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Cancer Research 67, 5498-5504, June 1, 2007. doi: 10.1158/0008-5472.CAN-07-0304
© 2007 American Association for Cancer Research
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Immunology

Recognition of Prostate and Melanoma Tumor Cells by Six-Transmembrane Epithelial Antigen of Prostate–Specific Helper T Lymphocytes in a Human Leukocyte Antigen Class II–Restricted Manner

Hiroya Kobayashi1, Toshihiro Nagato1, Keisuke Sato1, Naoko Aoki1, Shoji Kimura1, Masamoto Murakami2, Hajime Iizuka2, Makoto Azumi3, Hidehiro Kakizaki3, Masatoshi Tateno1 and Esteban Celis4

Departments of 1 Pathology, 2 Dermatology, and 3 Urology, Asahikawa Medical College, Asahikawa, Japan; and 4 Immunology Program and Department of Interdisciplinary Oncology, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida

Requests for reprints: Esteban Celis, H. Lee Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, Tampa, FL 33612. Phone: 813-745-1925; E-mail: ecelis{at}moffitt.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The six-transmembrane epithelial antigen of prostate (STEAP) protein is an attractive candidate for T cell–based immunotherapy because it is overexpressed in prostate cancer and various other tumor types. Several peptide epitopes capable of stimulating CTLs that killed STEAP-expressing tumor cells have been described. Our goal was the identification of helper T lymphocyte (HTL) epitopes of STEAP for the optimization of T cell–based immunotherapies against STEAP-expressing malignancies. Candidate HTL epitopes for STEAP were predicted using in silico algorithms for HLA class II–binding peptides and were tested for their ability to elicit HTL responses by in vitro peptide vaccination of CD4 T lymphocytes from healthy individuals and prostate cancer patients. Two peptides (STEAP102–116 and STEAP192–206) were effective in stimulating in vitro antitumor HTL responses in both normal individuals and prostate cancer patients. Notably, both STEAP HTL peptides behaved as promiscuous T-cell epitopes because they stimulated T cells in the context of more than one MHC class II allele. These newly described STEAP HTL epitopes could be of value for the design and optimization of T cell–based immunotherapy against STEAP-expressing tumors. [Cancer Res 2007;67(11):5498–504]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Prostate cancer is one of the most common types of malignancies and is the third leading cause of cancer death affecting adult men in the United States (1). In recent decades, its incidence has been increasing in Japan. Of concern is that limited therapeutic options exist for prostate cancer patients that are diagnosed at an advanced stage or when metastatic hormone-refractory disease has been established. In addition, not only the effectiveness of some of these therapies against advanced disease remains questionable but also their toxic effects and constraints in the patient's quality of life makes them undesirable. To overcome this situation, the development of novel nontoxic alternative therapies for prostate cancer such as immunotherapy using appropriate tumor-associated antigens (TAA) should be explored. Unfortunately, only a few TAAs for prostate carcinoma have thus far been identified and it still is unclear whether any of these will be capable of conferring effective antitumor immunity.

The six-transmembrane epithelial antigen of prostate (STEAP) protein, which is highly expressed in human prostate cancer and other tumors (bladder, colon, ovarian, and Ewing sarcoma), has been considered as a potential TAA for the development of T cell–based immunotherapy because it is almost absent in normal tissues (2). Our group and other investigators succeeded in inducing in vitro antitumor CD8 CTL responses using HLA-A2–restricted peptide epitopes (3–5). Moreover, in mouse tumor models, STEAP-specific CTLs were found to be effective in inhibiting the growth of transplantable prostate tumor cells that express the murine STEAP orthologue (4, 6).

