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The Journal of Immunology, 2001, 167: 1712-1719.
Copyright © 2001 by The American Association of Immunologists

The Generation of Both T Killer and Th Cell Clones Specific for the Tumor-Associated Antigen HER2 Using Retrovirally Transduced Dendritic Cells1

Christian Meyer zum Büschenfelde, Jochen Metzger, Christine Hermann, Nicole Nicklisch, Christian Peschel and Helga Bernhard2

Department of Hematology/Oncology, Technical University of Munich, Munich, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Induction of antitumor immunity involves the presence of both CD8+ CTLs and CD4+ Th cells specific for tumor-associated Ags. Attempts to eradicate cancer by adoptive T cell transfer have been limited due to the difficulty of generating T cells with defined Ag specificity. The current study focuses on the generation of CTL and Th cells against the tumor-associated Ag HER2 using autologous dendritic cells (DC) derived from CD34+ hematopoietic progenitor cells which have been retrovirally transduced with the human epidermal growth factor receptor 2 (HER2) gene. HER2-transduced DC elicited HER2-specific CD8+ CTL that lyse HER2-overexpressing tumor cells in context of distinct HLA class I alleles. The induction of both HLA-A2 and -A3-restricted HER2-specific CTL was verified on a clonal level. In addition, retrovirally transduced DC induced CD4+ Th1 cells recognizing HER2 in context with HLA class II. HLA-DR-restricted CD4+ T cells were cloned that released IFN-{gamma} upon stimulation with DC pulsed with the recombinant protein of the extracellular domain of HER2. These data indicate that retrovirally transduced DC expressing the HER2 molecule present multiple peptide epitopes and subsequently elicit HER2-specific CTL and Th1 cells. The method of stimulating HER2-specific CD8+ and CD4+ T cells with retrovirally transduced DC was successfully implemented for generating HER2-specific CTL and Th1 clones from a patient with HER2-overexpressing breast cancer. The ability to generate and expand HER2-specific, HLA-restricted CTL and Th1 clones in vitro facilitates the development of immunotherapy regimens, in particular the adoptive transfer of both autologous HER2-specific T cell clones in patients with HER2-overexpressing tumors without the requirement of defining immunogenic peptides.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The dynamic interaction of Ag-presenting dendritic cells (DC),3 T killer, and Th cells is required for the initiation of an immune response (1, 2, 3, 4) that is essential for Ag-specific tumor rejection. DC take up Ags released from dead tumor cells and subsequently process and present antigenic peptides in context with MHC class I and II molecules to CD8+ and CD4+ T cells, respectively (5, 6). In this scenario, Ag-specific CD4+ T cells provide direct and indirect help to CD8+ effector T cells (for review, see Ref. 7). Subsequently, activated CD8+ T cells can directly lyse tumor cells, since most tumors express MHC class I but not class II. However, Ag-specific CD4+ T cells can eliminate MHC class II-negative cancer cells in the absence of CD8+ T cells by indirect effects of released cytokines (8, 9, 10, 11, 12).

To date, attempts to treat human cancer by modulating this three-cell interaction have focused on stimulating tumor-reactive CD8+ T cells in vivo. Vaccination strategies have been based on the activation of MHC class I-restricted CD8+ T cells using autologous DC that have been loaded in vivo or ex vivo with tumor-associated peptide Ags (13, 14, 15, 16). Efficacy of these vaccine strategies was limited in part due to the inadequate induction of Th responses. Studies in animal models have already demonstrated that simultaneous vaccination with Th and CTL epitopes derived from the same tumor Ag resulted in a strong synergistic protection (17, 18). Recently, Th epitopes have been identified for human tumor Ags that had been previously defined by CTL recognition, e.g. tyrosinase, MAGE-3, and NY-ESO-1 (19, 20, 21, 22, 23, 24). The combination of multiple MHC class I- and II-binding peptide epitopes derived from tumor-associated Ags may improve the efficacy of active immunization in patients by recruiting both Ag-specific T killer and Th cells. In animal models, established tumors can be eradicated by the adoptive transfer of Ag-specific CD8+ and/or CD4+ T cells (8, 9, 11, 12, 25, 26, 27, 28). Greenberg and colleagues (29) have initiated a first clinical trial to evaluate the feasibility and efficacy of tumor-reactive CTL and Th cell clones directed against Melan-A/MART-1 for the treatment of patients with malignant melanoma.

