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III. Medizinische Klinik, Klinikum rechts der Isar, Technische Universität Müchen, Munich, Germany; and
I Medizinische Klinik, Section of Pathophysiology, Johannes Gutenberg Universität, Mainz, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The growing number of identified tumor-associated Ags serve as potential targets for adoptive therapy of Ag-specific T cells (for review, see Refs. 8, 9, 10, 11, 12). The human epidermal growth factor receptor 2, also known as HER2,3 neu, HER2/neu, and c-erbB2, represents a tumor-associated Ag that is an appealing immunological target for the following reasons (for review, see Ref. 13). The HER2 gene is selectively amplified, and the resulting HER2 protein is overexpressed by malignant cells, in contrast to the corresponding normal cells expressing only low levels of HER2 (14). Adenocarcinomas of different tissue origin overexpress HER2, such as adenocarcinomas of the breast, ovary, stomach, and lung (for review, see Ref. 15). In addition, HER2 overexpression is stable over time and at multiple metastatic sites (16). Some patients already have a pre-existing T cell and Ab response to HER2, indicating the immunogenicity of the molecule (17, 18). The HER2 gene encodes for a 185-kDa transmembrane protein, and portions of this protein are likely to be available to both class I and II Ag processing pathways. Naturally processed peptide epitopes recognized by autologous CD8+ cytotoxic T cells have already been identified for HLA-A*0201 and HLA-A*0301 (19, 20, 21, 22, 23, 24). As amplification of the proto-oncogene HER2 contributes to the malignant phenotype of the tumor (25), HER2-overexpressing tumors might not be able to escape from an HER2-targeted immunotherapy through immunoselection of Ag-loss variants, as observed after vaccination with peptides derived from melanoma-associated differentiation Ags (26, 27). Studies in an animal model have shown that vaccines with rat neu peptides can generate rat neu-specific T cell immunity (28). In the first clinical trial, vaccination with HER2-derived peptides elicited HER2-specific T cell immunity in women with HER2-overexpressing breast cancer (29). Beside the attempts to induce HER2-specific T cell immunity, Ab-based immunotherapy regimens have been developed to target the HER2 Ag. In clinical phase I/II trials, infusion of a humanized anti-HER2 mAb induced impressive tumor regression in some patients with HER2-overexpressing breast cancer (30, 31).
Attempts to transfer HER2-reactive CTL have been hampered by the difficulty to generate and clone autologous CTL directed against HER2. As preliminary studies of the development of an adoptive transfer of HER2-specific T cells, the current experiments examined whether HER2-reactive CTL can be generated, cloned, and expanded in vitro. Given that one of the major functions of dendritic cells (DC) is to initiate T cell responses (for review, see Refs. 32 and 33), DC were used as professional APCs for stimulating autologous T cells in vitro. The DC were genetically engineered to express the HER2 Ag through infection with a retrovirus encoding the HER2 gene. As retroviral transduction requires dividing cells, it was necessary to develop a system in which proliferating DC precursors could be efficiently transduced and further differentiated into mature DC. These retrovirally transduced DC expressed antigenic peptides and were able to elicit HER2-specific CTL that could be cloned and expanded in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following cell lines were obtained from American Type Culture Collection (Manassas, VA): ovarian cancer cell line SKOV3 (HLA-A*0201-, H2N++), breast cancer cell lines SKBR3 (HLA-A*0201-, H2N++) and MCF7 (HLA-A*0201+, H2N+), and the fibroblast cell line NIH-3T3. The EBV-transformed B cell line MZ-EBV1257 (HLA-A*0201+, H2N-) was generated as previously described (34). The HLA-A*0201+ transfectant cell line SKOV3tA*0201 was a gift from M. L. Disis (University of Washington, Seattle, WA). The HLA-A*0201+, TAP-deficient cell line, T2, was provided by P. Cresswell (Yale University, New Haven, CT). The amphotropic producer cell line GP+ envAM-12 was provided by B. Gansbacher (Technical University, Munich, Germany).
Tumor cell lines, EBV-transformed B cells, and T2 cells were cultured in RPMI 1640 (Life Technologies, Paisley, Scotland) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine. Packing cell line GP+ envAM-12 and NIH-3T3 cells were maintained in DMEM (Life Technologies) supplemented with FCS, penicillin, streptomycin, and L-glutamine at the concentrations stated above.
