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IMMUNOBIOLOGY
From the Second Department of Internal Medicine, the
Department of Obstetrics and Gynecology, and the Department of
Bioregulation, Mie University School of Medicine, Tsu; the First
Department of Surgery, Nagasaki University School of Medicine; the
Department of Synthetic Chemistry and Biological Chemistry, Graduate
School of Engineering, Kyoto University; and the Yokohama Research
Center, Tokyo-Mitsubishi, Kanagawa, Japan.
Recognition of the essential role of dendritic cells (DCs) as
professional antigen-presenting cells has prompted investigators to
search for methods to use DCs as natural adjuvants in immunotherapy. A
number of antigenic oligopeptides, recognized by CD8+
cytotoxic T lymphocytes (CTLs) specific for cancer cells, have been
applied in clinical trials using DCs. Such a monovalent vaccine with a
single epitope for a particular type of HLA class 1 molecule would be
effective. However, a polyvalent vaccine might be more potent. We
designed a novel protein delivery system consisting of hydrophobized
polysaccharides complexed with target proteins. The truncated HER2
protein encompassing 147 N-terminal amino acids, including the 9-mer
HER2p63-71 peptide (HER2p63), TYLPTNASL, the human homologue of an
antigenic murine tumor rejection peptide, was prepared. We report here
that HLA-A2402+ DCs could incorporate hydrophobized
polysaccharide-truncated HER2 protein complexes and process the
protein to present major histocompatibility complex class
1-binding HER2p63 peptide. The complexes enter DCs by
phagocytosis, and then the truncated protein is processed through a
pathway similar to that for endogenous proteins. DCs sensitized
by these complexes primed and boosted HER2p63-specific CD8+
T cells in the context of HLA-A2402. Vaccination with DCs incorporating these complexes completely suppressed lung metastases in a
HER2-expressing murine tumor model. We also generated 3 CD4+ clones reactive with different HER2- derived 25-mer
peptides from lymph node cells in mice treated with CHP/HER2-147. Thus, hydrophobized polysaccharide-protein complexes are promising
candidates for the construction of polyvalent vaccines.
(Blood. 2002;99:3717-3724) Recent studies have demonstrated that dendritic
cells (DCs) act as professional antigen-presenting cells (APCs) in host
immune responses and are particularly more important than other APCs in
priming naive T-cell populations for the cognate antigen
peptide.1-4 The use of DCs for cancer immunotherapy as
natural adjuvants for defined tumor antigen peptides has been explored.
In fact, vaccination of tumor-bearing mice with target antigen
peptide-pulsed DCs is effective in suppressing tumor
growth.5-9 Based on these encouraging results in
experimental tumor models, early-phase clinical trials with
peptide-pulsed DCs are under way for patients with melanoma and other
types of cancer.10-14
T cells recognize specific peptides bound to self-major
histocompatibility complex (MHC) molecules, a fundamental feature of antigen recognition by T cells. This unique characteristic of antigen recognition by T cells indicates that a particular oligopeptide can be antigenic, depending on HLA type. Despite the
identification of an increasing number of antigenic peptides that
bind to various HLA types, the use of these oligopeptides for
cancer vaccine is restricted by the expression of proteins with target
peptide sequences in tumor cells and the HLA type of patients.
In comparison with oligopeptides used as monovalent vaccine with a
single epitope, polyvalent vaccines with multiple epitopes are more
desirable. Protein molecules or their cDNAs may be used to load DCs
with multiple potential epitopes binding to various types of HLA.
However, exogenous soluble-protein antigens incorporated by APCs are in
general inefficient in sensitizing CD8+ T lymphocytes
because the proteins are hardly processed by the MHC class 1 pathway.