Increasing evidence from both human and murine studies indicates that TAA-specific CD4 helper T lymphocytes (HTL) play a central role in orchestrating immune response against malignancies and infectious diseases (7–9). Specifically, some studies have shown the essential role of HTLs in the elimination of tumors by (a) enhancing the generation of CTL during antigen priming; (b) facilitating the production and maintenance of memory CTL responses; and (c) by stimulating and maintaining the effector CTLs at the tumor site (10–13). Moreover, TAA-reactive HTLs are more effective than surrogate HTLs (those directed against non-TAA) in providing the antitumor effects (12). In view of this, we believe that only those vaccines capable of stimulating both TAA-reactive CTLs and HTLs will be effective in generating immune responses that will provide clinical benefit. Thus, because the identification of HTL epitopes from TAAs remains a priority for the design of clinically effective T cell–based vaccines, we have examined the capacity of STEAP to function as a TAA for HTL. Here, we report that two synthetic peptides from STEAP (STEAP102–116 and STEAP192–206) were capable of eliciting in vitro antigen-specific, MHC class II–restricted HTL responses. Most importantly, the STEAP-reactive HTLs were effective in recognizing the naturally processed STEAP protein on either the tumor cells directly or on antigen-presenting cells (APC) fed with dead tumor cells. In addition, the two STEAP peptides were also found to stimulate T-cell responses in prostate cancer patients. We believe that these HTL epitopes may be used in combination with previously described CTL epitopes to enhance the efficacy of vaccines directed against STEAP-expressing cancers.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines. EBV-transformed lymphoblastoid cell lines (EBV-LCL) were produced from peripheral blood mononuclear cells (PBMC) of HLA-typed volunteers using culture supernatant from the EBV-producing B95-8 cell line, obtained from the American Type Culture Collection. Mouse fibroblast cell lines (L cells) transfected and expressing individual human MHC-II molecules were kindly provided by Drs. Robert W. Karr (Idera Pharmaceuticals, Essex, CT) and Takehiko Sasazuki (Kyushu University, Fukuoka, Japan). The following tumor cell lines were obtained from the ATCC: LNCaP, PC3, and DU145 (prostate cancers); MCF7 and SKBr3 (breast cancers); SKmel28 (melanoma); WiDr and SW403 (colon cancers); KU812, K562 and KG1 (myeloid leukemias); Jurkat (T cell lymphoma); and Raji (lymphoma). The prostate cancer cell line LAPC4 was provided by Dr. Charles Sawyers (University of California at Los Angeles, Los Angeles, CA). The melanoma cell lines 624mel, 697mel, and 888mel were provided by Dr. Steven Rosenberg (Surgery Branch, National Cancer Institute, NIH, Bethesda, MD). The melanoma cell line MM33.1 was provided by Dr. Manabu Nakashima (Kyushu University, Fukuoka, Japan). The gastric carcinoma cell line MKN45 was supplied by the Japanese Cancer Research Resources Bank (Tokyo, Japan). The myeloid leukemia cell line KT1 was kindly provided by Dr. Masaki Yasukawa (Ehime University, Ehime, Japan). MT2 is an human T-cell lymphotrophic virus-1 (HTLV-1)–transformed T-cell line that was kindly provided by Dr. Y. Hinuma (Institute of Virus Research, Kyoto University, Kyoto, Japan). All cell lines were maintained in tissue culture as recommended by the supplier.

Synthetic peptides. Potential HLA-DR–restricted CD4+ T-cell epitopes were selected from the amino acid sequence of the STEAP protein using the peptide/MHC binding prediction algorithm tables derived for three HLA-DR alleles (DRB1*0101, DRB1*0401, and DRB1*0701) as described by Southwood et al. (14). The predicted peptide epitopes were synthesized by solid-phase organic chemistry and purified by high-performance liquid chromatography (HPLC). The purity (>80%) and identity of peptides were assessed by HPLC and mass spectrometry, respectively. The following synthetic peptides were used throughout this work: STEAP102–116 (HQQYFYKIPILVINK), STEAP131–145 (LPGVIAAIVQLHNGT), STEAP192–206 (LLNWAYQQVQQNKED), STEAP300–314 (VVLIFKSILFLPCLR), WT1124–138 (QARMFPNAPYLPSCL), and the Pan DR Epitope "PADRE" (aKXVAAWTLKAAa, where "a" is D-alanine, and "X" is L-cyclohexylalanine).