Synthetic peptides
Peptides were synthesized by standard solid-phase chemistry on a multiple peptide synthesizer and purified by reverse phase HPLC (MWG AG Biotech, Ebersberg, Germany). The purity of the peptides was >90%, as indicated by analytical HPLC. Lyophilized peptides were diluted in PBS/2% DMSO (Serva Electrophoresis, Heidelberg, Germany) and stored at -20°C.
HER2369–377-specific CTL line
Peptide-specific CTL were generated by repetitive stimulation with mature CD83+ DC as APC. DC were generated from monocytes using a protocol recently published by Jonuleit et al. (35). Monocyte-derived DC were incubated with 10 µg/ml HER2369–377 for 2 h at room temperature and then cocultured with autologous PBMC in RPMI 1640 medium supplemented with 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, and 5% autologous serum. The culture medium was further supplemented with 5 ng/ml rIL-7 (PharMingen International, Hamburg, Germany) on day 1 and 100 U/ml natural human IL-2 (nIL-2; Biotest Pharma, Dreieich, Germany) on day 3. Responding T cells were restimulated with peptide-pulsed DC at weekly intervals in the presence of nIL-2 and rIL-7. The ratio of stimulator to responder cells was 1:20 for priming and 1:50 for restimulation. Specificity analyses of proliferating T cells were performed after three restimulations.
Retroviral vector and virus production
The retroviral vector (NAPTK) used in this study was a gift from B. Gansbacher (Technical University of Munich). NAPTK is derived from the genome of Moloney murine leukemia virus containing the bacterial neomycin resistance (neo) gene as a selection marker and the herpes simplex virus thymidine kinase (HSV-TK) promoter (36). The plasmid pSC11-H2N, containing a cDNA encoding the human HER2, was provided by Dr. G. Spies (University of Washington, Seattle, WA). The cDNA encoding the full-length HER2 or the extracellular domain (ECD) of HER2 was cloned into a unique SnaBI restriction site of the retroviral vector NAPTK, and the resulting retroviral vector construct was transfected into the helper-free amphotropic packing cell line GP+ envAM-12 (37) using a liposomal transfection reagent (N-[1-(2,3-dioleoyloxylpropyl]-N,N,N-trimethylammonium methylsulfate, Roche, Mannheim, Germany). Colonies were isolated by neomycin selection (G418, Sigma-Aldrich, Steinheim, Germany) and expanded. Supernatants of cloned packing cells were harvested, filtered (0.45 µm pore size), and tested for the presence of virus. Viral titration was performed on NIH-3T3 cells in the presence of neomycin. Supernatants of cell clones secreting a high virus titer (105–106 neo CFU/ml) were used to infect proliferating DC precursors derived from CD34+ hemopoietic progenitor cells (HPC).
Generation of DC from CD34+ HPC
DC cultures were generated from CD34+ HPC
derived from peripheral blood stem cell collections from donors
following mobilization with G-CSF (38).
CD34+ HPC were isolated using positive selection
with an immunomagnetic bead system (Milteny, Bergisch Gladbach,
Germany). The purity of recovered CD34+ HPC was
determined by flow cytometric analysis to be 85–95%. After
purification, CD34+ cells were cryopreserved in
RPMI 1640 containing 10% DMSO (Serva Electrophoresis).
CD34+ cells were cultured at
106 cells/well in six-well plates (Greiner
Labortechnik, Oberschleissheim, Germany) using 3 ml of X-VIVO 15 medium
(BioWhittaker, Walkersville, MD) supplemented with 2 mM
L-glutamine, 100 IU/ml penicillin, 100 µg/ml
streptomycin, and 1% human AB serum (Life Technologies, Grand Island,
NY) in the presence of the following cytokines at the indicated
concentrations: 50 ng/ml stem cell factor (SCF; R&D Systems,
Minneapolis, MN); 75 ng/ml fetal liver tyrosine kinase 3-ligand
(Flt3-L; PeproTech EC, London, U.K.); 10 ng/ml HyperIL-6
(39); 50 ng/ml IL-4, 0.5 ng/ml TGF-ß1, and 2 or 100
ng/ml TNF-
(all from Strathmann-Biotech, Hannover, Germany); and 100
ng/ml GM-CSF (Novartis, Nürnberg, Germany).
