Higher than the negative signal control for each protein. Second, theImmunoglobulins Analysis by IsoelectrofocusingThin-layer isoelectrofocusing (IEF) was performed on a 5 acrylamide gel containing Bio-Lyte 3/10 ampholytes (Bio-Rad Laboratories, Mississauga, ON, Canada). Culture supernatants were compared to an in-house human monoclonal IgG and to a commercial intravenous immunoglobulin preparation (IVIg) (GamunexTM, Talecris Biotherapeutics ltd., Toronto, ON, Canada). A total of 100 ng IgG per samples and controls, according to IgG concentration determined by ELISA, were focused in three steps, consisting of 100V for 15 minutes, 200V for 15 minutes and 450V for one hour. Protein standards with pI ranging from 4.45 to 9.6 were used to monitor theFigure 3. Switched-memory B lymphocytes secrete mostly IgG. Cells from the same ten independent experiments shown in Fig. 2, were collected during the exponential phase, namely on days 28 (e, f), 33 (b, c, d) 35 22948146 (a), and 37 (g, h, i, j), and seeded in fresh IMDM at 1?6106 cells/mL for 20?2 h. For each experiment, secretion rates were determined for IgA, IgG and IgM (A, B) and IgG1, IgG2, IgG3 and IgG4 (C, D). Data are presented as the mean 6 SD. doi:10.1371/journal.pone.0051946.gLarge-Scale Expansion of Human B Lymphocytesnegative control signal value was less than 1000 RFU and finally, the replicate spot coefficient of variation (CV) was less than 50 .Results Large Expansion of Switched Memory B LymphocytesBlood CD19+ B lymphocytes were depleted for IgD+ and IgM+ cells, giving a switched memory B lymphocyte population comprised of IgG+ and IgA+ cells, 54 611 and 36 612 respectively (Fig. 1), namely the IgG/IgA B lymphocyte population. The ratio of IgG and IgA was approximately 1.5, which is close to what is observed for small B cells in human blood [24], possibly resulting from the elutriation process used to remove mononuclear cells during platelet collection [18]. For all 13 samples presented in this study, the residual frequency of IgD+ and/or IgM+ cells was always less than 3 at the initiation of the culture. During the culture period, residual IgD+ cells remained lower than 3 , however, the frequency of IgM+ cells reached 10 64 in some experiments. Ten independent IgG/IgA B lymphocyte samples were isolated and stimulated in the presence of high levels of CD154 interaction and a mix of IL-2, IL-4 and IL-10 for 36 to 65 days. In order to evaluate the total expansion Calyculin A factor (Fig. 2A) as well as viability (Fig. 2C and D), IgG/IgA B lymphocytes were maintained in the exponential growth phase and their number was determined at the indicated days. The regression analysis of these ten exponential 
growth curves (Fig. 2B) showed a 0.9965 coefficient, indicating that the expansion was similar in relation to time and consistent among these experiments. The Tgen period calculated between days 28 to 36 in all these cultures, corresponded to a mean of 51 h69 h (data not shown). Cell viability was also comparable from one BTZ-043 cultured sample to another and maintained at an acceptable level, ranging from 93 to 80 at the end of the culture period (Figs. 2C and D). Overall, these culture conditions allowed a final expansion factor, based on the expansion rate and seeding cell numbers, ranging from 107 to 109 after 50 to 65 days.the secretion rate mean values for the ten experiments (Fig. 3D), the relative proportions of IgG1 (67 ), IgG2 (24 ), IgG3 (6 ) and IgG4 (3 ) were comparable to those reported i.Higher than the negative signal control for each protein. Second, theImmunoglobulins Analysis by IsoelectrofocusingThin-layer isoelectrofocusing (IEF) was performed on a 5 acrylamide gel containing Bio-Lyte 3/10 ampholytes (Bio-Rad Laboratories, Mississauga, ON, Canada). Culture supernatants were compared to an in-house human monoclonal IgG and to a commercial intravenous immunoglobulin preparation (IVIg) (GamunexTM, Talecris Biotherapeutics ltd., Toronto, ON, Canada). A total of 100 ng IgG per samples and controls, according to IgG concentration determined by ELISA, were focused in three steps, consisting of 100V for 15 minutes, 200V for 15 minutes and 450V for one hour. Protein standards with pI ranging from 4.45 to 9.6 were used to monitor theFigure 3. Switched-memory B lymphocytes secrete mostly IgG. Cells from the same ten independent experiments shown in Fig. 2, were collected during the exponential phase, namely on days 28 (e, f), 33 (b, c, d) 35 22948146 (a), and 37 (g, h, i, j), and seeded in fresh IMDM at 1?6106 cells/mL for 20?2 h. For each experiment, secretion rates were determined for IgA, IgG and IgM (A, B) and IgG1, IgG2, IgG3 and IgG4 (C, D). Data are presented as the mean 6 SD. doi:10.1371/journal.pone.0051946.gLarge-Scale Expansion of Human B Lymphocytesnegative control signal value was less than 1000 RFU and finally, the replicate spot coefficient of variation (CV) was less than 50 .Results Large Expansion of Switched Memory B LymphocytesBlood CD19+ B lymphocytes were depleted for IgD+ and IgM+ cells, giving a switched memory B lymphocyte population comprised of IgG+ and IgA+ cells, 54 611 and 36 612 respectively (Fig. 1), namely the IgG/IgA B lymphocyte population. The ratio of IgG and IgA was approximately 1.5, which is close to what is observed for small B cells in human blood [24], possibly resulting from the elutriation process used to remove mononuclear cells during platelet collection [18]. For all 13 samples presented in this study, the residual frequency of IgD+ and/or IgM+ cells was always less than 3 at the initiation of the culture. During the culture period, residual IgD+ cells remained lower than 3 , however, the frequency of IgM+ cells reached 10 64 in some experiments. Ten independent IgG/IgA B 
lymphocyte samples were isolated and stimulated in the presence of high levels of CD154 interaction and a mix of IL-2, IL-4 and IL-10 for 36 to 65 days. In order to evaluate the total expansion factor (Fig. 2A) as well as viability (Fig. 2C and D), IgG/IgA B lymphocytes were maintained in the exponential growth phase and their number was determined at the indicated days. The regression analysis of these ten exponential growth curves (Fig. 2B) showed a 0.9965 coefficient, indicating that the expansion was similar in relation to time and consistent among these experiments. The Tgen period calculated between days 28 to 36 in all these cultures, corresponded to a mean of 51 h69 h (data not shown). Cell viability was also comparable from one cultured sample to another and maintained at an acceptable level, ranging from 93 to 80 at the end of the culture period (Figs. 2C and D). Overall, these culture conditions allowed a final expansion factor, based on the expansion rate and seeding cell numbers, ranging from 107 to 109 after 50 to 65 days.the secretion rate mean values for the ten experiments (Fig. 3D), the relative proportions of IgG1 (67 ), IgG2 (24 ), IgG3 (6 ) and IgG4 (3 ) were comparable to those reported i.