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The Journal of Immunology, 1998, 160: 820-830.
Copyright © 1998 by The American Association of Immunologists

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 Analysis1

Edward W. Schettino*, Andrea Cerutti*, Nicholas Chiorazzi{dagger} and Paolo Casali2,*

* Division of Molecular Immunology, Department of Pathology, Cornell University Medical College, and The Immunology Program, Cornell University Graduate School of Medical Sciences, New York, NY 10021; and {dagger} Department of Medicine, North Shore University Hospital and New York University School of Medicine, Manhasset, NY 11030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 V{kappa} and V{lambda} (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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic lymphocytic leukemia (CLL)3 is the most frequent form of adult leukemia in Western societies, accounting for 30% of all leukemias. In the recently proposed definition of the disease, the malignant B cells are surface CD5+ (1, 2), express mainly IgM, and are thought to represent the neoplastic expansion of a single clone that replaces the normal polyclonal B cell population in the peripheral blood (3, 4, 5, 6, 7, 8). CLL B cells putatively arise as a transformant of B-1a lymphocytes. In healthy humans, B-1a cells are committed mainly to the production of IgM, and account for 5 to 30% of the normal circulating, tonsillar, and splenic B lymphocytes (9, 10, 11). They also account for the majority of B cells in the fetus and neonate (10, 12, 13). B-1a cells have long been thought to be primordial elements that rearrange only a restricted selection of V(D)J genes, and that lack the machinery for Ig gene hypermutation. Consistent with this view, gained mainly from studies in the mouse (14, 15), CLL B cells have been thought to express a restricted set of Ig V(D)J genes, in general, in unmutated configuration (14, 15).

Recent findings from our laboratory, however, have shown that human B-1a cells can express different V(D)J genes in mutated configuration to encode for naturally occurring Abs and autoantibodies (16, 17, 18, 19, 20). Many of these somatically mutated Abs and autoantibodies display traces of an Ag-driven selection process that includes preferential segregation of somatic point mutations yielding an amino acid replacement (R mutations) within the complementarity-determining regions (CDRs), and various degrees of intraclonal diversification, as assessed by variation in the frequency and distribution of somatic point mutations among colinear Ig V(D)J gene sequences, that is different transcripts from the same clonotype. Thus, human B-1a lymphocytes rearrange different V(D)J genes, display the machinery necessary for somatic hypermutation, and can undergo a process of Ag-driven selection, somatic diversification, and, possibly, affinity maturation.

In light of the ability of normal B-1a cells to mutate their expressed Ig genes, we analyzed the sequences of V(D)J genes expressed by three CD5+IgM+CLLs for the presence of somatic point mutations, at multiple and sequential time points. We found that these leukemic B cells expressed Ig VHDJH and V{kappa}J{kappa} or V{lambda}J{lambda} genes that contained a number of somatic point mutations. These point mutations did not show any preferential enrichment for R mutations in the CDRs. In addition, they were absolutely conserved in multiple Ig cDNAs from the same time point and in different transcripts from multiple time points over a 2-yr period, and they were not associated with any translocation of the bcl-1 or bcl-2 proto-oncogenes. Thus, the absence of selection of R mutations together with the lack of V gene mutation over time in CD5+IgM+CLL cells suggests that Ag may no longer be capable of inducing clonal diversification in these leukemic cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
PBMCs and immunophenotyping

B lymphocytes were enriched from PBMCs by depletion of T cells and monocytes (21). Enriched B cells were reacted with FITC- or phycoerythrin-labeled mouse mAbs to human CD5, CD19, CD3, CD25, CD23, CD10, HLA-DR, {kappa} Ig, {lambda} Ig, IgG, IgM, IgD, or IgA (Coulter Immunology, Hialeah, FL, and Becton Dickinson Labware, Bedford, MA), in ice-cold sterile PBS, pH 7.4, containing 1% BSA and 1% human AB serum (Life Technologies, Gaithersburg, MD). After washing with the same buffer, the cells were applied to a Becton Dickinson FACScan fluorescence flow cytometer (Becton Dickinson, San Jose, CA) for analysis (21).

