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Immunology |
Institute for Immunology [M. S., P. D., B. W., F. S., E. P. R.] and Department of Surgery [D. O.], Medical Faculty, Technical University of Dresden, 01101 Dresden, and Department of Immunology, Institute for Cell Biology, University of Tuebingen, 72076 Tuebingen [S. S., H-G. R.], Germany
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The number of well characterized TAAs has steadily increased during the last years and has led to their classification into different groups (15 , 16) . One major group that seems particularly promising for immunotherapeutic approaches is represented by TAAs encoded by genes that are silent in most normal tissues, except testicular cells, yet are expressed in a variety of tumor types. Members of this group are the genes encoding MAGE (17) , BAGE (18) , GAGE (19) , and RAGE (20) . In addition, the telomerase catalytic subunit was identified as a widely expressed TAA, which can also serve as a target for tumor-specific cytotoxic T-cell responses (21) . Recently, survivin, a structurally unique new member of the IAP gene family, has been described to be selectively expressed in fetal tissue and in tumor cells including carcinomas of the lung, colon, pancreas, prostate, breast, and stomach (22) . This observation lately has been confirmed by analysis of human transcriptomes (23) , where survivin was described as one of the most prominent tumor-associated transcripts.
In the present study, we investigated whether human CD8+ T lymphocytes can be specifically activated against survivin in vitro. A major objective was to find out whether MHC class I-restricted immunogenic peptides derived from survivin can be predicted. Here, we demonstrate that monocyte-derived DCs pulsed with recombinant survivin protein efficiently induce antigen-specific CD8+ CTLs. In addition, two predicted survivin-derived peptides proved to elicit a peptide-specific CTL response; one of the peptides is shown to result from natural intracellular processing of survivin.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overexpression and Purification of Recombinant His-tagged
Survivin.
Survivin cDNA was subcloned into the BamHI and
HindIII restriction sites of the procaryotic expression
vector pQE30 (Qiagen, Hilden, Germany), which allows the expression of
recombinant proteins with a NH2-terminal 6x
His-tag. The correct sequence was checked using the ALF-express Auto
Read sequencing kit with cyanine-labeled dATP and pQE30-specific
primers. Overexpression was performed in Escherichia coli
M15[pREP4] (Qiagen), as recommended by the manufacturer. A control
culture was grown under the same conditions without induction by
isopropyl-1-thio-ß-D-galactopyranoside.
Cells were harvested by centrifugation at 2000 x g and 4°C for 10 min and resuspended in 7.5 ml of lysis
buffer [8 M urea, 100 mM
Na2HPO4, 10
mM Tris (pH 7.8), 0,1% Triton X-100, and 25
mM imidazole]. After sonication, cellular debris
were pelleted at 13,000 x g for 30 min.
Purification was carried on by mixing the cleared lysate with 2 ml of
the equilibrated Ni-NTA resin (Qiagen) on a rotator for 1 h,
followed by washing four times with lysis buffer and eluting with the
appropriate buffer containing 100 mM imidazole.
The His-tagged survivin was refolded by stepwise dialyzing against RPMI
1640 (Biochrom, Berlin, Germany). The protein yield was determined by
the Bradford protein assay (Bio-Rad Laboratories, Munich, Germany). The
lysates and eluates from both the expression and control culture were
analyzed by SDS-PAGE, followed by Coomassie Blue staining or Western
blotting. Western blotting was performed according to standard
protocols using anti-His antibody (mouse) as primary and alkaline
phosphatase-conjugated antimouse IgG as secondary antibody.
Transient Transfection of EBV-transformed B Lymphocytes with
Survivin.
The coding region of survivin cDNA was amplified by PCR using the
forward primer 5'-GAGAGAGAATTCACAACCATGGGTGCCCCGACGTTGCCC-3', the
reverse primer 5'-GAGAGAGGATCCTCAATCCATGGCAGCCAGGTGCTC-3' (both from
Genaxis Biotechnology, Spechbach, Germany), and pBsKS/survivin as
template under the same conditions as described above. EcoRI
and BamHI sites in the primers allowed the directional
cloning into pIRES2-EGFP (Clontech). This permits both survivin and
EGFP to be synthesized from a single bicistronic mRNA.
