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Immunology

WT1 peptide-specific T cells generated from peripheral blood of healthy donors: possible implications for adoptive immunotherapy after allogeneic stem cell transplantation

Abstract

The Wilms tumor antigen, WT1, is expressed at high levels in various types of leukemia and solid tumors, including lung, breast, colon cancer and soft tissue sarcomas. The WT1 protein has been found to be highly immunogenic, and spontaneous humoral and cytotoxic T-cell responses have been detected in patients suffering from leukemia. Furthermore, major histocompatibility complexes class I- and II-restricted WT1 peptide epitopes have been shown to elicit immune responses in patients with WT1-expressing tumors. As a consequence, WT1 has become an attractive target for anticancer immunotherapy. In this study, we investigated the feasibility of generating WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation. We analyzed the incidence of T cells specific for WT1 peptide epitopes in cancer patients and healthy volunteers. It is noted that we could generate WT1-specific responses in nine of ten healthy volunteer donors and established T-cell clones specific for two WT1-derived peptide epitopes. These in vitro expanded WT1-specific T cells effectively lysed WT1-expressing tumor cell lines, indicating the potential clinical impact of ex vivo expanded donor-derived WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation.

Introduction

Allogeneic stem cell transplantation has become an important treatment modality for patients with high-risk leukemia and is increasingly used to treat children and adolescents with high-grade or relapsed soft tissue sarcoma (STS).1 Especially in patients with a fatal prognosis such as relapsed alveolar rhabdomyosarcoma (RMS) or Ewing sarcoma (ES), cellular therapy might offer a feasible approach to improve survival.2 Even after allogeneic stem cell transplantation, however, relapse remains the major cause for treatment failure. Pre-emptive immunotherapy with donor lymphocyte infusions has been shown to eradicate minimal residual disease in patients with acute leukemia after allogeneic stem cell transplantation.3 Adoptive transfer of donor lymphocytes, however, induces an allogeneic reaction against the recipients human leukocyte antigens (HLA) rather than tumor-specific peptides. As a consequence, patients who receive donor lymphocytes are at risk to develop severe graft-versus-host disease. The application of specific effector cells that exert potent antitumor immunity with a lower risk of inducing clinical graft-versus-host disease might offer an alternative approach. For many years, potential tumor antigens such as minor histocompatibility antigens, proteinase 3, BCR-ABL, survivin and WT1 have been under investigation.4, 5, 6

WT1 has originally been described as a tumor suppressor gene but has also been found to be a key molecule for tumor proliferation in a large number of human malignancies, indicating a possible oncogenic function.7, 8 Overexpression of WT1 has been shown in a wide variety of leukemias and solid tumors, such as Wilms’ tumor, breast, lung, colon and skin cancer.4, 5, 9, 10, 11, 12, 13, 14, 15 Recently, WT1 has been proposed as a candidate antigen for tumor-directed immunotherapy. Expression of WT1 in healthy tissue is absent or very low, in contrast to its abundant expression in malignant cells. Moreover, spontaneous humoral and cellular immune responses against WT1 have been described in patients with leukemia.16, 17, 18

In this study, we investigated the feasibility of generating and expanding WT1 peptide-specific T cells from peripheral blood of healthy volunteer donors as a pre-requisite for adoptive T-cell therapy targeting WT1 after stem cell transplantation.

Materials and methods

Patient and healthy volunteer donor samples

Peripheral blood from HLA-A2 and HLA-A1 positive patients and healthy volunteers was collected after written informed consent was given according to the approval of the local ethics committee. Fifteen adult patients with solid tumors and leukemia (breast cancer n=10, melanoma n=4, leukemia n=1) and ten healthy volunteer donors were included in this study.

Peripheral blood mononuclear cells (PBMCs) were separated from whole blood using Ficoll density gradient centrifugation. HLA-typing was done by fluorescence activated cell sorting (FACS) analysis.

Cell lines and tumor samples

Soft tissue sarcoma cell lines, RMS13, Rh30, Rh41 (alveolar RMS), A204, TE671, RD (embryonal RMS) and Rh1 (ES), breast cancer cell line MDA-MB-231, CML cell line K562 and hybridoma T2 cells were obtained from DSMZ (Braunschweig, Germany).

