MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells to facilitate cytotoxic T lymphocyte-mediated tumor cell killing
Approximately half of patients with stage IV neuroblastoma are expected to relapse despite current therapy, and when this occurs, there is little likelihood of achieving a cure. Very few clinical trials have been conducted to determine whether cellular immune responses could be harnessed to fight this tumor, largely because potential tumor antigens for cytotoxic T lymphocytes (CTL) are limited. MAGE-A1, MAGE-A3, and NY-ESO-1 are cancer-testis (CT) antigens expressed on a number of malignant solid tumors, including neuroblastoma, but many tumor cell lines down-regulate the expression of CT antigens as well as major histocompatibility (MHC) antigens, precluding recognition by antigen-specific T cells. If expression of cancer antigens on neuroblastoma could be enhanced pharmacologically, CT antigen-specific immunotherapy could be considered for this tumor. We have demonstrated that the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells following exposure to pharmacologic levels of the demethylating agent 5-aza-2′-deoxycytidine (decitabine, DAC). Expression of NY-ESO-1, MAGE-A1, or MAGE-A3 was induced in 10/10 neuroblastoma cell lines after 5 days of exposure to DAC. Culture of neuroblastoma cell lines with IFN-γ was also associated with an increased expression of either MHC Class I or II by cytofluorometry, as reported by other groups. MAGE-A1, MAGE-A3, and NY-ESO-1-specific CTL were cultured from volunteer donors by stimulating peripheral blood mononuclear cells with dendritic cells pulsed with overlapping peptide mixes derived from full-length proteins, and these CTL preferentially lysed HLA partially matched, DAC-treated neuroblastoma and glioblastoma cell lines. These studies show that demethylating chemotherapy can be combined with IFN-γ to increase the expression of CT antigens and MHC molecules on neuroblastoma cells, and pre-treatment with these agents makes tumor cell lines more susceptible to CTL-mediated killing. These data provide a basis to consider the use of demethylating chemotherapy in neuroblastoma patients, in conjunction with immune therapies that facilitate the expansion of CT antigen-specific CTL.
KeywordsCancer-testis antigens Decitabine Interferon-γ Neuroblastoma MHC molecules
Neuroblastoma (NB) is the most common extracranial solid tumor in children and for those >1 year of age with advanced disease, the 3 year progression-free survival is only 30% . For patients with relapsed disease, there is little that can be done with curative potential, making the development of novel therapies of much interest in this patient group. Current therapies include autologous stem cell transplantation and are associated with a moderate to severe degree of toxicity, precluding further intensification of chemotherapy. Therefore, alternate therapies such as cancer antigen vaccines or cellular immunotherapy are of interest not only for relapsed patients but also as an adjuvant to prevent disease recurrence. Limitations in the practical application of adoptive immunotherapy for NB include a lack of thorough understanding of which tumor antigens can be reasonably targeted, as well as the mechanisms whereby tumor cells are able to escape immune surveillance. The latter includes down regulation of tumor antigens as well as MHC Class I and II molecules, preventing tumor cells from being recognized by cytotoxic T lymphocytes (CTL) . Cancer-testis (CT) antigens are expressed by a number of malignant solid tumors and leukemias, as well as a subset of NB [3, 4, 5]. Some of the most immunogenic CT antigens include NY-ESO-1 and MAGE family proteins, and previous studies have demonstrated clinical and immunologic responses following CT antigen vaccines in cancer patients [6, 7, 8, 9, 10, 11, 12]. Nevertheless, the level of CT antigen expression can vary widely on tumors, and not all cells may express these proteins, complicating CT antigen-based immunotherapeutic approaches.
DNA methylation is an epigenetic mechanism used by cells to decrease the expression of several genes, including tumor suppressor genes [13, 14]. Decitabine (5-aza-2′-deoxycytidine, DAC) is a potent inhibitor of DNA methylation, and the doses associated with the demethylating action of this drug are much lower than those required for cytotoxicity [15, 16, 17]. There have been multiple in vitro studies demonstrating that exposure to demethylating agents can lead to expression of several CT antigens by tumor cell lines, including melanoma, malignant glioma, and leukemia cells, but there is no such data for NB [18, 19, 20, 21, 22]. Current challenges in applying cellular immunotherapy for malignant solid tumors such as NB include the identification of suitable tumor antigens as well as the problem of MHC Class I and II downregulation on the surface of tumor cells [23, 24]. Exposure to interferon-γ (IFN-γ) or DAC can be associated with increased or new onset expression of MHC Class I/II antigens on these tumors [18, 20]. Previous studies have shown that demethylating chemotherapy can facilitate the expression of CT antigens on tumor cells, but this has not been studied in NB . If these antigens could also be upregulated on NB cells, then immunotherapy strategies such as vaccines or CTL infusions could be considered in high risk NB patients. We have demonstrated that DAC upregulates the expression of MAGE-A1, MAGE-A3, and NY-ESO-1 on the majority of NB cell lines, and treatment with IFN-γ increases MHC Class I expression. NB cells that are treated with these agents are more susceptible to killing by CT antigen-specific CTL-mediated killing.
