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Immunology |
Institut for Cell Biology, Department Immunology [S. P., M. S., A. M., H-G. R., S. Ste.], and Department of Obstetrics and Gynecology, University of Tübingen [B. G., S. Stu., S. K., D. W.], 72076 Tübingen, Germany
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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induction were analyzed by the "Predict-Calibrate-Detect" approach, which combines epitope prediction and high-performance liquid chromatography mass spectrometry. One of the predicted epitopes, MAGE-A1278–286 (KVLEYVIKV), was found to be presented by HLA-A*0201, with an estimated copy number of 18 molecules/cell. HLA-A*0201 transgenic mice (HHD mice) were used to generate CTL lines that stained positive with an HLA-A*0201 tetramer folded around the KVLEYVIKV peptide and killed peptide-loaded mouse target cells expressing HLA-A*0201. IFN--treated or -nontreated HLA-A*0201 expressing HeLa cells transiently transfected with a plasmid expressing the MAGE-A1 gene stimulated in vitro cytokine production by the CTL lines. Moreover, IFN--treated KS24.22 cells, but not IFN--treated HLA-A*0201+ MAGE-A1- cells or IFN--treated HLA-A*0201- MAGE-A1+ cells, were killed by these CTLs. Thus, the combination of HLA epitope prediction, peptide analysis, and immunological methods is a powerful approach for the identification of tumor-associated epitopes. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40% of melanoma and in some other tumors (12)
subsequent to a genome-wide demethylation process that occurs in many cancers (13)
. To confirm the presentation of this epitope at the surface of tumor cells and its relevance for antitumor immunity, we generated peptide-specific CTLs from HHD mice (14
, 15)
. These mouse CTL lines are stimulated in vitro by HLA-A*0201+ cells transfected with the MAGE-A1 gene and kill specifically HLA-A*0201- and MAGE-A1-positive human tumor cells. |
MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3, transmembrane, and cytoplasmic domains of H-2Db (14)
to give HeLa/A2A2Db transfectants. EL4S3-Rob/HHD cells are mouse ß2m-deficient EL4 cells expressing the HHD molecule (14)
. The human breast carcinoma KS24 cell line (16
, 17)
was transfected with a plasmid coding for Her2/neu generating the KS24.22 cell line. The human breast carcinomas GAKL and K1HE were also derived from malignant pleural effusions of breast carcinoma patients. For these breast carcinoma cells, the expression of HLA-A*0201 was tested by immunofluorescence using the mAb BB7.2 (American Type Culture Collection, Manassas, VA) and by genomic PCR. The expression of the MAGE-A1 gene was tested by RT-PCR using the pair of primers (5'-CGGCCGAAGGAACCTGACCCAG-3' + 5'-GCTGGAACCCTCACTGGGTTGCC-3') that amplifies a 421-bp DNA fragment from the MAGE-A1 sequence (18)
. The human lymphoblastoid cell line 721 was described previously (19)
. All cells were kept in RPMI 1640 enriched with 10% FCS, 2 mM L-glutamine and 1% penicillin/streptomycin (Life Technologies, Inc.) supplemented at 1 mg/ml G418 (Life Technologies, Inc.) for all transfectants (KS24.22, HeLa/A2A2Db, and EL4S3Rob/HHD).
Epitope Prediction.
Prediction of potential HLA-A*0201 ligands was carried out as described (20)
. Briefly, proteins were screened against a matrix pattern, which evaluates every amino acid within nonamer or decamer peptides fitting the HLA-A*0201 motif. Anchor residues are given values of 10; other residues, 0–10, reflect amino acid preferences for certain positions within the peptide. The theoretical maximum score for a candidate peptide is 36; scores for abundant natural ligands are typically between 32 and 34. Such motif predictions are available using the database SYFPEITHI.6
Peptides.
Peptides were synthesized in an automated peptide synthesizer 432A (Applied Biosystems, Foster City, CA) following the 9-fluorenylmethyloxycarbonyl/tert-butyl (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 volume) for 1 or 3 h (arginine-containing peptides), peptides were precipitated from methyl-tert-butyl ether, washed once with methyl-tert-butyl ether and twice with diethyl ether, and resuspended in water before lyophilization. Synthesis products were analyzed by HPLC (System Gold; Beckman Instruments, Fullerton, CA) and matrix-assisted laser desorption/ionization TOF MS (G2025A; Hewlett-Packard, Palo Alto, CA). Peptides of <80% purity were purified by preparative HPLC.
