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.

In patients suffering of chronic kidney disease (CKD), phosphates accumulation in blood is very dangerous and dialysis is the only way to remove them from blood, for those waiting for transplantation. Phosphate imbalance leads to a high cardiovascular mortality and to bone disorder in dialysis patients. Current procedures (hemodialysis (HM) and peritoneal dialysis (PD)) do not allow for the removal of sufficient phosphate amounts, as does normal kidney function. HD consists of extracorporeal blood purification with the help of a machine. During PD procedure, a designed dialysis solution is introduced into the peritoneal cavity of the patient and for a few hours, the composition of the liquid is balanced with that of the blood compartment. By diffusion and convection mechanisms through the capillaries, toxins and water in excess pass into the dialysate. The dialysate is then drained outside the body before the subsequent provision of new dialysate. The duration of the exchanges varies according to the needs of the patient. PHODIA project proposes to investigate the addition of iron oxide nanoparticles (IONPs) into dialysate used for PD to enhance phosphate removal from blood and possibly reduce the duration of PD for patients. It would allow establishing PD as the most adapted dialysis method. Indeed, in addition to being cheaper than HD, PD has many advantages for the patient: the possibility of being autonomous in the treatment, fewer hospital admissions needed (and therefore reduced transport costs), fewer secondary treatments such as anticoagulants, and the possibility of carrying out the treatment during the night. It allows for less diet and fluid restriction for the patient and a better vascular preservation compared to HD. This last point makes that PD is mainly used for infants and newborn babies. Therefore, the increase in phosphate removal efficiency by introducing IONPs in the dialysate would allow a very large number of patients to benefit from this more comfortable and less expensive PD technique. Such solution using IONPs for improving phosphate removal by PD was never tested before and is thus proposed for the first time. The first objective will be to synthesize IONPs with size and surface specific area optimized to ensure a good colloidal stability in dialysate, no transfer of IONPs from dialysis solution to blood vessels and a high phosphate capture. Three different IONPs synthesis methods and in particular one method leading to mesoporous IONPs, will be tested to screen IONPs with different mean diameter, pore size and surface specific area. We will then study the adsorption of phosphate of designed IONPs. We will pay attention to the possibility of IONPs removing other toxins and will verify that essential compounds will not be removed during the PD process. Another original objective is the building of an in vitro set-up mimicking the PD treatment to test and optimize IONPs design and extraction. It will aim at reproducing as closely as possible the exchanges that take place through the peritoneum during a dialysis session, as well as the conditions of exchange and at allowing feasibility tests without immediate recourse to animals trials. Chelating agent of 99mTc will be coupled at the surface of IONPs to follow their diffusion in the PD set-up by nuclear imaging with a gamma camera. Finally, we will check, by in vitro experiments, the interactions of IONPs with cells of peritoneal membrane and study their in vivo fate in dialysate and rats by nuclear imaging. The ultimate goal is to formulate dialysates containing a minimal and controlled amount of IONPs to extract a higher and controlled amount of phosphates during a PD procedure and possibly to reduce the duration of the PD treatment. This project would establish PD as an efficient procedure for controlling CKD and increase its use for better comfort in adult patients and better effectiveness in children, for whom it is the only possible treatment.