PCR amplification, cloning, and sequencing of expressed Ig V(D)J gene cDNA

mRNA was extracted from CLL B cells using the Mini RiboSep Ultra mRNA Isolation Kit (Becton Dickinson). First strand cDNA, synthesized using the SuperScript First Strand cDNA Synthesis Kit (Life Technologies) (16, 17, 22, 23, 24), was used as a template (100 ng) for PCR in a volume of 50 µl containing 200 mM of each dNTP, 2.5 U of Taq polymerase (Perkin-Elmer Cetus Corp., Norwalk, CT), and 10 pmol of each oligonucleotide primer. Six individual PCR amplifications were performed for the H chain. Each reaction included a sense leader VH primer, specific for the members of one of the six VH families, in conjunction with an antisense Cµ oligonucleotide primer. Each sense oligonucleotide primer consisted of a degenerate sequence encompassing an area of Ig gene leader region plus an EcoRI site, as follows: VH1, 5'-GGGAATTCATGGACTGGACCTGGAGG(AG)TC(CT)TCT(GT)C-3'; VH2, 5'-GGGAATTCATGGACATACT(GT)TG(GT)T(CT)CACGCT(CT)CT(GC)C-3'; VH3, 5'-GGGAATTCATGGAG(CT)TTGGGCTGA(CG)CTGG(CG)TTT(CT)T-3'; VH4, 5'-GGGAATTCATGAA(AG)CA(TC)CGTGGTTCTT(CT)(AC)T(CT)CT(CG)C-3'; VH5, 5'-ATGGGGTCAACCGCCATCCTCGCCCT-3'; and VH6, 5'-GGGAATTCATGTCTGTCTCCTTCCTCATCTTCC-3'. Due to the relatedness of the VH7 to the VH1 family (25), the VH7 family members can be amplified by the VH1 family primer. The H chain antisense oligonucleotide primer consisted of the reverse complement (5'-CCGAATTCAGACGAGGGGGAAAAGGGTT-3') of a 21-nucleotide Cµ sequence plus an EcoRI site. The L chains were amplified in five individual PCRs. Each PCR included a sense leader V{kappa} or V{lambda} primer specific for the members of the L chain gene families, in combination with an antisense C{kappa} or C{lambda} oligonucleotide primer. Each sense primer consisted of a degenerate sequence encompassing an area of the Ig gene leader region, as follows: V{lambda}1, 5'-ATG(GA)CC(TG)GCT(CT)CCCTCTCCTCCT-3'; V{lambda}2–6, 5'-ATG(AG)C(CT)TGGACCC(CT)(AT)CTC(CT)(TG)(TG)TT-3'; V{kappa}1,2, 5'-AGCTCCTGGG-GCT(GC)CT(AG)(AC)TGCTCT-3'; V{kappa}3, 5'-TCTCTTCCTCCTGCTACTCTGGCT-3'; and V{kappa}4, 5'-ATGGTGTTGCAGACCCAGGTCTTC-3'. The L chain antisense oligonucleotide primers consisted of the reverse complement of a 23-nucleotide C{lambda} sequence (5'-AGGAGACTCCTCGAAGTTCGGTT-3') or a 23-nucleotide C{kappa} sequence (5'-AGAAGGGCGGTAGACTACTCGTC-3'). PCRs for the amplification of both VH and VL gene cDNA consisted of 30 cycles of denaturation (95°C, 1 min), annealing (60°C, 1 min), and extension (72°C, 2 min). Amplified DNA was ligated into pCR II plasmid vectors (Invitrogen Corp., San Diego, CA), and dideoxy sequencing was performed using plasmid dsDNA prepared from selected bacterial clones, as reported (22, 24). Each V(D)J sequence was derived from the analysis of four to six independent bacterial isolates. Differences in nucleotide sequences among different recombinant clones were rarely observed, i.e., less than 0.0002 difference/base, a frequency that is consistent with the error rate of the Taq polymerase. The DNA sequences were analyzed using the BLAST algorithm, as found in the NCBI World-Wide-Web home page accessed through the Netscape Navigator. The MacVector v.5.0 sequence analysis software (International Biotechnologies, New Haven, CT) was used to analyze the current human Ig gene V-BASE database (MRC Centre for Protein Engineering, Cambridge, U.K.).

Genomic VH segment DNA analysis of CLL 1.19

Genomic DNA was extracted from the polymorphonuclear cells (PMNs) of patient 1.19, whose CLL B cells were used to generate the expressed 1.19 VH and VL gene sequences. The DNA was subjected to PCR amplification using the sense VH6 leader primer in conjunction with an antisense 23-bp primer, consisting of the reverse complement (5'-TTTGTGTCTGGGCTCACACTGACT-3') sequence of the 3' VH6 gene spacer-nonamer signal recognition sequence. PCR amplification of the germline VH6 gene consisted of 30 cycles of denaturation (95°C, 1 min), annealing (65°C, 1 min), and extension (72°C, 2 min).

