Abstract
T cell immunotherapy of prostate cancer (CaP) offers the potential for less toxic, more effective outcomes. A clinical trial was conducted in 28 patients with locally advanced or metastatic CaP to determine whether an HLA-A2 binding epitope of prostate-specific antigen, PSA146–154 (PSA-peptide), can induce specific T cell immunity. Patients were vaccinated either by intradermal injection of PSA-peptide and GM-CSF or by intravenous administration of autologous dendritic cells pulsed with PSA-peptide at weeks 1, 4 and 10. Delayed-type hypersensitivity (DTH) skin testing was performed at weeks 4, 14, 26 and 52. Fifty percent of the patients developed positive DTH responses to PSA-peptide. The size of the DTH induration progressively increased over time in the majority of responding patients. Skin biopsies from seven DTH-positive patients were available and T cells that developed in situ were also characterized. The phenotype of recovered T cells demonstrated variable proportions of CD4+CD8−, CD4−CD8+ and CD4+CD8+ T cell populations. Cytokine analysis of PSA-peptide stimulated T cells per bead array assay exhibited specific IFN-γ and TNF-α response in six of seven patients. Specific IL-4 response was observed in five patients, while IL-10 response was detected in one patient. Purified CD4−CD8+ T cells isolated from four patients demonstrated specific cytolytic activity per chromium release assay. In conclusion, immunization with PSA-peptide induced specific T cell immunity in one-half of the patients with locally advanced and hormone-sensitive, metastatic CaP. DTH-derived T cells exhibited PSA-peptide-specific cytolytic activity and predominantly expressed a type-1 cytokine profile.
Keywords: Cytolytic T cells, Dendritic cells, DTH, PSA-peptide, Type-1 and type-2 cytokines, Tumor immunity, Vaccine
Introduction
Prostate cancer (CaP) is the most common malignancy and the second leading cause of cancer-related mortality among the male population in the United States [22]. Patients who present with locally advanced or disseminated metastases usually succumb with hormone-refractory disease. Most affected individuals are elderly with limited tolerance for conventional treatment regimens. Current therapeutic approaches for metastatic CaP are limited to a palliative role. Hence, there is a strong need for the development of novel therapeutic strategies that are effective and well tolerated.
In patients with CaP, similar to other human malignancies, the immune system retains the potential to recognize native self-determinants associated with tumor cells [14]. Prostate-specific antigen (PSA) is a 34 kDa kallikrein-like serine protease, which is exclusively produced by ductal and acinar epithelial cells of the prostate gland in males [11, 24]. Several lines of evidence support the possibility that PSA may be a useful target antigen for specific T-cell immunotherapy of CaP. Firstly, the expression of PSA is highly restricted to normal and transformed prostatic epithelial tissues. Secondly, PSA is expressed at substantial levels by most adenocarcinomas of the prostate. Thirdly, the potential danger of developing debilitating autoimmune injury to normal tissues is limited because the prostate gland is not essential for survival and is often removed as part of conventional primary therapy.
The immunogenic potential of PSA administered as a whole-protein or expressed with DNA and RNA vectors has been evaluated in several phase 1 clinical trials [1, 6, 9, 12, 21]. These studies have shown modest immunological and/or clinical responses, encouraging further development of immunotherapies for patients with CaP. Vaccination with defined peptide epitopes of PSA may prove advantageous compared to methods that use whole PSA protein. Peptide-based vaccines are expected to elicit more focused responses, and thus, limit the risks of unexpected auto-immune injury.
Our laboratory has identified a 9-mer peptide of PSA (amino acid position 146–154, sequence KLQCVDLHV) which binds to the prevalent human leukocyte antigen, HLA-A2, and elicits specific cytotoxic T lymphocyte (CTL) responses in vitro from normal individuals and patients with CaP [17, 26]. A phase 1b pilot trial was conducted to determine whether vaccination with PSA 146–154 peptide (hereafter called PSA-peptide) can induce specific T cell immunity in patients with CaP. A total of 28 HLA-A2+ patients with high-risk, locally advanced (n=14) or metastatic, hormone-sensitive CaP (n=14) were randomly assigned to two different methods of administration of the PSA-peptide. One protocol entailed intradermal injection of PSA-peptide with recombinant GM-CSF. The second protocol consisted of intravenous administration of autologous dendritic cells (DC) pulsed with PSA-peptide. Vaccinations were performed on three occasions over a 10-week period. Strong delayed-type hypersensitivity (DTH) skin reactions to PSA-peptide were elicited by both vaccination methods in fifty percent of the patients. Seven of 14 patients with positive specific DTH reaction volunteered to undergo biopsy of the reaction site. Functional analysis of DTH derived T cells in terms of immuno-phenotyping, cytolytic activity and cytokine profile were examined.
Materials and methods
Patients
A total of 28 HLA-A2+ patients with pathologically confirmed CaP were enrolled in the phase 1b clinical trial. All subjects were between 50 and 80 years of age at the time of enrollment. Informed consent was obtained from all patients with authorization of the Institutional Review Board of the University of Illinois. All patients had undergone radio-therapy or surgical ablation of the prostate and had completed primary therapy a minimum of 6 weeks prior to enrollment in the vaccine study. Fourteen patients had high-risk, locally advanced disease (group A), and fourteen patients had metastatic, hormone-sensitive disease (group B). Group A-patients had either (1) T3, T4 disease, (2) serum PSA levels greater than 10 (ng/ml) or (3) a Gleason grade of at least 7. Group B-patients had stage D disease following primary therapy, with a declining serum PSA, and/or a stable or improving bone scan or CT scan in response to hormone therapy. All patients were tested against an anergy panel of mumps, measles and candida prior to vaccination and were found to be reactive.