The growing number of defined human tumor-associated Ags presented by MHC class I and II may serve as potential targets for Ag-specific T killer and Th cells (for review, see Refs. 30, 31, 32). The human epidermal growth factor receptor 2 (HER2/neu/c-erbB2) represents an appealing target for Ag-specific cellular immunotherapy. HER2 is expressed at high levels in a variety of human cancers (for review, see Ref. 33). Overexpression of HER2 contributes to the malignant phenotype of the tumor and is associated with poor prognosis (34, 35). Therefore, HER2-overexpressing tumors might not be able to easily escape HER2-targeted immunotherapy through immunoselection of Ag-loss variants, as observed after targeting the melanoma-associated differentiation Ag Melan-A/MART-1 by immunization or adoptive T cell transfer (29, 36). Naturally processed HER2 epitopes have already been identified for CTL that lyse HER2-overexpressing tumor cells in context with HLA-A*0201, HLA-A*0301, and HLA-A*2402 (37, 38, 39, 40, 41, 42, 43). In contrast, HER2 epitopes naturally presented with MHC class II have not yet been defined by class II-restricted Th cells. So far, the existence of Th epitopes is driven from proliferative Th cell responses to HER2 peptides with binding motifs to MHC class II (44, 45, 46, 47). Studies in an animal model have shown that vaccines with rat neu peptides can generate rat neu-specific Th cell immunity (48). In a pilot study, vaccination with HER2-derived peptides elicited HER2-specific Th cell immunity in women with HER2-overexpressing breast cancer (49).

Attempts to treat HER2-overexpressing tumors by adoptive transfer of HER2-reactive T cells have been limited due to the difficulty of generating autologous CTL and Th1 cells directed against the HER2 Ag. We have developed a protocol using retrovirally transduced DC with the HER2 gene for HER2-specific stimulation of autologous PBLs (50). Herein, we report that HER2-transduced DC presented multiple HER2 epitopes and subsequently induced HER2-specific cytotoxic and helper T cells in individual donors. Both HER2-reactive CD8+ CTL and CD4+ Th1 cells could be elicited and cloned from a patient with advanced HER2-overexpressing breast cancer, encouraging further development of adoptive T cell transfer for patients with metastatic disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines

The H2N++ ovarian cancer cell line SKOV3 (HLA phenotype: A3, A28, B18, B35), the H2N++ breast cancer cell line SKBR3 (HLA phenotype: A11, B40, B18), and the hybridoma cell lines secreting mAb w6/32 respective mAb HB55 were obtained from the American Type Culture Collection (Manassas, VA). B-lymphoblastoid cell lines (LCL) not expressing HER2 were generated from PBMC by EBV-transformation of B cells: HLA-A2+ LCL-InRi, HLA-A3+ LCL-MaBa, and MZ-LCL1257 (51). The HLA-A2 transfectant cell line SKOV3tA2 was a gift from M. L. Disis (University of Washington, Seattle, WA). Tumor cell lines, hybridoma cell lines, and LCL were cultured in RPMI 1640 (Life Technologies, Paisley, U.K.) supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine.

Flow cytometric analysis

The following PE-conjugated mAbs were used for phenotypic analyses: HLA-DR, CD80, CD86, CD54, and CD40 (all from BD Biosciences, Mountain View, CA); CD83, CD14, CD34, CD3, CD4, and CD8 (all from BD PharMingen, San Diego, CA). Conjugated isotype-matched mAb (all from BD Biosciences) were used as controls. HER2 expression of tumor cells was determined by sequential incubation with the unconjugated mAb c-neu-Ab6 recognizing the extracellular domain for HER2 (Oncogene Science, Boston, MA) and FITC-conjugated F(ab')2 goat anti-mouse Ig (Zymed Laboratories, San Francisco, CA). Fluorescence analyses were performed on an Epics Elite ESP flow cytometer (Coulter Electronics, Hialeah, FL).