Culture conditions for generating DC from CD34+
HPC consisted of three phases: expansion (days 0–7), differentiation
(days 8–26), and maturation (days 27–28). During the expansion phase,
extensive proliferation of primitive progenitor cells was induced in
the presence of HyperIL-6, SCF, Flt3-L, and TGF-ß1 (days 0–7).
Proliferating HPC were differentiated into immature DC with IL-4,
GM-CSF (days 8–26), and 2 ng/ml TNF- (days 15–26) added to the
cytokines used for expansion. Finally, maturation of DC was induced by
addition of high doses of TNF- (100 ng/ml) during the last 2 days
(days 27 and 28). Following 28 days of culture, cells were harvested,
phenotyped, and used for T cell stimulation.
Retroviral transduction of DC derived from CD34+ HPC
Proliferating CD34+ HPC were transduced twice with retroviral supernatant during the expansion phase. Cultured HPC were harvested on day 6, and 3 x 105–106 cells were resuspended in 3 ml of medium containing 1.5 ml of retroviral supernatant and 1.5 ml of X-VIVO medium supplemented with the cytokines necessary for cell expansion: HyperIL-6, SCF, Flt3-L, and TGF-ß1. HPC were transduced overnight in the presence of 4 µg/ml Polybrene (Sigma, Deisenhofen, Germany). On day 7 of the expansion phase, retroviral transduction was repeated to enhance the transduction efficiency (40, 41, 42).
Polymerase chain reaction
DNA from retrovirally transduced and nontransduced DC was isolated according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Equal amounts of DNA (100 ng) from retrovirally transduced and nontransduced DC were used for each PCR. As positive control, DNA was isolated from retroviral supernatant derived from the HER2-transfected packing cell line. The positive control contained less DNA, because equal amounts of DNA would have resulted in an overloaded gel. Primers used for amplification of HER2 were 5'-gagccgcgagcacccaagtgtgca-3' and 5'-ttgcagcgggcacagccaccggca-3'. PCR was performed for 33 min at 94°C, for 1.30 min at 58°C, and for 1.30 min at 72°C for 35 cycles, followed by a final extension time of 8 min at 72°C. The PCR product was resolved on a 1.5% agarose gel.
Western blot analysis
Cell lysates were made from DC, retrovirally transduced DC, and SKOV3tA*0201 cells, as recently described (28). Equal amounts of protein were loaded for the retrovirally transduced DC and the nontransduced DC, but lesser amounts for the HER2-overexpressing cell line SKOV3 which served as positive control. Protein samples were separated by SDS-PAGE and transferred to nitrocellulose. The HER2 protein was identified using the mAb c-neu-Ab3 recognizing the intracellular domain of HER2 (Oncogene Science, Uniondale, NY) as primary Ab and rabbit anti-mouse peroxidase-conjugated Fab2 fragment (Amersham Pharmacia Biotech, Freiburg, Germany) as secondary Ab. The blots were developed using a chemiluminescent reaction (ECL, Amersham).
Flow cytometric analysis
DC or T cells were harvested, washed with PBS, and resuspended in PBS containing 0.5% BSA. Because Fc receptors are highly expressed on DC, the DC were first incubated with Fc receptor-blocking reagent (Milteny) for 45 min at 4°C to reduce nonspecific fluorescence. Phenotypic analyses were performed by flow cytometry using saturating concentrations of the PE-conjugated mAb against following Ags: HLA-DR, CD80, CD86, CD54, CD40, CD19, and CD56 (all from Becton Dickinson, Mountain View, CA); CD83 (Coulter Immunotech, Miami, FL); and CLA, CD1a, CD14, CD34, CD3, CD4, and CD8 (all from PharMingen). Conjugated isotype-matched mAb (all from Becton Dickinson) were used as controls. For phenotype analyses all cells were gated with the exception of dead cells, which were excluded. For detection of HER2, cells were sequentially incubated with the unconjugated mAb c-neu-Ab6 recognizing the extracellular domain for HER2 (Oncogene Science) and FITC-conjugated F(ab')2 goat anti-mouse Ig (Zymed, San Francisco, CA). Fluorescence analyses were performed on an EPICS Elite ESP flow cytometer (Coulter Electronics, Hialeah, FL).