Instead they are internalized into endosomes and are then taken into
the MHC class 2 pathway.15-17 To overcome this issue, we
designed a novel simple protein delivery system consisting of
hydrophobized polysaccharide, pullulan (CHP) or mannan (CHM), complexed
with soluble protein molecules.18-20 As reported
previously in murine tumor systems, we prepared the truncated HER2
protein containing 147 amino acid residues from N-terminus (HER2-147),
including the 9-mer HER2p63-71 peptide (HER2p63), TYLPTNASL, the human
homologue of a murine tumor rejection antigen peptide, TYLPANASL, and
its complexes with CHP (CHP/HER2-147) or CHM
(CHM/HER2-147).21,22 When BALB/c mice were immunized with
CHP/HER2-147 or CHM/HER2-147 complexes, HER2-specific CD8+
cytotoxic T lymphocytes (CTLs) recognizing HER2p63 peptide with restriction of Kd were generated.21 Murine and
human MHC class 1 molecules, H-2Kd and HLA-A2402, have some
similarity in their anchor motifs for peptide
binding,23,24 and the HER2p63 amino acid sequence is compatible with binding to HLA-A2402. In fact, HER2-specific human CTLs
restricted with HLA-A2402 were induced by the use of HER2p63 peptide.25
With the aim of designing more advanced strategies for cancer vaccines,
we investigated in the present study whether human DCs prepared from
persons with HLA-A2402+ can incorporate and process
CHP/HER2-147 complexes and then present HER2-specific peptide on
HLA-A2402 molecules. The therapeutic effects of DCs incorporating
CHP/HER2-147 complexes were studied using in vivo murine models. We
also established 3 different HER2-specific CD4+ T-cell
clones from mice treated with the complexes. Our results suggest the
potential usefulness of CHP/HER2-147 complexes as a polyvalent vaccine.
Cell lines
Antibodies and immunostaining
Human.
Anti-HLA-A02 (MA2.1), anti-HLA-A24 (A11.1M), anti-HLA-A, -B, -C
(W6/32), and anti-HLA-DR (HDR1) monoclonal antibodies (mAbs) were
gifts from Dr K. Itoh (Kurume University, Japan). Anti-HLA-A30, -A31
(0273HA) mAb was purchased from One Lambda (Los Angeles, CA).
Anti-CD1a, anti-CD14, anti-CD80, and anti-CD86 mAbs were from Becton
Dickinson (San Jose, CA). Anti-CD83 mAb was purchased from Immunotech
(Marseilles, France). Anti-CD3 was from Ortho Biotech (Raritan, NJ).
Mouse IgG1, IgG2 (Becton Dickinson), or IgG2b (Coulter, Hialeah, FL) served as an isotype control.
Antihuman interferon- Murine.
Anti-L3T4/CD4 (GK1.5), anti-Lyt2.2/CD8 (19/178), anti-B220/CD45R
(RA3-3A/6.2), anti-H-2Kd (20-8-4), and
anti-I-Ad (MKD6) mAbs were purified from supernatant of
hybridomas. Anti-ICAM-1/CD54, anti-LFA-1, anti-Mac-1/CD11b,
anti-B7.1/CD80, and anti-B7.2/CD86 were purchased from PharMingen (San
Diego, CA). Anti-DEC205 (NLDC-145) mAb was a gift from Dr B. Kraal
(Leiden University, The Netherlands).
Reagents Human HER2-derived peptides, HER2p63 (amino acids 63-71, TYLPTNASL) and HER2p780 (amino acids 780-788, PYVSRLLGI), were synthesized by Sawady Technology (Tokyo, Japan).22 The truncated HER2 protein encompassing 147 N-terminal amino acids, HER2-147, was prepared as described previously.21 Complexes of CHP and either of recombinant HER2-147 or carbonic anhydrase 2 (CAB), CHP/HER2-147 and CHP/CAB, were synthesized as described previously.18-20 Ten 25-mer peptides of HER2, starting from the N-terminus and overlapping by 10 residues, were also synthesized by Sawady Technology.Lactacystin, a specific proteasome Preparation of human monocyte-derived DCs Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood of consenting healthy donors by density gradient centrifugation with Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). DCs were prepared from CD14+ cells in PBMCs, as described previously.36 Briefly, CD14+ cells were separated from PBMCs using the MACS CD14 Microbeads (Miltenyi Biotec, Auburn, CA). CD14+ cells were cultured at a density of 5 × 105 cells/mL in 6-well plates (Nunc, Roskilde, Denmark) in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS; Interoen, New York, NY) in the presence of GM-CSF and IL-4. After a 7-day culture period, the cells were harvested and used as immature DCs. Approximately 75% to 85% of the cells had typical immature DC morphology and displayed typical cell surface markers of immature DCs CD1a+, CD14 , CD80+,
CD86+, HLA-A, -B, -C+, HLA-DR+, and
CD83low. We also cultured CD14+ cells in the
presence of GM-CSF and IL-4 for 5 days, followed by TNF- for 2 additional days. The cells obtained expressed high levels of CD80,
CD86, HLA-A, -B, -C, HLA-DR, and CD83, a finding consistent with the
features of mature DCs.