Generation of DC from CD34+hemopoietic progenitor cells (HPC)

CD34+ HPC were isolated from peripheral blood stem cell collections (52) and differentiated into DC according to the recently described protocol (50), with a few modifications. Briefly, normal donors or patients were administered 5 µg/kg s.c. G-CSF twice a day for 4 days to mobilize CD34+ HPC into the peripheral blood, and leukapheresis was performed on day 5. CD34+ cells were isolated using the cell separation system Clinimax (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured with 3 ml of X-VIVO 15 medium (BioWhittaker, Walkersville, MD) supplemented with 2 mM L-glutamine, 100 IU 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), 100 ng/ml IL-3 (both from R&D Systems, Minneapolis, MN), 75 ng/ml fetal liver tyrosine kinase 3-ligand (Flt3-L; PeproTech EC, London, U.K.), 50 ng/ml IL-4, 0.5 ng/ml TGF-{beta}1, and 2 ng/ml or 100 ng/ml TNF-{alpha} (all from Strathmann-Biotech, Hannover, Germany), and 100 ng/ml GM-CSF (Novartis, Nürnberg, Germany). The culture condition for generating DC from CD34+ HPC consisted of three phases: expansion (days 0–7), differentiation (days 8–26), and maturation (days 27 and 28). During the expansion phase, extensive proliferation of primitive progenitor cells was induced in the presence of IL-3, SCF, Flt3-L, and TGF-{beta}1 (days 0–7). Proliferating HPC were differentiated into immature DC by adding IL-4 and GM-CSF (days 8–26) and 2 ng/ml TNF-{alpha} (days 15–26) to SCF, Flt3-L, and TGF-{beta}1 in the absence of IL-3. Finally, the maturation of DC was induced by addition of high doses of TNF-{alpha} (100 ng/ml) during the last 2 days (days 27 and 28). On day 28, 20–40% of all cultured cells displayed the typical phenotype of mature myeloid DC highly expressing CD83, MHC class II, as well as the accessory molecules CD40, CD80, CD86, and CD54.

Retroviral transduction of DC derived from CD34+ HPC

Proliferating dendritic progenitor cells were transduced with a retrovirus encoding the full-length HER2 gene, as previously described (50). In short, 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. 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 (53, 54, 55).

Generation of HER2-specific CD8+ cytotoxic T cells and CD4+ Th 1 cells 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. A total of 2 x 104 DC was cocultured with 7.5 x 105 PBMC in one well of a 24-well flat-bottom plate (Greiner, Nurtingen, Germany). To the DC-T cell coculture, 5 ng/ml rIL-7 was added on day 1 and 100 U/ml natural IL-2 on day 3. Proliferating T cells were restimulated weekly using HER2-transduced DC at a 1:50 stimulator:responder ratio. Following three cycles of weekly stimulations, HER2 specificity of T cells was analyzed by measuring cytotoxicity for CTL. HER2-specific CTL lines were cloned by limiting dilution as described below.

To enhance the efficiency of generating Th1 cells, CD4+ T cells were purified from PBMC and cocultured with autologous HER2-transduced DC in the presence of rIL-2 and rIL-7. CD4+ T cells were primed and restimulated twice with HER2-transduced DC. T cells secreting IFN-{gamma} upon stimulation with HER2-transduced DC were sorted using the cell surface affinity matrix technology as recently described (56, 57). Briefly, IFN-{gamma} released by stimulated T cells was captured on the surface of T cells by a bispecific Ab against CD45 and IFN-{gamma} (Miltenyi Biotec). After the cytokine capturing period, T cells were stained with a PE-conjugated IFN-{gamma}-specific mAb (Miltenyi Biotec) and PE-labeled cells were enriched by MACS using anti-PE microbeads (Miltenyi Biotec). T cells sorted by MACS were directly cloned by limiting dilution.

Isolation and expansion of HER2-specific CD8+ and CD4+ T cell clones

HER2-specific polyclonal T cells were cloned by limiting dilution according to the protocol published by Yee et al. (58). 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-LCL1257, and 50 U/ml rIL-2 (Chiron, Emeryville, CA). Proliferating CTL clones were screened for lytic activity in a microtoxicity assay using SKOV3tA2 as positive control and SKBR3 as negative control (see below). Th cell clones were screened for IFN-{gamma} production in an ELISPOT assay using autologous DC pulsed with either rECD or rICD of HER2 (see below). HER2-specific T cell clones were expanded in flasks (T30 or T60; Greiner) in the presence of anti-CD3 mAb, irradiated allogeneic PBMC, and irradiated allogeneic EBV-transformed B cells (MZ-LCL1257) as described (59). A total of 5 x 104 T cells resulted in 2–3 x 107 T cells after 2 wk of cell culture. Further expansion rounds start with 1 x 106 T cell clones and end up with 1–2 x 109 T cells after 2 weeks. Expanded T cell clones were phenotyped and further proofed for HER2 specificity.