Peptide Binding Assay.
T2 cells were used for binding studies. A 1 mM peptide stock solution in PBS 10% DMSO was made, and cells were incubated with the peptide at a final concentration of 50 µM in RPMI 1640 overnight at 37°C. HLA surface expression was monitored after staining with the primary antibody BB7.2 and a FITC-coupled goat antimouse IgG (Dianova, Hamburg, Germany) on a FACSCalibur cytometer (Becton-Dickinson, San Jose, CA). Results in Fig. 1
show the ratio of the mean fluorescence of the stained cells incubated with the peptide:the mean fluorescence of the stained cells incubated without peptide.
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1010 cells were analyzed by nanocapillary HPLC ESI MS.
HPLC-coupled MS.
Synthetic and natural peptide mixtures were analyzed as described (11)
by a reversed-phase HPLC system (ABI 140D; Applied Biosystems) coupled to a hybrid quadrupole orthogonal acceleration TOF tandem mass spectrometer (Q-TOF; Micromass, Manchester, United Kingdom) equipped with an ESI source. Solvent A was 4 mM ammonium acetate adjusted to pH 3.0 with formic acid. Solvent B was 2 mM ammonium acetate in 70% acetonitrile/water adjusted to pH 3.0 with formic acid. As a modification of the described set-up, loading of typical sample volumes of 100 µl was achieved by preconcentration on a 300-µm * 5-mm C18 µ-Precolumn (LC Packings, San Francisco, CA). A syringe pump (PHD 2000; Harvard Apparatus, Inc., Holliston, MA), equipped with a gastight 100-µl syringe (1710 RNR; Hamilton, Bonaduz, Switzerland), was used to deliver solvent and sample at a flow rate of 2 µl/min. For peptide separation, the preconcentration column was switched in line with a 75-µm * 250-mm, C18 column (LC Packings). A binary gradient of 25–60% B within 70 min was performed, applying a flow rate of 10 µl/min reduced to 300 nl/min with a precolumn split using a TEE-piece (ZT1C; Valco, Schenkon, Switzerland) and a 300-µm * 150-mm C18 column as a backpressure device. A gold-coated glass capillary (PicoTip; New Objective, Cambridge, MA) was used as the needle in the ESI source. The integration time for the TOF analyzer was 3 s with an interscan delay of 0.1 s. A blank run was performed before any subsequent HPLC MS run to ensure that the system was free of any residual peptide.
For on-line nanocapillary HPLC MS/MS experiments, fragmentation of the parent ion was achieved at the given retention time by collision with argon atoms. Q1 was set to the mass of interest ±0.5 Da and an optimized collision energy applied. Fragmentation was completed after 60 s.
Nanoflow ESI MS.
Gold-coated glass capillary nanoflow needles were obtained from Protana (normal type; Odense, Denmark). The needle was filled with 3 µl of the sample diluted in 50% methanol:water:1% formic acid and subsequently opened by breaking the tapered end of the tip under a microscope. A stable spray was observed applying a needle voltage of 800–1200 V, a backpressure of 2 psi, and a source temperature of 40°C. The estimated flow rate was 20–50 nl/min. For nanoflow ESI MS/MS experiments, fragmentation was achieved by collision with argon atoms. Q1 was set to the mass of interest ± 0.5 Da, and an optimized collision energy was applied. The integration time for the TOF analyzer was 1 s with an interscan delay of 0.1 s.
Generation of Mouse CTLs and Cytotoxicity Assays.
