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Immunology |
Department of Tumor Immunology, University Medical Center St. Radboud, Philips van Leydenlaan 25, 6525 EX Nijmegen [A. A. O. E., A. J. d. B., J. L. M. V., C. G. F., G. J. A.], Department of Immunohematology and Blood Bank, Leiden University Medical Centre, 2300 RC Leiden [T. v. H., R. O.], The Netherlands
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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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CTLs are considered as major effectors cells in the eradication of tumor cells in vivo (1) . At present, different vaccination strategies for the induction of CTL-mediated antitumor immunity are available, including the use of synthetic peptides, naked plasmid DNA, and antigen-loaded DCs. These strategies have been used successfully in experimental animal models to induce tumor-reactive CTL responses in vivo, however, primarily directed against relatively immunogenic foreign or mutated antigens (3, 4, 5) . In contrast, autologous nonmutated tumor antigens have proven to be poorly immunogenic, most probably because of the involvement of peripheral tolerance (6, 7, 8) . A potential candidate to break tolerance against nonmutated antigen and activate high avidity CTLs is a DC-based vaccine. It is currently well appreciated that DCs are highly immunostimulatory antigen-presenting cells capable of activating resting T cells. Indeed, both human and animal studies have demonstrated that DCs can efficiently mediate the induction of antitumor immunity (reviewed in Refs. 9 and 10 ).
The aim of this study was to evaluate DCs loaded with nonmutated CTL-defined epitopes derived from a melanocyte differentiation antigen as an antitumor vaccine in an autologous mouse model and compare its efficacy to other established vaccination strategies. The mouse TRP2 antigen (mTRP2), endogenously expressed by wild-type B16 melanoma as well as normal melanocytes, was chosen as a model differentiation antigen. Both human and mouse antimelanoma CTLs have been shown previously to recognize conserved nonmutated epitopes derived from TRP2 (11 , 12) , which renders mTRP2 an ideal model antigen to design antimelanoma vaccines in a fully autologous model for clinical application. The results demonstrate that, within this autologous tumor model, tolerance against mTRP2 can be overcome by peptide-loaded DCs but not by peptide in Freund’s adjuvant or plasmid DNA. Activation of high avidity CTLs mediating protective antitumor immunity in vivo without the development of adverse autoimmunity was observed upon DC vaccination, indicating the potency as well as the value of DC-based vaccines for immunotherapy of cancer.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The murine thymoma cell line EL4 (ATCC) was cultured in Iscove’s medium (Life Technologies, Inc., Paisley, United Kingdom), supplemented with 5% FCS and 50 µM ß-mercaptoethanol. The murine melanoma cell line B16, subline F10 (13) , was grown in MEM, supplemented with MEM nonessential amino acids, MEM vitamin mix, 1 mM sodium pyruvate, 0.15% sodium bicarbonate, and 5% heat-inactivated FCS (Life Technologies, Inc.). The immortalized C57BL/6 melanocyte line Melan-A (14) was grown as described for B16. Normal epidermal melanocytes were isolated from neonatal C57BL/6 mice and cultured as described (14 , 15) . Primary cultures of melanocytes are further referred to as MCT. H-2Kb-expressing HeLa cells (HeLa-Kb) were generated and cultured as described (16) .
For detection of mouse TRP2-reactive serum IgG, acetone-fixed cytospins
of Melan-A and B16 cells were incubated for 1 h with various
dilutions of serum isolated from vaccinated mice, 2 weeks after the
second vaccination. Specific staining was visualized by
immunoperoxidase substrate reaction as described previously
(4)
. Polyclonal rabbit antibody 
PEP8 (generously
provided by Dr. V. Hearing, National Cancer Institute, Bethesda, MD)
was used as a control for mouse TRP2-specific immunoperoxidase
staining.
Peptides and MHC Class I Binding Assays.
Peptides were synthesized with a free COOH terminus either by f-moc
peptide chemistry using a ABIMED Multiple Synthesizer or by t-boc
chemistry on a Biosearch SAM2 peptide synthesizer. Peptides were >90%
pure as indicated by analytical HPLC. The peptides were dissolved in
DMSO and stored at -20°C.
