PROJECT SUMMARY Lupus nephritis (LN) is a common manifestation of systemic lupus erythematosus that can lead to irreversible renal impairment. Current immunosuppressive therapies fail to reverse disease in more than half of treated patients and only 2 new drugs have been approved treatment, both of which confer only modest improvement. Renal myeloid cells are involved in renal injury in LN, both in humans and animal models. Macrophages are highly plastic and can mediate both pro-inflammatory and reparative functions. Because there are so many subsets of myeloid cells and some of them change their function over time, analysis of single cells is crucial to the study of these cells. Initial studies in Phase 1 and early Phase 2 of the Accelerating Medicines Partnerships-SLE (AMP-SLE), of which we are all members, have shown that there are multiple subsets of macrophages in LN kidneys that are analogous to myeloid subsets in mice but the function of these subsets and whether they are pathogenic, or protective is still not known. Study of kidney tissues in LN patients is hampered by the small size of the kidney biopsy and the infrequency of these biopsies in individual patients over the course of their disease. Therefore, animal models, when carefully and rigorously employed, are especially useful for addressing hypotheses generated by examining human data. Our overall hypothesis is that we will identify human-relevant myeloid cell subsets in LN models that reflect differences in disease pathophysiology, disease stage and responsiveness to treatments and that this information will help identify new pathways for therapeutic intervention and direct personalized treatment. Our approach is focused on identifying and testing the function of human-relevant cell subsets and pathways in vitro and under physiologic conditions in vivo. In Aim 1 we will complete the integration of single cell RNASeq analysis of myeloid cells from 5 different models of LN with that from AMP-SLE Phase 2 to map both shared and unique macrophage sub-populations. We will follow the fate of peripheral myeloid cells as they transition from the blood into the kidneys to define how the nephritis-specific profile of each subset and how they are related to each other. We will then determine whether there is a particular myeloid cell profile that is associated with response or non-response of LN patients to standard of care therapy. In Aim 2 we will determine how renal myeloid cells interact with renal stromal cells both in vitro and in vivo. We will analyze the fate of renal myeloid cells (in responder and non-responder mice) in human-relevant mouse models treated with remission induction therapies to identify reparative subsets and pathways. Our focus here will be on both standard of care therapy and on belimumab (anti-BAFF) since this is the only biologic drug so far approved for treatment of LN and for prevention of further renal flares. In Aim 3 we will use CRISPR libraries for in viv...