CLL cells that divided and up-regulated AID protein exhibited CSR and de novo SHM. (A) Sort strategy to obtain 20-cell and 1-cell aliquots of CD5⁺CD19⁺ cells from CLL PBMCs after up to 14 days of culture. (B) Comparison of the percentages and numbers of 20-cell/well aliquots that yielded α and γ (switched) CLL patient–specific IGHV-D-J transcripts from divided, undivided, and unstimulated CD5⁺CD19⁺ populations after 14 days of culture. ***P = .0002 by χ² test. (C) Representative FACS plots showing minimal surface IgG expression by CD5⁺CD19⁺ cells that had not divided after 14 days of stimulation compared with at least a 10-fold higher expression by multiply divided cells. (D) Comparison of the division number with the percentage of surface IgG expression by CD5⁺CD19⁺ cells derived from stimulated CLL PBMCs after 14 days of culture. (E) Change in number of unique mutated subclones identified by NGS from cells stimulated in the CD40 + IL-4 system compared with prestimulated samples. The change in unique subclone count is subdivided into mutations in IGHVDJ and IGHM; a negative number was generated for IGHM in both cases because less unique subclones were found in this region after stimulation compared with prestimulated cells.