Blood donors and cell lines
After informed consent, peripheral blood was obtained from healthy HLA-A2+ donors on a protocol approved by the Baylor College of Medicine Instutional Review Board. CTL and TAPC cultures were maintained in 45% RPMI 1640 (GIBCO), 45% Click (Eagle Ham amino acids; Irvine Scientific, Santa Anna, CA), 2 mM GlutaMAX-1 (Invitrogen, Carlsbad, CA), 5% human AB serum (Valley Biomed), referred to hereafter as T cell media (TCM-AB). Senma (HLA-A2+), Mel-1143 (HLA-A2-), K562, CEM.T2 and HSB-2 tumor cell lines were maintained in RMPI supplemented with 10% fetal calf serum (FCS; Hyclone, Logan, UT) and 2 mM GlutaMax-1.
Construction and production of IL-21 lentivirus
To clone human IL-21, CD4+ T cells were isolated from PBMC by MACS selection using CD4 microbeads (Miltenyi Biotech, Auburn, CA) and activated for 48 hours on non-tissue culture treated plates coated with 1 μg/ml anti-CD3 (OKT3; Ortho Pharmaceuticals, Raritan, NJ) and 1 μg/ml anti-CD28 antibody (Pharmingen, San Diego, CA). Following activation, RNA was isolated using the RNAeasy kit (Qiagen, Valencia, CA) and used as a template for One-Step RT-PCR (Qiagen) using gene-specific primers. The IL-21 forward PCR primer included a CACC tag (5'-CCACATGGGATCCAGTCCTGGCAACA-3') for directional insertion into pENTR/D-TOPO (Invitrogen, Carlsbad, CA), while the reverse PCR primer was made without the TGA stop codon (5'-TCAGGAATCTTCACTTCCGTGTGTTCTA-3') to allow translation of IL-21 with the fluorescent Lumio tag. Following insertion into pENTR/D-TOPO, the expression cassette was then transferred by LR recombination into the pLenti6.2/C-Lumio/V5-DEST plasmid per the manufacturer's protocol (Invitrogen), to create the lentiviral expression plasmids pLenti-6.2/IL21-C-Lumio/V5 (hereafter called pLenti-IL21-Lumio (). Lentiviral supernatant was produced by transiently tranfecting 293FT cells using Invitrogen's ViraPower Expression System (utilizing vesicular stomatis virus G glycoprotein (VSVG) packaging pseudotype). As a control, pLenti6.2/C-Lumio/lacZ (pLenti-lacZ) was used. Transient supernatant was harvested at 48 and 72 hrs after transfection, snap-frozen and stored at −80°C until use.
Gene-modification of TAPC with IL-21
Generation of IL-21-producing T cells
To examine the effects of IL-21, CD8+ T cells were selected from PBMC and activated on CD3/CD28-coated plates for 48 hours in TCM-AB supplemented with 100 U/ml IL-2 (Proleukin; Chiron, Emeryville, CA). To transduce with lentivirus, activated CD8+ T cells, non-tissue culture treated plates were first coated with 4 μg/ml Retronectin (TaKaRa, Otsu, Japan) at 4°C overnight, washed with phosphate buffered saline (PBS) and then coated with lentiviral supernatant. Activated T cells were then plated at 5×105 cells/well in supernatant supplemented with 100 U/ml IL-2 and incubated at 37°C/5% CO2 for 3 days. After incubation, TAPC were harvested, washed and subsequently expanded in TCM-AB plus IL-2. After expansion for a week, the cells were once again CD8 selected by MACS (Miltenyi) to eliminate confounding IL-21 production by residual CD4+ cells.
Analysis of IL-21 secretion by gene-modified T cells
Initial detection of trangenic IL-21 in T cells was performed using lentivirus generated from the pLenti-IL21-Lumio construct, where the IL-21 protein includes the 6 amino acid Lumio tag allowing fluorescent detection of recombinant protein within the cell (). Briefly, mock-transduced or T cells transduced with pLenti-IL21-Lumio virus were activated for 48 hours on CD3/CD28 plates in TCM-AB supplemented with IL-2. After activation, T cells were harvested and cultured for 24 hours at 37°C in Opti-MEM Reduced Serum Medium (Invitrogen) to reduce Lumio background. T cells were then washed in Hank's Buffered Saline Solution (HBSS) and then incubated in 1 μM Lumio Green Labeling Reagent (Invitrogen) for 15 minutes at room temperature in the dark. After incubation, cells were washed in HBSS and incubated with Disperse Blue 3 and analyzed by fluorescent microscopy and flow cytometry. IL-21 secretion was subsequently confirmed by ELISA. 1×106 IL-21 and lacZ gene-modified T cells were activated on CD3/CD28 plates and on days 0 (prior to activation), 3, 5 and 7, cells were harvested and replated in 24-well plates and incubated in fresh media for 24 hours at 37°C. ELISA plates were prepared by incubating 96-well protein binding plates (Immuno Plate Maxisorp, Nalge Nunc, Rochester, NY) with 2 μg/ml purified mouse monoclonal anti-IL-21 capture antibody (BD Pharmingen) overnight at room temperature. Plates were washed and then incubated with supernatants from activated gene-modified T cells and compared to a standard curve of serial diluted recombinant IL-21 protein (Biosource, Camarillo, CA). Plates were incubated for 24 hours at 4°C, washed and then incubated with biotinylated mouse anti-IL-21 antibody (BD Pharmingen) for 2 hours at room temperature. The ELISA was then developed using streptavidin, substrate and stop solution (all from R&D Systems, Minneapolis, MN). The optical density of each well was then determined at 450 nm using a microplate reader and concentration of IL-21 per 1×106 cells per 24 hours calculated from the standard curve.
