Frozen peripheral blood mononuclear cells (PBMC) from normal healthy leukophoresis donors were seeded at 1×105 cells/well and stimulated with SEB serially diluted 30-fold from 2.5 μg/mL. Anti-CTLA-4 (ipilimumab), anti-PD-1 (nivolumab), or huIgG4 isotype control (Bristol-Myers Squibb 1D12-g4) was present at a spike concentration of 10 μg/mL. IL-2 secretion was measured by ELISA (BD Biosciences) on day 3.
Mixed lymphocyte response assays were performed by co-culturing 1×105 cells CD4+ T cells with allogeneic monocyte-derived dendritic cells (DC) at a ratio of 10:1 (T:DC) in flat-bottom 96-well microtiter plates. CD4+ T cells and DC were incubated for 6 days in the presence or absence of nivolumab titrated 1:10 from 50 mg/mL to 5 ng/mL along with ipilimumab at 0, 5, or 50 μg/mL. Culture supernatants were harvested on day 5 for ELISA analysis of IFN-γ secretion.
To assess the potential of nivolumab or ipilimumab, alone or in combination, to induce nonspecific T cell activation, mAbs were mixed with samples of heparinized fresh human whole blood to measure cytokine release. After a 4-hour incubation at 37°C, the cells were pelleted and the plasma fraction collected for measurement of IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10 using a cytokine cytometric bead array assay (BD Biosciences). Comparison was made to responses generated by anti-CD3 or isotype control mAb treatment.
Mixed lymphocyte response assays were performed by co-culturing 1×105 cells CD4+ T cells with allogeneic monocyte-derived dendritic cells (DC) at a ratio of 10:1 (T:DC) in flat-bottom 96-well microtiter plates. CD4+ T cells and DC were incubated for 6 days in the presence or absence of nivolumab titrated 1:10 from 50 mg/mL to 5 ng/mL along with ipilimumab at 0, 5, or 50 μg/mL. Culture supernatants were harvested on day 5 for ELISA analysis of IFN-γ secretion.
To assess the potential of nivolumab or ipilimumab, alone or in combination, to induce nonspecific T cell activation, mAbs were mixed with samples of heparinized fresh human whole blood to measure cytokine release. After a 4-hour incubation at 37°C, the cells were pelleted and the plasma fraction collected for measurement of IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10 using a cytokine cytometric bead array assay (BD Biosciences). Comparison was made to responses generated by anti-CD3 or isotype control mAb treatment.