Direct regulation of Gata3 expression determines the T helper differentiation potential of Notch

Abstract

CD4(+) T helper cells differentiate into T helper 1 (Th1) or Th2 effector lineages, which orchestrate immunity to different types of microbes. Both Th1 and Th2 differentiation can be induced by Notch, but what dictates which of these programs is activated in response to Notch is not known. By using T cell-specific gene ablation of the Notch effector RBP-J or the Notch1 and 2 receptors, we showed here that Notch was required on CD4(+) T cells for physiological Th2 responses to parasite antigens. GATA-3 was necessary for Notch-induced Th2 differentiation, and we identified an upstream Gata3 promoter as a direct target for Notch signaling. Moreover, absence of GATA-3 turned Notch from a Th2 inducer into a powerful inducer of Th1 differentiation. Therefore, Gata3 is a critical element determining inductive Th2 differentiation and limiting Th1 differentiation by Notch.

Figure 1. Generation of Th2 mediated immunity in vivo is dependent on RBP-J

RBP-J deficient (RBP^flox/−/CD4Cre) (white bars and symbols) or heterozygous (RBP^flox/+/CD4Cre) littermates (black bars and symbols) were immunized with extract from Schistosoma mansoni eggs (SEA). Cytokine responses of purified splenic CD4+ T cells were measured upon in vitro restimulation with SEA (a). Each bar represents the response of CD4+ T cells from an individual mouse. In addition, sera from individual heterozygous control (closed symbols) or RBP-J deficient mice (open symbols) were tested for the presence of various isotypes of antibodies against SEA (b). Data in (a) and (b) are representative of 5 separate experiments. For IgE, values obtained from several experiments are shown combined, with each dot representing a single mouse.

Figure 2. Notch1 and 2 are required for physiological Th2 responses

(a) Naïve CD4+ T cells were isolated from AND TCR transgenic Notch1/2 double deficient mice (Notch1^flox/flox/Notch2^flox/flox CD4Cre) (open dots) or wild type (Notch1^flox/+/Notch2^flox/+ CD4Cre) (closed dots) littermates and stimulated in vitro with collagenase treated splenic APC in the presence of SEA (20μg/ml) and pigeon cytochrome C (10μg/ml). After 5 days viable effector cells were restimulated with PMA and ionomycin and IL4 and IFNγ production was measured by intracellular cytokine staining. Each dot represents percentage IL4⁺ or IFNγ⁺ cells in cells obtained from an individual mouse. Data represent cumulative results from 3 independent experiments. (b) Naïve CD4+ T cells were isolated from Notch1/2 double deficient (hatched bars) or wild type (filled bars) littermates (all positive for the CD4 Cre transgene) and activated in vitro with soluble anti CD3 and anti CD28 and splenic APC in the presence of Th1 (IL12 and anti IL4) or Th2 (IL4 and anti IFNγ) polarizing conditions. After 5 days viable effector cells were harvested, restimulated with plate bound anti CD3 (10μg/ml) and 48 hour supernatants were assayed for IL4 and IFNγ. Cytokine concentrations from cells obtained from individual mice are shown.

Figure 3. (a) Notch is required for Gata3 expression

Naïve CD4+ T cells from wild type (closed bar) or Notch1/2 deficient mice (open bar) were isolated and stimulated as in Figure 2(a). After 5 days in culture, cDNA was prepared and tested for the abundance of gata3 transcripts. (b) Notch ICD induces IL4 independent Th2 differentiation. Naïve STAT6 −/− CD4+ T cells were activated with splenic APC and soluble antibodies to CD3 and CD28 and transduced with a retrovirus encoding NICD linked to GFP through an IRES sequence (right panel) or with control virus (left panel). 3 days after transduction viable effector cells were restimulated with PMA and ionomycin and IL4 and IFNγ production was measured by intracellular cytokine staining. Numbers represent percentages in each quadrant. (c) Notch ICD induces IL4 independent Gata3 expression. Naïve STAT6 −/− CD4+ T cells were activated and transduced as in (b). 3 days after transduction GFP+ cells were isolated by FACSorting, cDNA was prepared and the abundance of gata3 transcripts was determined by real time PCR. Open bar represents Vector transduced and closed bar represents NICD transduced cells.

