Generation of DC, T cell and DC/myeloma fusion preparations
Peripheral blood was obtained from patients with multiple myeloma and normal volunteers in accordance with a protocol approved by the institutional review board. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density centrifugation. PBMCs were incubated in RPMI 1640 complete medium containing 2 mM L-glutamine (Mediatech, Herndon, VA) and supplemented with heat inactivated 10% human AB male serum (Sigma, St. Louis, MO), 100 U/ml penicillin and 100 μg/ml streptomycin (Mediatech) for 1 h at 37°C in a humidified 5% CO2 incubator. The nonadherent fraction rich in T cells was removed. The monocyte enriched adherent fraction was cultured in complete medium containing GM-CSF (1000 U/ml) (Berlex, Wayne/Montville, NJ) and IL-4 (1000 U/ml) (R&D Systems (Minneapolis, MN)) for 5 days to generate immature DCs. The DC preparation underwent maturation by culturing the cells for an additional 48-72 h in the presence of TNFα (25 ηg/ml). The cell preparation underwent flow cytometric analysis to confirm the presence of DC associated costimulatory and maturation markers (CD86 and/or CD83) and absence of myeloma antigens (CD38 and/or CD138).
Myeloma cells were obtained from bone marrow aspirates collected from patients with multiple myeloma in accordance with a protocol approved by the institutional review board. Bone marrow mononuclear cells were cultured in RPMI 1640 culture media containing 2 mmol/l L-glutamine (Lonza, Walkersville, MD) and supplemented with heat-inactivated 10% human serum, 100 U/ml penicillin and 100 μg/ml streptomycin (Mediatech). Myeloma preparations were stained with mouse anti-human CD38, CD138, CD86 and CD83 directly conjugated to FITC (BD Biosciences, San Jose, CA) to document expression of tumor associated markers CD38 or CD138, and the absence of DC associated markers CD83 and CD86.
Plasma cells were mixed with DC preparations at ratios of 1:1 - 1:3 (dependent on cell yields) and washed 3 times in serum-free RPMI 1640 culture media. After the final wash, the cell pellet was resuspended in 1 ml of 50% PEG solution. After 2 min at room temperature, the PEG solution was progressively diluted and cells were washed twice with serum free media. The DC-tumor fusions were cultured in RPMI complete media in the presence of GM-CSF, IL-4, and TNFα. Fusion cells were identified for subsequent analysis by FACS gating around the population of cells that co-express unique tumor and DC associated antigens as described below.
Characterization of PDL-1 expression on DCs, myeloma, and, DC/myeloma fusion cells by flow cytometric analysis
DC preparations were incubated with mouse anti-human PDL-1 directly conjugated to PE (eBioscience, San Diego, CA) and a matching isotype control for 45 minutes at 4°C. Patient derived myeloma cells isolated from bone marrow aspirates were stained with mouse anti-human CD38 or CD138 directly conjugated to FITC (BD Biosciences, San Jose, CA), and mouse anti-human PDL-1 directly conjugated to PE for 45 minutes at 4°C. Cells were fixed in 2% paraformaldehyde (Sigma) and underwent flow cytometric analysis using FACScan (Becton Dickinson, San Jose, CA) and CellQuest Pro software© (Becton Dickinson).
DC/myeloma fusion cell preparations were subjected to dual staining to quantify the percentage of cells that co-expressed unique DC (CD86-PE-Cy5) (BD Biosciences, San Jose, CA) and tumor antigens (CD38 or CD138-FITC) (BD Biosciences, San Jose, CA). Dual staining fusion cells were isolated by FACS gating and stained with PE-conjugated mouse anti-human antibodies directed against PDL-1. The percentage of fusion cells expressing PDL-1 was determined by multichannel flow cytometric analysis.
PD-1 expression on T cells isolated from patients with multiple myeloma
Nonadherent peripheral blood mononuclear cells and bone marrow mononuclear cells were obtained from patients with myeloma at various stages of disease presentation including at time of presentation, relapse, and following therapy mediated cytoreduction. The myeloma cell preparation was incubated with mouse anti-human anti-CD4 or anti-CD8 directly conjugated to FITC, and mouse anti-human anti-PD1 directly conjugated to PE (eBioscience, San Diego, CA). Cells were washed, fixed in 2% paraformaldehyde (Sigma), and analyzed by multichannel flow cytometry.
