| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Department of Immunohematology and Blood Bank, University of Leiden, Leiden, The Netherlands;
Department of Pharmaceutics, Faculty of Pharmacy, University of Utrecht, Utrecht, The Netherlands; and
Cancer Immunology Program, Cardinal Bernardin Cancer Center, Loyola University of Chicago, Maywood, IL 60153
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Peptide Ags can, however, also induce specific peripheral CTL nonresponsiveness (7, 8, 9). A peptide derived from lymphocytic choriomeningitis virus (LCMV)3 induces protective LCMV-specific CTL when given s.c. in IFA but tolerizes LCMV-specific CTL when given repeatedly i.p. at high peptide doses (10). Recently, we observed that s.c. vaccination with a peptide derived from the human adenovirus type 5 early region 1B (Ad5E1B) oncogene at doses ranging from 100 µg/mouse to as little as 1 µg/mouse evokes specific tolerance of Ad5E1B-directed CTL responses. This peptide-induced tolerization of Ad5E1B-specific CTL responsiveness was strongly associated with the inability of vaccinated mice (in contrast to nonvaccinated animals) to reject a challenge of Ad5E1B-expressing tumors (11). Similar findings were obtained in another tumor system. A single s.c. vaccination with 10 µg of a peptide representing a CTL epitope derived from the Ad5E1A oncogene induced Ad5E1A-specific CTL tolerance, causing enhanced outgrowth of Ad5E1A-transformed tumors (12). In both cases, tolerance induction was associated with rapid systemic diffusion of the peptides from the s.c. IFA depot (11, 12). These observations show that immunization with these particular peptides administered the same way and at the same doses as the aforementioned protective peptide vaccines (1, 2, 3) causes systemic peripheral CTL tolerance and indicate that the route and dose of peptide administration causing CTL priming cannot be generalized for all peptides delivered in adjuvants. More importantly, these findings indicate that clinical trials using synthetic peptide-based vaccines for the reinforcement of the host’s immune response against tumors or viral infections should be conducted with caution because they might lead to an effect opposite to that intended, namely, T cell tolerance causing enhanced tumor growth rather than protective T cell immunity.
Since it is of crucial importance to avoid T cell tolerization through peptide vaccination in a clinical setting, we set out to define vaccine formulations that also in the case of potentially tolerogenic peptides lead to protective antitumor immunity. To this end, the effectiveness was determined of different vaccine formulations reported to be capable of priming CTL responses, containing as antigenic component a peptide that readily induces specific CTL tolerance when administered s.c. in adjuvants. In this study, we show that peptide-loaded dendritic cells (DC) represent the only formulation tested that is capable of inducing CTL-mediated immunity, leading to tumor protection, whereas all other tested modes of synthetic peptide delivery cause enhanced tumor outgrowth.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
C57BL/6 (H-2b) mice were obtained from IFFA Credo (France). Mice were used at 7 to 10 wk of age.
Cell lines and culture conditions
Ad5E1A-transformed mouse embryo cells (MEC) of C57BL/6 origin were generated as previously described (12, 13). Cells expressing Ad5E1A and EJras (AR cell lines) were generated by transfection of MEC with pAd5E1A, pEJras, and pTKneo as described previously. (14) The Ad5E1A and EJras oncogenes are well known to efficiently cooperate in oncogenic transformation (15). In contrast to cells transformed by Ad5E1 alone, Ad5E1 + EJras-transformed cells are capable of forming tumors in immunocompetent mice (13, 14). All cell lines were tested for expression of the transfected Ad5E1A and Ad5E1B genes by Northern blotting (data not shown). All MEC and Ad5E1-transformed MEC were maintained in Iscove’s modified Dulbecco’s medium (IMDM) (Biocrom KG, Seromed, Berlin, Germany) supplemented with 4% FCS (HyClone Laboratories, Logan, UT), penicillin (100 IU/ml, Brocades Pharma, Leiderdorp, The Netherlands), and 2-ME (20 µM) at 37°C in a 5% C02 atmosphere.
