High-Affinity HLA-A(*)02.01 Peptides from Parathyroid Hormone-Related Protein Generate In Vitro and In Vivo Antitumor CTL Response Without Autoimmune Side Effects

Peptide synthesis

The peptides (PTR-2 and PTR-4) were synthesized using a solid phase automatic peptide synthesizer (model syto; MultiSynTec, Witten, Germany) and the F-moc/diisopropylcarbodiimide/1-hydroxybenzotriazole strategy. They were cleaved from the resins and defracted by treatment with trifluoroacetic acid containing ethandiethiol, water, trisbuthyl silone, and anisole (93/2.5/2/1.5/1). The crude peptides were purified by HPLC using a Vydac C18 column (25 × 1 cm; 10 μm). The products were dissolved in bidistilled water, sterile filtered, and frozen at −70°C at a concentration of 2 mg/ml. Peptide purity was >90% as analyzed by HPLC. The CAP-1 peptide was kindly donated by Dr. J. Schlom (Experimental Oncology Section, National Cancer Institute, National Institutes of Health, Bethesda, MD).

Cell cultures

The LNCaP (HLA-A(*)02.01⁺PTH-rP⁺) prostate carcinoma cell line, the MDA-MB 231 (HLA-A(*)02.01⁺PTH-rP⁺) breast carcinoma cell line, and the SW1463 (HLA-A(*)02.01⁺PTH-rP⁻) colon carcinoma cell line were purchased from the American Type Culture Collection (Manassas, VA). The murine lymphoma cell line EL-4-HHD (HLA-A(*)02.01⁺) has been previously described (34). The mycoplasma-free cultures were maintained in complete medium (RPMI 1640 for LNCaP and EL-4-HHD, and DMEM for SW1463 and MDA-MB 231) (Life Technologies, Grand Island, NY) supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies). The 174CEM-T2 (T2; transport deletion mutant) (35) and CIR-A2 (36) cell lines were provided by Dr. J. Schlom. The T2 and CIR-A2 cells were maintained in IMDM and RPMI 1640 complete medium, respectively.

T2-A2 and HLA up-regulation test

The binding of the PTR-2 and PTR-4 peptides to HLA-A(*)02.01 molecules was evaluated by means of flow cytometry using T2 cells and the method described by Nijman et al. (35). In this assay, increased stability (the accumulation of HLA-A(*)02.01 molecules on the surface of T2 cells as a consequence of peptide binding) is measured in terms of the increased binding of the Ab directed against HLA-A(*)02.01. Briefly, 10⁶ T2 cells in serum-free IMDM were incubated overnight with escalating concentrations (0, 5, 25, and 50 μg/ml) of the PTR peptides in 24-well culture plates at 37°C in an atmosphere containing 5% CO₂. The cells were then washed two times with DPBS and subsequently incubated for 1 h with an anti-HLA-A(*)02.01 (A2.69) specific mAb (A2.69, catalog no. 189HA-1; One Lambda, Canoga Park, CA), using 10 μl of the recommended dilution per 10⁶ cells. UPC-10 (Cappel/Organon Teknika, West Chester, PA) was used as an isotype control. The cells were again washed two times with DPBS, incubated with 100 μl of a 1/100 dilution of FITC-labeled anti-mouse IgG (BD Biosciences, San Jose, CA), and analyzed by means of FACScan (BD Biosciences) and single-color analysis. The cells were kept at 4°C during all of the manipulations unless otherwise stated.

Generation of DC and T cell lines

PBMCs were isolated from heparinized blood taken from two normal HLA-A(*)02.01 volunteers (one male and one female) using a lymphocyte separation medium gradient (Cappel/Organon Teknika) as previously described (37).

The PBMCs (10⁷ cells/ml) for dendritic cell (DC) enrichment were seeded in T75 flasks in a 10-ml volume of complete medium (RPMI 1640) with 10% FBS for 4 h at 37°C in 5% CO₂, after which the nonadherent cells were removed. The adherent cells were then maintained for 7 days in complete medium (RPMI 1640 with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) containing 50 ng/ml GM-CSF and 0.5 ng/ml IL-4 (both purchased from R&D Systems, Minneapolis, MN). The medium containing GM-CSF and IL-4 was replaced every 48 h. After 7 days of culture, the DC phenotype of the cells was investigated by means of flow cytometry direct immunofluorescence. To estimate DC enrichment before CTL stimulation, the cultures were examined for the expression of CD1a, HLA-I, HLA-DR, CD11c, CD80, CD86, and CD83 markers (38, 39, 40).

