PROJECT SUMMARY Sjögren’s disease (SjD), a systemic autoimmune disease that affects four million people in the United States and impairs health-related quality of life, has no FDA-approved disease-modifying treatments available. Current trials include only a narrow subset of SjD patients who are anti-SSA antibody positive (SSA+), excluding 25% of SjD patients who are anti-SSA antibody negative (SSA-). SSA- SjD patients have distinct clinical phenotypes from SSA+ patients. Recently, mesenchymal stromal cells (MSCs) have been implicated in SjD salivary gland pathobiology and represent a novel targetable cell type in this disease. Thus, there is a critical need to develop new targeted therapy that takes into account the heterogeneity of SSA+ and SSA- SjD patients and to further define the role of MSCs in the pathogenesis of SjD. Our long-term goal is to use SjD pathogenic insights to create new cell-targeted therapy. The goal of this project is to define the transcriptomics of MSCs within salivary gland tissue. We also will determine the lymphocyte subsets in proximity to salivary gland MSCs and determine how these subsets differ between control and SjD glands and, within SjD, between SSA+ and SSA- glands. We hypothesize that SjD has unique MSC subtypes and they will promote differential local cellular milieu from controls and between SSA+ and SSA- subjects. The rationale for this hypothesis is based on our data showing IFN𝛾-treated MSCs are capable of acting as antigen-presenting cells and promoting T-cell chemotaxis. The central hypothesis will be tested by pursuing two specific Aims. Aim 1 will provide an integral understanding of the SjD transcriptional phenotype of a novel cell type, the MSC, compared to controls. Aim 2 will define how the cellular molecular framework of MSCs and overall inflammatory cell composition differs between SSA+ and SSA- subjects. To accomplish these Aims, FISH combined with Hybridization Chain Reaction based signal amplification will be used to perform spatial genomics and rapid multiplexed immunofluorescence protein profiling of salivary gland tissue. MSC subtypes, lymphocyte interactions with MSCs, and differential lymphocyte composition between control, SSA+ and SSA- subjects will be determined using a series of spatially resolved cell state and interaction analyses. This project builds an interdisciplinary team integrating experts from single cell biotechnology, SjD and rheumatology, stem cell biology, pathology, and bioinformatics to create an atlas of MSC phenotypes that can be used to enhance SjD’s stem cell therapies. The proposed application is innovative because it uses cutting-edge technology to define spatial genomics and proteomics of salivary gland tissue and shifts from the traditional focus on SSA+ subjects alone, toward differences between SSA+ and SSA- salivary glands. This research is significant because it defines SjD MSCs, a potential novel therapeutic target, and because it details spatial profil...