Rh1 was originally categorized as an RMS cell line but has recently been reclassified as gene-expression profiling showed the expression of EWS-FLI1, a fusion transcript characteristic of ES family tumors.19 T2 cells suffer from defective presentation of endogenously synthesized antigens because of the deficiency of a transporter associated with antigen processing, but they can be exogenously loaded with cognate peptides.

Cell lines were maintained in RPMI 1640 (Invitrogen, Karlsruhe, Germany), DMEM (Invitrogen) or McCoy's medium (Sigma-Aldrich, Saint Louis, MO, USA) supplemented with 10% fetal calf serum (Biochrom, Berlin), penicillin and streptomycin (Invitrogen) at 37 °C with 5% CO2.

T cells were maintained in RPMI 1640 with 10% human serum and 100 U/ml IL-2 (Proleukin S, Novartis, Nürnberg, Germany), subsequently referred to as T-cell medium (TCM).

Real-time PCR

Total RNA of cell lines was extracted by using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA synthesis was performed using random hexamer primers (iScript cDNA Synthesis Kit, Bio-Rad, Munich, Germany) according to the instructions of the manufacturer.

WT1 transcripts were quantified by real-time PCR using the ABI Prism 7700 system (Applied Biosystems, Foster City, CA, USA). WT1 primers and probe were used as described earlier:20 forward primer: 5′-IndexTermACAGGGTACGAGAGCGATAACCA-3′, reverse primer: 5′-IndexTermCACACGTCGCACATCCTGAAT-3′, fluorescent probe: 5′-6-FAM-IndexTermCAACGCCCATCCTCTGCGGAGCCCA-TAMRA-3′ (TIB Molbiol, Berlin, Germany). WT1 expression levels were normalized to ABL as a control gene. Forward primer: 5′-IndexTermTGGAGATAACACTCTAAGCATAACTAAAGGT-3′, reverse primer: 5′-IndexTermGATGTAGTTGCTTGGGACCCA-3′, fluorescent probe: 5′-6-FAM-IndexTermCCATTTTTGGTTTGGGCTTCACACCAT-TAMRA-3′ (TIB Molbiol).21 WT1 and ABL copy numbers were determined using logarithmic plasmid dilution series and results are given as WT1 copy number per 10 000 ABL copies.

Western blot

For protein extraction, cell lines were harvested in lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.1% sodium dodecyl sulfate, 0.5% Na-deoxycholate, 1 mM phenylmethysulfonyl fluoride, protease inhibitor cocktail (Roche, Grenzach-Whylen, Germany)). Aliquots of soluble protein were electrophoresed on 12% sodium dodecyl sulfate polyacrylamide gels, electroblotted onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) and probed with monoclonal WT1-antibody (clone 6F-H2, 1:500, Dako, Glostrup, Denmark). Horseradish peroxidase-conjugated sheep anti-mouse antibody (Amersham Biosciences, Buckinghamshire, UK) was used as a secondary antibody. Membranes were developed using a chemiluminescence detection system (ECL, Amersham Biosciences).

Peptides and multimers

Four HLA-A*0201-restricted 9mer peptides were used for in vitro stimulation: WT1 p37–45: VLDFAPPGA,22, 23 WT1 p126–134: RMFPNAPYL,18, 22, 24, 25, 26, 27, 28 WT1 p187–195: SLGEQQYSV,13, 18, 22, 25 and WT1 p235–243: CMTWNQMNL.22, 24, 29 One HLA-A*01–restricted 11mer peptide was also used: WT1 p317–327: TSEKRPFMCAY.30 Influenza matrix protein peptides p58–66 GILGFVFTL for HLA-A231, 32 and p44–52 CTELKLSDY for HLA-A*01 32, 33 were used as positive controls. Peptides were manufactured by Biosyntan (Berlin, Germany) with >95% purity, dissolved in dimethylsulfoxide to a stock concentration of 10 mg/ml and further diluted in phosphate buffered saline.

HLA-A*0201 pentamer WT1 p126–134: RMFPNAPYL and HLA-A*0201 and A*2402 WT1 p235–243: CMTWNQMNL for FACS analysis were purchased from Proimmune (Oxford, UK). phycoerythrin (PE)-labeled pentamers as well as unlabeled pentamers with a PE-fluorotag were used for FACS analysis.