CTL cultures

The PBMCs for CTL primary cultures were washed two times in DPBS and then resuspended in AIM-V medium (Life Technologies) supplemented with 5% pooled human AB serum (Valley Biomedical, Winchester, VA), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies). The cells (2 × 10⁵) in a 100-μl volume of complete medium were added to each well of a 96-well flat-bottom assay plate (Corning Costar, Cambridge, MA). The irradiated autologous DC were pulsed with each one of the specific PTH-rP peptides (25 μg/ml) and added to the lymphocyte cultures at a final DC:PBMC ratio of 1:5. The cocultures were then incubated for 5 days at 37°C in a humidified atmosphere containing 5% CO₂. The cultures were then fed with human rIL-2 (20 U/ml; Cetus, Emmerville, U.K.) for 10 days, with the IL-2-containing medium being replenished every 3 days. Incubation for 5 days with peptide-pulsed DC plus 10 days with IL-2 constitutes one in vitro stimulation (IVS) cycle. The T cell cultures were restimulated with PTH-rP peptide (the same as that used for the first stimulation)-pulsed autologous DC. The irradiated (5000 rad) autologous DC were added in a 100-μl volume of complete medium (AIM-V) and used as APCs. After the second IVS, the cells were pooled and then seeded in 24-multiwell plates at a final concentration of 10⁵ cells/ml in a volume of 2 ml/well. The T cells were subsequently restimulated as described above using the same DC:T cell ratio.

Cytotoxic assays

Cytotoxic activity of CTLs derived from HHD mice against mouse target cells was tested in 6-h ⁵¹Cr release assays. Human CTL activity against human target cells could not be revealed in 6–8 h; 18-h ¹¹¹In release assays were performed as suggested by other authors (6). No explanation currently exists for this observation. It could be due to the partial ability of human tumor cell to escape the cytolytic effectors, to an intrinsic resistance to the cytolytic mediator released by human CTLs, or possibly to different mechanisms of killing used by human and mouse CTLs.

The human target cells were labeled with 50 μCi of [¹¹¹In]oxyquinoline (Medi Physics, Arlington, IL) for 15 min at room temperature, whereas the mouse EL-4-HHD target cells were labeled with 50 μCi of Na₂⁵¹CrO₄ (Amersham, Little Chalfont, U.K.) for 60 min at room temperature. The target cells (0.5 × 10⁴) in 100 μl of complete medium (see below) were added to each of the wells in 96-well flat-bottom assay plates (Corning Costar). The labeled targets were incubated at 37°C in 5% CO₂ before the addition of effector cells. The T cells were then suspended in 100 μl of AIM-V medium and added to the target cells. The plates were incubated at 37°C for 6 or 18 h, and the supernatants were harvested for gamma counting with harvester frames (Skatron, Sterling, VA). The determinations were made in triplicate and SDs were calculated. All of the experiments were repeated at least three times. Specific lysis was calculated as follows: percentage of specific lysis = (observed release (cpm) − spontaneous release (cpm))/(total release (cpm) − spontaneous release (cpm)) × 100.

Spontaneous release was determined from the wells to which 100 μl of complete medium were added instead of effector cells. Total releasable radioactivity was obtained after treating the target with 2.5% Triton X-100.

Blocking experiments

For the HLA blocking experiments, UPC-10 (Cappel/Organon Teknika) control mAb or anti-HLA-A(*)02.01 (A2.69, catalog no.189HA-1; One Lambda) or HLA class I A, B, C (W6/32; Scra, Sussex, U.K.), were incubated for 1 h before the cytotoxic assay.

