PROJECT SUMMARY The thymus, which is the primary site of T cell generation, is extremely sensitive to insult, but also has a remarkable capacity for endogenous repair. Even though there is likely continual thymic involution and regeneration in response to everyday insults like stress and infection, profound thymic damage caused by radiation injury leads to prolonged T cell lymphopenia. Consequently, identification of therapies that can boost T cell reconstitution in recipients after a dose of radiation is a clear priority. We have previously identified two distinct pathways of endogenous thymic regeneration, centered on the production of the regeneration factors IL-22 by innate lymphoid cells (ILCs), and BMP4 by endothelial cells (ECs); both of which mediate their regenerative effects by targeting thymic epithelial cells. More recently we have found that the trigger for these distinct regenerative pathways hinge on the balance between forms of cell death, with immunologically silent apoptosis (which is abundant in thymocytes during steady-state) suppressive to the regenerative program. On the other hand, after thymic damage caused by radiation injury, we found a switch toward immunogenic cell death, with the resulting release of damage-associated molecular patterns (DAMPs) sufficient to promote regeneration. Specifically, we identified that intracellular Zn was released after radiation injury, where it could signal through the G-protein coupled receptor 39 (GPR39) to stimulate production of BMP4 and IL-23, a key upstream regulator of IL-22 production. Separately, we also found that the release of the prototypical DAMP, ATP, was able to signal directly on thymic epithelial cells through purinergic (P2) receptors and promote their expression of Foxn1, key microenvironmental drivers of T cell development. Importantly, our preliminary data also suggests that each of these pathways can be therapeutically targeted to improve thymic recovery following radiation damage in young mice. Based on our preliminary data, we hypothesize that modulation of pathways associated with these DAMPs can be used as a countermeasure to improve immune function after radiation injury by stimulating the generation of new T cells in the thymus. Specifically, our proposal has the following aims: (1) To validate and optimize the targeting of GPR39 or P2 receptors to improve the production of new T cells and immune function after radiation injury; (2) to examine the ability of the thymus to respond to regenerative signals across mouse lifespan and sex; and (3) to comprehensively evaluate the potential for targeting GPR39 or P2 receptors to improve thymic function after radiation damage across mouse lifespan and sex. The studies outlined in this proposal not only have the potential to define important pathways underlying tissue regeneration across lifespan but could also result in innovative approaches to enhance T cell recovery after radiation damage.