Differential CTLs specific for prostate-specific antigen in healthy donors and patients with prostate cancer

Eyad Elkord1,4, Paul E. Williams1,2, Howard Kynaston3 and Anthony W. Rowbottom2

1 Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, UK
2 Department of Medical Biochemistry and Immunology and 3 Department of Urology, University Hospital of Wales, Cardiff, UK
4 Present address: CRUK Immunology Department, Paterson Institute for Cancer Research, Manchester M20 4BX, UK

Correspondence to: E. Elkord; E-mail: eelkord{at}picr.man.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Induction of CTL responses specific for prostate-specific antigen (PSA)-derived peptides in healthy individuals and patients with prostate cancer (PC) was investigated. Eight PSA-derived peptides that have the potential to bind HLA-A2 molecules were examined. Peripheral blood lymphocytes isolated from HLA-A2-positive volunteers were expanded using autologous mature, PSA-derived peptide-pulsed dendritic cells. The expansion of IFN-{gamma}-secreting CD8+ T cells specific for three of the eight PSA-derived peptides (PSA-2108–117, PSA-4141–150 and PSA-6146–154) was detected in healthy individuals, but not in patients with PC. Using HLA-A2/peptide tetramers, the PSA-specific CD8+ T cells were detectable at low frequency both in healthy individuals and patients with PC. Using flow cytometric cytotoxicity assays, the expanded effectors from healthy individuals were able to kill the PSA-expressing epithelial cell line LNCaP and the peptide-pulsed T2 cells in a MHC class I-restricted manner without involving NK activity. However, such killing by effectors expanded from prostatectomized patients involved a complete or a significant NK activity. Specific recognition of PSA-derived peptides in healthy individuals may occur by an adaptive CTL immune response, while such recognition in PC patients may additionally or alternatively be mediated by an innate NK immune response. In conclusion, our work indicates that the PSA-specific CD8+ T cells exist in both healthy individuals and PC patients, but they have impaired function in patients as they failed to release IFN-{gamma} and to kill targets without involving NK activity.

Keywords: cytotoxic T lymphocytes, dendritic cells, NK activity, prostate cancer, prostate-specific antigen


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Prostate cancer (PC) is the most common cancer and the second leading cause of cancer-related death among the male population in Western countries (1). Between 35 and 61% of PC patients undergoing radical prostatectomy or radical radiotherapy are afterwards found to have non-organ-confined disease or positive prostatic biopsies (2, 3). Currently, no adjuvant or salvage therapy has been confirmed to be of benefit for these patients. Clearly, new and improved therapeutic approaches are required for the management of patients with localized and advanced PC.

Identification of tumour-associated antigen-derived peptides able to elicit anti-tumour T cell responses is essential for the development of peptide-based cancer vaccines (4). Immunotherapy for PC is an attractive strategy in light of the potential role of the immune system, which is supported by the significant correlation of tumour-infiltrating lymphocytes with good prognosis (5). T cells against some epitopes of self-proteins exist, and the T cell repertoire directed against sequences in self-proteins can be primed in an attempt to eliminate cancers (6). Prostate-specific antigen (PSA) is a potential target of vaccine therapy for human PC since it is nearly exclusively expressed on prostatic epithelial cells, and its expression is conserved in nearly all advanced PCs (7, 8).

The aim of this work was to determine whether CTL immune responses specific for PSA-derived peptides could be induced in healthy individuals and patients with PC. The amino acid sequence of PSA was searched for putative peptides likely to bind HLA-A2 molecules. Eight potential peptides were tested for their ability to bind HLA-A2 molecules using the peptide stabilization assay, and to elicit HLA-A2-restricted CTL responses specific for PSA-derived peptides in normal volunteers and patients with PC using IFN-{gamma} enzyme-linked immunospot (ELISPOT), cytotoxicity, cytokine secretion profile and tetramer assays.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Healthy individuals and patients with PC
Blood was drawn with informed written consent from healthy individuals and patients with PC after obtaining the required ethical approval from the Bro Taf Local Research Ethics Committee. The characteristics of all normal volunteers and PC patients are shown in Table 1. All volunteers were determined to be HLA-A2 positive by whole HLA typing by the Welsh Transplantation and Immunogenetics Laboratory (Cardiff, Wales, UK). Blood samples from PC patients were taken after radical prostatectomy (serum PSA level had declined in all cases to below 0.1 ng ml–1). For one patient (P1), one set of experiments was conducted before radical prostatectomy (when serum PSA was elevated) and the other set was done 5 months after radical prostatectomy (when serum PSA had declined).


