The BCRs Expressed by Leukemia Cells from TCL1 Transgenic Mice Resemble Those of Unmutated B-CLL.

Introduction: Eμ-T cell leukemia-1 (TCL-1) transgenic (Tg) mice serve as models of human B-cell chronic lymphocytic leukemia (B-CLL). These animals develop oligoclonal expansions of CD5⁺ B cells, one of which transforms into a B-CLL-like cell at ~13 - 18 months of age. A major unanswered question is whether the IgV gene restrictions seen in the TCL1 Tg model resemble those identified in human B-CLL. Therefore we analyzed the DNA sequences of the expressed, rearranged VHDJH and VLJL from 11 TCL1 Tg mice for V gene use, association with specific D and J segments, and shared H and L CDR3 motifs.

Methods: Total RNA was isolated from spleens and lymph nodes of mice with obvious leukemia and reverse-transcribed to cDNA. To determine the Ig VH genes used by B cell clonal expansions, consensus FR1 primers and consensus JH primers were used for PCR. For Ig VL genes, Vκ consensus primers and Cκ primers were used. PCR products were either sequenced directly or cloned into vectors and then analyzed. DNA sequences were compared to the mouse Ig V gene germline genes deposited in NCBI GenBank and IMGT V-Quest. To confirm that nucleotide differences were actual point somatic mutations and not polymorphisms of known VH germline genes or heretofore unrecognized germline genes, PCR was performed on DNA from splenocytes of non-Tg mice using primers specific for the intron upstream of FR1 and the recombination signal sequences 3′ of the gene. PCR products were cloned and up to 60 colonies were sequenced. Using this approach, three new germline genes were identified and reported to GenBank. HCDR3 motifs were used to search both nucleotide and protein databases to identify similar sequences of known antigen specificity or B-cell subset origin.

Results: DNA sequences of the VHDJH and VLJL from all (n=11) TCL1 Tg mice studied were <2% different from the most similar germline counterpart. Eight of the 11 clones used VH 1 family genes and the other three used VH 3, 5 and 12 family genes. HCDR3 and LCDR3 of these sequences frequently contained charged amino acids at the V-(D)-J junctions. Database searches for sequences similar to those of the TCL1 clones revealed groups of non-B-CLL sequences with identical or very similar HCDR3 motifs; some of these groups used the same VH gene and others used different VH genes. These structurally similar antibodies were either autoantibodies or antibodies produced by B-1 cells. One anti-bacterial antibody also was included.

Conclusions: The clones that eventually become leukemic in TCL1 mice resemble those of human B-CLL cases with the worst clinical outcome in that they do not exhibit significant levels of Ig V gene mutations and they are structurally similar to autoantibodies and anti-microbial antibodies. Therefore, this model will be valuable in analyzing the development and progression of B-CLL cells from normal CD5⁺ B cells and the role that antigen-receptor engagement by autoantigens and microbial antigens plays in this process.