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*
Division of Urology and
Department of Surgery, Duke University Medical Center, Durham, NC 27710
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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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Although promising, peptide-loaded DC are capable of presenting single epitopes in the context with specific HLA molecules, most commonly HLA-A2, significantly limiting the patient population eligible for vaccine therapy. Although the efficacy of PSA-specific peptides pulsed onto DC to induce CTL has not been reported to date, a conceptually superior approach would entail the targeting of multiple class I and class II epitopes present on the PSA molecule, which are thought to further stabilize or maintain effective T cell responses in vivo (7, 8).
The primary objective of this study was to develop a broadly applicable DC-based immunization strategy for prostate cancer patients. Specifically, we examined the ability of DC transfected with mRNA encoding PSA to stimulate primary PSA-specific CTL responses in vitro. Ag in form of RNA carries the advantage to encode multiple epitopes for many HLA alleles, thus permitting induction of CTL among many cancer patients irrespective of their HLA repertoire (9). Because defective immune responses have been reported in cancer patients (10), we assessed and compared the efficacy of PSA mRNA-transfected DC to stimulate CTL responses among a large number of healthy male and female volunteers and among prostate cancer patients.
In this study, we show that PSA mRNA-transfected DC generated from a large number of individuals reproducibly led to successful in vitro generation of PSA-specific CTL without prior knowledge of the individual’s HLA haplotype. These PSA-specific CTL recognized and lysed cellular targets expressing PSA, but not kallikrein Ags, suggesting that autoimmunity with pathologic consequences may not be a serious problem with this approach. Furthermore, PSA RNA-transfected DC could serve as effective target cells in CTL assays, thus allowing immunological monitoring of PSA responses in humans, while obviating the need for culturing and labeling ex vivo the tumor cells from each patient. CTL responses of similar magnitude were observed among female and male healthy volunteers and cancer patients, suggesting that natural tolerance to PSA or tumor-mediated T cell anergy may not compromise the efficacy of this approach. This study provides the experimental foundation for further clinical investigation of a potentially effective and broadly applicable vaccine strategy for prostate cancer patients.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All cellular material used in these experiments was obtained
from human subjects following informed consent through protocols
approved by the Investigational Review Board (IRB) at our institution.
PBMC were harvested from 10 healthy volunteers and four prostate cancer
patients by peripheral blood leukapheresis. Cancer patients were
selected based on more aggressive tumor characteristics: Gleason sum
7 (all patients), extraprostatic tumor extension (stage pT3,
all patients), and positive nodal involvement (one patient).
DC generation from peripheral blood precursors
For DC culture, we adopted techniques previously described by
Romani (11) or Morse et al. (12),
implementing modifications to allow processing of cellular material
using defined serum-free media and supplements. Briefly, a concentrated
leukocyte fraction was generated through a 2-h restricted peripheral
blood leukapheresis processing 6–8 L of blood with each collection.
The leukapheresis product was further separated by density-gradient
centrifugation over polysucrose/sodium diatrizoate (Histopaque; Sigma
Diagnostics, St. Louis, MO), and cells were resuspended in serum-free
AIM-V medium (Life Technologies, Grand Island, NY). PBMC were incubated
in a humidified incubator for 2 h at 37°C to allow plastic
adherence. The adherent cell fraction was used for DC culture by
incubation in serum-free AIM-V medium supplemented with human rIL-4
(500 U/ml) and human rGM-CSF (800 U/ml) (R&D Systems, Minneapolis, MN).
After 7 days of culture, cells were harvested and phenotypically
characterized to assure they met the typical phenotype of immature DC
(Fig. 1
): CD3neg, CD14neg,
CD16neg, CD56neg,
CD19neg, MHC
Ipos, MHC IIpos,
CD40neg, CD80low,
CD86low, CD83neg
(13). DC preparations fulfilling these phenotypic criteria
were used for subsequent RNA transfection or pulsing with antigenic
peptides or proteins.