In vitro induction of antigen-specific HTLs with synthetic peptides. The procedure used for the generation of STEAP-reactive HTL lines using peptide-stimulated lymphocytes has been described in detail (15). Briefly, dendritic cells were produced in tissue culture from purified CD14+ monocytes (using antibody-coated magnetic microbeads from Miltenyi Biotech) that were cultured for 7 days at 37°C in a humidified CO2 (5%) incubator in the presence of 50 ng/mL granulocyte macrophage colony-stimulating factor (GM-CSF) and 1,000 IU/mL interleukin-4 (IL-4). Peptide-pulsed dendritic cells (3 µg/mL for 2 h at room temperature) were irradiated (4,200 rad) and cocultured with autologous purified CD4+ T cells (using antibody-coated magnetic microbeads from Miltenyi Biotech) in 96 round-bottomed-well culture plates. One week later, the CD4+ T cells were restimulated in individual microcultures with peptide-pulsed irradiated autologous PBMC, and 2 days later, human recombinant IL-2 was added at a final concentration of 10 IU/mL. One week later, the T cells were tested for antigen reactivity using a cytokine release assay as described below. Those microcultures exhibiting a significant response of cytokine release to peptide (at least 2.5-fold over background) were expanded in 24- or 48-well plates by weekly restimulation with peptides and irradiated autologous PBMC. Complete culture medium for all procedures consisted of AIM-V medium (Invitrogen/Life Technologies) supplemented with 3% human male AB serum. All blood samples were obtained after the appropriate informed consent.

Measurement of antigen-specific responses with HTL lines. CD4 T cells (3 x 104 per well) were mixed with irradiated APCs in the presence of various concentrations of antigen (peptides, tumor lysates) in 96-well culture plates. APCs consisted of either autologous PBMCs (1 x 105 per well), HLA-DR–expressing L cells (3 x 104 per well), MHC-typed EBV-LCL (3 x 104 per well), autologous dendritic cells (5 x 103 per well), or prostate, melanoma, and breast tumor cell lines (3 x 104 per well that were previously treated with IFN-{gamma} at 500 units/mL for 48 h to enhance MHC antigen expression). The expression of HLA-DR molecules on tumor cells was evaluated by flow cytometry using anti–HLA-DR (L243) monoclonal antibody (mAb) conjugated with FITC. Tumor cell lysates were prepared by three freeze-thaw cycles of 1 x 108 tumor cells, resuspended in 1 mL of serum-free RPMI 1640 medium. Tumor lysates were used as a source of antigen at 5 x 105 cell equivalents per milliliter. Culture supernatants were collected after 48 h for measuring antigen-induced lymphokine (IFN-{gamma} or GM-CSF) production by the HTL ELISA kits (BD PharMingen). To show antigen specificity and MHC restriction, blocking of antigen-induced responses were assessed by adding anti–HLA-DR mAb L243 (IgG2a, prepared from supernatants of the hybridoma HB-55 obtained from the ATCC) or anti–HLA-A/B/C mAb W6/32 (IgG2a, ATCC) at 10 µg/mL throughout the 48-h incubation period. All ELISA determinations were carried out in triplicate and results correspond to the mean values with the SD of the mean.

Western blot analysis. One million tumor cells and CD40-activated B cells were washed in PBS and lysed in NuPAGE LDS sample buffer (Invitrogen). The cell lysate was subjected to electrophoresis in a 4% to 12% NuPAGE bis-Tris SDS-PAGE gel (Invitrogen) under reducing condition and then transferred to Immobilon-P (Millipore) membrane. The membrane was then blocked in PBS containing 0.01% Tween 20 and 5% nonfat dry milk for 1 h at room temperature and incubated with antihuman STEAP (H-105) rabbit polyclonal antibody (1:200 in blocker; Santa Cruz Biotechnology) overnight at 4°C. After washing, the membrane was incubated with horseradish peroxidase–labeled donkey anti-rabbit IgG and subjected to the enhanced chemiluminescence (ECL) assay using the ECL detection system (Amersham). To generate CD40-activated B cells, purified CD19+ cells (using antibody-coated magnetic microbeads from Miltenyi Biotech) were cultured with either 1 µg/mL of a soluble CD40 ligand (PeproTech) or 10 µg/mL of an antihuman CD40 mAb (BD PharMingen) in the presence of 1,000 IU/mL IL-4 in complete medium consisting of Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 10% FCS (16). Fresh complete medium was added on day 3 in the presence of IL-4 with either soluble CD40 ligand or anti-CD40 mAb, and on days 7 to 10, the cells were harvested for Western blot analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of HTL epitopes from STEAP. To identify HLA class II promiscuous HTL epitopes from STEAP, we selected four peptides (STEAP102–116, STEAP131–145, STEAP192–206, and STEAP300–314) that displayed high prediction binding scores to HLA-DR1, HLA-DR4, and HLA-DR7 (data not shown). Combined, these three HLA class II molecules are commonly found in the general population (~68.3% in Caucasian and ~52.1% in Japanese; ref. 14). The in silico prediction system that was used is based on the algorithm tables described by Southwood et al. (14) and has been useful in identifying HTL epitopes from numerous TAAs (15, 17–25). In many instances, the predicted peptides were able to elicit HTL responses restricted by MHC class II alleles other than HLA-DR1, HLA-DR4, and HLA-DR7 (e.g., HLA-DR9, HLA-DR13, HLA-DR15, HLA-DR51, HLA-DR52, and HLA-DR53), indicating that this prediction system extends beyond the original three MHC alleles. Thus, we synthesized the four STEAP epitope peptides and proceeded to determine whether these peptides could induce HTL responses in vitro.

