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[Cancer Research 61, 5161-5167, July 1, 2001]
© 2001 American Association for Cancer Research

Immunology

Identification of T Helper Epitopes from Prostatic Acid Phosphatase1

Douglas G. McNeel2, Lan D. Nguyen and Mary L. Disis

Department of Medicine, Division of Medical Oncology, University of Washington, Seattle, Washington 98195


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Helper T cells (Th cells) play a central role in the initiation and maintenance of immune responses, including antitumor immunity. The ability of Th cells in murine models to maintain and enhance the cytolytic efficacy of CD8+ CTLs has led to a renewed interest in identifying human tumor antigens recognized by Th cells. Prostatic acid phosphatase (PAP) is a prostate cancer-associated tumor antigen. A rodent model has demonstrated that PAP-specific CTLs can induce destructive prostatitis. Human MHC class I epitopes derived from PAP have been identified previously, and peptide-specific CTLs have been shown to be able to lyse an MHC-restricted prostate cancer cell line. In the current study, we sought to identify Th epitopes derived from PAP that might be used to elicit PAP-specific Th responses, ultimately in the context of human vaccines targeting PAP. Using peripheral blood mononuclear cells (PBMCs) from subjects with and without PAP-specific Th responses, we screened a panel of 10 potential peptide epitopes for peptide-specific T-cell proliferation. Four peptides, p81-95, p199-213, p228-242, and p308-322, were identified for which peptide-specific T-cell proliferation occurred in the majority of patient PBMC samples that also exhibited PAP-specific T-cell proliferation. PBMCs from patients with prostate cancer and without PAP-specific Th immunity were then cultured in vitro with these four peptides. Peptide-specific T-cell lines could be generated from two of the four peptides, p199-213 and p228-242, that also proliferated in response to PAP protein stimulation. The ability of these two peptides to elicit PAP-specific Th responses suggests that they represent naturally processed PAP-specific MHC class II epitopes.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Th cells,3 typically CD4+ T cells, play a central role in the initiation of immune responses. Depending on the nature of the Th cells elicited, an immune response may be predominantly tolerizing or inflammatory (1) . This central role is of particular significance in antitumor immune responses, in which antigens recognized are typically autologous proteins, and therefore, the balance of inflammatory and tolerant immunity is of great importance (2 , 3) . Animal models have demonstrated the importance of CD4+ T cells in recruiting CD8+ CTLs (4) and other inflammatory cells such as macrophages and eosinophils (5) . CTLs have generally been believed to be the major participants in actual destruction of malignant tissue, and animal models suggest that the absence of a concurrent antigen-specific Th response can lead to CTL tolerance or ineffectiveness (1 , 6) . Other murine studies (7) have demonstrated that CD4+ T cells are required to maintain CTL memory and survival, as well as their functional ability to infiltrate tumors (8) . Similar results in human in vitro studies (9) have demonstrated that CD4+ T cells cross-prime dendritic cells to elicit tumor-specific CTLs. These roles of Th cells in antitumor immunity have led to a recent interest to define human tumor antigens recognized by Th cells (3 , 10 , 11) . To date, CD4+ T-cell responses have been identified specifically for several melanoma tumor antigens, including tyrosinase (12) , gp100 (13) , and MAGE-3 (14) . For prostate cancer, few human tumor antigens recognized by Th cells have been identified, and even less is known about the peptide epitopes recognized by these cells (15 , 16) .

Studies (17, 18, 19) in a rodent model of prostatitis have demonstrated that a CTL response directed at the prostate can destroy prostate tissue in vivo. These findings have led to an increased enthusiasm for the development of vaccines for treating patients with prostate cancer, specifically designed to elicit CTL immune responses to destroy malignant prostate cancer cells (20, 21, 22) . One means of eliciting CD8+ CTLs has been to immunize directly with antigen-specific MHC class I peptide epitopes recognized by CD8+ T cells. To that end, several groups have identified MHC class I epitopes from different potential prostate tumor antigens, including prostate-specific antigen (16 , 23, 24, 25) , prostate-specific membrane antigen (16 , 26) , and PAP (27) , with the concept of using these peptides as vaccine antigens. Because of the critical role of CD4+ T cells in eliciting effective CTLs and maintaining a CTL memory response, others (11 , 28, 29, 30) have advocated the incorporation of Th epitopes into the design of CTL epitope immunization strategies. Murine studies (31 , 32) , in fact, suggest that immunization with MHC class I peptide epitopes requires additional CD4+ T-cell help to elicit effective CTL responses. Consequently, identification of Th epitopes to defined tumor antigens may be important for inclusion in MHC class I peptide-based vaccines for prostate cancer, as well as other tumors, to enhance an antigen-specific CTL responses (11 , 13 , 16 , 33 , 34) .

