Ectopic expression of TIM-3 enhances activation of Jurkat cells.
(A) Flow cytometry analysis of human TIM-3 expression in parental (black) and TIM-3 lentivirus-transduced NF-κB GFP reporter Jurkat cells (red), detected with phycoerythrin-conjugated TIM-3 antibody. The inset shows Western blot analysis of TIM-3 expression in the same cells as marked — with three biological repeats of each. (B) Representative histogram of NF-κB-driven GFP expression in stimulated NF-κB reporter cells (black), and those expressing TIM-3 (red) — compared with unstimulated parental (gray) and TIM-3-expressing (pink) cells. Stimulation employed 1 μg/ml αCD3 plus 1 μg/ml αCD28 for 16 h to mimic antigen binding. T cell activation was quantified by flow cytometry analysis of the GFP reporter of NFκB transcriptional activity. Enhanced T cell activation in TIM-3-expressing cells requires cell stimulation, with no change being observed in unstimulated cells. (C) Mean GFP fluorescence intensity (MFI) values for NF-κB GFP reporter assays in cells stimulated as in (B) relative to parental NF-κB reporter cells (black). Data represent the mean ± standard deviation (SD) for four biological repeats for parental and TIM-3-expressing cells (red) and two repeats for PD-1-expressing cells (blue). P-value for the TIM-3/parental comparison was determined with a two-tailed, unpaired Student's t-test. (D) ELISA analysis of IL-2 secretion into the medium of parental and TIM-3+ NF-κB reporter cells following stimulation as in (B). Data represent mean ± SD across three biological repeats, with P-value determined using a two-tailed, unpaired Student's t-test. (E) IL-2 production by parental and TIM-3-expressing Jurkat NF-κB/GFP reporter cells stimulated with SEE-loaded Raji B cells to mimic the formation of the immunological synapse during T cell activation. IL-2 levels were quantified by ELISA. Data represent mean ± SD for three biological repeats, with P-value determined using a two-tailed, unpaired Student's t-test.