HTL responses to peptide epitopes from STEAP. CD4 T lymphocytes were isolated from PBMCs of five healthy MHC class II–typed individuals (HLA-DR1/15, HLA-DR4/15, HLA-DR4/9, HLA-DR9/14, and HLA-DR9/14) and were stimulated with autologous peptide-pulsed dendritic cells. After three to four peptide restimulation cycles using autologous irradiated PBMCs as APCs, two of the peptides (STEAP102–116 and STEAP192–206) were able to induce HTL responses as indicated by the production of IFN-{gamma} when stimulated with peptide-pulsed autologous PBMCs (data not presented). T-cell clones were isolated from these cultures by limiting dilution to assess the fine specificity of these responses. A total of five T-cell clones reactive with peptide STEAP102–116 (9C, TN14, MT12D, MT2E, and K9) and three T-cell clones specific for peptide STEAP192–206 (10E, 11C, and HK1) were isolated and in all cases responses to the peptides were observed in a dose-dependent manner (Fig. 1A and B ). All the T-cell clones were specific for the corresponding immunizing peptide because no responses were detected when an irrelevant synthetic peptide (WT1124–138; ref. 25) was used in similar assays (Fig. 1C).


Figure 1
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  		Figure 1. Induction of HTL responses using predicted peptide epitopes derived from STEAP. A, HTL clones induced with peptide STEAP102–116 (clone 9C from DR4/9 donor; clone TN14 from DR9/14 donor; clone MT12D, MT2E from DR9/14 donor; and clone K9 from DR1/15 donor) were tested for their capacity to recognize autologous PBMC as APCs in the presence of various concentrations of peptide. B, results with HTL clones obtained with peptide STEAP192–206 (clone 10E, 11C from DR9/14 donor and clone HK1 from DR4/9 donor). C, HTL responses against an irrelevant peptide (WT1124–138). Points, mean of triplicate determinations; bars, SD. Points without bars had SD <10% the value of the mean. Results are representative of at least two experiments that were done with the same samples.

 
MHC class II restriction analysis of STEAP-reactive HTLs. To determine which MHC molecules presented the STEAP peptides to the HTL clones, we first did blocking experiments using MHC-specific monoclonal antibodies (mAb). As shown in Fig. 2A , anti–HLA-DR mAb L243 substantially inhibited the IFN-{gamma} production from all the STEAP102–116-specific HTL clones that were stimulated with peptide pulsed APCs; however, no effective inhibition was observed with the anti-HLA class I mAb W6/32. Because antibody L243 does not react with the other MHC class II molecules (HLA-DP and HLA-DQ), these results suggested that peptide STEAP102-116 was presented to the HTL clones in an HLA-DR–restricted manner. Moreover, when mouse fibroblast (L cells) transfected with HLA-DR molecules derived from individual alleles were used as APCs, we observed that peptide STEAP102–116-reactive HTL clones 9C and MT2E recognized peptide in the context of the HLA-DR9 allele (Fig. 2B). Similarly, HTL clone K9 responded to peptide presented by APC-expressing HLA-DR1 and HTL clones TN14 and MT12D were restricted by the HLA-DR53 allele (Fig. 2B).


