Figure 3. Dividing CLL B cells express activation-induced cytosine deaminase (AID) protein that correlates with class switch recombination (CSR).

(A) AID expression in spleen-residing human (h) CD45⁺CD5⁺CD19⁺ cells increases as cells divide. Representative FC of single cell splenic suspensions. Significantly higher AID protein is found in the most divided chronic lymphocytic leukemia (CLL) B cells. Mean and SEM shown. Data from 3 independent experiments involving 12 mice; mice euthanized between days 14 and 28; Mann-Whitney test result. (B) AID⁺ cells are localized in CD20⁺PAX5⁺PVAs. ×20 images of CD20, PAX5, and AID; scale bars: 125 μm. (C) ×60 images of AID in areas indicated by arrows in B for U-CLL1122 and M-CLL1164 showing approximately 50% and 5% AID⁺ cells, respectively. Scale bar: 50 μm. For B and C, representative data of mice sampled from 11 independent experiments showing approximately highest and lowest extremes of AID⁺ cells identified in vivo. (D) Switching to IgG becomes evident after CLL B cells have divided multiple times. FC and matching IH of CFSE-labeled CD5⁺CD19⁺ cells for IgM and IgG. Undivided cells produce only IgM and not IgG (upper), whereas multiply divided CLL B cells make both isotypes (lower). Representative data of mice sampled from 5 independent experiments. Scale bar: 50 μm (E). FC confirms splenic-residing hCD45⁺CD5⁺CD19⁺ cells undergo CSR. hCD5⁺CD19⁺ cells with only minimal division (day 14) do not express smIgG, whereas CD5⁺CD19⁺ cells from mice receiving the same CLL clone express smIgG after multiple divisions (day 28). Representative data of mice sampled from 3 independent experiments. U-CLL, CLL clone with IGHV sequence differing ≤2% from most similar germline gene; M-CLL, CLL clone with IGHV sequence differing >2% from most similar germline gene; FC, flow cytometry; IH, immunohistology; sm, surface membrane; MFI, mean fluorescence intensity.