Flow cytometry

The flow cytometric analysis procedure has been previously described (41). The cells were first incubated with primary mouse anti-human mAbs against CD3, CD4, CD8, CD56, CD19, CD1a, HLA-I, HLA-DR, CD11c, CD80, CD86, and CD83 (all purchased from BD Biosciences), class I HLA (W6/32; Scra), and MOPC-21 (Cappel/Organon Teknika). Subsequently, they were stained with a FITC-conjugated goat anti-mouse Ig (1/100 dilution; Kirkegaard & Perry Laboratories, Gaithersburg, MD) and, finally, the samples containing 1 × 10⁶ cells in 1 ml of Ca²⁺/Mg²⁺-free DPBS were analyzed using a BD Biosciences FACScan equipped with a blue laser with an excitation level of 15 nW at 488 nm. The data gathered from 10,000 live cells were used to evaluate the results. The procedure for dual-color flow cytometry analysis closely resembles the single-color procedure with the following exceptions: the mAbs were anti-CD3 and anti-CD4 FITC conjugate, anti-CD56 and anti-CD8 PE conjugate, anti-IgG1 FITC conjugate, and anti-IgG3 PE conjugate (BD Biosciences); the cells were simultaneously stained for 1 h, after which they were washed three times, resuspended as above, and immediately analyzed using a BD Biosciences FACScan equipped with a blue laser with an excitation of level of 15 nW at 488 nm and the Lysis II program.

HLA typing

The PBMC HLA of the two donors (98/003263 and 98/0003668) was phenotyped by the Blood Bank of the Azienda Ospedaliera Senese (Policlinico “Le Scotte,” Siena, Italy) using a standard Ab-dependent microcytotoxicity assay and a defined panel of anti-HLA antisera for HLA class I determinations. The polymerase chain reaction was used to type HLA class II.

Animals

The HHD mice have been previously described (33). They are α₂m^−/− and D^b−/− and express a HLA-A(*)02.01 monochain consisting of the α₁ and α₂ domains of HLA-A(*)02.01, and the α₃ domain of D^b linked by its N terminus to the C terminus of human α₂m by a 15-aa peptide. The mice were housed in a temperature-controlled, light-cycled room. All of the in vivo experiments were performed in accordance with local ethical guidelines.

Vaccination of HHD mice

The HHD mice were injected at the base of the tail with 100 μg of the epitope of interest emulsified in IFA in the presence of 140 μg of the I-A^b-restricted hepatitis B virus core Ag-derived Th epitope (128–140; sequence TPPAYRPPNAPIL). The injection was repeated for an immunological boost after 11 days. Two weeks after peptide reboosting (25 days since the first injection), the HHD animals were sacrificed for histology study and in vitro spleen cell restimulation.

In vivo study of HHD mice injected with PTH-rP peptides

Two series of three mice were immunized with each one of the two PTH-rP peptides or a control peptide (hTRT). Two series of three animals for each peptide were used at different times. After 11 days, the mice were reinjected with cognate peptide for reboost. On days 11 and 25, the sera of the immunized HHD mice were collected from the retroorbital sinus for serum Ca ion level evaluation.

Human synthetic thyreocalcitonin (0.1 mg in 1 ml of 0.9% saline solution, catalog no. T3535; Sigma-Aldrich) injected s.c. in the dorsal neck was used as a positive control in a group of three mice, because it is capable of inducing hypocalcemia (0.64 ± 0.645 mmol/L) after 6 h; the normal value was 1.29 ± 25 mmol/L.

The mice were sacrificed 2 wk after the boost, and 4 μm-thick paraffin sections were cut from the sampled tissues and stained with H&E-safranin (Merck, Darmstadt, Germany). Spleen cells were collected and restimulated in vitro with 10 μM cognate peptide.

Generation of a PTH-rP plasmid/influenza virosomes

The PTH-rP gene was amplified from the prostate carcinoma DU-145 cell line by RT-PCR (42), starting from the specific mRNA using the sense primer 5′-TTGGATCCATGCAGCGGAGACTGGTT-3′ and the antisense primer 5′-CCGAATTCTCAATGCCTCCGTGAATCGA-3′, and then cloned in the BamHI-EcoRI sites of the pcDNA3 expression vector (Invitrogen, San Diego, CA) to obtain the recombinant plasmid GC90. The construct was grown in DH5 cells. Plasmid DNA was purified using the Qiagen Endo Free plasmid kit (Qiagen, Valencia, CA) as described by the manufacturer. The influenza virosomes were prepared as described elsewhere (43, 44). The nonencapsulated plasmids were separated by 0.1 gel filtration on a High Load Superdex 200 column (Amersham Pharmacia Biotech, Piscataway, NJ). The column was equilibrated with sterile PBS. The void volume fractions containing the virosomes with encapsulated plasmids were eluted with PBS and collected. For cell transfection, ∼10⁵ target EL-4-HHD cells were grown in six-well microplates at 37°C and infected with 0.3 μg of DNA virosomes or transfected with 1 μg of plasmid DNA using the Effectene Transfection reagent (Qiagen) as described by the manufacturer. After 2 days, PTH-rP Ag expression was analyzed by evaluating the presence of the specific mRNA by means of RT-PCR and immunofluorescence. Briefly, the cells were washed twice with PBS, fixed with cold methanol/acetone, and treated with a rabbit anti-PTH-rP serum (Calbiochem, La Jolla, CA) followed by FITC-conjugated goat anti-rabbit IgG (1/100). The coverslips were mounted on slides and examined using a Leitz Diaplan microscope.