Cytokines and peptides
Recombinant IL-4 and GM-CSF were obtained from Schering-Plough Research Institute (Kenilworth, NJ, USA) and Berlex Inc. (Richmond, CA, USA), respectively. PSA-peptide and Flu-M1 (GILGFVFTL) peptide were synthesized under GLP conditions by Multiple Peptide Systems (San Diego, CA, USA). The synthesized peptides were greater than 97% pure by RP-HPLC and mass spectral analysis. The final vialized dosage form was prepared by the pharmaceutical manufacturing facility at the University of Iowa, College of Pharmacy.
Vaccination
Patients were randomly assigned to treatment protocol 1 or protocol 2 as defined below. Protocol 1: PSA-peptide (100 μg) and GM-CSF (500 μg) mixed in 33% clinical grade DMSO (Edwards Lifesciences, Irvine, CA, USA) in a total volume of 1 ml were administered intradermally as five (0.2 ml aliquot) injections on the volar aspect of the right arm. Patients were immunized concurrently with the HLA-A2 binding Flu-M1 peptide in the same manner on the left arm at the same concentration. Protocol 2: Patients underwent 7–9 l leukapheresis at the Blood Donor Center of the University of Illinois. Monocyte-derived DC were cultured as per the method of Lau et al. [10] in a clinical grade sterile laminar airflow hood. PBMC were obtained by centrifugation over Ficoll-Hypaque (Amersham Biosciences, Uppsala, Sweden). 2×108 PBMC were cultured in T-150 flasks (Corning, Big Flats, NY, USA) containing 15 ml of AIM-V medium (Life Technologies, Grand Island, NY, USA) and incubated at 37°C for 2 h. Nonadherent cells were removed by gentle flushing and media was decanted and replaced with 25 ml of fresh medium containing IL-4 (1,000 U/ml) and GM-CSF (1,000 U/ml) and incubated for 7 days. On day 7, PSA-peptide and Flu-M1 peptide (20 μg/ml of each peptide) were added and incubated further for 16–18 h. On day 8, floating DC were recovered by pipetting, while adherent DC were harvested by gentle mechanical agitation. DC populations were pooled and washed three times with Dulbecco’s PBS (BioWhittaker, Walkersville, MD, USA) and then re-suspended in 50 ml of normal saline and aseptically transferred to an infusion bag (Baxter, Irvine, CA, USA). DC were then irradiated at 2,000 rads and aseptically transferred to freezing bags (Baxter) using a 10 cc syringe with a 19-gauge needle and cryopreserved in a freezing mixture containing 30% plasmalyte, 10% DMSO and 10% human serum albumin and stored in liquid N2. Release criteria for the final DC product included sterile bacterial, fungal and mycoplasma cultures, negative endotoxin per Limulus Amoebacyte lysate assay, viability of at least 90% and greater than 50% CD86, CD80, HLA-DR or CD1a positive cells and less than 10% CD14 positive cells by flow cytometric analysis. At the time of infusion, DC were rapidly thawed at 37°C, again checked for sterility and viability and administered intravenously to patients. Dose of DC was in the range of 0.94 to 2.02×108 cells per vaccine (average 1.499, median 1.555). Patients were vaccinated on weeks 1, 4, and 10 and were monitored for 4 h after every vaccination. One patient (UPIN88) received only a single vaccination due to fungal contamination of the second infusion DC product.
DTH skin testing
Immune responses were monitored by DTH skin testing on weeks 4, 14, 26, and 52 by intradermal injection of 0 (carrier only), 1, 10 and 20 μg of peptide in a total volume of 200 μl (sterile water with 33% DMSO). The FluM-1, positive control peptide, was injected on the volar aspect of the left arm, while the PSA-peptide was injected on the right arm. DTH reactions was measured at 48–72 h following injection and scored as 0 (<10 mm), 1 (10–14 mm), 2 (15–20 mm), or 3 (>20 mm). An induration score of 2 and 3 were considered as a positive reaction. A stringent cut-off value of 14 mm was taken into consideration for measuring true DTH skin responses and to avoid false positives.
A (10×10 mm2) punch biopsy of the skin was obtained from a strongly positive DTH site for each of the seven responding patients who volunteered to be biopsied. The biopsy was divided into two parts. One part was used for isolation of lymphocytes as described below and the second part was fixed in 10% buffered formalin for histology and further studies.
Isolation of lymphocytes from skin biopsies of DTH reaction sites
The dermal tissue was minced into small fragments and co-cultured in RPMI-1640 medium (BioWhittaker, Walkersville, MD, USA) containing 10% human AB serum (complete medium) and IL-2 (100 U/ml) along with CD3/CD28 beads (Dynal, Oslo, Norway) at a concentration of 2.5×105 per well in 1 ml of medium in 48-well plates (Nunc, Naperville, IL, USA). Out-growth of T cells was observed within 3 days. On day 4, 500 μl of the spent medium was aspirated and replenished with fresh medium plus IL-2 and cultured further for a total of 6–8 days, until the culture wells became confluent. Recovered T cells were tested directly (cycle 0) or after 1–4 cycles of in vitro stimulation with irradiated autologous PBMC pulsed with PSA-peptide and low-dose IL-2 (20 U/ml). Each cycle of stimulation was 7±1 days.
Cytokine bead array (CBA) analysis
Cytokines released into the culture supernatant, including IFN-γ, TNF-α, IL-4, IL-6 and IL-10, were measured concurrently by CBA analysis (BD Biosciences, San Diego, CA, USA) as described earlier [18]. Briefly, the antigen-presenting cell line, T2 (ATCC, Manassas, VA, USA) was used as a stimulator and was pulsed with 20 μg/ml of PSA-peptide or control HLA-A2 binding peptide, HIV-RT476-484 or diluent alone (0.4% volume by volume). T2 cells (25,000/well) were cultured with T cells (100,000/well) in complete medium containing 30 U/ml of IL-2 in a total volume of 1 ml per well in 48-well plates. This particular stimulator to responder ratio was found to be optimal for culture in 48-well plates. Cells were incubated at 37°C for 24 h in 5% CO2 atmosphere. Supernatants were harvested and stored in sterile vials at –80°C. At the time of assay, samples were thawed and cytokines were measured using a CBA kit as per the manufacturer’s protocol with a Calibure flow cytometer (Becton Dickinson, Mountain View, CA, USA). Results are represented as net cytokine levels (pg/ml) which was obtained by subtracting nonspecific background responses (T2 cells pulsed with HIV-RT476-484 or diluent).