Generation of dendritic cells (DC)
Bone marrow-derived DC were obtained as previously described (16, 17, 18). Briefly, bone marrow cells depleted for lymphocytes and MHC class II-positive cells were cultured overnight in IMDM supplemented with 10% FCS, 2-ME (20 µM), penicillin, sodium pyruvate (1 mM), and nonessential amino acids (10 µM) at 37°C in a 5% C02 atmosphere in 24-well plates (106 cells in 1 ml of culture medium per well). After 24 h, the cells were replated and cultured in the presence of granulocyte/macrophage colony-stimulating factor (103 U/ml, Schering-Plough, Kenilworth, NJ) and murine rIL-4 (900 U/ml, SanverTech/PeproTech, Rocky Hill, NJ). After 3 to 4 days the medium was exchanged with fresh medium containing cytokines. On day 8, the DC were pulsed for 2 h at 37°C in 1 ml of IMDM supplemented with 0.5% BSA and 10 µg of peptide. Peptide-pulsed DC were extensively washed before they were used.
Splenic DC were isolated according to the method described by Steinman et al. (19) with minor modifications. Spleens of B6 mice were injected in vitro with 0.5 ml of collagenase (100 U/ml) (Clostridium histolyticum, type IV, Sigma Chemical, St. Louis, MO), cut into pieces, and incubated at 37°C for 15 min in 1 ml of collagenase. A spleen cell suspension was made in IMDM supplemented with 15% FCS, penicillin (100 IU/ml), kanamycin (100 µg/ml), and 2-ME (2 x 10-5 M). The cells were spun on a discontinuous BSA gradient (Sigma Chemical) of 10, 28, and 35% BSA at 4°C for 30 min at 10,000 x g. The interphase between 10 and 28% BSA was removed, and the cells were cultured for 90 min at 37°C in glass petri dishes. Nonadherent cells were discarded, and the culture medium was replaced. After a further 18 h of culture, the nonadherent cells were harvested and used for peptide loading as described above.
Peptides
Synthetic peptides were constructed on an Abimed 422 multiple peptide synthesizer (Abimed, Langenfeld, Germany) at 10 µmol scale as previously described (20). Palmitoyl peptides were prepared by reaction with Pam-PyBop-NMM (palmitic acid-(benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate)-(N-methylmorpholine)) as the last step in the synthesis. For the preparation of bis-palmitoyl derivatives, peptides were elongated by coupling with Fmoc-Lys(Fmoc)-OH. After Fmoc removal, coupling was performed with Pam-PyBop-NMM. Palmitoyl peptides were purified by reversed phase HPLC to ensure the absence of nonpalmitoylated peptide material. In short, a solution of the crude material in 50% HOAc was applied to a semiprep reversed phase HPLC column. Gradient elution was perfomed in water, acetonitrile, 0.1% trifluoroacetic acid (TFA) with gradients of 1.5 to 2% acetonitrile increase per minute. The following palmitoyl-containing peptides were used in these studies: Pam-TSLTPRCNTAWNRLKSTDSCDSGPSNTPPEI, containing an I-Ab-restricted T helper epitope derived from Friend leukemia virus (21) and the immunodominant H-2Db-restricted CTL epitope expressed by Ad5E1A + ras-transformed tumor cells (12) linked to each other by the natural flanking sequences of the Ad5E1A epitope; Pam-TSLTPRCNTAWNRLKGQAEPDRAHYNIVTF, containing the Friend murine leukemia virus T helper epitope linked to a H-2Db-restricted CTL epitope derived from the human papilloma virus type 16 (HPV16) E7 protein (3) by the natural sequences of the HPV16 E7 epitope; PAM-STDSCDSGPSNTPPEI, containing the Ad5E1A-encoded CTL epitope and the natural flanking sequences of the Ad5E1A epitope; (Pam)2-KSSKOVAAWTLKAAASGPSNTPPEI, where O = cyclohexylalanine, containing the pan-T helper epitope "PADRE" (that enhances CTL induction in B6 mice) (22), linked to the Ad5E1A epitope by a three-alanine spacer as described elsewhere (23); (Pam)2-KSSKOVAAWTLKAAAVNIRNCCYI, containing the pan-T helper epitope "PADRE" linked to the H-2Db-restricted Ad5E1B-derived CTL epitope (14) by a three-alanine spacer.