Generation of HER2-specific CTL lines and clones using retrovirally transduced DC
DC retrovirally transduced with the HER2 gene were seeded
together with autologous PBMC in RPMI 1640 medium supplemented with 100
IU/ml penicillin, 100 µg/ml streptomycin, 2 mM
L-glutamine, and 5% autologous serum. DC (2 x
104) were cocultured with 7.5 x
105 T cells in one well of a 24-well flat-bottom
plate (Greiner Labortechnik). To the DC-T cell coculture, 5 ng/ml rIL-7
was added on day 1, and 100 U/ml nIL-2 was added on day 3.
Proliferating T cells were restimulated weekly using HER2-transduced DC
at a 1:50 stimulator to responder ratio. Following three to five cycles
of weekly stimulations, the HER2 specificity of T cells was analyzed by
measuring Ag-specific cytotoxicity and IFN-
production.
HER2-specific polyclonal T cells were cloned by limiting dilution
according to the protocol described by Greenberg et al. (43, 44). Briefly, T cells were plated at 0.3 cells/well in 96-well
round-bottom plates with 30 ng/ml anti-CD3 mAb (OKT-3; Janssen,
Cilag, Neuss, Germany), 5 x 104/well
allogeneic irradiated (30 Gy) PBMC, 104/well
irradiated (80 Gy) MZ-EBV1257, and 50 U/ml rIL-2 (Chiron, Emeryville,
CA). Proliferating T cell clones were screened for lytic activity in a
microtoxicity assay using SKOV3tA*0201 as a positive control and SKBR3
as a negative control. HER2-specific T cell clones were expanded in
flasks (T30; Greiner Labortechnik) in the
presence of anti-CD3 mAb, allogeneic PBMC, and allogeneic
EBV-transformed B cells as previously described (5).
Enzyme-linked immunospot (ELISPOT) assay
The presence of IFN--producing, HER2-specific T cells was
assessed in an ELISPOT assay, recently described by Herr et al.
(45). Stimulator and responder cells were seeded in a
96-well nitrocellulose filter plate coated with mouse anti-human
IFN- capture Ab Dm1.2 (Mabtech, Nacha, Sweden). Autologous DC
(20,000/well), HLA-matched tumor cells (20,000/well), or peptide-pulsed
T2 cells (20,000/well) were used as stimulator cells and cocultured
with CTL (1,000/well) overnight (12–18 h) at 37°C with 5%
CO2. Cells were then removed by washing with PBS,
and the presence of IFN- produced by Ag-specific T cells was
detected by sequential addition of biotinylated mouse anti-human
IFN- (Mabtech) and alkaline phosphatase-conjugated streptavidin
(Mabtech). The number of stained spots corresponding to the
IFN--producing cells was counted using a dissecting microscope
coupled with a computer-assisted video image analysis (Zeiss,
Göttingen, Germany). The data in the figures refer to the mean of
three replicates. SDs were generally within 5–20% of the
mean.
Chromium release assay
Cytolytic activity was determined as previously described (46). Briefly, 106 T2 cells were labeled in 100 µl of FCS with 200 µCi/ml 51Cr (ICN Biochemicals, Irvine, CA) for 1.5 h at 37°C and then loaded with 10 µg/ml peptide for 1 h at room temperature. Tumor cell lines and DC were labeled with 100 µCi/ml 51Cr for 1 h at 37°C. 51Cr-labeled target cells and graded doses of T cells were given in 200 µl of T cell medium/well of a V-bottom 96-well tissue culture plate (Costar, Cambridge, MA). For inhibition experiments, mAb W6/32, an Ab against a common MHC class I determinant, or mAb MA2.1 recognizing HLA-A*0201 was added to the coculture, as previously described (47). Cells were incubated for 4 h at 37°C. The plates were centrifuged at 200 x g for 5 min, 100 µl of supernatant was collected, and radioactivity was measured in a gamma counter. The percentage of specific 51Cr release was calculated as follows: % specific 51Cr release = (experimental 51Cr release - spontaneous 51Cr release) x 100/(maximum 51Cr release - spontaneous 51Cr release). Maximum 51Cr release was obtained by adding 100 µl of 1% Nonidet P-40 (Sigma, St. Louis, MO) to 100 µl of labeled target cells. Spontaneous 51Cr release ranged from 5 to 10% of the total counts incorporated. The data in the figures refer to the mean of two replicates. SDs were generally within 5–10% of the mean.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Given that efficient retroviral transduction requires dividing
cells, it was necessary to develop improved culture conditions that
allow retroviral transduction of proliferating dendritic progenitor
cells. We have previously shown that HyperIL-6 in the presence of SCF
leads to an expansion of CD34+ HPC capable of
differentiating into functional DC (48). Based on these
findings, initial experiments asked whether the proliferative capacity
of HyperIL-6 could be used for retroviral transduction of DC
progenitors. Dividing CD34+ HPC were retrovirally
transduced in the presence of HyperIL-6, SCF, and TGF-ß1, then