Immunophenotyping and FACS analysis
DTH-derived T cells were phenotyped for CD4 and CD8 markers. Briefly, 1×105 cells were labeled with CD4-PE and CD8-FITC and gated on side-scatter (SSC) and forward-scatter (FSC) to include lymphocyte population. Propidium iodide (Sigma, St. Louis, MO, USA) was added (1 μg/ml, final concentration) to each sample to exclude dead cells. All incubations were carried out at 4°C for 15 min in phosphate-buffered saline containing 2% normal mouse serum and 0.01% sodium azide (Sigma). The relative log fluorescence of viable cells was measured at 495 nm and the percentage of CD4+ and CD8+ T cells were quantified using the Calibure. MIgG1-FITC and MIgG2a-PE served as isotype controls. All antibodies and isotype controls were purchased from PharMingen (San Diego, CA, USA). In a separate experiment, CD4−CD8+ T cells were sorted and collected aseptically using a Vantage flow cytometer (Becton Dickinson, Mountain View, CA, USA).
Chromium release assay (CRA)
Specific cytolytic activity was analyzed by standard 4-h CRA as previously described [26]. Briefly, 1×106 targets, SW480-PSA+, SW480-PSA− and T2 cells, a peptide transport-deficient B-lymphoblastoid × T-lymphoblastoid cell line were labeled with 100 μCi of Na51CrO3 (Amersham Pharmacia Biotech, Piscataway, NJ, USA). SW480-PSA+ and SW480-PSA− target cells were generated as previously described [17]. Labeled T2 cells were then pulsed with PSA-peptide or HIV-RT476-484 control peptide (Research Genetics, Huntsville, AL, USA) or with diluent. Sorted CD4−CD8+ T cells (effector cells) were plated at indicated concentrations in 96-well ‘V’-bottom plates (Nunc, Naperville, IL, USA) in triplicates and incubated along with 1×103 target cells per well for 4 h. Supernatants recovered from CRA were assayed for gamma emission using a Top-count NXT scintillation counter (Packard, Meriden, CT, USA), and the percent-specific lysis was calculated as previously described [26].
Results
Specific DTH responses induced by vaccination with PSA-peptide
A total of 28 patients were vaccinated with PSA-peptide at three time points, i.e., weeks 1, 4 and 10 and DTH skin testing was performed on weeks 4, 14, 26 and 52. Increasing doses of peptide from 1 to 20 μg elicited increasing levels of DTH induration in responding patients (Fig. 1). FluM-1 peptide, a positive control peptide, showed comparable levels of DTH induration. Injection of carrier only, i.e. 33% DMSO, did not elicit significant induration.
Fig. 1.
Delayed-type hypersensitivity (DTH) skin reactions to PSA-peptide postvaccination. Increasing doses of peptide elicited increasing levels of DTH induration in responding patients (a–g, left to right indicate 1, 10 and 20 μg of peptide injected). FluM-1 peptide, a positive control peptide, showed comparable levels of DTH induration (h, left to right indicate 20, 10 and 1 μg of peptide injected) as that exhibited by PSA-peptide in the same patient (UPIN16). Injection of carrier only, i.e. 33% DMSO (200 μl), did not elicit significant induration (i, bold arrow )
Patients with both locally advanced and metastatic CaP responded to the vaccination (Table 1). Fifty percent of the patients (14 of 28) developed positive DTH responses to PSA-peptide. In 13 of 14 patients with positive DTH reactions, no response was detected at the initial testing on week 4, and specific responses only became evident at week 14 or later. Multiple cycles of peptide vaccination, therefore appeared to augment the recruitment of specific T cells to the DTH sites in the majority of the patients.
Table 1.
Delayed-type hypersensitivity skin reaction to PSA-peptide over various study time points
| Patient | Protocol | Group | DTH induration at weeks | |||
|---|---|---|---|---|---|---|
| 4 | 14 | 26 | 52 | |||
| UPIN13 | 1 | B | 0 | 0 | 1 | 2 |
| UPIN16 | 1 | A | 0 | 2 | a3 | 3 |
| UPIN28 | 1 | B | 0 | 0 | 3 | 2 |
| UPIN40 | 1 | B | 0 | 2 | a2 | 3 |
| UPIN45 | 1 | A | 0 | 1 | a2 | 3 |
| UPIN49 | 1 | B | 0 | 0 | 0 | a2 |
| UPIN50 | 1 | A | 1 | 1 | 3 | 3 |
| UPIN51 | 1 | A | 0 | 2 | 0 | 0 |
| UPIN53 | 2 | A | 0 | 3 | 3 | 0 |
| UPIN55 | 1 | A | 1 | 1 | a2 | 3 |
| UPIN69 | 2 | B | 3 | a3 | 3 | 3 |
| UPIN71 | 2 | A | 1 | a2 | 2 | 3 |
| UPIN81 | 2 | B | 0 | 2 | 0 | 0 |
| UPIN88 | 2 | A | 0 | 0 | 2 | 2 |
| UPIN2 | 2 | B | ND | 1 | 0 | 0 |
| UPIN21 | 2 | A | 0 | 0 | 0 | 1 |
| UPIN26 | 2 | A | 0 | 0 | 0 | 0 |
| UPIN27 | 2 | B | 0 | 1 | 1 | ND |
| UPIN32 | 1 | A | 0 | 1 | 0 | 0 |
| UPIN35 | 2 | B | 0 | 0 | 0 | 0 |
| UPIN37 | 1 | B | 0 | 0 | 0 | 1 |
| UPIN38 | 1 | A | 0 | 1 | 0 | 0 |
| UPIN43 | 2 | A | 0 | 0 | 0 | 0 |
| UPIN67 | 1 | B | 0 | 0 | 0 | 0 |
| UPIN70 | 2 | A | 0 | 0 | 0 | 1 |
| UPIN82 | 2 | B | 0 | 0 | 0 | 1 |
| UPIN85 | 1 | B | 0 | 1 | 0 | 0 |
| UPIN89 | 2 | B | 0 | 1 | 1 | 0 |
DTH reactions obtained at the highest dose of PSA-peptide (20 μg) were measured and scored as 0 (<10 mm), 1 (10–14 mm), 2 (15–20 mm), 3 (>20 mm). Indurations of 2 and 3 were considered positive reactions. ND not done. UPIN2 did not show up for week 4, UPIN27 was lost to f/u after week 26. aDTH skin biopsy was taken at these study time points. Fifty percent of the patients (14 of 28) developed positive DTH responses (upper panel) to PSA-peptide that progressively increased over time in majority (9 of 14) of the responding patients. Group A represents patients with high risk, locally advanced disease and group B represents patients with hormone-sensitive metastatic disease
The size of the DTH induration to PSA-peptide progressively increased over time in 9 of 14 responding patients. However, the size of the DTH induration decreased in four patients (UPIN28, UPIN51, UPIN53, UPIN81) while it was maintained steady in one patient (UPIN69).
