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Identification of Naturally Processed CD4 T Cell Epitopes from the Prostate-Specific Antigen Kallikrein 4 Using Peptide-Based In Vitro Stimulation¹

John A. Hural^2,*,, Rachel S. Friedman^2,*, Andria McNabb^*, Sean S. Steen^*, Robert A. Henderson^* and Michael Kalos^3,*

^* Corixa Corporation and Infectious Disease Research Institute, Seattle, WA 98104

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Kallikrein (KLK)4 is a recently described member of the tissue kallikrein gene family that is specifically expressed in normal and prostate tumor tissues. The tissue-specific expression profile of this molecule suggests that it might be useful as a vaccine candidate against prostate cancer. To examine the presence of CD4 T cells specific for KLK4 in PBMC of normal individuals, a peptide-based in vitro stimulation protocol was developed that uses overlapping KLK4-derived peptides spanning the majority of the KLK4 protein. Using this methodology, three naturally processed CD4 epitopes derived from the KLK4 sequence are identified. These epitopes are restricted by HLA-DRB1*0404, HLA-DRB1*0701, and HLA-DPB1*0401 class II alleles. CD4 T cell clones specific for these epitopes are shown to efficiently and specifically recognize both recombinant KLK4 protein and lysates from prostate tumor cell lines virally infected to express KLK4. CD4 T cells specific for these KLK4 epitopes are shown to exist in PBMC from multiple male donors that express the relevant class II alleles, indicating that a CD4 T cell repertoire specific for KLK4 is present and potentially expandable in prostate cancer patients. The demonstration that KLK4-specific CD4 T cells exist in the peripheral circulation of normal male donors and the identification of naturally processed KLK4-derived CD4 T cell epitopes support the use of KLK4 in whole gene-, protein-, or peptide-based vaccine strategies against prostate cancer. Furthermore, the identification of naturally processed KLK4-derived epitopes provides valuable tools for monitoring preexisting and vaccine-induced responses to this molecule.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Prostate cancer is the most commonly diagnosed cancer and the second leading cause of cancer deaths in American males (1). Although primary prostate cancer can be effectively treated at the early stages of disease, advanced and metastatic prostate cancer is much more difficult to treat and is usually fatal (2). Due to the limitations in the standard treatment of prostate cancer, much focus has been placed both on the discovery of prostate tissue- and/or tumor-associated molecules as well as on the development of novel immunotherapy- and vaccine-based treatments for cancer.

A number of molecules have been identified that are under consideration as reagents for the diagnosis and/or treatment of prostate cancer. In a few cases (3), immunological data have been generated validating the use of these proteins as immunotherapeutic agents. The development of effective vaccine strategies is likely to require the use of multiple Ags or combinations of Ags, because most Ags will not be expressed by all tumors, and immune escape mechanisms may select against expression of a particular vaccine Ag. Thus, the identification and immunological validation of additional prostate tumor- or tissue-specific molecules are critical for the development of immunotherapeutic strategies against prostate cancer.

Recently, an additional prostate-specific molecule has been described, termed kallikrein (KLK)⁴4 (prostase/KLK-L1). KLK4 was identified by multiple laboratories and, on the basis of sequence homology, was defined to be a serine protease and member of the tissue kallikrein gene family (4, 5, 6, 7). KLK4 is the fourth human tissue kallikrein gene identified in a locus that contains at least 15 potential tissue kallikrein genes (8, 9). Extensive cDNA microarray, quantitative real-time PCR, and immunohistochemistry analyses have shown that KLK4 mRNA and protein are expressed in normal prostate, benign prostate hyperplasia, and both primary and metastatic prostate tumors, with significantly lower levels of KLK4 mRNA expression detected in adrenal, salivary, and thyroid glands and breast, uterus, and colon tissues (4, 7). A number of splice isoforms have been identified for KLK4 (4, 10, 11, 12). While the majority of these isoforms appear to be localized intracellularly, a minor isoform has been identified that contains a signal sequence (12) and shares 72% identity with a secreted proteinase, pig enamel matrix serine protease I. The signal sequence containing isoform has been shown to represent a minor species of KLK4 (D. C. Dillon, unpublished observations). The tissue-specific expression profile of KLK4 suggests that this molecule is likely to be an excellent candidate Ag for the development of vaccine and immunotherapeutic strategies against prostate cancer.

The clinical utility of candidate vaccine Ags has typically been evaluated by the use of in vitro methodologies to demonstrate the existence of a peripheral CD8 and CD4 T cell repertoire specific for a candidate molecule. Recent experiments by a number of groups have demonstrated a critical role of CD4 T cells for both the establishment and maintenance of the anti-tumor response (13, 14, 15, 16).