Generation of tumor-specific CTL
All CTL experiments used cells from HLA-A2+ donors. Activated IL-21 gene-modified and non-modified TAPC were resuspended at 1×106
cells/mL of TCM-AB media and pulsed with a single HLA-A2 restricted peptide. These peptides were derived from two melanoma antigens: MelanA/MART-1 (ELAGIGILTV) and gp100 (IMDQVPFSV); or from a B-CLL antigen: RHAMM (ILSLELMKL)33
and were custom synthesized by Genemed Synthesis (San Francisco). During peptide loading of TAPC, each peptide was pulsed at 10 μg/mL of TAPC in the presence of β2 microglobulin at 3 μg/mL, for 2 hrs at 37°C with frequent agitation. Prior to use, TAPC were washed in TCM-AB and irradiated to 30 Gy. Autologous CD8+ cells were used as responders at a 1:2 stimulator to responder ratio (1×106
responder T cells in 2 ml in each well of a 24-well plate) in TCM-AB media. The CTL cultures were also supplemented with exogenous cytokines- rhIL-7 at 10 ng/mL and rhIL-12 at 10 ng/mL (both from R&D Systems) on day 0 to augment priming immune responses. The responder CD8+ cells were stimulated weekly with irradiated peptide pulsed TAPC for up to 3 stimulations, using the protocol described above. After the second stimulation the CTL cultures were supplemented with IL-2 at 50 U/ml twice weekly to enhance CTL expansion.
To determine antigen-specific T cell frequency and phenotype, cells were analyzed by flow cytometry (FACS Calibur; BD) using peptide pentamers for MART-1 (ELA) and gp100 (IMD) in combination with APC-labeled FluoroTag (Proimmune, Springfield, VA) and FITC, PE and PerCP conjugated antibodies for CD3, CD8, CD27, CD28, CD44, CD45RA, CD62L, CCR7 and CD127 (IL-7Rα) (all from BD). To determine the percentage of cells capable of secreting IFN-γ following activation, we performed intracellular cytokine flow cytometry (CFC). CTL generated were stimulated for 2 weeks with TAPC-lacZ or TAPC-IL21 and then rested for 1 week with media supplemented with 100 U/ml IL-2. Cells were harvested and resuspended in TCM-AB, and then non-specifically stimulated with 25 ng/ml phorbol myristate acetate (PMA; Sigma, St. Louis, MO) and 1 μg/ml ionomycin (Sigma). After incubation for 1 hour at 37°C, 10 μg/ml Brefeldin A (Sigma) was added and the cells were cultured for another 5 hours. Following incubation, cells were washed, fixed in 1% paraformaldahyde and permeablized with 0.1% saponin (Sigma) in PBS. Cells were then incubated with antibodies to CD3, CD8 and IFN-γ (all BD) and analyzed by flow cytometry.
ELIspot and cytotoxicity assays
Determination of frequency and function of tumor-specific CTL was determined using IFN-γ ELIspot and 51Chromium (Cr51)-release assays. For IFN-γ ELIspot analysis, 1×105
cells were resuspended in TCM-AB and plated in triplicate on Multiscreen 96-well plates (Millipore, Bedford, MA) coated with mouse anti-human IFN-γ antibody (Mabtech, Cincinnati, OH). CTL were stimulated for with cognate tumor peptide at 500 ng/ml diluted in TCM-AB and compared to CTL stimulated with an irrelevant control peptide, or CTL in media alone. After incubating the plate for 20 hours at 37°C, the plate was developed as previously described.34
To measure antigen-specific cytotoxicity, CTL were assayed against Cr51-labeled target cells in a standard 4 hour release assay. MART-1 (ELA)-specific CTL were tested for cytotoxicity against two different tumor cell lines, mel1143 (HLA-A2+; MART-1+) and Senma (HLA-A2−; MART-1+). To determine the cytotoxic potential of CTL, CTL were FACS sorted following labeling with ELA pentamer (>95%) and then used as effector cells against CEM.T2 cells pulsed with ELA peptide. CTL were also compared for cytotoxicity against K562 and HSB-2 cell lines for natural killer (NK) cell-mediated killing. Specific lysis was calculated as ([experimental release – spontaneous release]/[maximum release – spontaneous release]) × 100.
Cell death/apoptosis assays
To determine whether IL-21 has an affect on cell survival, we examined cell death and apoptosis of T cells exposed to IL-21 compared to control T cells by staining cells with Annexin-V and propidium iodide (PI; BD) followed by FACS analysis. Here, 1×106 T cells were plated in media alone or supplemented with 100 U/ml IL-2, 10 ng/ml IL-21 or both cytokines. Cells were analyzed for PI and Annexin staining by FACS analysis on days 0, 3, and 7.
To measure expression of Tcf7 and Lef1 gene expression in CTL exposed to IL-21, we performed RT-PCR. Tumor-specific CTL expanded with TAPC-lacZ or TAPC-IL21 were harvested after 14 days in culture and RNA was isolated and used as template for One-Step RT-PCR (Qiagen) with PCR primers for Tcf7, Lef1 and GAPDH (SABiosciences, Frederick, MD).
Paired, two-tailed Student's T-test was used to determine statistical differences between experimental groups. A P-value of less or equal to .05 was considered statistically significant.