The corresponding DTH responses to Flu-M1 peptide, a control peptide over various study time points are shown in Table 2. Thirteen of 14 patients who showed positive DTH responses to PSA-peptide were also positive for Flu-M1 peptide. On the other hand, 9 of 14 patients who showed negative DTH responses to PSA-peptide were positive for Flu-M1 peptide.
Table 2.
Delayed-type hypersensitivity skin reaction to Flu-M1, a control peptide, over various study time points
| Patient | Protocol | Group | DTH induration at weeks | |||
|---|---|---|---|---|---|---|
| 4 | 14 | 26 | 52 | |||
| UPIN13 | 1 | B | 0 | 2 | 2 | 2 |
| UPIN16 | 1 | A | 0 | 2 | 3 | 3 |
| UPIN28 | 1 | B | 0 | 1 | 2 | 0 |
| UPIN40 | 1 | B | 1 | 3 | 3 | 3 |
| UPIN45 | 1 | A | 1 | 2 | 0 | 2 |
| UPIN49 | 1 | B | 0 | 2 | 2 | 1 |
| UPIN50 | 1 | A | 1 | 2 | 3 | 3 |
| UPIN51 | 1 | A | 0 | 1 | 0 | 0 |
| UPIN53 | 2 | A | 2 | 1 | 0 | 1 |
| UPIN55 | 1 | A | 1 | 2 | 3 | 1 |
| UPIN69 | 2 | B | 1 | a2 | 1 | 1 |
| UPIN71 | 2 | A | 3 | 3 | 1 | 3 |
| UPIN81 | 2 | B | 0 | 3 | 3 | 0 |
| UPIN88 | 2 | A | 1 | 0 | 2 | 2 |
| UPIN2 | 2 | B | ND | 3 | 2 | 0 |
| UPIN21 | 2 | A | 0 | 2 | 2 | 1 |
| UPIN26 | 2 | A | 0 | 1 | 2 | 1 |
| UPIN27 | 2 | B | 0 | 2 | 2 | ND |
| UPIN32 | 1 | A | 1 | 0 | 0 | 0 |
| UPIN35 | 2 | B | 2 | 3 | 2 | 1 |
| UPIN37 | 1 | B | 0 | 0 | 0 | 0 |
| UPIN38 | 1 | A | 0 | 1 | 0 | 0 |
| UPIN43 | 2 | A | 1 | 0 | 0 | 0 |
| UPIN67 | 1 | B | 2 | 1 | 0 | 0 |
| UPIN70 | 2 | A | 0 | 0 | 0 | 1 |
| UPIN82 | 2 | B | 0 | 1 | 2 | 1 |
| UPIN85 | 1 | B | 0 | 2 | 0 | 2 |
| UPIN89 | 2 | B | 0 | 3 | 3 | 3 |
DTH reactions obtained at the highest dose of Flu-M1 peptide (20 μg) were measured and scored as 0 (<10 mm), 1 (10–14 mm), 2 (15–20 mm), 3 (>20 mm). Indurations of 2 and 3 were considered positive reactions. ND not done. UPIN2 did not show up for week 4, UPIN27 was lost to f/u after week 26. aDTH skin biopsy was taken at this study time point. Overall, 78.6% of the patients (22 of 28) developed positive DTH responses to Flu-M1 peptide. Upper panel represents patients who developed positive DTH responses to PSA-peptide and lower panel represents patients who showed negative DTH reaction to PSA-peptide (see also Table 1). Group A represents patients with high risk, locally advanced disease and group B represents patients with hormone-sensitive metastatic disease
Phenotype of T cells recovered from DTH sites
Skin biopsy of the DTH site was obtained from seven positive patients and provided the basis for the current report. T cells were recovered from the dermal tissue as previously described in “Materials and methods” using the CD3/CD28 micro-bead technique. Outgrowth of T cells was observed within 3 days, and wells of the culture plates became confluent by 6–8 days. Recovered T cells were phenotyped for CD4 and CD8 markers directly (cycle 0) or after stimulation with irradiated autologous PBMC pulsed with PSA-peptide and supplemented with low-dose IL-2 (20 U/ml) for 1–4 additional cycles.
