In initial studies we established that a large percentage of TCR-activated T cells co-stimulated by ZD-activated BMDCs differentiate into IL-10-producing T cells whereas T cells co-stimulated by TLR-activated BMDCs mainly differentiate into IFN-gamma-producing cells. The ZD-activated BMDC are not inducing a Th2 T cell response since only a small proportion of the co-stimulated T cells produce IL-4 alone or in combination with IL-10; nevertheless, the IL-10 response depends on activation of the IL-4/STAT6/GATA3 axis since it does not occur in STAT6- or GATA3-deficient T cells and requires the presence of small amounts of IL-4 in the BMDC/T cell co-culture. Performance of micro-array studies to further understand the molecular events driving the IL-10 response revealed via IPA analysis that the latter was associated with activation of CEBP-beta, a leucine zipper protein that binds to CCAAT motifs in promoter/enhancer regions. Indeed, in further studies we showed that T cells from mice in which CEBP-beta was deleted exhibit greatly reduced numbers of IL-10-producing cells when co-cultured with ZD-activated BMDCs. Related studies focusing on CEPB-beta function disclosed that this transcription factor is synthesized as a long isoform, termed LAP, and a short isoform, termed LIP. Whereas both forms bind to GATA-3, LAP binding results in increased GATA-3 proteolysis, whereas LIP binding results in increased GATA-3 survival. Extensive studies of IL-10 promoter activation in a luciferase reporter system indicated that optimal activation of the IL-10 promoter was achieved with a combination of LIP and GATA-3, as well as phosphorylated CREB, the latter a factor that binds to a site adjacent to the LIP-binding site. GATA-3 binds to an upstream binding site and probable mediates increased accessibility of the promoter to transactivation. Finally, c-MAF also participates in IL-10 transcription in these cells based on the luciferase reporter studies and studies of cells in which c-MAF was down-regulated by a lentivirus expressing c-MAF anti-sense oligonucleotides. Overall, these studies define the complex transcriptional regulation of IL-10 production in a unique subset of Dectin-1 induced regulatory T cells which we have termed Tr2 regulatory T cells. The relevance of these studies to fungal infection was shown in parallel studies in which C. albicans was used to stimulate BMDC rather than ZD. In these studies it was found that whereas stimulation of dendritic cells with the hyphal form of C. albicans elicits an IL-10 response in co-stimulated T cells, stimjulation of dendritic cells with the yeast form of C.albicans elicits an IL-17 response. These data thus suggest that during different phases of infection, C. albicans induces vastly different molecular programs in co-stimulated T cells and this, in turn, depends on different signaling between dendritic cells and T cells. In addition, these data suggest that C.albicans induces a regulatory response that prevents excessive inflammation during the mature phase of infection involving the hyphal form of C. albicans.
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