In vitro methodologies to demonstrate the presence of tumor Ag-specific CD4 T cells have generally relied on the use of computer algorithms to identify tumor Ag-derived peptide sequences predicted to bind to particular HLA molecules and the subsequent use of the peptides to stimulate and detect the presence of Ag-specific T cells in vitro. Such peptide-based approaches have been applied to demonstrate that a CD4 repertoire exists for a number of Ags including NY-ESO1 (17), Melan-A/MART-1 (18), and PAP (19). A major limitation of the algorithm-based peptide approach is the requirement for detailed knowledge of the peptide binding requirements for particular HLA class II molecules. Furthermore, these peptides may not represent naturally processed epitopes. Another approach that has been applied with some success has been to use whole recombinant protein for the in vitro stimulation protocols (20, 21, 22). Whole protein-based in vitro stimulation protocols also suffer from certain limitations; trace contaminants present in purified protein preparations can elicit potent T cell activity, the generation of recombinant protein can be difficult and time consuming, and, once T cells are generated, the relevant epitopes need to be identified.

To examine the presence of a CD4 T cell repertoire specific for KLK4, we developed an in vitro approach that overcomes the limitations associated with both existing peptide- and protein-based CD4 stimulation protocols. Specifically, CD4 T cells isolated from normal donors were stimulated in vitro using autologous APC loaded with pools of overlapping pentadecameric peptides derived from the KLK4 sequence. Using this approach, CD4 T cells are shown to exist in the peripheral circulation of normal donors that recognize naturally processed epitopes derived from KLK4. Three peptide epitopes are identified that correspond to amino acids 155–169, 160–174, and 125–139 of KLK4. T cells specific for these epitopes are shown to be restricted by HLA-DRB1*0404, HLA-DRB1*0701, and HLA-DPB1*0401 class II molecules. Furthermore, T cells specific for these epitopes are shown to exist in PBMC from multiple normal males that express the relevant class II molecules.

The demonstration that KLK4-specifc CD4 T cells exist in the peripheral circulation of normal donors and the identification of naturally processed KLK4-derived CD4 T cell epitopes support the use of KLK4 as a target for whole gene-, protein-, or peptide-based vaccine strategies against prostate cancer. Furthermore, the identification of naturally processed KLK4-derived epitopes provides valuable tools for monitoring preexisting and vaccine-induced responses to this molecule.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cells and cell lines

PBMC from normal individuals were isolated by apheresis followed by separation on a Lymphoprep (Nycomed, Oslo, Norway) Ficoll density gradient. The review of the research protocol by the Institutional Review Board and the performance of all aspects of the study, including the methods used for obtaining informed consent, were in accordance with the principles stated in Federal Regulations 21 CFR 21.50 (Protection of Human Subjects) and 21 CFR 21.56 (Institutional Review Boards). Isolated PBMC were frozen in RPMI 1640 (Life Technologies, Carlsbad, CA), 20% pooled normal human serum (HS), and 10% DMSO (Sigma-Aldrich, St. Louis, MO) and used for subsequent separations of dendritic cells (DC) and CD4 T cells. EBV-immortalized B cells (B-LCL) were generated from PBMC by culturing 1 x 10⁸ cells with 5 ml of EBV supernatant and 1 µg/ml cyclosporin A (Novartis, East Hanover, NJ). EBV supernatant was generated from conditioned medium of B95-8 cells (CRL-1612). The immortalized fibroblast line VA13 (CCL-75.1), the prostate cancer cell line MDA PCa 2b (CRL-2422), and the hybridomas L243 (HB-55), IVD12 (HB-144), and IVA12 (HB-145) were obtained from American Type Culture Collection (Manassas, VA).

Generation of overlapping peptides from the primary amino acid sequence of KLK4

Peptides used in this study were synthesized on a Rainin Symphony peptide synthesizer (Rainin Instrument, Woburn, MA) using O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (Nova Biochem, San Diego, CA) activation with batch F-moc chemistry. Cleavage of peptides from the solid support was conducted using the following cleavage mixture: trifluoroacetic acid (TFA; Baker, Phillipsburg, NJ):ethanedithiol:thioanisole:water:phenol (40:1:2:2:3). After cleavage for 2 h, the peptides were precipitated in cold ethyl ether (Aldrich, Milwaukee, WI). The peptide pellets were then dissolved in 10% v/v acetic acid (Baker, Phillipsburg, NJ) and lyophilized before purification by C18 reversed phase HPLC. A gradient of 5–60% acetonitrile (Baker) (containing 0.05% TFA) in water (containing 0.05% TFA) was used to elute the peptides. Peptide purity was verified by HPLC and mass spectrometry.