PAP is a potential tumor-associated antigen in prostate cancer, given that expression of PAP is essentially restricted to prostate tissue (35 , 36) , including metastatic prostate cancer (37 , 38) . Studies (19) in rats have demonstrated that a CTL response directed at the rat PAP homologue will destroy prostate tissue in vivo. As noted above, investigators have identified, in a human in vitro system, several HLA-A2 peptide epitopes derived from human PAP. PAP peptide-specific CTLs can be cultured from the peripheral blood of volunteer male blood donors and are able to lyse an MHC-restricted prostate cancer cell line (27) . The use of CTL peptide epitopes in a human vaccine study to elicit PAP-specific CTLs has been contemplated (27) . In the current study, we sought to identify PAP-specific Th peptide epitopes that might be used to elicit a PAP-specific Th response in the context of a peptide-based vaccine. We have identified previously (15) subjects with prostate cancer who have PAP-specific Th responses. Using PBMCs from subjects with and without PAP-specific Th responses, we screened a panel of 10 potential peptide epitopes for peptide-specific T-cell proliferation. Four peptides were identified for which peptide-specific T-cell proliferation occurred in the majority of patient PBMC samples that also exhibited PAP-specific T-cell proliferation. PBMCs from patients with prostate cancer and without PAP-specific T-cell responses were then cultured in vitro with each of these four peptides. Peptide-specific T-cell lines could be generated from two of the four peptides that also proliferated in response to PAP protein stimulation. The ability of these two peptides to elicit PAP-specific Th responses suggests that they represent naturally processed PAP-specific MHC class II epitopes. Therefore, these peptides are reasonable candidates for consideration for inclusion in peptide-based vaccines designed to elicit PAP-specific CD4+ T-cell responses.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects.
With informed consent, peripheral blood or leukapheresis products were obtained from male subjects (27 with prostate cancer of varying disease stages and five volunteer donors without prostate cancer) at the University of Washington Medical Center between 1997 and 1999. PBMCs were isolated by Ficoll-Paque centrifugation (Pharmacia AB, Uppsala, Sweden) and cryopreserved in liquid nitrogen.

Peptides.
The amino acid sequence of human prostatic acid phosphatase was scanned using the software analysis package T sites (39) . On the basis of this analysis, 10 15-18-mer oligopeptides derived from the amino acid sequence of PAP were synthesized and purified to >80% by high-performance liquid chromatography, and the identity and purity was confirmed by mass spectrum analysis (United Biochemical Research, Inc., Seattle, WA). Purified peptides were reconstituted in sterile water, filtered, and stored in aliquots at -20°C. Peptide sequences are shown in Table 1Citation .


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  		Table 1 PAP-derived peptides

 
Evaluation of Potential Immunogenic Epitopes by T-cell Proliferative Response.
T-cell proliferative responses to PAP protein or peptide stimulation were evaluated using a modified limiting dilution analysis designed for detecting low frequency-responding T cells (40 , 41) . Specifically, cryopreserved PBMCs were extensively washed and resuspended in assay medium consisting of equal parts of EHAA 120 (Biofluids, Rockville, MD) and RPMI 1640 (Life Technologies, Inc., Rockville, MD) with 10 mM L-glutamine, 2% penicillin/streptomycin, 50 µM ß-mercaptoethanol, and 10% human AB serum (Valley Biomedical, Winchester, VA). PBMCs (2 x 105)/well were plated into 96-well round-bottomed microtiter plates (Costar, Cambridge, MA) and cultured with highly purified 2.0 µg/ml PAP (Research Diagnostics Inc., Flanders, NJ; Chemicon International, Temecula, CA), 50 µg/ml of peptide, 2.5 µg/ml PHA (nonspecific T-cell mitogen; positive control), or no antigen in 24-well replicate cultures for each antigen tested. Cultures were incubated at 37°C in an atmosphere of 5% CO2 for 5 days. Eight h before termination of culture, cells were pulsed with 1 µCi of [3H]thymidine. The cultures were then harvested onto glass fiber filters, and the incorporated radioactivity was counted. The use of 24-well replicates, rather than standard 3-well replicates, permitted analysis by two different means. First, a standard SI was determined, defined as the mean of the response of the antigen-stimulated cells divided by the mean of the response of cells cultured without antigen. The second method of analysis involved scoring individual wells as positive if the [3H]thymidine uptake was greater than the mean and three SDs of the no-antigen control wells (40 , 41) . In this analysis, positive low-frequency events were defined if there was a significant difference at a 95% confidence level between the number of positive wells of the antigen-containing wells compared with that of the control, nonantigen containing wells. Operationally, because none of the no-antigen wells scored positive for any of the assays, >=3 of 24 antigen-containing wells was defined as a positive event (P <= 0.04; one-sided Student’s t test).

