Interactions between the immune system and the intestinal stem cell (ISC) compartment are poorly understood, as are the mechanisms by which ISCs respond to immune-mediated insults. In the previous R01 cycle, we made several major discoveries: 1) T cells infiltrate and directly target the ISC compartment in vivo. 2) T cells mediate Interferon-γ-dependent ISC killing in models of graft vs. host disease (GVHD) and autoimmunity. 3) Lymphocyte- derived cytokines also target ISCs to promote JAK/STAT-dependent epithelial regeneration, and this immunobiology can be translated clinically as demonstrated by our recently completed trial of Interleukin (IL)-22 treatment for patients with GVHD. 4) We have identified a new mechanism of epithelial regeneration whereby ISC-derived IL-33 induces the stem cell niche to augment epidermal growth factor (EGF) production after damage. Additionally, new unpublished data uncover the surprising finding that another EGF receptor (EGFR) ligand, Amphiregulin (Areg), which is thought to promote epithelial regeneration, can signal directly to T cells, drive their proliferation, and promote immune-mediated epithelial injury. Preliminary findings show that CD4 T cells upregulate Areg and EGFR upon activation, increasing their expansion, tissue infiltration, ISC loss, and intestinal pathology. The newly discovered IL-33-dependent epithelial regeneration pathway and our new data identifying a pathologic role for the T cell Areg/EGFR axis in tissue damage present a critical translational conflict: the IL-33/EGF regenerative circuit supports the therapeutic potential of EGF administration, but administering EGFR ligands also has the potential to drive further immune-mediated damage by stimulating T cell EGFR. The goals of this project are to investigate 1) the function of the EGFR pathway in activated T cells, 2) EGFR function within the ISC compartment in models of immune-mediated tissue damage, and 3) how these pathways can be targeted to reduce immune-mediated damage and enhance regeneration. Utilizing a combination of in vivo and ex vivo T cell activation models, as well as ex vivo modeling of tissue responses to T-cell-mediated damage using murine and human organoid culture systems, we will test the hypothesis that Areg produced by activated CD4 T cells drives autocrine EGFR signaling and T cell proliferation, leading to tissue infiltration and ISC compartment damage in the allogeneic setting. This represents a potential paradigm shift in the understanding of Areg and EGFR. We will also test the hypothesis that ISCs respond to this damage by enhancing the function of their own niche, identifying ISCs as not just the mediators but the regulators of regeneration. This study will thus mechanistically uncouple pathologic T cell EGFR signaling from regenerative epithelial EGFR signaling to enable rational design of novel therapies for protecting tissues from immune- mediated damage and promoting epithelial regeneration. In collabor...