PROJECT SUMMARY/ABSTRACT The long-term goal of this work is to facilitate the faithful regeneration of damaged human tissues. Regeneration in adult mammals is extremely limited; damaged tissue in most major organs fails to regenerate, and instead undergoes scar-based repair. The lack of adult regenerative capacity is an enormous burden on the healthcare system and society as a whole. Although both human and mouse digit tips can undergo a true regenerative response, this regeneration is positionally restricted to the terminal distal phalanx bone. Importantly, amputations with an axis point below the terminal distal phalanx bone or too close to the nail bed result in regeneration failure. Notably, salamander limbs have an anatomy similar to that of human limbs, but uniquely regenerate after amputation from any position throughout adult life. The biological mechanisms limiting regeneration in adult mammals is poorly understood. Although the immune system is a powerful regulator of wound repair, the exact role of immune-cell networks as a determinant of regenerative success has been grossly understudied. In our regeneration studies, including those proposed here, we use the mouse digit-tip model, examining regeneration following tissue removal at different digit locations. This is a powerful model, as regeneration can be measured non-invasively with high-resolution micro-computed tomography 3D-imaging (bone/soft tissue volume), and analyzed comprehensively using histology and molecular analysis. We identified several lymphoid immune-cell types that inhibit mouse digit-tip regeneration via cytotoxic activity against progenitor cells and showed that T- regulatory cells (Tregs) play a critical role in protecting progenitors from these cells. We also found that in mice lacking lymphoid immunity, novel regeneration is induced, providing new models to identify pro-regenerative cells and molecular pathways that can be exploited therapeutically. Importantly, we also identified several lymphoid-cell types that support regeneration, suggesting the potential to therapeutically enhance human repair through targeted immunomodulation. This project aims to identify and characterize the mechanisms by which lymphoid cells regulate adult regeneration. Specifically, we will: Aim 1: Dissect and characterize lymphoid-cell mechanisms inhibiting regeneration. We will use a range of mouse strains with mutations in cytotoxic function in ex vivo and in vivo analyses. Aim 2: Define mechanisms of pro-regenerative Treg suppression of lymphoid-cell cytotoxicity using Treg-specific deletion of functional genes in vivo. Aim 3: Test the hypothesis that targeted disarming of lymphoid cells could enhance regeneration in vivo. We will test tolerogenic molecules in ex vivo cytotoxicity assays and then evaluate tolerogenic antigen overexpression in vivo both direct transgenic and viral approaches and then via a modified Treg delivery strategy. This project will fuse developmental biology and immunological methods to identify the critical biological pathways and genetic modifiers required for transient immunomodulation strategies directed at inducing latent regenerative potential in adult tissues in mammals. This work will lay the groundwork for translation studies aimed at enhancing tissue repair in human patients.

PROJECT NARRATIVE/PUBLIC HEALTH RELEVANCE STATEMENT Humans and mice have very limited potential for scar-free repair or regeneration of adult tissues. Recent work in mice has identified components of the immune system as negative regulators of regeneration that can restrict adult regeneration. This project will identify the critical biological pathways required to develop new strategies to transiently modify components of the immune system with the goal of inducing the latent regenerative potential in mammals and thereby significantly improving human health.