No abstract

Chronic lymphocytic leukemia (CLL) usually involves the expansion of a clone of CD5ϩ B cells synthesizing IgM antibodies. These B cells appear to be blocked at the antigen receptor-expressing stage of B cell differentiation and are thought not to undergo an isotype class switch to IgG or IgA production. In vivo and in vitro studies suggest, however, that in some instances terminal differentiation and isotype switching can occur.To test the hypothesis that in vivo isotype class switching occurs in IgM

Peripheral blood mononuclear cells from five patients with IgG+ B-type chronic lymphocytic leukemia (B-CLL) were analyzed for the presence of clone-specific Ig H chain variable region gene mRNA transcripts linked to C mu and/or C alpha. This was assessed by (1) comparing the lengths of portions of the VHDJH of the IgG+ CLL clones with those of the mu and alpha isotype-expressing B cells, (2) performing clone-specific endonuclease digestion studies, and (3) determining the DNA sequences of the mu and alpha isotype-expressing cDNA. Thus, when B-cell mRNA from these five patients were reverse transcribed with C gamma-specific primers and then amplified by polymerase chain reaction, dominant cDNA were found with lengths corresponding to those of the IgG+ CLL B cell. In addition, in four cases, cDNA of lengths identical to those of the CLL B cell were detected when mRNA was reverse transcribed and amplified using c mu- and/or C alpha-specific primers, strongly suggesting clonal relatedness. These CLL-related mu- and alpha- expressing cDNA were present in greater amounts that unrelated (non- CLL) mu- and alpha-expressing cDNA from normal B cells that used genes of the same VH family. When the sequences of these CLL-related C mu- and C alpha-expressing cDNA were compared with those of the IgG+ CLL clones, it was clear that they were derived from the same ancestral gene as the IgG-expressing CLL B cell, thus documenting their common origin. Finally, nucleotide point mutations were observed in the mu- and alpha-expressing cDNA of certain patients, indicating divergence with the CLL. These data suggest that IgM+ B cells, which are precursors of the leukemic B cells, exist in increased numbers in the blood of most patients with IgG+ B-CELL and that these cells may differentiate, accumulate V genes mutations, and undergo isotype switching in vivo. In addition, the data are consistent with a sequential-hit model for the evolution of CLL.

In the present study, the complete sequences of the Ig H and L chain variable region genes of twelve RF+ B cell lines from two patients with RA were analyzed. Seven of the RF-producing B cells used VH3 family genes, four used VH4 genes, and one a VH1 gene. All but two of the cell lines expressing VH3 genes utilized different family members; among the VH4-expressing cells, a more restricted pattern was noted. V kappa gene use was restricted to the V kappa I and III families; V lambda gene use was more diverse, involving five different families. Computer comparisons of the expressed VH genes with their presumed germline progenitors indicated significant differences in every instance; eight of the corresponding VL genes also were significantly different. In many cases, assignment of the germline D segment(s) incorporated into the rearranged VH genes was impossible. These differences from the germline gene segments indicated the extensive changes induced by rearrangement, enzymatic activities, and somatic mutation. In hopes of defining a structural reason for the disparate antigen specificities of these cells, the CDR3 amino acid sequences of the multi- vs. the mono-reactive RF-producers were compared. Although CDR3 length was not appreciably different between these two sets of mAb, a greater than two-fold increase in charged amino acids was found in the H chain CDR3 of the multireactive RF. This relationship did not exist for the L chain CDR3. Thus, these sequence data indicate the use of a broad base of Ig V gene segments that have undergone extensive diversification. Based on the localization of R substitutions in the CDR of most of the V genes studied, the diversification appears to be antigen driven and selected. The significance of these findings for the evolution of these B cell clones into isotype-switched producers that are heterogeneous for antigen specificity (mono- vs. multi-reactivity) is discussed.