Peptide binding to H-2Kb was determined using the RMA/S-based MHC class I stabilization assay as described (17) . Briefly, RMA/S cells (ATCC), cultured as described for EL4, were incubated at room temperature for 36 h, pulsed with peptide at the indicated concentrations for 1 h at room temperature, and subsequently incubated for 4 h at 37°C to allow turnover of "empty" MHC class I molecules. MHC class I stabilization was determined with H-2Kb-specific monoclonal antibody B8-24-3 (ATCC), followed by FITC-conjugated goat-antimouse F(ab)2 (Zymed Laboratory, South San Francisco, CA) and analyzed by flow cytometry (FACScan; Becton Dickinson, Hamburg, Germany).
Peptide binding to HLA-A2.1 was determined using the JY-based MHC class I binding assay as described (18) . Briefly, HLA-A2.1-presented peptides were stripped from JY cells (ATCC), cultured as described for EL4, by mild acid elution. After washing, JY cells were incubated with a mixture of fluorescein-labeled hepatitis B virus core antigen-derived reference peptide (FLPSDC[-fl]FPSV) and different concentrations of competitor peptide for 24 h at 4°C, followed by flow cytometric analysis of fluorescence intensity. Binding capacity of competitor peptides was determined as the concentration needed for 50% reference peptide binding inhibition (IC50).
DC Culture and Vaccination Procedures.
DCs were generated as described previously (5
, 19)
with
minor modifications. Briefly, lymphocyte-depleted bone marrow was
cultured overnight in six-well plates (Costar, Badhoevedorp, The
Netherlands) at 3–4 x 106
cell/well in CM consisting of Iscove’s modified DMEM, supplemented
with 10% heat inactivated FCS, 50 µM
ß-mercaptoethanol, and antibiotics (Life Technologies, Inc.).
Nonadherent cells were harvested, resuspended in CM containing 20 ng/ml
recombinant mouse granulocyte/macrophage-colony stimulating factor and
IL-4 (kindly provided by Dr. G. Zurawski, DNAX, Palo Alto, CA) and
cultured in six-well plates at 0.5–1 x 106 cells/well. Fresh cytokines were given on day
3. Nonadherent and loosely adherent clusters of proliferating DCs were
harvested on day 6, resuspended in fresh CM containing 10 ng/ml
granulocyte/macrophage-colony stimulating factor and IL-4, and cultured
for 2 additional days in six-well plates. For experimental use,
nonadherent DCs were harvested on day 8. Prior to vaccination, DCs were
loaded in Optimem (Life Technologies, Inc.) for 1 h at 37°C,
followed by 2 h at room temperature with 25 µM
peptide in the presence of 3 µg/ml human
ß2-microglobulin (Symbus Bioscience,
Southhampton, United Kingdom). Peptide-loaded DCs were washed twice in
saline, irradiated (25 Gy), and injected s.c. in the left flank
(4 x 105
in 0.2 ml saline).
Construct pCMV-mTRP2 for genetic vaccination was generated by cloning the mouse TRP2 cDNA (generously provided by Dr. V. Hearing) in the coding orientation into the eukaryotic expression vector pCMV-neo (20) . Mice were injected intradermally in the abdominal skin with 100 µg of affinity purified (Qiagen Plasmid Mega kit, Westburg, Leusden, The Netherlands) plasmid DNA in 0.1 ml of saline. Synthetic peptides (100 µg in 0.1 ml of saline) were emulsified with an equal volume of incomplete Freund’s adjuvant (Difco Laboratory, Detroit, MI) and injected s.c. in the left flank.
All vaccinations were performed twice with a 2-week interval. Two weeks after the second vaccination, mice were challenged s.c. with 1 x 105 live B16 tumor cells in 0.1 ml of saline in the right flank. The size of growing tumors was monitored every 3 days.
CTL Culture and Chromium Release Assay.
