Generation of Tumor-Reactive CTL Against the Tumor-Associated Antigen HER2 Using Retrovirally Transduced Dendritic Cells Derived from CD34⁺ Hemopoietic Progenitor Cells

Cell lines

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.

HER2_369–377-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 HER2_369–377 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-β1, and 2 or 100 ng/ml TNF-α (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-β1 (days 0–7). Proliferating HPC were differentiated into immature DC with IL-4, GM-CSF (days 8–26), and 2 ng/ml TNF-α (days 15–26) added to the cytokines used for expansion. Finally, maturation of DC was induced by addition of high doses of TNF-α (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 × 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-β1. 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 × 10⁴) were cocultured with 7.5 × 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-γ 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 × 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-γ-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-γ 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-γ produced by Ag-specific T cells was detected by sequential addition of biotinylated mouse anti-human IFN-γ (Mabtech) and alkaline phosphatase-conjugated streptavidin (Mabtech). The number of stained spots corresponding to the IFN-γ-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 × 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) × 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.

CD34⁺ HPC expanded with HyperIL-6 and transduced with a retrovirus are capable of differentiating into mature CD83⁺ DC

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-β1, then further differentiated into immature DC by adding IL-4, GM-CSF, and low doses of TNF-α, and finally matured under the influence of high doses of TNF-α. 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-α 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. 1⇓B). 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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FIGURE 1.

Retrovirally transduced DC express critical costimulatory molecules and surface markers. The culture condition for DC development from CD34⁺ progenitor cells in vitro consisted of sequential expansion, differentiation, and maturation periods using the following cytokines: HyperIL-6, SCF, Flt3-L, and TGF-β1 (days 0–28); GM-CSF and IL-4 (days 8–28); and TNF-α at low (days 15–25) and high (days 26–28) concentrations. Primitive progenitor cells were retrovirally transduced on days 6 and 7 and were further differentiated into immature CD83⁻ DC (day 26). During maturation (days 26–28) CD83⁻ DC developed into CD83⁺ DC and up-regulated HLA-DR and costimulatory molecules. A, The phenotype of immature DC (□) 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; ▪) and isotype controls (□) are shown. Histograms represent the staining of all cultured cells, except the dead cells that were not gated.

DC transduced with HER2 retrovirus express HER2 protein and present HER2 peptides in context with MHC class I

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. 2⇓A). 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. 2⇓B). 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. 2⇓C). The transduction efficiency was ∼15%.

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FIGURE 2.

DC transduced with HER2 retrovirus express the HER2 gene. CD34⁺ progenitor cells were expanded in the presence of HyperIL-6, SCF, and TGF-β1 and retrovirally transduced with supernatant from a HER2-transfected packing cell clone secreting high titers of HER2 retroviruses. Retrovirally transduced progenitor cells were further differentiated into mature DC by adding GM-CSF, IL-4, and TNF-α. Retroviral transduction of HER2 in mature DC was determined by PCR (A), Western blotting (B), and FACS analysis (C) on day 28. A, HER2 gene (DNA) was detected in retrovirally transduced DC, but not in nontransduced DC; retroviral supernatant from HER2-transfected packing cell line served as a positive control. B, HER2 protein was only expressed by DC when previously transduced with HER2 retrovirus; the HER2-overexpressing ovarian cancer cell line SKOV3 served as a positive control. C, HER2 surface expression of retrovirally transduced DC (bold line) was compared with that of nontransduced DC (thin line) by FACS analysis; transduction efficiency was 15% as determined by an mAb against the extracellular domain of HER2.

Because stimulation of Ag-specific T cells requires correct processing and presentation of antigenic epitopes, the ability of HER2-transduced DC to present immunogenic HER2 epitopes in context with MHC class I molecules was determined. For detection of MHC class I-bound peptides on the surface of HER2-transduced DC, an HLA-A*0201-restricted CTL line specific for the immunodominant peptide HER2_369–377 was generated. The established peptide-specific CTL line NK1 lysed T2 cells pulsed with HER2_369–377, whereas T2 cells loaded with an irrelevant peptide derived from the influenza A matrix protein (INF-MP_58–66) were not recognized (Fig. 3⇓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-γ secretion of CTL line NK1 upon contact with T2 loaded with HER2_369–377 and SKOV3tA*0201 (Fig. 3⇓B).