Generation of recombinant KLK4 protein

Recombinant KLK4 protein was generated as described (4). Briefly, recombinant, His-tagged KLK4 containing the 159 C-terminal amino acids of KLK4 was expressed in Escherichia coli and purified by Ni-NTA column chromatography. Purified protein was shown to be low in endotoxin and to have a purity >90%, and was confirmed to be KLK4 by amino-terminal sequence analysis.

Generation of recombinant KLK4-expressing adenovirus

A plasmid containing the full-length KLK4 cDNA sequence, including the signal sequence, was generously provided by Dr. D. Dillon (Corixa). The KLK4 open reading frame was amplified using the proofreading thermostable polymerase Pwo (Roche Diagnostics, Indianapolis, IN) and standard PCR-based molecular techniques. The consensus Kozak sequence GCCGCCACC was included immediately 5' of the initiator ATG to maximize translational initiation. Recombinant adenovirus was generated using standard molecular biology methodologies essentially as described in Ref. 23 . Recombinant linearized adenovirus plasmid was transfected into HEK293 cells, and recombinant virus appeared 5–7 days later. Recombinant virus was amplified to large-scale culture and purified on CsCl gradients. Particle concentration was determined by optical density and biological activity was determined by a plaque-forming assay on HEK293 cells.

Generation of cell lysates

Cell lysates were generated by infection of VA13 or MDA PCA 2B cells with a recombinant adenovirus that expressed either KLK4 or an irrelevant Ag. VA13 cells were infected with 10 adenovirus PFU per cell in RPMI 1640, 10% FBS (HyClone Laboratories, Logan, UT), 50 U/ml penicillin (Life Technologies), 50 µg/ml streptomycin (Life Technologies), and 2 mM L-glutamine (Life Technologies). Adenovirus infection of MDA PCA 2B cells was aided by the preincubation of 10 PFU per cell of adenovirus with 2 µg/ml Lipofectamine (Life Technologies) and a 2-h concentrated infection in Optimem serum-free medium (Life Technologies), followed by the addition of RPMI 1640, 10% FBS, 50 U/ml penicillin, 50 µg/ml streptomycin, and 2 mM L-glutamine. Following 18–24 h of incubation at 37°C, 5% CO₂, cells were harvested by trypsinization (Life Technologies) and resuspended at 10⁷/ml in distilled water. Samples were subjected to three to four freeze-thaw cycles, and 1/10 volume of 10x PBS (Life Technologies) was added to the lysates.

Generation and peptide pulsing of DC

DC were generated essentially as described (24). Briefly, PBMC were isolated by separation on a Percoll (Amersham Pharmacia Biotech, Piscataway, NJ) density gradient followed by differential adherence and cultured for 5 days at 37°C, 5% CO₂ in DC medium (RPMI 1640, 1% HS, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine) supplemented with 30 ng/ml IL-4 and 50 ng/ml GM-CSF (Immunex, Seattle, WA). Following the 5-day culture, nonadherent and semiadherent DC were harvested by vigorous washing. Harvested cells were >90% DC as assessed by morphology and surface expression of CD13, CD33, CD54, CD80, CD86, and MHC class I and II, as well as expression of the DC marker CD83 upon maturation with CD40 ligand. DC were resuspended at 1 x 10⁶/ml in DC medium supplemented with 30 ng/ml IL-4, 50 ng/ml GM-CSF, and 1 ng/ml TNF- (BioSource, Camarillo, CA). Peptide pools, comprised of 4–10 15-mer peptides that overlapped by 10 amino acids, were pulsed onto DC at either 250 ng/ml each peptide or 10 µg/ml each peptide. Following overnight incubation, peptide-pulsed DC were harvested by vigorous pipetting and washed for addition to T cell cultures.

Isolation and in vitro stimulation of CD4 T cells

CD4 cells were negatively selected from thawed PBMC by magnetic beads, using CD4⁺ T cell isolation kit and VarioMACS magnet (Miltenyi Biotec, Auburn, CA). The resulting population generally consisted of >95% CD4⁺ T cells. Peptide-pulsed DC (1 x 10⁴/well) and CD4 T cells (1 x 10⁵/well) were plated in wells of 96-well round-bottom plates (Corning, Corning, NY) in a final volume of 200 µl/well CD4 medium (IMDM; Life Technologies), 10% HS, 50 U/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, 50 µg/ml L-asparagine (Life Technologies), and 200 µg/ml L-arginine (Life Technologies) supplemented with 60 ng/ml IL-6 (BioSource) and 10 ng/ml IL-12 (BD PharMingen, San Diego, CA). Stimulation cultures were incubated at 37°C, 5% C0₂. T cell cultures were restimulated weekly. Each restimulation consisted of DC isolation, peptide pulsing, and DC harvesting as described above. For the restimulations, medium was removed from wells and replaced with 1 x 10⁴ cells/well of peptide-pulsed DC in CD4 medium supplemented with 5 ng/ml IL-7 (PeproTech, Rocky Hill, NJ) and 10 µ/ml IL-2 (Chiron, Emeryville, CA).

