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Signal transducer and activator of transcription 3 (Stat3) is implicated in the pathogenesis of many malignancies and essential for IL-6–dependent survival and growth of multiple myeloma cells. Here, we demonstrate that the gene encoding oncogenic microRNA-21 (miR-21) is controlled by an upstream enhancer containing 2 Stat3 binding sites strictly conserved since the first observed evolutionary appearance of miR-21 and Stat3. MiR-21 induction by IL-6 was strictly Stat3 dependent. Ectopically raising miR-21 expression in myeloma cells in the absence of IL-6 significantly reduced their apoptosis levels. These data provide strong evidence that miR-21 induction contributes to the oncogenic potential of Stat3.

Transcriptional regulation of the interleukin-5 (IL-5) gene in T lymphocytes appears to be of central importance in the control of the eosinophilia characteristic of allergic responses and certain parasite infections. Previous studies of IL-5 gene regulation have been hampered by the lack of a transfection assay, which detects the antigen-responsive enhancer in the IL-5 promoter. Here we show that stable transfection of the Th2 clone D10.G4.1 and the T lymphoma EL4.23 with chloramphenicol acetyltransferase reporter gene constructs carrying the region to -3859 gives inducible expression with the known regulatory characteristics of the endogenous IL-5 gene. To facilitate detailed analysis of the promoter region, 3.9 kb of DNA sequence immediately up stream of the start of transcription was determined and the minimum upstream region required for inducible expression was further localized, by stable transfection studies in EL4.23 cells, to the region up to -1016. A CTF/NF1 site in the upstream enhancer at -940 to - 928 was shown to be required for regulated inducible expression. Mutation of this sequence motif abolished inducibility and also prevented binding of the sequence to a nuclear protein(s). A TCATTT-containing element in the proximal promoter region was also demonstrated to be essential for inducible expression of the IL-5 gene, similar to the role of this conserved element in the transcriptional regulation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 genes.

Abstract 3281 Regulatory γδT cells (γδTregs) are capable of suppressing T-cell activation and proliferation (Rita et al Journal of immunology 2009). Our previous studies demonstrated the immunosuppressive features of γδTregs and further supported the use of γδTregs as a promising therapeutic strategy for the adoptive immunotherapy of allograft rejection, post-transplantation graft-versus-host disease (GVHD) and autoimmune diseases (Hu et al ASH abstract 2011). To this end, large-scale, efficient expansion of γδTregs represents a fundamentally important prerequisite for their clinical application. Here we explored the efficacy of decitabine (a DNA hypomethylating agent) for the generation of functional γδTregs and its underlying mechanisms. Human peripheral blood mononuclear cells (PBMCs) were cultured with interleukin (IL)-2, IL-15, TGF-β1 and zoledronic acid (ZOL). Decitabine was added to aliquots of PBMCs. Frequency and absolute number of γδTregs were investigated by flow cytometry (FACS) and trypan blue live-cell counts after cultivation. Our results showed a higher frequency (61.9±10.3% versus 38.4±7.8%, p Next we analyzed the underlying mechanisms of decitabine on γδTreg induction. Genomic DNA was isolated from γδTregs sorted by FACS. The Foxp3 gene methyaltion status of upstream enhancer CpG islands from −5995 to −5778 and from −5031 to −4792, proximal promoter CpG island from -139 to -15 and non-coding DNA sequence 3 (CNS3) CpG island from 3748 to 3971 was analyzed by bisulfite sequencing. We found decitabine-induced γδTregs displayed efficient CpG demethylation of the upstream enhancer from −5031 to −4792, and of CNS3 from 3748 to 3971, but no differences were detected in the other CpG islands investigated in contrast to common γδTregs (Fig. 2A). We subsequently investigated if DNA demethylation of CpG islands could influence upstream enhancer or CNS3 activity to promote Foxp3 transcription. The upstream enhancer from −5031 to −4792 and CNS3 from 3748 to 3971 were methylated using SssI (CpG) methylase, and methylated or unmethylated sequences were inserted into demethylated pFoxP3Pro, respectively. Vectors were added to γδT cells and electroporated using the T-023 program of the Nucleofector. Dual fluorescence-based transient reporter assays showed an obvious reduction in luciferase activity after methylation of both CpG islands, but not with the demethylated CpG islands (Fig. 2B), indicating that demethylation of the upstream enhancer and CNS3 can promote Foxp3 expression via increased enhancer activity. Transcription factors (TF) play important roles in gene expression. To test whether decitabine enhanced specific TFs expression to influence γδTreg induction, we performed TF CHIP assays in FACS-sorted common γδTregs versus decitabine-induced γδTregs. The expression levels of 245 TFs were evaluated and the result showed that NF-κb expression in decitabine-induced γδTregs was up-regulated 50 times in contrast to that in common γδTregs. NF-κb up-regulation was further comfirmed by western blot analysis from 6 different healthy volunteers. Finally, We evaluated the immunosuppressive function of the enriched γδTregs on Carboxyfluorescein Diacetate (CFSE)-labeled hPBMC proliferation and activation stimulated with anti-CD3/anti-CD28 monoclonal antibodies for 5 days in vitro. As shown in Fig. 3, both common γδTregs and decitabine-induced γδTregs exerted dose-dependent suppression of proliferation, but decitabine-induced γδTregs had a greater suppressive effect than common γδTregs. In conclusion, these data demonstrate for the first time that decitabine facilitates the generation of functional γδTregs via foxp3 gene demethylation and NF-κb up-regulation. Futhermore, our results lay the foundation for large-scale availability of induced γδTregs for potential clinical applications, and reveal novel targets for regulating Foxp3 expression in γδT cells. Disclosures: No relevant conflicts of interest to declare.