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Table 1. Characteristics of normal volunteers and patients with PC involved in the in vitro induction of CTLs specific for PSA-derived peptides

 
Cell lines
T2 cells (American Type Culture Collection, LGC Promochem, Middlesex, UK) are HLA-A2 positive and TAP-deficient cells present only peptides from leader sequences and exogenous peptides (9, 10). These cells were cultured in RPMI-1640 with L-glutamine (Sigma, Poole, Dorset, UK) supplemented with 10% (v/v) heat inactivated FCS (FCSH, Sigma), 100 U ml–1 penicillin (Sigma) and 0.1 mg ml–1 streptomycin [referred to as complete medium (CM)]. LNCaP cells are positive for HLA-A2 and PSA. These cells were treated with 1x trypsin and EDTA solution (GIBCO, Paisley, UK) for subcultures, and were cultured in 10% CM. K562 cells, a NK-sensitive human erythromyelocytic leukaemia cell line, were cultured in 5% CM.

Peptides
SYFPEITHI prediction package (http://www.syfpeithi.de/) (11) was used for prediction of peptides derived from the mature PSA protein sequence (accession number NP_001639, from position 25 to 261) and presented on HLA-A2 molecules. Eight potential peptides were chosen for further investigation based on the ligation strength and hydrophilicity/hydrophobicity status. These peptides were enumerated from PSA-1 to PSA-8 based on the earlier position of the first amino acid in each peptide. Cytomegalovirus peptide (CMVpp65495–503) and Influenza matrix peptide (IMP58–66) were used as control peptides. They are derived from cytomegalovirus (12) and Influenza A matrix protein (13), respectively, and are highly immunogenic HLA-A2-restricted peptides. Relevant features of all controls and PSA-derived peptides (synthesised by Sigma-Genosys Ltd, Haverhill, UK) are shown in Table 2.


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Table 2. Description of PSA peptides and controls used in this study

 
Peptide-binding assay
Binding of all peptides to HLA-A2 molecules on T2 cells was evaluated by the HLA-A2 stabilization assay. The method described by Nijman et al. (14) with some modification was established and optimized. T2 cells were washed twice by RPMI, and 1 x 106 cells in serum-free IMDM medium were incubated overnight with different concentrations of peptides (final concentration from 0 to 100 µg ml–1) in the wells of 24-well culture plates at 37°C in a 5% CO2 incubator. A total of 105 cells were placed in a FACS tube, washed twice with PBS and incubated for 30 min with 0.15 µg ml–1 of FITC-labelled HLA-A2,28-specific mAb (BD PharMingen, Oxford, UK). Control cells were stained with IgG2b as an isotype-matched control antibody. The cells were then washed with PBS and analysed by flow cytometry.

Generation of immunogenic dendritic cells
Blood was drawn into heparinized vacutainer tubes (Becton Dickinson Vacutainer, Plymouth, UK), and PBMCs were isolated using Histopaque-1077 (Sigma chemicals, Poole, Dorset, UK) as previously described (15). Monocytes were isolated from PBMC preparations by plastic adherence in which 5 x 106 cells per well were distributed in 12-well flat-bottom plates (Bibby Sterilin Limited, Stone, Staffordshire, UK). Cells were allowed to adhere in a 5% CO2 incubator at 37°C for 2 h in 1 ml 5% CM. Non-adherent cells were removed and kept; the adherent cells were washed twice carefully with pre-warmed 5% CM.

For the generation of immature dendritic cells (DCs), monocytes were cultured in 1 ml 5% CM per well supplemented with 1000 U ml–1 recombinant human granulocyte macrophage colony-stimulating factor (R&D Systems, Abingdon, UK) and 1000 U ml–1 recombinant human interleukin hrIL-4 (R&D Systems). The cultures were supplemented with 200 µl fresh 5% CM containing cytokines every 2 days. After 6 days of culturing, cells were harvested, pooled together and counted. Immature DCs were aliquoted at 0.5 x 106 cells in 0.5 ml supernatant suspension into each well and incubated at 37°C with 0.5 µg ml–1 LPS from Escherichia coli 026:B6 (Sigma) for 15–20 min before adding 0.5 ml fresh 5% CM with cytokines, and then incubated further for 3 days.