B lymphocytes were transformed by supernatant of the EBV-producing cell line B95–8 [kindly provided by Dr. J. Endl (Roche, Penzberg, Germany)]. EBV-BLCLs were transfected with pIRES2-survivin/EGFP or pIRES2-EGFP by electroporation. Cell suspension (400 µl ; 5 x 106 cells/ml) was transfected with 50 µg of supercoiled plasmid DNA at 280 V/1050 µF (Easyject T Plus; Equibio, Kent, United Kingdom) and then cultivated in RPMI 1640 supplemented with 10% FCS (both from Biochrom). Eighteen to 24 h after transfection, cells expressing EGFP were selected by FACS and used as target cells.
Epitope Prediction.
Epitope prediction was done as described (24)
. Briefly,
potential HLA-A*0201 ligands from the sequence of survivin were
selected using a matrix pattern suitable for the calculation of nonamer
or decamer peptides fitting to the HLA-A*0201 motif. Such motif
predictions are available on our web
page.5
Peptides.
Peptides were synthesized in an automated peptide synthesizer 432A
(Applied Biosystems, Weiterstadt, Germany) following the Fmoc/tBu
strategy. After removal from the resin by treatment with
trifluoroacetic acid/phenol/ethanedithiol/thioanisole/water
(90:3.75:1.25:2.5:2.5 by vol) for 1 h or 3 h,
arginine-containing peptides were precipitated from methyl-tert. butyl
ether, washed once with methyl-tert. butyl ether and twice with diethyl
ether, and resuspended in water prior to lyophilization. Synthesis
products were analyzed by high-performance liquid chromatography
(system gold; Beckman Instruments, Munich, Germany) and matrix-assisted
laser desorption ionization-time of flight mass spectrometry (G2025A;
Hewlett-Packard, Waldbronn, Germany). Peptides of <80% purity were
purified by preparative high-performance liquid chromatography.
MHC Stabilization Assay.
The MHC stabilization assay was performed in 96-well microtiter plates
with 105
T2 cells (25)
per well
incubated overnight in 100 µl of RPMI 1640 without FCS. Peptide
concentration during incubation was 100 µM or 10
µM. Unbound peptide was washed away, and surface MHC
molecules were stained by incubation with the HLA class I-specific
antibody W6/32, followed by incubation with a FITC-labeled mouse
immunoglobulin-specific antibody (Dianova, Hamburg, Germany).
Fluorescence intensities were recorded in a FACSCALIBUR flow cytometer
(Becton Dickinson, Heidelberg, Germany).
In Vitro Generation of Survivin-specific T
Lymphocytes.
Blood was obtained from three healthy donors with the following HLA
types: JB: HLA-A*0201/0101, HLA-B*0801/1501, HLA-Cw7, HLA-Cw3,
HLA-DRB1*0301/0401, HLA-DQA1*0501/0303, HLA-DQB1*0201/0301; FS:
HLA-A*0201/2301, HLA-B*4403/2705, HLA-C*0102/0103, HLA-DRB1*0101/0701,
HLA-DQB1*0501/0202; MS: HLA-A*0206/0101, HLA-B*4402/0801,
HLA-C*0701–0712, HLA-DRB1*09012/1501, HLA-DQB1*0303/0602. Peripheral
blood mononuclear cells were purified from 100 ml of peripheral blood
by Ficoll Hypaque (Pharmacia, Freiburg, Germany) density centrifugation
with the informed consent of blood donors. Monocytes were isolated by
immunomagnetic cell separation with anti-CD14 antibody coupled to
magnetic microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). To
generate immature DCs, monocytes were cultured in the presence of 1000
units/ml granulocyte macrophage colony-stimulating factor and 1000
units/ml IL-4 (both from Strathmann Biotech, Hannover, Germany) in
X-VIVO medium (BioWhittaker, Walkersville, MD) supplemented with 10%
human serum (CC pro; Neustadt, Germany) for 7 days. After the
addition of 5–10 µg/ml recombinant survivin protein, DCs were
cultured for an additional 3 days in the presence of 1000 units/ml
granulocyte macrophage colony-stimulating factor, 1000 units/ml IL-4,
10 ng/ml IL-1ß, 10 ng/ml tumor necrosis factor-
(all from
Strathmann Biotech), and 1 µg/ml PGE2
(Sigma-Aldrich, Steinheim, Germany) in X-VIVO medium supplemented with
human serum for further maturation. For T-cell activation,
CD8+ T lymphocytes were enriched by positive
selection using an anti-CD8 antibody coupled to magnetic microbeads
(Miltenyi Biotech). Protein-loaded DCs (5 x 105
) were cocultured with 2 x 106 CD8+ T cells in 2 ml of
RPMI 1640 supplemented with 10% human serum per well of a 24-well
tissue culture plate (Greiner, Frickenhausen, Germany). Seven days
later, cultures were washed and restimulated with freshly prepared
survivin-loaded DCs at a responder to stimulator ratio of 2:1 and
supplemented with 25 units/ml recombinant human IL-2 and 10
ng/ml recombinant human IL-7 (both from Strathmann Biotech).
