Cell lines and human brain tissue samples
Five cell lines, DBTRG05-MG, SNB-19, U-251MG, U-373MG and T98G derived human glioblastoma cell lines were kindly provided by Dr. Carol Kruse (UCLA Department of Neurosurgery, Los Angeles, CA). They were maintained in DMEM (Mediatech, Inc., Herndon, Virginia) supplemented with 10% FBS (Gemini Bio-Products, West Sacramento, California), 1% (v/v) penicillin, streptomycin, and amphotericin B (Mediatech Cellgro, Manassas, Virginia) and kept in an atmosphere of 5% CO2 at 37°C. Normal human astrocytes (NHA) were kindly provided by Dr. Russ Pieper (UCSF Department of Neurosurgery, San Francisco, CA). A melanoma cell line, 624.38, was kindly provided by Dr. Steve Rosenberg (NIH/NCI). Both NHA and 624.38 cells were placed into identical culture conditions to glioblastoma cell lines.
Primary tumor cell cultures were derived from four patients with glioblastomas who had undergone surgical resection at the UCLA Medical Center. These tissues were cultured by digesting homogenized tumors in a collagenase DNAse mixture overnight as previously described [5
]. The digested tumor samples were filtered through a mesh and the cells placed into complete medium as described earlier. The cells were incubated in 5% CO2
at 37°C. Human brain tissue samples were also obtained from patients who had undergone resection at the UCLA Medical Center, CA. All patients provided written informed consent for these procedures.
Peripheral blood was obtained from healthy human volunteers provided by the Division of Hematology and Oncology at UCLA Medical Center, Los Angeles. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll gradient centrifugation as previously described [5
]. Written informed consent and institutional IRB approval was obtained for all studies involving human bloods and tissues.
Decitabine (5-aza-2'-deoxycytidine) was generously supplied by Eisai Pharmaceuticals (Woodcliff Lake, New Jersey). A 10 μM stock solution of it in DMSO was stored at -80°C.
In vitro treatment of cultured cells with decitabine
Cells were plated overnight at 106cells/ml at 37°C in a 5% CO2 incubator. The following day, the cell culture medium was replaced with either fresh medium or that supplemented with 1 μM decitabine. The cells were treated again the following day in new cell culture medium. At the end of treatment, the medium was replaced with fresh medium without decitabine, and the cells were cultured for an additional 48 hours before utilized in subsequent assays.
Conventional reverse transcription PCR analysis of NY-ESO-1 expression
Total RNA was isolated with the RNeasy Mini kit (Qiagen, Valencia, California) according to manufacturer's protocol. 3 × 106 human glioblastoma cancer cell lines or 25 mg of tumor tissue samples were used. cDNA was synthesized from 1 μg total RNA by using Omniscript Reverse Transcription kit (Qiagen), again according to the manufacturer's protocol. PCR was facilitated by using the Accuprime GC-rich kit (Invitrogen, Carlsbad, California). In each PCR, 2 μl cDNA was used in a 35-μl reaction volume containing 10 μM sense and antisense primers, 5 μL 5× PCR buffer (A for GC-rich genomic DNA target or B for non-GC-rich genomic DNA target), and 1 μl of Accuprime GC-Rich DNA polymerase (Invitrogen). The PCR primers were as follows: NY-ESO-1 forward primer: 5'-CATCACGGATCCATGCAGGCCGAAGGCCGG-3', reverse primer: 5'-ACCCGGGGTACCGCGCCTCTGCCCTGAGGG-3', GAPDH forward primer: 5'-GAAGGTGAAGGTCGGAGT-3', reverse primer: 5'-GAAGATGGTGATGGGATTTC-3' (Invitrogen). The PCR cycling parameters were as follows: denaturation at 95°C for 30 s, primer annealing at 60°C for 30 s, and 40 cycles of extension at 72°C for 60 s. PCR cycling was preceded by an initial denaturation at 95°C for 3 min, followed by final extension at 72°C for 10 min. The PCR products were electrophoresed on 1% agarose gels and analyzed after staining with ethidium bromide. Densitometry analysis was performed using the QuantityOne program (BioRad, Hercules, CA).