Generation of peptide-specific CD8+ T cells
We have shown earlier that DCs derived from adherence-isolated monocytes and activated by LPS are the most potent antigen-presenting cells for inducing antigen-specific CD8+ T cell responses (16). These cells were pulsed with one of the control or PSA-derived peptides at a final concentration of 50 µg /ml–1 for 2 h at 37°C, and used as stimulators for inducing peptide-specific CD8+ T cells. Non-adherent peripheral blood lymphocytes (PBLs) (~2 x 106) and the pulsed DCs (~1 x 105) were co-cultured at a ratio of 20:1 to 30:1 in 1 ml 10% AB (v/v) media (CM supplemented with heat-inactivated human AB serum instead of FCSH) in individual wells of a 24-well flat-bottom plate (Bibby Sterilin Limited) at 37°C. The cells were cultured with or without 25 U ml–1 hrIL-2 (R&D Systems). After 4 days, 1 ml hrIL-2-containing medium was added. Following another 3-day incubation, 1 ml culture medium was replaced with 1 ml fresh hrIL-2-containing medium and the cells were incubated for a further 4 days. Eleven days of incubation with pulsed DCs and hrIL-2 constituted one round of in vitro stimulation (IVS). After one round of IVS, CD8+ T cells were separated from the bulk cultures by positive selection with anti-CD8-coated microbeads (Miltenyi Biotech Ltd, Surrey, UK) according to the manufacturer's protocol, and introduced into the ELISPOT assay for IFN-{gamma}. After two rounds of IVS, effector cells were harvested without further separation and used as effectors in a flow cytometric cytotoxicity assay.

ELISPOT assays
The protocol for IFN-{gamma} ELISPOT assay (Immunodiagnostic Systems, Boldon, Tyne & Wear, UK) was used with some modifications, as described by the manufacturer (Diaclone Research, Besancon, France). Briefly, 96-PVDF-bottomed-well filtration plates were coated overnight at 4°C with an anti-cytokine capturing antibody. Unpulsed or peptide-pulsed DCs were used as stimulators, and incubated overnight with CD8+ T cells expanded with peptide-pulsed DCs. The captured cytokines were then revealed by a secondary biotinylated anti-cytokine antibody, which was in turn detected by streptavidin conjugated to alkaline phosphatase, and a blue precipitate produced by the enzymatic reaction.

Cytotoxicity assays
The ability of the expanded effector T cells to kill the human prostate carcinoma cell line (LNCaP) and the peptide-pulsed T2 cells was assessed in a flow cytometric cytotoxicity assay. In this assay, targets were first labelled with 4 µM of lipophilic membrane dye PKH26 (Sigma, St Louis, MO, USA) before incubating them with the effector T cells for 4 h at 37°C. For T2 cells, they were pulsed with peptides following their labelling with PKH26 dye. To discriminate dead cells from live ones by propidium iodide (PI) exclusion, 20 µg ml–1 PI dye (Sigma) was added to each tube prior to analysis by flow cytometry.

The level of cytotoxicity was expressed as the percentage of cell death within the PKH26-positive targets and calculated as:

The percentage of specific lysis was calculated as:

Cytokine assays
Cytokines were measured using the Bio-Plex protein array system (Bio-Rad Laboratories, Hercules, CA, USA). The pre-mixed multiplex beads of the Bio-Plex human cytokine Th1/Th2 panel (Bio-Rad, catalogue no. 171-A11081) were used, which can differentiate between Th1/Th2 or cytotoxic T cell (Tc)1/Tc2 phenotypes according to the pattern of cytokines present.