Generation and Evaluation of Peptide-specific T-cell Lines.
PBMCs from 12 patients with prostate cancer and with no detectable PAP protein- or peptide-specific T-cell responses were resuspended at 2 x 106 cells/ml in T-cell medium [RPMI 1640 (Life Technologies, Inc.) with 10 mM L-glutamine, 2% penicillin/streptomycin, 50 µM ß-mercaptoethanol, and 10% human AB serum (Valley Biomedical)] with peptide at a 10 µg/ml final concentration. Cells were cultured in 6-well sterile plates (Costar) with 15 x 106 starting cells. Proliferating lymphocytes were restimulated every 7–10 days with irradiated (3300 cGy) autologous PBMCs and peptide at 10 µg/ml. Media was exchanged every 3–4 days after stimulation with T-cell medium containing 10 units/ml recombinant human IL-2 (Chiron, Emeryville, CA). After three in vitro stimulations, T cells in lines were found to be predominantly CD4-expressing (50–94%); however, lines were not routinely assessed for phenotype. After three to five in vitro stimulations, T-cell cultures were assessed for cell proliferation in response to antigen stimulation. Cultured cells (5 x 104)/well were combined with 1.5 x 105 cells/well irradiated (3300 cGy) autologous PBMCs as antigen-presenting cells in assay medium. As before, cells were stimulated with 2.0 µg/ml PAP, 50 µg/ml of peptide, 2.5 µg/ml PHA, or no antigen in 4- to 8-well replicate cultures for each antigen tested. Cultures were incubated at 37°C in an atmosphere of 5% CO2 for 5 days, pulsed with 1 µCi of [3H]thymidine, harvested, and counted as described above. Results are reported as the mean and SD in cpm of [3H]thymidine uptake. Antigen-specific proliferation was defined as significant [3H]thymidine incorporation at a 95% confidence (with P <= 0.05; Student’s t test) in the antigen-stimulated wells compared with the no-antigen control wells.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Potential PAP-specific MHC Class II T-cell Epitopes Can Be Predicted by Peptide Motif Analysis.
Early studies (42 , 43) identified certain protein secondary structural motifs as common to MHC class II T-cell epitopes, typically amphipathic {alpha} helices. Consequently, to identify potential MHC class II epitopes for PAP, the amino acid sequence of PAP was analyzed using the software analysis package T sites (39) . This program identifies sequence motifs common to MHC class II epitopes according to the algorithm of Rothbard and Taylor (44) , as well as regions likely to form amphipathic {alpha} helices, as potential T-cell recognition sites (45 , 46) . Results from this analysis are shown in Fig. 1Citation . Ten potential T-cell antigenic regions were empirically chosen for study based on the prediction by both methods, as depicted by the brackets in Fig. 1Citation and as shown in Table 1Citation . Unlike MHC class I molecules, which typically bind peptides of 9 to 10 amino acids in length, MHC class II molecules are generally believed to be more permissive in the length and exact amino acid sequence of bound peptide (47) . In studies defining the Th epitopes of tetanus toxoid, panels of overlapping peptides were screened for their ability to stimulate T-cell proliferation in PBMCs from tetanus-immune subjects. Peptides of greater than 12 amino acids and less than 31 amino acids and typically in the range of 14 to 16 amino acids were found to be most efficient in defining recognized epitopes (48) . For that reason, 15 to 18 amino-acid peptides were constructed for the current study.



