Lack of intraclonal diversification in Ig heavy and light chain V region genes expressed by CD5+IgM+ chronic lymphocytic leukemia B cells: a multiple time point analysis

Abstract

To analyze the modalities of clonal expansion of chronic lymphocytic leukemia (CLL) cells, we sequenced at multiple time points the V(D)J genes expressed by CD5+IgM+CLL B cells in three patients. All three V(D)J gene sequences were found to be point mutated. The mutation frequency in the Ig VH (3.96 x 10(-2) and 2.41 x 10(-2) change/bp) and Vkappa and Vlambda (6.67 x 10(-2) and 1.74 x 10(-2) change/bp) genes of two CLLs (1.19 and 1.32, respectively) was similar, and higher than that in the corresponding gene segments of the third CLL (1.69; 3.4 x 10(-3) and 6.67 x 10(-3) change/bp). In all three CLLs, there was no preferential representation of nucleotide changes yielding amino acid replacement (R mutations), nor was there any preferential segregation of R mutations within the Ig V gene complementarity-determining regions. In all three CLLs, the somatic mutations were all identical in multiple Ig VHDJH transcripts at any given time point, and were all conserved at multiple time points throughout a 2-yr period. The lack of concentration of R mutations in the complementarity-determining regions and the lack of intraclonal heterogeneity suggest that Ag may no longer be able to play a significant role in the clonal expansion of these cells. This conclusion would be strengthened further by the germline configuration of the bcl-1 and bcl-2 proto-oncogenes that are translocated in neoplastic B cells that display significant traces of intraclonal diversification and Ag-dependent selection, such as B-prolymphocytic leukemia and low grade follicular non-Hodgkin lymphoma.

FIGURE 1

Surface expression of CD19 and CD5 by CLL B cells. The T cell-depleted circulating lymphocytes from one healthy adult subject (1–0) and from three patients diagnosed with CLL (1.19, 1.32, and 1.69) were reacted with phycoerythrin-labeled mAb to CD5 and FITC-labeled mAb to CD19, and then applied to the FACS for analysis of their fluorescence intensities. B cells were identified by their high levels of expression of CD19. Greater than 99% of the B lymphocytes were surface CD5 positive in the three CLLs, compared with a proportion of 10 to 25% in the healthy subject.

FIGURE 2

Southern blot analysis of genomic CLL B cell DNA digested with BamHI, HindIII, and EcoRI restriction enzymes and hybridized with a radiolabeled human J_H genomic probe. HL60 denotes the germline position in each of the digests. In both CLLs 1.19 and 1.69, one germline and one Ig gene rearrangement were detected (monoclonal). In CLL 1.32, a double Ig gene rearrangement was detected (monoclonal).

FIGURE 3

Nucleotide and deduced amino acid sequences of the V_H gene segments expressed by the three CLL B cell clones (1.19, 1.32, and 1.69). Dashes indicate identities. The top sequence in each cluster is that of the germline gene to which the remaining sequences of the cluster are compared. Solid lines on top of each cluster depict CDRs. Lower case letters denote untranslated sequences. Sequences encompassed by the V_H leader and V_H6 23-bp spacer primers are underlined. These sequence data are available from EMBL/GenBank/DDBJ under accession numbers U31936, U31937, and U31962.

FIGURE 4

Nucleotide and deduced amino acid sequences of the D and J_H gene segments expressed by the three CLL B cell clones (1.19, 1.32, and 1.69). Each expressed sequence represents a composite sequence of the multiple time point and multiple transcript analysis. Dashes indicate identities. The nucleotide sequences of relevant portions of the germline D and J_H genes are given for comparison, and appear above or below the expressed Ig D and J_H gene segments as underlined strings. Unencoded nucleotides (N) are segregated 5′ and/or 3′ of the D gene segment. The deduced amino acid sequences are divided in CDR3 and FR4.

FIGURE 5

Nucleotide and deduced amino acid sequences of the V_LJ_L gene segments expressed by the three CLL B cell clones (1.19, 1.32, and 1.69). Dashes indicate identities. A, V gene segments; the top sequence in each cluster is that of the germline gene to which the remaining sequences of the cluster were compared. Solid lines on top of each cluster depict CDRs. Sequences encompassed by the V_L leader primers are underlined. B, J gene segments; the nucleotide sequences of relevant portions of the germline J genes are given for comparison, and appear above the expressed J gene segments as underlined strings. The deduced amino acid sequences are divided in CDR3 and FR4. These sequence data are available from EMBL/GenBank/DDBJ under accession numbers U31963, U31964, and U31965.

FIGURE 6

Bcl-1 and bcl-2 proto-oncogene gene rearrangement analysis. Southern blot analysis of genomic CLL B cell DNA (1.19, 1.32, and 1.69) digested with BamHI, HindIII, and EcoRI restriction enzymes and hybridized with specific probes. A, MTC on the HindIII digest for the major translocation cluster of bcl-1 and p94PS on both EcoRI and BamHI digests for the second break point of bcl-1. B, pFL-1 and pFL-2 on both HindIII and BamHI digest for the major and minor break points of bcl-2, respectively. HL60 cells (promyelocytic leukemia cell line) were used as controls displaying unrearranged bcl-1 and bcl-2 proto-oncogenes.