Variable proportions of CD4+CD8−, CD4−CD8+ and CD4+CD8+ T cells were recovered per flow cytometric analysis. The number of CD4−CD8+ T cells was greater than CD4+CD8− T cells in four of seven patients at cycles 0, i.e., without peptide priming in vitro (Table 3). Following in vitro stimulation with PSA-peptide, the number of CD4−CD8+ T cells became greater than CD4+CD8− T cells in six of seven patients. However, in one patient, UPIN45, CD4+CD8− T cells were higher than CD4−CD8+ T cells. It is of interest to note that a significant percentage of CD4+CD8+ T cells (11–16%) was seen in one patient, UPIN55, and these cells were consistently observed from cycle 0 through cycle 4 of stimulation (Table 3). Attempts were made to isolate CD4+CD8+ T cells for further analysis, but sufficient cell numbers could not be recovered.
Table 3.
Phenotype of DTH-derived T-cells
| Patient | Cycle of culture | % Positive cells | ||
|---|---|---|---|---|
| CD4+CD8− | CD4−CD8+ | CD4+CD8+ | ||
| UPIN16 | 0 | 32 | 62 | 8 |
| 3 | 20 | 75 | 3 | |
| UPIN40 | 0 | 39 | 48 | 10 |
| 2 | 21 | 69 | 6 | |
| UPIN45 | 0 | 78 | 12 | 7 |
| 3 | 56 | 26 | 7 | |
| UPIN49 | 0 | 46 | 27 | 6 |
| 2 | 3 | 80 | 4 | |
| UPIN55 | 0 | 50 | 29 | 13 |
| 2 | 9 | 59 | 16 | |
| 4 | 12 | 65 | 11 | |
| UPIN 69 | 0 | 19 | 53 | 2 |
| UPIN71 | 0 | 39 | 46 | 6 |
| 3 | 14 | 69 | 2 | |
Skin biopsies from seven positive patients were available for in vitro analysis. Lymphocytes were isolated from biopsies by co-culturing the tissue in medium along with CD3/CD28 beads for 6–8 days (cycle 0). Additionally, T-cells were stimulated with PSA-peptide pulsed irradiated autologous PBMC for 1–4 cycles. Each in vitro culture cycle consisted of 7±1 days. 1×105 cells were labeled with CD4-PE and CD8-FITC and gated on SSC and FSC to include lymphocyte population. Percent positive cells were quantified by two-color staining as described in the Methods section
Cytokine profile of T cells derived from DTH sites
T cells recovered from skin biopsies of DTH-positive patients were examined to determine whether specific T cells develop in situ. Sufficient T cells were procured from one patient, UPIN55 for direct analysis at cycle 0, i.e., without peptide priming in vitro. T cells from this patient exhibited specific IFN-γ (174.4 pg/ml) and IL-4 (34.5 pg/ml) cytokines per CBA analysis.
The cytokine profile following in vitro stimulation with PSA-peptide for 1–3 cycles was studied in all the seven patients. As shown in Fig. 2, T cells derived from six out of seven patients showed specific IFN-γ (85–1,032 pg/ml) and TNF-α (23–96 pg/ml) responses. Specific IL-4 (18–486 pg/ml) response was observed in five patients, while IL-10 was detected in only one patient (37 pg/ml). Nonspecific expression of IL-6 was seen in one patient (data not shown).
Fig. 2.
Cytokine profile of T cells derived from DTH sites. Lymphocytes were isolated from skin biopsies by co-culturing the tissue in medium along with CD3/CD28 beads for 6–8 days (cycle 0). Additionally, T cells were stimulated with PSA-peptide for 1–3 cycles. Each in vitro culture cycle consisted of 7±1 days. T cells at 100,000 cells per well were incubated with T2 cells pulsed with PSA-peptide or HIV-RT476-484 or with diluent. Specific cytokines that secreted into the medium were assayed by CBA, and the net cytokine levels (pg/ml) after subtracting backgrounds are shown
A skin biopsy of a DTH reaction to the FluM-1 peptide, a positive control peptide, was also available for one patient, UPIN69. Flu-M1-peptide-specific T cells generated IFN-γ levels (1,090 pg/ml) that were comparable to that produced by PSA-peptide-specific T cells (1,032 pg/ml).
Specific cytotoxicity of T cells derived from DTH sites
CRA were performed to determine whether T cells derived from positive DTH reaction sites also exhibited specific cytotoxicity. Following the culture with CD3/CD28 beads, recovered T cells were further stimulated in vitro with irradiated autologous PBMC pulsed with PSA-peptide for 1–5 cycles. Since flow cytometric analysis revealed mixed phenotypic populations (see Table 3), CD4−CD8+ T cells were FACS sorted in four patients. Sorted CD4−CD8+ T cells showed peptide-specific lysis of PSA-peptide-pulsed T2 cells compared to T2 cells pulsed with irrelevant HLA-A2 binding peptide, HIV-RT476-484, or T2 cells pulsed with diluent. As shown in Fig. 3, T cells induced from patients UPIN45, UPIN55, UPIN49 were strongly cytolytic, while T cells from patient UPIN40 demonstrated modest cytolytic activity.
Fig. 3.
Delayed-type hypersensitivity derived T cells exhibit specific cytolysis of PSA-peptide pulsed target cells. Lymphocytes were isolated from skin biopsies by co-culturing the tissue in medium along with CD3/CD28 beads for 6–8 days (cycle 0). Recovered T cells were stimulated with PSA-peptide for 1–5 cycles. Each in vitro culture cycle consisted of 7±1 days. FACS-sorted CD4−CD8+ T cells (effectors) were plated in triplicates at indicated concentrations and incubated with T2 cells (targets) at 1,000 cells per well and assayed per 4-h CRA. Percent lysis of T2 cells pulsed with PSA-peptide (filled square) was significantly higher than T2 cells pulsed with irrelevant HLA-A0201 binding peptide, HIV-RT476-484 (filled circle), or with diluent (filled triangle)
In patient UPIN55, a comparison of cytolytic activity of prevaccine (Week 0) and postvaccine (Week 26) PBMC-derived T cells was also performed. PBMC samples were stimulated with PSA-peptide in vitro for 5 cycles before proceeding to CRA. Sorted postvaccine CD4−CD8+ T cells (upper panel) showed stronger specific lysis of PSA-peptide-pulsed T2 cells, especially at lower effector to target ratios compared to prevaccine T cells (lower panel). Similarly, postvaccine, but not pre-vaccine CD4−CD8+ T cells, demonstrated specific recognition of PSA+, SW480 target cells of the HLA-A2 phenotype (Fig. 4).