Two weeks after the second DC vaccination, spleens were isolated from
vaccinated mice, and 4 x 107
single-cell splenocytes were restimulated with 1 x 107 irradiated (25 Gy), peptide-loaded LPS blasts
in T25 culture flasks (Costar). LPS blasts were generated from
splenocytes during 3 days of culture in the presence of 25 µg/ml LPS
from Salmonella typhosa (Sigma) and 7 µg/ml dextran
sulfate and loaded with peptide as described above for DCs. Bulk CTLs
were isolated after 6 days restimulation by density gradient
centrifugation (Lympholyte-M; Cedarlane Laboratory, Sanbio, Uden, The
Netherlands). CTL lines were generated by weekly restimulation of
2–5 x 105
bulk CTLs/well with
1 x 105
recombinant rat IFN-
(TNO, Rijswijk, The Netherlands) treated (50 units/ml for 48 h),
irradiated (125 Gy) B16 cells in the presence of 1.5 x 106 irradiated (25 Gy) autologous splenocytes and
10 Cetus units/ml of recombinant human IL-2 (Cetus Corp., Emeryville,
CA) in 24-well culture plates (Costar).
Bulk CTLs and CTL lines were used as effectors in a chromium release
assay, performed as described previously (21)
. Briefly,
2 x 103
Na2[51Cr]O4
(Amersham, Buckinghamshire, United Kingdom)-labeled target cells were
cultured with various amounts of effector cells in triplicate wells in
U-bottomed microtiter plates (Costar). After 5 h of incubation,
the radioactive content of the supernatant was measured. When used in a
chromium release assay, B16, Melan-A, and MCT were pretreated with
IFN- as described above.
Analysis of Surface Expressed, Naturally Processed Peptides.
Surface expressed, MHC class I-associated peptides were isolated from
IFN--treated B16 melanoma cells by mild acid elution as described
(22)
. Eluted peptides were subjected to reverse-phase
HPLC-mediated fractionation using a protocol described previously
(23)
. Isolated peptide fractions were loaded onto
HeLa-Kb target cells and tested for stimulation
of CTL lytic activity in a chromium release assay as described above. A
mixture of synthetic mTRP2-derived peptides (10 µg each) was
subjected to HPLC fractionation using identical conditions as for
B16-eluted peptides.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The results indeed demonstrate a
significant increase in binding capacity for Kb
(Fig. 1A)
and HLA-A2.1 (Fig. 1B)
upon
NH2-terminal extension of VYDFFVWL to
SVYDFFVWL. Consequently, two nonmutated CTL epitopes with differential
binding capacity derived from a conserved region of the differentiation
antigen TRP2 are available to explore the induction of CTL-mediated
antitumor immunity with clinical relevance to the human setting.
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and B, after a single in vitro restimulation of
immune splenocytes, bulk CTLs against either the VYDFFVWL or the
SVYDFFVWL peptide specifically lyse target cells loaded with either of
the TRP2-derived epitopes. Moreover, bulk CTLs also lysed B16 melanoma
cells, indicating recognition of endogenously processed mTRP2-derived
epitopes. Bulk CTLs generated from mice vaccinated with DCs loaded with
an irrelevant peptide using similar conditions did not lyse the
indicated target cells above background level (not shown). Similar
results were obtained with
CD3+CD4-CD8+TCRß+
CTL lines LP8 (VYDFFVWL-induced) and LP9 (SVYDFFVWL-induced), generated
by identical repetitive in vitro restimulation of bulk CTLs
(Fig. 2, C
and D, respectively).
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. As
shown in Fig. 3
A, loading of target cells with an irrelevant
peptide did not result in detectable lytic activity. Recognition of the
VYDFFVWL peptide started to decrease at 1 µM
and was completely lost at 10-5
µM (Fig. 3B)
. In contrast, SVYDFFVWL
peptide concentrations as low as 10-5
µM still resulted in stimulation of lytic
activity by both CTL lines tested (Fig. 3C)
, in agreement
with the observed differential binding capacity of both peptides
depicted in Fig. 1
. These data point out that DC-induced CTLs against
nonmutated epitopes derived from autologous antigen can display highly
efficient lytic activity.
