Fig 2: TIM-3 cellular localization is dependent on CEACAM1 in primary CD8+ effector T cells.A CEACAM1 was assessed by flow cytometry (MFI) on days 4 and 14 post-activation; data normalized to day 4. B CEACAM1 mRNA was collected on days 4 and 14 post-activation and assessed by qPCR, normalized to 18S rRNA, and plotted as ΔCt values. C CEACAM1 and TIM-3 MFI were plotted for individual donors on day 4 post-activation. Dotted lines represent 95% confidence intervals. D Cells from five donors transfected with CEACAM1 siRNA on day 0 were assessed for both CEACAM1 and TIM-3 expression on day 4. Dotted lines represent 95% confidence intervals. E Representative western blot (of three donor replicates) probing for TIM-3 following either TIM-3 or CEACAM1 knockdown in newly activated cells (day 4). Statistical tests: A Mann–Whitney test, p = 0.0022; B paired t test, p = 0.6575; C simple linear regression and Pearson correlation, p = 0.0392; D simple linear regression and Pearson correlation, p = 0.0172.

Fig 3: TIM-3 ligand binding is required for mitigating RICD sensitivity in early stage effector T cells.Effector T cells at day 4 (A) or day 14 (C) were treated with soluble TIM-3 (+sTIM-3) or isotype control (-sTIM-3) for 1 h prior to restimulation with 100 ng/mL of OKT3 and death assessment 24 h later. Cells were treated day 4 (B) or day 14 (D) with TIM-3 blocking antibody or an isotype antibody control for 1 h prior to restimulation with 100 ng/mL of OKT3 and death assessment 24 h later. Asterisks denote statistical significance using paired t tests: A p = 0.0399; B p = 0.0003; C p = 0.9957; D p = 0.0563.

Fig 4: Co-expression of CEACAM1 promotes TIM-3 surface expression on T cells.A Jurkat T cells were transfected with empty vector (EV), TIM-3, CEACAM1, or TIM-3 and CEACAM1 FLAG-tagged expression plasmids and assessed for TIM-3 surface expression. Each point represents separate experiments in which the median fluorescence intensity (MFI) was assessed for each condition. Statistical tests: A one-way ANOVA with multiple comparisons; p < 0.0001 for EV against TIM-3/CEACAM1. B Cell lysates were assessed for total CEACAM1 and TIM-3 protein expression by western blot (anti-FLAG); β-actin served as a loading control. C Single experiment from (A) plotted as a histogram to highlight the increased % TIM-3+ cells with TIM-3 and CEACAM1 co-transfection. D Dot plot histogram overlay of Jurkats transfected with only TIM-3 (blue) versus both TIM-3 and CEACAM1 (teal). D Representative flow plots of Jurkats transfected as in (A).

Fig 5: Tim 3 protected DSCs from TLR-mediated proinflammatory response via NF-κB inhibition.(A) Quantitation (left) of flow cytometric analysis (right) of IFN-γ, TNF-α, IL-1β, and IL-2 production by DSCs treated with or without LPS (10 ng/ml) in the presence or absence of Tim-3 and anti–Tim-3 mAb. Data represent mean ± SEM. Flow cytometry plot shown is from one representative experiment; n = 14 DSCs in the first trimester of normal pregnancy. Treatment group numbers below histogram bars correspond to numbers on panels of flow cytometry plot. (B) In-cell western analysis (lower) and quantitation (upper) of phosphorylation/activation of NF-κB in DSCs stimulated with or without LPS, Tim-3, and U0126, alone or together. Images are representative of three individual experiments. *P < 0.5, **P < 0.01, ***P < 0.001, compared with group 1; ΔP < 0.5, ΔΔP < 0.01, ΔΔΔP < 0.001, compared with group 2; #P < 0.5, ##P < 0.01, ###P < 0.001, compared with group 3.