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* General Hospital St. Georg, Hamburg, Germany; Institutes of
Immunology and
Transfusion Medicine, University of Kiel, Kiel, Germany; and
Institute of Immunology, Carl Gustav Carus Technical University, Dresden, Germany
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, survivin-derived peptide epitopes could be identified capable of inducing specific cytotoxic T cell responses in both healthy donors (10, 11) and cancer patients (12, 13). Because survivin is also expressed in most of all leukemic blasts of patients with acute myeloid leukemia (AML)3 (7, 14, 15), we evaluated in the current study whether survivin is a suitable target for T cell-based immunotherapeutic strategies in patients with acute leukemia. We show that CTL generated from healthy HLA-A2+ individuals by primary in vitro immunization with autologous survivin-RNA-transfected dendritic cells (DC) elicited specific and MHC class I-restricted cytotoxic activity against several hematological tumor lines and primary AML blasts endogenously expressing survivin. In addition, the majority of mice treated with survivin-RNA-transfected DC are capable of rejecting an otherwise lethal dose of a murine B cell lymphoma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Peripheral blood and tumor samples were collected from healthy donors and patients at the II Department of Internal Medicine (University of Kiel, Kiel, Germany) and cryopreserved. Tumor samples used for cytotoxicity assay were obtained from patients with AML and chronic lymphocytic leukemia (CLL). All specimens were obtained after informed consent, and approval by our institutional review board were provided.
Human tumor cell lines and nonmalignant cell subsets
K562 (erythroleukemia), IM-9 and U-266 (plasmacytoma), REH, Karpas-422, Balm-3, Ramos (B lymphoblastic lymphoma), and T2 cells were obtained from American Type Culture Collection (Manassas, VA). MEC-1, a chronic lymphatic leukemia, was kindly provided by Dr. F. Caligaris-Cappio (University of Torino, Torino, Italy). Leukemic blasts (purity >80%) from AML patients were obtained by Ficoll (Amersham-Pharmacia, Piscataway, NJ) density gradient centrifugation. CD5+CD19+ CLL cells were obtained from peripheral blood of previously untreated CLL patients (Binet A). After Ficoll density gradient centrifugation, >95% of resulting cells coexpressed CD19 and CD5. Isolation of CD8+ T lymphocytes, CD19+ B cells, CD14+ monocytes, and CD34+ peripheral blood progenitor cells was conducted using MACS technology (Miltenyi, Bergisch-Gladbach, Germany) following the manufacturer’s instructions. FACS revealed a cell purity of >90%.
Immunophenotyping
For marker analysis, mononuclear cells (MNC) were incubated with appropriate primary and secondary Abs and analyzed using a flow cytometer (FACScan; BD Biosciences, Mountain View, CA). The following Abs for murine MNC were used: anti-Thy-1.2 (30-H12); anti-NK (DX5); anti-CD19 (1D3); anti-CD11c (HL3); anti-CD45 (30-F11); anti-CD18 (GAME-46); anti-CD80 (1G10); anti-CD86 (GL1); anti-MHC class I (34-2-12); and anti-MHC class II (AMS-32.1). All mAbs were obtained from BD PharMingen (San Diego, CA). For flow cytometric analysis of human MNC, FITC- or PE-conjugated mouse mAbs against CD4, CD3, CD8, CD56, CD14, CD40, CD80, CD86 (BD Biosciences, Heidelberg, Germany), HLA-DR and CD83 (Coulter-Immunotech, Hamburg, Germany) were used.
Intracellular staining of murine survivin was performed after fixation and permeabilization using the Fix & Perm kit (BD Biosciences, Heidelberg, Germany) following the manufacturer’s instructions. A rabbit polyclonal Ab against mouse survivin (ab8113; Abcam, Cambridge, U.K.) was used. FITC-labeled goat anti-rabbit Ig was used as a secondary reagent. For detection of human survivin, the rabbit polyclonal Ab against human survivin (DPC Biermann, Bielefeld, Germany) was used.