Statistical considerations

The between-mean differences were statistically analyzed using StatView statistical software (Abacus Concepts, Berkeley, CA). The results were expressed as the mean value ± SD of four determinations made in three different experiments, and the differences were determined using the two-tailed Student t test for paired samples. A value of p < 0.05 was considered statistically significant.

Peptide selection and HLA-A(*)02.01 binding

To test the immunogenicity of PTH-rP protein and the possible tolerance of the human T cell repertoire specific for it, we screened the PTH-rP molecule with the “Parker” algorithm (Bioinformatics and Molecular Analysis Section, Computational Bioscience and Engineering Lab Center for Information Technology) (45) to predict peptides having high theoretical HLA-A(*)02.01 binding motifs. The functional binding of the two peptides to HLA-A(*)02.01 molecules was tested in vitro by means of the T2 test. The PTH-rP peptides with similar or overlapping amino acid homology to the analog sequences of PTH were cut out of the screening. The peptide sequences included in the first 36 aa (propeptide) were ignored, although they were theoretically predicted with higher affinity for the HLA-A(*)02.01 molecule. We considered the peptide sequences included in the mature PTH-rP molecules and responsible for its biological activity (amino acids 37–177) (30). The two representative peptides selected for this study were designated PTR-2 (p59–68, FLHH LIAEIH) and PTR-4 (p165–173, TSTTSLELD). PTR-2 was found having the highest HLA-A(*)02.01 binding affinity, whereas PTR-4, although having noncanonic anchor motifs and an intermediate-low binding affinity, has been previously shown to be very immunogenic (46). Both PTR-2 and PTR-4 showed 100% amino acid divergence from the homolog sequence in the PTH (Table I⇓).

The results presented in the Table I⇑ also suggest that PTR-2 and PTR-4 are capable of binding HLA-A(*)02.01 molecules. A CEA-derived peptide (CAP-1), YLSGANLNL, was used as a positive control (Table I⇑).

Generation of PTH-rP peptide-specific CTL lines

To evaluate the PTH-rP-specific T cell repertoire availability in humans, CTL lines specific for each PTH-rP peptide were generated from PBMC of two healthy HLA-A(*)02.01⁺ donors by means of cyclic IVS with peptide-pulsed autologous DC and IL-2. Two different CTL lines were generated from each donor and designated as T-Donor-1 or T-Donor-2 followed by the specific peptide used for CTL stimulation (T-Donor-1-PTR-2, T-Donor-2-PTR-2, etc.). All of the lymphocyte cultures were investigated for immunophenotype expression and cytolytic activity after at least six stimulation cycles. Direct double-stain flow cytometry immunofluorescence showed that the CTL lines expressed a CD3-positive phenotype (90%) with different percentages of CD4⁺CD8⁻ (range, 22–40%) and CD4⁻CD8⁺ (range, 60–90%). The specificity of CTL lines was evaluated against PTH-rP peptide-pulsed CIR-A2 (HLA-A(*)02.01⁺) target cells. Cytotoxic assays demonstrated the peptide specificity of the CTL lines, which were able to kill target cells only when pulsed with the cognate peptide. The CTL lines were unable to kill the unpulsed target cells or those pulsed with the control CAP-1 peptide (Fig. 1⇓). These results demonstrate that a PTH-rP peptide-specific human CTL repertoire is available and mobilizable.

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FIGURE 1.