Mixed lymphocyte peptide culture

Peripheral blood was collected from HLA-A2+ and HLA-A1+ patients and healthy volunteers, and PBMCs were isolated by Ficoll density gradient centrifugation. CD8+ cells were isolated by MACS technology (Miltenyi Biotec, Bergisch Gladbach, Germany). For mixed lymphocyte peptide culture, the resulting negative fraction of CD8 PBMCs was used as autologous antigen presenting cells (APCs). APCs were irradiated (30 Gy), loaded exogenously with WT1-peptides (10 μg/ml) and β2-microglobulin (2.5 μg/ml, Sigma-Aldrich) for 1 h at room temperature, and subsequently used for in vitro stimulation of CD8+ cells.34 CD8+ cells and APCs were cultured at a ratio of 1:2 in TCM containing 10 ng/ml IL-7 (Richter-Helm Biologics, Hannover, Germany). Restimulation of effector cells with WT1 peptide (1 μg/ml) was carried out in weekly intervals.

T-cell cloning

Antigen-specific T-cell clones were generated from peptide-specific T-cell bulk cultures by limiting dilution assay as described earlier.34 CD8+ cells were diluted and distributed into round-bottom 96-well plates at a density of three cells per well in TCM with 50 U/ml IL-4. Irradiated (100 Gy) peptide-pulsed T2 cells (3 × 103/well) were used as APCs, and irradiated (100 Gy) EBV-transformed B cells (4 × 104/well) were used as feeder cells. T cells were restimulated once by adding peptide-pulsed T2 and feeder cells in TCM. Proliferating T-cell clones were tested for peptide-recognition in Elispot, and WT1-specific clones were further expanded. T-cell clones were restimulated weekly by adding irradiated peptide-pulsed T2 cells (2.5 × 104/48-well) and EBV-transformed B cells (1.5 × 105/48-well) in TCM substituted with IL-4 as described above.

Elispot assay

Peptide recognition was tested weekly in interferon-γ (IFN-γ) Elispot.34 96-well nitrocellulose plates (Multiscreen, Millipore) were pre-incubated with 3 μg/ml anti-INF-γ antibody at 4 °C overnight (Mabtech, Nacka Strand, Sweden). Plates were washed and blocked with TCM before pre-stimulated effector cells and T2 cells pulsed with WT1 peptides were incubated at an E/T-ratio of 1:2 at 37 °C for 16 h in TCM without IL-2.

After washing in phosphate buffered saline with 0.05% Tween 20, plates were incubated with 0.5 μg/ml biotinylated anti-IFN-γ detection antibody (Mabtech) for 2 h at 37 °C. Spots were detected by incubation with streptavidin-coupled alkaline phosphatase (1 h at room temperature) and substrate (5-Brom-4-chlor-3-indoxylphosphate (BCIP)/Nitrobluetetrazoliumchloride (NBT), Roche). Spots were counted under the microscope.

A positive response was recorded if the number of spots in the peptide-exposed wells was two times or more higher than the number of spots in the unstimulated wells and if there was a minimum of ten (after subtraction of background spots) peptide-specific spots per 25 000 CD8+ cells (or less if T-cell clones were used).

Cytotoxicity testing in 51Chromium-release and Europium-release assays

The lytic capacity of cytotoxic T lymphocytes (CTLs) was tested in 51Chromium (51Cr)- and Europium (Eu)-release assays as described earlier.34, 35 Briefly, target cells were labeled with 100 μ Ci 51Cr sodium chromate. After extensive washing to remove unincorporated 51Cr, cells were resuspended in RPMI/10% fetal calf serum, plated in 96 well V-bottom plates, and incubated with effector cells at different effector to target (E/T) ratios for 4 h at 37 °C. 51Cr-release into the supernatant was measured with a gamma-counter and specific cytotoxicity was calculated as follows: specific release (%)=100 × (experimental release-spontaneous release)/(maximum release-spontaneous release).

