In vitro–activated CLL PBMCs express AID protein. (A) Percentage of AID⁺ cells within the total CD5⁺CD19⁺ population derived from 16 cocultures of CLL PBMCs and CD32-transfected fibroblasts stimulated with CD40 + IL-4 for 72 hours. (B) Comparison of the presence or absence of AID mRNA prestimulation with the percentage CD5⁺CD19⁺ cells expressing AID protein 72 hours poststimulation. Means ± SEM of 16 samples are shown. *P = .01 by unpaired t test. (C) Detection of AID protein (white fill) compared with rat IgG2b isotype control mAb (gray fill) by FACS on CD5⁺CD19⁺ cells stimulated in the CD40 + IL-4 system at 0, 72, and 168 hours. (D) Confocal photomicrograph of CD23⁺ cells (red) from the CD40 + IL-4 system visualized at 168 hours demonstrating AID protein (green) localized only in the cytoplasm (replicating protein A-blue (RPA) is used as a nuclear stain). Original magnification was 600×. (E) Representative FACS plots of AID staining on CD5⁺CD19⁺ cells derived from unstimulated and CD40 + IL-4–stimulated, CFSE-labeled CLL PBMCs after 7 days. Percentages of AID⁺ cells in the total CD5⁺CD19⁺ population are shown. (F) Three patterns of AID up-regulation were observed after CD5⁺CD19⁺ cells were cultured for 7 days in the CD40 + IL-4 system: (I) no up-regulation (M-CLL922, M-CLL1227, and M-CLL1232), (II) up-regulation with each division cycle (M-CLL1082, M-CLL1201, U-CLL1238, M-CLL1252, and M-CLL1299), and (III) up-regulation after ≤ 2 cycles (M-CLL797, U-CLL976, and U-CLL1278). (G) Graphs comparing the percentage AID⁺CD5⁺CD19⁺ cells from 5 cultures at 7 and 14 days. Colors denote individual patient samples: purple: U-CLL1278, blue: M-CLL1082, green: M-CLL1299, black: M-CLL1252, and red: M-CLL922).