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PSA and kallikrein (Sigma) were used in the form of proteins for DC pulsing. Furthermore, the following HLA-A2-restricted peptides were used: influenza virus peptide M1 (FluM1; aa 58–66, GILGFVFTL; Research Genetics, Birmingham, AL) and the PSA-derived peptides PSA-1 (aa 141–150, FLTPKKLQCV) and PSA-3 (aa 154–163, VISNDVCAQV) (kindly provided by J. Schlom, National Cancer Institute, Bethesda, MD). Peptides were dissolved in serum-free AIM-V medium and stored at -20°C.
In vitro transcription of PSA RNA
To generate in vitro transcribed PSA RNA, the vector pGEM4/PSA/A64 was constructed. In brief, oligonucleotides containing 64 A-T bp followed by a SpeI restriction site were cloned into the EcoRI and NarI sites of pGEM4Z (Promega, Madison, WI) to create the plasmid pGEM4Z/A64. A 1347-bp HindIII fragment containing the entire coding sequence for PSA was isolated from pcDNA3.1 (kindly provided by J. Schlom) and was ligated into the HindIII site of pGEM4Z/A64 to generate the plasmid pGEM4Z/PSA/A64. Linearization with SpeI followed by in vitro transcription with T7 RNA polymerase (Ambion mMessage mMachine kit, Austin, TX) yields a transcript that contains 9 nt of vector-derived sequence, 36 nt of PSA 5' untranslated region (UTR), 786 nt corresponding to the coding region of PSA, 520 nt of PSA 3' UTR, 51 nt of polylinker sequence, and 64 A nt and 4 nt from the SpeI site. To exclude the possibility of anergy to PSA Ags, an irrelevant control RNA encoding jelly fish green fluorescent protein (GFP) was used (14).
Pulsing of DC
Pulsing of autologous DC with RNA, proteins, or peptides was performed by simple coincubation with DC. In brief, DC were washed twice in PBS, counted, and spun at 300 x g for 10 min. Subsequently, DC were resuspended at a concentration of 1 x 106 cells/ml in AIM-V medium and coincubated for 45 min with either 1.5 µg/ml RNA, 25 µg/ml peptide, or 20 µg/ml protein in a humidified incubator at 37°C/5% CO2. RNA, peptide, or protein-pulsed DC were not only used as stimulators for CTL, but also as cellular targets in cytotoxicity assays. For use as targets, RNA-pulsed DC were washed once after antigenic stimulation and incubated overnight to allow expression and presentation of the Ags.
In vitro cytotoxicity assay
The Ag-presenting function of the pulsed DC was assessed by
measuring the induction of primary CTL responses in a standard
51Cr cytotoxicity assay. The T cell-enriched
nonadherent fraction of PBMC obtained following the DC plastic
adherence step was used for CTL generation. Nonadherent PBMC were
cultured in RPMI cell culture medium supplemented with 20 U/ml human
IL-2 and 10 ng/ml human IL-7 (R&D Systems). Cells were stimulated
twice, 8 days apart with pulsed autologous DC at a stimulator to
effector ratio of 1:10. Following 16 days of culture, effector cells
were harvested without further separation and used in microcytotoxicity
assays. As determined by flow-cytometric analysis, 77 ± 10% of
these effector cells were CD3pos and 
40% were
of the CD3pos/CD8pos
phenotype. Target cells were labeled with
51sodium chromate in saline solution
(Na[51Cr]O4; NEN Life
Science Products, Boston, MA) by incubation of 2 x
106 transfected DC in 1 ml RPMI with 100 µCi of
51Cr for 1 h at
37°C/5%CO2. After three washes, 5 x
103 51Cr-labeled targets
and serial dilutions of effector cells at various E:T ratios were
incubated in 200 µl of RPMI in 96-well U-bottom plates. These plates
were incubated for 6 h at 37°C/5%CO2.
Then, 50 µl of the supernatant was harvested, and released
51Cr was measured with a scintillation counter.