Sensitization of human monocyte-derived DCs by CHP/HER2-147 complexes or peptides DCs (1 × 106) were incubated in 10% FCS RPMI 1640 with 20 µg/mL CHP/HER2-147, CHP/CAB, HER2-147 protein, or CAB protein for 3 hours at 37°C in a humidified atmosphere flushed with 5% CO2, followed by washing 3 times with 10% FCS RPMI 1640 and reculturing in 10% FCS RPMI 1640 with GM-CSF and IL-4 for 18 hours until CTL assay.37 DCs, PBMCs, or T2-A24 cells (1 × 106) were pulsed with 10 µM peptides for 1 hour at room temperature and an additional hour at 37°C and were used as stimulator and target cells.Treatment of sensitized DCs with inhibitors DCs were cultured with 30 µg/mL cytochalasin D, 0.2 mM amiloride, 100 mM chloroquine, 100 µM lactacystin, 5 µg/mL brefeldin A, or 10 µg/mL cycloheximide for 1 hour at 37°C in a serum-free AIM-V medium (Gibco-BRL, Grand Island, NY) and were incubated with or without CHP/HER2-147 in the presence of the inhibitors for 3 hours. Then they were washed 3 times with PBS and cultured in 10% FCS RPMI 1640 supplemented with GM-CSF and IL-4 for 18 hours. DCs cultured without CHP/HER2-147 were pulsed with HER2p63 for 1 hour at room temperature and an additional hour at 37°C. They were then harvested and used for antigen presentation.Generation of human killer cells and CD8+ T-cell clones PBMCs (1 × 106 cells/well) were cultured with autologous HER2p63 peptide- or CHP/HER2-147-pulsed and irradiated (46 Gy) DCs (5 × 105 cells/well) in 24-well plates (Nunc) in RPMI 1640 medium supplemented with 10% heat-inactivated human AB serum. After a 7-day culture period, the cells were restimulated with HER2p63 peptide- or CHP/HER2-147-pulsed, irradiated DCs and irradiated (33 Gy) PBMCs (1 × 106 cells/well). IL-2 was added on days 1 and 4 after restimulation. Subsequent restimulation was performed weekly with HER2p63 peptide-pulsed, irradiated PBMCs and irradiated (80 Gy) allogeneic Epstein-Barr virus-transformed B lymphoblastoid cell lines (LCLs; 1 × 106 cells/well). Responder populations were tested for their cytotoxic activity against HER2-expressing cells at the end of the second restimulation culture period. Before the fourth stimulation, CD8+ T cells were separated using the MACS CD8 Microbeads (Miltenyi Biotec).CTL clones were established as described previously, with some modifications.9,25,38 Cloning was performed by limiting dilution at 0.3 to 0.5 cells/well of CD8+ killer T cells in 96-well round-bottom plates (Nunc) in the presence of irradiated autologous PBMCs (5 × 104 cells/well), irradiated LCLs (1 × 104 cells/well), IL-2 (20 IU/mL), and anti-CD3 mAb (30 ng/mL). CD8+ killer T-cell clones that specifically lysed HER2-expressing cells were expanded in the presence of irradiated PBMCs, irradiated LCLs, and anti-CD3 mAbs. Chromium Cr51 release cytotoxicity assay Cytotoxicity assays were performed, as described previously.39 Target cells labeled with 100 µCi chromium (3.7 × 106 Bq) Cr51 (1 × 104 cells) were incubated with serial numbers of effector cells in 96-well V-bottom plates (Nunc) at 37°C. After 5 hours, 100 µL supernatant was collected, and the mean percentage of specific lysis of triplicate wells was calculated as follows: % specific lysis = [(cpm experimental release cpm spontaneous
release)/(cpm detergent release cpm spontaneous release)] × 100.
Antigen presentation assay We fixed DCs onto the flat bottoms of 96 wells by incubation in 1% paraformaldehyde PBS for 10 minutes at room temperature and quenched the well with 0.1 M glycine PBS for 10 minutes. The wells were washed thoroughly with medium. CTLs (5 × 104 cells/well) were cultured with DCs (105 cells/well) fixed in 96-well round-bottom plates in 200 µL 10% FCS RPMI 1640 for 18 hours. IFN- production by CTL was measured in duplicate using an ELISA kit
(Endogen, Woburn, MA).
