Caractérisation de deux déficits immunitaires primitifs suggérant le rôle de RECQL4 dans l'expansion lymphocytaire T au cours des infections herpétiques sévères et le rôle de l'activation de PLC-γ2 dans la lymphomagenèse B

Epstein-Barr virus (EBV) is an oncogenic virus that infects mostly B lymphocytes. Mostly asymptomatic, the primary infection may be complicated by non-malignant, malignant B-Iymphoproliferative disorders (LPD) and hemophagocytic lymphohistiocytosis (HLH). Infection resolution requires an appropriate cytotoxic T-cell response, which depends on sustained T-cell expansion of EBV-specific T-cells. Several Inborn Errors of Immunity (IEIs) are associated with a high risk of developing severe EBV infection particularly during childhood. These IEls are mostly characterized by defective T-cell expansion and defects in cytotoxic functions of lymphocytes, and their characterization has revealed important factors and pathways required for the immune control of EBV infection. However, there are patients who develop severe EBV infections in the infancy in whom the genetic basic of their disease is unknown. I have studied three individuals with severe EBV infection, one with severe CMV infection and one with a combined immunodeficiency with no genetic diagnosis who were found to be carriers of biallelic variants in RECQL4 by Whole Exome Sequencing (WES), RECQL4 encodes the RECQL4 helicase involved in the initiation of replication and the associated DNA repair process. RECQL4 cleficiency has been previously reported causing a malforrnative syndrome without susceptibility to EBV infection. Four patients displayed severe EBV (in three) or CMV (in one) infections complicated with HLH or LPD. Analysis of RECQL4 expression showed that it was strongly up-regulated in healthy control T-cells in response to CD3/TCR activation but not IL-2. In activated T-cells of patients, RECQL4 was absent or strongly diminished, which was associated with an impaired expansion along with an apoptosis excess. In primary healthy control T-cells or in the Jurkat T-cell line, transduction of three different shRNA targeting RECQL4 silenced its expression resulting in a cell cycle blockage, decreased proilferation defect and an apoptosis excess. Conversely, restoration of RECQL4 expression in primary T-cells of a RECQL4-deficient patient restored their capacity to expand upon CD3/TCR. On the other hand, I also studied four children and one adult with B-cell lymphoma identified carriers of a monoallelic variant in PLCG2 by WES. PLCG2 encodes the phospholipase gamma 2 (PLC-γ2), an important molecule involved in the BCR signaling and required for proper activation of B lymphocytes. These variants are rare and are predicted to be deleterious. Two autoinflammatory syndromes (PLAID and APLAID) with no lymphoma have been previously reported to be associated with gain-of-function variants in PLCG2. The five variants identified were evaluated in a PLCG2-deficient B-cell line. Different cell lines expressing the different variants and PLAID and APLAID variants were obtained and analyzed for Cat2+ flux in response to BCR stimulation. Four out of five variants exhibited an ability to trigger an increased Ca2+ flux compared to wild type PLCG2, suggesting that these variants behave as GoF. However, this GoF effect was significantly lower than that of PLAID or APLAID variants. Thus, this could suggest that PLCG2 variants with high GoF are associated with autoinflammatory diseases, while PLCG2 variants with milder GoF are involved in lymphomagenesis, leading to lymphoma. The ability of the milder GoF PLCG2 variants should now be tested in animal models. My thesis project aimed to characterize variants in RECQL4 and PLCG2 associated with novel clinical phenotypes. My results extend 1) the clinical spectrum associated with RECQL4 showing that severe immunodeficiency features can develop in RECQL4 deficient patients, and showing the critical role of RECQL4 in the proliferation of activated T-cells; 2) the clinical spectrum associated with GoF variants in PLCG2 showing an association with B-cell lymphomas that requires investigations in animal models.