Fig. 4.
Specific recognition of PSA+ and PSA-peptide pulsed targets of HLA-A2 phenotype by PBMC-derived T cells. Pre- and postvaccine PBMC (UPIN55) were stimulated in vitro with PSA-peptide for 1–5 cycles. Each culture cycle consisted of 7±1 days. FACS-sorted CD4−CD8+ T cells (effectors) were plated in triplicates at indicated concentrations and incubated with 51Cr-labeled targets, SW480-PSA+ (filled daimond), SW480-PSA− (open diamond) or T2 cells pulsed with PSA-peptide (filled square), or pulsed with irrelevant HLA-A0201 binding peptide, HIV-RT476-484 (filled circle), or with diluent (filled triangle) at 1,000 cells per well and assayed per 4-h CRA. Postvaccine CD4−CD8+ T cells (upper panel) showed relatively stronger specific lysis of PSA-peptide pulsed T2 cells compared to prevaccine T cells (lower panel). Similarly, postvaccine, but not prevaccine CD4−CD8+ T cells, demonstrated specific recognition of PSA+, SW480 target cells of HLA-A2 phenotype
Discussion
A pilot clinical trial involving a previously identified HLA-A2 restricted epitope of PSA was conducted in 28 patients with high risk, localized (group A) or hormone-sensitive metastatic CaP (group B). Patients were vaccinated on three occasions over a 10-week period by intradermal injection of PSA-peptide plus GM-CSF or by intravenous administration of autologous DC pulsed with peptide. Both methods of vaccination were immunogenic. Strong DTH skin reactions to the PSA-peptide became detectable in 50% (14 of 28) of patients over time. While DTH responses to Flu-M1 peptide, a positive control peptide, was observed in 78.6% (22 of 28) of the patients.
DTH reactions to immunizing peptides have been observed in other peptide-vaccination protocols, particularly melanoma vaccine studies, and are often used as an indicator of antitumor immunity and vaccine efficacy [5, 15, 23]. In the current study, 13 of 14 patients with positive DTH reactions showed no response when initially tested at week 4, and specific responses became evident at week 14 or later. The size of the DTH induration progressively increased over time in the majority (9 of 14) of the responding patients. These results therefore indicate the induction of specific T cell immunity in CaP patients post vaccination.
Although, injection of PSA-peptide for DTH skin testing might immunologically resemble another round of vaccination, low doses (1–20 μg) of naked peptide alone were unlikely to have a confounding impact on the vaccine outcome. This argument is further supported by the observation that, in 4 of 14 responding patients, the size of DTH induration to PSA-peptide either decreased or positive DTH responses that were initially seen totally disappeared by week 52 (Table 1).
Direct recovery of large number of T cells from small clinical specimens, such as punch biopsy of the skin, is often very difficult with traditional culture techniques involving only IL-2. In the current study, we, therefore, cultured dermal tissues in the presence of CD3/CD28 micro-beads and low-dose IL-2 for 6–8 days. T cells were tested directly or following further stimulation with PSA-peptide in vitro. The expanded T cells exhibited specificity to PSA-peptide per CRA and CBA analysis, presumably reflecting corresponding responder T cells that prevail in vivo although the actual frequency of specific T cells are undoubtedly distorted. Similarly, the frequency of PSA-peptide reactive T cells is fairly low in peripheral circulation and therefore required in vitro stimulation of PBMC prior to performing cytolytic assays. Such observations have also been made by Meidenbauer and co-workers in a vaccine study involving CaP patients [12].
Functional analysis of T cells from pretreatment skin biopsy was not possible due to the logistics of the prewritten clinical protocol and also partly due to patient’s reluctance to submit to multiple biopsies. Hence, in one patient, cytolytic activity of pre- and postvaccine PBMC sample were compared. Sorted CD4−CD8+ T cells showed relatively stronger specific lysis of PSA-peptide-pulsed T2 cells compared to prevaccine T cells. Similarly, postvaccine but not prevaccine CD4−CD8+ T cells demonstrated specific recognition of PSA+, SW480 target cells of HLA-A2 phenotype. Such analysis is currently being examined in circulating T cells of several other patients. These results are consistent with data obtained from Gulley et al. [8], who have shown the induction of PSA-specific T cell lines from postvaccine PBMC that lysed PSA+, LNCaP prostate cancer cells but not the PSA−, breast cancer or melanoma cells.
Vaccination with PSA-peptide, therefore, at least in this patient leads to the expansion of specific CD8+ cytolytic T cells. However, direct ex vivo analysis of pre- and postvaccine frozen PBMC for more sensitive tetramer staining together with other markers such as, CD8, CD45RA, CD27 and CCR7, may determine the expansion of long-term memory T cells in peripheral circulation [25].
The strong immunogenic potential of the PSA-peptide is underscored by the finding that T cells obtained from the skin biopsy of a DTH reaction to FluM-1 peptide, a positive control peptide, exhibited comparable levels of peptide-induced cytokine levels as those exhibited by PSA-peptide-specific T cells obtained from the same patient.
The optimal type and route of DC vaccination is still debatable, i.e., immature versus mature DC and subcutaneous or intradermal versus intravenous injection of DC [16, 19]. A recent study has shown that mature DC are essential for optimal peptide loading and are superior in expanding CD8+ primary T cell responses [4]. In our current study, patients were vaccinated with immature monocyte-derived DC, which were cultured with GM-CSF and IL-4 for 7 days followed by pulsing with PSA-peptide and Flu-M1 peptide overnight. This procedure induced PSA-peptide-specific DTH responses in 5 of 14 patients, and specific IFN-γ responses were observed in two of the positive patients tested. This indicates that, probably, maturation of DC was achieved at least in some of the patients. Similarly, in a melanoma study, peptide-pulsed immature DC vaccination was able to induce peptide-specific DTH responses [15].