Materials and methods
Treatment of tumor cell lines with DAC and IFN-γ
BE2C, NBL-S, Kelly, NGP, SHSY5Y, EB2M17, and IMR32 (Human NB cell lines, kindly provided by Dr. Rani E. George, Pediatric Hematology-Oncology, Children’s Hospital of Boston) were grown in RPMI (GIBCO BRL). SKN-AS, SKN-SH, CHP134 (human NB cell lines purchased from American Type Culture Collection (ATCC), (Rockville, MD), and SKN-MC (human neuroepithelioma cell line, ATCC HB-10) were grown in Dulbecco Modified Eagle Medium (DMEM)(GIBCO BRL). T98G (human glioblastoma cell line; ATCC CRL-1690), A172 (human glioblastoma cell line; ATCC CRL-1620), U251 (Human glioma, kindly provided by Dr. Song Lee at Penn State Hershey Medical Center, Hershey, PA) and were grown in DMEM, and CCF-STTG1 (human astrocytic glioblastoma cell line; ATCC CRL-1718) cells were grown in RPMI 1640 (GIBCO BRL). All media were supplemented with 10% fetal bovine serum. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO2.
Cells were counted and plated in 10-cm dishes 1 day prior to treatment, and then the media was removed and replaced with fresh media containing 1 μM of DAC (Sigma–Aldrich, St. Louis, MO) or 100 ng/ml human recombinant IFN-γ (R&D Systems, Minneapolis, MN). After incubation with DAC or IFN-γ for 5 days, the cells were harvested, counted, and then prepared for molecular and functional assays.
Reverse transcription polymerase chain reaction
Total RNA was prepared from 2 to 5 × 106 cells using the RNA-Bee-RNA Isolation reagent (TEL-TEST, Inc. Friendswood, TX) as per the kit instructions and was quantified by absorbance at 260 nm. A 1-μg aliquot of total RNA was converted to cDNA using the RETROscript (Ambion, Austin, TX), and PCR was performed on 1 μl of cDNA using Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA). Primers were synthesized using previously published sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , MAGE-A1 , MAGE-A3 , NY-ESO-1 . After the PCR mixes were heated to 95°C for 10 min, 30 cycles of amplification were performed (1 cycle = 60 s denaturation at 94°C, 60 s annealing at the temperature as indicated below, 60 s extension at 72°C), and a final extension was completed at 72°C for 6 min. Annealing was 62°C for MAGE-A1 and NY-ESO-1, 70°C for MAGE-A3, and 55°C for GAPDH (25 cycles). Relative expression levels were determined by visualizing DNA bands on ethidium bromide-stained 1.5% agarose gels.
Cells were lysed with M-PER mammalian protein extraction reagent buffer (Pierce Biotechnology Inc., Rockford, IL) with proteinase inhibitors (Pierce Biotechnology Inc., Rockford, IL). Protein concentrations were determined according to the manufacturer’s protocol using the BIO-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Fifty micrograms of whole cell lysates were separated on 10% SDS denaturing polyacrylamide gels and transferred onto nylon membrane (Millipore) at 30 V for 1 h at room temperature. Membranes were blocked in TBS containing 0.5% Tween 20 plus 5% nonfat dried milk for 1 h at room temperature and probed with primary antibodies at 4°C overnight. Blots were incubated with antibodies to MAGE A1 (MA545, Santa Cruz Biotechnology), MAGE A3 (mouse polyclonal, Abcam), NY-ESO-1 (monoclonal, E978, Invitrogen), and actin (Sigma), the latter as a loading control. Membranes were washed 3 times for 10 min each in TBS containing 0.5% Tween 20 and incubated with horseradish peroxidase–conjugated goat anti-rabbit or anti-mouse secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Targeted proteins were visualized using an enhanced chemiluminescence detection system (Pierce Biotechnology Inc., Rockford, IL).
