Detection of de novo IGHV mutations by ultra-deep sequencing from in vitro activated B-cell chronic lymphocytic leukemia cells: Evidence for activation-induced deaminase function

摘要

Human B-cell chronic lymphocytic leukemia (CLL) is a clonal CD5+CD19+ B-lymphocyte whose B-cell receptor may be classified as unmutated (U-CLL) or mutated (M-CLL) depending on the level of IGHV mutations. Aggressive CLL associates with acquisition of new gene mutations and cytogenetic aberrations, not necessarily in the IGHV or IGLV loci and perhaps caused by activation-induced deaminase (AID). To test if CLL cells can produce functional AID, CLL cells were activated in vitro with CD32-transfected murine L cells, anti-CD40 and interleukin-4 (7 and 14 days U-CLL1278, 0.0% mutated IGHV3-30; 14 days M-CLL1299, 4.9% mutated IGHV3-23), plus irradiated T lymphocytes (10 or 14 days M-CLL1299). CLL cells in these cultures produced detectable AID protein. To evaluate mutational activity, CLL IGHV cDNA was ultra-deep sequenced using the 454 FLX system (Roche) prior to (day 0) or after activation. The resulting 458,124 sequence reads were processed to generate fixed sequence length datasets. Individual subclone sequences occurring at least twice were extracted and unique de novo subclones not shared between day 0 and activation were analyzed for new mutations. All culture conditions showed increases in IGHV mutation frequencies relative to the IGHM constant region. U-CLL1278 showed increased mutation at AID hotspots and a lower transition mutation frequency. M-CLL1299 showed an overall high frequency of transitions and an increase in mutation at AID hotspots in T cell cultures. Thus, de novo mutations consistent with AID activity were found, with some differences between U-CLL and M-CLL. Mutationally-active AID in CLL could lead to adverse consequences.