Figure 4. (a) Notch stimulates transcriptional activity of the upstream but not the downstream Gata3 promoter

Naïve STAT6 −/− CD4+ T cells were transduced, sorted for GFP+ cells and cDNA was prepared as in 3(c). The abundance of exon1a or exon 1b containing transcripts was determined by real time PCR using a common 3′ primer annealing in exon 2 and specific primers annealing in exon 1a (left) or exon 1b (right).(b) Notch induced activity of the upstream Gata3 promoter is dependent on RBP-J. Naïve CD4+ T cells from wild type or RBP-J deficient mice were transduced and analyzed as in (a) for the abundance of exon 1a (top) or exon 1b (bottom) containing transcripts. Neutralizing antibody to IL4 (10μg/ml) was included in the cultures.

Figure 5. The upstream Gata3 promoter contains a bona fide conserved RBP-J binding element

(a) diagram of the Gata3 locus showing upstream and downstream Gata3 promoters each driving expression of a unique exon 1, which is spliced to the common exon 2. Underneath, the consensus RBP-J binding element is shown and its conservation between mouse and human. (b) EMSA using radiolabeled double strands probe containing the RBP^G3P1a and lysates from CHO cells transfected with vector alone (1st lane), RBP-J (2nd lane and 4th to 10^th lane) or RBP-J R218H (3rd lane). A probe carrying a point mutation mRBP^G3P1a was used as specificity control (10^th lane). The specific RBP-J-RBPG3P1a complex was competed away by addition of (10-fold or 3-fold) excess unlabeled RBP^G3P1a (lanes 4 and 5) or the previously described RBP-J binding element RE (lanes 8 and 9), but not by the mutant probe mRBP^G3P1a (lanes 6 and 7). (c) EMSA using lysates from CD4+ T cells (lanes 2 to 5) and radiolabeled RBP^G3P1a, (lanes 1,2 and 4,5) or mRBP^G3P1a(lane 3). Antibody to RBP-J (lane 5) or control mouse Ig (lane 4) was added to the reaction to supershift the RBP-J- RBP^G3P1a complex.

Figure 6. RBP-J binds the upstream Gata3 promoter in vivo

(a) ChIP was performed on chromatin from CD4+ T cells using control antibody (white bars) or antibody to RBP-J (black bars). Precipitation of the upstream Gata3 promoter (USG3P) or the downstream Gata3 promoter (DSG3P) was determined by quantitative PCR. Samples were normalized to Input values. (b) ChIP was performed as in (a) using chromatin from wild type (black bars) or RBP-J deficient (white bars) CD4 T cells.

Figure 7. Gata3 is necessary for Notch induced Th2 responses

(a) Naïve CD4+ T cells from Gata3^flox/− mice were activated with splenic APC and antibodies to CD3 and CD28 and infected with control GFP virus or virus encoding Cre linked through an IRES sequence to GFP. After 3 days, GFP+ cells were isolated by FACSorting and RNA was made. Relative abundance of Gata3 message was determined by real time PCR using a primer probe set that detects the floxed exon 4 of gata3. (b) Naïve CD4+ T cells from Gata3^flox/− mice were activated as in (a) and infected after 20 hours first with control IRES-GFP (black bars) or Cre-IRES-GFP (gray bars). 6 hours later, cells were transduced with control IRES-Thy1.1 virus (left) or NICD-IRES-Thy1.1 (right). Neutralizing antibodies to IL4 and IFNγ (10μg/ml each) were added to the cultures at the time of the second transduction. 3 days after the last transduction cells were sorted for GFP and Thy1.1 expression and restimulated with plate bound anti CD3 (10μg/ml). 48 hour supernatants were tested for IL4 and IFNγ concentrations by ELISA. These results are representative of 3 independent experiments.