Overexpression of Activation-Induced Deaminase in TCL1 Mice Leads to the Development of IGHV -Mutated and -Unmutated CLL Clones That Resemble Unique Subsets of Human CLL

Introduction. Somatic hypermutation (SHM) and class-switch recombination (CSR) are critical physiologic events in an effective normal B-cell immune response. Notably, both SHM and CSR are initiated by activation-induced cytidine deaminase (AID). In CLL, IGHV clonal mutations correlate strongly with better clinical outcomes. Eμ-T-cell leukemia-1 (TCL1) transgenic (Tg) mice are a valuable model of CLL. However because SHM and CSR occur rarely in the leukemic clones from these animals, TCL1 Tg mice mimic only IGHV -unmutated CLL and hence may not provide an understanding of the roles of SHM and CSR in disease evolution. To address these issues, we developed two new TCL1 strains by interbreeding mice over-expressing AID in all cells (Em-TCL1xActin-AID) or only in B lymphocytes (Em-TCL1xVκ-AID).

Methods. B-cell clonal expansions were identified in spleen cells from 22 TCL1 and 33 TCL1xAID Tg (10 Em-TCL1xActin-AID plus 23 Em-TCL1xVκ-AID) mice at 10-20 months of age. This was done by amplifying cDNAs by PCR using consensus IGHV FR and IgM-, IgG-, and IgA-specific primers for IgH chains and Vκ FR and Cκ for IgL chain genes. DNA sequences of these amplicons were compared to murine germline IGHV s and IGKV s, and IGHV-D-JH and IGKV-JK rearrangements by IMGT V-Quest. Because there were no major differences in the parameters listed below for the two TCL1xAID Tg mouse strains, data were combined.

Results.

Clonal expansions: Monoclonal/oligoclonal expansions were detected in all TCL1 mice; these used only µ H and κ L chains. Similar expansions were detected in 31 of 33 TCL1xAID mice; each animal bore an IgMκ⁺ clone and 7 also exhibited an IgGκ⁺ clone.

IGHV gene use in IgMκ⁺ clones: IGHV use did not differ significantly between IgMκ⁺ TCL1 and IgMκ⁺ TCL1xAID clones. Approximately 50% used VH1-55, VH11-2, or VH12-3, some of which encoded stereotyped anti-phosphatidylcholine antibodies.

SHM: Among the IgM⁺ TCL1 clones, there was a mutation frequency of 0.05% for IGHV and 0.09% IGKV. In contrast, the IGHV and IGKV mutation frequencies were considerably higher in TCL1xAID mice. Specifically, for IGHV, the TCL1xAID animals displayed a frequency of 0.47% in IgM⁺ and 3.0% in IgG⁺ clones; for IGKV, the frequency was 0.9% for IgM⁺ and IgG⁺ combined. Consistent with these mutations being mediated by AID, mutations localized more frequently in AID hotspots than coldspots at a ratio of ≥7:1.

However, SHM did not affect all clones equally. Although the mutation frequency in VH12-3 and VH11-2 clones was higher (0.38%, range: 0-1.9%) than the TCL1 level (0.05%), it was considerably less than that found in the TCL1xAID clones using other IGHV s (0.80%, range: 0-3.8%). In addition, no mutations were detected in VH1-55 clones. Consistent with a reduced AID mutational activity in clones expressing these specific genes, none of the genes were found in the isotype-switched, IgG-expressing clones.

IGHV gene use and SHM in clones that underwent CSR: Notably, in only 2 of 9 instances was the same IGHV-D-J rearrangement found in IgMκ⁺ and IgGκ⁺ clones from the same mice; these IgM⁺ and IgG⁺ shared clones used VH5 genes. For the remaining 7, only the IgG⁺ version was detected; all but one of these used a VH1 gene. Also, within the IgG-only group, IGHV1-47 was used by 2 different clones and these were highly mutated (8.9%).

Stereotyped IGHV-D-J and IGκ V-Jκ rearrangements: Among 43 IgM clones from TCL1xAID mice, we found 8 clones expressing VH11-2 and Vκ14-126 and 10 clones expressing VH12-3 and Vκ4-91; these IGHV-D-J and IGκV-Jκ rearrangements were very similar to canonical anti-phosphatidylcholine-producing clones.

Summary and conclusions.Over-expression of AID in TCL1 mice leads to markedly increased SHM and CSR. However, SHM is not equivalent for all IGHV genes since despite AID over-expression certain IGHVs and IGKVs appeared less sensitive to major increases in the extent of SHM and the occurrence of CSR. This property resembles some human CLL IGHV s that rarely develop SHMs or undergo CSR despite the B-cell's ability to synthesize AID (e.g., many IGHV1-69⁺ clones). AID overexpression also led to IgG⁺ clones for which an IgM precursor was not found. This resembles those human stereotyped CLL clones that are only found as IgGs (e.g., stereotyped subsets 4 and 8). Finally, the two new TCL1xAID mouse strains described provide new models to study IGHV -mutated and IGHV -unmutated CLL and represent novel tools to evaluate the role of AID in leukemic progression.