Quantitative real-time analysis of NY-ESO-1 expression
Quantitative RT-PCR was performed with the LightCycler real-time RT-PCR system (Roche, Mannheim, Germany), using the LightCycler SYBR green mastermix (Roche). Primers specific for NY-ESO-1 were as follows: forward primer: 5'-TGTCCGGCAACATACTGACT-3', reverse primer: 5'-ACTGCGTGATCCACA TCAAC-3'. GAPDH forward primer: 5'-AGCCACATCGCTCAGACAC-3', reverse primer: 5'-CGCCCAATACGACCAAATC-3' (Invitrogen). Each sample was amplified as follows: 1 cycle at 95°C for 10 min, 40 cycles at 95°C for 15 s, and 60°C for 1 min. Triplicate samples were tested.
Total RNA was extracted from decitabine treated and untreated U-251 MG cells using the RNeasy mini kit (Qiagen). cDNA was generated, quantified and hybridized to U133 Plus 2.0 arrays at the UCLA DNA Microarray Facility using standard Affymetrix protocols. CEL files were normalized using GeneSpring GX 11.5.1 software (Agilent Technologies) with LiWong. To evaluate the baseline expression of NY-ESO-1 in heterogeneous human brain tumor tissues, we analyzed the relative expression of NY-ESO-1 using microarray gene expression profiling in over 300 human brain tumor samples. The relative expression of NY-ESO-1 was normalized and tested. Dots plotted underneath the black line are considered to have at or under background relative expression levels.
FACS analysis and antibodies
Cells (105) obtained from decitabine-treated and untreated U-251 MG cells were used for flow cytometric analysis. Cells were washed twice with FACS staining buffer and stained with antibodies to HLA-A, B, C (clone DX17) and HLA-A2 (clone BB7.2) (BD Pharmingen, San Diego, California). A Becton Dickinson LSRII was used for acquisition and the acquired data were analyzed using FlowJo (TreeStar, Ashland, Oregon). Gates were set based on singly-stained isotype-control antibodies (data not shown).
Generation and maintenance NY-ESO-1 TCR-transduced lymphocytes
Six well cluster plates were coated with Retronectin (RN) (Takara, Madison, Wisconsin) at a concentration of 10 μg/ml overnight at 4°C. The next day, blocking buffer (PBS containing 2% Human Serum Albumin) was added for 30 min and washed. Supernates were retrieved from a stable PG13-based retroviral producer cell line encoding a HLA-A*0201-restricted NY-ESO157-165
specific T cell receptor (TCR) generated in the MSGV1 retroviral vector backbone, as described [30
]. The PG13-based NY-ESO-1 stable retroviral packaging cell line was obtained from Dr. Paul Robbins (Surgery Branch, NCI/NIH). Anti-CD3 (clone OKT-3; 50 ng/ml) was used to stimulate human PBMCs for 48 hours in X-VIVO 15 medium supplemented with 5% human AB serum in the presence of 300 IU IL-2/ml prior to spin-infection. Stimulated PBMCs were transduced twice as described [15
]. Transduced T cells were maintained in X-VIVO 15 medium supplemented 5% human AB serum and 300 IU IL-2/ml. The cells were expanded for 3 days in the presence of IL-2 and then rested for 2 days in the absence of IL-2.
Transduction efficiency was evaluated 48 hr post-transduction by NY-ESO-1 T cell receptor staining of the CD3+
gated population using antihuman antibodies to CD3-PerCP (clone SK7; BD Biosciences, San Diego, CA), CD8-FITC (clone SFCI21Thy2D3; Beckman Coulter, Brea, CA), and TCR Vβ13.1-PE (clone IMMU 222; Beckman Coulter). Detection of the NY-ESO-1 peptide-MHC complex was accomplished by NY-ESO-1 tetramer staining as described [31
CD107A marker staining of NY-ESO-1 TCR transduced PBMC after co-culture with human glioma cells
NY-ESO-1 TCR-transduced PBMCs and untransduced PBMCs were harvested and resuspended at a concentration of 1 × 107 cells/ml. Treated and untreated HLA-0201+ T98G glioma cells were harvested using trypsin and reconstituted to 1 × 107 cells/ml. 1 × 106 PBMCs were co-incubated with target T98G cells in 96-well round-bottomed plates at a 1:1 ratio with total medium volume of 200 μl. Golgi plug (1×) and 5 μl of CD107A-AF647 (clone H4A3) (BD Biosciences) were added into each well and incubated for 6 hr at 37°C. Cells were washed twice with FACS staining buffer (PBS + 2% FBS) and stained with surface antibodies to CD4-PE (clone RPA-T4; BD Biosciences), CD8-PacBlue (clone RPA-T8; BD Biosciences), and CD3-PE-Cy5 (clone UCHT1; BD Biosciences) on ice for 25 min. Cells were washed, fixed with Fixation Buffer (eBioscience, San Diego, CA), and kept at 4°C until analysis. Data were collected using a Becton Dickinson LSRII and analyzed with FlowJo software (TreeStar).
