Table 1.
EMSA oligonucleotide sequences.
Fig 1.
CRTh2 expression on Jurkat T cells and freshly isolated CD4+ cells.
(A) Representative staining for CRTh2 staining in T cells. Empty histogram indicates CRTh2 staining and filled histogram indicates corresponding isotype control. Jurkat T cells and freshly isolated CD4+ cells express low levels of CRTh2 by surface staining. After 7 days in Th2 differentiation conditions, 24% of cells are CRTh2+. In vitro differentiated CRTh2+ Th2 cells express CRTh2 (78%) following the proliferation phase (B) Box and whisker plots summarizing CRTh2 surface staining from 3–18 experiments. (C-E) Using intracellular staining, CRTh2+ Th2 cells were IL-4 and IL-13 positive, but low IL-13 and IFNγ (2.5%) double positive (D) following mitogenic stimulation. In vitro differentiated CRTh2+ Th2 cells highly express CD45RO and CD62L (E) following the proliferation phase. (F) Box and whisker plots summarizing intracellular and surface staining from 3 independent cell lines.
Fig 2.
CRTh2 expression decreases following activation.
(A) CRTh2 surface expression on in vitro differentiated CRTh2+ Th2 cells decreases following T cell stimulation (n = 8). (B) CD62L is equally expressed following the resting and activation phases (n = 6–8; 3 independent cell lines). (C and D) CRTh2 surface expression is dynamic, showing high (after resting) and low (after activation) over numerous cycles. (E) Surface CRTh2 expression was maintained after 4hr of stimulation but falls significantly by 24hr (n = 4). (F) CRTh2 mRNA is significantly decreased following 4hr of treatment with αCD3/αCD28. N = 3–4 independent experiments. (G) Following removal of TCR stimulation, mRNA (top) and surface staining (bottom—MFI to isotype surface expression by flow cytometry) for CRTh2 recovers over 3–4 days. (H) Return to surface expression of CRTh2 is highly correlated with mRNA levels. * p< 0.05, determined by Student’s t-test.
Fig 3.
Predicted and observed transcription factor binding to the CRTh2 proximal promoter.
(A) The CRTh2 proximal promoter contains putative NFAT and GATA binding sites, which show varying degrees of evolutionary conservation. (B) Site-directed mutagenesis of the CRTh2pro reporter vector to delete the GATA/NFAT/GATA containing-region. (C) Specific deletion of the conserved (-125/-94) base pairs reduced both overall luciferase activity of the vector and response to activation with PMA/Ionomycin. (D) This region was used as a probe for EMSA. Nuclear extracts were made from in vitro differentiated CRTh2+ Th2 cells following the resting phase (IL-2) and were re-stimulated (3h PI). NFAT1 and GATA3 bound to the CRTh2 promoter, although NFAT1 binding is more prominent after PMA/ionomycin stimulation Competitors and super-shifting antibodies used are noted above the corresponding lanes. Antibodies denoted as α. Gels are representative of n = 8 from 6 independent cell lines. *p< 0.05, determined by Student’s t-test.
Fig 4.
Determining the functional GATA3 sites in the CRTh2 proximal promoter.
Recombinant GATA3 and NFAT1 were used to characterize binding. Mutated CRTh2 proximal oligonucleotides were used as competitors (A) and labelled as probes (B). Competitors and super-shifting antibodies used are noted above the corresponding lanes. Antibodies denoted as α. G1, G2, and G3 refer to the GATA3 site that has been mutated in each case.
Fig 5.
NFAT1 inhibits GATA3-induced CRTh2 promoter activation.
(A) GATA3, but not NFAT1, over-expression in Jurkat T cells increased transcriptional activity of CRTh2-pro/Luc. When GATA3 (G) and NFAT1 (N) were co-expressed this led to inhibition of GATA3-mediated activity. (B) Transcriptional activity of the CRTh2 proximal promoter (CRTh2-pro/Luc) was measured by fold increase of relative luciferase activity (RLA) in stimulated (16h P/I) over unstimulated samples. When NFAT1 was co-transfected in increasingly lower amounts (up to 1/8th GATA3) CRTh2 promoter activity was recovered n = 3–5, *p< 0.05, determined by ANOVA with post hoc analysis using Holm-Sidak method.
Fig 6.
NFAT inhibition reverses the loss of CRTh2 expression following TCR stimulation.
(A) Inhibiting NFAT with a cell permeable inhibitor peptide (VIVIT) maintains CRTh2 surface expression following 3 days of priming with αCD3/αCD28, control peptide (CP, VEET) did not affect the CRTh2 surface expression (n = 3). (B) CRTh2 mRNA was also recovered following 3 days of priming with αCD3/αCD28 in the presence of VIVIT, though IL-13 mRNA was increased. (C-F) GATA3 and NFAT1 binding to the promoters of CRTh2 and IL-13 was confirmed by ChIP (n = 4), *p< 0.05, determined by Student’s t-test, activation vs media.
Fig 7.
Schematic representation of molecular regulation of CRTh2.
Although GATA3 can bind the CRTh2 promoter probe, NFAT1 binds with greater intensity following activation and displaces GATA3, resulting in lower CRTh2 expression. Dashed line represents that one or two GATA3 molecules may bind.