Europium-release assays were conducted in a similar manner, following the manufacturer's instructions. Target cells were incubated with BATDA reagent (Perkin Elmer, Waltham, MA, USA) for 30 min at 37 °C. After extensive washing, target cells were resuspended in medium and incubated with effector cells at different E/T-ratios for 2 h at 37 °C. Afterwards, supernatant was harvested and incubated with europium solution (Perkin Elmer) for 15 min under continuous rotation. The concentration of EuTDA-complex, a highly fluorescent and stable chelate complex between europium and TDA-ligand, was measured in a time-resolved fluorometer (1420-018 Victor, Perkin Elmer). Specific release was calculated as indicated above. Tumor cell lines were pre-incubated with IFN-γ (100 U/ml) before cytotoxicity testing.

Flow cytometry

Fluorescein isothiocyanate (FITC), PE, peridinin chlorophyll protein (PerCP) and allophycocyanin (APC)-labeled mouse anti-human antibodies were used for FACS staining. Data acquisition was performed on FACS Calibur (Becton Dickinson Biosciences, San Jose, CA, USA) and analyzed with CellQuest and CellQuest pro software (Becton Dickinson).

For pentamer staining, lymphocytes were incubated with unlabeled pentamer for 10 min at room temperature. After washing twice, PE-labeled fluorotag and further antibodies were added and incubated on ice for 20 min.

Results

WT1 expression in STS and leukemia cell lines and primary tumor samples

The WT1 expression level of cell lines and primary tumor samples was assessed by quantitative real-time PCR. WT1 copy number was normalized to ABL gene expression and copy numbers were given as a ratio of WT1-copies/10 000 ABL-copies. Expression of WT1 protein in cell lines was confirmed by non-quantitative western blot (data not shown), and results were correlated with real-time PCR results. The cell lines tested showed WT1 expression compared with ABL ranging from 1.47 × 10−1 to 5.87 × 103 (median 1.5 × 10−1) for STS cell lines and from 9.08 × 101 to 5.39 × 104 (median 9.4 × 103, mean 1.4 × 104) for leukemic cell lines (Figure 1a). Primary RMS tumor samples expressed WT1 at levels comparable to those of cell lines with only one negative sample (range 0–1.6 × 104, median 3.4 × 103) (Figure 1b).

Figure 1
figure1

WT1 expression of STS and leukemia cell lines and primary RMS tumor samples. WT1 and ABL copy numbers were assessed by real-time PCR using a logarithmic plasmid dilution series. WT1 expression is given in copy numbers normalized to the expression of the ABL control gene (WT1-copies/10 000 ABL-copies).

HLA expression on cell lines

HLA class I/II expression and HLA-A2+ phenotype of the cell lines was confirmed by FACS staining with HLA-A, B, C, HLA-DR and HLA-A2 antibodies and/or PCR.

WT1-expressing leukemic cell lines THP-1 and BV173, as well as the ES cell line Rh1 and breast cancer cell line MDA-MB-231 were positive for HLA-A2 and were consequently used in subsequent experiments (data not shown).

Generation of WT1-specific CD8+ T cells from peripheral blood

Four HLA-A*0201 restricted WT1-peptide epitopes (WT1 p37, p126, p187, p235) and one HLA-A*01-associated WT1 epitope (WT1 p317) were applied to generate peptide-specific T cells from the peripheral blood of HLA-A2+ and HLA-A1+ cancer patients and healthy volunteer donors.

Six out of ten adult breast cancer patients, one patient with acute myeloid leukemia and one out of four melanoma patients showed a positive reaction in the IFN-γ Elispot assay after two or three restimulation cycles (Table 1). A WT1-specific reaction could not be induced from any of the HLA-A1+ patients after in vitro stimulation with WT1 p317. Positive T-cell responses could be detected in an individual patient against up to three different WT1 epitopes. Recognition of one WT1 peptide was the most common finding (four patients), followed by a positive reaction against two peptide epitopes (three patients). One breast cancer patient showed recognition of three HLA-A2+ WT1-peptide epitopes after in vitro stimulation.

Table 1 Results of interferon-γ Elispot

Next, we analyzed a group of ten HLA-A2+ healthy volunteer donors. Remarkably, nine of them displayed a positive response after in vitro stimulation of T cells with WT1 peptides. Up to three different T-cell cultures specific for a single WT1-peptide epitope could be generated from the peripheral blood of a healthy volunteer donor. The most common finding was the generation of T cells specific for one (four donors) or two peptides (four donors). Only one healthy donor did not display a T-cell response towards any of the WT1 epitopes tested (Table 1). None of the patients or healthy donors stimulated with WT1 p187 (HLA-A*0201) or WT1 p317 (HLA-A*01) showed a positive reaction.

