Project Summary Innate lymphoid cells (ILC) are new important players in mounting innate immune responses to various pathogens as well as in the development of diverse immunological diseases. For example, increased levels of group 2 ILCs (ILC2s) has been linked to asthma and allergic illnesses in the respiratory system and skin. How ILCs are formed is just beginning to be understood. Current dogma states that ILCs are derived from common lymphoid progenitors (CLP) in the bone marrow. Several subsets of ILC progenitors that give rise to all ILC types without producing B or T lymphoid cells have been identified and thought to lie downstream of CLP. However, it is not known if ILCs can also originate from other multipotent progenitors and at different locations besides the bone marrow. We have obtained compelling evidence which allows us to propose that the thymus is capable of producing ILC2s, and preferentially supply them to the lung. In this proposal, we will devote Aim1 to further characterizing this phenomenon. We will first determine if ILC2 counts in the lung decrease as thymic output declines with age. We will then compare lung ILC2 levels in mice with or without thymus (nu/+ vs. nu/nu). We will next obtain gene expression profiles of ILC2 cells in the bone marrow and thymus, as well as lung ILC2s derived from the bone marrow and thymus. Finally, we will identify the progenitors in the thymus that are capable of generating ILC2s. We have also garnered strong evidence that down-regulating E protein activities promotes ILC2 differentiation in the thymus. This is consistent with the essential role of Id2, the inhibitor of E proteins in ILC differentiation. Since the underlying mechanisms by which Id2 controls ILC differentiation is unknown, we will designate Aim2 for probing the impact of altered E protein activities on ILC2 differentiation from multipotent progenitors of the bone marrow and thymus, as well as on the proliferation and survival of ILC2s. We are also interested in testing the idea that Id2 down-regulates E proteins to enhance IL-2/IL-7 and CD28 signaling, which are important for ILC2 proliferation and survival. We will determine if E protein inhibition could alleviate the need for IL-2 in ILC2 differentiation in vitro and rescue ILC2 development in IL-7-/-, IL-2Rγc-/- and Icos-/- mice. Finally, we will augment E protein activities in ILC2 cells to search for target genes that are responsible for suppressing the innate lymphoid fate and promoting the adaptive lymphoid fate. In summary, we believe our studies will lead to paradigm-shifting discoveries that will enrich our knowledge about the ontogeny of ILC2 cells and provide insight into diseases such as childhood asthma, in which thymus-derived ILC2s likely play a causative role.