A central challenge in autoimmunity is to discover the cells and pathways that drive pathological immune responses in humans. Previous studies of human samples were limited by low-dimensional single cell methods or high-dimensional bulk methods confounded by cell mixtures. By sequencing RNA of thousands of single cells from lupus nephritis kidneys, we found 21 unique immune cell states, many of which were also found in synovial tissue of RA patients. In Project 2, we will focus on 3 monocyte states that we observe in lupus nephritis kidneys, which likely arise from patrolling blood monocytes that enter the kidney. While these monocytes share expressed genes with the previously described M1/M2 spectrum of monocyte states, they express distinct functional modules and do not map directly to those states. To better understand these disease-associated monocyte states, we will perform experiments to address three hypotheses. First, we hypothesize that monocyte inflammation, phagocytosis, and tissue repair programs in lupus nephritis kidneys are regulated by fibroblasts and tissue-derived factors. With Project 3, we will co-culture primary monocytes with activated/inflammatory fibroblasts together with known local pathogenic factors and determine changes in cellular functions, including phagocytosis, cytokine secretion, endothelial extravasation, T/B cell activation (with Project 1) and other functions. Preliminary data show that fibroblasts and a proposed pathogenic factor, necrotic cells, strongly induce monocyte differentiation. Second, we hypothesize that TFs expressed in lupus monocytes will induce differentiation and expression of disease-associated gene inflammation and tissue repair programs. We will overexpress candidate TFs (based on their expression and genetic association with lupus) in monocytes and assess inflammation, phagocytosis, and differentiation. Preliminary studies show that the TF overexpression is feasible, impacts differentiation, and if successful, would allow reliable in vitro generation of differentiated monocytes for functional studies. Third, to address monocyte roles in lupus nephritis kidneys, we hypothesize that the proximity of monocytes, fibroblasts and tissue lesions, along with expression of fibroblast-induced monocyte gene programs, will reflect cell-cell interactions and functions of monocytes in patient kidneys. Using automated staining, microscopy and image analysis (with the Computational Systems Immunology Core) of kidney sections from up to 205 clinically-annotated lupus nephritis patients, we will visualize and assess co- localization of monocytes, fibroblasts, tissue structures/lesions. Preliminary data shows close contacts between monocytes and fibroblasts and feasibility of scaling our imaging studies to the full cohort. By building on a more accurate definition of monocytes in human lupus nephritis kidneys, developing methods to study them in vitro and in human tissues, we will define the origin, differen...