The purity of the peptides was determined by analytical reversed phase HPLC using a water-acetonitrile gradient containing 0.1% TFA and proved to be at least 80% (UV 214 nm). The integrity of the peptides was determined by laser desorption time-of-flight mass spectrometry on a lasermat mass spectrometer (Finnigan MAT, Hemel Hempstead, U.K.). About 5 pmol of the peptide in 0.5 µl of water-acetonitrile containing 0.1% TFA were mixed with 0.5 µl of matrix solution (10 mg/ml in acetonitrile-water, 60:40 (v/v) containing 0.1% TFA) and applied to the instrument. Calibration was performed with peptides of known molecular mass, either as external or as internal references. The amount of free Ad5E1A peptide was less than 1%.
Liposome preparation
Partially hydrogenated egg phosphatidylcholine with an iodine value of 40 was obtained from Asahi Chemical Industry, (Ibarakiken, Japan). Egg phosphatidylglycerol was a gift from Lipoid (Ludwigshafen, Germany). Cholesterol (Chol) was from Sigma Chemical. A lipid mixture of partially hydrogenated egg phosphatidylcholine-egg phosphatidylglycerol-Chol (10:1:4 molar ratio) was prepared in chloroform-methanol (10:1) and dried to a thin film by rotary evaporation. After evaporation for at least 1 h, the lipid film was hydrated with 5% acetic acid (210 µmol of phospholipid per ml) containing the Ad5E1A peptide (5 mg/ml). The resulting peptide-containing liposome dispersion was subsequently diluted 50-fold with HEPES buffer (50 mM HEPES, 73 mM NaCl, pH 7.6). Nonentrapped peptide was removed by ultracentrifugation at 160,000 x g during 20 min. After decantation of the supernatant, the pellet was resuspended and subsequently washed three times with HEPES buffer (10 mM HEPES, 140 mM NaCl, pH 7.4). The liposomes were diluted with 10 mM HEPES buffer to a final phospholipid concentration of 17 µmol/ml. The liposomes was stored at 4°C under nitrogen atmosphere and used to immunize mice the next day.
To determine the amount of pepide encapsulated in liposomes, a mixture of 0.5 ml of liposomes and 0.5 ml of acetonitrile was vortexed for 5 min. The mixture was centrifuged (13,000 rpm), and 0.5 ml of peptide-containing supernatant was lyophilized. The peptide was reconstituted in 1.0 ml of water, and aliquots were applied to reversed phase HPLC. Peptide content was determined by comparison of HPLC peak areas to those of known amounts of peptide. Peptide leakage from liposomes was determined 48 h after construction. Liposomes were washed with PBS, and after concentration of the washings the peptide content of the washings was determined as described above. Peptide leakage was <1%.
Immunizations and tumor cell challenge
Peptide immunizations were performed as described in the legends
to the figures. Two weeks after immunization, the mice were challenged
s.c. with Ad5E1A + ras-transformed tumor cells in PBS. When a
vaccination protocol was studied for its ability to induce
Ad5E1A-specific CTL tolerance, the mice were challenged with AR6 cells
(107 cells). This tumor cell clone causes growth of
small tumors in 80 to 100% of naive B6 mice. After 
10 wk, 20 to
30% of the animals have died because of a progressively growing
tumors. The other animals that have developed a tumor still carry it or
have eradicated the tumor (12). The outgrowth of this tumor can no
longer be controlled by the animals after s.c. injection of 10 µg of
Ad5E1A peptide in IFA (12). AR5 cells (0.5 x 106
cells/mouse) were used to analyze the ability of peptide-loaded DC to
induce protective immunity against an otherwise lethal challenge with
this Ad5E1A + ras-transformed tumor cell. Injection of 0.5 x
106 cells of this tumor cell clone kills 75 to 100% of the
animals in 5 to 6 wk.
Generation and analysis of CTL bulk cultures
Ad5E1-specific CTL in bulk culture were generated as follows:
5 x 106 spleen cells per well derived from B6
mice taken 3 wk or more after the second i.v. immunization with
0.5 x 106 peptide-loaded bone marrow-derived DC were
cocultured for 6 days with 10% irradiated (25 Gy) IFN-
(10
U/ml)-treated Ad5E1-transformed stimulator cells in 24-well plates.
Next, effector cells were harvested, and dead cells were removed by
density centrifugation on Lympholyte M (Cedarlane, Hornby, Canada).