ELISPOT assay

The presence of IFN-{gamma}-producing, HER2-specific CD4+ Th 1 cells was assessed in an ELISPOT assay as recently described (60). Autologous DC pulsed with the recombinant protein of the intracellular domain (ICD) or extracellular domain (ECD) of HER2 were used as stimulator cells. Isolated proteins were >98% pure, as verified by silver staining after separation with SDS-PAGE and by positive staining with anti-ICD respective anti-ECD mAb (Oncogene Science) after immunoblotting. Immature DC were generated from monocytes using GM-CSF and IL-4 loaded with 50 µg/ml soluble protein and further matured to CD83+ DC according the protocol recently published by Jonuleit et al. (61). Stimulator and responder cells were seeded in a 96-well nitrocellulose filter plate coated with mouse anti-human IFN-{gamma} capture Ab Dm1.2 (MABTECH, Nacha, Sweden). Protein-loaded DC (20,000/well) were cocultured with CTL (1,000/well) overnight (12–18 h) at 37°C with 5% CO2. For inhibition experiments, 10 µg/ml purified mAb HB55 (IgG2a) directed against HLA-DR or 10 µg/ml mAb w6/32 (IgG2a) were added to the coculture. Cells were then removed by washing with PBS, and the presence of IFN-{gamma} produced by Ag-specific T cells was detected by sequential addition of biotinylated mouse anti-human IFN-{gamma} (MABTECH) and alkaline phosphatase-conjugated streptavidin (MABTECH). The number of stained spots corresponding to the IFN-{gamma}-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 (62). Briefly, 106 tumor cells or DC were labeled in 100 µl of FCS with 100 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. 51Cr-labeled target cells and graded doses of T cells were given in a V-bottom 96-well tissue culture plate (Costar, Cambridge, MA). For inhibition experiments, mAb MA2.1 recognizing HLA-A*0201 and HLA-B17 (63) was added to the coculture as previously described (51). Murine mAb IgG1 (clone 679.1 MC7; Immunotech, Luminy, France) was used as isotype control. Following incubation for 4 h at 37°C, the supernatant was collected and radioactivity was measured in a gamma counter. The percentage of specific 51Cr release was calculated as follows: percent 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) to 100 µl of labeled target cells. Spontaneous 51Cr release ranged from 5 to 10% of total counts incorporated. The data in the figures refer to the mean of two replicates. SDs were generally below 5% of the mean.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
DC retrovirally transduced with the HER2 gene elicit tumor-reactive CD8+ CTL recognizing HER2 epitopes in the context of different HLA class I alleles

We have previously shown that DC transduced with a HER2 retrovirus elicit tumor-reactive CTL recognizing HER2 in the context with HLA-A2 (50) known to be a dominant restriction element. Based on these findings, we were led to question whether HER2-transduced DC are capable of inducing CTL recognizing HER2 in the context of restricting alleles other than HLA-A2. Proliferating CD34+ HPC derived from the HLA-A2- and HLA-A3-positive donor designated FS were transduced with a retrovirus encoding the HER2 Ag and further differentiated into mature CD83+ DC (data not shown). These HER2-transduced DC were used as APC to stimulate autologous PBMC in vitro. Following three stimulations with retrovirally transduced DC, proliferating T cells were evaluated for HER2-specific cytotoxic activity in a standard chromium release assay (Fig. 1GoA). This T cell line FS lysed the HLA-A3-matched, HER2-overexpressing ovarian cancer cell line SKOV3. Moreover, SKOV3tA2 cells transfected with the HLA-A2 allele were better lysed (48% lysis at E:T ratio of 90:1) than the parental SKOV3 cells which do not express HLA-A2 (20% lysis at E:T ratio of 90:1). In contrast, the HER2-overexpressing breast cancer cell line SKBR3, which is negative for HLA-A2 and HLA-A3, was not lysed by CTL FS. This lysis pattern suggested the presence of distinct CTL recognizing HER2 epitopes in the context of either HLA-A2 or HLA-A3. Lysis of SKOV3tA2 by CTL line FS was partially inhibited by mAb MA2.1 directed against HLA-A2 (Fig. 1GoB). Blockade of the HLA-A2 allele resulted in a reduced lysis of SKOV3tA2 that was comparable to the lysis of SKOV3 cells, confirming HLA-A2 and HLA-A3 as the restriction elements for the lytic activity of the HER2-specific CTL line FS.