Analysis of Ig V gene mutations

The number of expected R mutations in the Ig V segment CDRs and FRs was calculated using the formula R = n x CDR Rf or FR Rf x CDRrel or FRrel, in which n is the total number of observed mutations, Rf is the replacement frequency inherent to CDR or FR sequences, and CDRrel and FRrel are the relative size of the CDRs or FRs (26, 27). The CDR Rf and FR Rf inherent to the respective progenitor germline genes are as follows: V6-1, CDR = 0.7935, FR = 0.7404; V4-59, CDR = 0.8218, FR = 0.7217; DP-88, CDR = 0.7801, FR = 0.7477; 02/012, CDR = 0.7991, FR = 0.7533; lv318, CDR = 0.8128, FR = 0.7319; and A23, CDR = 0.7901, FR = 0.7463. A binomial probability model was used to evaluate whether the excess of R mutations in CDRs or their scarcity in the FRs was due to chance only: p = {n!/[k! (n-k)!]} x qk x (1-q)n-k, in which q is the probability that an R mutation will localize to CDRs or FRs (q = CDRrel x CDR Rf or FRrel x FR Rf), and k is the number of observed R mutations in the CDRs or FRs (26).

Clonality of the CLL B cells

In addition to the single PCR amplification product, the clonality of the CD5+ B cells was assessed by Ig gene rearrangement analysis using a genomic JH DNA probe on HindIII, EcoRI, and BamHI DNA digests (28). Briefly, B cell genomic DNA (5 µg) was digested with a fivefold excess of EcoRI, BamHI, or HindIII (Boehringer Mannheim Corp., Indianapolis, IN) in appropriate buffer, loaded onto a 0.8% agarose gel (Life Technologies), and electrophoresed at 22 V for 24 h. Size-fractionated DNA was transferred overnight onto Hybond-N nylon membranes (Amersham Life Sciences, Arlington Heights, IL) and prehybridized at 37°C for 4 h. Membranes were incubated overnight at 37°C in hybridization solution containing a {gamma}-32P-labeled 2.2-kb genomic JH DNA probe (28), and then washed four times before being autoradiographed at -70°C for 16 to 48 h with Kodak XAR film (Eastman Kodak Co., Rochester, NY).

bcl-1 and bcl-2 proto-oncogene configuration in the CLL B cells

To analyze the configuration of the bcl-1 and bcl-2 proto-oncogenes, the nylon membranes blotted with DNA from the CLL B cells, and previously hybridized with the {gamma}-32P-labeled JH DNA, were stripped of the JH probe, according to the manufacturer’s protocol, and then reacted with the following probes: MTC on the HindIII digest for the major translocation cluster of bcl-1 (29); p94PS on both EcoRI and BamHI digests for the second break point on bcl-1 (29); and pFL-1 and pFL-2 on HindIII and BamHI digests for the major and minor break points of bcl-2, respectively (30, 31). After four washings, the membranes were autoradiographed at -70°C for 16 to 48 h with Kodak XAR film.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotypic analysis and clonality of the CLL B cells

PBMCs were obtained from three male patients (69 to 75 yr of age: one African-American, 1.19; and two Caucasians, 1.32 and 1.69), who fulfilled the established diagnostic criteria for CLL (1, 2, 30, 32), ranging from Rai stage I to stage II (Table IGo). Direct immunofluorescence analysis of the T cell-depleted PBMCs from each CLL patient showed that virtually all of these cells were surface IgM+, {kappa}+ or {lambda}+, CD19+, HLA-DR+, and CD23+ (Table IGo), with less than 1% being CD3+ and/or CD10+ (data not shown). More than 99% of these CD19+ cells expressed surface CD5 at high density (Fig. 1Go), consistent with a virtually complete replacement of the PBL by the clonally expanded neoplastic B cells.


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  		Table I. CLL patients: medical history and cellular morphology

 


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  		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.

 
The monoclonality of these cell populations was verified by isolating genomic DNA from each of the CLL B cells and separately digesting it with BamHI, HindIII, and EcoRI for Southern blot hybridization analysis. The reaction of the filter-immobilized DNA with the radiolabeled genomic JH probe yielded, in each CLL, a hybridization pattern consistent with a monoclonal Ig gene rearrangement (Fig. 2Go). Consistent with a monoclonal B cell composition, only one of the six VH gene family primers, VH6, VH4, or VH1, yielded a product of appropriate size (approximately 500 bp) when used in conjunction with the antisense Cµ primer to amplify cDNA reverse transcribed from mRNA isolated from each CLL sample, 1.19, 1.32, and 1.69, respectively (Fig. 3Go). Accordingly, only one of the five L chain primers, V{kappa}1,2 or V{lambda}2–6, yielded an amplification product of appropriate size (approximately 350 bp) in each CLL sample when used in conjunction with the antisense C{kappa} or C{lambda} primer (data not shown).