Figure 2
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  		Figure 2. MHC restriction analysis of STEAP-reactive HTL clones. A, peptide-reactive STEAP102–116 HTL clones. MHC class II restriction molecules were identified by antibody blocking using anti-DR L243 or anti-MHC class I W6/32 (negative control) mAbs (both used at 10 µg/mL). These experiments were done using irradiated peptide-pulsed autologous PBMCs as APCs. B, in addition, responses of the STEAP102–116 HTL clones were also evaluated using L cells transfected with individual HLA-DR genes as APCs to define the restricting MHC class II alleles. HTL clone 9C derived from the DR4/9 donor; clone MT2E derived from the DR9/14 donor; clone K9 derived from the DR1/15 donor; clone TN14 derived from the DR9/14 donor; and clone MT12D derived from the DR9/14 donor. C, MHC restriction analysis of peptide STEAP192–206 HTL clones. D, antigen presentation by transfected L cells to the STEAP192–206 HTL clones. Experimental conditions were identical to those described for (A) and (B). HTL clone HK1 derived from the DR4/9 donor; clones 11C and 10E derived from the DR9/14 donor. Columns, mean of triplicate determinations; bars, SD. Columns without bars had SD <10% the value of the mean. Results are representative of two experiments that were done with the same samples.

 
In the case of the peptide STEAP192–206-specific HTL clones, we observed that HLA-DR molecules also presented this epitope (Fig. 2C) and that the restriction alleles were HLA-DR9 for clone HK1 and HLA-DR53 for clones 11C and 10E (Fig. 2D). The results thus far indicate that peptides STEAP102–116 and STEAP192–206 behave as typical promiscuous epitopes because they can be presented to HTL by more than one MHC class II allele.

Peptides STEAP102–116 and STEAP192–206 are naturally processed epitopes on STEAP-expressing tumor cells. Although the data presented above show that the peptides STEAP102–116 and STEAP192–206 could elicit HTL responses, it was critical to assess whether these epitopes were produced by STEAP-expressing tumor cells via the MHC class II antigen–processing pathway. To select the appropriate tumor cells for HTL recognition assays, we first assessed the expression of the STEAP protein and cell surface HLA-DR molecules in several tumor cell lines. Expression of the STEAP protein was observed in several prostate tumor cell lines (LNCaP, LAPC4, PC3, and DU145), melanomas (624mel, 697mel, 888mel, and MM33.1), gastrointestinal tumors (MKN45, WiDr), and in a breast tumor cell line (MCF7). On the other hand, very little STEAP was detected in SKmel28 (melanoma), SW403 (colon), and SKBr3 (breast) cells (Supplementary Fig. S1). Because it is necessary for tumor cells to express cell surface MHC class II molecules to react directly with HTLs, we evaluated the expression level of cell surface HLA-DR molecules in some of the STEAP-expressing cell lines. In these experiments, the cells were cultured with IFN-{gamma} for 48 h before the assays to enhance the expression of MHC molecules on the cell surface. HLA-DR expression was observed in two STEAP-expressing tumors (PC3 and 697mel), which express the HLA-DR53 molecule (Supplementary Fig. S1). Substantial expression of cell surface HLA-DR molecules was also observed in the STEAP-negative SKBr3 cells (also of the HLA-DR53 allele), and, as expected, HLA-DR expression was high in an EBV-LCL, which was included as a positive control (Supplementary Fig. S1). In view of these findings, PC3 and 697mel cells were used as relevant APCs and SKBr3 and EBV-LCL were included as negative controls in assays that examined the direct recognition of STEAP+ tumor cells by the HLA-DR53–restricted HTL clones. The data presented in Fig. 3 shows that STEAP102–116-specific HTL clones TN14 and MT12D, and STEAP192–206-specific HTL clone 10E, were all capable of recognizing antigen directly on DR53+, STEAP+ PC3, and 697mel cells but not the DR53+, STEAP-negative SKBr3 cells. The reactivity against PC3 and 697mel by the HTL clones was inhibited by anti–HLA-DR mAb, indicating that the interaction of the HTL and the tumor cells was via the T-cell receptor and the tumor MHC class II molecules. Quite unexpectedly, the HTL clones exhibited strong responses against autologous EBV-LCL (included in these assays as additional negative controls), suggesting the possibility of STEAP expression by these cells. In view of this, we examined the expression STEAP protein by EBV-LCL by Western blot analysis. STEAP was clearly observed in 6 of the 11 EBV-LCLs that were tested (Supplementary Fig. S2). However, STEAP was not detected in several hematologic malignant cell lines (Burkitt's lymphoma Raji, myeloid leukemia, histiocytic lymphoma, T-cell lymphoma, and HTLV-1+ T-cell line), or nontransformed B lymphocytes whether these were stimulated with anti-CD40 mAb, CD40 ligand, or infectious EBV (Supplementary Fig. S2). These results suggest that STEAP expression in transformed B lymphocytes may be induced by EBV, but seems to be limited to the latency III stage.