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  		FIGURE 1. HER2-transduced DC elicit both HLA-A2- and HLA-A3-restricted HER2-specific CTL. Retrovirally transduced DC expressing the HER2 gene were used as APC for stimulating autologous T cells. Following three stimulations, the resulting CTL line FS was tested for HER2-specific lysis using the HER2-overexpressing tumor cell lines SKOV3, SKOV3tA2, and SKBR3 as targets. A, The CTL line FS lysed HLA-A2-/A3+ SKOV3 ({blacktriangleup}) and HLA-A2+/A3+ SKOVtA2 ({blacksquare}), but not HLA-A2-/A3- SKBR3 (•). B, Lysis of SKOV3tA2 by the CTL line FS was partially inhibited by mAb MA2.1 directed against HLA-A2, but not by an IgG1 isotype control.

 
To validate the induction of both HLA-A2- and HLA-A3-restricted CTL by HER2-transduced DC, the CTL line FS was cloned by limiting dilution. Proliferating CTL clones were screened for lytic activity using SKOV3tA2 and SKBR3 as target cells (Fig. 2GoA). Of 50 screened CTL clones, CD8+ CTL clones FS-1 and FS-32 could be expanded 400-fold during a culture period of 2 wk. Expanded CTL clones FS-1 and FS-32 were tested for cytotoxicity against the HER2-overexpressing tumor cell lines SKOV3tA2, SKOV3, and SKBR3 (Fig. 2GoB). Lytic activity of CTL clone FS-1 was dependent on the presence of HLA-A2, since SKOV3tA2 cells were lysed very efficiently, whereas SKOV3 and SKBR3 were not lysed, even at a high E:T ratio. CTL FS-1 did not lyse HLA-A2-matched HER2- LCL-InRi. In contrast, HLA-A3 was the restricting allele for the CTL clone FS-32. SKOV3tA2 and SKOV3, both expressing HLA-A3, were lysed by FS-32, whereas SKBR3, which is negative for HLA-A3, was not lysed. HLA-A3-matched HER2- LCL-MaBa were not lysed by the CTL clone FS-32.



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  		FIGURE 2. HER2-specific HLA-A2- and A3-restricted CTL clones can be isolated from CTL stimulated with retrovirally transduced DC. A, CTL line FS was cloned by limiting dilution and proliferating CTL clones were screened for lytic activity in a microtoxicity assay. The dots represent the lytic activity of each CTL clone using the HER2-overexpressing tumor cell lines SKOV3tA2 (HLA-A2+ /A3+ ) and SKBR3 (HLA-A2- /A3- ) as targets. B, CD8+ CTL clone FS-1 lysed HLA-A2+ /A3+ SKOV3tA2 ({blacksquare}), but not HLA-A2- /A3+ SKOV3 ({blacktriangleup}) or HLA-A2- /A3- SKBR3 (•). CTL clone FS-1 did not lyse HER2- HLA-A2-matched LCL-InRi ({circ}). In contrast, CD8+ CTL clone FS-32 lysed HLA-A2+ /A3+ SKOV3tA2 ({blacksquare}) and HLA-A2- /A3+ SKOV3 ({blacktriangleup}), but not HLA-A2- /A3- SKBR3 (•) or HLA-A3-matched LCL-MaBa ({triangleup}).

 
Generation of CD8+ cytotoxic T cell clones from a patient with HER2-overexpressing breast cancer

Since HER2-specific T cell clones could be successfully generated from healthy donors, we next addressed the question of whether CTL clones could be generated from a patient with HER2-overexpressing breast cancer. The 59-year-old patient HR had progressive metastatic breast cancer despite polychemotherapy. One of the metastases was biopsied and immunohistochemistry revealed a score +3 for HER2 expression using the HercepTest (Dako, Copenhagen, Denmark). Therefore, she was treated with a combination of anti-HER2-mAb trastuzumab (Herceptin; Genentech, South San Francisco, CA) and anthracycline epirubicine (Farmorubicin; Pharmacia and Upjohn, Erlangen, Germany). After the first treatment cycle, CD34+ HPC were mobilized into the peripheral blood and harvested by leukapheresis. Purified CD34+ HPC were retrovirally transduced with the HER2 gene and cultured into DC as described in Materials and Methods. HER2-transduced DC were used as stimulator cells for autologous PBMC derived from the patient after the completion of four therapy cycles.