ELISA and Cytokine Bead Array
To determine cytokine concentrations, supernates were collected from the Golgi-plug free overnight co-cultures. Cell-free supernates were analyzed for interferon-γ using an ELISA kit (eBiosciences) or for simultaneous levels of IFN-γ, TNF-α, IL-2, IL-4, IL-5, and IL-10 using a cytokine bead array (CBA) system (BD Biosciences), following the manufacturer's instructions. Co-cultures at 1:1 (E:T) ratios were placed into complete medium (X-VIVO 15 with 5% human AB serum) in 96-well round-bottomed plates and incubated overnight at 37°C. All samples were in triplicate and supernates were harvested for cytokine detection. IFN-γ ELISAs were performed according to the manufacturer's specification (ELISA Ready-set-go, eBioscience). Color development was stopped after 2-5 minutes. The colorimetric density of each well was measured at 450 nm by a plate reader, and the final concentration of each sample was calculated (pg/ml) based on the standard curve. Triplicate assays were performed for each case and the results were expressed as the mean of 3 separate assays.
Data acquisition for CBA experiments was performed with the FCAP Array software (BD). Once the FCAP Array experiment was created, the files were exported to the BD FACSArray software for sample acquisition and analysis following manufacturer's protocol.
Apoptosis was induced in T98G glioma cells or 624.38 melanoma cells by the addition of an agonistic anti-CD95 antibody (clone CH-11; Medical and Biological Laboratories). Cells were analyzed for Fas-expression using a CD95-PE mAb (clone DX2) (BD Pharmigen). On the day of harvest, the target cells were cultured in DMEM with FBS containing 200 ng/ml of CH-11 for 24 hr. Cells were washed and resuspended in 1× binding buffer (Invitrogen). Cells were then stained with FITC-conjugated Annexin V (Invitrogen) and the vital dye, propidium iodide (PI, Invitrogen), for 20 min at 37°C. Cells negative for both PI and Annexin V staining were considered live cells; early apoptotic cells were PI-negative, Annexin V-positive; and late apoptotic/dead cells were PI-positive, Annexin V-positive. Each specimen was analyzed in triplicate, and the results shown were the mean ± SE of 3 assays.
Mixed lymphocyte tumor cell cultures stimulated with antagonistic Fas mAb
PBMCs transduced with the NY-ESO-1 TCR were co-cultured with tumor target cells and blocked with an antagonistic anti-CD95 antibodies (clone ZB4; Medical and Biological Laboratories). Treated and untreated HLA-0201+ T98G glioma cells were harvested using trypsin and reconstituted to 4 × 105 cells/ml and blocked with antagonistic anti-CD95 antibodies for 1 hr on ice. Cells were washed and reconstituted to 1 × 107 cells/ml in complete medium. Transduced PBMCs were plated with target cells in 96-well round-bottomed plates at a 1:1 ratio with total medium volume of 200 μl. Golgi plug (1×) and 5 μl of CD107A-AF647 (clone H4A3) (BD Biosciences) were added into each well and incubated for 6 hr at 37°C. Cells were washed twice with FACS staining buffer and stained with surface antibodies to CD4-PE, CD8-PacBlue, and CD3-PE-Cy5 on ice for 25 min. Cells were fixed with Fixation Buffer and kept at 4°C until analysis. Data were collected using a Becton Dickinson LSRII and analyzed using FlowJo software (TreeStar).
Continuous variables were compared using a paired Student's t-test. Results comparing more than two groups were analyzed by ANOVA followed by Kruskal-Wallis statistics. Values were considered significant at P < 0.05. Resulting data files from the gene expression profiling experiments were analyzed using RMA from BIOconductor. All statistical analysis and graphs were constructed using GraphPad software.