Ability of human PTH-rP peptide-specific CTL lines generated from donor 1 (circles) and donor 2 (squares) to lyse CIR-A2 target cells previously pulsed with the cognate peptide. T cell lines generated using PTR-2 (A) or PTR-4 (B) peptides were tested for their ability to lyse ¹¹¹In-labeled CIR-A2 pulsed with PTR-2 or PTR-4 peptides (• and ▪ with bold continuous lines, respectively), the control CEA peptide (CAP-1; • and ▪ with dashed lines, respectively), or no peptide (○ and □ with dashed lines, respectively). An 18-h assay was performed with the peptides being used at a concentration of 25 μg/ml. The results are expressed as the percentage of specific lysis at different E:T ratios. ¹¹¹In release in the presence of culture medium without effectors was <15%. Mean values from triplicate determinations in individual experiments (performed at the 6th, 8th, 10th, and 12th CTL line IVS), with SDs. There was a significant difference (p < 0.05, two-tailed t test) of values when results from cognate peptide-pulsed CIR-A2 target cells and controls were compared (CIR-A2 unpulsed or pulsed with the control peptide).

To investigate the functional avidity of these lymphocytes, we investigated their lytic activity against CIR-A2 target cells pulsed with different concentrations of the cognate peptide at the fixed E:T ratio of 25:1. These experiments showed that, in comparison with PTR-2, the PTR-4-specific CTL lines induced 25% of killing in target cells pulsed with lower concentrations of cognate peptide (∼10 vs 25 μg/ml) (Fig. 2⇓). No differences were instead observed against unpulsed or control peptide-pulsed target cells.

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FIGURE 2.

Ability of human PTH-rP peptide-specific CTL lines generated from donor 1 (circles) and donor 2 (squares) to lyse CIR-A2 target cells previously pulsed with different concentrations of the cognate peptide. T cell lines generated using PTR-2 (A) or PTR-4 (B) peptides were tested for their ability to lyse ¹¹¹In-labeled CIR-A2 pulsed with PTR-2 or PTR-4 peptides (• and ▪ with bold continuous lines, respectively), or the control CEA peptide (CAP-1; ○ and □ with dashed lines, respectively). An 18-h assay was performed with the peptides being used at the concentration of 0, 5, 10, 20, 25, and 50 μg/ml, at the E:T ratio of 25:1. ¹¹¹In release in the presence of culture medium without effectors was <15%. Mean values from triplicate determinations in individual experiments (performed at the 8th, 10th, and 15th CTL line IVS), with SDs. There was a significant difference (p < 0.05, two-tailed t test) of values when results from cognate peptide-pulsed CIR-A2 target cells and CIR-A2 pulsed with the control CAP-1 peptide were compared.

Antitumor activity of the PTH-rP peptide-specific CTL lines

To test the antitumor activity of CTL lines specific for PTR-2 and PTR-4, we chose the MDA-MB 231 (breast) and LNCaP (prostate) HLA-A(*)02.01⁺PTH-rP⁺ tumor cell lines as targets. Cytotoxic assays showed that the PTH-rP peptide-specific CTL lines were able to kill both MDA-MB 231 and LNCaP cells (Fig. 3⇓). In contrast, no lysis was observed against the HLA-A(*)02.01⁺PTH-rP⁻ cell line SW-1463 (colon carcinoma). Furthermore, cytotoxic assays also showed that the CTL-mediated killing of MDA-MB 231 and LNCaP target cells was HLA-A(*)02.01 restricted insofar as the addition of an Ab directed against HLA-A(*)02.01 molecules (A2.69) abrogated the cytotoxic effect (Fig. 3⇓); similar results were also obtained using an anti-class I HLA (Fig. 3⇓) mAb (W6/32) (data not shown). Conversely, the addition of an A2.69 isotype-negative control mAb did not interfere with the process of tumor cell killing (Fig. 3⇓).

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FIGURE 3.