Effect of non-antigenic and antigenic stimulation of T cells on expression of PD-1
T cells were activated for by exposure to the immobilized monoclonal antibodies, anti-CD3 (clone-UCHT1; Pharmingen) and anti-CD28 (clone-CD28.2; Pharmingen; CD3i/CD28i) for 1-4 days. Twenty-four-well non-tissue culture-treated plates (Falcon, Fisher) were coated with each of the antibodies (1 ug/ml in PBS) at 0.5 ml/well and left overnight at 4°C. The plates were washed in 1x PBS, and T cell preparations were added at a density of 2×106 cells/well. T cells were stimulated with anti-CD3/CD28 (48 hours). Alternatively, T cells were stimulated by the mitogen PHA (2 ug/ml) for 4 days, or DC/myeloma fusion cells at a ratio of 10:1 for 5 days. PD-1 expression on T cells before and after stimulation was assessed by flow cytometric analysis.
Effect of PD-1 blockade on T cell polarization and cytokine expression following stimulation by DC/myeloma fusions
DC/myeloma fusions were co-cultured for 5 days with autologous T cells at a 1:10 ratio in the presence or absence of 5ug/ml anti-PD1 (CT-011, CureTech Ltd., Israel). Co-cultures were then pulsed with GolgiStop (1 μg/ml; Pharmingen) for 4-6h at 37°C prior to analysis. Cells were harvested and cultured with murine anti-human FITC conjugated anti-CD4 or anti-CD8 (BD Biosciences). Cells were then permeabilized by incubation in Cytofix/Cytoperm plus™ (containing formaldehyde and saponin) (Pharmingen) for 45 min at 4°C, washed twice in Perm/Wash™ solution (Pharmingen), and incubated with PE-conjugated IFNγ (Caltag Laboratories), IL-10 (Caltag Laboratories), or a matched isotype control antibody for 45 min. Cells were washed in 1x Perm/Wash™ solution and fixed in 2% paraformaldehyde (Sigma). Labeled cells were analyzed by flow cytometry using FACScan (Becton Dickinson, San Jose, CA) and CellQuest Program.
Effect of PD-1 blockade on vaccine mediated expansion of regulatory and activated T cells
Autologous T cell preparations were co-cultured with DC/myeloma fusions for 5 days at a 10:1 ratio in the presence and absence of 5ug/ml anti-PD-1 (CT-011, CureTech Ltd., Israel). The cell preparations were incubated with FITC conjugated anti-CD4, tricolor conjugated anti-CD25 (Caltag Laboratories), and PE-conjugated anti-CD69 (BD Biosciences). Alternatively, cells were permeabilized and cultured with PE conjugated antibody directed against FoxP3 (eBioscience). Cells were subsequently analyzed by multichannel flow cytometry.
Effect of PD-1 blockade on CTL response following stimulation with DC/myeloma fusions
DC/myeloma fusions were cocultured with autologous T cells at a ratio of 1:10 for 5 days in the presence and absence of 5ug/ml anti-PD1 (CT-011, CureTech Ltd, Israel). Cell mediated cellular cytotoxicity was evaluated using the GranToxiLux cell-based fluorogenic cytotoxicity assay (OncoImmunin, Inc., Gaithersburg MD). Autologous tumor cells were used as target cells. Target cells were incubated in PE labeled Medium T (1μL of reconstituted TFL2 in PBS at 1:3000 ratio) at 2×106 cells/ml for 45 minutes at 37°C. Labeled cells were washed twice in PBS. T cells stimulated by fusions alone or in the presence of PD-1 blockade were co-incubated with labeled target cells in the presence of a fluorogenic granzyme B substrate for 1-2 hours at 37°C. Cells were washed and analyzed by flow cytometry. Dead target cells are identified by cells that dually stain for granzyme B and PE label (right upper quadrant). As a control, killing of targets by unstimulated T cells was assessed.
Results are expressed as mean ± SEM. 2 tailed students t test was used for comparisons, and values of p<0.05 were considered as significant