antigen capture is not the only mechanism underlying the functional abnormalities described. Three previously published papers have shown that CML-DCs are able to capture, process, and present antigen. However, the sensitivity to detect antigen processing defects may have been lower in those reports because responder cells were purified polyclonal resting T lymphocytes rather than a homogeneous population of established antigen-specific T-cell clones.11,12,31 In addition, Chen et al do not provide a comparison between CML-DCs and DCs derived from healthy individuals.31 The role of cytoskeletal proteins in antigen capture and processing is not yet completely understood. Macropinocytosis has emerged as a key mechanism of antigen capture by DCs and is believed to arise from deformations of the plasma membrane known as ruffles driven by constant remodeling of the actin cytoskeleton.7,8 To distinguish between antigen processing and antigen presentation, T-cell responses to well-defined peptides were studied. T-cell proliferation to the presentation of an optimal concentration of either HA307-319 peptide or TT947-967 was similar between CML-DCs and normal DCs. In contrast, for some of the CML patients T-cell responses to limiting doses of peptide presented by CML-DCs were impaired. These results were particularly evident with CML-DCs that had a significantly abnormal morphology, and they were further confirmed using the same DC preparations as stimulators in MLRs. Altogether, these results are consistent with the observation that DCs actively polarize F-actin and fascin upon clustering with T cells and suggest an important role for the DC cytoskeleton in the establishment of the immunologic synapse.32 An increase in the incidence of infection has not been reported in patients with CML, and a number of reasons may underlie this. Estimates of blood DC numbers range from 0.1% of mononuclear cells (MNCs) to 1% of MNCs.33-35 Recently, Mohty et al reported a tremendous expansion (1.36% to 41%) of myeloid DCs in 59% of patients with AML. DC numbers have not been recorded in patients with CML, but in light of the expansion of myeloid and monocytic lineages, an increase in the number of myeloid DCs would be expected. Such an increase in vivo may serve to compensate for the functional impairment observed in vitro in DCs generated from patients with CML.36 Furthermore, plasmacytoid DCs have been shown to play an important role in the host defense against viral infection. Plasmacytoid DCs are of lymphoid origin,6 and lymphocytes in CML patients are rarely derived from a Ph+ progenitor.37 Altogether, these data raise questions concerning the suitability of
CML-DCs for vaccination. Much depends on whether or not their ability
to present intracellular proteins, such as BCR-ABL, with MHC
class II molecules is intact. The data of Choudhury et al38 suggest that this may be the case; however, much of
their work was focused on CD8+ T-cell responses. This issue
is the subject of further investigation. If a defect in the
presentation of intracellular proteins does exist, this may be
reversible by in vitro treatment with interferon-
We thank Ms Nicola Foot and Emma Walker for their help with the FISH analysis and separation of CD34+ cells, respectively.
Submitted June 21, 2002; accepted December 17, 2002.
Prepublished online as Blood First Edition Paper, December 27, 2002; DOI 10.1182/blood-2002-06-1841.
Supported by an MRC Component Grant. K.C. is the holder of an MRC CSC Training Fellowship.
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: Giovanna Lombardi, Department of Immunology, Faculty of Medicine, Imperial College at Hammersmith Hospital, DuCane Road, London, W12 ONN, United Kingdom; e-mail: g.lombardi{at}ic.ac.uk.
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Top
Abstract
Introduction
) and from CML patients
(
): patient 1 (A,D), patient 2 (B,E), and patient 7 (C,F). Immature
HLA-DR7-expressing DCs were pulsed overnight with either whole TT
(0.008 IU/mL) (A-C) and HA307-319 peptide (10 µg/mL) (D-E) or
TT947-967 peptide (10 µg/mL) (F). Different numbers of irradiated and
prepulsed DCs were cultured in the presence of either TT-specific
T-cell clones ([A] clone TT1; [B] clone TT1, solid lines, and clone
CG1, dotted lines; [C] clone JNI27) or HA307-319-specific T-cell
clone ([D-E] clone 7P61) or TT947-967-specific T-cell clone ([F]
clone JNI27). After 2 days, 3HTdR was added and
thymidine incorporation was measured after 20 hours. The data are
expressed as counts per minute (cpm) ± standard deviations (SD).
Results are representative of 15 experiments obtained with DCs derived
from different patients as indicated in Table 
[A-B]; dotted lines
[C-D]) and from CML patients (
[A-B]; solid lines [C-D]):
patient 1 (A,C) and patient 6 (B,D). (A-B) Immature DCs were added to
the 8 µm pore size filter in a transwell cell culture chamber. In the
lower chamber MIP-1
namely, their ability to migrate
and their capacity to capture and process antigen.