PROJECT SUMMARY/ABSTRACT This proposal for an NRSA Individual Fellowship is centered on two principal goals: 1) afford the candidate the necessary time and resources required to develop into an independent physician-scientist, and 2) investigate the mechanisms governing the stability of induced regulatory T cells (iTregs) in experimental influenza viral pneumonia. The candidate and his mentors have outlined a comprehensive and progressive research plan to achieve these objectives while laying the foundation for a successful, independent research career. Despite decades of clinical experience and research, severe viral pneumonia and ARDS remain a leading cause of morbidity and mortality worldwide. Regulatory T-cells are a subset of CD4+ T-cells critical to maintaining immune homeostasis and coordinating lung tissue repair after injury. Tregs require stable expression of the Foxp3 transcription factor. Stable, long-lived FoxP3+ Tregs that originate from the thymus are referred to as natural Tregs (nTregs). In vitro, TGF-β induces transient Foxp3 expression and imparts temporary suppressive function to naïve CD4+ T-cells, generating cells defined as iTregs. The inherent instability of iTreg phenotype and function poses a concern for their clinical use as a cellular therapy, as reversion to a CD4+ effector T-cell phenotype promotes inflammation. The stability of Treg-specific transcriptional programs is known to be regulated by DNA methylation, a process mediated by DNA methyltransferases and their adapter protein, UHRF1. iTregs generated from UHRF1-null naïve CD4+ T-cells possess enhanced suppressive function. Hence, we hypothesize that UHRF1 expression in iTregs destabilizes acquired suppressive and reparative transcriptional programs, leading to loss of pro-recovery function following viral pneumonia. The long- term hope of this proposal is to identify determinants of maintenance of transcriptional and functional stability in iTregs and provide validation for their use as cellular therapy in patients suffering from viral pneumonia-induced ARDS. In Specific Aim 1, we will determine whether UHRF1 is necessary to destabilize iTreg suppressive and reparative transcriptional programs both in vitro and in vivo via tandem RNA-seq and DNA methylation analysis. In Specific Aim 2, we will determine whether loss of UHRF1 in iTregs is sufficient to promote recovery following viral pneumonia. We will use cutting-edge techniques for adoptive cell transfer, severity assessment of lung injury, tamoxifen-based inducible systems in mice, flow cytometry, transcriptional profiling with RNA-sequencing, and DNA methylation profiling with modified reduced representation bisulfite sequencing as the primary methods to support the experimental design of this proposal.