Figure 3
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  		Figure 3. Antigen specificity and direct tumor recognition by HLA-DR53–restricted, STEAP-reactive HTLs. STEAP102–116-reactive HTL clones TN14 and MT12D, and STEAP192–206-reactive HTL clone 10E, were tested for their capacity to recognize antigen directly (without additional APCs) on HLA-DR53+/STEAP+ tumor cells (PC3 and 697mel) and an HLA-DR53+, STEAP-negative tumor (SKBr3). The ability of anti–HLA-DR mAb L243 to inhibit T-cell recognition was also assessed. The HTL clones were also tested against autologous EBV-LCLs. Columns, means of triplicate determinations; bars, SD. Columns without bars had SD <10% the value of the mean. Results are representative of two experiments that were done under the same conditions.

 
In view of the above, we examined the ability of STEAP-expressing EBV-LCLs to be recognized by the STEAP-specific HTL. As shown in Fig. 4A , the HLA-DR9–restricted STEAP102–116-specific HTL clones 9C and MT2E were effective in reacting with the STEAP+ HLA-DR9+ EBV-LCL (LCL-HK, LCL-TN and LCL-Ky) but not with the DR9+, STEAP-negative EBV-LCL (LCL-YA) or with the DR9 negative, STEAP+ EBV-LCL (LCL-KO). Similar patterns of reactivity and specificity were obtained with the HLA-DR9–restricted STEAP192–206-specific HTL clone (Fig. 4B) and with the two HLA-DR53–restricted HTL clones specific for STEAP102–116 (Fig. 4C) and STEAP192–206 (Fig. 4D). These results indicate that the expression of STEAP in EBV-LCL results in the generation of MHC class II–binding peptides that can be directly recognized by HTL.


Figure 4
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  		Figure 4. Recognition of EBV-LCLs by STEAP-reactive HTL clones. A, HLA-DR9–restricted and STEAP102–116-reactive HTL clones 9C and MT12E were tested for their ability to recognize STEAP+ and STEAP-negative LCLs in an MHC-restricted manner. The following LCLs were used as APCs: HLA-DR9+/STEAP+ EBV-LCLs (LCL-HK, LCL-TN, and LCL-Ky), HLA-DR9+/STEAP-EBV-LCL (LCL-YA), and HLA-DR9-/STEAP+ LCL (LCL-KO). B, STEAP192–206-reactive HTL clone HK1 was tested for its capacity to recognize HLA-DR9+/STEAP+ EBV-LCLs (LCL-HK, LCL-TN, and LCL-MT) and HLA-DR9+/STEAP-EBV-LCL (LCL-YA). Similar determinations were done with the HLA-DR53–restricted, STEAP102–116-reactive HTL clone MT12D (C), and the STEAP192–206-reactive HTL clone 10E (D) using HLA-DR53+/STEAP+ EBV-LCLs (LCL-TN, LCL-HK, LCL-MT, LCL-Ky, and LCL-KO) and an HLA-DR53+/STEAP– LCL (LCL-Wa). Columns, means of triplicate determinations; bars, SD. Columns without bars had SD <10% the value of the mean. Results are representative of two experiments that were done under the same conditions.