Ag-specific T cell lines were cloned in 96-well round-bottom plates at 2 cells/well and 0.5 cells/well, with 1 x 10⁴ gamma-irradiated B-LCL/well, 7.5 x 10⁴ gamma-irradiated PBMC/well, 30 ng/ml anti-CD3 Ab (OKT3; Ortho Biotech, Raritan, NJ), and 50 U/ml IL-2 in 150 µl/well RPMI 1640, 10% HS, 50 µ/ml penicillin, 50 µg/ml streptomycin, 2 mM L-glutamine, and 5.5 x 10^-5 M 2-ME (Life Technologies). Cultures were incubated at 37°C, 5% CO₂. Following 10–14 days of culture, wells were screened for cell growth and specificity to KLK4.

Measurement of T cell activity

T cell activity was measured by IFN- secretion and proliferation assays. Initial peptide specificity was assessed following three to four in vitro stimulation cycles. Adherent cells from 2 x 10⁵ autologous PBMC were plated in 96-well flat-bottom tissue culture plates (Corning) and pulsed with peptide pools (0.25 or 10 µg/ml). One-fourth of each well of cultured CD4 T cells was added to the peptide-pulsed APC and cultures were incubated at 37°C, 5% CO₂.

To test peptide pool-reactive lines for specificity to individual peptides from the stimulating pools adherent cells from 2 x 10⁵ autologous PBMC were plated in 96-well flat-bottom plates in the presence of individual peptides (250 ng/ml or 10 µg/ml) and 10⁴ CD4 T cells/well in CD4 medium. Cultures were incubated at 37°C, 5% CO₂.

To measure specific T cell activity against KLK4 peptide, protein, and lysates from KLK4-expressing cells, DC were plated at 10⁴/well into 96-well flat-bottom plates (Corning) in 100 µl/well DC medium plus 30 ng/ml IL-4 plus 50 ng/ml GM-CSF plus Ag. Recombinant Ag was relevant or irrelevant peptide, E. coli-derived recombinant KLK4, an irrelevant E. coli-generated Ag, or whole cell lysates generated from cells infected with an adenovirus expressing KLK4 or an irrelevant Ag. Following incubation of DC with peptide, recombinant protein, or lysate at 37°C, 5% CO₂, for 18–24 h, medium was removed from the wells and 10⁴ CD4 T cells/well were added to Ag-pulsed DC in 200 µl/well CD4 medium. Cultures were incubated at 37°C, 5% CO₂.

For Ab blocking assays, autologous adherent cells and 250 ng/ml peptide were incubated for at least 30 min with 25 µg/ml of the following HLA-blocking Abs before the addition of CD4 T cells: pan-class II blocking (isolated from hybridoma IVA12), HLA-DR blocking (isolated from hybridoma L243), HLA-DQ blocking (isolated from hybridoma IVD12), mouse IgG2a isotype control (03020D; BD PharMingen), and mouse IgG1 isotype control (03211D; BD PharMingen).

HLA mismatch analyses were performed using a panel of peptide-pulsed, partially HLA-matched APC to stimulate T cells in IFN- and proliferation assays as described above.

T cell assays

IFN- ELISA. EIA/RIA plates (Corning) were coated with 100 µl/well PBS (Life Technologies) plus 4 µg/ml IFN- mAb (18891D; BD PharMingen) and incubated overnight at 4°C. Plates were blocked for 1 h at room temperature (RT) with 5% nonfat dry milk in PBS. Following washes (12 times with PBS/0.1% Tween and six times with PBS), 50 µl CD4 medium plus 50 µl assay culture supernatant (from the 24- to 48-h time point) were added to Ab-coated wells and incubated overnight at RT. Following washes, 100 µl/well rabbit anti-human IFN- polyclonal Ab (generated in house) diluted 1/2000 in PBS plus 10% normal goat serum (Life Technologies) was incubated at RT for 2 h. Following washes, 100 µl/well donkey anti-rabbit HRP (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1/2000 in PBS plus 5% nonfat dry milk was incubated at RT for 1–2 h. Following washes, the assay was developed with tetramethylbenzidine peroxidase substrate plus peroxidase solution (Kirkegaard & Perry Laboratories, Gaithersburg, MD) and stopped with 1 N H₂SO₄. ELISA plates were read at 450 - 570 nm using the Benchmark Microplate Reader and Microplate Manager 4.0 (Bio-Rad, Hercules, CA).