HHD mice (14)
were injected s.c. in the neck with 25 µg of synthetic KVLEYVIKV peptide mixed with 25 µg of the HBV core128–140 helper epitope (23)
and 50 µl of Titermax (Sigma). Ten days later, spleens were removed, and splenocytes were put in culture in 10 ml of Iscove’s modified Dulbecco’s medium supplemented with 10% FCS, 2 mM L-glutamine, 1% penicillin/streptomycin (Life Technologies, Inc.), 50 µg/ml Gentamycin (Life Technologies, Inc.), 5 x 10-5 M ß-mercaptoethanol, and 1 mM sodium pyruvate (Sigma). Peptide was added at a final concentration of 1 µM. Three days later, 25 units of recombinant IL-2 (Proleukin; CHIRON) were added. At day 6, the cytotoxicity of the cultures was tested on 5000 unloaded or peptide-loaded EL4S3Rob/HHD cells in a 6-h 51Cr release assay. Specific lysis was calculated as follows: (experimental release - spontaneous release)/(total release - spontaneous release) x 100. CTL cultures exhibiting detectable peptide-specific cytotoxicity were restimulated weekly using 2 x 107 million peptide-loaded (1 µM peptide for 1 h), fresh irradiated (200 Gy) HHD splenocytes. Long-term cultures were used for cytolytic assays on 5000 human tumor cells previously treated or not treated for 48 h with 250 units/ml of human IFN- (Boehringer Mannheim).
Cell Staining Using HLA-peptide Tetrameric Complexes.
HLA-peptide tetrameric complexes were produced as described previously (6)
. In brief, the heavy chain was modified by deletion of the transmembrane domain and COOH-terminal addition of a sequence containing the BirA enzymatic biotinylation site. The HLA-A2 heavy chain and ß2-microglobulin were produced using a prokaryotic expression system (pET/HLA-A2 plasmid and bacteria kindly provided by Dr. Vincenzo Cerundolo), purified, and refolded in vitro by limiting dilution with the HLA-A*0201-binding peptides. The HLA-A*0201-binding peptides used were MAGE-A1278–286 KVLEYVIKV and CEA694–702 GVLVGVALI. The refolded complexes were purified by gel filtration (Superdex 75, Pharmacia) using fast protein liquid chromatography, biotinylated by BirA (Avidity, Denver, CO) in the presence of biotin (Sigma Chemical), ATP (Sigma Chemical), and Mg2+ (Sigma Chemical). The biotinylated product was separated from free biotin by gel filtration and ion exchange (monoQ Pharmacia) using fast protein liquid chromatography. Tetramers were made by mixing biotinylated protein complexes with streptavidin-PE (Molecular Probes) at a molecular ratio of 4:0.8. Mouse CTLs (2 x 105) were incubated with 10 µg/ml of tetrameric complexes on ice; after 15 min incubation, anti-CD8 antibody (Pharmingen) was added, and the samples were incubated on ice for an additional 15 min. After extensive washing with PBS containing 1% FCS, the samples were analyzed using a FACScalibur (Becton Dickinson).
Plasmids, Transient Transfections, and Intracellular Cytokine Staining.
The MAGE-A1 cDNA (kindly provided by Dr. Pierre van der Bruggen) and the CEA cDNA (kindly provided by Dr. Cécile Gouttefangeas) were cloned in a pIRES-CD4t vector (kindly provided by Dr. Peter Gaines; Ref. 24
). These plasmids were introduced into tumor cells using electroporation mixing 2 x 107 cells with 25 µg of DNA and pulsed at 225 V, 1050 µF. Forty-eight h after transfection, untreated or IFN--treated cells were harvested and incubated with antihuman CD4-coated magnetic beads (Miltenyi Biotec). CD4-positive cells were sorted as recommended by the manufacturer (Miltenyi Biotec). CD4+ cells (50,000) were incubated with 500,000 anti-MAGE-A1278–286 CTLs overnight in a medium supplemented with GolgiStop (Pharmingen). Then, cells were permeabilized and stained using the Cytofix/Cytoperm Plus kit and anti-CD4-FITC, anti-IFN- PE, and anti-CD8 cytochrome antibodies, according to the manufacturer’s recommendations (Pharmingen). FACS analysis was performed using a FACSCalibur cytometer (Becton-Dickinson). Data in Fig. 4
show the IFN- and CD8 expression in gated CD4-negative lymphocytes.
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RESULTS |
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shows the results obtained for peptides predicted from MAGE-A1 and for the immunodominant Influenza Matrix58–66 HLA-A*0201 epitope as positive control. Although three of the MAGE-A1 peptides show no experimental binding, all of them were used to calibrate the HPLC MS system, because, to our knowledge, the stabilization assay is not sensitive enough to detect the binding of all natural ligands.
Detection of a Predicted MAGE-A1 Epitope by an HPLC MS System.
