Chronic allograft rejection remains a major obstacle to long-term success of solid organ transplantation despite improvements in immunosuppression and advances in costimulation blockade. Following immune injury, vascular smooth muscle cells (VSMCs) switch from a differentiated contractile to a synthetic phenotype and proliferate, resulting in intimal expansion and vascular compromise, often causing ischemic organ failure. Allograft-specific regulatory T cells (Treg) may inhibit immune cells without immunosuppression but their direct effects on the pathologic VSMC phenotype switch have been insufficiently studied. Moreover, Treg heterogeneity and plasticity associated with complex post-transcriptional regulation of gene expression by microRNAs represent challenges for cellular therapies requiring expansion of Treg ex vivo. We hypothesize that mRNA- specific enhancement of translation can skew the secretome of the alloantigen-specific Tregs toward promoting VSMC homeostasis. This exploratory, developmental (R21) project will design and evaluate a novel approach to promoting vascular smooth muscle cell homeostasis in the context of immune responses to vascular allografts through modulation of the Treg secretome. In Aim 1, we will identify Treg-enriched secreted products promoting VSMC homeostasis. We will expand alloantigen-specific Treg and identify clones that secret factors with favorable effects on VSMC phenotype. Using two complementary secretome analyses (multianalyte immunoassays from Isoplexis and pulsed SILAC followed by LC/MS-MS), we will select 2-3 Treg-enriched factors for modulation. In Aim 2, we will design and evaluate locked nucleic acid (LNA)-modified mRNA target site blockers (TSB) that specifically relieve miRNA-mediated translational repression and enhance production of IL-10, as our model candidate, and newly identified Treg-enriched targets, to promote VSMC homeostasis. The effects of the TSB on Treg and VSMC phenotype will be evaluated in vitro, followed by validation in 2 in vivo models: (1) factor-enhancing effect on secondary lymphoid organ antigen-activated Treg, and (2) vascular- stabilizing effect on minor antigen (HY) mismatch aortic allografts. The results of this exploratory study will newly reveal specific regulatory networks of Treg-VSMC interactions and provide a proof of concept for a novel class of therapeutics directed at improving long-term vascularized allograft outcomes.