(A) Representative histograms of NF-κB-driven GFP expression in parental NF-κB GFP reporter Jurkat cells. Cells were starved for 4 h — including a 1 h pre-treatment with anti TIM-3 (F38.2E2) at the concentrations marked — and were then stimulated with 1 μg/ml αCD3 plus 0.5 μg/ml αCD28 for 16 h or left unstimulated (gray). GFP expression was analyzed by flow cytometry, and quantitation is shown in (D). (B) Representative histograms for TIM-3-expressing NF-κB GFP reporter Jurkat cells, stimulated as in (A), with from 0 μg/ml to 10 μg/ml F38.2E2. Unstimulated TIM-3⁺ cells are shown in pink. Quantitation is shown in (E). (C) Representative histograms from TIM-3-expressing NF-κB GFP reporter Jurkat cells stimulated with 0 μg/ml (red) or 10 μg/ml F38.2E2 (blue), showing that 10 μg/ml F38.2E2 does reduce the level of NF-κB-driven GFP expression seen in stimulated TIM-3 cells. (D,E) Quantitation of data in (A) and (C) over three independent biological repeats, plotting mean values for relative mean GFP fluorescence intensity (MFI) and SD. P-values were determined with two-tailed, unpaired Student's t-tests. (F) IL-2 secreted into cell medium was measured by ELISA for parental NF-κB GFP reporter Jurkat cells (black bars) and TIM-3-expressing cells (red bars). Adding 10 μg/ml F38.2E2 reduces enhanced IL-2 production in TIM-3 expressing cells. Bars represent the mean concentration of IL-2 (± SD) for three biological replicates. P-values were determined with two-tailed, unpaired Student's t-tests.