further differentiated into immature DC by adding IL-4, GM-CSF, and low
doses of TNF-, and finally matured under the influence of high doses
of TNF-. During the culture period of 4 wk, the total cell count
increased 60- to 80-fold depending on the individual cell culture (data
not shown). On day 26, 55–85% of all cultured cells displayed the
typical phenotype of immature DC expressing low levels of HLA-DR, CD80,
and CD86 (Fig. 1
A). With
TNF- treatment, immature CD83- DC developed into
mature DC, as determined by the detection of CD83, a molecule known to
be expressed by mature, myeloid-derived DC (Fig. 1B). Mature
CD83+ DC highly expressed MHC class II as well as
the accessory molecules CD40, CD80, CD86, and CD54 (ICAM-1). DC did not
express CD1a or CLA (data not shown), molecules related to Langerhans
cells. The phenotype of retrovirally transduced DC did not differ from
that of nontransduced DC (data not shown). Cultured cells did not
display lineage markers for B cells (CD19), T cells (CD3), NK cells
(CD56), or monocytes (CD14).
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DC progenitors were infected with the HER2 retrovirus on days 6
and 7 of the expansion phase (days 0–7). Following culture periods of
DC differentiation (days 8–26) and maturation (days 27 and 28), DC
were harvested on day 28 and assessed for successful retroviral
transduction. Integration of HER2 DNA was determined by PCR using
HER2-specific primers (Fig. 2A). Retrovirally transduced
DC, but not native DC, were positive for HER2 DNA. DNA isolated from
virus particles served as a positive control. HER2 protein synthesis
was analyzed by Western blotting using an mAb directed against the
intracellular domain of HER2 (Fig. 2B). Transduced DC
expressed the HER2 protein, as documented by positive staining and
correct size of 185 kDa. The ovarian cancer cell line SKOV3, which is
known to overexpress HER2, served as a positive control; nontransduced
DC served as a negative control. The efficacy of retroviral
transduction was analyzed by FACS analysis using an mAb against the ECD
of HER2 (Fig. 2C). The transduction efficiency was
15%.
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A). In addition, the CTL line
NK1 lysed the HER2-overexpressing SKOV3 cells when transfected with the
relevant MHC allele HLA-A*0201 (SKOV3tA*0201), whereas SKOV3 cells
naturally not expressing HLA-A*0201 were not lysed. Peptide specificity
was confirmed by measuring IFN- secretion of CTL line NK1 upon
contact with T2 loaded with HER2369–377 and
SKOV3tA*0201 (Fig. 3B).
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secretion by the CTL line NK1 were used to analyze the correct peptide
presentation of retrovirally transduced DC. HLA-A*0201-positive DC
infected with HER2 retrovirus were lysed by the CTL line NK1,
demonstrating that the peptide HER369–377 had
been endogenously processed and presented with HLA-A*0201 upon
retroviral transduction (Fig. 3
A). In addition, CTL line NK1
secreted IFN- upon contact with HER2-transduced HLA-A*0201-positive
DC (Fig. 3B). In contrast, retrovirally transduced DC from
HLA-A*0201-negative donors and nontransduced DC from
HLA-A*0201-positive donors did not serve as targets for CTL line
NK1. HER2-specific, tumor-reactive CTL can be generated and cloned using HER2-transduced DC as APC
Because retrovirally transduced DC were capable of presenting
HER2-derived T cell epitopes, HER2-transduced DC were used as
professional APC to induce a cytotoxic T cell response in vitro. PBMC
from a normal donor were stimulated with autologous HER2-transduced DC
at weekly intervals. Following four stimulations, the resulting CTL
line, PS, was investigated for HER2-specific cytotoxic activity in a
standard chromium release assay (Fig. 4A). CTL line PS lysed
HER2-overexpressing SKOV3tA*0201 cells in an HLA-A*0201-restricted
manner, whereas HLA-A*0201-negative SKOV3 cells were not lysed. An
HLA-A*0201-positive breast cancer cell line, MCF7, expressing low
levels of HER2 was not lysed by CTL line PS. HER2 specificity was
confirmed using autologous DC as target cells in an IFN- ELSPOT
assay (Fig. 4B). The CTL line PS released IFN- upon
stimulation with HER2-transduced DC, whereas nontransduced DC did not
induce IFN- secretion. CTL lines generated from two additional
HLA-A*0201-positive donors displayed a similar specificity pattern as
the CTL line PS lysing HER2-overexpressing, HLA-A*0201-positive tumors
(data not shown). Lysis of SKOV3tA*0201 cells (38% lysis at an E:T
cell ratio of 30:1) was inhibited in the presence of mAb W6/32 (10%
lysis) or mAb MA2.1 (12% lysis), confirming HLA-A*0201 as a
restriction element.
