TIM-3 Suppresses Anti-CD3/CD28-Induced TCR Activation and IL-2 Expression through the NFAT Signaling Pathway

Abstract

TIM-3 (T cell immunoglobulin and mucin-domain containing protein 3) is a member of the TIM family of proteins that is preferentially expressed on Th1 polarized CD4+ and CD8+ T cells. Recent studies indicate that TIM-3 serves as a negative regulator of T cell function (i.e. T cell dependent immune responses, proliferation, tolerance, and exhaustion). Despite having no recognizable inhibitory signaling motifs, the intracellular tail of TIM-3 is apparently indispensable for function. Specifically, the conserved residues Y265/Y272 and surrounding amino acids appear to be critical for function. Mechanistically, several studies suggest that TIM-3 can associate with interleukin inducible T cell kinase (ITK), the Src kinases Fyn and Lck, and the p85 phosphatidylinositol 3-kinase (PI3K) adaptor protein to positively or negatively regulate IL-2 production via NF-κB/NFAT signaling pathways. To begin to address this discrepancy, we examined the effect of TIM-3 in two model systems. First, we generated several Jurkat T cell lines stably expressing human TIM-3 or murine CD28-ECD/human TIM-3 intracellular tail chimeras and examined the effects that TIM-3 exerts on T cell Receptor (TCR)-mediated activation, cytokine secretion, promoter activity, and protein kinase association. In this model, our results demonstrate that TIM-3 inhibits several TCR-mediated phenotypes: i) NF-kB/NFAT activation, ii) CD69 expression, and iii) suppression of IL-2 secretion. To confirm our Jurkat cell observations we developed a primary human CD8+ cell system that expresses endogenous levels of TIM-3. Upon TCR ligation, we observed the loss of NFAT reporter activity and IL-2 secretion, and identified the association of Src kinase Lck, and PLC-γ with TIM-3. Taken together, our results support the conclusion that TIM-3 is a negative regulator of TCR-function by attenuating activation signals mediated by CD3/CD28 co-stimulation.

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).