STAT3 deficiency impairs the generation of Ag-specific memory B cells in vivo. B cells from healthy controls or STAT3_MUT patients were cultured with CD40L, CpG, SAC, IL-10, and IL-21 for 4 d and were recultured for 3 d. The proportion of cells secreting (A) IgM+IgG or (C) antitetanus IgG was determined by ELISPOT. (B) Levels of total Ig (IgM+IgG+IgA) secreted during the secondary culture were determined by ELISAs; the results are expressed as micrograms of Ig secreted per 10⁵ input cells in the secondary culture. Each symbol represents the response from an individual control or patient; the horizontal bars represent means. (D and E) Levels of antitetanus IgG in culture supernatants (Sn; D) or serum (E) from normal controls or STAT3_MUT patients were determined by ELISA. (F) Sera from controls or STAT3_MUT patients (1:40 dilution) were incubated with decreasing amounts of immobilized tetanus toxoid. Binding of antitetanus IgG was determined by ELISA. The results are expressed as the percentage of maximum binding, which is defined as the binding to the highest concentration of tetanus (i.e., 3 μg/ml). In D, the data for the controls represent means ± SEM from eight control donors; each blue line represents data from an individual STAT3_MUT patient (n = 8). The data in E and F represent means ± SEM from four control donors and five STAT3_MUT patients.