T-cell response to influenza

T-cell responses to influenza matrix protein p58–66 GILGFVFTL (HLA-A*0201) or p44–52 CTELKLSDY (HLA-A*01) were analyzed in Elispot as a positive control to test the general efficiency of expansion of pre-existing T cells from peripheral blood. Only one patient did not show an influenza-specific T-cell response (Table 1).

Cytotoxicity and peptide-specificity of WT1-specific CTLs generated from cancer patients

Functional characteristics of WT1-specific CTL lines established from cancer patients were further analyzed in cytotoxicity assays. To test peptide reactivity and lytic activity, T2 cells were pulsed with the specific WT1-peptide epitope and then exposed to CTL lines. Non-specific HLA-A*0201 WT1 epitopes, influenza or CMV peptides were used as negative controls to determine specificity and exclude cross-reactivity. Peptide-specific T-cell cultures showing tumor recognition of WT1+ tumor cell lines could be generated from the peripheral blood of three breast cancer patients. Data from a T-cell bulk culture generated from the peripheral blood of breast cancer patient NW-585 are shown in Figure 2. These T cells exhibited lytic activity restricted to the specific peptide, WT1 p37 (Figure 2a). Subsequent experiments with tumor cell lines that express WT1 endogenously as targets showed specific lysis of HLA-A2+ breast cancer cell line MDA-MB-231 (Figure 2b).

Figure 2
figure2

Peptide-specificity and tumor-reactivity of in vitro pre-sensitized WT1 p37 CTLs generated from the peripheral blood of breast cancer patient NW-585. (a) T2 cells pulsed with specific peptide WT1 p37 were used as target cells in a 51Cr-release assay to test peptide-specificity. Unpulsed T2 cells and irrelevant peptide WT1 p126 were used as controls to exclude cross-reactivity and non-specific lysis. (b) Cytotoxicity of the WT1 p37 specific CTL line was also tested against unpulsed HLA-A2+ WT1+ tumor cell lines Rh1 and MDA-MB-231. An HLA-A2+ WT1 pancreas carcinoma cell line was used to confirm the dependence of tumor cell recognition on WT1 expression. No antigen independent NK-activity could be observed when using K562 as target cells. Killing of target cells is shown at different effector to target (E/T) ratios. Results are given as mean and standard error of the mean.

Generation of WT1 CTL clones from peripheral blood of healthy volunteer donors

We investigated the possibility of establishing WT1-specific T cells from healthy volunteer donors. In vitro stimulation and testing of peptide recognition was carried out as described above. Generation of T-cell clones was performed by limiting dilution from T-cell cultures that tested positive in the IFN-γ Elispot after 2 or 3 weeks of in vitro stimulation. Proliferating clones were tested for specific peptide recognition in the IFN-γ Elispot. Clones that tested positive were expanded further. WT1 CTL clones targeting WT1 epitopes p37 or p126 were generated from four healthy donors. Clones from all four donors exhibited specific cytotoxic activity against peptide-pulsed T2 cells (Figures 3a and b) and against WT1+ tumor cell lines in three out of four donors (Figures 3c–f). Clones showing cross-reactivity against non-specific WT1 peptides were excluded from further analysis.

Figure 3
figure3

Peptide-specificity and tumor-reactivity of in vitro generated CTL clones from healthy volunteer donors. Specific lytic activity of WT1 p37 (a) and WT1 p126 (b) CTL clones from three healthy volunteer donors was assessed using peptide-pulsed T2 cells as target cells. To test non-specific killing, irrelevant WT1 and/or influenza peptides were used. No cross-reactivity could be observed. Cytotoxic activity of WT1 p37 (c) and p126 (d, e, f) CTL clones against WT1+ tumor cell lines were assessed in 51Cr- and Eu-release assays. Killing of Rh1 (c, d) and MDA-MB-231 (e) (both HLA-A2+ WT1+), but not of an HLA-A2+ WT1 pancreas carcinoma cell line, was detectable in three of four healthy volunteer donors. (f) Leukemia cell lines, THP-1 and BV173 (HLA-A2+ WT1+) were only recognized after exogenous peptide loading, in spite of high levels of WT1-RNA and protein expression as confirmed by real-time PCR and western blot. Results are shown as mean and standard error of the mean.