The cell line KS24.22 is a Her2/neu-transfected variant of the KS24 human breast carcinoma cell line described previously (16)
. It expresses HLA-A*0201 as checked by mAb staining using BB7.2 and RT-PCR and the MAGE-A1 gene as checked by RT-PCR.7
IFN--treated (250 units/ml; 48 h treatment) KS24.22 cells (3 x 1010) were used for HLA-A*0201 purification and peptide extraction as described (21)
. Analysis of the peptide mixture eluted from HLA-A*0201 showed coelution of peptides with molecular masses corresponding to the self-ligands p68168–176 (YLLPAIVHI) and PP2A402–410 (SLLPAIVEL) selected as internal controls, as well as the predicted tumor-associated peptide MAGE-A1278–286 (KVLEYVIKV; Fig. 2
). The amino acid sequences of these three peptides were confirmed in a second experiment by on-line nanocapillary HPLC MS/MS, shown in Fig. 2
for MAGE-A1278–286. Nanoflow ESI MS/MS of synthetic MAGE-A1278–286 under identical conditions as in the nanocapillary HPLC MS/MS experiment confirmed further the identity of the peptide (data not shown). The self-peptide p68168–176 was present at 2.5 pmol/1010 cells, as estimated by comparison of signal intensities of eluted (3.81 x 103 counts/scan) and 3 pmol of synthetic peptide (4.61 x 103 counts/scan). This corresponds to 600 copies/cell assuming an overall yield of 25% of natural peptides after peptide extraction and HPLC. Signal intensities for MAGE-A1278–286 were 4.19 x 103 counts/scan for 720 fmol of synthetic peptide and 450 counts/scan for the eluted peptide. MAGE-A1278–286 was therefore present at 300 fmol/1010 cells, corresponding to 18 copies/cell.
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. After few restimulations in vitro using irradiated, peptide-loaded fresh splenocytes from HHD mice, CTL lines consisting essentially of CD8+ cells were obtained. These lines can be stained using a tetrameric fluorescent HLA-A*0201 molecule folded around the MAGE-A1278–286 epitope (Fig. 3B)
. A control HHD mouse-derived CTL line generated against a CEA HLA-A*0201 epitope described previously (11)
is not stained by this tetramer, and an irrelevant HLA-A*0201 tetramer folded around a cytomegalovirus epitope does not stain the anti-MAGE-A1278–286 CTL lines. Altogether, these results show that we obtained homogeneous anti-MAGE-A1278–286 cytotoxic lines from HHD mice.
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1 and 2 domains of HLA-A*0201 and the 3, transmembrane, and cytoplasmic domains of H2-Db to stimulate in vitro the anti-MAGE-A1278–286 CTL lines. These HeLa cells were transfected with pIRES plasmids coding for both a truncated version of the human CD4 molecule and either the CEA or the MAGE-A1 antigens. Thirty-six h after transfection, CD4-positive cells were sorted using magnetic beads coated with antihuman CD4 antibodies. Sorted cells were then coincubated with anti-MAGE-A1278–286 HHD-derived CTL lines for 12 h before the cells were permeabilized and stained for intracellular IFN-. Fig. 4
shows that IFN--treated (or -nontreated; data not shown) HeLa cells expressing a chimeric HLA-A*0201 molecule and the MAGE-A1 protein can stimulate interferon- production by CD8+ anti-MAGE-A1278–286 CTL lines. Peptide-loaded HeLa/A2A2Db cells induce a stronger stimulation of the CTL line, and, on the contrary, HeLa/A2A2Db cells transiently expressing the human CD4 molecule and the CEA antigen induce a background expression of IFN- in some cells. This assay shows that MAGE-A1278–286 is exposed at the cell surface after the processing of the cytoplasmic MAGE-A1 protein and loading on HLA-A*0201 molecules.
Anti-MAGE-A1278–286 HLA-A*0201-restricted Mouse CTLs Kill Specifically HLA-A*0201 and MAGE-A1-positive Human Tumor Cells.
We tested the recognition of human tumor cells by the anti-MAGE-A1278–286 mouse CTL lines using cytotoxicity assays. Results of a representative experiment are shown in Fig. 5
. All peptide-loaded HLA-A*0201+ tumor cells are efficiently killed by the mouse anti-MAGE-A1278–286 CTL lines, but only the IFN--treated MAGE-A1+ and HLA-A*0201+ KS24.22 tumor cells are killed without prior incubation with the peptide. Neither IFN--treated or -untreated HLA-A*0201+ MAGE-A1- (GAKI; 721) nor IFN--treated or -untreated HLA-A*0201- MAGE-A1+ (KLHE) tumor cells are lysed by the HLA-A*0201-restricted anti-MAGE-A1278–286 mouse CTL line. These results demonstrate that the MAGE A1278–286 epitope is presented at the cell surface of human tumor cells expressing HLA-A*0201 and the MAGE-A1 tumor antigen.
