N human serum, namely IgG1 60 615 , IgG2 30 65 , IgG3 7 62 and IgG4 3 61 [25,26]. The IgG total secretion recorded for each culture experiments ranged from 30 to 115 mg/mL (data not shown). Overall these data showed that CP21 web long-term culture of IgG+ human B lymphocytes did not induce a bias in the secretion of IgG isotypes, which was consistent with the reported proportions in human blood.Expanded IgG+ B Lymphocytes are Polyclonal PopulationsThe degree of heterogeneity of secreted IgG molecules was assessed during the long-term culture by sampling culture supernatant at various time points and analyzing their patterns by isoelectrofocusing. Analysis of secreted IgG from day 16 to day 49 (fig. 4A) showed smears of IgG bands, which are characteristic of polyclonal IgG and similar to the IgG IEF pattern of IVIg. All ten experiments (a to j), showed similar patterns of polyclonality indicating that the expanded IgG+ B-lymphocyte population maintained its diversity, even after long-term culture. The presence of EBNA1 was determined only on samples e to j (Fig. 2) and 3 of them (e, f, h) were found positive (data not shown).Validation of Expansion of Switched-memory B LymphocytesThe ability of switched-memory B lymphocytes to expand in larger culture AN-3199 volumes was assessed by serial passaging of three long-term cultures from 6-wells plates to petri dishes. A culture period of 35 to 40 days could be easily achieved, and allowed to reach real culture volumes up to 300 to 450 ml. 25837696 Cellular densities were maintained between 46105 cells/ml and 36106 cells/ml, which added up to more than 109 total switched-memory B lymphocytes at the end of the culture experiment (Fig. 5 A and B). The three independent samples presented in Figure 5 (k, l and m) 
expanded in larger volumes, showed expansion rates similar to those observed above (Fig. 1) and to experiments done with the same samples cultured in 6-well plates (data not shown). The presence of EBNA1 was detected in expanded cells originated from sample l, whereas those generated with samples k and m were found negative (data not shown). In these experiments, the cumulated supernatants were separately pooled and the total secretion of IgG, IgA, and IgM ranged from 30 to 116 mg/mL, 8 to 49 mg/mL and 1 to 3700 ng/mL, respectively (Fig. 5C). As above, IgM concentration represented less than 4 while IgG consisted of 67 to 93 of total Ig secretion. IEF analysis of these cumulated supernatants, separately and as a pool, also showed a polyclonal IgG distribution (Fig. 4B), which was similar to that of IVIg as well as IgG present in human serum. Flow cytometry analysis of the expanded cells showed acceptable proportions of kappa and lambda light chain (Fig. 5D) [27] and, as above, all four gamma isotypes were present with frequencies declining from IgG1 to IgG3/ IgG4 (data not shown). Lastly, the total IgG secretion of the pooled culture supernatants reached 30 mg to 100 mg of human IgG in final volumes of 0.7L to 1L.Expanded Switched-memory B Lymphocytes Contained Functional Ig-secreting CellsIn order to estimate the differentiation status of the expanded IgG/ IgA B lymphocytes, the secretion rates for IgG and IgA were determined during the exponential phase, i.e. between day 28 and day 37. IgM secretion was also measured in the supernatant as a supplemental control for negative selection efficiency and to verify whether the frequency of IgM+ B lymphocytes increased (Fig. 3A and B). As expected, all ten expe.N human serum, namely IgG1 60 615 , IgG2 30 65 , IgG3 7 62 and IgG4 3 61 [25,26]. The IgG total secretion recorded for each culture experiments ranged from 30 to 115 mg/mL (data not shown). Overall these data showed that long-term culture of IgG+ human B lymphocytes did not induce a bias in the secretion of IgG isotypes, which was consistent with the reported proportions in human blood.Expanded IgG+ B Lymphocytes are Polyclonal PopulationsThe degree of heterogeneity of secreted IgG molecules was assessed during the long-term culture by sampling culture supernatant at various time points and analyzing their patterns by isoelectrofocusing. Analysis of secreted IgG from day 16 to day 49 (fig. 4A) showed smears of IgG bands, which are characteristic of polyclonal IgG and similar to the IgG IEF pattern of IVIg. All ten experiments (a to j), showed similar patterns of polyclonality indicating that the expanded IgG+ 
B-lymphocyte population maintained its diversity, even after long-term culture. The presence of EBNA1 was determined only on samples e to j (Fig. 2) and 3 of them (e, f, h) were found positive (data not shown).Validation of Expansion of Switched-memory B LymphocytesThe ability of switched-memory B lymphocytes to expand in larger culture volumes was assessed by serial passaging of three long-term cultures from 6-wells plates to petri dishes. A culture period of 35 to 40 days could be easily achieved, and allowed to reach real culture volumes up to 300 to 450 ml. 25837696 Cellular densities were maintained between 46105 cells/ml and 36106 cells/ml, which added up to more than 109 total switched-memory B lymphocytes at the end of the culture experiment (Fig. 5 A and B). The three independent samples presented in Figure 5 (k, l and m) expanded in larger volumes, showed expansion rates similar to those observed above (Fig. 1) and to experiments done with the same samples cultured in 6-well plates (data not shown). The presence of EBNA1 was detected in expanded cells originated from sample l, whereas those generated with samples k and m were found negative (data not shown). In these experiments, the cumulated supernatants were separately pooled and the total secretion of IgG, IgA, and IgM ranged from 30 to 116 mg/mL, 8 to 49 mg/mL and 1 to 3700 ng/mL, respectively (Fig. 5C). As above, IgM concentration represented less than 4 while IgG consisted of 67 to 93 of total Ig secretion. IEF analysis of these cumulated supernatants, separately and as a pool, also showed a polyclonal IgG distribution (Fig. 4B), which was similar to that of IVIg as well as IgG present in human serum. Flow cytometry analysis of the expanded cells showed acceptable proportions of kappa and lambda light chain (Fig. 5D) [27] and, as above, all four gamma isotypes were present with frequencies declining from IgG1 to IgG3/ IgG4 (data not shown). Lastly, the total IgG secretion of the pooled culture supernatants reached 30 mg to 100 mg of human IgG in final volumes of 0.7L to 1L.Expanded Switched-memory B Lymphocytes Contained Functional Ig-secreting CellsIn order to estimate the differentiation status of the expanded IgG/ IgA B lymphocytes, the secretion rates for IgG and IgA were determined during the exponential phase, i.e. between day 28 and day 37. IgM secretion was also measured in the supernatant as a supplemental control for negative selection efficiency and to verify whether the frequency of IgM+ B lymphocytes increased (Fig. 3A and B). As expected, all ten expe.