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. The
data demonstrate that target cells loaded with peptide fractions
104–109 are recognized by TRP-specific CTL lines LP8 and LP9. Analysis
of a mixture of synthetic VYDFFVWL peptide and SVYDFFVWL peptide
fractionated using identical HPLC conditions as for the B16-presented
epitopes indicates that the majority of VYDFFVWL peptide peaks in
fraction 105, whereas the majority of SVYDFFVWL peptide peaks in
fraction 109 (Fig. 4B)
. These results indicate that both
mTRP2-derived peptides are presented at the surface of B16 melanoma
cells, suggesting that both VYDFFVWL and SVYDFFVWL are naturally
processed by B16 melanoma. The observed difference in lytic activity
against fractions 104–109 in Fig. 4A
remains to be
investigated but probably involves differential processing as well as
elution efficiency of both epitopes.
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Fig. 5
shows the induction of protective
antitumor immunity in vivo by different vaccination
strategies, as determined by challenge with a lethal s.c. dose of
wild-type B16F10 melanoma cells. Vaccination with DCs loaded with an
irrelevant H-2Kb binding peptide as a negative
specificity control did not mediate protection against a B16 tumor
challenge in any of the mice tested. In contrast, partial tumor
protection of 20% was observed upon prophylactic vaccination with
VYDFFVWL-loaded DCs (Fig. 5A)
. The observed tumor protection
could be enhanced up to 80% when mice were vaccinated with
SVYDFFVWL-loaded DCs (Fig. 5A)
. These data indicate that the
observed protective immunity depends on loading of DCs with the
relevant, TRP2-derived peptides prior to vaccination. Furthermore, in
line with the results obtained in vitro as depicted above in
Figs. 1
and 3
, the difference in binding capacity of the peptides used
to load DCs prior to vaccination also appears to affect the level of
protective antitumor immunity in vivo. In contrast, both
peptide in Freund’s adjuvant (Fig. 5B)
and plasmid DNA
(Fig. 5C)
as alternative vaccination strategies failed to
induce significant protective immunity against B16 melanoma.
Apparently, peptide in adjuvant and plasmid DNA vaccination used as
such are not sufficiently effective to overcome tolerance against self
antigen in the current autologous tumor model. Notably, the successful
use of both vaccine procedures, as described in "Materials and
Methods," has been reported previously by us, eliminating the
involvement of suboptimal vaccination procedures (4
, 24)
.
These data emphasize the potency of DCs as a vaccine adjuvant to induce
tumor-reactive immunity in vivo directed against autologous
tumor-associated antigen.
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, both VYDFFVWL- and
SVYDFFVWL-induced CTLs (Fig. 6, A
and B,
respectively) display in vitro lytic activity against the
melanocyte cell line Melan-A as well as against normal melanocytes
isolated from epidermis (MCT), whereas the nonmelanocytic cell line EL4
is lysed at background levels. The level of melanocyte lysis observed
in Fig. 6
appears relatively low, despite sufficient expression levels
of both MHC class I and mTRP2 of the used melanocyte target cells as
determined by flow cytometric and immunocytochemical analysis (not
shown). In contrast to the observed in vitro melanocyte
destruction, in vivo coat depigmentation reflecting the
destruction of hair bulb-residing melanocytes was detected only
sporadically. Mild depigmentation developed in a minor fraction,
representing <10% of DC/TRP2 peptide-vaccinated mice, and was
primarily restricted to the vaccination site (not shown). The use of
nominal CTL epitopes for vaccination will primarily result in
activation of specific CTLs, however, and may also result in a
secondary antibody response. To exclude this, serum isolated from
vaccinated mice was analyzed for the presence of mTRP2-reactive IgG.
Immunoperoxidase staining of B16 and Melan-A failed to detect
mTRP2-specific IgG in the serum of vaccinated mice, suggesting that the
observed low level melanocyte destruction in vivo is CTL
mediated and that peptide-loaded DC vaccination did not result in the
production of potentially harmful autoantibodies (not shown).
