Adipose tissue (AT) regulatory T cells (T regs) control inflammation and metabolism. Diet-induced obesity causes hyperinsulinemia and diminishes visceral AT (VAT) T reg number and function, but whether these two phenomena were mechanistically linked was unknown. Using a T reg–specific insulin receptor (Insr) deletion model, we found that diet-induced T reg dysfunction is driven by T reg–intrinsic insulin signaling. Compared with Foxp3 cre mice, after 13 wk of high-fat diet, Foxp3 cre Insr fl/fl mice exhibited improved glucose tolerance and insulin sensitivity, effects associated with lower AT inflammation and increased numbers of ST2 + T regs in brown AT, but not VAT. Similarly, Foxp3 cre Insr fl/fl mice were protected from the metabolic effects of aging, but surprisingly had reduced VAT T regs and increased VAT inflammation compared with Foxp3 cre mice. Thus, in both diet- and aging-associated hyperinsulinemia, excessive Insr signaling in T regs leads to undesirable metabolic outcomes. Ablation of Insr signaling in T regs represents a novel approach to mitigate the detrimental effects of hyperinsulinemia on immunoregulation of metabolic syndrome.

Strong T cell receptor (TCR) signaling largely induces cell death during thymocyte development, whereas weak TCR signals induce positive selection. However, some T cell lineages require strong TCR signals for differentiation through a process termed agonist selection. The signaling relationships that underlie these three fates are unknown. RasGRP1 is a Ras activator required to transmit weak TCR signals leading to positive selection. Here, we report that, despite being dispensable for thymocyte clonal deletion, RasGRP1 is critical for agonist selection of TCRαβ + CD8αα intraepithelial lymphocyte (IEL) progenitors (IELps), even though both outcomes require strong TCR signaling. Bim deficiency rescued IELp development in RasGRP1 −/− mice, suggesting that RasGRP1 functions to promote survival during IELp generation. Additionally, expression of CD122 and the adhesion molecules α 4 β 7 and CD103 define distinct IELp subsets with differing abilities to generate TCRαβ + CD8αα IEL in vivo. These findings demonstrate that RasGRP1-dependent signaling underpins thymic selection processes induced by both weak and strong TCR signals and is differentially required for fate decisions derived from a strong TCR stimulus.

T regulatory (Tr) cells are essential for the induction of peripheral tolerance. Several types of Tr cells exist, including CD4 + T cells which express CD25 constitutively and suppress immune responses via direct cell-to-cell interactions, and type 1 T regulatory (Tr1) cells, which function via secretion of interleukin (IL)-10 and transforming growth factor (TGF)-β. The relationship between CD25 + CD4 + T cells and Tr1 cells remains unclear. Here, we demonstrate at the clonal level that Tr1 and CD25 + CD4 + T cells are two distinct subsets of regulatory cells with different cytokine production profiles. Furthermore, CD25 − CD4 + T cells can be rendered anergic by IL-10 and differentiated into Tr1 cells in the absence of CD25 + CD4 + T cells. Cloned human CD25 + CD4 + T cell populations are heterogeneous and only a subset of clones continues to express high levels of CD25 and is suppressive. The intensity of CD25, cytotoxic T lymphocyte antigen (CTLA)-4, and glucocorticoid-induced tumor necrosis factor (TNF) receptor expression correlates with the suppressive capacity of the T cell clones. None of the CD25 + CD4 + T cell clones with suppressive function produce IL-10, but all produce TGF-β. Suppression mediated by CD25 + CD4 + T cell clones is partially dependent on TGF-β, but not on constitutive high expression of CD25. Together these data indicate that naturally occurring human CD25 + CD4 + T cells are distinct from IL-10–producing Tr1 cells.

Active suppression by T regulatory (Tr) cells plays an important role in the downregulation of T cell responses to foreign and self-antigens. Mouse CD4 + Tr cells that express CD25 possess remarkable suppressive activity in vitro and in autoimmune disease models in vivo. Thus far, the existence of a similar subset of CD25 + CD4 + Tr cells in humans has not been reported. Here we show that human CD25 + CD4 + Tr cells isolated from peripheral blood failed to proliferate and displayed reduced expression of CD40 ligand (CD40L), in response to T cell receptor–mediated polyclonal activation, but strongly upregulated cytotoxic T lymphocyte–associated antigen (CTLA)-4. Human CD25 + CD4 + Tr cells also did not proliferate in response to allogeneic antigen-presenting cells, but they produced interleukin (IL)-10, transforming growth factor (TGF)-β, low levels of interferon (IFN)-γ, and no IL-4 or IL-2. Importantly, CD25 + CD4 + Tr cells strongly inhibited the proliferative responses of both naive and memory CD4 + T cells to alloantigens, but neither IL-10, TGF-β, nor CTLA-4 seemed to be directly required for their suppressive effects. CD25 + CD4 + Tr cells could be expanded in vitro in the presence of IL-2 and allogeneic feeder cells and maintained their suppressive capacities. These findings that CD25 + CD4 + Tr cells with immunosuppressive effects can be isolated from peripheral blood and expanded in vitro without loss of function represent a major advance towards the therapeutic use of these cells in T cell–mediated diseases.