 
Recognition of naturally processed STEAP antigen presented to HTL by autologous dendritic cells. The results presented thus far show that STEAP-reactive HTL can recognize antigen directly on MHC class II–expressing STEAP+ transformed cells. Another way that TAA-specific HTL can recognize antigen is on conventional APCs (e.g., dendritic cells, macrophages) that have ingested dead tumor cells (or tumor lysates). Thus, we assessed whether autologous dendritic cells would be able to capture the STEAP antigen derived from dead STEAP+ tumor cells (freeze-thaw cell lysates) and appropriately process the antigen through the MHC class II pathway to stimulate STEAP-specific HTL clones. As shown in Fig. 5 , the DR53-restricted STEAP102–116-reactive HTL clones TN14, MT12D, and the DR9-restricted STEAP102–116-reactive HTL clone MT2E were efficient in recognizing STEAP+ tumor cell lysates (produced from LNCaP and 888mel cells) when presented by autologous dendritic cells. These HTL responses were antigen specific and via the interaction of the TCR with MHC class II molecules because lysates prepared from a STEAP-negative tumor (Jurkat) were not stimulatory and the response to STEAP+ tumor lysates was inhibited by anti–HLA-DR mAb (Fig. 5). Paradoxically, none of the HTL specific for epitope STEAP192–206 were able to recognize antigen in the form of tumor lysates presented by autologous dendritic cells (data not shown), although as previously shown, these HTLs reacted well against antigen presented directly by MHC class II–expressing tumors and EBV-LCL (Figs. 3 and 4).


Figure 5
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  		Figure 5. STEAP-reactive HTLs recognize naturally processed exogenous antigen presented by autologous dendritic cells. STEAP102–116-reactive HTL clones TN14, MT2E, and MT12D were tested for reactivity against STEAP+ tumor cell (LNCaP, 888mel) lysates (Lys) and a STEAP-negative cell (Jurkat) lysate when presented by autologous dendritic cells (DC). The capacity of anti-DR mAb (L243) was assessed to determine that T-cell reactivity was through TCR/MHC interactions. Columns, mean of triplicate determinations; bars, SD. Columns without bars had SD <10% the value of the mean. Data are representative of two experiments that were done under the same conditions and with the same samples.

 
Recognition of peptides STEAP102–116 and STEAP192–206 by PBMCs from prostate cancer patients. Last, we examined whether T cells from prostate cancer patients would recognize peptides STEAP102–116 and STEAP192–206. Because only small blood samples from these patients were available, we were unable to establish long-term T-cell lines and to perform HLA typing for some of the patients. PBMCs from four advanced prostate cancer patients were stimulated twice (at a 1-week interval) with the STEAP peptides and with the PADRE peptide, which was used as a positive control because it elicits strong HTL responses in the majority individuals regardless of their MHC class II alleles (26). Seven days after the second stimulation with peptide, culture supernatants were collected and analyzed for lymphokine production (IFN-{gamma} and GM-CSF) resulting from T-cell activation by the peptide antigens. The data presented in Fig. 6 show that significant T-cell responses to both STEAP peptides and to PADRE were evident in three of four prostate cancer patients as indicated by the production of IFN-{gamma} and GM-CSF. These results indicate that T-cell precursors reactive with the newly described STEAP peptide epitopes exist in the peripheral blood of prostate cancer patients.