Ability of human PTH-rP-specific CTL lines to lyse in vitro HLA-A(*)02.01⁺ tumor cells capable of producing PTH-rP. T cell lines generated using PTR-2 (A and C) or PTR-4 (B and D) peptides were tested for their ability to lyse ¹¹¹In-labeled prostate (LNCaP; A and B) and breast carcinoma (MDA-MB-231; C and D) target cells. HLA-A(*)02.01⁺ colon carcinoma SW-1463 cells incapable of producing PTH-rP were used as a negative control target. The cytotoxic activity of the T cell lines against LNCaP and MDA-MB-231 cells in fresh medium is represented by • and a bold line for the donor 1 and by ▪ and a bold line for the donor 2. The cytotoxic activity of the T cell lines against LNCaP and MDA-MB-231 cells in the presence of the UPC-10 mAb is represented by • and a continuous line for the donor 1 and by ▪ and a continuous line for the donor 2. The cytotoxic activity of the T cell lines against LNCaP and MDA-MB-231 cells in the presence of mouse anti-human HLA-A(*)02.01 mAb is represented by ○ and a dashed line for the donor 1 and by □ and a dashed line for the donor 2. The cytotoxic activity of the T cell lines against SW-1463 target cells is represented by ▴ and ▪ and a bold dashed line for the donor 1 and ▵ and □ and a bold dashed line for the donor 2. The target cells (1 × 10⁶) were labeled with ¹¹¹In, incubated for 1 h in the presence of medium containing no Ab, negative control Ab UPC-10 (10 μg/ml), or anti-HLA-A2,69 (1/100 dilution), and then used as targets in 18-h cytotoxic assays. The results are from single experiments with triplicate determinations, expressed as the percentage of specific release at different E:T ratios. This experiment was performed three times with similar results (performed at the 8th, 10th, and 12th CTL line IVS). There were significant differences (p < 0.05, two-tailed t test) between the values obtained from the same T cell line against LNCaP plus UPC-10 vs LNCaP plus A2.69; MDA-MB-231 plus UPC-10 vs MDA-MB-231 plus A2.69; LNCaP plus UPC-10 vs SW1463; MDA-MB-231 plus UPC-10 vs SW1463. No significant differences were found when the same T cell line was tested against LNCaP in fresh medium vs LNCaP plus UPC-10 or MDA-MB-231 in fresh medium vs MDA-MB-231 plus UPC-10. The spontaneous release in these assays in the presence or absence of mAbs without effectors was always <15%.

The CTL immunophenotype and CTL activity against these target cells were investigated at the 6th, 8th, 10th, and 12th IVS, the results being very similar in terms of phenotype stability and PTH-rP/PTH-rP peptide-specific cytotoxic activity (data not shown).

Cold competition assay

Cold competition assays were performed to check Ag involvement and haplotype restriction of target cell killing. The CTL cytotoxic assays were performed using T2-A2 cells (unpulsed or pulsed with different peptides including PTR-2 and -4) as cold competitors and, respectively, ¹¹¹In-loaded MDA-MB 231 and LNCaP cells, respectively, as labeled CTL targets. The results showed efficient competition (measured as a reduction in ¹¹¹In release) when cognate peptide-pulsed T2 cells were used as cold competitors of MB-MDA-231 and LNCaP target cells. The CTL-mediated killing of breast and prostate carcinoma cells was, in fact, mainly abrogated by the addition of cold competitors pulsed with the specific peptide used to generate the CTL line. The addition of cold T2 cells, unpulsed or pulsed with a control HLA-A(*)02.01-binding CAP-1 peptide, did not interfere with the CTL-mediated killing of cancer cells (Fig. 4⇓). Fig. 4⇓ also shows the ability of CTL lines to recognize and kill T2 cells exposed for 24 h to the specific peptide used for the CTL line generation. The addition of cold CEA⁻HLA-A2.1⁻ K562 cells did not interfere with the CTL-mediated target cell killing, showing no competition (data not shown).

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FIGURE 4.

