B cells are precursors of antibody-producing cells. They are also professional antigen presenting cells (APCs) that can activate both CD4+ and CD8+ T cells through presentation of peptides in the context of major histocompatibility (MHC) class II and class I molecules, respectively. B cells are targets of the g-herpesvirus family like Epstein-Barr Virus (EBV) and Kaposi Sarcoma Herpesvirus (KSHV). This can lead to severe immune hyperactivation of primarily CD8+ T cells, exemplifying the superior ability of especially EBV infected B cells to expand these cytotoxic lymphocytes. Autophagy is a catabolic process executed through ATG (autophagy-related) molecules allowing the translocation of cytoplasmic material into lysosomes, after sequestration in double membrane vesicles. Autophagy is a master regulator of B cell biology. It allows memory B cell survival, long-lived plasma cell maintenance, and consequently immune homeostasis. The importance of autophagy in long-term B cell responses is illustrated by its necessity to maintain humoral protection against viruses like influenza. It also plays pathogenic roles contributing to continuous autoantibody production during systemic autoimmunity in mice. MHC class II presented peptides were initially thought to come mainly from endocytosis and phagocytosis, resulting in lysosomal degradation. More recently, autophagy has also been shown to generate MHC class II presented peptides. The contribution of autophagy in antigen processing of endogenous molecules onto MHC-II molecules is highly relevant for non-phagocytic cells such as B cells. One of the consortium partners was pioneer in the original discovery that autophagy generates MHC class II presented peptides. These initial studies showed that under some circumstances, intracellular antigens could be processed by the autophagy machinery for presentation to CD4+ T cells. Nevertheless, autophagy’s impact on antigen presentation by B cells has to date mainly been studied in vitro. The role of autophagy dependent antigen presentation by B cells in vivo is poorly known but it is highly probable that it allows to maintain CD4+ T cell activation but might dampen immune surveillance by cytotoxic T cells like in the case of EBV infection. Moreover, the role played by ATG molecules might be more complex that initially thought. Indeed, some endocytic processes use part of the autophagy machinery and are also involved in antigen presentation. Furthermore, they could be necessary for the entry of some viruses in target cells, or conversely to limit infection. Thus, dissecting the roles played by ATG molecules in B cells by using in vivo approaches and viral infection models is crucial. This issue will be addressed by (i) describing the functional role of the ATG machinery in endocytosis in B cells, including during g-herpesvirus entry, (ii) by characterizing in vivo the role played by ATG molecules in the germinal center (GC) reaction and antigen presentation, as well as by (iii) defining the impact of autophagy proteins on EBV infection and protective virus specific T cell priming in vivo.

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.

Tissue-resident macrophages (TMs), including epidermal Langerhans cells (LCs), are long-lived cells able to proliferate. Their ontogeny and adaptation to different organs have been studied in great details, yet their physiological maintenance deserves further investigations. Autophagy, a catabolic process regulated by autophagy-related (Atg) genes, prevents accumulation of harmful cytoplasmic components and mobilizes energetic reserves in long-lived and self-renewing cells. Recently, we found that Atg5-deficient LCs undergo apoptosis as a result of lipid metabolism dysregulation. Here, we propose that autophagy allows TMs to manage lipid stocks and ensure long-term maintenance. We will first validate this in murine and human LCs, then verify whether it holds true for TMs of the lung, liver and lymph nodes. Finally, we will test whether autophagy could permit TMs to adapt to cellular stress and limit inflammation induced by metabolic alterations, irradiation, aging and viral infection. Altogether, our results will introduce a new paradigm on the maintenance of TMs in a broad range of organs under physiological and inflammatory conditions.

Intracellular cholesterol trafficking is essential for the maintenance of vascular integrity. However, the mechanisms by which cholesterol is transported into the cell are poorly understood. Recently, we showed that a ligand of Wnt signaling, Wnt5a, inhibits intracellular cholesterol accumulation in vascular smooth muscle cells (VSMCs). We show that Wnt5a interacts with lysosomal Niemann-Pick C1/C2 proteins and cholesterol, facilitates its export from the lysosome and protects against atherosclerosis. We also generated antibodies that activate Wnt signaling. We show that these antibodies decrease cholesterol accumulation in VSMCs. The aim of the present project is 1) to study the physiological mechanisms by which Wnt5a facilitates intracellular cholesterol trafficking and interacts with NPCs, 2) to test the therapeutic potential in atherosclerosis of the antibodies we generated and that activate Wnt signaling. 3) In a pilot study, we assayed several Wnt ligands in the sera of 86 patients with familial hypercholesterolemia (FH) and 32 controls. Our results show a marked and significant negative correlation of several of these ligands with the presence of coronary lesions, suggesting that they can be used as biomarkers during atherosclerosis. In a larger study, these markers will be evaluated by Elisa in sera of HF patients from the Rhu Chopin (CHOlesterol Personalized Innovation) registry. This national registry aims to identify new markers of cardiovascular risk. In addition, by sequencing our candidate Wnt genes in FH probands without mutations in the LDLR/APOB/PCSK9/APOE genes, a genetic study will establish the spectrum of variants of these Wnt genes in these patients.