After six to eight cycles of stimulation, the cultures were tested for
survivin-reactive T cells.
For activation of CD8+ T cells against selected survivin peptides, mature autologous monocyte-derived DCs were pulsed with a mixture of two predicted peptides, each at a concentration of 50 µg/ml in serum-free X-VIVO medium for 4 h at 37°C. Stimulation of enriched CD8+ T cells was performed as described above. After four to six cycles of stimulation, the cultures were assayed for survivin peptide-reactive T cells.
Chromium Release Assay.
Cytotoxic activity of the in vitro-stimulated CTLs was
tested against survivin-transfected autologous EBV-BLCLs as targets in
a 4-h standard 51Cr- release assay. Briefly,
EBV-BLCLs were transfected with pIRES2-survivin/EGFP or pIRES2-EGFP and
were selected by FACS. After washing, 1 x 106 EBV-BLCLs were labeled for 1 h at 37°C
with 100 µCi 51Cr (sodium chromate; NEN,
Zaventem, Belgium) in 1 ml of RPMI 1640. To assay the cytotoxic
activity of cultured T cells against predicted survivin peptides, the
HLA-A2-positive mutant cell line T2 was incubated for 4 h with
individual peptides at a concentration of 50 µg/ml, washed three
times, and then used as target. Chromium-labeled target cells were
washed three times and plated in round-bottomed 96-well plates at
5 x 103 cells/well. Effector
cells were added as triplicates at various E:T ratios. After 4 h
of incubation, 100 µl of supernatant was collected from each well and
the released 51Cr was determined in a ß-plate
scintillator (Wallac, Freiburg, Germany). Maximal and spontaneous
release were measured by treating labeled cells with 1% NP40 or medium
alone, respectively. The specific cytotoxicity was calculated according
to the formula:
Percent specific lysis = 100 x [(cpm experimental release - cpm spontaneous release)/(cpm maximal release - cpm spontaneous release)].
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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20 kDa, as detected
by Coomassie Blue staining and by Western blotting using anti-His
antibody (Fig. 1)
. In this experimental setting, DCs exposed to survivin were supposed
to take up and process the protein for cross-presentation as MHC class
I-bound peptides. To prove that CTLs were raised against survivin
peptides resulting from intracellular processing and not against
contaminating peptides contained in the survivin preparation, we had to
choose target cells exposing peptides that are bona fide derived from
survivin by intracellular processing. To verify survivin specificity of
activated CTLs, survivin-negative homologous target cells had to be
included. To fulfill these requirements, we decided to transfect
autologous EBV-BLCLs with the cDNA for survivin because in
nontransfected EBV-BLCLs survivin-mRNA was not detectable by PCR (data
not shown). To evaluate the transfection efficiency and to enrich the
survivin-expressing cells, cDNA coding for EGFP was included by using
the bicistronic vector pIRES2-survivin/EGFP. Transfection efficiencies
were between 10% and 20%, as determined by flow cytometry (Fig. 2A)
, and survivin-positive target cells could be enriched to a
purity of 80–90% by FACS (Fig. 2B)
. EBV-BLCLs transfected
with the control vector pIRES2-EGFP were treated in the same way,
resulting in similar transfection and sorting efficiencies (Fig. 2, C and D)
. The transfected cells served as target
cells in a chromium release cytotoxicity assay. Fig. 3
shows marked lysis of pIRES2-survivin/EGFP-transfected and enriched
EBV-BLCLs by survivin-activated CD8+ CTLs of both
donors, whereas only background activity was seen against
pIRES2-EGFP-transfected EBV-BLCLs. These results reveal that
survivin-specific CTLs can be generated from T-cell populations of
healthy donors when cultured with monocyte-derived DCs pulsed with
soluble survivin.
