Fig 2: TIM-3 modulates CD28 phosphorylation after αCD3/αCD28 stimulation.Changes in CD28 phosphorylation following TCR activation with αCD3/αCD28 as in Figure 1 were monitored by Western blotting using phospho-specific antibodies. (A) Representative Western blot of pY191 in CD28 for TIM-3+ and parental NF-κB reporter Jurkat cells. Grb2 was used as a loading control as described [44]. Quantification bar graphs indicate mean band intensity ± SD (n = 6 biological repeats). Two-tailed, unpaired Student's t-test was used to determine P-values. (B) Representative Western blot of pY218 in CD28 for TIM-3+ and parental NF-κB reporter cells, with quantitation below the blot. Grb2 was used as a loading control. Bars indicate mean band intensity ± SD for the 5 min time point (seven biological repeats) with P-values determined by two-tailed, unpaired Student's t-test. (C) Parental, TIM-3+, and PD-1-expressing NF-κB reporter Jurkat cells were stimulated for 5 min with αCD3/αCD28 (1 μg/ml each) with- or without the PI3K inhibitor, wortmannin (0.2 μM) to show that PTEN deficiency does not account for the effect of TIM-3 expression on CD28 phosphorylation.

Fig 3: TIM-3 antibody treatment selectively reduces co-stimulatory signaling.(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.

Fig 4: 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.

Fig 5: Mutated TIM-3 variants with impaired PS-binding lose co-stimulatory signaling.(A) Details of the PS-binding site in murine TIM-3 (PDB: 3KAA — blue) overlaid on human TIM-3 (PDB: 6TXZ — red), revealing key interacting residues [34,82]. Inset at the right shows the PS-binding pocket, where short-chain PS is sandwiched by L99/M100 and W41 in mTIM-3 (I96/M97 and F40 in human TIM-3). A calcium ion (gray sphere) also sits in the binding pocket and interacts with the negatively charged PS headgroup. (B) PS binding of wild-type sTIM-3 (solid red curve), sTIM-3F40A (solid magenta curve), and sTIM-3I96A/M97A (dotted magenta curve) were analyzed using SPR by flowing the purified sTIM-3 variants over 20% DOPS/80% DOPC lipid vesicles immobilized on an L1 chip (in the presence of 1 mM CaCl2). Binding curves are representative of at least three independent experiments. Kd values for sTIM-3F40A and sTIM-3I96A/M97A were too high to measure, both appearing to exceed ∼200 μM (although PS binding was detectable). (C) A GFP reporter was used to measure NF-κB transcriptional activity downstream of TCR activation with 1 μg/ml αCD3 plus 0.5 μg/ml αCD28 for 16 h. Representative histogram of NF-κB-driven GFP expression is shown for parental NF-κB GFP reporter cells (black), and those expressing TIM-3WT (solid red curve), TIM-3F40A (solid magenta curve), and TIM-3I96A/M97A (dotted magenta curve) variants. (D) Relative mean GFP fluorescence intensity (MFI), expressed as fold change over parental cells within each experiment, was determined across eight biological replicates for parental NF-κB GFP reporter cells (black) or those expressing TIM-3WT (solid red line, open bar), TIM-3F40A (solid magenta line, open bar and data points), or TIM-3I96A/M97A (dotted magenta line, gray filled bar and data points). Means ± SD are plotted, with P-values determined by two-tailed, unpaired Student's t-tests. (E) ELISA analysis of IL-2 secreted into the cell culture medium by parental NF-κB GFP reporter cells, and those expressing, TIM-3WT (solid red line, open bar), TIM-3F40A (solid magenta line, open bar and data points), or TIM-3I96A/M97A (dotted magenta line, gray filled bar and data points) following stimulation by αCD3/αCD28, as described in (C). Means ± SD are plotted for three biological repeats. P-values comparing TIM-3WT with the mutated TIM-3 variants were determined using two-tailed, unpaired Student's t-tests. (F) Analysis of TNFα secretion into the cell culture medium was performed using a Luminex assay. Cells were stimulated with αCD3/αCD28 (1 μg/ml each) for 16 h. Mean values ± SD are plotted for parental (black line, open bar), TIM-3WT (solid red line, open bar), TIM-3F40A (solid magenta line, open bar and points), and TIM-3I96A/M97A (dotted magenta line, gray filled bar and data points). Two-tailed, unpaired Student's t-tests were used to compare TIM-3WT with mutated TIM-3 variants for three biological repeats. (G) SEE-loaded Raji B cells were used to stimulate NF-κB GFP reporter Jurkat cells to recapitulate the formation of an immunological synapse. IL-2 production following Raji B cell stimulation was determined by ELISA analysis for parental NF-κB GFP reporter cells (solid black line, open bar) and cells expressing TIM-3WT (solid red line, open bar), TIM-3F40A (solid magenta line, open bar and data points), or TIM-3I96A/M97A (dotted magenta line, gray filled bar and data points). Mean ± SD are plotted for three biological repeats, with P-values comparing TIM-3WT with the mutated TIM-3 variants determined using two-tailed, unpaired Student's t-tests.