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DISCUSSION |
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treatment, putting this peptide into the family of epitopes produced both by constitutive and immuno-proteasomes (26)
. Strikingly, the MAGE-A1278–286 epitope is not the best HLA-A*0201 binder compared with other predicted MAGE-A1 epitopes (Fig. 1)
. Limiting steps in the antigen processing (proteasome-mediated degradation or generation of the correct epitope COOH terminus, TAP transport, loading on neosynthesized class I molecules) might favor the presentation of this MAGE-A1 epitope. Accordingly, our recently described cleavage prediction algorithm (27)
indicates that proteasomes would perform a cleavage of the MAGE-A1 protein after the valine 286, which is the COOH-terminal anchor amino acid of the MAGE-A1 HLA-A*0201 epitope (data not shown).
Besides reporting a clinically important new tumor epitope, the first described HLA-A*0201-restricted epitope from the MAGE-A1 protein, our results demonstrate the potential of our PCD approach for high through-put identification of class I epitopes. Moreover, the CTL-independent characterization of HLA-class I restricted epitopes might allow the description of subdominant or cryptic CTL epitopes (28)
. These epitopes are often ignored by the immune system. As opposed to epitopes recognized during the development of the tumor, cryptic epitopes might be used in therapy to trigger the activation of antitumor cytotoxic T cells derived from the pool of naive CD8-positive cells. These effector cells might have a higher antitumor potential than tolerized or previously stimulated and possibly anergized CTLs (29
, 30)
. Thus, such epitopes appear to be interesting targets for immunotherapy. In order to classify the MAGE-A1 epitope as a dominant or cryptic epitope, we stained PBLs with fluorescent HLA-A*0201 tetramers. We could not unambiguously detect CTLs specific for the MAGE-A1278–286 epitope in the blood of healthy people or tumor patients. Functional assays indicating the presence of effector or memory anti-MAGE-A1278–286 CTLs in blood or tumors are still required. Nevertheless, we found that the repertoire of human T cells contains precursors that can recognize the MAGE-A1278–286 epitope in the context of HLA-A*0201. Indeed, it was possible to generate in vitro human anti-MAGE-A1278–286 CTLs using antigen-presenting cells (mature DCs or activated B cells) loaded with 1 µM synthetic peptide. Unfortunately, these CTLs killed only peptide-loaded T2 cells but not MAGE-A1+ HLA-A2+ tumor cells treated or not with IFN-.8
We do not know yet whether this negative result is due to a too-low affinity of the CTLs for their epitope or to the inhibition of killing through either KIRs expressed by the CTLs or specific anti-CTL-mediated killing mechanisms developed by the tumor target cells, e.g., heat shock protein expression (31)
, Fas ligand (32)
, or serpin proteases (33)
. The recognition of the MAGE-A1278–286 epitope by the human immune system and its clinical relevance will be evaluated in breast carcinoma patients vaccinated using DCs loaded with a cocktail of tumor epitopes which include the MAGE-A1 HLA-A*0201 peptide.9
We demonstrated the presentation of the MAGE-A1278–286 epitope at the cell surface through the utilization of HLA-A*0201-restricted CTLs derived from HLA-class I transgenic mice (14)
. Injection of synthetic KVLEYVIKV in HHD mice allowed the fast generation of CTL lines that can be stained using an HLA-A*0201 tetramer folded around the MAGE-A1278–286 peptide. This indicates that these mouse HLA class I-restricted CTLs have a high avidity for the peptide (34)
. Such CTL lines are able to kill peptide-pulsed, HLA-A2-positive human cells as well as IFN--treated HLA-A*0201+ and MAGE-A1+ human tumor cells. Previously, many groups used HLA class I-restricted CTLs from transgenic mice to identify or to study human class I epitopes (15
, 25
, 34, 35, 36)
. Usually, one drawback of these HLA-A*0201-restricted mouse CTL lines is that they do not kill human cells expressing a wild-type class I molecule (37)
. This phenomenon might be attributable to the lack of interaction between mouse CD8 molecule and human class I molecules, resulting in weak interaction between effector and target cells. In contrast, we observed that CTL lines derived from HHD mice kill HLA-A*0201-positive human cells in an epitope-specific and CD8-independent way. This characteristic of HHD-derived CTLs might be attributable to the low expression level of the HHD molecule in the transgenic mice, which results in the development of T cells expressing high avidity T-cell receptors. These CTLs killed human peptide-loaded target cells with high efficiency; moreover, antigen-expressing KS24.22 cells were killed after IFN- treatment.
