Regulation of T cell Differentiation

National Institute Of Allergy And Infectious Diseases

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Abstract

Project 1: In previous studies we showed that Tr2 cells, regulatory T cells that are demonstrably distinct from Tr1 cells or Foxp3 cells(Tregs), are induced by dendritic cells (DCs) stimulated by zymogen-depleted yeast extracts (ZD) and by the hyphal form of C. albicans, both of which express 1,3-beta glucan, the ligand of Dectin-1. The T cells so stimulated undergo two interlocking molecular processes that together result in Tr2 cells. The first involves activation of GATA3, a factor that binds to the IL-10 promoter at two sites, i.e., at a distal site where it acts as a direct transcription factor and at the proximal site where it acts indirectly on transcription as a epigenetic factor that augments histone acetylation. The second involves activation of the TORC1 arm of the mTOR signaling pathway and the resulting generation of a particular C/EBP-beta isoform known as LIP. The latter contributes to IL-10 transcription by forming a complex with CREB1 that binds to adjacent sites in the IL-10 promoter. Whereas the above findings established that mTOR (TORC1)activation was central to the induction of IL-10 synthesis in Tr2 cells, further explanation was necessary to elucidate how such activation was induced. Recent studies have shown that the metabolic profile of a T cell is a key determinant of its subset differentiation pathway. Accordingly, we performed metabolome studies to identify a metabolic profile that might be unique to Tr2 cells and influence its differentiation. Such studies revealed that Tr2 cells were clearly different from Th2 and Th0 cells according to partial least-squares discriminant analysis (PLS-DS) and showed a distinct metabolic signature . Among the various metabolic pathways expressed in Tr2 cells, we focused on glutamate metabolism due to its known connection to the mTOR activation pathway. It has been shown that glutamine taken up by T cells is subject to a process known as glutaminolysis in which the glutamine is concerted into glutamate and then into alpha-ketoglutarate; the latter then either enters the TCA cycle for support of mitochondrial respiration or translocates to a lysosome to induce mTOR activity. On this basis, we first measured the total glutamine consumption as well as the glutaminolysis-linked oxygen consumption rate (OCR) and ATP production of Tr2 and Th2 cells previously generated in vitro. Whereas we found no difference between these two T cell subsets with respect to total consumption, Tr2 cells exhibited a significantly lower level of OCR and ATP production than Th2 cells. This suggested that in Tr2 cells glutaminolysis-generated alpha-KG is utilized for a process other than mitochondrial respiration, most likely for the activation of the mTOR pathway. We next determined the mTOR activity (as evaluated by the phosphorylation status of S6, a downstream signaling component of mTOR) of comparable Th2 and Tr2 cell populations and found that Tr2 cells exhibited greater mTOR activity than Th2 cells by this criterion. In addition, Tr2 cells exhibited greater ablation of IL-10 production than Th2 cells when exposed to a glutaminase inhibitor, 6-Diazo-5-oxo-L-norleucine (DON), and the latter also caused specific inhibition of C/EBPbeta-LIP expression in Tr2 cells. Finally, we found that cell-permeable dimethyl alpha-KG (DMK) induced IL-10 production in Tr2 cells previously subjected to prior glutamine-deprivation. Taken together, these data strongly suggested that the mTOR pathway is activated in Tr2 cells by glutaminolysis and its downstream induction of alpha-KG. Furthermore, given the centrality of glutaminolysis to Tr2 T cell development it seems likely that the soluble factor produced by dendritic cells that induces Tr2 cells is a factor that induces glutaminolysis. A key question concerning the generation of Tr2 cells was the identity of the factor produced by Dectin-1-stimulated DCs that induces these cells. To address this question we first conducted RNAseq studies in which we determined differentially expressed genes (DEGS) in cells stimulated with a Dectin-1 ligand vs. unstimulated cells or cells stimulated with C. albicans yeast vs. cells stimulated with C. albicans hyphae, both conditions in which Tr2 cells are differentially induced. We found that under both conditions the substance exhibiting the highest DEG was inhibin beta-A, a component of either activin-A or inhibin-A. In subsequent studies we showed that inhibin-A is highly produced by DCs stimulated by Dectin-1 under Tr2 T cell generating conditions and that Tr2 T cell induction is virtually completely blocked by an activin-A (or inhibin-A receptor ALK4/5 inhibitor, vactosertib. Finally, we found that Tr2cell induction is not blocked by follistatin, a specific activin A inhibitor, strongly suggesting that DC induction of Tr2 cells is uniquely induced by inhibin-A itself. In further studies we explored the possible clinical functions of Tr2 cells. Inasmuch as Tr2 cell induction requires IL-4 production by the nascent Tr2 cell itself we reasoned that the development and function of these cells are best tested in the context of a Th2-driven inflammation such as asthma. Accordingly, we conducted studies of Tr2 cell regulation of experimental asthma induced by house-dust mite antigen (HDM). In a first study we found that repeated ZD administration (IP) during asthma induction by IP and IN HDM administration gives rise to a dramatic reduction in total BAL cells, BAL eosinophils and CD4-positive cells; in addition, total IgE and HDM-specific IgE in the circulation are dramatically reduced as is histologic evidence of pulmonary inflammation. In a second study, we administered Tr2 cells generated in vitro (IP) to mice at time of asthma initiation by IN HDM administration and again found that such administration led to great decreases in the various parameters of asthmatic inflammation noted above. These studies thus showed that Tr2 can be induced by ZD during a Th2-driven inflammation such as asthma and may therefore have efficacy in treating asthma. Project 2: In previous studies we showed that experimental AIP that closely resembles human AIP can be induced in MRL/MpJ mice by repeated injections of polyinosinic-polycytidylic acid. Furthermore, we that AIP development in this model is accompanied by and is dependent on the generation of type I IFN (e.g. IFN-alpha) and IL-33 by plasmacytoid dendritic cells (pDCs). In current studies we explored the possibility that AHR activation prevented the devrelopment of experimental AIP and, if so, how. We found that activation of the AHR by indole-3-pyruvic acid or indigo naturalis, which were both administered p.o. as a dietary supplement did indeed inhibit the development of experimental AIP, and, furthermore, this therapeutic effect was independent of the activation of plasmacytoid dendritic cells producing IFN-alpha and IL-33. Additional studies revealed that interaction of indole-3-pyruvic acid and indigo naturalis with AHRs robustly augmented the production of IL-22 by pancreatic islet alpha cells and that blockade of IL-22 signaling pathways completely canceled the beneficial effects of AHR ligands on experimental AIP. This observation applied to human AIP because serum IL-22 concentrations were found to be elevated in patients with AIP after the induction of remission with prednisolone treatment. These data suggest that AHR activation suppresses the fibroinflammatory reactions that characterize AIP via induction of IL-22 produced by pancreatic islet alpha cells.