Regulation of T cell Differentiation

National Institute Of Allergy And Infectious Diseases

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Abstract

In previous studies we showed that Tr2 cells 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, we found, contributes to IL-10 transcription by forming a complex with CREB1 that then 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 for how such activation was induced. Recent studies have shown that the metabolic profile of a T cell is one 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 unique to Tr2 cells, we focused on glutamate metabolism due to its known connection to the mTOR activation pathway. It is known that glutamine taken up by T cells is converted into glutamate by a process known as glutaminolysis and is then converted into alpha-ketoglutarate; the latter then either enters the TCA cycle for support of mitochondrial respiration or translocates to the 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 generated in vitro. Whereas we found no difference between these two 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 stronger 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), 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. Given the fact that Tr2 cell IL-10 production depends on mTOR signaling we explored the possibility that IL-10 production could be enhanced pharmacologically by agents that affect mTOR signally. Indeed, we found that culture of developing Tr2 cells in the presence of Celecoxib, a COX2 inhibitor greatly enhanced induction of Tr2 cells and that such enhancement was nullified by co-treatment with rapamycin. In addition, we found that Celecoxib greatly enhanced the expression of p-4EBP1, the factor that facilitates mTOR induction of LIP and correspondingly, Celecoxib enhanced the expression of LIP. In further studies of the mechanism of Celecoxib enhancement we found that only inhibition of the EP2 prostaglandin receptor among the four known prostaglandin receptors reversed enhancement and that such reversal led to major downregulation of both LIP and LAP expression; furthermore, the IL-10-enhancing activity of Celecoxib was mimicked by H-29, a PKA inhibitor, indicating that the Celecoxib effect was attributable to EP2 receptor-mediated inhibition of PKA. Finally, we determined if the enhancing effect of Celecoxib on Tr2 induction in vitro could be observed in vivo. Here, we treated mice undergoing C. albicans infection with Celecoxib and found that such treatment enhanced C.albicans toxicity and reduced mouse survival. This, negative effect on survival is best attributed to increased intra-renal generation of Tr2 cells that in this case are facilitating cytokine storm. What are 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.