Flow cytometry was performed with a FACScan (BD Biosciences, San Jose, CA) for MHC Class I and Class II expression. MHC Class I and Class II expression levels were determined by staining with directly conjugated monoclonal antibodies (mAb): PE conjugated anti-human HLA-ABC mAb to detect MHC Class I and FITC conjugated anti-human HLA-DR, DP, DQ mAb (BD Biosciences, San Jose, CA) to detect MHC Class II.
Generation of dendritic cells for CTL stimulation
PBMC from normal volunteers were isolated by Ficoll-Paque (Amersham Biosciences) gradient centrifugation of 50 ml heparinized whole blood. Cells were seeded at 1 × 107 cells/2 ml/well into 6-well plates (Corning) in CellGenix™ DC medium. PBMC were incubated in a humidified incubator for 2 h at 37°C to allow plastic adherence. After 2 h of incubation, the nonadherent cells were removed and cryopreserved at −80°C, to be used later for cell isolation. The adherent blood monocytes were cultured 5 days in CellGenix™ DC medium supplemented with human recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF; 1,000 U/ml, Bayer, Seattle, WA) and interleukin 4 (IL-4; 10 ng/ml; R&D Systems, Minneapolis, MN). Immature DC were matured for 48 h in the presence of 10 ng/ml of TNF-α, 10 ng/ml of IL-1β, 10 ng/ml of IL-6 (R&D Systems, Minneapolis, MN), and 1 μg/ml of PGE2 (Sigma, St. Louis, MO).
CT antigen-specific T-cell cultures
To generate CT antigen-specific T cells, PBMC from healthy donors were stimulated with DC pulsed with MAGE-A1, MAGE-A3, and NY-ESO-1 overlapping peptide mixes, consisting of pooled, 11 amino acid overlapping 15mers derived from the full-length protein (JPT Peptide Technologies, Berlin, Germany). These cells were stimulated an additional two times (weekly) with DC pulsed with these peptide mixes. IL-7 and IL-15 (10 ng/ml; R & D Systems, Minneapolis, MN) were added to these cultures initially and replaced weekly, and IL-2 (50 units/ml, Novartis, East Hanover, NJ) was added every 3 days, starting at day 3.
To select antigen-specific T cells based on CD137 (human CD137 MicroBead Kit, Miltenyi Biotec Inc. Auburn, CA) production, we performed preliminary studies to determine the time at which CD137 reached its peak level of expression post-stimulation. CMV pp65 CTL were examined for CD137 expression, which was at its maximum from 18 to 24 h post-stimulation. Therefore, T cells that had already been stimulated twice with tumor antigen were re-exposed with DC pulsed peptide mixes for 24 h. After CD137 selection, activated T cells were further cultured for 14 days in a cocktail containing the anti-CD3 monoclonal antibody OKT3 (30 ng/ml; Ortho-Biotech, Raritan, NJ), irradiated (3 Gy) allogeneic feeder PBMC (1 × 106 cells/ml), irradiated (10 Gy) BLCL (0.1 × 106 cells/ml), and IL-2 (50 units/ml, Novartis, East Hanover, NJ). This cell culture was placed in a T25 flask, OKT3 was removed on day 5, and 50% of the culture medium (RPMI 1640 with 10% FBS) was replaced every 3 days or as needed with fresh medium and IL-2 [28, 29]. The tumor-specific function of expanded CTL was characterized in chromium release assays described below.
Chromium release assays
Cytotoxicity were determined by 51Cr release assays using the following target cells: autologous B cell blasts (BB; used as a negative control), BB pulsed with the MAGE-A1, MAGE-A3, and NY-ESO-1 peptide mixes, as well as tumor cells before and after DAC and IFN-γ treatment. Targets were labeled overnight with 51Cr (100 μCi/106 cells; PerkinElmer Life and Analytical Science), washed in PBS, and dispensed in triplicate into 96-well V-bottom plates (ICN) at 4 × 103 cells/well, as previously described . CTL were added at a responder-to-target ratio (20:1), and after pelleting and incubation for 4 h, the supernatant was analyzed in a gamma counter. Spontaneous and total release for each target were used to calculate percent-specific release by the following formula: % specific release = (experimental cpm − spontaneous cpm)/(total cpm − spontaneous cpm).
