Three-day neonatal thymectomy was performed as described (34). maintain immune homeostasis by limiting T-cell responses to self-, environmental-, and pathogen-associated antigens and by modulating innate immune responsiveness. The critical role of Treg in preventing autoimmunity is highlighted when Treg are developmentally absent or depleted (1C3). In vivo, the modulatory effects of Treg on adaptive immunity are likely mediated through suppression of both CD4+ and CD8+ T cells. However, although Treg suppression of CD4+ T-cell responses has been widely studied both in vitro and in vivo, modulation of CD8+ T-cell responses by Treg is less well understood. Depleting Treg promotes CD8+ T-cellCdependent responses, such as virus and tumor clearance (4, 5). In many of these studies, however, whether Treg depletion affects primarily the BST2 afferent phase of CD8+ T cell expansion and effector differentiation or the later efferent phases has been largely undistinguished. In CD4+ T-cellCdependent autoimmune responses, Treg limit both T cell priming in lymph node (LN) (6) and effector activity at sites of inflammation (7). For CD8+ T cells, it has been elegantly shown in a tumor setting that Treg directly inhibit CD8+ T cell-mediated cytolysis through, for example, TGF-Cdependent inhibition of degranulation (8, 9) without modulating effector differentiation. However, in settings that lead Vitamin E Acetate to strong priming, such as vaccination, Treg can restrain CD8+ T Vitamin E Acetate cell expansion (10). Such disparate observations could reflect differences between T cell activation occurring under either weak or strong innate immune cell or antigen-presenting cell activation, respectively. It is, however, also possible that Treg function is enhanced by T cells undergoing effector differentiation in response to strongly immunogenic T cell priming, facilitating Treg control under these conditions. Mechanisms of CD4+ T cell Vitamin E Acetate inhibition by Treg in vitro have been well described (reviewed in refs. 11 and 12) but in vivo mechanisms of CD8+ T cell suppression are poorly understood. Consumption of IL-2 is often proposed as a mechanism of Treg suppression. Evidence is strong that CD4+ T-cell effector function is inhibited in vitro by this mechanism (13, 14), but direct experimental evidence for this mechanism in vivo is lacking. Existing evidence is correlative, which most likely reflects the technical challenge of blocking IL-2 activity without disturbing T-cell developmental homeostasis and Vitamin E Acetate studies have relied on knockout or immunodeficient mice in which homeostasis of T cells and cytokines is highly perturbed. Recent studies have shown that both Treg and CD8+ T cells are exquisitely sensitive to modulation of IL-2 homoeostasis in vivo (15C17). Emerging evidence implicates alterations in IL-2 homeostasis in development of a range of autoinflammatory responses (18C20). Given the importance of CD8 T-cell responses in tumor clearance and autoimmunity, we investigated the interrelationship of IL-2, Treg, and CD8+ T-cell effector differentiation. We demonstrate, in vivo, regulation of CD8+ effector differentiation by Treg-mediated modulation of IL-2 bioavailability. Results Regulatory T Cells Restrain CD8+ T-Cell Expansion and Inhibit Effector Differentiation. To define the influence of Treg on CD8+ T cells, ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic CD8+ T cells (OT-I) were transferred and recipient mice immunized with or without Treg depletion. Immunization with OVA/QuilA led to rapid expansion of OT-I T cells in the draining (inguinal) LN, which was followed by accumulation of differentiated effectors in spleen (Fig. 1 and and and and and and and 0.05, ** 0.01, *** 0.001. Treg modulate immune responses to self-antigens and, in so doing, prevent Vitamin E Acetate autoimmune disease (2, 3). Current paradigms suggest steady-state dendritic cell (DC) tolerise T cells, but loss of Treg converts.