Western blot analysis
Whole cell protein extracts were prepared from 1 x 107 cells using a lysis buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40 (Sigma-Aldrich, Diesenhofen, Germany), 2 mM EDTA (pH 8.0), and Complete mini (protease inhibitor; Roche, Basel, Switzerland). Cell lysates were separated by 12% gels in SDS-PAGE under reducing conditions for 60 min at 140 V, and the proteins were transferred onto nitrocellulose membrane (Machery & Nagel, Düren, Germany) in 25 mM Tris base, 0,2 M glycine, 20% methanol for 60 min at 100 V and blocked in 20 mM Tris (pH 8.0), 137 mM NaCl, 0.1% Tween 20 (TBST), and 5% powdered milk overnight. Immunoblottings were performed using the rabbit polyclonal anti-survivin (DPC-Biermann, Bad-Neuheim, Germany) Ab at a concentration of 1 µg/ml (in TBST and 5% BSA) as primary and HRP-linked anti-rabbit Ig (diluted to 1/5000 v/v in TBST and powdered milk; Amersham, Little Chalfont, U.K.) as secondary Ab. All immunoblots were performed using ECL (Amersham).
Stimulation of B-CLL cells with CD40 ligand
Primary tumor cells from CLL patients (Binet A) were obtained by Ficoll densitiy centrifugation. Cells were washed twice in PBS (CellConcepts, Umkirch, Germany), resuspended in RPMI 1640 supplemented with 10% FCS (Biochrom, Cambridge, U.K.) and 1% penicillin/streptomycin (Sigma-Aldrich), and cultured in six-well plates. For B cell activation, 100 ng/ml CD40 ligand and 1 µg/ml Enhancer (Alexis, Lausen, Switzerland) were added to 1 x 106 cells/ml and incubated for 3 days at 37°C. FACS revealed that >90% of the stimulated CLL cells were survivin positive (data not shown).
Peptides and loading of T2 cells
Survivin5–14, survivin95–104, and FluM158–66 were purchased from Biosyntan (Berlin, Germany) and provided at >90% purity, as verified by HPLC and MS analysis. The HLA-A2+ mutant cell line T2 was separately incubated with peptides for 2 h at a concentration of 50 µg/ml, washed three times, and used as target in a 51Cr release assay.
Isolation of human and murine survivin-RNA
The coding sequence for human survivin was ligated into BamHI/XhoI site of the pCITE-2a(+) vector (Novagen, Madison, WI). The coding sequence for murine survivin was obtained by extraction of total RNA from murine A20 B-cell lymphoma cell line by using the RNeasy Kit (Qiagen, Hilden, Germany). cDNA was performed by using the First-Strand cDNA Synthesis Kit (Amersham) after DNA amplification by PCR with specific primer for murine survivin (murine survivin forward primer 5'-gCggAggTTgTggTg and reverse primer 3'-AAAggCTCAgCATTAggc). The coding sequence for murine survivin was first ligated into the pCR2.1-TOPO vector (Invitrogen, Karlsruhe, Germany) by using the TOPO TA Cloning Kit (Invitrogen) and transformed into Escherichia coli XL-1 blue. The coding sequence was cut out with BamHI and NotI and ligated into BamHI/NotI site of the pCITE-2a(+) vector (Novagen). Survivin vectors were transformed into E. coli JM83 by heat shock transformation. Correctness of human survivin sequence and murine survivin sequence were verified by sequencing. Resulting plasmid DNA was linearized and in vitro transcribed using the T7 RiboMAX Large Scale Transcription Kit (Promega, Mannheim, Germany). The resulting RNA was purified with RNeasy columns (RNeasy Kit) and visualized on a denaturated agarose gel. Quantity and purity were determined by UV spectrophotometry. The human survivin RNA translation product (STP3 T7 Kit; Novagen, Madison, WI) was visualized on a Western blot.
Cell isolation and generation of human immature DC
Immature DC (iDC) were generated from PBMC of healthy HLA-A2+ volunteers following the protocol of Feuerstein et al. (16). Briefly, a concentrated leukocyte fraction was generated through a 2-h restricted peripheral blood leukapheresis processing 6–8 liters of blood with each collection. The leukapheresis product was further separated by density gradient centrifugation over polysucrose-sodium diatrizoate (Histopaque; Sigma-Aldrich, St. Louis, MO), and cells were resuspended in serum-free AIM-V medium (Life Technologies, Gaithersburg, MD). PBMC were incubated at 2 x 108 cells/30 ml in T-150 culture flasks in a humidified incubator for 2 h at 37°C to allow plastic adherence. The adherent cell fraction was used for DC culture by incubation in serum-free AIM-V medium supplemented with recombinant human IL-4 (500 U/ml) and recombinant human GM-CSF (800 U/ml; CellConcepts).