Proliferation assay. To measure T cell proliferation, 1 µCi/well [³H]thymidine (Amersham Pharmacia Biotech) was added to assay cultures at 48 h. Following incubation for 12–16 h, cultures were harvested onto Unifiter Plates (Packard Instrument, Meriden, CT). Microscint 20 scintillation fluid (Packard Instrument) was added to each well, and plates were counted on the TopCount Scintillation Counter (Packard Instrument).

HLA typing. HLA typing of cell lines was performed by the Puget Sound Blood Center (Seattle, WA) using the Micro SSP HLA DNA typing tray PCR typing kit (One Lambda, Canoga Park, CA).

FACS analysis. Cells were stained with a mixture of CD4 FITC, CD8 PE, and CD3 PerCP (BD PharMingen). Following washes, cells were analyzed using a FACSCalibur (BD Biosciences, San Diego, CA).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of CD4⁺ T cell lines and clones specific for KLK4-derived peptides from normal donor PBMC

To stimulate and expand KLK4 peptide-specific CD4⁺ T cells from peripheral blood, a series of 15-mer peptides were synthesized that overlapped by 10 amino acids and covered 208 of the 254 amino acids of the full-length KLK4 protein. Peptides were combined into pools of 4–10 ten peptides and pulsed onto autologous DC generated from normal donors. The peptide-pulsed DC were then used to stimulate and expand CD4 T cells isolated from PBMC in vitro. These experiments were performed with cells generated from normal female donors because we reasoned that KLK4-specific CD4 T cells might be more easily expanded from individuals in which the peripheral T cell repertoire specific for prostate Ags had not been tolerized or eliminated. T cells were stimulated weekly for a total of four stimulation cycles and then assayed for KLK4 peptide-specific activity using ³H proliferation and IFN- production assays. In five different experiments, using different donors and a range of peptide concentrations, a total of 28 independent lines specific for KLK4 peptide pools were identified with at least a 3-fold stimulation index (SI) in both proliferation and IFN- ELISA. Fig. 1 presents the data identifying one such KLK4-specific line, 1C3. From 96 total wells stimulated with one pool of KLK4-derived peptides, line 1C3 was identified in both IFN- ELISA and ³H proliferation assays to show specific activity for the pool of KLK4 peptides compared with a pool of irrelevant peptides.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. CD4+ T cell lines specific for KLK4 peptide pools can be expanded from peripheral blood. A total of 1 x 104 peptide pool-pulsed DC were plated with an aliquot (25 µl) of T cells from each well and incubated for 48 h at 37°C. An aliquot (50 µl) of supernatant was removed for IFN- ELISA and cells were pulsed with 20 mCi/ml [3H]thymidine and incubated overnight for proliferation assays. The solid line indicates a SI of 1. The dashed line indicates a SI of 3. Line 1C3 is indicated with an arrow.

View larger version (38K): [in this window] [in a new window]   FIGURE 2. CD4+ T cell lines can be identified that respond to naturally processed epitopes of KLK4. Autologous DC were pulsed overnight with recombinant KLK4 or an irrelevant protein. A total of 1 x 104 pulsed APC were plated with 1 x 104 T cells and incubated for 48 h at 37°C. An aliquot (50 µl) of supernatant was removed for IFN- ELISA, and cells were pulsed with 20 mCi/ml [3H]thymidine and incubated overnight for proliferation assays. Each condition was assessed in triplicate. SIs represent the mean of the T cell activity against APC pulsed with KLK4 divided by the mean of the T cell activity against APC pulsed with the irrelevant Ag (background). SDs of the triplicates were always <15% of the mean. Filled bars represent the SI for proliferation and hatched bars represent the SI for IFN- production. n/d, Proliferation assay was not performed for lines 1A1 and 2C4.