Enzyme-linked immunospot assay Ninety-six-well nitrocellulose enzyme-linked immunospot (ELISPOT) plates (MAHA S4510; Millipore, Bedford, MA) were coated with 75 µL antihuman IFN- mAb (1-D1K) at a concentration of 2 µg/mL
in PBS overnight at 4°C. The plates were washed 6 times with PBS and
treated with 10% human AB serum RPMI 1640 for 2 hours to block
nonspecific binding. PBMCs were stimulated with autologous DCs at
1-week intervals for 3 weeks. After stimulation, CD8+ T
cells were separated from cultured PBMCs, using MACS CD8 Microbeads (Miltenyi Biotec), and were used as the effector cells. In the next
step, 5 × 104 effector cells and 1 × 105
peptide-pulsed T2/A24 cells were placed in each well of the ELISPOT plate. After incubation for 18 hours at 37°C with 5%
CO2, the plates were extensively washed 6 times with PBS
containing 0.05% Tween 20 (PBS-Tween). They were then incubated with
75 µL biotinylated antihuman IFN- mAb (7-B6-1) at a concentration
of 0.2 µg/mL in PBS for 2.5 hours at 37°C, washed 6 times
with PBS-Tween, and reacted with 1.0 µg/mL streptavidin-alkaline
phosphatase conjugate (Mabtech) in 75 µL PBS for 90 minutes at room
temperature. Wells were washed 6 times with PBS-Tween and then stained
with an alkaline phosphatase conjugate substrate kit (BioRad, Hercules,
CA). The reaction was stopped by rinsing the plates with distilled
water. After drying the plates, the spots were counted using the
Axioplan 2 imaging system (Carl Zeiss Vision, Hallbergmoos, Germany).
In vivo tumor model Seven-week-old female BALB/c mice were obtained from Japan SLC (Hamamatsu, Japan). Bone marrow-derived DCs were prepared, as described previously.14,40 Briefly, single-cell suspensions were obtained from pooled femurs and tibiae, and the marrow cells were enriched for progenitors by negative selection using a cocktail of mAbs including anti-CD4, anti-CD8, anti-B220, and anti-Ia antibodies and rabbit complement (Cedarlane, Ontario, Canada). The enriched cells were cultured at a density of 1 × 106 cells/mL in 6-well plates in RPMI 1640 medium supplemented with 10% FCS in the presence of murine GM-CSF. Cultures were incubated for 7 days at 37°C in a humidified atmosphere flushed with 5% CO2. On days 3 and 5 of culture, floating cells were removed and half the culture media were replaced with fresh medium containing murine GM-CSF. Nonadherent and loosely adherent cells were collected and replated at a density of 106 cells/mL in a 100-mm Petri dish (Nunc). After a 10-day culture period, the cells were harvested. Approximately 80% to 90% of the cells had DC morphology. These cells expressed high levels of Kd, Ia, CD86, CD54, and LFA-1 and moderate levels of CD80, CD11b, and DEC-205, which is typical for murine mature DCs. Seven-week-old female BALB/c mice were inoculated with 1 × 106 human HER2-expressing tumor cells, CMS5mHE cells, in a total volume of 0.1 mL through the tail vein. Four days later, subcutaneous vaccination with 4 × 105 of DCs alone, DCs pulsed with HER2p63, or DCs incorporating CHP/HER2-147 was performed twice at a 1-week interval. One week after the second vaccination, the mice were killed. Lungs were rinsed with PBS and fixed in 10% neutral-buffered formalin. Tumor nodules in the lungs were counted using a dissecting microscope. Lungs were prepared for histopathologic examination using standard methods.Generation of murine CD4+ T-cell lines and clones and their proliferation assay Seven-week-old female BALB/c mice were used. CHP/HER2-147 at 10 µg was subcutaneously administered through footpads. After 10 days, single-cell suspensions were prepared from popliteal lymph nodes of the mice. First, the lymph node cells (1 × 106 cells/well) were cultured with 20 µg/mL CHP/HER2-147 alone in 24-well plates in 10% FCS RPMI 1640 for 1 week. Cells were restimulated in 10% FCS RPMI 1640 with syngeneic CHP/HER2-147-pulsed spleen cells (5 × 106 cells/well) that had been treated with 50 µg/mL mitomycin