Chronic Lymphocytic Leukemia B Cells Variably Express Functional Activation-Induced Cytosine Deaminase Protein

Abstract 378

During the course of CLL, ongoing genetic changes occur within the leukemic clone and such changes associate with disease progression. Activation-induced cytidine deaminase (AID), the enzyme required for IGV gene somatic hypermutation and isotype class switching in B cells, is a candidate enzyme for causing such changes. Depending upon the detection method, circulating CLL cells express mRNA for AID in 40 – 100% of patients, although at any point only a very small percentage of cells within the clone express this message. Because B lymphocytes must be in the cell cycle for AID-induced DNA changes to occur, we hypothesized that AID protein would be contained within recently divided CLL cells and that these cells would exhibit new IGV mutations and/or class switching.

Using appropriate markers for recently divided cells, we found that AID mRNA is enriched in/limited to this subset. Furthermore, because dividing cells in CLL are principally found within bone marrow and secondary lymphoid tissues, we analyzed such cells in lymph nodes (LNs) for AID protein expression and activity. In 50% of LNs infiltrated with CLL (n=10), AID protein was detected in large cells expressing a CLL phenotype; these cells were predominantly in the cell cycle. Nevertheless, even in those cases where CLL cells expressed AID, most cycling cells were AID protein negative. FACS analysis of dispersed LN cells confirmed the presence of AID protein-expressing cells and such cells had the phenotype of recently divided cells.

To demonstrate that AID protein was functionally competent, we co-cultured peripheral blood CLL cells with anti-CD40 mAb and IL-4 in the presence of irradiated CD32-transfected fibroblasts, a model that mimics the tissue microenvironment. In 16 patients, we showed that peripheral blood leukemic cells could express AID protein, although the degree of upregulation was highly variable between cases. Using the dye CFSE to track CD5⁺CD19⁺ cell division, we found that AID protein always occurred when multiply divided cells were present. Some cases showed immediate AID production prior to division, while others exhibited no or little expression until passing through several cell cycles. AID protein causes double strand breaks (DSBs) within DNA, for example in IG switch regions during class switch recombination. We therefore used confocal microscopy to detect the presence of phospho-histone H2A.X (pH2AX), which localizes to DSBs, in CFSE-labeled cells stimulated for 14 days by the conditions mentioned above. At least 10 × 60 magnification images from 3 cultures showing cell division and AID upregulation were obtained, and the fluorescent signals for CFSE and pH2AX quantified from greater than 250 CD23⁺ CLL cells in each case. A mean of 20.4% of cells (range 10.4 – 38.2%) showed increased fluorescence with anti-pH2AX compared to unstimulated cells. Moreover, stimulated cultures demonstrated increased anti-pH2AX signal in a significantly greater number of cells with diminished CFSE intensity, which are the most divided cells, as compared to less/undivided cells with higher CFSE intensity (p<0.0001 in all cases analyzed, Fisher's exact test). In addition, 20 cell aliquots of unstimulated CLL cells and stimulated CFSE-labeled CLL cells were sorted after culture, yielding pure populations of either undivided cells or cells that had undergone 5 – 6 divisions. While all sorted populations yielded unswitched mu IG transcripts (≥75% wells positive in all groups), switched gamma transcripts with the same V-D-J rearrangement as the leukemic clone were only obtained from divided cells (range 4–9% wells positive), and not present in either undivided or unstimulated cells (0% wells positive). Taken together, the presence of heightened numbers of DSBs in the most divided cells compared to no/minimally divided cells and evidence of IG class switching in the former, indicate that AID protein was functional in these activated CLL peripheral blood cultures.

In all, these data demonstrate that in CLL functional AID protein predominates in cells that are dividing or have a recently divided phenotype, although cases vary in the number of cells expressing AID as well as the relative amounts of enzyme expression. Differential AID activity between discrete CLL cases may relate to the development of new DNA mutations leading to clonal evolution and the variable nature of disease progression seen in this disease.