Apparently, differentiation antigenspecific CTLs can eradicate
tumor cells in vivo without significantly affecting the
integrity of normal tissue.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Here, we present a fully autologous mouse tumor model to dissect essential requirements of effective induction of antitumor immunity against the melanocyte differentiation antigen TRP2, which is involved in mouse as well as human antimelanoma immunity. Two nonmutated TRP2-derived peptides, VYDFFVWL and the NH2terminal extended SVYDFFVWL, were used, both of which are fully conserved in mouse and human TRP2 and bind with different affinity to mouse H-2Kb and human HLA-A2.1. We used the poorly immunogenic, wild-type B16F10 mouse melanoma, which dictates a relatively stringent setting to accomplish protective antitumor immunity. It also facilitates relevant comparative analyses of established vaccination strategies. Both synthetic peptide in adjuvant and naked DNA have proven potent vaccination strategies for tumor-reactive CTL induction in mice and are currently evaluated in clinical studies. In the present autologous model, however, synthetic VYDFFVWL or SVYDFFVWL peptide in Freund’s incomplete adjuvant as well as mTRP2 encoding plasmid DNA failed to induce detectable protective immunity in vivo. Possibly, both vaccination strategies may not have reached the level of CTL activation and/or expansion required for protective immunity against B16 melanoma, although we have reported previously that both vaccination approaches can be successfully applied to induce CTL-mediated immunity in mice using more immunogenic antigens (4 , 24) . Our results obtained in this poorly immunogenic TRP2 model are in agreement with other mouse tumor models in which tumor-protective CTL induction against an autologous antigen failed, irrespective of the use of multiple vaccination strategies including synthetic peptides and naked DNA (6 , 8 , 28) or recombinant adenovirus and vaccinia virus (7 , 25 , 29) . In contrast, we show in this report that peptide-loaded DCs as a vaccine in this model did result in significant protective immunity, in line with a recent report that effective antimelanoma immunity could be induced by vaccination using DCs transduced with mTRP2-encoding adenovirus (30) . The negative specificity control, DCs loaded with an irrelevant peptide, further indicates that the observed protective immunity is fully dependent of DC loading with the relevant TRP2-derived peptides. Consequently, tumor protection resulting simply from immunity induced against irrelevant antigens, including FCS components, can be excluded. Because all three vaccination modalities were used at similar levels of optimalization, these results emphasize the immunogenic potential of DCs and point out the superiority and value of DCs as vaccine adjuvant when comparatively used in a fully autologous, clinically relevant setting.
Both TRP2-derived epitopes used in this study, VYDFFVWL and the NH2-terminal extended SVYDFFVWL, could be eluted from the surface of B16 melanoma cells, pointing out the truly autologous nature of the current tumor model. The observation that both epitopes are naturally processed and presented could result from differential NH2-terminal trimming of proteasome-processed TRP2. Aminopeptidases present in the endoplasmic reticulum may be responsible for generating both length variants upon transporter associated with antigen processing (TAP)-mediated translocation of a precursor peptide from the cytosol (31) . Alternatively, the presence of a subdominant proteolytic cleavage site between the NH2-terminal serine and valine residues may explain the presentation of both epitopes. Elucidation of these peptide-processing mechanisms would greatly enhance future prediction of dominant epitopes.
Analysis of the immune response induced by vaccination with TRP2 peptide-loaded DCs demonstrates the activation of high avidity CTLs after a single round of in vitro stimulation for both antigenic TRP2 peptides. These results indicate that the lack of endogenous immunogenicity of the TRP2 antigen can be overcome when DCs are used as vaccine adjuvant. Both epitopes mediated the induction of protective immunity against B16 melanoma in vivo when loaded onto DCs prior to vaccination. In contrast with the comparable avidity of VYDFFVWL- and SVYDFFVWL-induced CTLs in vitro, the level of protective immunity in vivo was higher for the high-affinity Kb binding peptide SVYDFFVWL. This may reflect the stringent conditions of the current autologous setting, comparable as encountered in the human setting. One may hypothesize that in the current model an increase in peptide binding capacity enhances the in vivo quantity of tumor-reactive CTLs upon vaccination, rather than the quality. Possibly, the increased influx of DCs still presenting the high-affinity TRP2 peptide in the draining lymph node positively affects the magnitude of the immune response induced. In agreement with this hypothesis, phenotypic analysis of both CTL lines LP8 and LP9 did not show significant differences in expression levels of T-cell receptor, adhesion, and other accessory molecules (not shown). However, previous data have indicated that qualitative differences in mTRP2-specific CTLs can be critical to their in vivo antitumor efficacy (32) . Most probably an interplay between CTL quality and quantity will ultimately dictate the efficacy of antitumor immunity in vivo. The recently developed MHC tetramer technology will provide a valuable tool to investigate the relative contribution of CTL quality and quantity in antitumor immunity.
