T-bet, a Th1 transcription factor regulates the expression of Tim-3

Abstract

T-cell immunoglobulin, mucin domain-3 (Tim-3) is a membrane protein expressed at late stages of IFN-gamma secreting CD4(+) Th1 cell differentiation and constitutively on DC. Ligation of Tim-3 on Th1 cells terminates Th1 immune responses. In addition, Tim-3 plays a role in tolerance induction, although the mechanism by which this is accomplished has yet to be elucidated. While it is clear that Tim-3 plays an important role in the immune system, little is known regarding the molecular pathways that regulate Tim-3 expression. In the current study, we examine the role of Th1-associated transcription factors in regulating Tim-3 expression. Our experiments reveal that Tim-3 expression is regulated by the Th1-specific transcription factor T-bet. This introduces a novel paradigm into the generation of a Th1 response, whereby a transcription factor responsible for effector Th1 cell differentiation also increases the expression of a specific counter-regulatory molecule to ensure appropriate termination of pro-inflammatory Th1 immune responses.

Figure 1. Impaired Tim-3 expression in T-bet and STAT-4 deficient Th1 cells

(A) Naïve CD4⁺ (CD4⁺ CD62L^high) T cells from wild type, T-bet^−/−, STAT-4^−/− and T-bet^−/− × STAT-4^−/− mice were stimulated in vitro under Th1 polarizing conditions. After each round of polarization, cells were harvested and total RNA prepared for analysis of Tim-3 expression by real-time PCR. Expression of Tim-3 RNA relative to control HPRT expression is shown. Error bars indicate SEM of two replicate experiments. Data shown are representative of 2–3 independent experiments. (B) CD4⁺ T cells from (A) were activated with PMA/Ionomycin and then stained with mAbs to Tim-3, CD4, IFN-γ, IL-4, IL-10 and IL-17. Histograms represent Tim-3 expression (filled histogram, FMO control; solid black line, Tim-3 staining). Dot plots represent cytokine expression in CD4⁺ cells. Numbers indicate frequency of cytokine positive cells. Similar results were obtained in 2–3 independent experiments. (C) Median fluorescence intensity (MFI) of Tim-3 staining on CD4⁺ T cells from wildtype, T-bet^−/−, STAT-4^−/− and T-bet^−/− × STAT-4^−/− T cells after each round of in vitro Th1 differentiation. Data shown are representative of 2–3 independent experiments.

Figure 2

T-bet can drive the expression of Tim-3 independently of transactivation of IFN-γ. Naïve (CD4⁺CD62L^highCD25⁻) CD4⁺ T cells from T-bet^−/− × IFN-γ^−/− mice were transduced at 48 hours post activation with plate bound CD3 and CD28 with either empty (RV GFP) or T-bet (RV T-bet) expressing retrovirus and sorted for CD4 and GFP expression at day 5. The cells were rested overnight in IL-2 (100 U/ml) and RNA was extracted 24 hours later. Tim-3 RNA expression was quantified by real time PCR and normalised to β-actin. Error bars represent SEM from triplicate PCRs. Data shown are representative of 3 independent experiments.

Figure 3. Impaired Tim-3 expression in T-bet and STAT-4 deficient dendritic cells

CD11c⁺ dendritic cells were isolated by cell sorting from the collagenase digested spleens of wild type, T-bet^−/−, STAT-4^−/− and T-bet^−/− × STAT-4^−/− mice. Expression of Tim-3 RNA relative to control HPRT expression is shown. Error bars indicate SEM of two replicate experiments. Data shown are representative of 2–3 independent experiments.

Figure 4. T-bet controls Tim-3 expression in vitro

Naïve CD4⁺ (CD4⁺CD62L^highCD25⁻) T cells from T-bet CD2-Tg mice (and BALB/c wild type controls) were stimulated in vitro under Th0 (A) and Th1-polarizing conditions (B). After one round of restimulation, T cells were stimulated and then stained with mAbs to Tim-3 and CD4, and cytokine expression was detected by intracellular staining. Histograms represent Tim-3 expression (dotted line, isotype control; solid line, specific staining). Dot plots represent cytokine expression in CD4⁺ T cells (Th1 condition). This experiment was repeated 3 times (n=4 for both wildtype and Tg mice in each experiment). Representative data are shown.

Figure 5. T-bet controls Tim-3 expression in vivo

1 × 10⁶ naive (CD4⁺CD62L^highCD44^lowCD25⁻) CD4⁺ T cells from wildtype (Balb/c) or T-bet^−/− (Balb/c) were adoptively transferred i.v. into Rag2^−/− (Balb/c) mice (n=5/group). Transferred mice were administered Brdu in the drinking water (0.8mg/ml). Two weeks later, splenic T cells populations were harvested. (A) Frequency of CD4⁺Brdu⁺ cells in transferred mice. (B) Frequency of CD4⁺CD25⁺ cells in transferred mice. (C) Frequency of total CD4⁺Tim-3⁺ cells in transferred mice. Error bars indicate SEM. Data shown are representative of 2 independent experiments.

Figure 6. T-bet binds to the Tim-3 promoter in vivo

ChIP was performed on murine Th1 CD4⁺ T cells with a polyclonal antibody to T-bet, and the resulting genomic DNA was analyzed by real-time PCR. (A) IFN-γ promoter, (B) IL-4 promoter, (C) Tim-3 promoter and (D) Tim-3 promoter after ChIP with a STAT-4 antibody. Enrichment is expressed as the fold enrichment of IP DNA over whole cell extract. Error bars indicate SEM of triplicate reactions. Data shown are representative of 2 independent experiments.