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* Division of Medical Oncology,
Institut National de la Santé et de la Recherche Médicale Unité d’Immunité Cellulaire Antivirale, Institut Pasteur, Paris, France; and
Blood Bank and
Department of Molecular Biology, Siena University School of Medicine, Siena, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of these sequences have been described for the most common human HLA isotypes. CTL epitope peptides with HLA-A(*)02.01 binding consensus motifs, which are capable of generating Ag-specific CTL lines with antitumor activity, have been derived from the most common tumor-associated Ags (TAA)3 (5, 6, 7, 8, 9, 10, 11, 12).
In humans, the large majority of TAA investigated as targets of anticancer vaccine therapy trials are nonmutated self proteins expressed during development (carcinoembryonic Ag (CEA)) and differentiation (tyrosinase, gp100, TRP-1 and -2), or self proteins overexpressed in tumors, such as HER-2/neu, p53, and hTRT (13, 14). All these trials are based on the use of dominant epitopes with a high affinity for HLA, derived from self protein expressed in tumors and in normal tissues. Despite some positive results, increasing evidence indicates that the lack of clear efficacy is probably due to tolerance to self-Ags that concerns dominant determinants for self Ags (15, 16, 17, 18).
Experimental evidence demonstrates that the survival and growth of
tumor cells in bone tissue is promoted by their interaction with the
normal cells involved in bone turnover, and particularly by the
abnormal activation of osteoclasts, which makes the bone
microenvironment favorable for tumor cell implantation and growth
(19, 20, 21, 22). A protein produced by tumor cells, designated as
parathyroid hormone-related protein (PTH-rP), is mainly responsible for
osteoclast cell activation (23). This is a 175- to 177-aa
protein that is mainly produced by tumor cells in the bone
microenvironment under promotion by TGF-
and other cytokines, whose
levels are normally very high in the bone matrix (24, 25).
PTH-rP is expressed in 90% of primary prostate and lung spindle cell
carcinomas and 50% of primary breast cancers (26, 27, 28, 29). It
is highly expressed in fetal tissues, where it is involved in bone
tissue differentiation (28). Low levels of PTH-rP have
been sporadically detected in keratinocytes, uterus, and mammary glands
during lactation (29). PTH-rP has functional and amino
acid analogies with the parathyroid hormone (PTH) and is capable of
acting on the same cell membrane receptor(s) (29, 30).
As a result of these PTH mimetic actions, when PTH-rP is produced in large amounts by tumor cells, it can be responsible for the hypercalcemia of malignancy syndrome (30, 31, 32). Because PTH-rP plays a critical role in the occurrence and development of bone metastases, it could be investigated as a biological target for the active TAA-specific immunotherapy of epithelial malignancy bone metastases.
We screened PTH-rP peptides having amino acid sequences with HLA-A(*)02.01 binding consensus motifs and not overlapping the analog peptide sequences of native human PTH.
Two PTH-rP peptides, designated PTR-2 and PTR-4, were chosen for this
study and synthesized in the laboratory. The aim of this study was to
investigate the ability of PTH-rP peptides to generate peptide-specific
CTL lines capable of antitumor activity from
HLA-A(*)02.01+ PBMC. Furthermore, the
immunological activity as well as the autoimmune and toxic potential of
PTH-rP peptides were evaluated in a transgenic HHD mouse model
engineered to express human 
1 and
2 HLA-A(*)02.01 domains (33). The
aim of our study was to demonstrate the possibility of raising a
CTL-mediated immune response to PTH-rP protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 x 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 x 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 x 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)) x 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 x 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 2m-/- and
Db-/- and express a HLA-A(*)02.01 monochain consisting of
the 1 and 2 domains
of HLA-A(*)02.01, and the 3 domain of
Db linked by its N terminus to the C terminus of human
2m 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-Ab-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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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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10 vs 25
µg/ml) (Fig. 2). No differences were
instead observed against unpulsed or control peptide-pulsed
target cells.
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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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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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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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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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We studied the capacity of two PTH-rP-derived peptides that bind HLA-A(*)02.01 molecules to elicit a strong antitumor response. The vaccination of HLA-A(*)02.01 transgenic mice with the PTH-rP peptides also showed their in vivo ability to generate an effective PTH-rP-specific CTL response with no parallel signs of cell-mediated normal tissue degeneration. We chose target peptides capable of binding HLA-A(*)02.01 because this HLA haplotype is widely expressed in the human population.
We have previously shown that autologous DC pulsed with PTR-4 can generate a PTH-rP-specific CTL response with antitumor activity against autologous metastatic prostate cancer cells and prostate carcinoma cell lines when used to stimulate in vitro tumor-infiltrating lymphocytes isolated from a HLA-A(*)02.01+ patient with metastatic prostate carcinoma (46). This finding was intriguing in relation to the definition of PTH-rP as a valid TAA and to the significance of T cell repertoire available for this protein. The particular sequence of the PTR-4 peptide shows an atypical HLA-A(*)02.01 binding motif. We hypothesized that the lack of the canonic anchor amino acid in position two of the peptide sequence (SP2) and in position 9 (DP9) probably leads to an instability of the MHC molecule that underlies the lack of tolerance for this PTH-rP peptide epitope. To investigate this point, and clarify the possibility of inducing a CTL-mediated immune response using different epitope peptides with potential clinical implications, we also selected another peptide, theoretically with higher-affinity HLA-A(*)02.01 binding motifs, after screening of the PTH-rP amino acid sequences using the HLA peptide binding prediction analysis described by Parker et al. (45). Further reasons for choosing the PTH-rP p59–68 FLHHLIAEIH (PTR-2) peptide for the comparative study with p165–173 TSTTSLELD (PTR-4) were its effective binding to HLA-A(*)02.01 molecules (measured by means of the T2 class I HLA up-regulation test) and the high degree of divergence from the analogous peptide sequences belonging to human PTH.