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  		Fig. 1. T sites analysis of amino acid sequence of human PAP, and selection of potential T-cell epitopes. Shown in the top line is the amino acid sequence of human PAP. X, the residues predicted to represent T-cell epitopes based on motif algorithms of Rothbard and Taylor (RT) or predicted amphipathic {alpha} helical structure (AM). Ten 15-18-mer oligopeptides were constructed based on the predictions of both methods, denoted in the brackets.

 
T-cell Proliferative Responses to PAP-derived Peptides Can Be Detected in Patients with Proliferative T-cell Responses Specific for PAP.
To determine whether the peptides chosen could represent PAP-specific Th epitopes, the peptides were used as stimulator antigens in proliferation assays in male subjects with and without PAP-specific T-cell responses. We have found previously (15) that some men with prostate cancer have detectable T-cell proliferative responses specific for PAP, and although these responses are generally low-level (SI < 4), the prevalence of responses is higher in patients with prostate cancer than in controls. Similar methods have been used previously (49 , 50) in other systems to identify helper epitopes from large panels of overlapping peptides using PBMCs from antigen-immunized patients. Fig. 2Citation shows an example in which T cells obtained preoperatively from a 63-year-old male with clinically localized prostate cancer were found to proliferate in response to stimulation with PAP, with a SI of 3.3. In this subject, T-cell proliferation in response to individual peptide stimulation with a SI greater than 2.0 was not detected; however, responses with 3 of 24 and 4 of 24 positive wells were detected to peptides p228-242 and p199-213, respectively. This type of analysis was applied to PBMCs from 20 subjects, as shown in Fig. 3Citation . Thirteen of these subjects had no detectable immunity to PAP (eight of which had prostate cancer and five of which were male control blood donors without prostate cancer), and seven were patients with prostate cancer with detectable immunity to PAP. In Fig. 3ACitation , few peptide-specific responses could be detected in PBMCs from the 13 subjects who had no detectable T-cell responses specific for PAP, both patients with prostate cancer and male control donors. In Fig. 3BCitation , however, some level of peptide-specific T-cell immunity could be detected to each of the peptides using PBMCs from the seven prostate cancer subjects with detectable T-cell responses specific for PAP.



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  		Fig. 2. T-cell proliferative responses to PAP-derived peptides can be detected. This is an example of a modified dilution proliferative T-cell assay using PBMCs from a 63-year old with early stage prostate cancer. Responses to individual peptides were tested in 24-well replicates, and individual wells were scored positive if the cpm were greater than the mean and three SD of the 24 no-antigen control wells. Positive responses were defined as >=3 of 24 wells, indicated by the line. Numbers above the columns represent the calculated SI of the 24-well replicates.

 


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  		Fig. 3. T-cell proliferative responses to PAP-derived peptides can be detected in patients with prostate cancer who have proliferative T-cell responses specific for PAP. PBMCs from 20 males with and without prostate cancer were assessed for proliferative T-cell responses to PAP, PHA, and the 10 peptides in 24-well replicates. The number of individual wells scoring positive is shown, with positive responses defined as >=3 of 24 wells, indicated by the line. Results are shown for (A) those subjects with <3 of 24 wells specific for PAP (n = 13) and (B) those subjects with >=3 of 24 wells positive for PAP (n = 7).

 
p199-213 and p228-242 Represent Th Epitopes of PAP.
We next wanted to determine whether any of the peptides constructed could represent naturally processed PAP-derived helper epitopes, capable of priming T-cell responses in vitro to respond to PAP protein. Given that the experiments were done irrespective of binding to specific MHC class II types, we chose to prioritize for study only those peptides identified above to which responses occurred in the majority of subjects with PAP-specific T-cell responses compared with subjects without PAP-specific T-cell responses. Consequently, four peptides were chosen, p81-95, p199-213, p228-242, and p308-322, given that at least 4 of 7 of the PAP-specific PBMC specimens proliferated in response to these peptides (Fig. 3)Citation . In addition, because our ultimate goal was to identify peptides that might be capable of eliciting Th responses to PAP in patients with prostate cancer, PBMCs from 12 patients with advanced stage prostate cancer and without PAP-specific proliferative responses (data not shown) and of presumably diverse MHC class II backgrounds were chosen for limited T-cell culture experiments. PBMCs from these 12 patients were stimulated in vitro with each of the four peptides (p81, p199, p228, and p308). After three to five in vitro stimulations with peptide, T-cell cultures were assessed for proliferation in response to either the stimulating peptide or PAP protein. Fig. 4Citation shows an example of results from one individual patient. Before culture (Fig. 4A)Citation , this patient did not have evidence of PAP-specific or peptide-specific T-cell proliferation. After four in vitro stimulations with either p199-213 peptide (Fig. 4B)Citation or p308-322 peptide (Fig. 4C)Citation , peptide-specific T-cell proliferation could be detected. No specific proliferation could be detected after culture with p81-95 or p228-242 in this particular patient (data not shown). In addition, whereas no PAP-specific T-cell proliferation could be detected in the p308-322 culture (Fig. 4C)Citation , PAP-specific T-cell proliferation could be detected in PBMCs cultured with p199-213 peptide (Fig. 4B)Citation . A summary of similar results for the 12 patients is shown in Table 2Citation . As demonstrated, peptide-specific lines could be generated to each of the peptides in at least 1 of the 12 patient samples. Of the peptide-specific lines, however, only p199- and p228-specific lines also proliferated in response to PAP protein, suggesting that these two peptides represent naturally processed PAP-specific MHC class II epitopes.



