The TIM family ligand PS is present on Jurkat cells and binds TIM-3 with a ∼10 μM dissociation constant.
(A) PS exposure on Jurkat cells in culture, detected by staining with fluorescently-tagged (PerCP-CyTM5.5) annexin V, and quantified by flow cytometry analysis of parental NF-κB GFP reporter cells (black) and those expressing TIM-3 (red) — compared with unstained cells (gray shaded). A representative histogram from five biological repeats is shown. (B) Fluorescence imaging of parental and TIM-3+ NF-κB GFP reporter cells stained with DAPI (left panels) and fluorescently-tagged (AlexaFluor 647) annexin V (middle panels). Cells were stained with annexin V prior to fixation and staining with DAPI. (C) PS binding of sTIM-1 (teal), sTIM-3 (red), and sTIM-4 (green) was analyzed using SPR by flowing purified TIM ECRs over 20% DOPS/80% DOPC lipid vesicles immobilized on an L1 sensorchip. Binding was measured in the presence of 1 mM CaCl2. The curves indicate the fit of the data in Prism 9 to a one-site specific binding equation. Binding curves are representative of at least five independent experiments. Mean Kd and SD values are listed in the figure (n = 6 for sTIM-1, n = 11 for sTIM-3, and n = 5 for sTIM-4). Binding to additional lipids and effects of calcium are shown in Supplementary Figure S7.