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). CTL clone PS-D10 displayed the
same specificity pattern as the parental CTL line PS.
HER2-overexpressing, HLA-A*0201-positive cells (SKOV3tA*0201) were
lysed, whereas HER2-overexpressing HLA-A*0201-negative cells (SKOV3 and
SKBR3) were not lysed. The HLA-A*0201-positive tumor cells expressing
low levels of HER2 (MCF7) and HLA-A*0201-positive cells negative for
HER2 (MZ-EBV1257) were not recognized by CTL clone PS-D10. Because the
CTL clone PS-D10 recognized DC transduced with a retrovirus encoding
the ECD of HER2, two immunodominant peptides,
HER2369–377 (20) and
HER2435–443 (24), both derived from
the ECD HER2 and known to be presented with HLA-A*0201, were tested.
These two tested HER2 peptides were not able to sensitize T2 cells for
lysis by CTL clone PS-D10 (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study we have improved the culture conditions for
CD34+ HPC, allowing retroviral transduction of
proliferating precursors that are capable of differentiating into
mature DC. The expansion phase of the cell culture was based on the
proliferative signal of HyperIL-6, a fusion protein of IL-6 linked to
its soluble IL-6R (39). HyperIL-6 associates with the
signal transduction protein gp130 that is expressed by every cell
(54) and, in contrast to IL-6 (55), can
stimulate cells known to be negative for membrane-bound IL-6R,
including primitive HPC (39, 48, 56). Simultaneous
stimulation of gp130 via HyperIL-6 together with c-kit via
SCF or Flt3 via Flt3-L synergizes for expansion of primitive
CD34+ HPC capable of forming multilineage
colonies (39) (C. Peschel, manuscript in preparation).
TGF-ß1 was added to proliferating HPC, because TGF-ß1 mediates
protection of DC progenitors from apoptotic cell death
(57). Hemopoietic progenitor cells were further
differentiated into DC using GM-CSF, IL-4, and TNF-, cytokines known
to promote DC differentiation (58, 59, 60, 61, 62, 63).
The sequence of expansion, differentiation, and maturation (64) of the culture procedure described here resulted in a high percentage of mature DC (55–85%), in contrast to other culture conditions (40, 41, 42, 57, 61). These CD34-derived DC did not express CD1a or CLA, molecules known to be expressed by CD34-derived DC that are related to Langerhans cells (32, 40, 41, 42, 57, 61). The DC grown with the method described here belonged to the interstitial type of DC expressing myeloid markers, in contrast to plasmacytoid DC (65). Cultured DC were highly homogeneous for MHC class II; costimulatory molecules CD40, CD80, and CD86; as well as the adhesion molecule CD54. Based on the proliferative capacity of HyperIL-6 in the presence of SCF and Ft3L, we achieved a stable expression of HER2 on retrovirally transduced DC. Retroviral transduction did not alter the phenotype of CD83+ DC expressing unchanged levels of the critical accessory molecules CD40, CD80, CD86, and CD54. This is in accordance with a previous report that CD1a+ DC did not alter the phenotype following retroviral transduction (40). In contrast, vaccinia viruses might down-regulate molecules, including those that are critical for immunostimulatory activity of DC in vitro (66). Adenoviral gene transfer into DC may also lead to a suppression of T cell stimulation, as recently described (67).