Recognition of recombinant KLK4 and lysates from KLK4-expressing cells by CD4⁺ T cell clones

To determine the relative sensitivity of KLK4-specific T cell clones to respond to DC pulsed with recombinant KLK4, each of the clones was stimulated by APC pulsed with titrations of the relevant peptide or recombinant KLK4, and Ag-specific responses were measured in ³H proliferation and IFN- ELISA. Each of the clones demonstrated distinct degrees of sensitivity to the pulsed APC; while J1F11 recognized APC pulsed with 250 pg/ml peptide or 2 µg/ml protein (Fig. 3A), R2C7 specifically recognized APC pulsed with as little as 250 pg/ml peptide or 20 ng/ml recombinant protein (Fig. 3B), and A2F6 recognized APC pulsed with 25 ng/ml of either peptide or protein (Fig. 3C). The differences of the relative ability of these clones to recognize recombinant protein may reflect differences in the affinity of the T cells for Ag or, alternatively, differences in the efficiency of processing and/or presentation of these epitopes by the APC.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. CD4+ T cell clones exhibit varied degrees of sensitivity to DC pulsed with peptide or recombinant protein. Autologous DC were pulsed overnight with the indicated dilution of relevant or irrelevant peptide or recombinant E. coli-generated KLK4 or irrelevant protein. A total of 1 x 104 pulsed DC were plated with 1 x 104 T cells and incubated for 48 h at 37°C. Cultures were assessed for proliferation and IFN- production as described in Fig. 2. SDs of the triplicates were always <15% of the mean. For reference, absolute proliferation values against APC loaded with irrelevant Ag ranged from 136 to 1475 cpm. Results from the IFN- assays correlated in all cases with the proliferation data and are not shown. Peptide and protein final concentrations are indicated.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. CD4+ T cell clones exhibit varied degrees of sensitivity to DC pulsed with lysates of cells expressing KLK4. VA13-transformed fibroblasts and the prostate tumor cell line MDA PCa 2b were infected with recombinant adenovirus that expressed either KLK4 or an irrelevant protein. Equivalent multiplicities of infection of virus were used for each infection. Infected cells were harvested and resuspended at 1 x 107 cells/ml, and lysates were generated by hypotonic lysis and freeze thaw. Autologous DC were pulsed overnight with the indicated dilutions of lysates from infected cells. A total of 1 x 104 pulsed DC were plated with 1 x 104 T cell clones and incubated for 48 h at 37°C. Cultures were assessed for proliferation and IFN- production as described in Fig. 2. SDs of the triplicates were always <15% of the mean. For reference, absolute proliferation values against APC loaded with irrelevant Ag ranged from 211 to 1956 cpm. Results from the IFN- assays correlated in all cases with the proliferation data and are not shown.

To determine the HLA molecules responsible for presenting the KLK4-derived epitopes to CD4 T cells, Ab blocking and MHC mismatch assays were performed. For the Ab blocking analyses, T cells were incubated with autologous APC pulsed with the relevant peptides in the presence of anti-class II, anti-HLA-DR, anti-HLA-DQ, or control isotype-matched Abs. T cell activity was measured using ³H proliferation and IFN- production assays. The specific ability of clones J1F11 (Fig. 5A) and R2C7 (Fig. 5B) to recognize peptide-pulsed APC was blocked by both the anti-class II and anti-HLA-DR Abs, but not by control Abs or Abs to the HLA-DQ molecule. These results demonstrate that the reactivity of clones J1F11 and R2C7 is restricted by an HLA-DR molecule. The specific ability of clone A2F6 to recognize peptide-pulsed APC was blocked by anti-class II Ab but not by the anti-HLA-DR or anti-HLA-DQ Abs (Fig. 5C), suggesting that the response of this clone is restricted by an HLA-DP molecule.

View larger version (16K): [in this window] [in a new window]   FIGURE 5. KLK4-specific CD4 T cell clones are restricted by HLA-DR and HLA-DP class II alleles. KLK4-specific CD4 T cell clones were incubated with autologous monocytes pulsed with 250 ng/ml of the relevant or irrelevant peptide in the presence of 50 µg/ml pan-class II-, HLA-DR-, or HLA-DQ-blocking Ab or isotype-matched control Abs. A total of 1 x 104 pulsed monocytes were plated with 1 x 104 T cells and incubated for 48 h at 37°C. Cultures were assessed for proliferation and IFN- production as described in Fig. 2. SDs of the triplicates were always <15% of the mean. For reference, absolute proliferation values against APC loaded with irrelevant Ag ranged from 190 to 803 cpm. Results from the IFN- assays correlated in all cases with the proliferation data and are not shown.

View larger version (37K): [in this window] [in a new window]   FIGURE 6. KLK4-reactive CD4+ clones are restricted by HLA-DRB1*0404, HLA-DRB1*0701, and HLA-DPB1*0401 HLA class II alleles. KLK4-specific T cells were incubated with a panel of partially HLA-matched monocytes (genotypes listed in Table II) pulsed with the relevant or irrelevant peptide. A total of 1 x 104 pulsed monocytes were plated with 1 x 104 T cells and incubated for 48 h at 37°C. Cultures were assessed for proliferation and IFN- production as described in Fig. 2. SDs of the triplicates were always <15% of the mean. For reference, absolute proliferation values against APC loaded with irrelevant Ag ranged from 195 to 720 cpm. Results from the IFN- assays correlated in all cases with the proliferation data and are not shown.