Analysis of cytotoxicity against WT1+ tumor cell lines

Further analysis of the cytotoxic potential against HLA-A2+ tumor cell lines with endogenous WT1 expression was tested in standard 51Cr- and/or Eu-release assays. Experiments were carried out in duplicates, and positive results were confirmed at least twice.

WT1 p126 specific CTL clones generated from healthy donors recognized the HLA-A2+ WT1+ breast cancer cell line MDA-MB-231 and ES cell line Rh1. Rh1 was also recognized by WT1 p37-specific CTL clones, which showed lower cytotoxic capacity against MDA-MB-231. All tumor-reactive T cell clones proved to be peptide specific. No killing of HLA-A2+ WT1 or HLA-A2 WT1+ tumor cell lines could be observed, further confirming WT1-specific recognition of tumor cell lines. No antigen independent NK-activity was detectable when using K562 as target cells (Figures 3c–e).

HLA-A2+ WT1+ leukemic cell lines THP-1 and BV173 were not recognized by WT1 p37 or p126 CTL clones generated from healthy donors unless exogenously loaded with WT1-peptides (Figure 3f). These results indicate that, in principle, lysis of these cell lines by WT1-specific T cells is possible but that recognition by CTLs is possibly constrained because of a lack of expression of WT1-peptide epitopes p37 and p126 on the cell surface.

Antigen specificity and HLA restriction of CTL responses

The amount of peptide presented by target cells might represent a limiting factor for efficient recognition by T cells. Therefore, we analyzed the peptide affinity of CTL clones from four healthy donors by peptide-dose titration on T2 cells in the Eu-release and Elispot assays. T2 cells were pulsed with peptide concentrations ranging from 10 μM to 0.001 nM. CTL clones from four donors were then incubated at E/T-ratios of 10:1 with peptide-pulsed T2 cells for analysis of cytotoxicity and IFN-γ secretion, respectively. WT1 p37 and p126 CTL clones from healthy donors recognized peptide-pulsed T2 cells at concentrations as low as 0.0256 nM (WT1 p37) and 0.64 nM (WT1 p126) (Figures 4a and b).

Figure 4
figure4

HLA restriction of restriction of tumor cell lysis by WT1-specific CTL clones generated from healthy donors. The affinity of TCR-peptide binding was analyzed by peptide-titration on T2 cells in Eu-release assays. Specific lysis of peptide-pulsed T2 cells could be detected at concentrations as low as 0.0256 ng/ml for WT1 p37 (a) and 0.64 ng/ml for WT1 p126 CTL clones (b). Peptide titration was carried out with WT1-specific CTL clones from four healthy volunteer donors. Squares and circles represent clones from different healthy volunteers. Inhibition of WT1 p37 (c) and p126 (d) CTL-mediated antigen recognition by blocking with HLA-A2, HLA-class I and CD8 antibodies in IFN-γ Elispot. HLA-DR, CD4 and CD56 were used as negative controls. White and gray bars represent different WT1-specific T cell clones from four healthy volunteer donors. The allo-reactive potential of WT1 p37 CTL clones generated from the peripheral blood of three healthy donors was tested in Eu-release assays against HLA-A2 PBMCs, HLA-A2+ PBMCs and peripheral HLA-A2+ CD34+ enriched stem cells from healthy donors. No lysis could be observed (e). Results are shown as mean and standard error of the mean.

HLA-class I-restriction of WT1 CTLs was confirmed by blocking cytotoxicity with specific antibodies for HLA-A2 (MA2.1), HLA-class I (W6/32) and CD8. HLA-DR, CD4 and CD56 antibodies were used as negative controls. Inhibition of peptide recognition could be shown in the IFN-γ Elispot (HLA-A2, HLA-class I, CD8) (Figures 4c and d) and Eu-release assays (CD8) in WT1-specific CTL clones from four healthy volunteer donors (data not shown). HLA-class I antibody W6/32 could not be used in our Eu-release assays because of interference with the fluorimetric detection system. Results showed a distinct reduction in the number of IFN-γ spots (to a level close to background signals) or a reduction in specific lysis either when T cells were blocked by CD8 antibody or T2 target cells were blocked by HLA-A2 or HLA-class I antibodies. Hence, HLA restriction of antigen recognition could be shown for WT1 p37 and p126 CTL clones.