Spontaneous release was less than 15% of the total release by
detergent in all assays. SD of the means of triplicate wells was less
than 5%.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The goal of these experiments was to develop a sequential protocol
for DC generation and RNA loading to produce large amounts of PSA
RNA-transfected DC from cancer patients. We have adapted the techniques
described by Romani (11) and Morse (12),
implementing minor modifications to allow human cell processing on a
large scale. To develop a clinically compatible protocol, DC were
generated from leukapheresis-derived PBMC and cultured for 7 days in
low protein, serum-free medium (AIM-V) solely supplemented with
GMP-grade GM-CSF and IL-4. The phenotypic characteristics of these DC
were similar to those previously described (13),
exhibiting the typical phenotype of immature DC:
CD3neg, CD14neg,
CD16neg, CD56neg,
CD19neg, MHC
Ipos, MHC IIpos,
CD40neg, CD80low,
CD86low, CD83neg (Fig. 1
). The typical yield of PBMC following a
2-h restricted leukapheresis in cancer patients (n = 4)
and healthy volunteers (n = 10) was at least 3 x
109 cells, resulting in an average yield of
3.1 x 108 viable DC (cancer patients) and
3.3 x 108 DC (healthy volunteers) following
the 7-day culture period. The differences among groups were
statistically not significant. We next determined whether these
phenotypically immature DC can be successfully transfected with mRNA
encoding PSA. PSA mRNA was generated by in vitro transcription of a
cDNA plasmid (Fig. 2). Transfection was
performed by coincubation of DC with naked RNA without the use of
transfection agents used for eucaryotic cell transfection. Incubation
of 1 x 106 DC with 1.5 µg of PSA RNA for
at least 30 min resulted in successful integration and translation of
RNA, as evidenced by the successful amplification of the PSA gene
product from transfected DC (Fig. 3).
Interestingly, even a small amount of 15 ng PSA mRNA transfected onto
1 x 106 DC for 45 min resulted in
detectable PSA expression (Fig. 3). Amplification from transfected DC
rather then from adsorbed PSA RNA was demonstrated by the absence of
the 710-bp product in DC incubated with PSA RNA for less than 30 min
(Fig. 3) as well as by the demonstrated capability of PSA
RNA-transfected DC to prime CTL, as presented below. The transient
expression of the PSA gene product was demonstrated by complete
disappearance of the 710-bp PCR product 24 h following
transfection presumably related to intracellular mRNA enzymatic
degradation (Fig. 3, lane 11). In our hands, RNA
transfection of human DC using cationic liposomes was associated with
significant toxicity and considerable cell loss (data not shown).
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To assess the APC function of the DC generated under these
conditions, we next determined whether PSA peptide-pulsed DC are
capable of stimulating PSA-specific CTL responses in vitro. For these
experiments, DC from HLA-A2+ healthy volunteers
were pulsed with the PSA-specific peptides PSA-1 or PSA-3 and used to
stimulate CTL from PBMC in vitro. As target cells, peptide-pulsed T2
cells (HLA-A2+) or the human prostate cancer cell
line LNCaP, which expresses both PSA and HLA-A2, was used. To control
for possible anergy to PSA as a self Ag, we tested the ability of DC to
stimulate CTL against a HLA-A2-restricted peptide derived from
influenza M1 nucleoprotein (flu M1). As shown in Fig. 4, PSA-3 peptide-pulsed DC stimulated a
robust CTL response against PSA-3 peptide-pulsed T2 cells and also
against human LNCaP cells, while no lysis of M1 peptide-pulsed T2 cells
was observed (Fig. 4A). Consistent with studies reported by
Correale et al. (2), DC pulsed with PSA-1 peptide were
also able, albeit less effectively than PSA-3 peptide-pulsed DC, to
induce PSA-specific CTL responses (Fig. 4B). Stimulation of
PBMC with flu M1 peptide-pulsed DC reproducibly generated CTL that
lysed flu M1-pulsed, but not PSA-3 peptide-pulsed T2 cells. These data
demonstrate that the DC generated under clinical conditions and pulsed
with PSA-specific peptides are functionally active by stimulating
potent PSA-specific CTL responses in vitro.