Labeling studies have shown that intravenously given DC are trapped initially in the lungs before they migrate to the spleen and liver, while DC administered via intradermal or intranodal injection effectively migrate to draining lymph nodes [3, 13]. However, previous vaccine trials administered by intravenous route have shown immunological and clinical responses, and hence we opted for this method [19]. As observed in the current study, 64% of the patients vaccinated by subcutaneous injection as opposed to 36% vaccinated via intravenous DC arm developed specific DTH responses to PSA-peptide. Therefore, we feel that vaccination by subcutaneous method is more effective.
Interestingly, the method of vaccination also appeared to impact the pattern of cytokine responses. DTH-derived T cells from two patients who exhibited only type-1 responses (IFN-γ and TNF-α) were vaccinated by protocol 2. On the other hand, five out of seven patients who exhibited type-1 and type-2 cytokine responses (Fig. 2) were vaccinated by protocol 1. Although, the study population is small, previous studies have shown that (1) route of vaccine administration, (2) method of antigen loading and (3) the type of adjuvant administered influences the polarization and efficacy of antitumor T cell responses [2, 7, 20].
In conclusion, our results indicate that vaccination with soluble peptide or DC-bound peptide elicits strong specific T cell immunity to the PSA-peptide in one-half of patients with locally advanced or hormone-sensitive metastatic CaP. The determinants that distinguish responding versus nonresponding subjects are currently being examined. The impact of successful immunization on disease course will be examined in an expanded phase 2 study.
Acknowledgements
This work was supported by the National Cancer Institute (CA88062), the Department of Army (DAMD17-98-1-8489), the American Cancer Society (IRG 99-224-01), the Illinois Department of Public Health (prostate cancer research fund 4328301), the Cancer Research Institute, New York City, and the General Clinical Research Center of University of Illinois, Chicago, funded by NIH grant M01-RR-13987. The contents in the manuscript are solely the responsibility of the authors and do not necessarily reflect the official views of the respective funding agencies. The authors would like to thank Susan Nishimura and Susan Cascio for their excellent clinical assistance.
Abbreviations
- CaP
Prostate cancer
- CBA
Cytokine bead array
- CRA
Chromium release assay
- DC
Dendritic cells
- DMSO
Dimethyl sulfoxide
- DTH
Delayed-type hypersensitivity
- PSA
Prostate-specific antigen
- PSA
Peptide-PSA146–154 peptide
References
- 1.Barrou B, Benoit G, Ouldkaci M, Cussenot O, Salcedo M, Agrawal S, Massicard S, Bercovici N, Ericson ML, Thiounn N. Vaccination of prostatectomized prostate cancer patients in biochemical relapse, with autologous dendritic cells pulsed with recombinant human PSA. Cancer Immunol Immunother. 2004;53:453–460. doi: 10.1007/s00262-003-0451-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Constant SL, Bottomly K. Induction of Th1 and Th2 CD4+ T cell responses: the alternative approaches. Annu Rev Immunol. 1997;15:297–322. doi: 10.1146/annurev.immunol.15.1.297. [DOI] [PubMed] [Google Scholar]
- 3.De Vries IJ, Krooshoop DJ, Scharenborg NM, Lesterhuis WJ, Diepstra JH, Van Muijen GN, Strijk SP, Ruers TJ, Boerman OC, Oyen WJ, Adema GJ, Punt CJ, Figdor CG. Effective migration of antigen-pulsed dendritic cells to lymph nodes in melanoma patients is determined by their maturation state. Cancer Res. 2003;63:12–17. [PubMed] [Google Scholar]
- 4.Dieckmann D, Schultz ES, Ring B, Chames P, Held G, Hoogenboom HR, Schuler G. Optimizing the exogenous antigen loading of monocyte-derived dendritic cells. Int Immunol. 2005;17:621–635. doi: 10.1093/intimm/dxh243. [DOI] [PubMed] [Google Scholar]
- 5.Disis ML, Schiffman K, Gooley TA, McNeel DG, Rinn K, Knutson KL. Delayed-type hypersensitivity response is a predictor of peripheral blood T-cell immunity after HER-2/neu peptide immunization. Clin Cancer Res. 2000;6:1347–1350. [PubMed] [Google Scholar]
- 6.Eder JP, Kantoff PW, Roper K, Xu GX, Bubley GJ, Boyden J, Gritz L, Mazzara G, Oh WK, Arlen P, Tsang KY, Panicali D, Schlom J, Kufe DW. A phase I trial of a recombinant vaccinia virus expressing prostate-specific antigen in advanced prostate cancer. Clin Cancer Res. 2000;6:1632–1638. [PubMed] [Google Scholar]
- 7.Galea-Lauri J, Wells JW, Darling D, Harrison P, Farzaneh F. Strategies for antigen choice and priming of dendritic cells influence the polarization and efficacy of antitumor T-cell responses in dendritic cell-based cancer vaccination. Cancer Immunol Immunother. 2004;53:963–977. doi: 10.1007/s00262-004-0542-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Gulley JL, Arlen PM, Bastian A, Morin S, Marte J, Beetham P, Tsang KY, Yokokawa J, Hodge JW, Menard C, Camphausen K, Coleman CN, Sullivan F, Steinberg SM, Schlom J, Dahut W. Combining a recombinant cancer vaccine with standard definitive radiotherapy in patients with localized prostate cancer. Clin Cancer Res. 2005;11:3353–3362. doi: 10.1158/1078-0432.CCR-04-2062. [DOI] [PubMed] [Google Scholar]
