Fig. 1. Kinetics of wildtype B-cell clonal expansion, CSR, and PC differentiation in 40LB culture.

B6 splenic B cells co-cultured with 40LB feeder cells and IL-4 and IL-21 were periodically analyzed by flow cytometry, while culture supernatants were analyzed by ELISA. A, Growth kinetics of cultured B cells. B, Expression of key transcription factors (TFs) in cultured B cells and control B cells. Ex vivo B6 splenocytes were the positive control for Pax5 and Bach2 staining, ex vivo B6 Peyer’s patch GC B cells were the positive control for Bcl6 staining, and cultured J558L plasmacytoma cells were the positive control for IRF4 and Blimp1 staining. C and D, Dynamics of transcription factor expression in cultured B cells, normalized to control cells (as shown in B). E, Frequency of B cells expressing surface IgG1. F, Aggregate data for IgG1-CSR (gated as in E) at all time points. G, Frequencies of B cells expressing the PC markers CD43 or CD138 at various time points during 40LB culture. H, Aggregate data for expression of CD43 and CD138 (gated as in G) at all time points. I, The average rate of IgG and IgM secretion by 40LB-cultured B cells at various time points. GeoMFI, geometric mean fluorescence intensity. LOD, limit of detection. Flow cytometry analyses were pre-gated on viable H2-K^d−CD19⁺ cells (B-H). Error bars depict geometric mean ± 95% CI (A, I) or arithmetic mean ± 95% CI (C, D, F, H) of three replicates. Data are representative of one (B, C, D, I) or ≥3 independent experiments (A, E, F, G, H) with similar results.