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. Accordingly, we performed metabolomic studies to identify a metabolic profile that might be unique to Tr2 cells and influence its mTOR-mediated differentiation. Such studies revealed that metabolically, Tr2 cells were clearly different from Th2 and Th0 cells 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 glutamine is converted 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. However, in subsequent studies we showed that whereas activin-A is produced by DCs stimulated by Dectin-1 under Tr2 T cell generating conditions its activity is not blocked by follistatin, a specific activin A inhibitor nor by anti-activin. In addition, Tr2 T cell generation is augmented by addition of inhibit beta-A. Finally, Tr2 T cell generation was completely and repeatedly blocked by vactosertib, a specific inhibitor of both ALK5 and ALK4, components of the TGF-b and activin-A receptors respectively. We therefore concluded that Tr2 cells are induced by cell signaling by inhibit beta-A linked to a co-factor that enables interaction with ALK4 component of the activin receptor. In this case, inhibit beta-A has a unique positive, rather than a negative interaction with the latter receptor. In further studies we explored the possible clinical functions of Tr2 cells. Inasmuch as Tr2 cell induction requires IL-4 production we reasoned that the 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 murine experimental AIP model that closely resembles human AIP can be induced in MRL/MpJ mice by repeated injections of polyinosinic-polycytidylic acid. Furthermore, we showed 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). origin of the major pathogenic cells, pDCs and how these cells interact with effector T cells causing disease. In the present studies we explored how pDCs and effector T cells develop in the nascent MRL/MpJ AIP model. We found first that pancreatic TLR3+ conventional DCs (cDCs) are the poly(I:C)-responsive cells that initiate murine AIP. Moreover, we found that these cells, via secretion of C-X-C motif chemokine ligand 9 and 10 (CXCL9 and CXCL10), attract C-X-C motif chemokine receptor 3+ (CXCR3+) T cells into the pancreas and, under the influence of IFN- secreted at this site, the latter cells produce C-C motif chemokine ligand 25 (CCL25) that attracts disease-sustaining pDCs expressing C-C chemokine receptor 9 (CCR9) into the pancreas. This train of events sets up a positive chemokine and cytokine cascade or feedback loop wherein pancreatic pDCs, upon stimulation with TLR9 ligands, become the source of the IFN- that induces further rounds of migration of CXCR3+ T cells and pDCs into the pancreas. We also found that CXCR3+ T cell interaction with pDCs is critical for high level IFN- production by pancreatic pDCs so that these T cells have the dual roles of both attracting pDCs into the pancreas and of stimulating pDCs that have arrived at this site. Inasmuch as the cytokine and chemokine components of the AIP-generating feedback loop found in the murine model are elevated in the serum of AIP/IgG4-RD patients, these conclusion apply to human AIP/IgG4