These cells were used in a cell-mediated lymphocyte cytotoxicity assay
as described previously (3, 14).
Primary in vitro induced Ad5E1-specific CTL bulk cultures were obtained by coculturing 5 x 106 spleen cells derived from a naive B6 mouse with 10% Ad5E1 peptide-loaded bone marrow-derived DC. After 7 days, the cells were harvested and, after removal of dead cells by density centrifugation, were restimulated with 10% Ad5E1-transformed stimulator cells. Six days thereafter, the bulk CTL cultures were used in a TNF production assay as previously described (24). In short, 5 x 103 bulk CTL were added to 2 x 104 stimulator cells in a total volume of 100 µl of medium containing 10 Cetus U rIL-2 (Cetus, Emeryville, CA) in wells of a 96-well U-bottom plate. After 24 h, 60 µl of supernatant were collected, and its TNF content was determined by measuring its cytotoxic effect on WEHI-164 clone 13 cells. The percentage of WEHI cell death was calculated by the formula: [(OD550–650 in experimental wells - OD550–560 in wells containing medium only)/(OD550–560 in wells containing 50 pg/ml TNF - OD550–560 in wells containing medium only)] x 100.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Previously, we have shown that immunization with synthetic peptides can lead to enhanced outgrowth of tumors. A single s.c. (local) injection of as little as 10 µg of a peptide derived from the Ad5E1A oncoprotein (sequence, SGPSNTPPEI) in IFA led to enhanced outgrowth of Ad5E1A-transformed tumor cells, whereas injection of a peptide derived from the Ad5E1B oncoprotein (sequence, VNIRNCCYI) induced enhanced outgrowth of Ad5E1B-expressing tumor cells (11, 12). In both cases, enhanced tumor outgrowth was accompanied by functional deletion of Ad5E1A- and Ad5E1B-specific CTL, respectively, and associated with rapid systemic distribution of the peptides from the s.c. IFA depot. Since such tolerizing effects op peptide vaccination are unacceptable in a clinical setting, we set out to define vaccine formulations that would not induce CTL tolerance but instead would lead to protective antitumor immunity. We focused on vaccination strategies that 1) have been shown to be able to induce CTL-immunity in other model systems and 2) are likely to retain the peptide locally or are able to deliver the peptide to phagocytes such as macrophages or to DC, because of the possible correlation between systemic distribution of CTL epitope-containing peptides and their ability to induce tolerance (9, 10, 11, 12).
Ags encapsulated in liposomes are known to be immunogenic to CTL and to
be presented by macrophages (25, 26, 27, 28). To test whether peptide
encapsulated into liposomes induces protective antitumor immunity
instead of tumor-specific CTL tolerance, we entrapped the Ad5E1A
peptide (the immunodominant CTL epitope expressed by Ad5E1A +
ras-transformed tumor cells (12) into liposomes. Mice immunized with 10
µg of Ad5E1A peptide in liposomes given i.v. in PBS, challenged 2
weeks later with Ad5E1A + ras-transformed tumor cells, showed
enhanced tumor outgrowth and increased mortality compared with naive
animals or animals that had received empty liposomes (Fig. 1
A). Similar effects
were observed when liposome entrapped Ad5E1A-peptide was given s.c.
(Fig. 1B). These results show that encapsulation of
the Ad5E1A peptide into liposomes still leads to enhanced outgrowth of
Ad5E1A + ras-transformed tumor cells, indicating that this
vaccination strategy does not prevent Ad5E1A-specific CTL tolerance
induction.
|
Synthetic lipopeptide-based vaccines can induce peptide-specific
CTL responses that cross-react on targets expressing endogenous Ag.