RNA transfection and maturation of human DC
Transfection of iDC was performed by using the liposomal transfection reagent dioleoyloxy-trimethylammonium-propane-methylsulfate (DOTAP; Roche). DOTAP (20 µl) and in vitro-transcribed-RNA (10 µg) were mixed in 500 µl of Opti-MEM (Life Technologies, Karlsruhe, Germany) and incubated for 20 min at room temperature. The RNA-lipid complex was added to 1 x 106 iDC/ml (in Opti-MEM) and incubated at 37°C for 3 h. Maturation of the transfected iDC was performed by culturing for 2 days in serum-free medium supplemented with GM-CSF, IL-4, IL-1
, TNF-
(10 ng/ml; CellConcepts), IL-6 (1000 U/ml; CellConcepts), and PGE2 (1 µg/ml; Sigma-Aldrich, Deisenhofen, Germany). Flow cytometric analysis revealed that survivin-RNA-transfected and maturated DC expressed 
20% of survivin and high levels of CD80, CD83, CD86, MHC class I, and MHC class II molecules.
Generation of human CTL
CTL were generated using a protocol adopted from Heiser et al. (17); in brief, the T cell-enriched nonadherent fraction of PBMC obtained after the DC plastic adherence step was used for CTL generation. Nonadherent PBMC were cultured in serum-free medium supplemented with 20 U/ml human IL-2 and 10 ng/ml human IL-7 (CellConcepts). Cells were stimulated weekly at least four times with autologous survivin-RNA-transfected DC.
CTL assay
Target cells were labeled with 200 µCi of NaCrO4 (Amersham-Buchler, Braunschweig, Germany) in 0.5 ml of complete medium for 1 h. They were washed three times with complete medium and added at a concentration of 5 x 103 cells/well in round-bottom microtiter plates (Nunc, Roskilde, Denmark). Effector cells were added at various E:T ratios in a final volume of 200 µl/well. The plates were incubated for 4 h at 37°C in a humid atmosphere with 5% CO2. Maximum chromium release was ensured by the addition of 10% Triton-X, and spontaneous release was assessed by adding complete medium (RPMI 1640 plus 10% FCS) to the target cells. The culture supernatant was harvested semi automatically with a Scatron Titertek System (Scatron, Suffolk, U.K.) and counted in a gamma counter (Beckmann, Heidelberg, Germany). The percentage of specific lysis was calculated as [(experimental cpm - spontaneous cpm)/(maximum cpm - spontaneous cpm)] x 100. All determinations were made in triplicate.
Cold target inhibition assay
Specificity of tumor cell lysis was determined in a cold target inhibition assay by analyzing the capacity of unlabeled HLA-A2+ survivin-negative and HLA-A2+ survivin-positive CLL cells to block lysis of tumor cells at a ratio of 20:1 (inhibitor-target ratio).
Ab blocking inhibition assay
For Ab-blocking experiments, human CTL were generated as described in Generation of human CTL. For blocking experiments, cells were incubated with 10 µg/ml mAbs (anti-HLA-A2 (BB7.2); BD Biosciences, Heidelberg, Germany; CD8 (T8); anti-Pan-TCR (BMA 031), CD4 (T4), isotype-matched controls MAM-6 and HMFG-1, Beckman Coulter) for 30 min. Cell suspensions were washed twice with complete medium and tested in a chromium release assay.
Murine tumor cell lines
A20 is a nonimmunogeneic B cell leukemia/lymphoma that occurred spontaneously in a 15-mo-old BALB/c mouse and was used for in vivo tumor challenge experiments. The murine EL-4 thymoma cell line derived from C57BL/6 mice were grown in RPMI 1640 (Life Technologies, Karlsruhe, Germany) containing 5% FCS (Biochrom) and was used as a fully MHC-fully mismatched target for CTL analysis. Both cell lines were obtained from American Type Culture Collection (Manassas, VA).
Isolation of total A20-RNA
A20 total RNA was obtained by using the RNeasy Kit following the manufacturer’s instructions. Isolated RNA was visualized on agarose gel. Quantity and purity of RNA were determined by UV spectrophotometry. Total RNA samples were stored at -80°C.
Generation of murine survivin-RNA-transfected DC
For in vitro restimulation, bone marrow-derived DC were used as professional APC and generated as previously described (18). iDC were transfected using 50 µg of DOTAP (Roche) and 25 µg of in vitro-transcribed RNA or total RNA mixed in 500 µl of Opti-MEM. The complex was incubated for 20 min at room temperature and then added to 5 x 106 immature DC. DC Maturation was achieved by adding LPS (2 µg/ml for 5 x 106 iDC/ml; Sigma-Aldrich, Deisenhofen, Germany) for 4 h at 37°C. Afterward, matured DC were washed three times and applied for vaccination studies. Flow cytometric analysis demonstrated that these matured DC expressed 15–25% of survivin and high levels of CD45 (96 ± 1.9%), CD11c (92 ± 2.9%), CD18 (95 ± 1.8%), CD80 (89 ± 2.2%), CD86 (83 ± 2.9%), class I MHC (98 ± 0.3%), and class II MHC (92 ± 1.0%) Ags.