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To test whether human DC can be effectively sensitized with
RNA-encoded Ags, we assessed the capability of DC transfected with mRNA
encoding PSA to stimulate primary CTL responses in vitro. PSA mRNA was
generated by in vitro transcription of the cDNA plasmid pGEM4Z/PSA/A64.
DC from a HLA-A2+ individual were transfected
with PSA RNA and used to stimulate CTL responses in vitro against the
following targets: 1) human LNCaP prostate carcinoma cells; 2) PSA
RNA-transfected DC; 3) PSA-3 peptide-pulsed DC. DC pulsed with the Ags
influenza nucleoprotein M1 peptide, or transfected with GFP mRNA were
used as control targets. As shown in Fig. 5A, PSA RNA-transfected DC
stimulated a robust CTL response, which recognized LNCaP, PSA RNA, or
PSA-3 peptide-pulsed DC target cells. DC transfected with the control
RNA (GFP RNA) or pulsed with flu M1 peptide were not lysed. The primary
nature of this response was suggested by the fact that PSA
RNA-transfected DC failed to stimulate significant CTL activity
following only one round of stimulation (data not shown). To our
knowledge, these findings represent the first demonstration that CTL
priming against PSA Ags can be achieved by stimulating PBMC with either
PSA peptide-pulsed or PSA RNA-transfected DC. Importantly, these data
also demonstrate that PSA RNA-transfected DC can serve as effective
targets in CTL assays, thus allowing immunological monitoring of PSA
responses in humans, while obviating the need for culturing and
labeling the patient’s tumor cells (15).
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B, a RNA concentration-dependent CTL response was
observed. Based on the observed lytic levels, this response was optimal
by transfecting 1 x 106 DC with 1.5 µg of
PSA mRNA (Fig. 5B). Higher RNA concentrations reproducibly
did not augment this response (data not shown). In a separate set of
experiments, we determined that the function of DC to serve as targets
is similarly dependent on the RNA concentration pulsed onto DC. Similar
to the previous experiment, DC generated under optimized RNA
transfection conditions stimulated CTL, which optimally recognized and
lysed DC targets transfected with 1.5 µg of PSA mRNA/1 x
106 DC (data not shown). PSA RNA-transfected DC stimulate CTL specific for PSA, but not for the structurally related kallikrein
Although the use of a well-defined tumor Ag such as PSA may
greatly reduce the risk of inducing immune responses against other self
Ags, the potential induction of autoimmunity with pathologic
consequences remains a concern. In particular, serum kallikrein is a
ubiquitous protein found in various normal tissues, which shares about
80% structural homology with PSA (3). The purpose of the
following experiment was to investigate whether DC transfected with PSA
mRNA activate CTL against epitopes shared with kallikrein. For CTL
generation, the following DC preparations were generated: 1) DC
transfected with PSA RNA, 2) DC pulsed with PSA protein, or 3) DC
pulsed with kallikrein protein. These DC were used both as stimulators
and as targets in cytotoxicity assays. Similar to the previous
experiments shown in Fig. 5, PSA RNA-transfected DC (Fig. 6A) and also PSA
protein-pulsed DC (Fig. 6B) were capable of stimulating CTL
responses recognizing and lysing DC targets presenting PSA, but not
kallikrein Ags, suggesting the absence of CTL recognizing epitopes
shared between these structurally related proteins. Interestingly, as
shown in Fig. 6C, kallikrein protein-pulsed DC were also
capable of stimulating kallikrein-specific, but not PSA-specific CTL,
despite the fact that kallikrein is a ubiquitously expressed self Ag.