Although they are sensitive to the cytotoxic effector functions of the MAGE-A1278–286-specific mouse CTL lines, human cells could not efficiently stimulate IFN- production in these killer cells (data not shown). Indeed, cytotoxicity and activation of effector CTLs are separate functions, cytotoxicity being most readily observed (38)
. In our case, full activation of the mouse effector cells required either very high epitope density (reached using peptide-loaded human cells; data not shown) or coactivation by CD8. This latter requirement is fulfilled using transfected human cells expressing the chimeric HLA-A*0201 molecule and which contain a mouse class I 3 domain. Thus, only HeLa cells expressing such a chimeric HLA-A*0201 molecule and the MAGE-A1 tumor antigen can fully activate the mouse peptide-specific CTL lines to produce IFN-. Altogether, these studies allowed us to describe further the advantages and the limitations of mouse CTL lines for the identification of HLA-class I-restricted epitopes and for the recognition of human tumor cells. Because of the low HLA-A*0201 expression, HHD mice-derived CTLs might have a higher affinity for their antigen than other HLA-A*0201 transgenic mice-derived CTLs. Direct comparison of the affinity of CTL lines derived from both transgenic mice is in progress. Nevertheless, by combining intracellular cytokine staining after CD8-dependent stimulation of the effectors with CD8 independent cytotoxicity assays using HHD-derived CTL lines, we unambiguously confirmed the presentation of the PCD-identified MAGE-A1278–286 epitope at the surface of human cells. This combination of MS-based analysis and class I transgenic mice utilization is now used extensively for the identification of bacterial, viral, and tumoral epitopes.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by grants from the Deutsche Krebshilfe (10-1258-St I), MERCK Darmstadt and European Union (BMH4-CT98-3589). S.P. is a recipient of a fellowship from the Human Frontier Science Program Organisation. S.K. and B.G. were supported by grants from the Deutsche Krebshilfe. 
2 These authors contributed equally to this work.
3 To whom requests for reprints should be addressed, at Institut for Cell Biology, Department Immunology, Auf der Morgenstelle 15, 72076 Tübingen, Germany. Phone: 00-49-7071-29-80-997; Fax: 00-49-7071-29-5653; E-mail: steve.pascolo{at}uni-tuebingen.de
4 Present address: University Hospital Zürich, Department of Pathology, Institute for Experimental Immunology, Schmelzbergstrasse 12, CH-8091 Zürich, Switzerland.
5 The abbreviations used are: DC, dendritic cell; PCD, Predict-Calibrate-Detect; MS, mass spectrometry; HPLC, high-performance liquid chromatography; HHD, HLA-A*0201 transgenic H2 class I-deficient; CEA, carcinoembryonic antigen; mAb, monoclonal antibody; TOF, time of flight; ESI, electrospray ionization; RT-PCR, reverse transcription-PCR; FCS, fetal calf serum; PE, phycoerythrin; FACS, fluorescence-activated cell sorter; IL-2, interleukin-2.
6 Available on our web page at http://www.uni-tuebingen.de/uni/kxi.
7 B. Gückel, manuscript in preparation.
8 B. Gückel, unpublished results.
Received 10/11/00. Accepted 3/15/01.
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) or peptide loaded (•) EL4S3-Rob/HHD as target cells. B, FACS analysis of the mouse HLA-A*0201-restricted CTL lines specific for the MAGE-A1278–286 epitope (HHD MAGE) or for the CEA694–702 epitope (HHD CEA) using CD8 antibodies and HLA-A*0201 tetramers folded around either the MAGE-A1278–286 epitope (TA2 MAGE) or a CMVpp65-derived epitope (TA2 pp65).