Figure 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).
Figure 2.
Long-term expansion of switched-memory B lymphocytes.
(A) Ten samples of switched-memory B lymphocytes were cultured for 35 to 65 days in the presence of IL-2, IL-4, IL-10 and CD154+ cells (L4.5 cells) at a ratio of five B cells per L4.5 cell. Expansion factors for the ten independent samples were plotted as a function of time (days) in culture. (B) Regression analysis of the ten exponential growth curves presented in (A) resulted in a correlation coefficient of 0.9965 corresponding to the equation y = 10 (0.1344x+0.5415). (C) The proportion of viable cells during long-term culture was monitored on a regular basis for each culture. (D) The mean value and standard deviation of viability (%) showed a similar evolution for each samples.
Figure 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 (a), and 37 (g, h, i, j), and seeded in fresh IMDM at 1–2×106 cells/mL for 20–22 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 ± SD.
Figure 4.
Switched-memory B lymphocytes secrete polyclonal IgG.
IgG’s polyclonality was determined by IEF for experiments a to j. A representative pattern is presented in panel A. (A) Briefly, IEF standard (Std), IVIg, a human monoclonal IgG (mAb) and culture supernatants from experiment a sampled on days 16, 21, 30, 41 and 49 are shown. This polyclonal IEF pattern is similar for the ten independent cultures presented in Fig. 1. (B) IEF pattern was determined for the cumulated supernatants of the three experiments presented in Fig. 5 as well as the pooled supernatants of 13 representative experiments. As mentioned above, Std, IVIg and mAb were used as controls and a sample from a healthy human serum was also used for the same purpose. For all samples in (A) and (B), analyses were done on 100 ng IgG per well.
Figure 5.
Validation of expansion during long-term culture.
Three switched-memory B lymphocyte samples were cultured as described in Fig. 1 and transferred in petri dishes to test the feasibility of increasing the culture volume up to 500 mL. (A) Expansion factors were similar to those obtained in 6-well plates (Fig. 1). (B) Culture volumes are shown as a function of time. (C) IgA, IgG and IgM concentrations were determined in supernatants of the three independent samples at the end of the culture. (D) Flow cytometry analyses for kappa and lambda chain expression was similar for all three independent samples.
Table 1.
Human polyclonal IgG interacting proteins using a protein array.