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  		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 JH 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).

 


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  		FIGURE 3. Nucleotide and deduced amino acid sequences of the VH 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 VH leader and VH6 23-bp spacer primers are underlined. These sequence data are available from EMBL/GenBank/DDBJ under accession numbers U31936, U31937, and U31962.

 
Sequences of the CLL B cell VHDJHgenes

The PCR-amplified Ig VHDJH gene DNAs were cloned and sequenced. Each sequence was derived from the analysis of four to six independent bacterial isolates, that is, discrete Ig VHDJH gene cDNAs. The nucleotide and deduced amino acid sequences of the VHDJH gene segments are depicted in Figures 3Go and 4Go, and summarized in Table IIGo. The sequence of the Ig VH gene expressed by CLL 1.19 contained 12 nucleotide differences when compared with that of the germline V6-1 gene, the single member of the VH6 family; the sequence of the VH gene expressed by CLL 1.69 displayed one nucleotide difference when compared with that of the closest reported germline gene DP-88 (an allelic variant of the V1-69 gene) (33); and the sequence of the Ig VH gene expressed by CLL 1.32 displayed seven nucleotide differences when compared with the closest reported germline gene V4-59 (formerly referred to as VH4-15). These VH gene nucleotide differences were assumed to result from somatic point mutations because of the following considerations. First, the whole human Ig H chain locus has now been fully characterized, and all VH germline gene segments have been sequenced (34, 35, 36, 37). Like many other human VH genes, including V3-23 (formerly referred to as VH26) and V4-34 (formerly referred to as VH4.21), the V6-1 gene displays no allelic polymorphism, and recurs in an absolutely conserved form within each individual ethnic population (38, 39, 40, 41, 42). To further confirm the conserved nature of V6-1 in CLL 1.19, we utilized DNA from the patients’ autologous PMNs and a pair of primers encompassing sequences identical in the expressed 1.19 and the reported V6-1 gene to amplify an approximately 400-bp DNA segment for cloning. Sequences from multiple independent isolates were identical to each other and throughout the overlapping area to that of V6-1 (Fig. 3Go), therefore pointing at this gene segment as the template of the expressed and mutated 1.19. The remaining two germline genes, DP-88 and V4-59, putatively utilized by the CLLs analyzed in this study, have been reported to occur in polymorphic variants. DP-88 is one of 13 related variants (variant 7) of the V1-69 gene. Each of these variants has been sequenced, thereby producing a complete profile of this gene’s polymorphism (41, 42). The sequences of the V1-69 allelic set differ by a limited number of nonconservative substitutions that may have functional significance. Sasso and coworkers concluded that 6 of these 13 elements, variants 1, 5, 7, 10, 12, and 13, comprise the alleles present in most people (33, 41). When compared with the sequences of all V1-69 variants, the transition (C to T) at position 20 was found to be unique to the expressed CLL 1.69 VH gene segment, suggesting that this substitution that yields an amino acid replacement is the result of a somatic point mutation. The polymorphic variants of the V4-59 gene, although not yet fully characterized, are believed to consist of only few nucleotide differences. When compared with the sequences of all known V4-59 variants (38, 43, 44), each of the seven nucleotide differences detected in the expressed CLL 1.32 VH gene segment was found to be unique to the CLL B cell clone; thus, the likelihood that these differences were due to allelic polymorphism is highly improbable. Finally, the contention that the differences detected in the VH gene segment sequences expressed by 1.19, 1.32, and 1.69 CLL cells represented somatic mutations, as compared with their respective germline VH gene sequences, was supported further by the presence of point mutations in the sequences of the JH gene segments of CLLs 1.19 and 1.32, and in the sequences of the J{kappa} and J{lambda} gene segments of each CLL B cell clones.



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  		FIGURE 4. Nucleotide and deduced amino acid sequences of the D and JH 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 JH genes are given for comparison, and appear above or below the expressed Ig D and JH 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.