As discussed above, to maximize the potential of identifying CD4 T cell responses to KLK4, in vitro stimulation experiments to generate KLK4-specific CD4 T cells were initially conducted using T cells and APC from normal female donors because such donors were less likely to be tolerized or deleted of T cells specific for prostate Ags. To determine whether responses to naturally processed KLK4-derived epitopes could be identified in PBMC from normal males, in vitro stimulation experiments were established with the relevant peptides KLK4_155–169 and KLK4_160–174, using APC and CD4 T cells generated from normal male donors that expressed the HLA-DRB1*0404 or HLA-DRB1*0701 alleles. Because our donor pool is not routinely typed for HLA-DP alleles, male donors with the appropriate allele to present the peptide epitope KLK4_125–139 were not identified. Following three stimulation cycles, T cell lines were assayed for KLK4 peptide and lysate specificity in proliferation and IFN- assays as above. KLK4 peptide-specific CD4 T cell responses were detected in all male donors (Table II) and, additionally, the responding T cells lines were capable of recognizing APC pulsed with cell lysates from KLK4-expressing cells. The specific SI for these lines was at least 4 in proliferation assays and at least 4 in IFN- ELISA. These results demonstrate that CD4 T cells specific for naturally processed KLK4-derived epitopes exist in the T cell repertoire of normal males.

KLK4 is a member of a multigene family that includes at least 15 family members with sequence homology that are expressed in a variety of normal and transformed tissues. To examine the homology between the newly identified KLK4 CD4 T cell epitopes and other tissue kallikreins, a comparative amino acid alignment was performed. The results of this analysis are presented in Fig. 7. KLK4_125–139, the peptide epitope recognized by clone A2F6, showed very low homology with other kallikreins; the closest identity (4 of 15 residues) was with hk8. KLK4_155–169, the peptide epitope recognized by clone J1F11, also showed low homology with other kallikreins; the closest identity (7 of 15 residues) was with hk14. KLK4_160–174, the peptide epitope recognized by clone R2C7, showed moderate homology with other kallikreins; the closest identity (9 of 15 residues) was with hk5. No significant homology was detected between the 50-aa fragment that contains the CD4 T cell epitopes and any sequences present in the GenBank database (data not shown). These data demonstrate that T cells specific for KLK4_125–139, KLK4_155–169, or KLK4_160–174 are not likely to recognize epitopes derived from other tissue kallikreins. Therefore, a vaccine designed to elicit a CD4 T cell response to these epitopes is unlikely to induce an immune response to tissues other than prostate. The epitopes KLK4_125–139, KLK4_155–169, and KLK4_160–174 are also present in the major KLK4 isoform localized intracellularly and shown to be expressed in prostate and ovarian tumors (4, 10), suggesting that T cells specific for these epitopes would potentially recognize tumor cells that express the intracellular KLK4 isoforms.

View larger version (40K): [in this window] [in a new window]   FIGURE 7. KLK4-derived CD4+ T cell epitopes are not conserved among the tissue KLK gene family. Protein sequence alignments were conducted with MegAlign and EditSeq (DNAstar, Madison, WI).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A growing body of evidence has demonstrated the requirement of a tumor-specific CD4 T cell response for the development of an effective antitumor response (27, 28). In animal models, tumor-specific CD4 T cells have been shown not only to be important for providing classical helper function but also to be critical for recruitment of activated, tumoricidal macrophages and eosinophils (14). Additionally, CD4 T cells have been shown to be required for allowing tumor infiltration of the CD8⁺ population, maintenance of CD8 numbers, and maintenance of a functional cytolytic CD8 phenotype (15). Recently, a direct role for CD4 T cells in tumor rejection has been demonstrated, as CD4 T have been shown to both directly produce and facilitate the production of IFN- with the consequent inhibition of tumor angiogenesis (13, 16).