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, peptide-stimulated CD8+ T lymphocytes from donor
JB efficiently lysed T2 target cells loaded with either peptide
ELTLGEFLKL or peptide TLPPAWQPFL. CTLs from donor FS showed marked
lysis of T2 cells only when pulsed with peptide ELTLGEFLKL, but failed
to recognize peptide TLPPAWQPFL. To determine whether the predicted
survivin peptides were endogenously produced,
CD8+ T cells were tested against EBV-BLCLs
transfected with pIRES2-survivin/EGFP or pIRES2-EGFP. As shown in Fig. 5A
, responder T cells of both donors efficiently lysed
EBV-BLCLs transfected with pIRES2-survivin/EGFP, whereas no
cytotoxicity was observed against EBV-BLCLs transfected with
pIRES2-EGFP. Because CTLs of both donors recognized the survivin
peptide ELTLGEFLKL it can be concluded that at least this peptide
results from intracellular processing. Its presentation by HLA-A*0201
molecules is evidenced by partial inhibition of lysis in the presence
of HLA-A2-specific monoclonal antibody MA 2.1 (Fig. 5B)
. In
addition, we tested whether the survivin-reactive CTLs induced by
protein-loaded DCs can recognize the two survivin-derived peptides. As
shown in Fig. 6
, CTLs of donor JB efficiently lysed T2 cells loaded with peptide
ELTLGEFLKL, but not T2 cells loaded with the peptide TLPPAWQPFL. In
contrast, CTLs of donor FS failed to recognize survivin peptide-pulsed
T2 cells. The lysis of T2 cells pulsed with peptide ELTLGEFLKL by CTLs
of donor JB after induction by protein-pulsed DCs confirm the
endogenous processing and MHC class I-restricted presentation of this
survivin peptide.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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After incubation with whole tumor proteins, DCs process the endocytosed proteins to peptides that are bound to MHC class II molecules and are presented on the cell surface for stimulation of CD4+ T helper lymphocytes. In addition to this classical mechanism of antigen presentation, DCs turned out to be capable of processing exogenous proteins by an alternative pathway leading to peptide presentation on MHC class I molecules, which is referred to as cross-presentation (7 , 26) . The efficient induction of antigen-specific CTL responses by protein-pulsed DCs was demonstrated by recent in vivo experiments (27 , 28) . In the present study, DCs incubated with soluble recombinant survivin were shown to induce specific MHC class I-restricted CTLs. To prove that these CTLs were induced by survivin peptides cross-presented by DCs, we chose autologous EBV-BLCLs as targets that had been transfected with cDNA for both survivin and EGFP using a bicistronic vector construct. We reasoned that this approach might be of 2-fold advantage. First, it provided appropriate control target cells because EBV-BLCLs not transfected or only transfected with the EGFP-cDNA did not express survivin. Second, the transiently transfected EBV-BLCLs could be enriched by FACS to obtain a suitable survivin-expressing target cell population. Transient transfection with the bicistronic EGFP-cDNA-containing vector combined with FACS seems to be particularly useful in experiments where high-frequency expression of proteins is attempted without establishing a stably transfected cell line. When assayed against these transfected EBV-BLCL targets, CTLs generated by stimulation with survivin-pulsed DCs performed their cytotoxicity in a survivin-dependent manner. From these results we conclude that monocyte-derived DCs cross-present survivin peptides and are cabable to efficiently induce survivin-specific CTLs from lymphocytes of healthy donors.
A whole array of strategies in experimental immunotherapy of cancer are based on peptide vaccination. Because survivin seems to be an almost universal TAA (22 , 23) it seemed an important task to identify relevant HLA class I-restricted survivin peptides. In a first approach we made a search for survivin peptides displaying the HLA A*0201 peptide motif, because HLA A*0201 is the most frequent HLA-A allele in Caucasian individuals. Among five scoring peptides, two peptides (ELTLGEFLKL and TLPPAWQPFL) were identified that showed significant stabilization of HLA A*0201 molecules at the surface of T2 cells. Both peptides proved to be immunogenic because they induced specific CTLs in vitro. The peptide-induced CTLs of both donors included in the experiments were found to lyse T2 cells pulsed with the peptide ELTLGEFLKL as well as EBV-BLCLs transfected with pIRES2-survivin/EGFP. From these data we conclude that at least this peptide is generated by natural processing of endogenously synthesized survivin. The finding that CD8+ T cells of one donor (FS) were activated only against one of the predicted peptides (ELTLGEFLKL) and not against the other peptide (TLPPAWQPFL) is in line with the general observation that individual healthy blood donors and also tumor patients often fail to show an in vitro CTL response against a selected peptide. This selective unresponsiveness might be due either to the low frequency of cognate precursors in the blood or to an overall affinity of the T-cell receptors being too low to induce T-cell activation. Finally, the failure of T cells to be activated by a peptide in vitro can be caused by a specific T-cell anergy or tolerance that may be induced by homologous or altered peptides derived from constitutively expressed proteins.