A PTH-rP peptide-specific cytotoxic T cell cold competition assay. Cognate peptide-pulsed T2 target cells used as cold competitors abrogate the CTL-mediated lysis of prostate and breast carcinoma cells. Shown are the results of a competition assay using an 18 ¹¹¹In release assay expressed as the percentage of specific lysis at an E:T ratio of 25:1. CTL lines from donor 1 (open bar) and donor 2 (filled bar) were tested against labeled LNCaP (A and B) or MDA-MB-231 (C and D) in presence of unlabeled T2 used at a ratio of 1:10. The T2 cells were incubated with or without the cognate PTH-rP peptide (25 μg/ml) in serum-free medium for 24 h at 37°C before being added to the assay. There was a significant difference (p < 0.05, two-tailed t test) between the value from LNCaP plus cold T2 pulsed with the specific peptide and the data derived from labeled LNCaP plus unlabeled T2 not pulsed with peptide or pulsed with the CEA (CAP-1) control peptide. There was a significant difference (p < 0.05, two-tailed t test) between the value for MDA-MB-231 plus cold T2 pulsed with the specific peptide and the data derived from labeled MDA-MB-231 plus unlabeled T2 not pulsed with peptide or pulsed with the CEA (CAP-1) control peptide. Each experiment was repeated three times with similar results (performed at the 8th, 12th, and 15th CTL line IVS). Numbers below the graphs represent the following: 1, ¹¹¹In-labeled T2-A2 cells with no cold competitors; 2, ¹¹¹In-labeled T2-A2 cells pulsed with CAP-1 targets with no cold competitors; 3, ¹¹¹In-labeled T2-A2 targets pulsed cognate-PTH-rP peptide (described in the figures) with no cold competitors; 4, ¹¹¹In-labeled LNCaP (Fig. 3⇑, A and B) or MDA-MB-231 (Fig. 3⇑, C and D) targets with no cold competitors; 5, ¹¹¹In-labeled LNCaP (Fig. 3⇑, A and B) or MDA-MB-231 (Fig. 3⇑, C and D) with T2-A2 as cold competitors; 6, ¹¹¹In-labeled LNCaP (Fig. 3⇑, A and B) or MDA-MB-231 (Fig. 3⇑, C and D) with T2-A2 pulsed with CAP-1 as cold competitors; 7, ¹¹¹In-labeled LNCaP (Fig. 3⇑, A and B) or MDA-MB-231 (Fig. 3⇑, C and D) targets with T2-A2 pulsed with the cognate PTH-rP peptide (described in the figures) as cold competitors.

Target cell PTH-rP production and cell membrane HLA-A(*)02.01 expression were evaluated by immunoradiometric assays (IRMA) (47) and indirect immune fluorescence flow cytometry, respectively (Table II⇓).

Taken together, these results demonstrate that killing of HLA-A(*)02.01⁺PTH-rP⁺ tumor lines was a peptide-mediated and HLA-A(*)02.01-restricted phenomenon.

Ag-specific CTL in HHD transgenic mice vaccination with PTH-rP peptides

To evaluate the immunogenic potential of PTH-rP peptides in vivo, HLA-A(*)02.01 transgenic mice (HHD) mice were injected with PTR-2, PTR-4 peptide, and an irrelevant control peptide. PTR-2 and PTR-4 showed 100 and 60% amino acid homology, respectively, with the respective mouse peptide sequences. Twenty-five days after after the first peptide injection, the spleen cells were isolated and restimulated in vitro with the cognate peptide for 6 days. The spleen cell cultures were then tested against autologous EL4-HHD target cells pulsed with the cognate peptide or expressing PTH-rP after infection with PTH-rP gene plasmids included in influenza virosomes (GC90V).

The construct GC90V used in this study was formerly generated and characterized in our laboratory (44).

The expression of PTH-rP in the GC90V-infected EL4-HHD target cells was evaluated by means of IRMA, RT-PCR detection of specific mRNA, and immunofluorescence using a rabbit anti-PTH-rP serum (Table II⇑).

Significant PTH-rP and PTH-rP peptide-specific cytotoxic activity was observed only for the spleen cells derived from mice immunized with PTR-2 or PTR-4. These T cells were able to kill EL4-HHD pulsed with the cognate peptide (PTR-2 and PTR-4, respectively) (data not shown), and the same cells producing PTH-rP after infection with the GC90V (Fig. 5⇓). Spleen cells derived from mice immunized with PTR-2 and -4 could not kill the same target cells pulsed with the control CAP-1 peptide or pulsed with the PTR peptide not used for mouse immunization (data not shown). Target cell lysis was class I MHC restricted, because CTL activity was abrogated when the anti-HLA-A(*)02.01 mAb was added to the cytotoxic assays (Fig. 5⇓), whereas the addition of a negative isotype control mAb (UPC-10) did not affect the killing (data not shown). No cytotoxic activity was detected against parental uninfected EL4-HHD cells (data not shown) or those infected with the virosome including the plasmid backbone pcDNA3 (Fig. 5⇓). The spleen cells derived from HHD mice immunized with the control peptide, or not injected at all, did not give rise to any PTH-rP-specific CTL activity; they had slight lytic activity against the target cells at a high E:T ratio, but it was not specific for PTH-rP or class I MHC restricted (Fig. 5⇓), thus suggesting the presence of a residual NK activity. Taken together, these results suggest that the vaccination of HHD mice with PTR-2 and PTR-4 peptides generates a peptide-specific CTL response capable of recognizing naturally processed peptides on tumor cells expressing PTH-rP.