The allo-reactive potential of WT1 CTLs against normal hematopoietic cells was assessed using PBMCs as target cells in cytotoxicity assays. PBMCs from HLA-A2 healthy donors, HLA-A2+ PBMCs and peripheral HLA-A2+ CD34+ enriched cells were tested. No killing of normal hematopoietic cells could be observed in WT1-specific CTL clones from three healthy volunteer donors (Figure 4e).

Phenotype of WT1 CTLs

WT1 p126-stimulated T-cell lines and clones were stained with PE-labeled HLA-A*0201/WT1 p126 multimers and anti-CD8 antibody. HLA-A*0201/WT1 p235 and HLA-A*2401/WT1 p235 multimers were used as negative controls. To our knowledge, no HLA-A2/WT1 p37 multimer was available at the time these analyses were performed.

Specific expansion of WT1 p126 T cells from the bulk culture could be shown for healthy donor D4 by pentamer staining. Multimer HLA-A*0201/WT1 p126 staining of CD8+ bulk culture after 2 weeks of in vitro stimulation showed about 1.7% positive cells (numbers given as percentage of live lymphocyte gate) (Figure 5a). The proportion of stained cells increased during the following 2 weeks and resulted in 91.6% positive cells after four restimulation cycles (Figures 5b and c). Pentamer binding was sustained until day 70 of in vitro culture (Figure 5d). T-cell clones generated from this donor's bulk culture harbored more than 90% multimer-binding T-cells (Figure 5e). Nevertheless, other T-cell lines and clones generated from the same and other donors with comparable functional characteristics and peptide-specificity did not bind to HLA-A*0201/WT1 p126 multimers.

Figure 5
figure5

Expansion of WT1-specific T cells from the peripheral blood of healthy volunteer donors. FACS multimer staining of WT1 p126 T-cell bulk culture generated from healthy donor D4 during in vitro stimulation at day 14 (a), day 21 (b), day 28 (c) and day 70 (d). Cells were stained with CD8-FITC and PE-labeled HLA-A*0201/WT1 p126–134 pentamers. HLA-A*0201/WT1 p235–243 pentamer was used as a negative control and resulted in less than 0.05% positive cells (not shown). (e) WT1 p126 CTL clones generated from this bulk culture by limiting dilution consisted of more than 95% multimer binding CD8+ T-cells. A representative example is shown.

Multimer+ T-cell clones showed expression of CD45R0 although being negative for CD45RA and CCR7. Thus, they can be considered effector-memory T cells, a T-cell subset that confers immediate immune protection in peripheral tissues.

Discussion

Wilms tumor antigen 1 has been detected in various human hematopoietic malignancies and solid tumors, with only low expression in normal tissues such as kidney and ovaries and transient expression in hematopoietic stem cells.36, 37, 38, 39 Little was known about the expression of WT1 in STS. We measured WT1 expression in RMS/ES cell lines in comparison to leukemia cell lines by quantitative real-time PCR and western blot. WT1 was expressed in three out of seven (43%) RMS/ES cell lines at levels in the range of leukemic cell lines, which are known to express substantial amounts of WT1 mRNA and protein as confirmed in this study.4, 5 Four out of seven (57%) RMS/ES cell lines expressed low levels of WT1 RNA or were negative in western blot analysis. In contrast, WT1 expression in primary RMS tumor samples was higher than in the cell lines analyzed. Only one tumor specimen was WT1-negative in spite of good RNA/cDNA quality, as confirmed by analysis of ABL gene expression. WT1 positivity could be shown by PCR and western blot assays, which is in accordance with earlier published data showing strong cytoplasmic WT1 immunostaining in RMS, suggesting this antigen a potential target for immunotherapy.15, 40, 41 As shown by us and others, RMS and ES cell lines and primary tumors show increased expression of HLA class I molecules compared with normal striated muscle or chemotherapy-sensitive cells,42, 43 which may render RMS and ES tumors ideal candidates for T cell based immunotherapy targeting WT1. Although earlier studies had shown that CTLs exhibiting cytotoxic effects against WT1-expressing tumor cells and leukemic blasts could be expanded from patients with various malignancies10, 12, 13, 23, 27, 29 it remained unclear whether such WT1-specific T cells could also be reliably generated from the peripheral blood of healthy volunteer donors. This is a prerequisite for the use of WT1 peptide-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation. In this study, we were able to show that functionally active WT1-specific T cells can be isolated from the peripheral blood of healthy volunteers and expanded by in vitro stimulation.

