Media and reagents
T cells and tumor cell lines were cultured in Iscove’s Modified Dulbecco’s Medium (IMDM) containing 10% fetal bovine serum and 1% L-glutamine and Penicillin/Streptomycin (all from Gibco, Invitrogen, Grand Island, NY). IMDM containing 5% human serum (Gemini Bio-Products, West Sacramento, CA) was used as the base medium for the outgrowth of T cell cultures. Two different serum-free medium types were used as the base medium for short-term stimulation of human NK cells as well as to generate DCs: AIM-V medium (Gibco, Invitrogen, Grand Island, NY) and CellGenix DC medium (CellGenix Technologie Transfer GmbH, Freiburg, Germany). The following factors were used throughout the study: granulocyte macrophage colony-stimulating factor (GM-CSF) and IL-4 (Schering-Plough, Kenilworth, NJ); IFNα (Intron A-IFN-α-2b; Schering-Plough); TNFα and IFNγ (both from Miltenyi Biotech, Bergisch Gladbach, Germany); IL-6 (Thermo Scientific, Waltham, MA); PGE2 (Sigma-Aldrich, St. Louis, MO); IL-18 (MBL International, Woburn, MA); IL-2 (Chiron, Emeryville, CA); IL-7 (PeproTech, Rocky Hill, NJ); and poly-I:C (Sigma-Aldrich, St. Louis, MO). The R24 anti-GD3 monoclonal antibody (mouse IgG3) used in this study was prepared at CellTech (London, UK) and provided by the National Cancer Institute (NCI) BRMP, and was stored at −80°C until use.
NK cell and CD8+ T cell isolation
Peripheral blood from patients with advanced melanoma (stage III and stage IV) and healthy donors was harvested by venipuncture under IRB-approved protocols. NK cells and CD8+ T cells (>95% pure) were isolated by negative magnetic selection using the StemSep system (StemCell Technologies Inc., Vancouver, British Columbia, Canada).
Generation of DCs
Peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of either healthy donors or melanoma patients (all stage III and IV donors) by density gradient separation using Lymphocyte Separation Medium (Cellgro Mediatech, Herndon, VA). Monocyte fractions were further isolated by CD14 positive selection (Miltenyi Biotech, Bergisch Gladbach, Germany). Immature DCs were generated from monocytes cultured for 6 days in 24-well plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) at 4×105
cells per well in GM-CSF and IL-4 (both 1,000 IU/ml). To generate “standard” mature DCs, day 6 immature DCs were cultured for an additional 48 h with IL-1β (10 ng/ml), TNFα (25 ng/ml), IL-6 (1,000 IU/ml), and PGE2
mol/L) as previously described37
Induction of IFNγ production by NK cells
NK cells were isolated and plated in 96 well plates at 1×105 cells/well. NK cells were stimulated with IFNα (1,000 IU/ml) together with either IL-18 (1 µg/ml), K562 cells (2×104 cells/well), or melanoma (FEM-X) cells (1×104 cells/well) in a final adjusted volume of 200 µl. When stated, anti-GD3 antibody (R24) was used to opsonize FEM-X cells. To accomplish this, 1×106 FEM-X cells were placed in 1 ml of tumor culture media and exposed to the R24 antibody at 1 µg/ml for 30 min at room temperature. Cells were then washed three times to remove excess antibody before use.
DC and NK cell co-cultures
Previously isolated and cryopreserved autologous NK cells were thawed and added to DC cultures either directly or separated by Transwell culture inserts (Costar-3413; 0.4µm pore size) at 1.5×105 cells/well to day 6 DC cultures in the presence of IFNα (1,000 IU/ml) and IL-18 (1 µg/ml). When stated, poly-I:C(20 µg/ml) was also added 20 h after co-culture initiation, which was previously determined to be optimal for enhancing its effects.