The shared expression of melanocyte differentiation antigens between normal melanocytes and melanoma tumor cells may result in adverse autoimmunity mediated by differentiation antigen-directed immunity. Despite the observed in vitro lysis of normal melanocytes, albeit of relatively low level, by TRP2-specific CTLs in the current model, in vivo melanocyte destruction occurred only sporadically. In contrast to these findings, induction of immunity against mouse TRP1 (gp75) using human TRP1-encoding DNA (28) or mouse TRP1-encoding recombinant vaccinia virus (29) resulted not only in antitumor immunity but also in extensive coat depigmentation. In both studies, high titers of autoantibodies specific for mouse TRP1 were detected, and CD4+ but not CD8+ lymphocytes were shown to play an integral part in the development of TRP1-specific immunity. The results presented in this report indicate CD8+ CTLs as the predominant effector cells in the development of TRP2-specific immunity, appearing unable to mediate extensive autoimmunity. Recently, CTL-mediated depigmentation resulting from autoimmune destruction of normal melanocytes was reported in mice (8) . In the latter study, autoimmunity depended on perforin-mediated CTL lytic activity. Alternatively, skin-homing human melanocyte-reactive CTLs were shown to require expression of the skin homing receptor cutaneous lymphocyte-associated antigen for CTL-mediated melanocyte destruction in vivo (33) . The absence of extensive depigmentation in the current model may involve lack of CTL-expressed cutaneous lymphocyte-associated antigen and/or different CTL effector mechanisms.
Collectively, our results demonstrate that DCs loaded with nonmutated, naturally processed epitopes derived from a melanocyte differentiation antigen enable efficient activation of high avidity CTLs and induction of antitumor immunity in vivo without extensive autoimmunity against normal tissue. Within the same autologous model, alternative well-established vaccination strategies failed to induce protective immunity. As a result, DC vaccination holds its promise. It is a superior vaccine for clinical application to treat cancer patients, despite the relatively labor-intensive procedures required to generate DC vaccines. Encouraging results have already been obtained in melanoma patients injected with peptide-loaded DCs (34 , 35) . The autologous tumor model described herein will contribute to further optimalization of immunotherapy using DCs as vaccine adjuvant to break peripheral CTL tolerance and allow the use of unmodified self-antigen as an antitumor vaccine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported by Grant KUN 95-911
from the Dutch Cancer Society and Grant ERB FMRX CT960053 from the
European Community. 
2 Present address: Department of Immunology, The
Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The
Netherlands.
3 To whom requests for reprints should be
addressed, at Department of Tumor Immunology, University Hospital
Nijmegen St. Radboud, Philips van Leydenlaan 25, 6525 EX Nijmegen, The
Netherlands. Phone: 31-24-3617600; Fax: 31-24-3540339; E-mail: G.Adema{at}dent.kun.nl
4 The abbreviations used are: TRP,
tyrosinase-related protein; mTRP2, mouse TRP2; DC, dendritic cell;
ATCC, American Type Culture Collection; IL, interleukin; LPS,
lipopolysaccharide; HPLC, high-pressure liquid chromatography; CM,
culture medium; CMV, cytomegalovirus.
5 Internet address:
http://bimas.dcrt.nih.gov/molbio/hla_bind.
