Primary antibody deficiencies (PADs) are the most common primary immunodeficiency (PID) in humans. We showed recently that hyper-activation of PI3K signalling is a frequent cause for PADs. Autosomal dominant mutations in PIK3CD encoding p110d, the catalytic subunit of PI3K, specifically expressed in lymphocytes, and PIK3R1, encoding p85a, the regulatory subunit of PI3K, ubiquitously expressed, have been shown to result in Activated PI3K-d Syndromes (APDS1 and APDS2, respectively). APDS patients presented often, already in early childhood, with recurrent sino-pulmonary infections. Benign lymphoproliferation are common and some APDS patients developed B cell lymphomas. Predominant immunological features are antibody deficiency, most frequently hyper-IgM (HIGM) syndrome, increased transitional B cells, decreased naive B and naive T cells and increased frequency of effector T cells especially CD8+CD57+ T cells, so-called senescent T cells. APDS patient-derived T cells have increased level of phosphorylated AKT and ribosomal protein S6, and are sensitive to activation-induced cell death. In contrast, an immunodeficient patient lacking the p85a subunit of PI3K was described as suffering from a profound defect in B cell development and Ig production. Together, these studies suggest that PI3K signal levels should be strictly regulated in lymphocytes for an optimal immune response. The research proposal aims to further elucidate the mechanisms responsible for the immunodeficiency in APDS1 and APDS2 patients. We hypothesize that hyper-active PI3K activity might affect naive and effector lymphocyte subsets differently in their function. Thus, we will quantify phosphorylation of AKT and S6 in various lymphocyte subsets (e.g. transitional, naive and memory B cells, naive, memory and senescent CD8 T cells) present in the blood of healthy donors and APDS1 and APDS2 patients. Furthermore we will characterize the impact of hyper-activated PI3K signalling on gene expression by gene expression profiling of patients’ and controls’ T lymphocytes. The antibody deficiency present in APDS patients could be explained by an intrinsic B cell defect associated with disturbed survival and/or differentiation of naïve B cells into plasma cells. To investigate this hypothesis we will analyze in vitro differentiation of B cells into plasma blasts. Besides an intrinsic B cell defect, extrinsic defects could occur, such as impaired T follicular helper (TFH) cell function. Thus the frequency and function of TFH-like cells in the blood of APDS patients and controls will be examined. In addition, we will characterize the phenotype and function of T regulatory cells in APDS patients. The clinical and immunological features observed in APDS patients are variable from one patient to another, even in the same family, suggesting the influence of other factors (environmental or modifier genes). To identify genetic components for the phenotypic variability we will perform RNA-Sequence analysis with whole blood from several APDS1 and APDS2 patients to identify single nucleotide variation (SNV) in « modifier » gene(s) (collaboration with Sergey Nejentsev, Cambridge, UK). Our work might thus result in the identification of biomarkers and functional tests to evaluate and monitor treatment efficiency with potential drugs, e.g. mTOR- and p110d-inhibitors. We presume that genetic defects associated with hyper-activation or misbalanced PI3K/AKT/mTOR signalling could be also responsible for other PADs presenting with similar clinical and immunological phenotypes. Thus we attempt to identify and characterize these novel molecular causes for PIDs patients. Together, from this project we expect to better understand PI3K function in lymphocytes, the physiopathogeny of APDS and the identification of biomarkers for diagnosis and monitoring of treatment efficiency. It also aims to define new gene defects responsible for APDS-like patients.