Retrovirally transduced DC correctly processed and presented the Ag, because HLA-A*0201-positive DC served as targets for CTL recognizing the HLA-A*0201-binding immunodominant peptide HER2369–377. Of note, retrovirally transduced DC were lysed very efficiently, in contrast to SKOV3tA*0201 tumor cells. Differential lysis by HER2-specific CTL NK1 might be due to different processing pathways of DC and tumor cells. In tumor cells, HER2 peptides might compete for MHC class I processing with peptides derived from proteins not present in HER2-transduced DC. Alternatively, nontransduced DC may phagocytose apoptotic bodies of HER2-transduced DC and subsequently cross-present peptides to Ag-specific CTL (68).
In this paper we demonstrate the feasibility of using retrovirally transduced DC for generation of HER2-specific and tumor-reactive CTL that can be cloned and expanded in vitro. The HLA-A*0201 HER2-specific CTL clone PS-D10 did not detect one of the previously described immunodominant HLA-A*0201-binding HER2 peptides (20, 24). These findings support the hypothesis that patients sharing an HLA allele and an Ag may not always use common antigenic epitopes, but may have individual T cell epitopes. The mechanisms involved might be the presence of other HLA alleles that compete for the processing of certain peptide epitopes (69) or serve as ligand for killing inhibitory receptors present on Ag-specific CTL (70). In contrast to peptide-loaded DC, retrovirally transduced DC take advantage of the processing, presentation, and recognition of individual T cell epitopes. In addition, the use of defined peptides to generate tumor-reactive T cells may lead to peptide-specific CTL that fail to recognize HER2-overexpressing tumors (71). Due to the low Ag expression level of retrovirally transduced DC, stimulated T cells are confronted with low amounts of peptides that might support the generation of T cells with a sufficiently high affinity to kill tumor cells. Of note, the DC were cultured in human serum instead of FCS (40, 41), consecutively circumventing the presentation of xenogenic protein Ags.
Recognition of peptide epitopes by CTL has been shown to require the expression of the encoding gene above a certain threshold (72). Given the fact that the established HER2-specific CTL clone PS-D10 lysed tumor cells expressing high levels of HER2, but not cells with low level expression of HER2 in vitro, adoptive transfer of these T cells with intermediate affinity might lead to tumor rejection without damage in normal tissues in vivo. Similar observations have been made using a humanized anti-HER2 mAb that inhibits the growth of HER2-overexpressing tumor cells. Administration of this Ab as a single agent produced tumor responses without evidence of autoimmune disease (31).
The ability to generate CTL against HER2-overexpressing tumors using retrovirally transduced DC allows generation of CTL without the knowledge of HLA alleles or peptide epitopes. Successful screening and cloning of CTL lysing HER2-overexpressing tumor cells facilitates the further development of adoptive transfer of HER2-specific T cells for patients with HER2-overexpressing tumors. Current experiments focus on the generation of HER2-specific Th cells, because long-term survival of CTL is dependent on the presence of T cell help (5).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Helga Bernhard, III. Medizinische Klinik, Hämatologie und Onkologie, Klinikum rechts der Isar, Technische Universität München, Ismaningerstrasse 22, 81664 Munich, Germany.
3 Abbreviations used in this paper: HER2, neu, HER2/neu, H2N, c-erbB2, human epidermal growth factor receptor 2; DC, dendritic cells; DC tH2N, HER2-transduced DC; ECD, extracellular domain; Flt3-L, fetal liver tyrosine kinase 3 ligand; HPC, hemopoietic progenitor cells; INF-MP, influenza A matrix protein; nIL-2, natural IL-2; SCF, stem cell factor; ELISPOT, enzyme-linked immunospot.
Received for publication July 5, 2000. Accepted for publication July 14, 2000.