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FIGURE 3.

HLA-A*0201-positive DC transduced with a HER2 retrovirus serve as targets for the HLA-A*0201-restricted CTL NK1 specific for HER2_369–377. The CTL line NK1 was generated by repetitive stimulation with autologous monocyte-derived DC loaded with HER2_369–377, which is known to be naturally presented with HLA-A*0201. The lytic activity of the CTL line NK1 was tested in a standard chromium release assay (A), and IFN-γ release was detected in an ELIPOT assay (B). A, The CTL line NK1 lysed T2 cells loaded with HER2_369–377 (▪), but did not lyse T2 cells pulsed with the irrelevant peptide INF-MP_58–66 (□). HER2-overexpressing SKOV3tA*0201 cells (•) were lysed when transfected with HLA-A*0201, whereas the HLA-A*0201-negative cells from parental cell line SKOV3 (○) 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-γ secretion by the CTL line NK1 (1,000 CTL/well) seeded with the relevant and irrelevant stimulator cells (20,000 cells/well) as indicated.

HER2-specific, HLA-A*0201-restricted lytic activity and IFN-γ 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 HER_369–377 had been endogenously processed and presented with HLA-A*0201 upon retroviral transduction (Fig. 3⇑A). In addition, CTL line NK1 secreted IFN-γ upon contact with HER2-transduced HLA-A*0201-positive DC (Fig. 3⇑B). 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. 4⇓A). 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-γ ELSPOT assay (Fig. 4⇓B). The CTL line PS released IFN-γ upon stimulation with HER2-transduced DC, whereas nontransduced DC did not induce IFN-γ 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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FIGURE 4.

HER2-specific, HLA-A*0201-restricted CTL are generated by repetitive stimulation with autologous DC retrovirally transduced with HER2. Retrovirally transduced DC expressing the HER2 gene were used as stimulator cells for autologous T cells. Proliferating T cells were restimulated three times with HER2-transduced DC at weekly intervals in the presence of nIL-2. The resulting CTL line PS was tested for HER2-specific lysis (A) and IFN-γ release (B) using a standard 4-h chromium release assay and an ELISPOT assay, respectively. A, The CTL line PS lysed HER2-overexpressing HLA-A*0201-positive SKOV3tA*0201 (▪), but not HLA-A*0201-negative SKOV3 (□). HLA-A*0201-positive MCF-7 cells (▵) expressing low levels of HER2 were not lysed. B, HER2 specificity and HLA-A*0201 restriction were confirmed by detecting IFN-γ secreted from the CTL line PS (1,000 CTL/well) upon stimulation with HER2-transduced autologous DC and SKOV3tA*0201, but not with SKOV3 (20,000 stimulator cells/well).

The HER2-specific CTL line PS was cloned by limiting dilution and screened for HER2 specificity using autologous HER2-transduced and nontransduced DC as target cells for cytotoxicity analyses. Of four screened HER2-specific CTL clones, the CTL clone PS-D10 could be expanded and tested for cytotoxicity against a panel of cell lines (Fig. 5⇓). 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, HER2_369–377 (20) and HER2_435–443 (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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FIGURE 5.

The CTL clone PS-D10 lyses HER2-overexpressing tumor cells in an HLA-A*0201-restricted manner. The CTL clone PS-D10 was generated by limiting dilution and was tested for HER2-specific lysis. HLA-A*0201-positive tumor cells expressing high levels of HER2 (SKOV3tA*0201; ▪) were lysed, whereas HLA-A*0201-positive cell lines expressing low levels of HER2 (MCF7; ▿) or negative for HER2 (MZ-EBV1257; ▵) were not lysed. HER2-overexpressing tumor cells, such as SKOV3 (□) and SKBR3 (○), both negative for HLA-A*0201, were not recognized.