Figure 6
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  		Figure 6. Assessment of T-cell responses to the STEAP102–116 and STEAP192–206 epitopes in prostate cancer patients. PBMCs from four prostate cancer patients were stimulated with peptides and T-cell responses (production of IFN-{gamma} and GM-CSF) were measured as described in Materials and Methods. Columns, means of triplicate determinations; bars, SD. Columns without bars had SD <10% the value of the mean.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The identification of a reliable TAA for prostate cancer containing immunogenic CTL and HTL epitopes will be critical for the development of effective antitumor immunotherapy. Thus far, clinical trials in cancer patients using only CTL epitope peptide-based vaccines have been ineffective in triggering strong and long-lasting immune responses. Thus, we believe that vaccines capable of stimulating both tumor-reactive CTL and HTL would be more effective in the clinic. In addition, it is clear that a single TAA will not be sufficient to offer population coverage and that it would be advisable for a vaccine to include CTL and HTL epitopes from multiple antigens (27). In the case of prostate cancer, we and others have previously identified CTL and HTL epitopes from other TAAs such as prostate-specific membrane antigen and T-cell receptor alternative reading frame protein (21, 23, 28–32). Because STEAP is another attractive TAA for prostate cancer, significant efforts were first devoted toward the identification of CTL epitopes (3–5). Here, we have described two HTL epitopes for STEAP that could be used to complement the previously described STEAP CTL epitopes and the CTL/HTL epitopes from prostate-specific membrane antigen and T-cell receptor alternative reading frame protein for the development of a comprehensive T cell–based immunotherapy for prostate cancer. It should be noted that both of the STEAP epitopes described here could be presented to HTL in the context of more than one MHC class II allele, which is a characteristic of promiscuous HTL epitopes that are highly desired for clinical use because of their increased population coverage.

TAA-reactive HTL can recognize antigen (MHC class II/peptide complexes) directly on MHC class II–expressing tumor cells, or indirectly on conventional APCs that have captured and processed exogenous antigens (derived from tumor cells). It is not clear whether these two modes of antigen recognition by tumor-reactive HTL lead to antitumor effects. It is conceivable that direct tumor recognition by HTL, if it occurs in vivo, could lead to the production of lymphokines with antitumor activity (e.g., tumor necrosis factor) and perhaps in some cases to cell-mediated cytotoxicity; however, this remains to be shown. On the other hand, indirect recognition of TAA by HTL though antigen presentation by conventional APCs will facilitate CTL responses during the antigen-priming phase occurring at the lymph node and later during the effector phase of the immune response at the tumor site. HTLs play a critical function during the initiation of the immune response by providing signals to the APCs and to the CTLs that result in effective CTL expansion, maturation to effector CTL, and establishment of memory CTL (33–35). In addition, HTL at the tumor site continue to assist CTL by providing costimulation allowing the CTL to continue to expand and prevent cell death (10–13). Our results show that both of the newly identified HTL epitopes were presented directly to HTL by STEAP+ HLA-DR+ tumor cells, but only one of the epitopes (STEAP102–116) was presented to HTL by conventional APCs. Although the data is somewhat limited, these results raise the possibility that these differences could be due to the nature of antigen-processing variances between MHC class II+ tumor cells and conventional dendritic cells.

The use of STEAP as a TAA is not limited to prostate cancer because it is also frequently found in other tumor types such as pancreas, colon, breast, testicular, bladder, ovarian, melanoma, and Ewing sarcoma (2, 3). Most importantly, the expression level in normal tissue is very low (except in prostate and bladder epithelium), which suggests that significant autoimmune pathology may not be elicited when this TAA is applied to cancer immunotherapy. An interesting and unexpected finding was the expression of STEAP protein in EBV-LCLs but not in normal resting or activated B-lymphocytes, or in hematologic malignancies. Moreover, STEAP102–116- and STEAP192–206-specific HTL clones had the ability of directly recognizing STEAP-expressing EBV-LCLs, indicating that the endogenous MHC class II molecules of EBV-LCLs presented STEAP-derived peptides. Recently, Tassi et al. (36) reported that EBV-LCLs were recognized by renal cell carcinoma–reactive CD4+ HTLs, suggesting that EBV-LCLs can express epithelial solid cancer antigens. In addition, we reported that the WT1 tumor antigen, which is predominantly expressed by leukemias, was also found in EBV-LCLs (25). Thus, it seems that under some circumstances during latent III EBV infection, some genes that are usually silent in B lymphocytes may be reactivated and expressed. These observations open the possibility of using EBV-LCLs that abnormally express TAAs to expand tumor-reactive CTL and HTL ex vivo for adoptive immunotherapy.


   Acknowledgments
 
Grant support: NIH grants P50CA91956, R01CA80782, and R01CA103921 (E. Celis), and Ministry of Education, Sports, and Culture of Japan grant-in-aid 18590360 (H. Kobayashi).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 1/23/07. Revised 3/ 6/07. Accepted 3/16/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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