Tumor challenge experiments
Healthy BALB/c mice 8–12 wk old received three s.c. injections of 5 x 105 syngeneic DC transfected with murine survivin-RNA at weekly intervals. Control groups were treated with untransfected DC or PBS (tumor control group). One week after the last vaccination, animals were inoculated s.c. with 1 x 105 A20 lymphoma cells.
Murine CTL assay
BALB/c mice were treated as described above. On day 100, DC-treated animals were killed under ether anesthesia. The spleens of two mice per group were removed, pooled, and pressed through wire mesh screens to obtain single-cell suspensions. MNC were isolated by Lympholite-M (Cedarlane, Hornby, Ontario, Canada) gradient centrifugation. Splenocytes were restimulated at a concentration of 2 x 106 cells/ml with 5 x 105/ml DC transfected with survivin-RNA or total A20-RNA in 24-well plates. After 5 days, viable cells were harvested and tested in a 51Cr release-assay for their ability to lyse A20 and EL-4 cells.
Statistical analysis
Survival of animals was calculated according to the method of Kaplan and Meier. Group comparisons were made by the Wilcoxon test. The calculations were done on a PC with Statistica data analysis software (StatSoft, Tulsa, OK).
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Results |
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The expression of survivin on malignant hemopoietic cells was assessed by FACS and Western blot using the polyclonal survivin-specific Ab. As illustrated in Tables I and II, survivin expression could be detected in all tumor cell lines and primary AML blasts tested (Fig. 1). Fig. 1A gives an example of survivin expression on AML blasts from two patients. In contrast, in none of the CLL cells obtained from newly diagnosed B-CLL patients (Binet A) was expression of survivin protein found (Table II). We could not detect any expression of survivin on cell subpopulations purified from peripheral blood like CD19+ (B lymphocytes), CD3+CD8+ (T lymphocytes), CD14+ (monocytes) cells. In addition, we observed that purified CD34+ progenitor cells were survivin negative, as well as iDC and matured DC (Table II).
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We next generated polyclonal CTL in vitro using autologous survivin-transfected DC as Ag-presenting cells obtained from healthy HLA-A2+ individuals. CTL obtained after four weekly restimulations elicited efficient T cell responses recognizing and lysing endogenously survivin-expressing tumor cell lines. As depicted in Fig. 2A, the survivin-positive, HLA-A2-expressing cell lines Karpas-422, Balm-3, IM-9, REH, and U266 were killed whereas no lysis of the hematological cell lines K562, Ramos, and Mec-1 (survivin+/HLA-A2-) occurred (Fig. 2B). In addition, survivin-specific CTL elicited significant cytotoxic activity against survivin RNA-transfected autologous DC (Fig. 2A) but showed no lysis of untransfected DC, purified monocytes, or CD34+ hemopoietic progenitor cells (Fig. 2C).
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To examine whether T cell lines generated against survivin protein could kill not only tumor cell lines but also fresh malignant targets from patients, cytotoxicity assays were performed by using survivin-specific CTL against survivin-expressing blasts from AML patients. In four cases, T cell lines were capable of lysing HLA-A2+/survivin-expressing AML blasts (Fig. 4A), whereas HLA-A2- targets were not lysed (Fig. 4B).
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In a next step, we investigated whether polyclonal survivin-specific CTL contain T lymphocytes capable of recognizing two recently identified HLA-A2-binding peptides (survivin5–14 and survivin95–104) derived from the survivin protein (3, 5). As shown in Fig. 5, T2 cells pulsed with the cognate survivin peptides could be recognized and efficiently killed by survivin-specific CTL, whereas T2 cells coated with an irrelevant control peptide (FluM158–66) were not lysed. These findings suggest that at least two clonotypes were induced specific for different survivin epitopes.