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  		Fig. 4. PAP-specific T-cell lines can be established after repetitive in vitro stimulation with PAP-derived peptides. As an example, PBMCs obtained from a patient with prostate cancer were assessed before culture (A) and after four in vitro stimulations with either p199-213 peptide (B) or p308-322 peptide (C). Shown are the mean and SD of cpm of [3H]thymidine incorporation of cultures after antigen stimulation with peptide, PAP protein, PHA, or no antigen.

 

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  		Table 2 T-cell lines specific for p199 and p228 proliferate in response to PAP protein

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Th cells are of central importance in the generation of immune responses (3) . Animal models have underscored the necessity of tumor-specific Th cells directly or indirectly by recruiting other effector cells for maintenance of therapeutic antitumor immune responses (10 , 51 , 52) . CTLs, in particular, have been shown to require CD4+ Th cells for long-term memory, survival, and cytolytic efficacy (7 , 8) . In the current study, we used PBMCs from patients with detectable Th immunity to PAP to identify potential PAP-specific Th peptide epitopes. We report that epitopes can be identified by motif analysis and that peptides identified in this fashion could be used to prime PAP-specific Th responses in vitro. By this means, we identify two peptide epitopes, p199-213 and p228-242, that are capable of eliciting Th responses to PAP protein in vitro and, thus, likely represent naturally processed MHC class II epitopes.

The identification of Th epitopes has traditionally been done either by eluting peptides bound to specific MHC class II molecules or by screening panels of overlapping peptides derived from the antigen being studied. The first method, eluting and sequencing peptides bound to specific MHC class II molecules from antigen-presenting cells, has been used to identify MHC class II epitopes associated with insulin-dependent diabetes (53 , 54) , multiple sclerosis, and rheumatoid arthritis (55) , as well as from the melanoma tumor antigens tyrosinase (12) and gp100 (13) . Obviously, elution of peptides from specific MHC class II molecules requires selection of specific MHC restriction elements for study. This method is also potentially cumbersome, identifying epitopes from multiple processed antigens and not specifically a single antigen under study (56) . Moreover, it is generally believed that MHC class II epitopes are less length- and sequence-constrained compared with MHC class I epitopes and, in fact, that specific Th epitopes may bind multiple MHC class II molecules (53 , 56 , 57) . Because we wanted to identify epitopes that might be common to many patients of diverse MHC backgrounds, we chose to screen peptides using PBMCs irrespective of specific MHC class II types. In that regard, studies identifying Th epitopes of the diphtheria toxin identified epitopes that were common to several patients of diverse MHC class II backgrounds (50) .