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FIGURE 5.

PTH-rP specific cytotoxic activity of spleen cells derived from HHD mice immunized with the PTH-rP peptides. Cultured spleen cells derived from HHD mice immunized with PTH-rP peptides recognize and kill HLA-A(*)02.01⁺ EL-4-HHD target cells expressing PTH-rP. PTH-rP-specific cytotoxic activity of mouse spleens pooled from different mouse groups was tested against EL-4-HHD and EL-4-HHD target cells infected with the virosome including the PTH-rP gene plasmids (GC90V). Shown are the cytotoxic effects of the spleen cells derived from mice immunized with control peptide (A), PTR-2 peptide (B), or PTR-4 peptide (C) against 1) EL-4-HHD target cells infected with GC90V (• and bold lines); 2) EL-4-HHD target cells infected with the GC90V and exposed to the CTL in the presence of anti-A2.69 mAb (□ and dashed lines); 3) EL-4-HHD infected with influenza virosomes including the plasmid backbone (pcDNA3; ○ and dashed lines). The addition of a negative isotype control mAb (UPC-10) did not affect the killing of EL-4-HHD target cells infected with the GC90V mediated by spleen cells from mice receiving PTR-1 and PTR-2 peptides (data not shown). The data are from three different experiments with similar results. The differences between EL-4-HHD target cells infected with the virosome including the PTH-rP gene plasmids (GC90V) and both EL-4-HHD target cells infected with the GC90V and exposed to the CTL in the presence of anti-A2.69 mAb and EL-4-HHD infected with influenza virosomes including the plasmid backbone (pcDNA3) were statistically significant only for the groups B and C (p < 0.05).

In vivo study of HHD transgenic mice after vaccination with PTH-rP peptides

To evaluate the tissue-specific toxicity and autoimmunity induced by PTH-rP peptide vaccination, a postmortem histology study of tissues chosen because of their reported physiologic expression of PTH or low levels of PTH-rP was performed 25 days after the first peptide injection. At the end of the vaccination cycle, 4-μm-thick paraffin sections taken from the parathyroid, skin, derma, and bone tissues of PTH-rP peptide injected HHD mice were collected and stained with H&E-safranin. The tissues were chosen because of their potential expression of PTH-rP proteins in amounts that are theoretically detectable in vivo by means of a secondary PTH-rP-directed T cell response. Our study failed to demonstrate any PTH-rP mRNA expression in these tissues, in the control as well as in the vaccinated group of mice, probably because of the extremely low production of this protein in normal conditions (data not shown).

Histology showed the absence of pathological microscopic lymphocyte infiltration and no abnormal inflammatory tables of stained tissues including the parathyroids (data not shown).

Furthermore, to exclude the possibility that the PTH-rP-directed immune response may cross-react with PTH-expressing cells in the parathyroids or in some way affect the osteoclast activity in the bone tissue, we investigated the effects of PTH-rP peptide vaccination on the Ca²⁺ turnover of HHD mice. To this end, serum Ca²⁺ ion levels were evaluated during treatment in blood samples collected 11 and 25 days after the first PTH-rP peptide injection. To exclude a delayed effect, blood samples were also drawn 52 days after the first peptide administration in a different set of animals receiving the same above-described treatments. Control blood samples were collected from HHD mice not injected or injected with an irrelevant peptide.

The results demonstrate absence of serum Ca²⁺ ion fluctuations in any of the transgenic animals, thus indicating that the vaccination with PTR peptides elicits a PTH-rP-specific CTL response without affecting parathyroid functions or bone osteoclast activity (Table III⇓), and without inducing direct cell-mediated bone tissue damage. Taken together, these results suggest that the injection of PTH-rP peptides in HHD transgenic mice generates a PTH-rP-specific response in vivo without affecting normal tissues.