PBMC were stimulated three times with HER2-transduced DC and tested for HER2-specific lysis. Since the patient HR was HLA-A2+, the HER2-overexpressing tumor cell line SKOV3tA2 was used as a HLA-A2-matched target, and the HLA-A2- tumor cell line SKOV3 served as a negative control. The DC-stimulated T cells lysed SKOV3tA2 (22% lysis at an E:T cell ratio of 30:1), but did not SKOV3 (3% lysis) nor SKBR3 (2% lysis; data not shown). These T cells were further cloned by limiting dilution. Of 50 screened CTL clones, the two CTL clones HR-4 and HR-9 displayed the highest lytic activity for SKOV3tA2 and did not lyse SKOV3 (Fig. 3GoA). Both CTL clones could be further expanded and tested for specificity. CTL clone HR-4 displayed the same specificity pattern as CTL clone HR-9, lysing SKOV3tA2, but not lysing SKOV3 nor SKBR3, indicating the HLA-A2-restricted recognition of HER2-overexpressing tumor cells (Fig. 3GoB). HLA-A2-matched LCL-InRi being negative for HER2 were not lysed by HR-4 or HR-9.



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  		FIGURE 3. HLA-A2-restricted HER2-specific CD8+ CTL clones can be generated from patient HR with HER2-overexpressing breast cancer using HER2-transduced DC. A, HER2-transduced DC were used as APC for stimulating autologous PBMC. Following three stimulations, the resulting CTL line HR was cloned by limiting dilution and proliferating CTL clones were screened for lytic activity in a microtoxicity assay. The dots represent the lytic activity of each CTL clone using the HER2-overexpressing tumor cell lines SKOV3tA2 (HLA-A2+) and SKOV3 (HLA-A2-) as targets. B, CTL clone HR-4 and HR-9 both lysed HLA-A2+ SKOV3tA2 ({blacksquare}), but did not lyse HLA-A2- SKOV3 ({blacktriangleup}), HLA-A2- SKBR3 (•), or HLA-A2+ LCL-InRi ({circ}).

 
DC retrovirally transduced with the HER2 gene elicit HER2-specific CD4+ Th1 cells from patient HR

It has been previously shown in a mouse model, that retrovirally transduced DC were able to stimulate Ag-specific CD4+ T cells (18). Therefore, we were led to question whether HER2-transduced human DC were able to induce HER2-specific CD4+ T cells. CD4+ cells were isolated from PBMC and stimulated with autologous HER2-transduced DC. After the second stimulation, T cells were sorted based on HER2-specific IFN-{gamma} secretion and cloned by limiting dilution. Growing T cell clones were tested for HER2-specific IFN-{gamma} release upon stimulation with autologous DC pulsed with the rECD of HER2 or the rICD of HER2 in an ELISPOT assay (Fig. 4GoA). Of the 11 growing T cell clones, two clones released IFN-{gamma} upon stimulation with DC pulsed with the rECD of HER2. Interestingly, none of the Th1 clones released IFN-{gamma} when stimulated with DC loaded with rICD of HER2. HER2 specificity of expanded Th1 clones HR-1 and HR-8 was confirmed using DC loaded with the relevant (rECD) or irrelevant protein (keyhole limpet hemocyanin (KLH)) as stimulator cells (Fig. 4GoB). Both T cell clones released IFN-{gamma} upon stimulation with autologous DC loaded with rECD, but not to the irrelevant protein Ag KLH. The IFN-{gamma}-producing T cell clones HR-1 and HR-8 expressed CD4 and were negative for CD8 (data not shown). None of these two T cell clones displayed lytic activity, when autologous rECD-pulsed DC were used as targets in a chromium release assay (data not shown). Therefore, T cell clones HR-1 and HR-8 can be defined as type 1 of CD4+ Th cells (Th1 cells). Further characterization of Th1 clone HR-8 demonstrated that the Ag recognition was restricted to MHC class II (Fig. 4GoC). IFN-{gamma}-release by HR-8 was completely inhibited in the presence of anti-HLA-DR mAb HB55, but not in the presence of anti-MHC class I mAb w6/32, defining HLA-DR as restriction element.