Lipopeptide-based vaccines are even considered to be superior in
priming CTL responses compared with vaccination with free synthetic
peptide in adjuvants, or other vaccination strategies using recombinant
viruses and bacteria (23, 29, 30, 31). To study the in vivo effects of a
lipopeptide containing the Ad5E1A epitope, we immunized mice s.c. with
Ad5E1A lipopeptide in PBS. Two weeks later, these mice were challenged
with Ad5E1A + ras cells. Lipopeptide-immunized mice are no longer
able to control the outgrowth of Ad5E1A-expressing tumor cells (Fig. 2A). Also, addition of
a known T helper epitope derived from Friend murine leukemia virus (21)
did not prevent the outgrowth of Ad5E1A + ras tumors, but instead,
led to enhanced tumor outgrowth (Fig. 2A). A similar
observation was made when the lipopeptide vaccine was administered
together with rIL-2 (105 Cetus U/mouse), or administered
i.v. in PBS (data not shown). Likewise, vaccination with an
Ad5E1B-containing lipopeptide vaccine caused an accelerated outgrowth
of Ad5E1B-expressing tumors (but not of Ad5E1A + ras-transformed
tumors lacking the relevant Ad5E1B-Ag) on challenge with an
Ad5E1B-expressing tumor cell (data not shown). Linkage of two lipid
molecules and a pan-T helper epitope (a T helper epitope reported to
enhance CTL induction by peptide vaccination in B6 mice (22) to the
Ad5E1A epitope, likewise, did not lead to protective antitumor
immunity. Instead, the use of this lipopeptide (of similar design as
that for clinical use (22, 23)) resulted in the inability of vaccinated
mice to control the outgrowth of Ad5E1A + ras cells (Fig. 2B). Thus, addition of a lipid tail (and/or a T
helper epitope) to the Ad5E1A peptide does not lead to protective
antitumor immunity, nor does it prevent the induction of
Ad5E1A-specific CTL tolerance. Moreover, immunization with such a
lipopeptide vaccine still leads to the inability of vaccinated mice, in
contrast to control animals, to repress the outgrowth of
Ad5E1A-transformed tumor cells.
|
Peptide-pulsed DC have been shown to induce protective and
therapeutic antitumor immunity in several model systems, but they have
also been reported to present Ag in a tolerogenic fashion (18, 32, 33, 34, 35, 36).
These findings prompted us to evaluate whether DC loaded with the
Ad5E1A peptide are able to initiate protective antitumor immunity.
Therefore, DC generated out of bone marrow cultures were loaded with
the Ad5E1A or Ad5E1B peptide and analyzed for their potential to induce
a primary peptide- and tumor-specific CTL response in vitro. Spleen
cell cultures derived from naive B6 mice primed in vitro with Ad5E1A
peptide-loaded DC recognize Ad5E1A peptide-loaded target cells and
Ad5E1-transformed cells (expressing both the Ad5E1A and Ad5E1B
oncogenes), whereas spleen cell cultures activated in vitro with Ad5E1B
peptide-loaded DC recognize Ad5E1B peptide-loaded target cells as well
as Ad5E1-transformed cells (Fig. 3).
Spleen cell cultures incubated with freshly isolated peptide-loaded
bone marrow cells do not recognize the Ad5E1 peptides or
Ad5E1-transformed cells (data not shown). Taken together, these
findings indicate that the nonadherent cells present in the bone marrow
cultures consisted of DC, because they exhibited one of the most
important features of DC: the ability to induce primary
peptide-specific CTL responses in vitro that cross-react on tumor cells
endogenously presenting the relevant Ag.
|
). Bulk
CTL cultures obtained from mice immunized with Ad5E1B peptide-pulsed DC
kill Ad5E1B-loaded target cells and Ad5E1-transformed cells, but not
Ad5E1A + ras tumor cells (not expressing the Ad5E1B oncogene).
Similar results were obtained when peptide-loaded DC isolated from
spleen were used for immunization (data not shown). These data show
that immunization with Ad5E1A or Ad5E1B peptide-loaded DC does not
induce specific CTL tolerance but leads to a strong peptide- and
tumor-specific CTL-mediated immune response in vivo.
|
). The tumor cell clone AR5 has a
much higher growth rate in vivo than AR6. Therefore, an accelerated
tumor growth rate after Ad5E1A peptide immunization in IFA is not
observed, because the animals in the control group must be euthanized
before the differences with the Ad5E1A peptide-immunized group become
apparent. Mice immunized with freshly isolated bone marrow cells pulsed
with the Ad5E1A peptide are not protected against a challenge with AR5
cells (Fig. 6), illustrating the
necessity to use DC for the induction of protective antitumor immunity.
