Project Summary Biomaterials are studied broadly for regenerative immunotherapies. Despite the major need for such therapies in the elderly, most preclinical work is performed in young mice. Volumetric muscle loss (VML) - traumatic muscle loss that exceeds the body’s natural regenerative capacity – is one area where traditional surgical methods are insufficient and immune modulating biomaterials have shown promise in mouse models. Yet, sufficient muscle regeneration in humans remains a challenge, one which is compounded in the elderly. Understanding and addressing the efficacy gap of regeneration in the elderly is a critical unmet clinical need. The role of the adaptive immune system in extracellular matrix (ECM) scaffold-based VML repair has recently been shown. Adaptive responses are characterized by phenotype and antigen specificity. Studies by the Elisseeff Lab and others have shown T helper 2 (Th2) cells boost repair. Natural scaffolds from different source tissues and species have been shown to promote regeneration. Together, these indicate antigen specificity of T-cell responses to these materials is less significant than phenotype for regeneration. We hypothesize that enhancing Th2 responses during ECM implantation will promote enhanced regeneration regardless of antigen specificity. We have observed viral-specific T cells from past immune responses in tissue near human biomaterial implants. Using single cell RNA sequencing, we identified CD4+ and CD8+ T cells specific to cytomegalovirus (CMV), Epstein-Barr virus, and influenza. The presence of these cells suggests the ability to leverage pre-existing T cell populations to enhance ECM-mediated wound repair. We propose encapsulation of previously encountered T- cell antigens present broadly across the population – such as CMV – as a strategy to enhance ECM mediated repair. In mice, we will model pre-existing viral T cells by utilizing T-cell antigen-conjugated vaccinia virus. Further, the Elisseeff Lab has recently investigated the effects of aging and wound healing, finding that old mice have enhanced IL-17 mediated inflammation, fibrosis, and CD8+ T cell responses after ECM treatment of VML. We propose to enhance responses specifically in aged animals by combining our strategy with anti-IL-17 antibody therapy to ameliorate age-associated dysregulation of healing. Additionally, we will utilize CD4+ peptide epitopes in place of full protein antigen to specifically enhance CD4+ responses and dampen the enhanced CD8+ frequency in aged animals. We will investigate our hypotheses via two specific aims: Aim 1: Enhance ECM-mediated muscle regeneration by leveraging pre-existing T-cell populations. Aim 2: Investigate the effects of combination immunotherapy strategies in aged mice. We will use a murine quadriceps VML model and investigate immune responses with a combination of methods including multiparameter spectral flow cytometry, quantitative PCR, and histology. Additionally, we will tetramer...