Received 3/13/00. Accepted 10/17/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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S. Nair, D. Boczkowski, B. Moeller, M. Dewhirst, J. Vieweg, and E. Gilboa Synergy between tumor immunotherapy and antiangiogenic therapy Blood, August 1, 2003; 102(3): 964 - 971. [Abstract] [Full Text] [PDF]
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E. Davila, R. Kennedy, and E. Celis Generation of Antitumor Immunity by Cytotoxic T Lymphocyte Epitope Peptide Vaccination, CpG-oligodeoxynucleotide Adjuvant, and CTLA-4 Blockade Cancer Res., June 15, 2003; 63(12): 3281 - 3288. [Abstract] [Full Text] [PDF]
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A. E. M. Dirkx, M. G. A. oude Egbrink, M. J. E. Kuijpers, S. T. van der Niet, V. V. T. Heijnen, J. C. A. B.-t. Steege, J. Wagstaff, and A. W. Griffioen Tumor Angiogenesis Modulates Leukocyte-Vessel Wall Interactions in Vivo by Reducing Endothelial Adhesion Molecule Expression Cancer Res., May 1, 2003; 63(9): 2322 - 2329. [Abstract] [Full Text] [PDF]
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 J. W. Hodge, D. W. Grosenbach, W. M. Aarts, D. J. Poole, and J. Schlom Vaccine Therapy of Established Tumors in the Absence of Autoimmunity Clin. Cancer Res., May 1, 2003; 9(5): 1837 - 1849. [Abstract] [Full Text] [PDF]
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 L. Samady, E. Costigliola, L. MacCormac, Y. McGrath, S. Cleverley, C. E. Lilley, J. Smith, D. S. Latchman, B. Chain, and R. S. Coffin Deletion of the Virion Host Shutoff Protein (vhs) from Herpes Simplex Virus (HSV) Relieves the Viral Block to Dendritic Cell Activation: Potential of vhs- HSV Vectors for Dendritic Cell-Mediated Immunotherapy J. Virol., March 15, 2003; 77(6): 3768 - 3776. [Abstract] [Full Text] [PDF]
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S. Oh, J. W. Hodge, J. D. Ahlers, D. S. Burke, J. Schlom, and J. A. Berzofsky Selective Induction of High Avidity CTL by Altering the Balance of Signals from APC J. Immunol., March 1, 2003; 170(5): 2523 - 2530. [Abstract] [Full Text] [PDF]
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M. Kalat, Z. Kupcu, S. Schuller, D. Zalusky, M. Zehetner, W. Paster, and T. Schweighoffer In Vivo Plasmid Electroporation Induces Tumor Antigen-specific CD8+ T-Cell Responses and Delays Tumor Growth in a Syngeneic Mouse Melanoma Model Cancer Res., October 1, 2002; 62(19): 5489 - 5494. [Abstract] [Full Text] [PDF]
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W. Paster, M. Kalat, M. Zehetner, and T. Schweighoffer Structural Elements of a Protein Antigen Determine Immunogenicity of the Embedded MHC Class I-Restricted T Cell Epitope J. Immunol., September 15, 2002; 169(6): 2937 - 2946. [Abstract] [Full Text] [PDF]
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D. W. Mullins, T. N. J. Bullock, T. A. Colella, V. V. Robila, and V. H. Engelhard Immune Responses to the HLA-A*0201-Restricted Epitopes of Tyrosinase and Glycoprotein 100 Enable Control of Melanoma Outgrowth in HLA-A*0201-Transgenic Mice J. Immunol., November 1, 2001; 167(9): 4853 - 4860. [Abstract] [Full Text] [PDF]
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 R. P.M. Sutmuller, L. M. van Duivenvoorde, A. van Elsas, T. N.M. Schumacher, M. E. Wildenberg, J. P. Allison, R. E.M. Toes, R. Offringa, and C. J.M. Melief Synergism of Cytotoxic T Lymphocyte-Associated Antigen 4 Blockade and Depletion of Cd25+ Regulatory T Cells in Antitumor Therapy Reveals Alternative Pathways for Suppression of Autoreactive Cytotoxic T Lymphocyte Responses J. Exp. Med., September 17, 2001; 194(6): 823 - 832. [Abstract] [Full Text] [PDF]
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A. van Elsas, R. P.M. Sutmuller, A. A. Hurwitz, J. Ziskin, J. Villasenor, J.-P. Medema, W. W. Overwijk, N. P. Restifo, C. J.M. Melief, R. Offringa, et al. Elucidating the Autoimmune and Antitumor Effector Mechanisms of a Treatment Based on Cytotoxic T Lymphocyte Antigen-4 Blockade in Combination with a B16 Melanoma Vaccine: Comparison of Prophylaxis and Therapy J. Exp. Med., August 20, 2001; 194(4): 481 - 490. [Abstract] [Full Text] [PDF]
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