? Interleukin-4 is a prototypic type I cytokine that is the central regulator of allergic inflammatory responses. It controls the polarization of naive CD4 T cells to the TH2 phenotype and Ig class switching to IgE. The Cytokine Biology Unit has characterized the signaling mechanisms utilized by the IL-4 receptor. It has shown that activation of the latent transcription factor, Stat 6, controls both TH2 polarization and IgE class switching. In addition, IL-4-mediated Stat6 signaling rescues activated naive CD4 T cells from apoptosis. Scientists in the Unit have established that both GATA3 and Stat5 are essential for acquisition of IL-4 producing capacity. It has now been shown that T cell receptor-mediated activation of the erk signaling pathway prevents transcription of GATA3 and desensitizes the IL-2 receptor. Since GATA3 and IL-2/ STat5 are essential for early transcription of IL-4, which in turn controls TH2 differentiation, erk activation prevents Th2 commitment. It has been shown that high concentrations of antigen strongly stimulate erk phosphorylation, thereby preventing TH2 differentiation while low concentrations of antigen, which activate erk only weakly, are permissive for early IL-4 production and TH2 differentiation. Recently, it has been proposed that activation of the Notch signaling pathway is essential for TCR-mediated GATA3 activation. However, using mice that are conditionally deleted of presinilin 1 and 2, which are essential for Notch signaling, no abnormalities in TCR-induced GATA3 could be shown, implying that Notch effects on TH2 polarization are mediated at a different level. Utilizing conditional GATA3 knockout mice prepared by scientists in the Unit, it has been shown that GATA3 is essential for priming for IL-4-production both in vitro and in vivo but that cells that have already differentiated into Th2 cells can sustain their IL-4 producing capacity even when GATA3 is eliminated. By contrast, both IL-5 and IL-13 production are strictly dependent on GATA3. ? It has been further demonstrated that GATA3 is a potent growth regulator of TH2 cells and that GATA3 and Gfi-1 act together to induce this striking stimulation of TH2 cell growth. Indeed, Gfi-1 conditional knockout mice show a major diminution in Th2 responses in S. mansoni infections due to diminished growth of these cells. Gfi-1, in its role as a transcriptional repressor, is critical for robust growth to IL-2, since it appears to repress expression of genes such as SOCS1 and SOCS3 that inhibit IL-2 mediated growth. On the other hand, Gfi-1 diminishes growth in response to IL-7 since it mediates TCR- and cytokine-mediated down regulation of the IL-7R alpha chain. These studies, utilizing Gfi-1 conditional knockout mice developed in the Unit, establish that Gfi-1 plays a critical role in lymphocyte homeostasis and that precise regulation of Gfi-1 expression is central to a balanced population of CD4 and CD8 T cells. Further, Gfi-1 plays a central role in the phenotype of memory CD4 and CD8 cells induced by viral infection.? p38 has now been shown to play a major role in IL-4 expression. Its function is mediated, at least in part, through its regulation of IL-4 mRNA half-life. Th2 cells in which p38 has been activated display long IL-4 mRNA half-lives but if p38 fails to be activated or if its activity is blocked, most IL-4 mRNA has been degraded within 30 minutes. Unit scientists have shown that recently activated Th2 cells robustly activate p38 in response to stimulation but that resting memory cells show poor activation, suggesting that effector cells have a striking advantage in the capacity to produce IL-4, at least in part due to their capacity to sustain IL-4 mRNA. Ionomycin alone can initiate IL-4 production by memory Th2 cells. This appears to result from the phosphorylation of MAP kinases upstream of p38 including ASK and TAC, resulting in p38 activation, as well as the activation of the NFAT signaling pathway. A further in depth analysis of the distinctiveness of the various differentiated populations of CD4 T cells including TH2, TH1 and TH17 cells has been undertaken through a genome-wide analysis of distinctive sites for histone acetylation in each of these differentiated populations. Using new technology for high throughput sequencing combined with chromatin immunoprecipitation, an analysis has been done of those sites that show high levels of association with acetylated histone and they have been compared to genes that show evidence of activation using microarray analysis. The correlation has been very good. Additional use of this extremely powerful approach to understand global gene activation during differentiation will be undertaken.? Laboratory scientists have also demonstrated that the transcription of the two alleles at the Il4 locus is governed stochastically and that the likelihood of transcription is associated with the degree of chromatin accessibility of the two alleles. Furthermore, although there are shared requirements for the transcription of IL-4 and IL-13, in individual cells and individual clones, the expression of these cytokines is independently determined. To study this in greater detail, scientists in the Cytokine Biology Unit are using the techniques of recombineering to develop mice that bear fluorescent protein reporters for expression of IL-4 and IL-13. ? In addition, an in depth effort to understand the distinctive signaling mechanisms used for IL-4 and IL-13 responses, with emphasis on the differential roles of the type I and type II IL-4 receptors and how different cell types show very different relative sensitivity to these congeners. For this purpose, responses of various macrophage populations from wild-type and gamma common-deficient mice to IL-4 and IL-13 have been analyzed in detail. Both the phosphorylation of STAT6, as measured by glow cytometric analysis, and the induction of arginase-1, a gene that is rapidly induced through the IL-4 receptor, has been evaluated. Bone marrow derived macrophage (BMDM) populations and monocytes are exquisitely sensitive to IL-4 but require 100 to 1000 fold more IL-13 to achieve a comparable response. Paradoxically, BMDM from gamma common-deficient mice respond well to IL-13 but poorly to IL-4 and anti- gamma common antibody essentially abolishes responses of monocytes to IL-4. Peritoneal macrophages and fibroblasts respond well to both IL-4 and IL-13 and gamma common deletion has very little effect on either IL-4 or IL-13 responses. Using information regarding the numbers of receptor subunits and equilibrium constants for each interaction, a model has been derived to explain these results. From the analysis, it has been concluded that IL-4 plays a central role as an inducer of differentiation while IL-13 is a major effector molecule.? LI scientists have also developed new insights into the production of IL-4 by basophils. They have shown that these cells are massively recruited into the tissues, spleen and blood in helminth infection and that this recruitment is T cell dependent. IL-3 appears to play a major role in basophil recruitment. ? Efforts are now underway to use both small molecule libraries and a retroviral cDNA library expression system to identify new pathways and genes that play an important role in Th2 priming and maintenance.

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Dewas, Cedric; Chen, Xi; Honda, Tetsuya et al. (2015) TSLP expression: analysis with a ZsGreen TSLP reporter mouse. J Immunol 194:1372-80
Paul, William E (2014) Endless fascination. Annu Rev Immunol 32:1-24
Ben-Sasson, S Z; Wang, K; Cohen, J et al. (2013) IL-1? strikingly enhances antigen-driven CD4 and CD8 T-cell responses. Cold Spring Harb Symp Quant Biol 78:117-24
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