Our results showed that the CTL lines generated using PTH-rP peptide-pulsed DCs had epitope peptide specificity, HLA-A(*)02.01 restriction, and the ability to kill HLA-A(*)02.01+/PTH-rP-producing cancer cells, indicating that PTR-4 and PTR-2 peptides have corresponding human CTL precursor repertoire with Ag-specific antitumor activity ex vivo. Our hypothesis was that the particular sequence feature of the peptides allows a specific T cell repertoire response against a TAA that is mainly expressed during the fetal life, when it is involved in the chondrocyte differentiation. Very small amounts of PTH-rP have also been detected: in cultured skin cells, in the epithelial layer of skin and respiratory mucosa as a result of inflammatory insults and chronic degenerative diseases, in breast tissue during lactation, and in the uterus during pregnancy (29, 48, 49, 50). In our study, PTR-2 had the highest predicted affinity for HLA-A(*)02.01, which was confirmed by functional tests in vitro. Its capacity to generate a strong CTL response in PBMC from diverse sources indicated that PTH-rP is not a completely tolerated protein.
Our data also suggest that the CTL lines specific for PTR-4 peptide have a greater functional avidity than the PTR-2-specific CTL lines, because they could recognize target cells pulsed with lower concentrations of cognate peptide. The observation that PTR-2 has a higher binding affinity for HLA-A(*)02.01 excludes the possibility that these results reflect MHC/PTR-2 peptide complex instability on the target cells. The equal capacity of PTR-4 to generate antitumor CTL lines from different healthy donors adds more evidence concerning its immunologic features. Our results demonstrate that it is as immunogenic as PTR-2, and so, given their immunogenic and antitumor capacity, PTR-2 and PTR-4 peptides must be considered two PTH-rP epitopes.
To demonstrate the possibility of raising a CTL immune reaction against PTH-rP protein in vivo, we injected the two peptides into HLA-A(*)02.01 transgenic mice. PTR-2 and PTR-4 show 100 and 60% homology, respectively, with the respective murine PTH-rP peptides. The fact that PTR-4 overlaps only five of the nine amino acids of murine PTH-rP peptide allows this epitope to be used as a positive control. After vaccination and in vitro restimulation with PTR-2 and PTR-4, we could collect spleen cells capable of killing PTH-rP-expressing tumor cells and cognate peptide-pulsed syngeneic target cells with no apparent signs of in vivo tolerance toward PTH-rP. Interestingly, histology showed no signs of tissue degeneration or abnormal lymphocyte infiltration in comparison with analogous samples taken from control mice. PTH and PTH-rP hormone activity targets, such as bone and kidneys, also showed no anomalies. On the basis of previously described results and considerations, we can exclude the possibility that the absence of autoimmunity was due to TCR degeneracy or target escape phenomena. PTH is a peptide hormone produced by the parathyroids that has a high amino acid and functional homology with PTH-rP and is involved in the control of calcium metabolism. In this context, an efficient immune reaction to PTH-rP may possibly affect calcium turnover or cross-react with PTH, thus leading to parathyroid damage. Pathologic studies excluded any parathyroid involvement or degeneracy, and the monitoring of serum ionized calcium levels in mice previously vaccinated with PTH-rP peptides detected no anomalies in peripheral blood calcium levels. These data may be explained by the fact that PTR-2 and PTR-4 do not show any homology with the corresponding peptide sequences in the human and murine PTH.
The results from human PBMC and HHD transgenic mice indicate the possibility of raising a CTL-mediated immune response against PTH-rP and that PTR-2 and PTR-4 are immunogenic in vivo and capable of generating a strong antitumor response. In our preliminary study, we were unable to detect PTH-rP mRNA in the examined tissues (data not shown); therefore, we have attributed the absence of autoimmune effects to an Ag presentation that is too weak in the normal tissue cells to be targeted by Ag-specific CTL precursors.
Our results suggest that the two PTH-rP peptides examined in this study may be useful in peptide-based cancer vaccines. It may be useful to perform a further study in which all its of the determinants of PTH-rP can be screened for the amplification of a potential panel of epitopes for immunotherapy. One interesting point is the possibility of directing a large immune response toward PTH-rP for preventive purposes after primary debulking by means of surgery, radiotherapy, or chemotherapy. Tumor cells expressing PTH-rP have greater metastatic potential and could be targeted before their implantation in bone tissue, thus potentially reducing the risk of metastatic spreading.
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2 Address correspondence and reprint requests to Dr. Guido Francini, Division of Medical Oncology, Faculty of Medicine, University of Siena, Viale Bracci 11, 53100 Siena, Italy. E-mail address: francini{at}unisi.it
3 Abbreviations used in this paper: TAA, tumor-associated Ag; DC, dendritic cell; PTH, parathyroid hormone; PTH-rP, PTH-related protein; CEA, carcinoembryonic Ag; IVS, in vitro stimulation; IRMA, immunoradiometric assay.
Received for publication January 23, 2002. Accepted for publication June 13, 2002.
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signaling blockade inhibits PTHrP secretion by breast cancer cells and bone metastases development. J. Clin. Invest. 103:197.[Medline]
2 microglobulin (2m) HLA-A2.1 monochain transgenic H-2Db 2m double knockout mice. J. Exp. Med. 185:2043.. Cancer Res. 50:6248.This article has been cited by other articles:
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with bold continuous
lines, respectively), the control CEA peptide (CAP-1; • and 
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).
and 
and 