Up-regulation of CT antigen expression on neuroblastoma cells by DAC
The effect of the various doses of DAC and duration of treatment on MAGE-A1, MAGE-A3, and NY-ESO-1 expression
Enhancement of MHC Class I and Class II expression on NB cell lines after IFN-γ treatment
Generation of CT antigen-reactive T lymphocytes, and recognition of DAC-treated tumor cells
Epigenetic regulation has several effects on cancer cells, including the expression of tumor antigens, facilitating tumor cell apoptosis by methylation of tumor suppressor genes, and modulating caspase-8 activation [13, 14, 32]. There are several mechanisms by which tumor cells can escape immune surveillance, including the down regulation of tumor antigens and MHC Class I/II molecules [2, 23, 24]. Recently several groups have demonstrated the fact that the use of demethylating agents and histone deacetylase inhibitors can be used to increase the expression of CT antigens on tumor cell lines, but there is no data for NB or other tumors that primarily affect children [18, 19, 20, 21, 22]. Current therapy for stage IV NB includes the sequential use of high-dose chemotherapy, which cannot be further intensified. Therefore, further study is needed into the biologic feasibility to targeting CT antigens on NB cells by specific CTL. We have demonstrated that DAC and IFN-γ can have positive effects on the expression of CT antigens and MHC Class I/II molecules by NB cell lines. The majority of NB cells treated with DAC had either new onset or increased expression of MAGE-A1, MAGE-A3, or NY-ESO-1 as early as 24 h post-exposure. The fact that CT antigen-specific CTL have improved recognition/killing of DAC-treated tumor cell lines indicates that pharmacologic upregulation of specific antigens could be used prior to immunotherapy to maximize the likelihood of a clinical response.
One of the most significant challenges to the practical application of cellular immunotherapy is the identification of tumor antigens that are preferentially expressed on tumor cells. CT antigens are expressed on a limited number of tissues, including male germ line cells, placenta, melanoma, bladder, breast, prostate, non-small cell lung cancers, and NB [33, 34]. Three CT antigens that can be expressed on solid tumors include NY-ESO-1, MAGE-A1, and MAGE-A3 [3, 35, 36]. From previous studies, the frequency of NY-ESO-1 expression has been reported to range from 30 to 82% for NB, and IFN-γ producing NY-ESO-1-specific T cells have been detected from NB patients with NY-ESO-1 positive tumors [3, 37]. Screening NB for these antigens by RT-PCR has revealed that 44% are positive for MAGE-A1 and 21% for MAGE-A3, and immunohistochemical analysis showed a good correlation between protein and gene expression . There is no data on the detection of MAGE-specific T cells in NB patients, but studies in adult patients have demonstrated that MAGE-A1- and MAGE-A3-specific T cells were present and can be augmented following a vaccine, or by stimulating these T cells in culture [6, 7, 8]. There has also been a single report of the detection of IFN-γ producing NY-ESO-1-specific T cells in NB patients , as has been demonstrated in adult patients with NY-ESO-1 positive tumors. There have been several trials with MAGE-A1, MAGE-A3, and NY-ESO-1 vaccines in adult cancer patients with malignant solid tumors, using either whole tumor protein or individual epitopes. Clinical trials have been reported using DC-based vaccines as well as using whole tumor proteins or HLA-restricted, CT antigen epitopes, and several of these studies have demonstrated immune responses to MAGE-A1, MAGE-A3, and NY-ESO-1 post-vaccination [9, 10, 11, 12, 38]. Some subjects have had regressions of metastatic lesions and improved survival following CT antigen vaccines, but many patients receiving immunotherapy experience disease progression [12, 38]. Therefore, strategies which facilitate the recognition of tumors by antigen-specific T cells are of interest in improving the clinical outcomes following immune-based therapies.
Some studies have shown that DAC can upregulate the expression of CT antigens and MHC Class I molecules on tumor cells, but data are lacking on whether CT antigens can be epigenetically upregulated on NB cells [18, 20]. Serrano et al.  demonstrated that MHC Class I antigens could be upregulated on DAC-treated melanoma cells, restoring MAGE-A1 antigen-specific killing of the DAC-treated cells. Teitz et al.  showed that epigenetic upregulation of the pro-apoptotic gene caspase-8 can also occur in NB as a result of demethylation with DAC, restoring capsase-8 expression and making these cell lines more susceptible to apoptosis. Fulda reported that DAC could be combined with IFN-γ to upregulate caspase-8, enhancing TRAIL-induced apoptosis in NB and medulloblastoma cell lines . Our studies confirm previous reports showing that IFN-γ can induce expression of MHC Class I and Class II antigens on NB cells in vitro and in vivo [40, 41]. Our findings and previous studies demonstrated that MHC Class I was upregulated to a greater degree than MHC Class II, but some of the changes in MHC antigen expression were relatively small. IFN-γ has been used safely as an immunostimulant in numerous clinical situations, including cancer vaccines , and could be combined with a regimen that includes a demethylating agent such as DAC, to maximize antigen presentation.