Two and three-colored cell surface immunostaining analyses were performed using a Beckman Coulter Epics XL Flow Cytometer. FITC-labeled anti-human CD86, CD40, and CD3 monoclonal antibodies and the corresponding FITC-isotype (mouse IgG1) control antibodies were purchased from BD Biosciences (San Jose, CA). PE-labeled anti-human CD83 and the corresponding PE-isotype (mouse IgG2b) control monoclonal antibodies were purchased from BD Biosciences (San Jose, CA). PE-Cy5-labeled anti-human HLA-DR and the corresponding PE-Cy5-isotype (mouse IgG1) control monoclonal antibodies were purchased from Beckman Coulter (Brea, CA). PE-labeled anti-human CCR7 monoclonal antibody was purchased from R&D Systems (Minneapolis, MN) and the corresponding PE-isotype (mouse IgG2a) control antibody was purchased from BD Biosciences (San Jose, CA). PE-labeled MART-1 tetramer (ELAGIGILTV) and the control influenza virus tetramer (GILGFVFTL) were purchased from Beckman Coulter (Brea, CA). Before staining, the cells were treated for 20 min at 4°C in PBS buffer containing 0.1% NaN3, 2% human serum, 0.5% BSA, and 1 µg/ml of mouse IgG (Sigma-Aldrich, St. Louis, MO) to block non-specific Fc receptor binding sites. Cells were stained for 40 min at 4°C followed by washing with PBS buffer containing 0.1% NaN3 and 0.5% BSA, then fixed and stored in 2% paraformaldehyde until analysis.
DC production of IL-12p70
Dendritic cells were harvested, washed, and plated in 96-well plates at 2×104
cells/well. To mimic the interaction with CD40L-expressing Th cells, CD40L-transfected J558 cells (a gift from Dr. P. Lane, University of Birmingham, United Kingdom), which in previous studies proved equivalent to activated CD4+
T cells and soluble CD40L15,38
, were added at 5×104
cells/well. Supernatants were collected after 24 h and analyzed by IL-12p70 ELISA (Endogen, Woburn, MA).
Dendritic cell migration was induced by CCL21 (6C-Kine-Biosource, Camarillo, CA) and measured using a 96-well 8um pore ChemoTx system (Neuro Probe, Gaithersburgh, MD). 25×103 DC in AIM-V medium were placed on the top of the membrane and permitted to migrate for 90 min at °C. Enumeration of migrated DC was determined by counting four random areas in the bottom chamber. Results are expressed as mean DC numbers ± SD of four random areas in duplicate wells.
HLA-A2+ melanoma patient-derived CD8+ T cells (5×105 cells) were plated in 48-well plates and sensitized by autologous DCs (5×104 cells) that were pulsed with the HLA-A2-restricted peptides MART-1 (26–35), gp100 (209–217), and tyrosinase (368–376). Added to the mix were γ-irradiated (3,000 rad) CD40L-transfected J558 cells (5×104), which acted as a surrogate for CD40L-expressing CD4+ Th cells. At day 4, T cell cultures were supplemented with IL-2 (50 IU/ml) and IL-7 (10 ng/ml). The CD8+ T cells were expanded following an additional in vitro stimulation (day 12) with irradiated peptide-pulsed autologous PBMCs (1:1 T cell:PBMC ratio). At day 24, the differentially-induced CD8+ T cell lines were stimulated with target cells to determine the generated frequency of melanoma-specific CD8+ T cells by IFNγ enzyme-linked immunospot (ELISPOT), using either T2 cells (pulsed with the relevant individual antigenic melanoma peptides or the irrelevant HPV-E7 peptide (43–62), or left unpulsed as an additional nonspecific control) or the HLA-A2+ and HLA-A2− melanoma cell line targets FEM-X and MEL-397, respectively. The pan-MHC class I blocking antibody (W6/32) was used to determine MHC class I restriction. CTL activity was further assessed by standard 4 h 51Cr-release cytotoxicity assays using the antigen relevant HLA-A2+ and irrelevant HLA-A2− melanoma cell lines FEM-X and MEL-397, respectively.
Data was analyzed using unpaired and paired t tests (two-tailed) and one-way and two-way ANOVA, where appropriate. Significance was judged at an α of 0.05.