Ental pressure on their supply [4]. IVIg consist of a large repertoire of polyclonal human IgG showing reactivity to pathogens as well as to human self-proteins [5]. Extensive investigations aiming at identifying specific IVIg immunomodulatory properties in order to eventually create substitutes to treat autoimmune and 
inflammatory diseases are currently being performed by several groups. Currently, a unique preparation of 25 monoclonal anti-RhD antibodies [6] is in phase II of clinical trials for the treatment of immune thrombocytopenic purpura (ITP) [7]. Further success in those clinical trials could qualify this monoclonal mix as a substitute for IVIg in ITP-treatment. However, polyclonal preparations for clinical applications are still the exception. Essentially, patient’s accessibility to IVIg depends exclusively upon volunteer blood donation and there are no in vitro proceduresallowing the preparation for these polyclonal human antibodies. Therefore, the development of an in vitro method for the production of large quantities of human IgG that could substitute for 15481974 IVIg is highly relevant. As introduced above, in vitro generated human therapeutic antibodies are monoclonal and are mostly generated from transgenic mouse or genetic engineering such as chimeric, humanized or recombinant antibodies [8,9,10,11]. Nevertheless, long-term cultures of human B lymphocytes have been proposed 20 years ago by Banchereau and collaborators while designing the CD40-CD154 culture system [12]. This coculture model is based upon interactions between CD40 present on all B lymphocytes and CD154+ purchase Eledoisin adherent cell line. The model was expected to allow the generation and clonal expansion of human B cell lines [13]. Since then, many groups have used this culture system to activate human B lymphocytes to study their physiological characteristics in relation to the immune response (reviewed in [14]). However, the concept of large expansion of B lymphocytes was not developed nor relevant until recently, when antigen-presenting capacity of B lymphocytes were viewed as an asset for cancer treatment [15,16,17]. Here, we report a model based upon CD40-CD154 interactions, enabling high levels of expansion as well as differentiation of human switched memory B lymphocytes. This long-term culture model could be a critical step toward a large-scale production of human IgG as well as ex vivo expansion of human memory B lymphocytes.Large-Scale Expansion of Human B LymphocytesFigure 1. Selection of switched-memory B lymphocytes. In all experiments, purified CD19+ B lymphocytes (A) were depleted for IgD+IgM+ and IgM+ cells in a two-step selection process (B). Analysis of the resulting cell populations showed a relatively similar proportion of cells with surface IgA (C) and IgG (D). doi:10.1371/journal.pone.0051946.gMaterials and Methods Preparation of Human Mononuclear CellsThis study has been approved by Hema-Quebec’s Research ??Ethics Committee and every regular platelet donors who agreed to participate in this study, have signed an ITI007 informed consent. Leukoreduction system (LRS) chambers from Trima AccelTM collection systems (Gambro BCT, Lakewood, CO, USA) were collected after routine apheresis. Leukocytes were recovered from LRS chambers, as previously described [18], and used to isolate peripheral blood mononuclear cells (PBMNCs) by centrifugation on Ficoll-Paque following manufacturer’s instructions (GE Healthcare, Baie d’Urfe, QC, Canada). PBMNCs were stored, frozen, ?un.Ental pressure on their supply [4]. IVIg consist of a large repertoire of polyclonal human IgG showing reactivity to pathogens as well as to human self-proteins [5]. Extensive investigations aiming at identifying specific IVIg immunomodulatory properties in order to eventually create substitutes to treat autoimmune and inflammatory diseases are currently being performed by several groups. Currently, a unique preparation of 25 monoclonal anti-RhD antibodies [6] is in phase II of clinical trials for the treatment of immune thrombocytopenic purpura (ITP) [7]. Further success in those clinical trials could qualify this monoclonal mix as a substitute for IVIg in ITP-treatment. However, polyclonal preparations for clinical applications are still the exception. Essentially, patient’s accessibility to IVIg depends exclusively upon volunteer blood donation and there are no in vitro proceduresallowing the preparation for these polyclonal human antibodies. Therefore, the development of an in vitro method for the production of large quantities of human IgG that could substitute for 15481974 IVIg is highly relevant. As introduced above, in vitro generated human therapeutic antibodies are monoclonal and are mostly generated from transgenic mouse or genetic engineering such as chimeric, humanized or recombinant antibodies [8,9,10,11]. Nevertheless, long-term cultures of human B lymphocytes have been proposed 20 years ago by Banchereau and collaborators while designing the CD40-CD154 culture system [12]. This coculture model is based upon interactions between CD40 present on all B lymphocytes and CD154+ adherent cell line. The model was expected to allow the generation and clonal expansion of human B cell lines [13]. Since then, many groups have used this culture system to activate human B lymphocytes to study their physiological characteristics in relation to the immune response (reviewed in [14]). However, the concept of large expansion of B lymphocytes was not developed nor relevant until recently, when antigen-presenting capacity of B lymphocytes were viewed as an asset for cancer treatment [15,16,17]. Here, we report a model based upon CD40-CD154 interactions, enabling high levels of expansion as well as differentiation of human switched memory B lymphocytes. This long-term culture model could be a critical step toward a large-scale production of human IgG as well as ex vivo expansion of human memory B lymphocytes.Large-Scale Expansion of Human B LymphocytesFigure 1. Selection of switched-memory B lymphocytes. In all experiments, purified CD19+ B lymphocytes (A) were depleted for IgD+IgM+ and IgM+ cells in a two-step selection process (B). Analysis of the resulting cell populations showed a relatively similar proportion of cells with surface IgA (C) and IgG (D). doi:10.1371/journal.pone.0051946.gMaterials and Methods Preparation of Human Mononuclear CellsThis study has been approved by Hema-Quebec’s Research ??Ethics Committee and every regular platelet donors who agreed to participate in this study, have signed an informed consent. Leukoreduction system (LRS) chambers from Trima AccelTM collection systems (Gambro BCT, Lakewood, CO, USA) were collected after routine apheresis. Leukocytes were recovered from LRS chambers, as previously described [18], and used to isolate peripheral blood mononuclear cells (PBMNCs) by centrifugation on Ficoll-Paque following manufacturer’s instructions (GE Healthcare, Baie d’Urfe, QC, Canada). PBMNCs were stored, frozen, ?un.