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  		FIGURE 4. HER2-specific CD4+ Th1 cell clones can be isolated from patient HR with HER2-overexpressing breast cancer using HER2-transduced DC. CD4+ T cells were isolated from PBMC and stimulated three times with autologous DC retrovirally transduced with the HER2 gene. HER2-specific T cells were enriched based on their ability to secrete IFN-{gamma} upon stimulation with HER2-transduced DC and then cloned by limiting dilution. A, HER2 specificity of growing T cell clones was determined by detecting IFN-{gamma} released by the T cells upon stimulation with autologous DC pulsed with either rICD or rECD. B, Expanded CD4+ Th1 clones HR-1 and HR-8 recognized rECD presented by autologous DC. Both Th1 clones did not secrete IFN-{gamma} when DC were loaded with the irrelevant protein KLH. C, IFN-{gamma} release by Th1 clone HR-8 was completely inhibited in the presence of anti-HLA-DR mAb HB55, but not in the presence of anti-MHC class I mAb w6/32.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Results obtained from the current immunotherapy studies lead to the conclusion that both tumor-specific helper and cytotoxic T cells are required for optimal eradication of cancer. In this study, we have imitated the physiological interaction of DC and T cells in an in vitro priming model to generate HER2-specific CD8+ and CD4+ T cells. DC retrovirally transduced with the HER2 gene were used as APC, because DC genetically modified to express Ags take advantage of processing and presenting individual T cell epitopes in the context of different MHC alleles. In addition, due to the low Ag expression level of retrovirally transduced DC, stimulated T cells are confronted with low amounts of peptides that support the generation of T cells with a high enough affinity for naturally processed peptides. HER2-transduced DC from the HLA-A2+ and HLA-A3+ donor FS simultaneously elicited autologous CTL recognizing HER2-overexpressing tumor cells in the context of HLA-A2 and HLA-A3. Induction of both HLA-A2- and A3-restricted HER2-specific CTL was confirmed on a clonal level. This indicates that, despite the presence of the immunodominant HLA-A2 allele (37, 38, 39, 40, 41, 50), HER2 peptides are presented in the context of HLA class I alleles other than HLA-A2. Related studies have shown, that DC engineered to express melanoma-associated Ags using different vector systems can process and present multiple epitopes along with MHC class I (58, 64, 65, 66, 67). In this study, HLA-A2- and A3-matched tumor cells were used as targets for screening HER2-specific tumor-reactive CTL. To take advantage of all HLA molecules of an individual, current screening procedures use autologous fibroblasts as targets (68) that had been transfected with a modified vaccinia virus Ankara (69), resulting in an HER2 overexpression of fibroblasts, and, therefore, mimicking HER2-overexpressing autologous tumor cells.

The clinical goal of our studies is the adoptive T cell transfer in tumor patients. We therefore addressed the question of whether Ag-specific CTL and Th1 cells could be generated from patients with advanced cancer. Herein, we demonstrate that it is feasible to generate HER2-specific CTL clones from a patient with HER2-overexpressing breast cancer, who had been treated with several chemotherapy regimens for metastatic disease. HER2-transduced DC derived from the HLA-A2+ patient HR induced HER2-reactive CTL that lysed HER2-overexpressing tumor cells in an HLA-A2-restricted manner. The two isolated CTL clones displayed the same specificity pattern as the parental T cell line. It has been previously shown that murine DC transduced with a retrovirus encoding native OVA protein were able to present OVA-derived peptides in the context of MHC class II (18). Based on these findings, we examined whether human DC retrovirally transduced with the HER2 gene were capable of stimulating HER2-specific CD4+ T cells. The development of Th cells was directed into the Th1 pathway by using a high APC:responder ratio (70). In addition to the mode of stimulation, HER2-specific Th1 cells were screened by the ability to specifically release the Th1 cytokine IFN-{gamma}. For specificity analyses, autologous DC pulsed with the recombinant HER2 protein were used to imitate the physiological situation of DC presenting processed protein Ags to Ag-specific T cells. HER2-transduced DC derived from patient HR elicited autologous HER2-specific CD4+ Th1 cells that produced IFN-{gamma} upon stimulation with HER2-pulsed DC. Two Th1 clones were isolated that released IFN-{gamma} upon stimulation with autologous DC pulsed with the rECD of HER2, but not with an irrelevant protein Ag. Ag recognition by Th1 clone HR-8 was restricted to HLA-DR. The CD4+ T cell clones did not lyse Ag-pulsed DC, indicating the presence of nonlytic IFN-{gamma}-producing Th 1 cells recognizing naturally processed HER2 peptides in context with MHC class II. Up until now, the existence of CD4+ T cells against HER2 was driven from proliferative responses of polyclonal T cells against peptides with a binding motif for MHC class II (44, 45, 46, 47).