Tetramers
Fluorescent peptide–MHC class I tetramer reagents were synthesised by Proimmune Limited, Oxford, UK. Four tetramers for HLA-A2-restricted peptides were synthesized; three of them are derived from PSA protein (PSA-2108–117, PSA-4141–150 and PSA-6146–154) and one derived from Influenza virus matrix protein (IMP58–66) as a control. These tetramers were labelled with commercially manufactured streptavidin–PE. The IMP–CD8+ clone was used as a positive control to optimize the staining conditions and tetramer and antibody concentration used. Briefly, 0.5 x 106 of the expanded T cells were stained first with PE-labelled tetramer for 10 min at 37°C, washed twice with buffer and then incubated with anti-CD8–FITC mAb (clone LT8, Proimmune Limited) for 45 min on ice before flow cytometric analysis.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Binding of the peptides to HLA-A2 molecules
In the peptide stabilization assay, binding of peptide leads to increased stability of HLA-A2 molecules, and consequently increase in their expression, which can be measured by fluorescence intensity. Control peptides (IMP58–66, CMVpp65495–503), PSA-4, PSA-6 and PSA-8 peptides bound strongly to HLA-A2 molecules, while PSA-5 peptide showed slightly positive binding results (data not shown). Despite the inability of some candidate peptides (PSA-1, PSA-2, PSA-3 and PSA-7) to bind to HLA-A2 molecules, none was excluded from further investigation since peptide binding is an in vitro assay and might not be sensitive enough to detect binding of all natural ligands (17).

IFN-{gamma}-secreting CD8+ T cells specific for PSA-derived peptides are detected in healthy donors, but not in patients with PC
To investigate if PC patients respond differently from healthy individuals, the number of peripheral blood CD8+ T cells responding to IMP58–66 peptide was assessed in healthy individuals and PC patients. IFN-{gamma}-secreting CD8+ T cells specific for IMP58–66 peptide were detected in both healthy donors and PC patients (Fig. 1); however, they were significantly lower in PC patients, as determined by the unpaired two-tailed Student's t-test (P = 0.0177).



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Fig. 1. Plot of CD8+ T cell spots specific for IMP58–66 peptide. The spots were measured, with IFN-{gamma} ELISPOT assays, in healthy donors (n = 4) and in patients with PC (n = 4). The mean ± SD for healthy donors was 704 ± 243 spots per 100 000 T cells or 1 in 142 and for PC patients 277 ± 52 spots per 100 000 T cells or 1 in 361.

 
At the same time, to assess the response of the volunteers to another peptide, detection of the precursors for CMVpp65495–503 peptide was assessed in three healthy donors and one PC patient (P1). None of the tested volunteers responded to this peptide by secreting IFN-{gamma}, as measured by IFN-{gamma} ELISPOT assays. Further investigation showed that all individuals were seronegative for CMV IgG antibodies, as tested by the Public Health Laboratory Service/University Hospital of Wales, Cardiff, UK.

To evaluate CD8+ T cell responses for PSA peptides, the mature DCs were pulsed with each of the eight PSA-derived peptides. CD8+ T cells were then positively selected and assayed by IFN-{gamma} ELISPOT versus all the peptides. The number of IFN-{gamma}-secreting cells in the absence of peptide (stimulators: unpulsed DCs) was subtracted from the number of IFN-{gamma}-secreting cells in the presence of peptide (stimulators: peptide-pulsed DCs) in order to calculate the number of IFN-{gamma}-secreting CD8+ T cells specific for that peptide. Table 3 shows that CD8+ T cells specific for three of the eight PSA peptides (PSA-2108–117, PSA-4141–150 and PSA-6146–154) were detected in three out of four healthy donors. One donor (NV1) showed response to seven of the eight peptides. By contrast, no ELISPOT responses were detected in PC patients.


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Table 3. IFN-{gamma}-secreting CD8+ T cells specific for PSA-derived peptides in four normal volunteers and four patients with PC

 
PSA-expanded effectors from healthy donors specifically kill LNCaP and T2 targets pulsed with PSA-derived peptides
A flow cytometric cytotoxicity assay was optimized as described in Methods, and used to measure cytotoxicity ability of the expanded effectors. A FACS analysis of this method is explained in Fig. 2. We found that the addition of IL-2 after 4 days of culture resulted in larger numbers of IMP58–66-specific IFN-{gamma}-producing CD8+ T cells than the addition of IL-2 into culture at day 0. Therefore, IL-2 was added on day 4 for all subsequent generation of peptide-specific CD8+ T cells. The ability of the expanded effectors specific for IMP58–66 peptide to kill T2 cells pulsed with IMP58–66 peptide was investigated. PBLs expanded from both healthy individuals and PC patients were able to kill significant percentages of IMP58–66-pulsed T2 targets, as shown in Table 4. This killing was not inhibited by the addition of K562 cells as confirmed by K562 competition assays. For one patient (P1), the expanded effectors were able to kill a significant percentage of these targets pre- and post-radical prostatectomy (Table 4).



