Fig 1.
Ectopic TIM-3 expression suppresses TCR-induced activation of NF-κB and NFAT.
(A) Flow cytometric analysis of stably transfected, sorted pools shows expression of TIM-3 on the surface of cells. (B) Cells were stimulated with Cell Stimulation Cocktail or anti-CD3/CD28 beads and NF-kB activity, as determined by GFP expression, was measured on an Accumen (left panel). NFAT activation was monitored by transient transfection of cells with NFAT-luciferase reporter plasmid. 72h post-transfection, cells were stimulated and luciferase activity was monitored after 6h using the One-Glow Assay System (right panel). Results are presented as fold change over base line. Results shown in panels B are the average ± SD for quadruplicate samples from a single experiment, representative of at least three independent experiments. (*, P < 0.01; ns: not significant as determined by two-way t-test).
Fig 2.
Ectopic Expression of TIM-3 does not suppress CD3/CD28 induced Ca2+ Flux.
(A) Cells pre-loaded with the fluorescence, calcium-sensitive dye, Cal-520 AM were treated with PMA/Ionomycin, or anti-CD3/CD28 beads, (B) soluble anti-CD3 (OKT3) and anti-CD28 (CD28.2), (C) OKT3 only or (D) CD28.2 only. Calcium flux was measured immediately, in real-time, using the FDSS/μCELL kinetic plate reader. Results are presented as the average ratio of Max-Min for Ex480:Em540, for quadruplicate replicates ± SD, representative of at least three independent experiments.
Fig 3.
Ectopic Expression of TIM-3 Suppresses CD69 expression and IL-2 secretion.
(A) Parental or TIM-3 over-expressing Jurkat T cells were stimulated over night with anti-CD3/CD28 beads or Cell Stimulation Cocktail. Flow cytometric analysis was performed using a CD69-PE conjugated antibody as a positive control and IgG-PE isotype for negative control staining. Red: CD69 for Jurkat-parental; Blue: CD69 for Jurkat-TIM3; Black: Isotype. (B) Cells were stimulated under the same conditions, supernatant was collected and subjected cytokine multiplex analysis. Data represents fold change in observed cytokines for quadruplicate replicates ± SD, representative of at least three independent experiments. (*, P < 0.01; ** P < 0.05 as determined by two-way t-test).
Fig 4.
The cytoplasmic domain of human TIM-3 suppresses TCR-induced NF-κB and NFAT Activity.
(A) Flow cytometric analysis demonstrating the relative cell surface expression of chimeric protein using an anti-murine CD28 antibody for detection. (B) Cells were stimulated overnight with anti-CD3/CD29 beads or Cell Stimulation Cocktail, NF-κB activity was measured by monitoring GFP expression. (C) Using similar stimulation conditions, NFAT activity was measured 6 hours post-stimulation. Data represents fold change in reporter activity for quadruplicate replicates ± SD, representative of at least three independent experiments. (*, P < 0.01 as determined by two-way t-test).
Fig 5.
TIM-3 Association with Intracellular Kinases in CD8+/MART-1+ T cells.
TIM-3 co-immunoprecipitation analysis of unactivated and 15 min. stimulation with anti-CD3/CD28 beads (activated). Equivalent amounts of protein (~2mg) were co-immunoprecipitated with pAb anti-TIM-3 antibody and western blot was performed using capillary electrophoresis. Cleared lysate served as a loading control for individual antibody reactivity.
Fig 6.
TCR-induced NF-κB/NFAT promoters and cytokine secretion in CD8+/MART-1+ T cells.
Cells were stimulated overnight with cell stimulation cocktail (CSC) or anti-CD3/CD28 beads. Cell supernatents and protein lysates were collected. To evaluate promoter activity, lysates were subjected to (A) NF-kB (left panel: p50 sub-unit; right panel: p65 sub-unit) or (B) NFAT analysis. Data represents fold change in reporter activity for quadruplicate replicates ± SD. (*, P < 0.01 as determined by two-way t-test).
Fig 7.
(C) Supernatents from stimulated cells were subject to multiplex analysis for evaluation of cytokine secretion.
Data shown for fold change in TNF-α, IFN-γ, and IL-2 secretion, relative to unstimulated control cells, for quadruplicate replicates, ± SD. (*, P < 0.01 as determined by two-way t-test).