Different cells of the innate and adaptive immune system cooperate to achieve an equilibrium of immune responses that maintains tolerance to self-antigens (Ags), nutrients and commensal bacteria, but clears foreign Ags and eliminates tumor cells. T lymphocytes and their innate counterparts, the innate lymphoid cell (ILCs) are tightly regulated by a coordinate program of transcription factors. Moreover, tissue microenvironments contribute to the outcome of local immune responses such that immunopathology is avoided. Group 3 ILC3s are abundant in Ag entry sites like intestine, secondary lymphoid organs (SLOs) and are also found in postnatally developing tertiary lymphoid organs (TLOs). They act as first-line responders to tissue injury, infection and inflammation. Small intestinal (SI) ILC3s prevent T cell responses to commensal Ags, whereas splenic (SP) ILC3s promote T cell responses to foreign Ags emphasizing their tissue-specific properties. How SP and SI ILC subsets and T cells collaborate during inflammation and infection is not understood. A detailed analysis of SP and SI ILC subsets under homeostatic and inflammatory conditions will help to understand the mutual interaction of lymphoid tissues, ILC3s and T cells that are required for immune homeostasis in tissues. Here we propose a system-wide approach to investigate transcriptional landscapes of SP and SI-specific ILC3 subsets in mice under homeostatic and inflammatory conditions and in mice with disturbed T cell compartments. In particular, we seek to identify transcripts in ILC3s, which are tissue-specific or depend on interactions with CD4+ T cells. Moreover, we want to identify key pathways in ILC3s, which regulate their immune-regulatory function for CD4+ T cells. Finally, we want to elucidate how TLOs modulate adaptive immune responses. Working hypothesis: This research project is based on the hypothesis that the collaboration of ILC3 subsets with CD4+ T cells differentially regulates innate inflammation and adaptive immune responses in a tissue-dependent manner.Specific Aims: Here we propose a novel project aimed at (1) dissecting the molecular signatures of ILC3s, (2) characterizing the mutual interactions of ILC3s and CD4+ T cells and their impact for innate and adaptive immunity in vivo and (3) generating an inducible IL-7 tg mouse to test the effect of TLOs on ILC3s and adaptive immune cells.Experimental Design and Methods: Advances in single cell analysis, gene targeting and generation of inducible mouse models together with state of the art bioinformatics will be used to address the specific aims. We will map ILC3 signature in SP and SI of normal and CD4+ T cell deficient mice and in mouse model with acute intestinal or splenic inflammation. The function of tissue-specific ILC3 genes will be tested in loss of function models using CRISPR-Cas9-mediated deletion in hematopoietic stem cells (HSCs) and by generating ROR?Cre-lox recombination mouse models. Using various Ags we will test the capacity of ILC3 subsets to regulate T cell responses in vivo and in vitro. Finally will study whether the induction of TLOs by ILC3s has an impact on adaptive immune cell homeostasis. Expected Value of the proposed project: Data obtained from this study will help to identify target genes and pathways that control the induction or suppression of T cell responses. These targets are laying the foundation for the development of new therapeutic strategies to prevent chronic inflammation and immune stimulation and to promote protective immune responses against pathogens as well as tolerance against commensal bacteria.