In the current study, we have succeeded in isolating and expanding HER2-reactive CTL and Th1 clones to numbers that allow adoptive T cell transfer. The ability to adoptively transfer Ag-specific T cell clones with defined Ag specificity provides the optimal basis for effective tumor elimination. In contrast, previous studies for adoptive T cell transfer based on polyclonal T cells showed limited efficacy, most likely due to the low frequency of Ag-specific CTL and Th1 cells present in the transferred T cell population (71, 72). Adoptive transfer of Th1 cells and CTL, both specific for HER2, should result in a strong synergistic tumor immunity, since Ag-specific Th cells are superior to Th cells recognizing an unrelated epitope, as previously shown in a mouse model (17). Furthermore, it has been shown by Greenberg and colleagues (73) that the survival of adoptively transferred virus-specific CTL clones is dependent on the presence of virus-specific T cell help. In addition to the regulatory helper role of CD4+ Th1 cells, adoptively transferred HER2-specific CD4+ T cells may eliminate HER2-overexpressing tumor cells by CTL-independent mechanisms (8, 9, 10, 11, 12). Up until now, HER2-directed immunotherapies, such as vaccine therapy with HER2-derived peptides (16, 49) or treatment with mAb trastuzumab (74, 75) have not induced autoimmune disease in patients with HER2-overexpressing tumors. However, it cannot be ruled out that the transfer of large amounts of T cells specific for the self-Ag HER2 might lead to autoimmunity. Greenberg and colleagues (29) have recently reported that adoptive transfer of CTL clones directed to the melanocyte differentiation Ag Melan-A/MART-1 led to melanocyte destruction in melanoma patients. Given the fact that HER2 is an Ag overexpressed by malignant cells in contrast to the corresponding normal cells expressing only low levels of HER2, adoptive transfer of HER2-specific T cells with low avidity (50) might lead to tumor rejection without damage in normal tissues in vivo (76).

In this study, we have succeeded in generating CD8+ CTL and CD4+ Th1 cell clones that recognize different HER2 epitopes. The ability to generate HER2-reactive T killer and helper 1 cell clones even from a patient with advanced stage cancer further encourages the development of T cell therapy. Ongoing studies focus on the feasibility, toxicity, and efficacy of adoptively transferred HER2-specific CTL and Th1 cell clones in patients with HER2-overexpressing breast cancer.


   Acknowledgments
 
We thank Dr. Martin Cheever (Corixa Corp., Seattle, WA) for providing rECD; Dr. Bernd Gänsbacher (Department of Experimental Oncology, Technical Universiy of Munich) for providing the retroviral vector system; and Dr. Ekkehard Albert (Laboratory for Immunogenetics, Ludwig Maximilian-Universität München) for HLA typing.


   Footnotes
 
1 This work was supported by the German Research Council (Sonderforschungsbereich 456), the Wilhelm Sander-Stiftung, and the Deutsche Krebshilfe. Back

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. E-mail address: helga.bernhard{at}lrz.tum.de Back

3 Abbreviations used in this paper: DC, dendritic cell; Flt3-L, fetal liver tyrosine kinase 3 ligand; HER2, human epidermal growth factor receptor 2; HPC, hemopoietic progenitor cell; ECD, extracellular domain; ICD, intracellular domain; LCL, B-lymphoblastoid cell line; SCF, stem cell factor; KLH, keyhole limpet hemocyanin. Back

Received for publication January 9, 2001. Accepted for publication May 24, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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