Abstract Foxp3+ regulatory T (TR) play a requisite role in peripheral immunological tolerance and in restraining exuberant immune responses. Elucidating mechanisms that control TR cell responses is of pivotal importance to the restoration of tolerance in diseases of chronic inflammation and autoimmunity. Relevant to this goal is our recent determination of a critical role for the Notch pathway in regulating the peripheral TR cell compartment. TR cell-specific deletion of Notch1 antagonized T helper (Th)1 responses and protected against graft versus host disease, while TR cell-specific constitutive expression of the intracellular domain of Notch1 led to lymphoproliferation, Th1 cell dysregulation and autoimmunity. We have also demonstrated that TR cell- specific loss of function Notch pathway mutations that incapacitate signaling via all Notch receptors (Pofut1 deletion) or canonical pathway signaling (Rbpj deletion) but not deletion of Notch1 or Notch2, protected against allergic airway inflammation, indicating that individual Notch receptors on TR cells mediated regulation of distinct Th cell programs. Some of the effects of Notch signaling in TR cells involved transcriptional activation via the canonical Notch pathway co-factor RBPJ. Yet others appeared to involve activation of the non- canonical mechanisms. Accordingly, We hypothesize that Notch signaling fulfills an important physiological role in negatively regulating TR cells to ensure optimal immune homeostasis. Excessive activation of this pathway would result in TR cell dysfunction, leading to immune dysregulation, inflammation and autoimmunity. We further hypothesize that individual Notch receptors on TR cells fulfill non-redundant roles in controlling Th cell responses, with Notch1 regulating Th1 and Notch4 Th2 cell responses, respectively. We also hypothesize that the capacity of individual Notch receptors on TR cells to mediate distinct immune regulatory outcomes is reflected by hierarchal Notch receptor-ligand interactions and by distinct transcriptional circuitries activated by the respective Notch receptors. We propose under Aim 1 to elucidate mechanisms by which sustained Notch1 signaling in TR cells promotes dysregulated Th1 cell responses by enabling complete Th1 cell reprogramming of TR cells, and the role of T follicular regulatory (TFR) cell subversion by Notch1 signaling in inciting intense humoral autoimmunity. Under Aim 2, we will examine mechanisms by which Notch signaling in TR cells, most notably that by Notch4 (and possibly Notch3), regulates Th2 cell responses in allergic airway inflammation, while Aim 3 will explore mechanisms by which signaling via different Notch receptors in TR cells leads to distinct functional outcomes, including ligand specificity of the respective receptor expressed on TR cells and the nature of the downstream transcriptional programs activated upon Notch1 versus Notch4 signaling. The proposed studies will identify novel mechanisms by which N...