PROJECT SUMMARY Innate lymphoid cells (ILCs) include conventional natural killer (cNK) cells and a broad spectrum of cells that belong to three groups based on their development and production of signature cytokines. ILC1s secrete IFN-γ, ILC2s produce IL-5 and IL-13, and ILC3s secrete IL-22 and IL-17. In this proposal, we identify a novel tissue-resident ILC subset in the salivary gland (SG) that deviates functionally from other ILCs and cNK cells and may have a regulatory function in protecting the SG from inappropriately activated T and B cells that cause inflammation and tissue damage. Thus, this proposal is highly significant for Sjögren's syndrome (SS), an autoimmune disease typified by chronic inflammation and tissue destruction of the SG; the identification of an ILC subset with potential regulatory function in the SG may help identify new therapeutic avenues for SS. Our preliminary data show that SG ILCs express markers typical of tissue residency - CD103, CD49a and CD69 - and do not rely on transcription factors that are necessary for the development of cNKs and other ILCs, including Nfil3, T-bet and Eomes. Importantly, SG ILCs do not produce IFN-γ, but rather express the cell death molecule TRAIL, which endows SG ILCs with the ability to kill activated T cells that express the receptor for TRAIL. We demonstrate that these features are due to ILC exposure to TGFβ, which is abundant in the SG. In Aim 1 we will establish when and for how long SG ILCs require TGFβ to develop. We hypothesize that SG ILCs originate from hematopoietic progenitors that seed the SG, proliferate, and acquire irreversible tissue resident/regulatory features after the SG has completed development. To test this, we will examine reconstitution of SG ILCs after transfer of congenically marked ILC progenitors into lymphopenic mice. We will also track SG ILC progenitors at various stages of hematopoiesis in inducible reporter mice as well as in parabiotic pairs and define the timing of TGFβ action in mice in which TGFβ signaling can be inducibly ablated. In Aim 2 we will identify the signals required for SG ILC development downstream of TGFβ. We will test the hypothesis that TGFβ drives progenitor cells towards ILCs rather than cNKs by suppressing Eomes. As TGFβ signals via SMAD-dependent and -independent paths, we will ascertain which route suppresses Eomes. In Aim3 we will assess the regulatory capacity of SG ILCs. Our preliminary data indicate that depletion of SG ILCs augments T and B cell infiltration in the mouse model of submandibular duct ligation. We will determine whether SG ILCs regulate immune responses via TRAIL-TRAIL-R interactions. Because SG ILCs express the IL-21 receptor (IL-21R), we will ask whether IL-21R signaling enables additional regulatory functions, such as the release of granzyme B and IL-10. Moreover, we will examine the potential regulatory function of SG ILCs in NOD.H-2h4 mice, which contain SG ILCs and are relevant to SS. We have also...