Having demonstrated that NB cells upregulate CT antigens on a molecular as well as a protein level, we next utilized overlapping peptide pools derived from full-length MAGE-A1, MAGE-A3, and NY-ESO-1, so that the resulting CTL would be able to recognize CT epitopes in the context of multiple HLA-alleles. The degree of HLA matching and tumor antigen expression impact tumor cell recognition/killing by the CTL, and these factors are variably affected in tumor cell lines by pre-treating with DAC or IFN-γ. In the setting of using a panel of partially HLA matched tumor cell lines as we have done, it is difficult to ascertain precisely which HLA antigen represents the restricting element in terms of tumor cell killing. We used multiple tumor lines to permit cytotoxicity assays with targets that partially matched with the CTL donor. Donor 1 CTL had more cytotoxicity against U251 than IMR32, which could have been related to the fact that the U251 tumor line is matched for more HLA antigens with this donor than IMR32. While the U251 cell line had detectable tumor antigens by RT-PCR before DAC treatment, these antigens could not be detected by western blot before DAC, and were expressed at a protein level after DAC, which could be the reason for enhanced cytotoxicity. Although the Kelly cell line expressed MAGE-A1 before and after DAC treatment by both PCR and Western blot, MHC Class I expression was detectable only after IFN-γ treatment (from 0.01 to 18.85%), which could have been responsible for the increased cytotoxicity. Among all the targets tested with donor 2 CTL in this study, cytotoxicity against SKNMC was increased the most after exposure to both IFN-γ and DAC. It is not clear why some tumor cells become more sensitive to antigen-specific CTL than others after treatment with DAC or IFN-γ, and there are possibly other variables involved that could be making some cell lines more or less susceptible, requiring further investigation of these mechanisms.
The practical application of tumor antigen-specific cellular immunotherapy can be limited by the fact that these CTL exist at exceptionally low frequencies, precluding culture with methods typically used for memory CTL. Several authors have described MAGE-A1-, MAGE-A3-, and NY-ESO-1-specific epitopes that are restricted to certain HLA types [8, 9]. For practical purposes in clinical immunotherapy, stimulating T cells with individual epitopes restricts the patients that can potentially benefit from this strategy. Limitations in expanding CT antigen-specific CTL include having an advanced knowledge of all relevant CT antigen epitopes in the context of multiple HLA antigens. For this reason, we used a pooled mixture of overlapping peptides from full-length MAGE-A1, MAGE-A3, and NY-ESO-1. Wofl and group demonstrated that IFN-γ producing, antigen-specific T cells could be isolated by selecting cells expressing the activation marker CD137 . Considering the low precursor frequencies of CT antigen-specific CTL, we decided to select these cells based on expression of CD137 and demonstrated that overlapping peptide mixes can be used to generate these cells. The resulting MAGE-A1-, MAGE-A3-, and NY-ESO-1-specific CTL preferentially lysed NB and GBM cells that have been exposed to DAC and IFN-γ. These data provide the proof of concept that tumor cells can be made more susceptible to CTL killing by increasing the expression of CT antigens and MHC Class I/II molecules. This result is consistent with the findings that chemotherapy enhances tumor cell susceptibility to CTL-mediated killing in mice . Further studies are needed to determine the mechanism of killing of tumor cells which have had the expression of CT antigens upregulated following exposure to demethylating chemotherapy.
Several clinical trials have been conducted using pharmacologic agents to epigenetically modify the expression of genes by tumor cells [17, 19]. Studies have shown that the clinical administration of doses of DAC in the range of 15–20 mg/m2/day was associated with demethylation of the MAGE-A1 promoter . In one series of 10 patients with refractory solid tumors, DAC was given at 2 mg/m2/day for 7 days, with significant MAGE-A1 promoter hypomethylation by 14 days post-exposure in all subjects studied . Genomic DNA methylation reverted to baseline levels by 28 days after initial exposure to DAC, indicating the potential need for cyclic administration of this drug to achieve sustained CT antigen expression. Blum and group reported global DNA hypomethylation at a DAC dose of 15 mg/m2/day, with an average maximal plasma concentration of 1.3 μM, compatible with the concentrations used to treat the tumor cell lines were exposed to in this study. Further studies are needed to determine how long MAGE-A1, MAGE-A3, and NY-ESO-1 are expressed after the cessation of DAC and whether the same DAC dose can be used to restore CT antigen expression after several cycles. Since DAC has a low toxicity profile in this dose range, it could be considered to enhance immune responses to CT antigen vaccines or adoptively transferred antigen-specific CTL.
This work was supported by a grant from the Four Diamonds Fund of The Pennsylvania State University College of Medicine.
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