The white arrows (left) denote CD5+Foxp3+pStat5+ cells, whereas the yellow arrows (right) indicate CD5+Foxp3+pStat5? cells (representative staining from 3 sections per tissue obtained from 3 animals in 1 experiment; bars, 25 m). cells in lymphoid and nonlymphoid sites is critical for developing effective strategies to manipulate T reg cell activity to promote allograft tolerance and treat autoimmunity, chronic contamination, and malignancy. Like conventional CD4+Foxp3? helper T cells, T reg cells are phenotypically and functionally heterogeneous, with unique populations associated with different tissue sites and inflammatory conditions (Campbell and Koch, 2011; Cretney et al., 2013). However, despite the considerable body of literature highlighting the specialization of T reg cell subsets, the homeostatic mechanisms that A-889425 maintain such complexity remain poorly comprehended. As a populace, T reg cells undergo quick homeostatic proliferation in vivo (Fisson et al., 2003), which is usually thought to be due to their high degree of self-reactivity and their constitutive expression of the A-889425 high-affinity IL-2 receptor component CD25, which signals through phosphorylation of the transcription factor Stat5 (Hsieh et al., 2004; Setoguchi et al., 2005). In the constant state, IL-2 is usually produced Rabbit Polyclonal to ZNF695 by activated CD4+CD25+Foxp3? T cells and is thought to take action in a paracrine fashion to link the size of the T reg cell compartment to the number of these activated T cells (Setoguchi et al., 2005), thereby ensuring that autoimmunity and inflammatory diseases do not develop as a result of uncontrolled T cell activation. The importance of IL-2 in controlling T reg cell function in the periphery is usually indicated by the lymphoproliferative disease that evolves in mice deficient for IL-2 or its receptor (Sadlack et al., 1993; Willerford et al., 1995), and IL-2 is usually thought to control T reg cell homeostasis through regulation of genes involved in cell proliferation, metabolism, and apoptosis (Fontenot et al., 2005a). However, mice deficient in either IL-2 or CD25 contain near-normal numbers of T reg cells that are functionally suppressive in vitro, indicating that the role of IL-2 in controlling T reg cell large quantity and activity is usually more complicated than currently appreciated and that the homeostasis of T reg cells A-889425 is at least partially IL-2 impartial (Fontenot et al., 2005a; Burchill et al., 2007; Soper et al., 2007). In addition to IL-2, T reg cell large quantity is tightly linked to the number and activity of antigen-presenting DCs (Darrasse-Jze et al., 2009), and the co-stimulatory receptors CD28 and inducible co-stimulator (ICOS) have been shown to influence T reg cell homeostasis in vivo (Tang et al., 2003; Burmeister et al., 2008). However, an integrated understanding of how IL-2 and TCR/co-stimulatory signals combine to control the homeostasis of different T reg cell populations in unique tissue locations is lacking. For instance, one possibility may be that IL-2 just potentiates antigen-driven proliferation/selection of highly self-reactive T reg cells in the periphery. Alternatively, IL-2 and TCR/co-stimulatory signals could drive parallel pathways of T reg cell homeostasis (Zou et al., 2012). Indeed, the presence of functionally specialized T reg cell subsets and their common tissue distribution raises the possibility that different T reg cell populations are subject to individual homeostatic constraints. In this study, we identify a fundamental subdivision in T reg cells associated with differential tissue localization and engagement of unique homeostatic pathways. Instead of acting as a panCT reg cell growth/survival factor, we found that IL-2 was uniquely required to maintain quiescent CCR7hiCD44loCD62Lhi T reg cells and that loss of IL-2 signaling was not associated with impaired T reg cell proliferation. Furthermore, we recognized the chemokine receptor CCR7 as a key factor that provides these cells access to IL-2 in secondary lymphoid tissues. In contrast, although they remain IL-2 responsive, we found that CD44hiCD62LloCCR7lo cells have reduced IL-2 signaling in vivo and that the maintenance of these cells is usually IL-2 impartial but relies on signals delivered by DCs and ICOS. Together, these data provide a new framework for understanding T reg cell homeostasis in different tissue sites that will be useful in developing and assessing strategies to therapeutically manipulate T reg cell function in a variety of immune-mediated diseases. RESULTS Central T reg (cTR) and effector T reg (eTR) cell subsets with unique homeostatic characteristics Conventional.