A delayed outgrowth of Ad5E1A + ras tumors is observed in mice
vaccinated with control peptide-loaded DC compared with mice that did
not receive DC (Figs. 5 and 7). This is most likely due to
"nonspecific" T cell reactivity against FCS components as also
observed by others (37) (both tumor cells and DC are cultured in
FCS-containing medium), since no delayed outgrowth of tumors is
observed when Ad5E1A + ras cells that have been cultured for 3
weeks in serum-free medium are used for tumor challenge (data not
shown). DC derived from bone marrow cultures are as potent in eliciting
tumor-protective immunity as DC isolated from the spleen (Fig. 7). Both DC populations induce optimal
tumor protection when given i.v., whereas only partial protection is
induced after s.c. administration of Ad5E1A peptide-loaded DC (Fig. 7).
These data indicate that i.v. administration of DC-based vaccines is
more effective than s.c. injection of this type of vaccine, as also
reported by others (38). Taken together, these results show that the
Ad5E1A peptide is able to induce protective antitumor immunity when
loaded on DC and point to the superior qualities of DC in the induction
of effective antitumor immunity associated with CTL memory responses
over several other vaccine vaccine formulations that may lead to
specific CTL tolerance.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Peptides that are given systemically or that disperse throughout the
body after local s.c. injection have been shown to be able to induce
specific CTL tolerance (7, 9, 10, 12). Vaccine approaches capable of
controlling the diffusion of Ags would, therefore, have distinct
advantages over methods that can lead to systemic distribution of Ags.
For this reason, we have focused in this study on vaccine modalities
that are likely to retain the peptide locally or that have been shown
to deliver the peptides to phagocytes. Immunization of mice with
lipopeptide vaccines encoding influenza virus nucleoprotein-derived CTL
epitopes leads to the induction of influenza virus-specific CTL (23, 29). In this model, immunization with an influenza epitope-containing
lipopeptide was shown to be superior over immunization with free
peptide. Moreover, the CTL response was significantly enhanced when a T
helper epitope was incorporated into the vaccine (23, 29, 41, 42).
Likewise, in a Plasmodium berghei model, a stronger CTL
response was induced after immunization with a lipid-tailed peptide
compared with vaccination with free peptide in adjuvant (30). Despite
the encouraging results obtained in these models, immunization with an
Ad5E1A-containing lipopeptide results in the inability of vaccinated
mice to control a challenge with Ad5E1A-expressing tumor cells (Fig. 2
A), whereas vaccination with an Ad5E1B-containing
lipopeptide leads to the inability of immunized animals to reject a
challenge with Ad5E1B-expressing tumor cells (data not shown). Also a
lipopeptide mimicking a lipopeptide vaccine designed for use in a
clinical setting (23) equipped with two lipid molecules containing the
Ad5E1A epitope in addition to a pan class II-binding T helper epitope
that has been shown to enhance peptide-induced CTL responses in B6 mice
(22) did not induce protective antitumor immunity. Instead, vaccination
resulted in the enhanced outgrowth of Ad5E1A-expressing tumor cells
(Fig. 2B), indicating that also this vaccine
formulation does not prevent tolerization by the Ad5E1A peptide.
Previously, we have shown that s.c. administration of Ad5E1A- or Ad5E1B-peptide in IFA, together with i.v. injection of a CTL clone specifically recognizing the injected peptide, induces a severe respiratory syndrome, most likely due to activation of Ad5E1-specific CTL that are trapped in the vascular bed of the lungs (11, 12). This severe immunopathologic damage induced by peptide Ag treatment in the presence of a substantial amount of memory CTL located in a particular organ can be used as a readout to determine whether the peptides rapidly diffuse out of the s.c. depot into the periphery. Mice given the combination of Ad5E1A lipopeptide and i.v. injection of Ad5E1A-specific CTL (but not of Ad5E1B-specific CTL) suffered from severe respiratory problems. The same was observed when injection of Ad5E1B lipopeptide was combined with i.v. injection of Ad5E1B-specific CTL (data not shown), indicating that s.c. administered Ad5E1-peptides linked to lipid tails are not retained locally but disperse throughout the animals. These findings may be explained by cleavage of the lipopeptides by proteases and/or lipases in the body, liberating free Ad5E1 peptide, which then diffuses throughout the body, instigating Ad5E1-specific CTL tolerance. Moreover, they point to a similar erratic behavior of lipopeptide vaccines in vivo as peptides delivered in adjuvants. In contrast, vaccination with Ad5E1 peptide-loaded DC, followed by infusion of Ad5E1-specific CTL, does not induce lung congestion (data not shown).