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Fig. 2. An illustration of a flow cytometric cytotoxicity assay performed by dual parameter analysis of PKH26-labelled targets counterstained with PI. When all targets and effectors were gated (A), four different populations are clear (B). Cells double negative for PKH26 and PI are live effectors, while cells negative for PKH26 and positive for PI are dead effectors. Double-positive cells are dead targets, while cells positive for PKH26 and negative for PI are live targets. When only labelled targets are gated (C), dead targets are clearly distinguished from live ones (D). Figures are for the 50:1 E:T ratio and ~18% of the targets were killed.

 

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Table 4. Percentages of IMP58–66-pulsed T2A2 targets killed by IMP-expanded effectors from healthy donors and patients with PC

 
Three PSA-derived peptides were chosen for cytotoxicity studies because three out of four healthy donors responded to them by secreting IFN-{gamma} as detected by ELISPOT. Different target to effector (T:E) ratios were used, and the expanded effectors from a healthy donor were able to kill LNCaP and peptide-pulsed T2 targets in a T:E ratio-dependent manner, as shown in Fig. 3. Optimum killing was observed at T:E ratio of 1:50; therefore, this ratio was fixed for all subsequent cytotoxicity assays.



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Fig. 3. Cytotoxicity assays for PSA-expanded PBLs from a healthy donor. PBLs isolated from NV1 healthy donor were expanded using mature autologous DCs pulsed with one of the three PSA-derived peptides. The ability of the expanded cells, at different T : E ratios, to kill LNCaP cells and PSA-pulsed T2 cells was measured using flow cytometric cytotoxicity assays.

 
We performed blocking studies using anti-HLA-ABC mAb (clone W6/32, from Serotec Ltd, Oxford, UK) to determine if the observed cytotoxicity of the expanded effectors from healthy donors was MHC class I restricted. Killing of LNCaP targets was shown to be MHC class I restricted, as confirmed by the inhibition of killing by anti-HLA-ABC antibody (Fig. 4). The specificity of LNCaP killing was investigated in the presence of a 10-fold excess of unlabelled, cold K562 cells. As shown in Fig. 4, there was no considerable reduction in the killing of the hot LNCaP targets. These results confirmed that the killing of LNCaP targets by all three PSA-expanded effectors grown from healthy donors did not include any NK activity. The cytolytic activity of the expanded effectors against PSA peptide-pulsed T2 targets was also investigated. As shown in Fig. 4, the expanded effectors were able to kill considerable percentages of T2 cells pulsed with the matching PSA-derived peptides.



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Fig. 4. Ability of PSA-expanded effectors from healthy donors to kill various targets. PBLs from healthy donors were expanded for two stimulation cycles using mature DCs pulsed with PSA-2 (A), PSA-4 (B) and PSA-6 (C). Effector cells from the four healthy donors were mixed as the cytotoxicity patterns presented by these volunteers were similar. Killing activity at T:E ratio of 1:50 against LNCaP and T2 targets in the absence or presence of the corresponding peptide was measured using flow cytometric cytotoxicity assays. Graphs shown are representative of experiments performed in triplicate with cells from four different healthy donors.

 
Cytolytic activity of PSA-expanded effectors from patients with PC
Unlike healthy donors, the cytotoxicity patterns of the expanded effectors from PC patients against various targets varied from one patient to another. For PC patient pre-radical prostatectomy (P1), none of the expanded effectors for the three PSA-derived peptides was able to kill a significant percentage of LNCaP targets and PSA peptide-pulsed T2 targets (Fig. 5). The whole experimental set for this patient was performed twice at two time points, 5 months apart, and the expanded effectors were unable to kill targets on both the occasions. Interestingly, for the same patient post-radical prostatectomy, the ability of the expanded effectors to kill was restored and they killed significant percentages of LNCaP and PSA-pulsed T2 targets (Fig. 5). When K562 cells were added in an excess of 10-fold, the ability of expanded effectors to kill targets was significantly reduced, which supports the involvement of NK activity. Blocking of MHC class I on LNCaP targets did not reduce the cytotoxicity level, and this confirms that the killing was not MHC class I mediated. For the other PC patients, PBLs taken post-radical prostatectomy and expanded for two rounds of IVS were able to kill significant percentages of targets in two out of three patients (P2 and P4). The NK activities in these patients were very evident, and K562 addition resulted in a complete inhibition of LNCaP killing, as shown in Fig. 5.



