Dysregulation of normal tissue repair processes can lead to fibrosis, a pathological feature of many diseases, including chronic inflammatory conditions, autoimmune diseases, and cancer. Excessive accumulation of extracellular matrix can occur in all tissues, and if progressive, can be fatal. The long-term goal of our group is to understand the underlying cellular and molecular interactions that contribute to fibrosis, and to use this insight to develop effective therapeutics to combat this condition, which accounts for up to 45% of all deaths in the United States each year. Although long assumed to be irreversible, recent evidence from both preclinical studies and clinical trials demonstrates that fibrosis can be halted and even reversed in vivo. However, there is a significant gap in the development of safe and effective therapeutic interventions that directly target the mediators of fibrotic pathogenesis. To address this gap, we have assembled an interdisciplinary team with distinct expertise to develop and assess the in vivo efficacy of a novel cellular immunotherapy to combat fibrosis. Fibrosis drives pathology in the chronic autoimmune disease systemic sclerosis (SSc). SSc has the highest case fatality rate of any systemic autoimmune disease with no validated biomarkers or curative treatments. Multi- tissue bioinformatic analyses implicate alternatively activated macrophages (MØs) as key drivers of SSc in multiple end-target organs, suggesting these cells are a common feature across organs and subsets in SSc patients. Thus, we hypothesize that targeting these pathogenic MØs directly will reduce fibrosis in SSc patients. To test this hypothesis, we will engineer chimeric antigen receptor (CAR) T cells to secrete anti-fibrotic mediators and evaluate their therapeutic efficacy in vivo using multi-omic and spatial transcriptomic technologies developed by the parent award. The development of this therapy, which will target pro-fibrotic MØs that drive fibrosis and key secreted mediators of fibroblast activation, has potentially significant therapeutic benefit for patients that suffer from many fibrotic conditions, including cancer. This proposal integrates the diverse expertise of each co- Project Leader, with Dr. Pioli contributing MØ, fibrosis and SSc expertise, Dr. Huang contributing CAR T cell therapy engineering expertise, and Dr. Kolling bringing expertise in the development and application of single cell multi-omics and spatial transcriptomics approaches.

Project Narrative Our work and that of others implicate a type of white blood cell, macrophages, in the activation and maintenance of fibrosis, a pathological condition that accounts for up to 45% of all deaths annually in the United States. The studies we propose will target macrophages using a novel cellular therapeutic approach that has been used with success in the treatment of cancer, in which we will engineer T cells to attack macrophages directly and secrete anti-fibrotic molecules. We will use multiomic and spatial transcriptomic approaches to investigate the pathological relationship between macrophages, fibroblasts, and other cells that contribute to fibrosis, and will determine how these interactions are altered by our novel therapeutic intervention.