Soluble Ags entrapped in liposomes induce CTL responses after i.v. injection (27, 28). Depletion of macrophages in vivo abolishes the induction of Ag-specific CTL in this system, indicating that liposomes are able to deliver the encapsulated Ags to cells important for proper CTL induction (28). Nonetheless, i.v. or s.c. immunization with Ad5E1A peptide encapsulated in liposomes leads to enhanced outgrowth of Ad5E1A-transformed tumor cells. Although other types of liposomes may be more effective in eliciting Ad5E1A-specific CTL responses, these observations indicate that also this mode of Ag delivery does not prevent the induction of peptide-specific CTL tolerance, most likely due to release of free Ad5E1A peptide in vivo.
On the basis of these results, we applied the Ad5E1 peptides directly to ex vivo generated DC and used these as vaccine vehicles. Peptide-loaded DC have been shown to induce both protective and therapeutic antitumor immunity when used for vaccination (18, 33, 34, 37, 43). Although DC are unmatched in their ability to activate naive T cells (44), they have also been reported to be able to present Ag in vivo in a tolerogenic fashion (35, 36). The latter is, however, not the case in the Ad5E1-tumor system. DC loaded with Ad5E1A or Ad5E1B peptide are able to induce strong Ad5E1A- and Ad5E1B-specific CTL-responses, respectively, both in vivo and in vitro. More importantly, mice immunized with Ad5E1A peptide-loaded DC are protected against an otherwise lethal challenge with Ad5E1A-transformed tumor cells. These findings indicate that the Ad5E1 peptides do not act as antagonist peptides. Therefore, antagonism of these peptides in vivo is an unlikely explanation for the observed CTL tolerance induction. Also, these peptides can apparently induce protective Ad5E1-specific CTL responses, instead of Ad5E1-specific tolerance, when given in the right context. In this regard, costimulation seems to be particularly important for the induction of protective antitumor immunity, since it has been shown that blockade of CD80 and CD86 by CTLA4-Ig abrogated the tumor-protective effects of peptide-pulsed DC in vivo (34).
Thus, in summary, immunization with Ad5E1A peptide-loaded DC induces protective antitumor immunity against Ad5E1A-positive tumor cells, in contrast to other synthetic peptide vaccination strategies. These vaccination strategies include vaccination with Ad5E1A lipopeptides given s.c. in PBS, with or without known T helper epitopes, with or without IL-2, Ad5E1A lipopeptide given i.v., Ad5E1A peptide given s.c. in adjuvants (like IFA or CFA (12)), Ad5E1A peptide given i.v. in PBS, or Ad5E1A peptide encapsulated in liposomes given s.c. or i.v. Likewise, immunization with Ad5E1B peptide-loaded DC (a CTL epitope presented by different tumor cells and recognized by different CTL) induces Ad5E1B-specific CTL responses both in vitro and in vivo. In contrast, s.c. vaccination of this peptide in adjuvant or Ad5E1B-peptide linked to a lipid tail injected s.c. or i.v. in PBS evokes a specific functional deletion of Ad5E1B-directed CTL ((11), and this report). These data show that DC can revert the CTL-tolerizing potential of a synthetic peptide vaccine into a vaccine inducing protective antitumor immunity and provide the rationale for using DC as the primary vehicles of choice for the development of synthetic peptide-based vaccines.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. René E. M. Toes, Department of Immunohematology and Blood Bank, University of Leiden, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
3 Abbreviations used in this paper: LCMV, lymphocytic choriomeningitis virus; Ad5E1B, adenovirus type 5 early region 1B; Pam, palmitic acid; DC, dendritic cells; MEC, mouse embryo cells; IMDM, Iscove’s modified Dulbecco’s medium; TFA, trifluoroacetic acid; HPV16, human papilloma virus type 16; Chol, cholesterol.