The frequency of antigen-specific T cells constitutes a major limitation for effective retrieval of functionally active effector cells. Ex vivo expansion of specific T cells has been established for antiviral or antifungal treatment approaches targeting adenoviral, cytomegalovirus or aspergillus antigens.44 The frequency of antigen-specific T cells, however, is usually higher for viral or fungal antigens than for tumor antigens that are expressed in malignant cells but may also be expressed at a low level in some healthy tissues. Therefore, strategies to generate and effectively expand T cells in vitro are urgently needed to render this approach feasible for anti-cancer immunotherapy.

In a first step, we analyzed the effectiveness of in vitro stimulation methods to generate and expand WT1 antigen-specific T cells from the peripheral blood of cancer patients. In six of eight HLA-A2+ breast cancer patients, one melanoma and one leukemia patient, a CD8+ response to WT1-peptide stimulation could be detected after in vitro pre-sensitization. The high incidence of positive responses to WT1 peptide stimulation in vitro in cancer patients may be a consequence of endogenous immune responses against WT1 tumor antigen in vivo.

In a second step, we successfully isolated and expanded functionally active WT1-specific T cells from the peripheral blood of nine out of ten HLA-A2+ healthy volunteer donors. T-cell lines and clones recognizing WT1 p37 and p126 could be generated. Although other reports have showed peptide-specific responses in healthy donors for only a single WT1-peptide, here several T-cell cultures with diverse specificities against up to three different WT1 epitopes could be established from one individual donor. This more closely resembles observations in cancer patients that have frequently shown recognition of several epitopes.22, 26 Furthermore, clinical application of adoptive immunotherapy demands ex vivo expansion of effector cells. We were successful in generating and expanding T-cell clones specific for WT1 p37 and p126. We could also show anti-tumor reactivity against the WT1+ breast cancer cell line MDA-MB-231 and the ES cell line Rh1 in vitro.

As WT1 is also expressed at lower levels by CD34+ hematopoietic stem cells and non-malignant mesenchymal cells, adoptive transfer of WT1 peptide-specific T cells after allogeneic stem cell transplantation might induce adverse effects like marrow aplasia or graft-versus-host disease-like tissue damage. We could show that WT1-specific CTL clones established from healthy volunteers do not kill allogeneic PBMCs or CD34+ peripheral stem cells. These findings are in agreement with animal studies and ongoing clinical trials with WT1 peptide vaccines that have shown no adverse effects regarding hematopoiesis or other autoimmune phenomena, with a considerable increase in the frequency of WT1-specific T cells after vaccination.12, 16, 24, 45, 46, 47, 48

In summary, we could show that the generation and in vitro expansion of functionally active WT1-specific T cells from the peripheral blood of healthy volunteer donors is feasible. These in vitro expanded effector cells displayed peptide-specific cytotoxicity against WT1-expressing solid tumor cell lines. No allo-reactive effects against HLA-different PBMCs or HLA-A2+ CD34+ peripheral stem cells could be observed. These findings indicate that WT1 peptide-specific T cells are ideal candidates for post-transplant immunotherapy in patients with WT1-expressing malignancies such as leukemia or STS.

Conflict of interest

The authors declare no conflict of interest.

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Acknowledgements

This work was supported by ‘Patenschaftsmodell’ of the University of Frankfurt/Main, Germany (GW) and is a sub-project of TranSaRNet, Translational Sarcoma Research Network of the Bundesministerium für Bildung und Forschung, Germany (PB).

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Correspondence to P Bader.

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Weber, G., Karbach, J., Kuçi, S. et al. WT1 peptide-specific T cells generated from peripheral blood of healthy donors: possible implications for adoptive immunotherapy after allogeneic stem cell transplantation. Leukemia 23, 1634–1642 (2009). https://doi.org/10.1038/leu.2009.70

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Keywords

  • WT1
  • Wilms' tumor gene
  • cytotoxic T lymphocytes
  • immunotherapy
  • stem cell transplantation

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