Meredith A. Buchholz

Effect of Promoter Methylation on the Regulation of IFN-γ Gene During In Vitro Differentiation of Human Peripheral Blood T Cells into a Th2 Population

The carefully orchestrated events that result in a protective immune response are coordinated to a large extent by cytokines produced by Th1 and Th2 cell subsets. Th1 cells preferentially produce IL-2 and IFN-γ, resulting in a cellular response that helps to eliminate infected cells. In contrast, Th2 cells produce IL-4, IL-5, IL-6, and IL-10, stimulating an Ab response that attacks extracellular pathogens, thereby preventing the cells from becoming infected. To elucidate the mechanisms of differential regulation of cytokine genes by these two different subsets of T cells, we established an in vitro differentiation model of freshly isolated human peripheral blood T cells in which IFN-γ was used as an index gene to study the transcriptional regulation. The data presented here demonstrate that the IFN-γ promoter undergoes differential methylation during in vitro differentiation: the promoter becomes hypermethylated in Th2 cells, whereas it is hypomethylated in Th1 cells. Hypermethylation in Th2 cells results in chromatin condensation and exclusion of CREB proteins from the IFN-γ promoter. Treatment with 5-azacytidine, a demethylating agent, causes Th2 cells to reverse histone condensation and enables CREB recruitment to the hypomethylated promoter. This results in the increased production of IFN-γ. These data indicate the importance of promoter methylation in the regulation of the IFN-γ gene during differentiation.

Interleukin-12-induced Interferon-γ Production by Human Peripheral Blood T Cells Is Regulated by Mammalian Target of Rapamycin (mTOR)

Depending on the type of external signals, T cells can initiate multiple intracellular signaling pathways that can be broadly classified into two groups based on their sensitivity to the immunosuppressive drug cyclosporin A (CsA). Interleukin (IL)-12-mediated interferon (IFN)-γ production by activated T cells has been shown to be CsA-insensitive. In this report, we demonstrate that the IL-12-induced CsA-resistant pathway of IFN-γ production is sensitive to rapamycin. Rapamycin treatment resulted in the aberrant recruitment of Stat3, Stat4, and phospho-c-Jun to the genomic promoter region resulting in decreased IFN-γ transcription. IL-12-induced phosphorylation of Stat3 on Ser-727 was affected by rapamycin, which may be due to the effect of rapamycin on the IL-12-induced interaction between mammalian target of rapamycin (mTOR) and Stat3. In accordance with this, reduction in the mTOR protein level by small interfering RNA resulted in suppression of Stat3 phosphorylation and decreased production of IFN-γ after IL-12 stimulation. These results suggest that mTOR may play a major role in IL-12-induced IFN-γ production by activated T cells.