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* Corixa Corporation and
Infectious Disease Research Institute, Seattle, WA 98104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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). The six lines recognized five
distinct peptides; lines 1F9 and 1C3 both recognized p160
(RMPTVLQCVNVSVVS), line 3a9H recognized p155
(LLANGRMPTVLQCVN), and line 2C4 recognized p125
(SVSESDTIRSISIAS). Line 1A1 and 3aF9 recognized peptides p110
(RPLLANDLMLIKLDE) and p145 (GNSCLVSGWGLLANG), respectively.
Line 3aF9 was not pursued further because it lost activity following
restimulation on the relevant peptide, and line 1A1 was not pursued
further because it did not recognize APC pulsed with mammalian-derived
KLK4 (see below). Each of the epitopes RMPTVLQCVNVSVVS,
LLANGRMPTVLQCVN, and SVSESDTIRSISIAS is contained within both secreted
and intracellular forms of KLK4.
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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.
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ELISA, using autologous DC pulsed with
lysates from cells that expressed KLK4 as APC. Lysates were generated
from either the VA13 fibroblast line or the MDA PCA 2b prostate tumor
cell line infected with an adenovirus vector that expressed either KLK4
or an irrelevant protein. The lysates were generated at a cell density
of 107 cells/ml. In titration experiments, each
of the clones was specifically and efficiently stimulated by APC pulsed
with lysates from either VA13 or MDA PCA 2b cells expressing KLK4 (Fig. 4). Clones A2F6 and R2C7 recognized DC
pulsed with at least a 625-fold dilution of the lysates (Fig. 4, A and C), while J1F11 recognized DC pulsed with
at most a 125-fold dilution of lysate (Fig. 4B). These
results demonstrate that, while the sensitivities are variable, the
epitopes recognized by the CD4 T cell clones are efficiently processed
from both recombinant, E. coli-generated KLK4 protein as
well as mammalian-derived KLK4. The use of adenovirus-KLK4-infected
cell lysates was necessary in these experiments because we were unable
to demonstrate T cell recognition of DC pulsed with lysates from the
established tumor cell lines, MDA PCA 2b and LnCap, or lysates from a
primary prostate tumor sample passaged in SCID mice (data not shown).
Although KLK4 mRNA is detected in each of these samples by
real-time PCR analysis, KLK4 mRNA levels are significantly
lower (as much as 25-fold less) compared with primary prostate tumor
tissue (data not shown).
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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.
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production assays. The HLA class II DR and DP
genotypes for J1F11 (d366), R2C7 (d326), and A2F6 (d207), as well as
for a panel of partially matched donors, are presented in Table I. HLA-DR4 and -DR7 individuals express
DR alleles from DRB1 and DRB4 loci. As indicated
in Table I, donor 366 expresses the DRB1*0401,
DRB1*0404, and DRB4*0103 alleles, donor 326
expresses the DRB1*0301, DRB1*0701, and
DRB4*0101 alleles, and donor 207 expresses
HLA-DPA1*0103, DPB1*0401, and
DPB1*2301 alleles. Clone J1F11 specifically recognized APC
derived from the autologous donor (d366) and donor d150 (Fig. 6, filled bars). Both of these donors
share the HLA-DRB1*0404 and HLA-DRB4*0103
alleles; however, the inability of clone J1F11 to recognize other
DRB4*0103-expressing APC (d207, d72, d104, d140, d208, and
d379) pulsed with the relevant peptide indicates that the response of
clone J1F11 is restricted by the HLA-DRB1*0404 molecule. Clone R2C7
specifically recognized APC derived from the autologous donor (d326) as
well as APC derived from donors d72, d150, d207, and d379 (Fig. 6, open
bars). Because the only HLA-DR allele uniquely shared between these APC
is HLA-DRB1*0701, these data show that the response of clone
R2C7 is restricted by the HLA-DRB1*0701 molecule. Clone A2F6
specifically responded to APC derived from the autologous donor (d207)
as well as APC derived from donors d150 and d326 (Fig. 6, stippled
bars). Because the only HLA-DP allele uniquely shared by these donors
and not expressed by the nonresponding donor (d366) is
HLA-DPB1*0401, these data show that the response of clone
A2F6 is restricted by the HLA-DPB1*0401 molecule.
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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
KLK4155–169 and
KLK4160–174, 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
KLK4125–139 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.
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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.
KLK4125–139, the peptide epitope recognized by
clone A2F6, showed very low homology with other kallikreins; the
closest identity (4 of 15 residues) was with hk8.
KLK4155–169, the peptide epitope recognized by
clone J1F11, also showed low homology with other kallikreins; the
closest identity (7 of 15 residues) was with hk14.
KLK4160–174, 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
KLK4125–139, KLK4155–169,
or KLK4160–174 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 KLK4125–139,
KLK4155–169, and
KLK4160–174 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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, KLK4155–169, KLK4160–174, and KLK4125–139. 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 KLK4155–169, the epitope recognized by clone J1F11, and clone J1F11 demonstrated reduced reactivity (20% compared with clone 1C3) to peptide KLK4160–174, 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.
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Acknowledgments |
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Footnotes |
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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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-dependent inhibition of tumor angiogenesis by tumor-infiltrating CD4+ T cells requires tumor responsiveness to IFN-. J. Immunol. 166:2276. receptor expression by nonhematopoietic cells. Immunity 12:677.[Medline]
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