C (Sigma Chemical) for 30 minutes at 37°C. For approximately 3 months, these cells were stimulated every week with the mitomycin C-treated spleen cells that had been pulsed with CHP/HER2-147. Responding cells were screened for the expression of CD4 by flow cytometry. Cloning of the proliferating population was performed by limiting dilution in 96-well round-bottom plates containing CHP/HER2-147-pulsed and mitomycin-treated spleen cells. Three CD4+ clones, A6, A8, and C8, were generated.In the next step, 5 × 106 mitomycin C-treated spleen cells were reacted in 10% FCS RPMI 1640 with 20 µg/mL CHP/HER2-147 or CHP/CAB for 3 hours at 37°C in a humidified atmosphere flushed with 5% CO2, washed 3 times with 10% FCS RPMI 1640, recultured in 10% FCS RPMI for 18 hours, and used as stimulator cells in the proliferation assay. Furthermore, 5 × 106 mitomycin C-treated spleen cells were also pulsed with 10 µM 25-mer peptides for 1 hour at room temperature and an additional hour at 37°C and were used as stimulator cells. The stimulator cells (5 × 105) were cultured in 96-well tissue culture plates (Nunc) in 100 µL RPMI 1640 supplemented with 10% FCS with 1 × 105 CD4+ clones as responding cells. Plates were incubated for 5 days at 37°C with 5% CO2. To assess the proliferation of CD4+ clones, tritiated thymidine (3H-TdR; specific activity, 25 Ci/mM [0.925 MBq]) uptake was carried out by using a modification of the method described previously.37 On day 5, the cells were pulsed with .037 MBq/well of 3H-TdR for the last 18 hours of incubation. Cells were harvested onto nitrocellulose filters using an automatic cell harvester, and radioactivity was measured in a scintillation counter. The results were expressed as the mean count per minute of duplicate experiments. Statistics Student t test was used to determine statistical significance.
Monocyte-derived DCs can incorporate CHP/HER2-147 and present HER2p63 peptide with HLA-A2402 Primary CTLs were established by PBMCs from a person with HLA-A2402+ using autologous DCs pulsed with HER2p63. A T-cell clone, Y.I.1a, which recognizes HER2p63 peptide in the context of HLA-A2402, was generated, as described in "Materials and methods." Monocyte-derived DCs were prepared from CD14+ cells in PBMCs obtained from persons with HLA-A2402+ and HLA-A2402. DCs were incubated with CHP/HER2-147 or
CHP/CAB and were used as target cells of HER2p63 peptide-specific
Y.I.1a cells. HLA-A2402+ DCs were treated with 0.001 to 20 µg/mL CHP/HER2-147, CHP/CAB, HER2-147 protein, CAB protein, HER2p63,
or HER2p780 peptides, and their stimulatory activity on the CTL clone,
Y.I.1a, was examined. DCs treated with CHP/HER2-147 or HER2p63
stimulated the CTL clone, Y.I.1a, in a dose-dependent manner, as
determined by the production of IFN- (Figure
1A). The stimulatory effects of
CHP/HER2-147 and HER2p63 reached a plateau at the concentrations of 2 and 0.1 µg/mL, respectively. This finding is in agreement with the
fact that the relative concentration of amino acids in CHP/HER2-147 is
approximately 0.06 (9 of 147) because CHP/HER-147 and HER2p63 contain
147 and 9 amino acids, respectively. DCs treated with CHP/CAB, HER2-147
protein, CAB protein, or HER2p780 peptides did not increase the
production of IFN- by Y.I.1a. As shown in Figure 1B,
HLA-A2402+ DCs treated with CHP/HER2-147, but not those
treated with CHP/CAB, were lysed by the CTL clone at an effector-target
ratio of 2.5. HLA-A2402 DCs treated with
CHP/HER2-147 or CHP/CAB were not susceptible to Y.I.1a cells. Similar
results were obtained at effector-target ratios of 5 and 10 (data not
shown). These findings suggest that DCs incorporating CHP/HER2-147
present HER2p63 peptide on HLA-A2402 molecules. Treatment of DCs with
CHP/HER2-147 or CHP/CAB did not modify the cell surface markers,
including CD80, CD86, HLA-A, B, C, HLA-DR, and CD83 (data not shown),
implying that DCs incorporating CHP/HER2-147 or CHP/CAB retained the
phenotype of immature DCs.