Received for publication October 8, 1997. Accepted for publication December 22, 1997.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  D. Atanackovic, N. K. Altorki, Y. Cao, E. Ritter, C. A. Ferrara, G. Ritter, E. W. Hoffman, C. Bokemeyer, L. J. Old, and S. Gnjatic Booster vaccination of cancer patients with MAGE-A3 protein reveals long-term immunological memory or tolerance depending on priming PNAS, February 5, 2008; 105(5): 1650 - 1655. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. B. Gendron, A. Rodriguez, and D. A. Sewell Vaccination with human papillomavirus type 16 e7 Peptide with CpG oligonucleotides for prevention of tumor growth in mice. Arch Otolaryngol Head Neck Surg, March 1, 2006; 132(3): 327 - 332. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. F. H. M. Boonman, G. J. D. van Mierlo, M. F. Fransen, R. J. W. de Keizer, M. J. Jager, C. J. M. Melief, and R. E. M. Toes Maintenance of Immune Tolerance Depends on Normal Tissue Homeostasis J. Immunol., October 1, 2005; 175(7): 4247 - 4254. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Th. den Boer, G. J. D. van Mierlo, M. F. Fransen, C. J. M. Melief, R. Offringa, and R. E. M. Toes The Tumoricidal Activity of Memory CD8+ T Cells Is Hampered by Persistent Systemic Antigen, but Full Functional Capacity Is Regained in an Antigen-Free Environment J. Immunol., May 15, 2004; 172(10): 6074 - 6079. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S.-C. Yang, S. Hillinger, K. Riedl, L. Zhang, L. Zhu, M. Huang, K. Atianzar, B. Y. Kuo, B. Gardner, R. K. Batra, et al. Intratumoral Administration of Dendritic Cells Overexpressing CCL21 Generates Systemic Antitumor Responses and Confers Tumor Immunity Clin. Cancer Res., April 15, 2004; 10(8): 2891 - 2901. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Ramirez, Y. Ghani, and H. Stauss Incomplete tolerance to the tumour-associated antigen MDM2 Int. Immunol., February 1, 2004; 16(2): 327 - 334. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Schmidt, C. Ziske, A. Marten, S. Endres, K. Tiemann, V. Schmitz, M. Gorschluter, C. Schneider, T. Sauerbruch, and I. G. H. Schmidt-Wolf Intratumoral Immunization with Tumor RNA-Pulsed Dendritic Cells Confers Antitumor Immunity in a C57BL/6 Pancreatic Murine Tumor Model Cancer Res., December 15, 2003; 63(24): 8962 - 8967. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Dadaglio, S. Morel, C. Bauche, Z. Moukrim, F. A. Lemonnier, B. J. Van den Eynde, D. Ladant, and C. Leclerc Recombinant adenylate cyclase toxin of Bordetella pertussis induces cytotoxic T lymphocyte responses against HLA*0201-restricted melanoma epitopes Int. Immunol., December 1, 2003; 15(12): 1423 - 1430. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zwaveling, S. C. F. Mota, J. Nouta, M. Johnson, G. B. Lipford, R. Offringa, S. H. van der Burg, and C. J. M. Melief Established Human Papillomavirus Type 16-Expressing Tumors Are Effectively Eradicated Following Vaccination with Long Peptides J. Immunol., July 1, 2002; 169(1): 350 - 358. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Parmiani, C. Castelli, P. Dalerba, R. Mortarini, L. Rivoltini, F. M. Marincola, and A. Anichini Cancer Immunotherapy With Peptide-Based Vaccines: What Have We Achieved? Where Are We Going? J Natl Cancer Inst, June 5, 2002; 94(11): 805 - 818. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. G. Chikh, S. Kong, M. B. Bally, J.-C. Meunier, and M.-P. M. Schutze-Redelmeier Efficient Delivery of Antennapedia Homeodomain Fused to CTL Epitope with Liposomes into Dendritic Cells Results in the Activation of CD8+ T Cells J. Immunol., December 1, 2001; 167(11): 6462 - 6470. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Th. den Boer, L. Diehl, G. J. D. van Mierlo, E. I. H. van der Voort, M. F. Fransen, P. Krimpenfort, C. J. M. Melief, R. Offringa, and R. E. M. Toes Longevity of Antigen Presentation and Activation Status of APC Are Decisive Factors in the Balance Between CTL Immunity Versus Tolerance J. Immunol., September 1, 2001; 167(5): 2522 - 2528. |