These experiments show that DC transfected with PSA RNA can stimulate
CTL responses, which are specific for PSA Ags and do not cross-react
with kallikrein, suggesting that the induction of harmful autoimmunity
may not be a serious issue with this approach.
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The experiments described to date have demonstrated that DC
transfected with PSA RNA are capable of inducing CTL responses in
HLA-A2+ healthy volunteers. The concern was
raised that tumors could tolerize the immune system against Ags
expressed on the tumor cells (16). Therefore, cancer
patients may not respond to a particular vaccine strategy in a similar
fashion as healthy volunteers. The purpose of the following experiments
was several-fold. First, we sought to assess the efficacy of PSA
RNA-transfected DC to stimulate primary CTL responses among cancer
patients. Second, to assess the general applicability of this vaccine
strategy, we tested whether in vitro stimulation of PSA-specific CTL
can be achieved from a large number of randomly selected individuals
irrespective of their HLA composition. Finally, we tested whether males
exhibit immunological tolerance to PSA by comparing the ability to
stimulate CTL between males and females. DC were generated from a total
of 14 randomly selected individuals consisting of 4 healthy males, 6
healthy females, and 4 prostate cancer patients. Following PSA mRNA
transfection, DC from all groups were analyzed and compared for their
ability to generate PSA-specific CTL responses. Fig. 7 demonstrates the results from three
representative candidates within each group. Although minor variances
in the lytic levels were observed among and within the three groups, we
could reproducibly generate CTL responses against PSA Ags from all
individuals tested. Interestingly, CTL responses of a similar magnitude
were observed between healthy males and cancer patients. Thus, we find
no evidence that the T cell population of healthy males or cancer
patients is deficient in PSA-specific CTL precursors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It was argued that tumors could tolerize the immune system against Ags expressed on tumor cells (17). In one study, tumor Ag-specific CD4pos T cell anergy was observed in tumor-bearing mice as an early event in the course of tumor progression (16). In contrast, others have not found any impairment of tumor Ag-specific CD8pos responses (18, 19). In this study, we have not seen evidence that in vitro stimulation of CTL against the self Ag PSA is significantly blunted in cancer patients as compared with healthy volunteers. Although this could be attributed to an insufficient level of tumor burden in the patients examined, the issue as to how or to what extent progressing tumors impact negatively on tumor-specific immunity is yet unresolved and awaits further investigation.
Ags presented by thymic APC cause clonal deletion of autoreactive T lymphocytes, whereas the response to peripherally expressed proteins with no access to the thymus can result in either their physical elimination or functional anergy. Because healthy women lack significant PSA expression, whereas healthy males or prostate cancer patients are exposed to higher levels of circulating PSA, we initially hypothesized that significant differences may exist in our ability to induce CTL against PSA among these groups. Surprisingly, we found that PSA RNA-transfected DC generated from healthy males, females, and cancer patients stimulated CTL responses of similar magnitude. One possible explanation is the fact that PSA expression, albeit at very low levels, can be found in some tissues in normal females (20, 21). Indeed, anti-PSA Abs have been detected in some healthy females, suggesting that PSA can be the target of a humoral autoimmune response (22). The present study shows that PSA-specific CTL can be generated to a comparable extent from healthy males and females, or from prostate cancer patients, suggesting that PSA-specific CTL precursors are present at comparable frequency among those groups, which can be activated by stimulation with PSA RNA-transfected DC. Although in this study we could not identify any evidence for immunological tolerance or tumor-mediated anergy to PSA, the critical questions remain as to whether the increase in the observed PSA CTL frequency will ultimately lead to a clinical benefit for cancer patients. Such questions will need to be carefully determined in a clinical trial setting.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Johannes Vieweg, Duke University Medical Center, MSRB, Room 464, Box 2626, Durham, NC 27710.
3 Abbreviations used in this paper: PSA, prostate-specific Ag; DC, dendritic cell; GFP, green fluorescent protein; neg, negative; pos, positive.
Received for publication November 23, 1999. Accepted for publication March 7, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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