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  M. Barve, J. Bender, N. Senzer, C. Cunningham, F. A. Greco, D. McCune, R. Steis, H. Khong, D. Richards, J. Stephenson, et al. Induction of Immune Responses and Clinical Efficacy in a Phase II Trial of IDM-2101, a 10-Epitope Cytotoxic T-Lymphocyte Vaccine, in Metastatic Non-Small-Cell Lung Cancer J. Clin. Oncol., September 20, 2008; 26(27): 4418 - 4425. [Abstract] [Full Text] [PDF]
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 H. Conrad, K. Gebhard, H. Kronig, J. Neudorfer, D. H. Busch, C. Peschel, and H. Bernhard CTLs Directed against HER2 Specifically Cross-React with HER3 and HER4 J. Immunol., June 15, 2008; 180(12): 8135 - 8145. [Abstract] [Full Text] [PDF]
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M. J. Palmowski, L. Lopes, Y. Ikeda, M. Salio, V. Cerundolo, and M. K. Collins Intravenous Injection of a Lentiviral Vector Encoding NY-ESO-1 Induces an Effective CTL Response J. Immunol., February 1, 2004; 172(3): 1582 - 1587. [Abstract] [Full Text] [PDF]
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K. Fleischer, B. Schmidt, W. Kastenmuller, D. H. Busch, I. Drexler, G. Sutter, M. Heike, C. Peschel, and H. Bernhard Melanoma-Reactive Class I-Restricted Cytotoxic T Cell Clones Are Stimulated by Dendritic Cells Loaded with Synthetic Peptides, but Fail to Respond to Dendritic Cells Pulsed with Melanoma-Derived Heat Shock Proteins In Vitro J. Immunol., January 1, 2004; 172(1): 162 - 169. [Abstract] [Full Text] [PDF]
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V. Renard, L. Sonderbye, K. Ebbehoj, P. B. Rasmussen, K. Gregorius, T. Gottschalk, S. Mouritsen, A. Gautam, and D. R. Leach HER-2 DNA and Protein Vaccines Containing Potent Th Cell Epitopes Induce Distinct Protective and Therapeutic Antitumor Responses in HER-2 Transgenic Mice J. Immunol., August 1, 2003; 171(3): 1588 - 1595. [Abstract] [Full Text] [PDF]
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 J. L. Murray, M. E. Gillogly, D. Przepiorka, H. Brewer, N. K. Ibrahim, D. J. Booser, G. N. Hortobagyi, A. P. Kudelka, K. H. Grabstein, M. A. Cheever, et al. Toxicity, Immunogenicity, and Induction of E75-specific Tumor-lytic CTLs by HER-2 Peptide E75 (369-377) Combined with Granulocyte Macrophage Colony-stimulating Factor in HLA-A2+ Patients with Metastatic Breast and Ovarian Cancer Clin. Cancer Res., November 1, 2002; 8(11): 3407 - 3418. [Abstract] [Full Text] [PDF]
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 H. J. Bontkes, T. D. de Gruijl, G. J. Schuurhuis, R. J. Scheper, C. J. L. M. Meijer, and E. Hooijberg Expansion of dendritic cell precursors from human CD34+ progenitor cells isolated from healthy donor blood; growth factor combination determines proliferation rate and functional outcome J. Leukoc. Biol., August 1, 2002; 72(2): 321 - 329. [Abstract] [Full Text] [PDF]
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 C. M. zum Buschenfelde, C. Hermann, B. Schmidt, C. Peschel, and H. Bernhard Antihuman Epidermal Growth Factor Receptor 2 (HER2) Monoclonal Antibody Trastuzumab Enhances Cytolytic Activity of Class I-restricted HER2-specific T Lymphocytes Against HER2-overexpressing Tumor Cells Cancer Res., April 1, 2002; 62(8): 2244 - 2247. [Abstract] [Full Text] [PDF]
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C. Meyer zum Buschenfelde, J. Metzger, C. Hermann, N. Nicklisch, C. Peschel, and H. Bernhard The Generation of Both T Killer and Th Cell Clones Specific for the Tumor-Associated Antigen HER2 Using Retrovirally Transduced Dendritic Cells J. Immunol., August 1, 2001; 167(3): 1712 - 1719. [Abstract] [Full Text] [PDF]
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) on day 26 was compared with that of
mature DC (
) on day 28. B, Expression of various cell
surface molecules on mature DC (day 28; 
) were not
lysed. HLA-A*0201-positive DC retrovirally transduced with HER2 were
lysed (
), but not retrovirally transduced DC from an
HLA-A*0201-negative donor (
) or nontransduced autologous DC from an
HLA-A*0201-positive donor (
). B,
HLA-A*0201-restricted HER2-recognition was confirmed by measuring
IFN-