Other groups have identified Th epitopes by screening panels of overlapping peptides derived from the antigen being studied using PBMCs from immune individuals. Th epitopes from tetanus toxoid (48) and diphtheria toxin (50) , as well as the MAGE-3 melanoma tumor antigen (14) , have been identified by this method. In the case of tetanus and diphtheria toxins, panels of peptides of varying lengths were used, spanning the entire length of the proteins, as stimulator antigens in T-cell proliferation assays to determine which peptides stimulated proliferation using PBMCs from tetanus-immune individuals (48 , 49) . The advantage to this type of approach is that the results are exhaustive, spanning the entire length of the immunogen, and have identified peptide epitopes common to most immune patients of presumably diverse MHC class II types. In addition, this method identified peptides of 14–16 amino acids in length as efficient in eliciting Th proliferation. We adopted a similar strategy to define Th epitopes from PAP, given that we had identified previously (15) that some patients with prostate cancer have detectable Th immune responses to PAP. A comprehensive study of overlapping peptides would have been less feasible in our system, however, given that Th responses to PAP were found previously to be rare among patients and are generally of low magnitude. Consequently, an exhaustive search of overlapping peptides would have necessitated extremely large numbers of PBMCs (>109 cells) to detect low-level responses. Therefore, we purposefully chose to bias our selection of peptides, using algorithms to identify potentially antigenic regions of the PAP protein of 15–18 amino acids in length. Although this method may certainly have missed potential epitopes, the fact that we identified two Th epitopes of 10 peptides studied demonstrates the feasibility and utility of this method.

In the current study, we identified peptides derived from PAP, Th proliferative responses to which were associated with responses to soluble PAP protein. In fact, responses to each of the peptides were found in at least one of the seven patients with previously identified Th responses to PAP. This suggests that all of these peptides could represent PAP Th epitopes presented by different MHC class II epitopes present in some individuals but not in others. We were particularly interested in identifying epitopes that might be capable of binding multiple MHC class II types and, therefore, be more "universal" as Th epitopes, similar to peptides identified for diphtheria toxin (50) . Consequently, we prioritized for study only those four peptides identified that stimulated T-cell proliferation in the majority of PBMC samples that also exhibited PAP protein-specific T-cell proliferation. By repetitive in vitro stimulation, peptide-specific T-cell lines could be generated from each of these four peptides from at least 1 of 12 PBMC specimens obtained from patients with prostate cancer who had no detectable preexisting peptide-specific or PAP-specific Th responses. Peptide-specific lines derived from two peptides, p199-213 and p228-242, were also able to proliferate in response to PAP protein stimulation, suggesting that these represent MHC class II epitopes naturally presented after antigen processing.

The development of vaccines capable of eliciting prostate-specific immunity for the treatment of prostate cancer is an active area of research (20) . Clinical trials underway include cytokine-transfected whole cell vaccines (58) , protein-based vaccines (21) , viral-based vaccines (59 , 60) , and dendritic cell vaccines pulsed with either MHC class I-restricted peptide epitopes (61) or proteins, including PAP (22 , 62) . Immunization directly with MHC class I peptides represents another vaccination approach that has shown success in eliciting antigen-specific CD8+ T cells in human clinical trials, in some cases with the suggestion of clinical benefit (63, 64, 65) . Similar trials (16 , 23, 24, 25, 26, 27) have been contemplated in prostate cancer, particularly because MHC class I epitopes have already been identified for PAP, prostate-specific antigen, and prostate-specific membrane antigen. Animal studies (19) have suggested the importance of CD8+ T-cell immunity for actual immune-mediated destruction of prostate tissue. Consequently, antigen-specific Th cells, and Th1 cells in particular, may be particularly important for the effectiveness and maintenance of prostate-specific CTLs (9 , 15 , 66) . Data presented here represents the first report of PAP-specific Th epitopes and a demonstration that PAP-specific Th responses can be elicited in vitro after culture with these epitopes. Ongoing studies will evaluate whether peptide-specific lines exhibit a Th1- or Th2-type bias with respect to cytokine secretion. Given the importance of Th cells in effective antitumor immune responses, vaccine strategies, and MHC class I peptide-based vaccine strategies in particular, targeting this protein might be further enhanced by inclusion of these epitopes capable of inducing PAP-specific Th responses.


   FOOTNOTES
 
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.

1 Supported by Grants from the Berlex Oncology Foundation and the Department of Defense/United States Army (DAMD17-99-1-9529; to D. G. M.) and by Grants from the NIH, National Cancer Institute (K24 CA85218-01 and R01 CA75163; to M. L. D.). Back

2 To whom requests for reprints should be addressed, at Division of Medical Oncology, Box 356527, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195-6527. Phone: (206) 543-8557; Fax: (206) 685-3128; E-mail: dmcneel{at}u.washington.edu Back

3 The abbreviations used are: Th, helper T cell; PAP, prostatic acid phosphatase; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; SI, stimulation index. Back

Received 2/16/01. Accepted 4/24/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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