 

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  		Table II. Structure of the expressed CLL B cell Ig V(D)J genes

 
The nucleotide and deduced amino acid sequences of the expressed junctional VHDJH gene segments from the CLL B cell clones were compared with those of the reported germline D, DIR, and JH genes (Fig. 4Go) (45, 46, 47, 48, 49). The best-fit D and/or DIR genes were identified according to the two following criteria: first, priority was given to the VH or JH gene sequence when VH and D, or D and JH gene sequences overlapped; and second, if more than one candidate D and/or DIR gene was identified, the germline D gene sequence, displaying the longest stretch of identity (with a minimum match of six nucleotides in a stretch of 7 bp, or with a minimum match of five nucleotides in row) to that of the expressed D gene segment, was assigned. One of the three clones, 1.69, expressed a D segment that resulted from the utilization of a single germline D gene; the second, 1.19, resulted from the utilization of two different germline D genes, possibly as result of a D-D fusion, one of which was inverted; and the third, 1.32, from the utilization of three different D genes (Fig. 4Go and Table IIGo). Thus, the D gene segments expressed by the CLL B cells represented the rearrangement of a heterogeneous assortment of germline D genes in conventional, fused, or inverted configurations. Two of the three CLL-expressed D genes (1.19 and 1.32) were 5' flanked by nontemplated residues (N additions); and all but that encoding CLL 1.32 were also 3' flanked by nontemplated nucleotides. CLL 1.19 and 1.32 utilized JH6b and JH4b genes, respectively, that were both truncated and mutated (Fig. 4Go and Table IIGo). CLL 1.69 utilized a JH6b gene that was both intact and unmutated. The deduced amino acid sequences of the DJH segments of the three CLLs were divided into CDR3 and FR4 stretches (Fig. 4Go), according to Kabat et al. (50). The CDR3 sequences were highly divergent in composition and ranged in length from 15 to 22 amino acids; the FR4 sequences were invariable in length and displayed little diversity.

Sequences of the CLL B cell V{kappa}J{kappa} and V{lambda}J{lambda} genes

The nucleotide and deduced amino acid sequences of the VLJL gene segments expressed by the three CLL B cells are depicted in Figure 5Go, A and B, respectively, and summarized in Table IIGo. Like the VH gene locus, the human Ig {kappa}- and {lambda}-chain loci have been characterized recently in detail, and virtually all germline V{kappa} and V{lambda} gene segments have been sequenced (50, 51, 52, 53), thus providing a representative assortment of unmutated germline templates to which the expressed V{kappa} and V{lambda} genes can be compared.



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  		FIGURE 5. Nucleotide and deduced amino acid sequences of the VLJL 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 VL 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.

 
The Ig V{kappa} gene expressed by CLL 1.19 B cells was of the V{kappa}1 subgroup, and its sequence contained 19 somatic point mutations as compared with that of the germline gene 02/012. The CLL 1.69 Ig V{kappa} gene sequence contained two somatic point mutations as compared with that of the V{kappa}2 germline gene A23. Finally, the CLL 1.32 Ig V{lambda} gene sequence contained five somatic point mutations as compared with that of the V{lambda}3 germline lv318 gene.

Comparison of the expressed Ig JL gene nucleotide and deduced amino acid sequences with those of the germline J{kappa} and J{lambda} genes (Fig. 5GoB) showed that CLL 1.19 utilized a mutated J{kappa}5 gene; CLL 1.69 utilized a mutated J{kappa}2 gene; and CLL 1.32 utilized a mutated J{lambda}2/J{lambda}3 gene that was 5' flanked by a CCT triplet coding for a Pro residue. This additional CCT triplet could have resulted from the direct juxtaposition of the two deoxcytosine nucleotides (CC) 5' of the heptamer/nonamer sequences of the donor V{lambda} gene segment and the deoxythymidine residue (T) 3' of the heptamer/nonamer sequences of the donor J{lambda} gene segment (53).

Analysis of the somatic point mutations in the CLL B cell Ig V(D)J genes

In the absence of negative or positive selective pressure on a gene product, R and S mutations distribute randomly throughout the coding sequence. If a DNA segment displays a number of R mutations higher than that expected by chance alone, it is likely that a positive pressure was exerted on the gene product to select for those mutations, as it occurs in the V segment CDRs of affinity mature Abs in which nucleotide changes yield a high R:S mutation ratio. Conversely, if a DNA segment displays a number of R mutations lower than that expected by chance, it is likely that a negative pressure was exerted on the gene product to select against mutations such that the protein structure is preserved, as it is in the FRs of a functional Ab, in which nucleotide changes yield a low R:S mutation ratio. We calculated the number of expected R mutations in the CLL VH and V{kappa} or V{lambda} gene segments, as proposed by Chang and Casali (26). In our calculations, the general replacement frequency (Rf) value of 0.75, originally calculated by Jukes and King (54) for random hypermutation of a gene product that need not be conserved in structure, was substituted with the Rf values derived from the analysis of each individual germline VH, V{kappa}, or V{lambda} gene sequence, as listed in Materials and Methods. The introduced correction yields a more accurate estimate of the frequency of R mutations expected by chance only, given the demonstration by Chang and Casali (26) that a significant number of human Ig V gene segments display a CDR codon composition that is inherently more prone to R mutations.