Induction of a HER2p63 peptide-specific, HLA-A2402-restricted CTL clone by HLA-A2402+ DCs incorporating CHP/HER2-147 We next attempted to induce a CTL clone specific for HER2 using HLA-A2402+ DCs incorporating CHP/HER2-147. PBMCs obtained from a person with HLA-A2402+ were stimulated with autologous DCs treated with CHP/HER2-147. A CTL clone, PHYI8/63, was generated, and its specificity for HER2 and restriction to HLA-A2402 were examined. The CTL clone, PHYI8/63, lysed the human HER2-expressing ovarian cancer cell line, SKOV3, transfected with HLA-A2402 cDNA (SKOV3-A24) but not SKOV3 transfected with HLA-A0201 cDNA (SKOV3-A2) or HLA-A2402-negative parental SKOV3 (Figure 2A). We also confirmed using flow cytometry that the expression level of HLA-A2402 on SKOV3-A24 was almost identical to that of HLA-A0201 on SKOV3-A2 (data not shown). In addition, PHYI8/63 cells also lysed HER2 T2-A24 pulsed
with HER2p63 but not T2-A24 pulsed with HER2p780 or without peptides
(Figure 2B).
Comparative assessment of HER2p63-specific T-cell induction by DCs incorporating CHP/HER2-147 or DCs pulsed with HER2p63 We compared the induction of HER2p63-specific CD8+ T cells by DCs incorporating CHP/HER2-147 and pulsed with HER2p63. HLA-A2402+ PBMCs were stimulated at 1-week intervals for 3 weeks with autologous DCs incorporating CHP/HER2-147 or pulsed with HER2p63. As shown in Figure 3A, DCs incorporating CHP/HER2-147 or pulsed with HER2p63 induced HER2p63-specific IFN- spot-forming CD8+ cells. In the
experiments following 1, 2, or 3 stimulations, the potential of DCs
incorporating CHP/HER2-147 to induce HER2p63-specific IFN-
spot-forming CD8+ cells was apparently high, compared with
that of DCs pulsed with HER2p63. In contrast, the number of nonspecific
IFN- spot-forming CD8+ cells induced by DCs
incorporating CHP/HER2-147 was lower than that induced by DCs pulsed
with HER2p63 when control HER2p780-pulsed T2-A24 cells were used as
target cells. These findings indicate that CHP/HER2-147-treated DCs
induce HER2p63-specific T cells more efficiently than
HER2p63-pulsed DCs.
The capacity to induce HER2p63-specific IFN- Effect of vaccination using DCs incorporating CHP/HER2-147 and HER2p63-pulsed DCs in an in vivo murine tumor model To examine the effects of vaccination using DCs incorporating CHP/HER2-147 and HER2p63-pulsed DCs on tumor growth in vivo, we used a murine model with human HER2-expressing tumor cells, CMS5mHE cells. As described in "Materials and methods," the phenotype of prepared DCs was that of mature DCs. BALB/c mice were intravenously injected with syngeneic CMS5mHE cells and were examined for lung metastases. Mice bearing CMS5mHE cells were treated twice by weekly subcutaneous injection with DCs alone, DCs incorporating CHP/HER2-147, or HER2p63-pulsed DCs. DCs alone reduced lung metastases, though the extent was approximately 20% (Figure 4). Treatment of mice with HER2p63-pulsed DCs resulted in more than 85% reduction in the number of metastatic foci in the lungs, compared with mice treated with DCs alone. Interestingly, treatment with DCs incorporating CHP/HER2-147 resulted in complete absence of lung metastases, which was confirmed by histologic examination.