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Fig. 5. Ability of PSA-4-expanded effectors from PC patients to kill various targets. PBLs from PC patients were expanded for two stimulation cycles using mature DCs pulsed with PSA-4 peptide. Killing activity at T:E ratio of 1:50 against LNCaP and T2 targets in the absence or presence of the corresponding peptide was measured using flow cytometric cytotoxicity assays. K562 cells were added in excess to detect any NK activity. A total of five experiments were performed for four patients [two experiments for one patient (P1), pre- and post-radical prostatectomy]. The patient code is shown above each figure. Expanded effectors for PSA-4 peptide is shown as representative, and the same killing patterns were obtained for PSA-2 and PSA-6 peptides.

 
PSA-stimulated CD8+ T cells from healthy donors and PC patients display the phenotype of effector/memory and are of Tc1 subsets
Peptide-stimulated CD8+ T lymphocytes were further characterized. Cells were co-stained with an anti-CD8 mAb (Dako Corporation, Cambridge, UK) and anti-CD45RA, anti-CD45RO, anti-CD27 or anti-CD28 mAbs (Dako Corporation), and flow cytometry analysis was performed. Expanded cells for IMP58–66, as a control, and the three PSA-derived peptides expressed a phenotype of CD45RACD45RO+CD27CD28, suggesting that these cells belonged to the effector/memory subset according to the classification outlined by Hamann et al. and Sallusto et al. (18, 19).

In order to investigate whether adherence/monocyte-derived dendritic cell (MoDC) stimulators induced the development of Tc1 or Tc2/Tc0 effectors, peptide-expanded CD8+ T cells were isolated by positive selection, stimulated by phorbol myristate acetate and ionomycin for 72 h, and the cytokine profiles were determined. The peptide-specific CD8+ T cells grown from healthy donors and PC patients produced high concentrations of IFN-{gamma} and no significant amounts of IL-4 (Table 5). From these cytokine profiles, mature DCs promoted the induction of Tc1 cells specific for IMP58–66 and PSA-derived peptides.


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Table 5. Th1/Th2 cytokine panel for CD8+ T cells specific for IMP58–66, as a control, and PSA-derived peptides (PSA-6 is shown as a representative)

 
PSA-specific CD8+ T cells are detectable at low frequency both in healthy individuals and patients with PC
Using tetramer complexes, we investigated the frequency of CD8+ T cells specific for PSA and Influenza A virus. PBMC samples from one healthy donor (NV1) and three PC patients (P2, P3 and P4), with one round of either pre- or post-IVS (with LPS-activated, peptide-pulsed DCs), were stained using tetramers. IMP/tetramer was used as a control to enumerate CD8+ T cells specific for Influenza A virus. Staining patterns using four different tetramers of CD8+ T cells pre- and post-peptide stimulation from a PC patient (P4), as a representative experiment, are shown in Fig. 6. The results of experiments involving tetramer staining are shown as a summary in Table 6. CD8+ tetramer+ T cells specific for IMP58–66 peptide were detected at a range of 0.06–0.67% (1 of 1667 to 1 of 149) amongst the CD8+ T cells of both healthy individuals and patients with PC. Upon stimulation with peptide-pulsed DCs, IMP58–66-specific CD8+ T cells were significantly amplified (19- to 303-fold), mainly for the healthy individual (303-fold). For CD8+ tetramer+ T cells specific for any of the three PSA-derived peptides, they were detected at a lower frequency both in the healthy individual and PC patients. A range of 0.02–0.06% (1 of 5000 to 1 of 1667) was found. Upon peptide stimulation, these precursors were increased by different factors, but not as great as for IMP58–66 peptide. Maximum amplification of 15.5-fold was achieved for the expanded PBMCs for PSA-2 peptide from the healthy individual. For all other expanded cells, an amplification of 2.33- to 9.5-fold was achieved, as shown in Table 6.