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To evaluate the therapeutic potential of survivin as a tumor rejection Ag healthy BALB/c mice were vaccinated with syngeneic DC transfected with RNA coding for the murine survivin protein. After three weekly s.c. injections of 5 x 105 DC, animals were subsequently challenged with an otherwise lethal dose of 1 x 105 A20 lymphoma cells. As depicted in Fig. 6, vaccination of animals with unpulsed DC did not significantly improve survival compared with the tumor control group. In contrast, animals receiving vaccinations with DC transfected with survivin-RNA or with total RNA derived from the A20 leukemia experienced tumor protection in 10 of 12 (83%) and 11 of 12 (91%) animals being tumor free after an observation period of 140 days (Fig. 6A). CTL analysis from animals treated with survivin-RNA- or A20-RNA-transfected DC demonstrated efficient tumor-specific killing of the A20 leukemia, whereas the fully MHC-mismatched control target EL-4 was not lysed (Fig. 6B).
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Discussion |
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First experimental evidence that survivin can be regarded as a potential tumor-associated Ag came from Schmitz et al. (10), demonstrating that survivin-peptide-specific cytotoxic T cell lines generated from healthy HLA-A2+ donors can recognize and selectively kill autologous EBV-transformed B lymphocytes transfected with survivin-coding cDNA. Using an ELISPOT assay, Andersen et al. (12, 13) detected CTL responses against two survivin-deduced peptide epitopes in three of six melanoma patients and three of four CLL patients, whereas no T cell reactivity could be measured in PBL from six healthy HLA-A2+ individuals. In a more recent report, the same author isolated survivin-peptide-specific T cells from HLA-A2+ melanoma patients capable of eliciting cytotoxic reactivity against some established HLA-A2-matched tumor cell lines (13).
To develop a vaccination strategy for the treatment of human hematological neoplasms potentially representing multiple human survivin-epitopes, we investigated the capability of human DC transfected with human survivin-RNA to stimulate primary CTL responses in vitro. We were able to demonstrate powerful cytotoxic responses against a broad range of both hematological tumor cell lines and primary AML blasts, whereas nonmalignant cell subpopulations derived from the hemopoietic system were not affected (monocytes, DC, CD34+ cells). HLA-A2-matched CD34-purified peripheral blood progenitor cells obtained from an healthy donor were not lysed by survivin-specific CTL, suggesting that nonmalignant hemopoietic cells might not be the target for T cell-based immunotherapeutic approaches using survivin as a target structure. However, because in nonmalignant CD34+ peripheral blood progenitor cells survivin is detectable at low levels (14), but can be up-regulated in the presence of hemopoietic growth factors (3), induction of autoimmune reactions cannot be completely excluded.
In the current study, polyclonal survivin-specific CTL generated with autologous survivin-RNA-transfected DC contain T lymphocytes capable of recognizing two recently described epitopes (survivin5–14 and survivin95–104) as demonstrated by their potential to kill survivin-peptide-pulsed T2 cells. Using DC loaded with whole survivin protein, Schmitz et al. (10) generated CTL from one HLA-A2-matched donor unable to recognize either one of the two survivin peptides, whereas T cells obtained from another healthy donor revealed a specific reactivity against survivin95–104 but not against survivin5–14.
In an established murine B cell leukemia model, we were able to demonstrate efficient antitumoral CTL responses in vitro and a complete tumor rejection in most of the treated animals receiving survivin-RNA-transfected DC. Although survivin is expressed at low levels on normal epithelial cells of the gastrointestinal tract (4) and on hemopoietic progenitor cells (3, 14), no visible signs of any autoimmune manifestations occurred after this type of DC therapy.
Taken together, these findings advance the notion that survivin could serve as a useful Ag for T cell-based immunotherapeutic strategies in the treatment of hematological malignancies, especially of AML. Similar to the catalytic subunit of telomerase (19), the use of survivin as an Ag for vaccination purposes has the advantage that down-regulation or Ag loss as a mechanism for immune escape would hinder the progression of the tumor. Future studies will have to investigate this as well as from what proportion of cancer patients it is possible to induce survivin-specific CTL and whether immunization against survivin could lead to autoimmune reactions.
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Footnotes |
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2 M.Z. and S.S. contributed equally to this manuscript.
3 Address correspondence and reprint requests to Dr. Matthias Zeis, General Hospital St. Georg, Lohmühlenstrasse 5, 20099 Hamburg, Germany. E-mail address: mzeis47{at}hotmail.com
4 Abbreviations used in this paper: AML. acute myelogenous leukemia; DC, dendritic cell; CLL, chronic lymphatic leukemia; MNC, mononuclear cell; iDC, immature DC; DOTAP, dioleoyloxy-trimethylammonium-propane-methysulfate.
Received for publication December 20, 2002. Accepted for publication March 20, 2003.
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