CD4+ clones generated with CHP/HER2-147 recognize the 25-mer peptides of truncated HER2 protein, HER2-147 CD4+ lines were generated in response to CHP/HER2-147 from lymph node cells of mice treated with CHP/HER2-147. Cloning of the lines gave rise to 3 CD4+ clones: A6, A8, and C8. The responses of these 3 CD4+ clones to 10 different 25-mer peptides of HER2, which were continuous from the N-terminus and overlapped by 10 residues, were tested. The sequences of the 25-mer peptides are shown in Figure 5A. When A6 cells were incubated with syngeneic spleen cells pulsed with the 10 different 25-mer peptides, A6 cells were reactive against spleen cells pulsed with the peptide p16-40 but not with those of the other peptides (Figure 5B). A8 and C8 cells showed reactivity to spleen cells pulsed with peptides p1-25 and p136-160, respectively, though the specific reactivity of C8 cells was low compared with that of A6 and A8 cells. Moreover, A6, A8, and C8 cells proliferated in response to spleen cells pulsed with CHP/HER2-147, but not with those pulsed with CHP/CAB.
Processing pathway of CHP/HER2-147 in DCs for presentation of HER2p63 bound to HLA-A2402 The processing pathway of CHP/HER2-147 in DCs was examined using various inhibitors. Cytochalasin D was used as an inhibitor of phagocytosis, amiloride as an inhibitor of macropinocytosis, chloroquine as an inhibitor for protein hydrolysis, lactacystin as an inhibitor of proteasome, brefeldin A as an inhibitor of exocytosis, and cycloheximide as an inhibitor of protein synthesis. As shown in Figure 6, the stimulatory activity of DCs incorporating CHP/HER2-147 on the CTL clone, Y.I.1a, was inhibited when DCs were treated with cytochalasin D, lactacystin, brefeldin A, or cycloheximide. The stimulatory activity of DCs incorporating CHP/HER2-147 was, however, persistent when treated with amiloride or chloroquine. Neither of these inhibitors suppressed the stimulation of Y.I.1a cells by DCs pulsed with HER2p63. These results indicate that DCs incorporating CHP/HER2-147 process HER2-147 and present MHC class 1-binding HER2p63 through the conventional processing pathway for endogenous protein molecules.
To develop an effective immuno-cell therapy, different approaches
for loading DCs with antigenic peptides have been sought. The use of
proteins containing multiple antigenic peptides is one such approach.
It is, however, well known that professional APCs Our antigen molecule delivery system using the hydrophobized
polysaccharide-protein complex has several advantages. DCs
incorporating hydrophobized polysaccharide-protein complex can process
and present MHC class 1-binding peptides, as shown clearly by the fact
that DCs incorporating CHP/HER2-147 complexes can specifically
stimulate a HER2p63-specific CTL clone and also can generate a CTL
clone with similar specificity. Experiments with inhibitors for a
variety of molecules that are required for processing and presenting
antigen molecules indicated that CHP/HER2-147 complex is incorporated into DCs by phagocytosis because the pretreatment of DCs by
cytochalasin D completely abrogated antigen presentation. Although we
still do not know how CHP/HER2-147 or HER2-147 protein alone enters the
cytosol through the phagosomes, HER2-147 protein was subsequently processed like endogenously synthesized proteins for MHC class 1-binding peptides because pretreatment of DC with lactacystin, brefeldin A, or cycloheximide totally abolished their capability for
peptide presentation. Other strategies for delivering peptides derived
from exogenous proteins to MHC class 1 molecules have been
reported.48-55 These include the use of
particle-associated antigens,48-50 the use of antigen-IgG
complexes internalized through Fc In the ELISPOT assay for IFN- Taken together, hydrophobized polysaccharide-protein complexes appear to be able to load DCs with antigenic peptide epitopes that can bind to MHC class 1 and class 2 molecules as well, implying that these complexes could be used for immuno-cell therapy as polyvalent vaccine. Hydrophobized polysaccharides of a simple structure are expected to be nonhazardous; in fact, no adverse effects have been observed in animals treated with CHP alone or with CHP/HER2-147.21 An early-phase clinical trial of immuno-cell therapy with CHP/HER2-147-treated DCs will be launched in our hospital in the near future in patients with HER2-expressing advanced cancer refractory to conventional therapy.
Submitted February 26, 2001; accepted January 14, 2002.
Supported in part by a Grant-in Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Science, Sports, and Technology and from the Ministry of Health, Labour, and Welfare, Japan.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Hiroshi Shiku, The Second Department of Internal Medicine, Mie University School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan; e-mail: shiku{at}clin.medic.mie-u.ac.jp
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Abstract
Introduction
subunit inhibitor, was purchased
from Kyowa Medix (Tokyo, Japan).
Differences are significant at P < .01.