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Fig. 6. Enumeration of peptide-specific CD8+ T cells using tetramers. PBLs, isolated from a PC patient (P4), were stimulated for one cycle of IVS using LPS-activated DCs pulsed with IMP58–66, or with any of the three PSA-derived peptides (PSA-2, PSA-4 and PSA-6). CD8+ T lymphocyte populations (purity >95%, as shown in A) were obtained by positive selection using MACS following one cycle of IVS. The positively selected CD8+ T lymphocyte preparations, obtained either directly from PBMC (B–E, pre-stimulation) or after peptide stimulation (F–I, post-stimulation) were then stained with IMP, PSA-2, PSA-4 or PSA-6/tetramers together with anti-CD8 mAb, and analysed by flow cytometry. Percentages of CD8+ tetramer+ T cells are shown on the top of each dot plot.

 

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Table 6. Frequencies of CD8+ tetramer+ T cells

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
PC might be an ideal candidate for immunotherapy for the following reasons. First, PCs express several characteristic proteins, which could be potential targets for immunotherapy. Second, the prostate gland is not an essential organ. Third, the significant failure rate after the treatment of the primary tumour, and the lack of an effective chemotherapy for metastatic PC, make it important to investigate alternative therapies such as immunotherapy (20).

In this study, two highly immunogenic HLA-A2-restricted peptides were used as controls. IMP58–66 peptide worked as a positive control as IFN-{gamma}-secreting CD8+ T cells specific for this peptide were expanded in four healthy donors and four PC patients. However, amplification of these precursors was significantly higher in healthy volunteers than in PC patients. This supports the concept that the immune system of healthy individuals is intact and they respond in a stronger manner, while patients with cancers, including PC, have impaired immune responses. On the other hand, CMVpp65495–503 peptide worked as a relevant negative control as no IFN-{gamma}-secreting CD8+ T cells specific for this peptide were detected in any of the three healthy donors and one PC patient tested (all were seronegative for CMV IgG antibodies).

CD8+ Tc responses against a prostate carcinoma cell line (LNCaP) and peptide-pulsed T2 cells were induced in both healthy individuals and PC patients following stimulation with LPS-activated DCs pulsed with PSA-derived peptides. Killing of targets by effectors expanded from healthy individuals was MHC class I mediated as confirmed by blocking the killing using MHC class I antibody, and was not NK mediated as shown by cold target K562 competition assays. CTL responses specific for PSA-derived peptides were induced in cells isolated from two female healthy volunteers. This was not surprising as PSA was detected in female sera with the availability of highly sensitive methods for measuring minute amounts of PSA (21, 22). The main source of female PSA has been suggested to be the ‘female prostate’, Skene's glands and ducts, which are located paraurethrally (23, 24). Furthermore and in agreement with our finding, Heiser et al. showed that DCs transfected with PSA RNA generated from male or female healthy volunteers or from PC patients were equally effective in stimulating PSA-specific CTLs in vitro (25). Expanded effectors from three out of four patients with PC post-radical prostatectomy exhibited a cytotoxic activity against LNCaP and peptide-pulsed T2 targets. Interestingly and unlike healthy individuals, a complete or a significant part of the killing was found to be NK mediated. The mechanism of NK activation in PC patients is not known. To some extent, this might be explained by the critical role that the innate immune response of NK cells can play where the adaptive CTL immune response is limited, mainly in individuals with impaired immune responses such as cancer patients. Clearly, further investigation is required to assess whether this killing was mediated by classical CD3CD56+ NK cells, CD3+CD56+ NKT cells or lymphokine-activated killer cells. Both CTLs and NK cells are important anti-tumour effectors. However, the common loss of MHC class I expression in PC cells may enable these cells to escape from CTLs. In contrast, NK killing activity is not MHC restricted, and therefore NK cells are able to kill targets that have down-regulated or lost the expression of MHC molecules (26). The innate NK activity we report in PC patients can be an alternative or an additional mechanism besides the adaptive CTLs. In recent studies, it has been suggested that DCs, especially MoDCs, are useful for directly stimulating NK and NKT cells and as a result mobilizing the additional power of the innate immune system to attack tumour cells (27, 28). Notably, the NK activity shown in this study gains special importance since in two recent studies it was possible to expand NK cells with strong cytotoxic activity against PC cells in patients with PC (29, 30). Our results demonstrate the activation of NK cells by LPS-activated MoDCs in patients with PC.