- 9.Heiser A, Coleman D, Dannull J, Yancey D, Maurice MA, Lallas CD, Dahm P, Niedzwiecki D, Gilboa E, Vieweg J. Autologous dendritic cells transfected with prostate-specific antigen RNA stimulate CTL responses against metastatic prostate tumors. J Clin Invest. 2002;109:409–417. doi: 10.1172/JCI200214364. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Lau R, Wang F, Jeffery G, Marty V, Kuniyoshi J, Bade E, Ryback ME, Weber J. Phase I trial of intravenous peptide-pulsed dendritic cells in patients with metastatic melanoma. J Immunother. 2001;24:66–78. doi: 10.1097/00002371-200101000-00008. [DOI] [PubMed] [Google Scholar]
- 11.Li TS, Beling CG. Isolation and characterization of two specific antigens of human seminal plasma. Fertil Steril. 1973;24:134–144. doi: 10.1016/s0015-0282(16)39496-1. [DOI] [PubMed] [Google Scholar]
- 12.Meidenbauer N, Harris DT, Spitler LE, Whiteside TL. Generation of PSA-reactive effector cells after vaccination with a PSA-based vaccine in patients with prostate cancer. Prostate. 2000;43:88–100. doi: 10.1002/(SICI)1097-0045(20000501)43:2<88::AID-PROS3>3.0.CO;2-G. [DOI] [PubMed] [Google Scholar]
- 13.Morse MA, Coleman RE, Akabani G, Niehaus N, Coleman D, Lyerly HK. Migration of human dendritic cells after injection in patients with metastatic malignancies. Cancer Res. 1999;59:56–58. [PubMed] [Google Scholar]
- 14.Nair SK, Hull S, Coleman D, Gilboa E, Lyerly HK, Morse MA. Induction of carcinoembryonic antigen (CEA)-specific cytotoxic T-lymphocyte responses in vitro using autologous dendritic cells loaded with CEA peptide or CEA RNA in patients with metastatic malignancies expressing CEA. Int J Cancer. 1999;82:121–124. doi: 10.1002/(SICI)1097-0215(19990702)82:1<121::AID-IJC20>3.0.CO;2-X. [DOI] [PubMed] [Google Scholar]
- 15.Nestle FO, Alijagic S, Gilliet M, Sun Y, Grabbe S, Dummer R, Burg G, Schadendorf D. Vaccination of melanoma patients with peptide- or tumor lysate-pulsed dendritic cells. Nat Med. 1998;4:328–332. doi: 10.1038/nm0398-328. [DOI] [PubMed] [Google Scholar]
- 16.Nestle FO, Farkas A, Conrad C. Dendritic-cell-based therapeutic vaccination against cancer. Curr Opin Immunol. 2005;17:163–169. doi: 10.1016/j.coi.2005.02.003. [DOI] [PubMed] [Google Scholar]
- 17.Perambakam S., Xue B-H, Sosman JA, Peace DJ. Induction of Tc2 cells with specificity for prostate-specific antigen from patients with hormone-refractory prostate cancer. Cancer Immunol Immunother. 2002;51:263–270. doi: 10.1007/s00262-002-0281-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Perambakam SM, Srivastava R, Peace DJ. Distinct cytokine patterns exist in peripheral blood mononuclear cell cultures of patients with prostate cancer. Clin Immunol. 2005;117:94–99. doi: 10.1016/j.clim.2005.06.011. [DOI] [PubMed] [Google Scholar]
- 19.Reichardt VL, Brossart P, Kanz L. Dendritic cells in vaccination therapies of human malignant disease. Blood Rev. 2004;18:235–243. doi: 10.1016/j.blre.2003.12.001. [DOI] [PubMed] [Google Scholar]
- 20.Romagnani S. The Th1/Th2 paradigm. Immunol Today. 1997;18:263–266. doi: 10.1016/S0167-5699(97)80019-9. [DOI] [PubMed] [Google Scholar]
- 21.Sanda MG, Smith DC, Charles LG, Hwang C, Pienta KJ, Schlom J, Milenic D, Panicali D, Montie JE. Recombinant vaccinia-PSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer. Urology. 1999;53:260–266. doi: 10.1016/S0090-4295(98)00539-1. [DOI] [PubMed] [Google Scholar]
- 22.Stewart SL, King JB, Thompson TD, Friedman C, Wingo PA. Cancer mortality surveillance—United States, 1990–2000. MMWR Surveill Summ. 2004;53:1–108. [PubMed] [Google Scholar]
- 23.Waanders GA, Rimoldi D, Lienard D, Carrel S, Lejeune F, Dietrich PY, Cerottini JC, Romero P. Melanoma-reactive human cytotoxic T lymphocytes derived from skin biopsies of delayed-type hypersensitivity reactions induced by injection of an autologous melanoma cell line. Clin Cancer Res. 1997;3:685–696. [PubMed] [Google Scholar]
- 24.Watt KW, Lee PJ, M’Timkulu T, Chan WP, Loor R. Human prostate-specific antigen: structural and functional similarity with serine proteases. Proc Natl Acad Sci USA. 1986;83:3166–3170. doi: 10.1073/pnas.83.10.3166. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Walker EB, Haley D, Miller W, Floyd K, Wisner KP, Sanjuan N, Maecker H, Romero P, Hu HM, Alvord WG, Smith JW, II, Fox BA, Urba WJ. Gp100(209-2 M) peptide immunization of human lymphocyte antigen-A2+ stage I-III melanoma patients induces significant increase in antigen-specific effector and long-term memory CD8+ T cells. Clin Cancer Res. 2004;10:668–680. doi: 10.1158/1078-0432.CCR-0095-03. [DOI] [PubMed] [Google Scholar]
- 26.Xue B-H, Zhang Y, Sosman JA, Peace DJ. Induction of human cytotoxic T lymphocytes specific for prostate-specific antigen. Prostate. 1997;30:73–78. doi: 10.1002/(SICI)1097-0045(19970201)30:2<73::AID-PROS1>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]