To determine the presence of KLK4-specific CD4 T cells in peripheral blood, we developed a methodology that uses overlapping Ag-derived peptides to stimulate and expand T cells in vitro. This approach has significant advantages over existing peptide- and whole gene-based methodologies, because it is not limited by knowledge of HLA binding motifs, nor is it dependent on the availability of highly purified recombinant protein preparations. Our approach presents a high throughput method of stimulating CD4 T cells essentially with the whole gene, without the difficulties of protein expression and the presence of trace contaminants, and without any bias in either the peptides used or the class II alleles involved in the presentation. Using this approach, we have rapidly validated the immunogenicity of a self-Ag by demonstrating the presence of KLK4-specific CD4 T cells in the periphery, as well as identified multiple KLK4-derived naturally processed epitopes recognized by distinct HLA class II alleles. To our knowledge this is the first account in which Ag-specific CD4 T cells were generated by a protocol that exclusively involves overlapping peptides that span the majority of a molecule. These results demonstrate that circulating CD4 T cells specific for KLK4 exist in normal male individuals, thus validating KLK4 as a potential vaccine candidate for prostate cancer.

Our approach has allowed us to identify three naturally processed KLK4-derived CD4 T cell epitopes restricted by HLA-DRB1*0404, HLA-DRB1*0701, and HLA-DPB1*0401 alleles. Each of the T cell lines that were generated responded to one of three individual KLK4 peptides, KLK4_155–169, KLK4_160–174, and KLK4_125–139. While two of these peptides overlap by 10 amino acids, the T cells that respond to these peptides recognize distinct epitopes because clone 1C3 demonstrated no reactivity with peptide KLK4_155–169, the epitope recognized by clone J1F11, and clone J1F11 demonstrated reduced reactivity (20% compared with clone 1C3) to peptide KLK4_160–174, the epitope recognized by clone 1C3 (data not shown). Clones isolated from each of the KLK4-specific lines were shown to efficiently recognize APC pulsed with both recombinant E. coli-generated KLK4 protein as well as lysates from KLK4-expressing cells. These results demonstrate that the epitopes recognized by these T cells are naturally processed from both unmodified, bacterial-generated KLK4 protein as well from KLK4 produced and posttranslationally modified by prostate tumor cells. It was necessary to infect the tumor lines to express KLK4 because none of the KLK4-specific CD4 clones could recognize APC pulsed with MDA PCa 2b or LnCap cell lysates, or lysates from a SCID-passaged prostate tumor sample, despite the fact that these tumor cell lines have been shown to express KLK4 mRNA. This result is most likely explained by the fact that established prostate tumor cell lines express very low levels of KLK4 mRNA. Indeed, real-time analysis demonstrates that primary prostate tumor tissue expresses up to 25-fold higher levels of KLK4 message compared with established prostate tumor cell lines (data not shown).

We initially set out to identify naturally processed epitopes from KLK4 in normal female donors because we reasoned that such donors would be less likely to be tolerized to a prostate-specific Ag. Nonetheless, for both epitopes tested, we were able to demonstrate the existence of CD4 T cells specific for those epitopes in normal male donors. T cells derived from male donors were recovered at a frequency similar to the frequency in females. Furthermore, T cell lines derived from males were of sufficient affinity to efficiently recognize APC pulsed with lysates of KLK4-expressing cells. Thus, the KLK4-specific T cell repertoire in males appears, at least at this level of analysis, to be qualitatively similar to the repertoire in females. These results indicate that high-affinity KLK4-reactive CD4 T cells exist in the peripheral blood of normal males. A recent report using transgenic mice has demonstrated that peripheral Ag-specific T cells can be shown to exist in a tolerant state as a result of anergy but can be effectively activated to express effector functions (29). Because the KLK4-specific T cells described in this report are potentially tolerized in vivo, immunotherapeutic strategies to induce an anti-KLK4-specific CD4 T cell response will likely require the use of potent vaccine delivery protocols to activate the tolerized cells.

The presence of KLK4-specific CD4 T cells in the peripheral circulation, and the demonstration that tumor cells can process and present epitopes derived from KLK4 to such T cells, suggest that KLK4 is likely to be a useful target Ag for vaccination against prostate cancer.

	Acknowledgments

 
We thank Gregory Spies for production of recombinant adenovirus, Tom Vedvick for production of recombinant proteins, AnnaMarie Beckman and Melodee Smith for assistance with apheresis procurement and blood sample analyses, and Mark Alderson, Davin Dillon, and Eden Zasloff for critical review of the manuscript.

	Footnotes

 
¹ This work was supported in part by a grant from the Cancer Research Institute.

² J.A.H. and R.S.F. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Michael Kalos, Corixa Corporation, 1124 Columbia Street, Suite 200, Seattle, WA 98104. E-mail address: kalos{at}corixa.com

⁴ Abbreviations used in this paper: KLK, kallikrein; DC, dendritic cell; TFA, trifluoroacetic acid; HS, human serum; SI, stimulation index; RT, room temperature.

Received for publication January 9, 2002. Accepted for publication April 24, 2002.

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