In this study, generation of PSA-specific effectors from PC patients was performed from T cells grown from peripheral blood collected after prostatectomy. For one patient, these experiments were performed on such T cells collected before and after prostatectomy. Most importantly, cells expanded before prostatectomy were unable to kill LNCaP and peptide-pulsed T2 targets. Interestingly, ability of the expanded effectors to kill targets was restored after prostatectomy. The mechanism of this restoration is not clear but the operation itself might alter cytokine levels and possibly change NK activity. In an in vitro study, Kennedy-Smith et al. showed that PSA protein suppressed lymphocyte proliferation in a dose-dependent manner (31). Our results support the hypothesis that high concentration of serum PSA may hamper immune responses.

The ability to expand potent PSA-specific CTLs using peptide-pulsed DCs in healthy individuals supports the potential use of PSA-derived peptides in the design of prophylactic cancer vaccines. As proposed by Finn and Forni, the justification for using prophylactic cancer vaccines is quite strong (32). There is some evidence that patients with cancer can be particularly immunosuppressed to their tumour, and cancer vaccines are more efficient in generating potent immune responses in healthy individuals. The ability to expand potent effectors from patients after radical prostatectomy that are able to kill PC cells supports the concept of using immunotherapy as an adjuvant therapy after surgery.

We reported that PSA-expanded effectors from some healthy individuals and PC patients were able to kill LNCaP and peptide-pulsed T2 targets despite the negative IFN-{gamma}-secreting cells obtained by ELISPOT. It has been reported that cytotoxicity and IFN-{gamma} production are regulated independently (33). More specifically, IFN-{gamma} secretion is not an absolute assessment of cytotoxic activity of CTLs because non-cytotoxic cells are able to produce IFN-{gamma} and, in addition to that, CTLs with killing activity do not always produce IFN-{gamma}, and it is unclear how the numbers of IFN-{gamma}-specific CD8+ T cells relate to cytotoxic activity (34, 35).

Indeed, some of the investigated peptides in this study were examined by others (36, 37, 38). However, in these studies, the induction of CTLs specific for PSA-derived peptides was only investigated by cytotoxicity assays. In this study, a relatively high number of PSA-derived peptides (eight) was examined, and variety of assays including IFN-{gamma} ELISPOT, cytotoxicity, cytokine secretion profile and tetramer complexes were employed together to further characterize the PSA-expanded T cells. Both normal donors and PC patients were examined, and different CTL responses were reported. Moreover, one patient was examined prior to and after radical prostatectomy and only cytotoxicity activity was restored after prostatectomy. Finally, one new PSA-derived peptide (PSA-2) presented in this study was found to induce T cell responses. Finally, our study is the first to describe the use of tetramers to directly enumerate CD8+ T cells specific for PSA-derived peptides. Although IFN-{gamma}-secreting CD8+ T cells specific for PSA-derived peptides were not detected in PBMCs isolated from PC patients, it was possible to detect CD8+ T cells specific for these peptides at low frequency both in healthy individuals and PC patients, and to expand them using LPS-activated, peptide-pulsed DC.


    Acknowledgements
 
The authors thank Graham Read and Peter L. Stern for critical reading of the manuscript. This work was supported by the Tom Owen Memorial Fund and the Karim Rida Said Foundation.


    Abbreviations
 
CM   complete medium
DC   dendritic cell
ELISPOT   enzyme-linked immunospot
FCSH   FCH heat inactivated
IMP   influenza matrix protein
IVS   in vitro stimulation
MoDC   monocyte-derived dendritic cell
PBL   peripheral blood lymphocyte
PC   prostate cancer
PI   propidium iodide
PSA   prostate-specific antigen
Tc   cytotoxic T cell
T:E   target to effector

    Notes
 
Transmitting editor: E. Simpson

Received 9 May 2005, accepted 15 July 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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