When CD8+ T cells of donor FS were activated by DCs loaded with whole survivin protein they turned out to be unable to recognize either one of the two peptides presented on T2 cells, whereas T cells from donor JB subjected to an identical activation procedure revealed a specific reactivity against the peptide ELTLGEFLKL, but not against the second predicted peptide TLPPAWQPFL. Because CTLs of both donors stimulated by survivin-loaded DCs were able to lyse survivin-expressing target cells the failure of donor FS to recognize the two selected HLA A*0201-bound peptides may be due to an individually different pattern of survivin-derived peptides, which are presented by various HLA-class I molecules on DCs after processing for cross-presentation.
To further substantiate the survivin peptide ELTLGEFLKL as a potent candidate antigen for immunotherapy of tumors it remains to be shown that it is naturally exposed on tumor cells for recognition by CTLs. Because most prepared tumor tissues (22) and all tumor cell lines (29) seem to express survivin, a survivin-negative target cell that could serve as an appropriate specificity control is missing. For this reason, specific recognition of survivin on tumor cells can only be proven by the use of cloned CTLs that are not yet available.
In summary, we show here for the first time the specific activation of CD8+ CTLs against the widely occurring tumor-associated protein survivin. Thus, survivin recommends itself as a universal antigen for the design of protein- and peptide-based anticancer vaccines.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by Grant 99.009.1 from the Wilhelm
Sander-Stiftung (to M. S. and E. P. R.) and by the Medical Faculty,
Technical University, Dresden (to M. S. and P. D.). 
2 These authors contributed equally to this
work.
3 To whom requests for reprints should be
addressed, at Institute for Immunology, Technical University of
Dresden, P.O. Box 800115, 01101 Dresden, Germany. Phone:
49-351-8832777; Fax: 49-351-8832778; E-mail: rieber{at}rcs.urz.tu-dresden.de
4 The abbreviations used are: TAA,
tumor-associated antigen; DC, dendritic cell; EGFP, enhanced green
fluorescent protein; EBV-BLCL, EBV-B lymphoblastoid cell line; IL,
interleukin; FACS, fluorescence-activated cell sorting.
5 http://www.uni-tuebingen.de/uni/kxi.
Received 2/14/00. Accepted 7/ 5/00.
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 A. Temme, M. Rieger, F. Reber, D. Lindemann, B. Weigle, P. Diestelkoetter-Bachert, G. Ehninger, M. Tatsuka, Y. Terada, and E. P. Rieber Localization, Dynamics, and Function of Survivin Revealed by Expression of Functional SurvivinDsRed Fusion Proteins in the Living Cell Mol. Biol. Cell, January 1, 2003; 14(1): 78 - 92. [Abstract] [Full Text]
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C. Xia, Z. Xu, X. Yuan, K. Uematsu, L. You, K. Li, L. Li, F. McCormick, and D. M. Jablons Induction of Apoptosis in Mesothelioma Cells by Antisurvivin Oligonucleotides Mol. Cancer Ther., July 1, 2002; 1(9): 687 - 694. [Abstract] [Full Text] [PDF]
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Y. Hirohashi, T. Torigoe, A. Maeda, Y. Nabeta, K. Kamiguchi, T. Sato, J. Yoda, H. Ikeda, K. Hirata, N. Yamanaka, et al. An HLA-A24-restricted Cytotoxic T Lymphocyte Epitope of a Tumor-associated Protein, Survivin Clin. Cancer Res., June 1, 2002; 8(6): 1731 - 1739. [Abstract] [Full Text] [PDF]
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A. Yagihashi, K. Asanuma, M. Nakamura, J. Araya, Y. Mano, T. Torigoe, D. Kobayashi, and N. Watanabe Detection of Anti-Survivin Antibody in Gastrointestinal Cancer Patients Clin. Chem., September 1, 2001; 47(9): 1729 - 1731. [Full Text] [PDF]
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M. H. Andersen, L. O. Pedersen, B. Capeller, E.-B. Brocker, J. C. Becker, and P. thor Straten Spontaneous Cytotoxic T-Cell Responses against Survivin-derived MHC Class I-restricted T-Cell Epitopes in Situ As Well As ex Vivo in Cancer Patients Cancer Res., August 1, 2001; 61(16): 5964 - 5968. [Abstract] [Full Text] [PDF]
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