The chemokine receptor CXCR4 is a protein of great interest in basic science and medical research., because it is expressed by many cell types and has multiple biological roles, including cell migration and signaling. Expression levels of CXCR4 are increased in lupus and arthritis and correlate with disease activity. We recently discovered a novel biological function of CXCR4: indeed, binding of monoamines to the extracellular pocket of CXCR4 drives an important signal through the modulation of type I interferon (IFN-I) and pro-inflammatory cytokine production by Toll-Like Receptor (TLR) activated innate immune cells, including monocytes and plasmacytoid dendritic cells (pDCs) We identified several CXCR4 Minor Pocket Agonists (MiPAs) and evaluated their potential activity. This consortium published together that MiPAs control IFN-I secretion in TLR-7 activated primary pDCs and inhibit spontaneous IFN-I production from lupus patients’ cells and drastically reduces disease progression in a pre-clinical lupus animal model. We further demonstrated that MiPAs control pro-inflammatory cytokine production from juvenile arthritic patients’ cells, and in a collagen-induced arthritis mouse model, extending our findings to other diseases. Remarkably, MiPAs treatment of mice with lupus resulted in a significant reduction of circulating anti-dsDNA antibodies suggesting an effect on adaptive immunity. In addition, preliminary results of Team 2 show that MiPAs inhibit TLR-mediated activation of purified human B cells in vitro. We thus identified the CXCR4 minor pocket as a regulator of innate immune activation and potentially of adaptive immunity. However, the intracellular mechanism leading to CXCR4-induced immunomodulation remain to be characterized, as well as its effects on adaptive immunity. Furthermore, Team 3 previously identified a strong difference in CXCR4 expression between men and women, potentially revealing sex associated different immunoregulatory effects of MiPAs. This could be highly relevant for clinical applications, as recent studies showed overexpression of CXCR4 in lupus and rheumatoid arthritis patients who are mostly women. The main goal of our project is to deeply characterize the molecular and cellular mechanisms behind the novel CXCR4 minor pocket immunomodulation pathway. In Aim 1 we will focus thoroughly on the CXCR4 minor pocket signaling cascades induced by MiPAs in innate immune cells. Our preliminary results demonstrate that MiPAs also directly impact the adaptive immune response. Thus, we will precisely address in Aim 2 the impact of CXCR4 activation by MiPAs on purified B and T cells in a normal context, and in a pathological situation in lupus patients’ cells and in a lupus murine model. Finally, as we showed that gene expression of CXCR4 is highly heterogeneous in human populations, with a significant difference between men and women, we will study how such variability in CXCR4 cell surface expression on both innate and adaptive immune cells impacts its immunoregulatory function in Aim 3. We will analyze healthy male and female donors, as well as a cohort of lupus patients who are mostly female and naïve to therapy or under low conventional treatment. While these aims are technically independent insuring their feasibility, they have strong scientific connections regarding CXCR4 dependent immune regulation in different biological and clinical settings. Whereas the role of CXCR4 in promoting inflammation and in the development/migration of B cells has been extensively studied, this project will provide, from a fundamental point of view, new insights into the physiological role of the CXCR4 minor pocket engagement and its role in the inhibition of inflammation and autoimmunity, and could open new therapeutic options for autoimmune diseases as lupus, but also for other inflammatory/autoimmune diseases.