The observed distribution of the R and S mutations in the VH and VL gene segment CDRs and FRs is summarized in Table IIGo, which also reports the expected number of R mutations in the CDRs and FRs. In the CLL 1.19 B cells, the expressed VH gene contained five R mutations, two of which were located in the CDR2, and seven S mutations, all of which were located in the FRs, yielding an R:S ratio of 2:0 in the CDRs and 3:7 in the FRs; the V{kappa} gene contained 11 R mutations, six of which were located in the CDRs, and five S mutations, two of which were located in the FRs, yielding an R:S mutation ratio of 6:3 in the CDRs and 5:2 in the FRs. In CLL 1.32 B cells, the expressed VH gene contained three R mutations, all of which were located in the FRs, and four S mutations, three of which were located in the FRs, yielding an R:S mutation ratio of 0:1 in the CDRs and 3:3 in the FRs; the V{lambda} gene contained four R mutations, three of which were located in the CDRs, and one S mutation located in the FR1, yielding an R:S mutation ratio of 3:0 in the CDRs and 0:1 in the FRs. In the CLL 1.69 B cells, the expressed VH gene contained only one R mutation located in the FR1, yielding an R:S mutation ratio of 0 in the CDRs and 1:0 in the FRs; the V{kappa} gene contained one R mutation, located in the FR1, and one S mutation, located in the CDR3, yielding an R:S mutation ratio of 0:1 in the CDRs and 1:0 in the FRs. In all three VH genes, the number of R mutations in the CDRs was lower than expected (Table IIGo). In one VL gene segment (1.69), it also was lower then expected; in the two others (1.19 and 1.32), it was higher than expected (Table IIGo); however, this excess can be due to chance only. Thus, these results demonstrate that in each of the CLLs analyzed, both the mutated Ig VH and VL gene segments did not show any preferential enrichment for R mutations in the CDRs. In addition, in all CLL VH and VL gene segments but one (1.19 VH), the number of R in the FRs was not different from that expected on the basis of chance only. In the 1.19 VH gene segment, the scarcity of R mutations in the FRs reached a level of significance, demonstrating that a negative pressure was probably exerted on the gene product so that the protein structure is preserved.

Lack of intraclonal diversification in the CLL B cell clones

To determine whether selective forces were influencing the clonal expansion of the three CLL B cells in vivo at times different from that initially considered, we analyzed the intraclonal diversity of each case by selecting multiple time points during a 11/2- or 2-yr time period. The expressed Ig VHDJH gene from CLL 1.19 was analyzed over a 1–1/2-yr period at four separate time points (Figs. 3Go and 4Go, and Table IGo). Each of the VH gene segments as well as the sequences of the CDR3 and FR4 were absolutely identical at each of the four time points, and no additional point mutations were present. The expressed Ig VHDJH genes from CLLs 1.32 and 1.69 were both analyzed over a 2-yr period by selecting three separate time points (Figs. 3Go and 4Go, and Tables I and II). Like CLL 1.19, all of the somatic point mutations in the VH gene segment (1.32 and 1.69, respectively), as well as the sequences encoding the CDR3 and FR4, were absolutely identical at each of the three time points, and no additional point mutations were observed.

bcl-1 and bcl-2 proto-oncogene configuration

Translocation of the bcl-1 and bcl-2 proto-oncogenes has been associated with other B cell neoplasia that display significant traces of intraclonal diversification and Ag-dependent selection of somatic point mutations. bcl-1 is translocated in mantle zone lymphoma and B-prolymphocytic leukemia (2), and bcl-2 is translocated in low grade follicular non-Hodgkin lymphoma (55). In both cases, translocation of these proto-oncogenes has been associated with the high degree of clonal expansion and cellular division characteristic of these B cell disorders. To analyze whether these proto-oncogenes were translocated in the three CLLs, B cell genomic DNA from each of the CLL B cells was subjected to Southern blot analysis using four different probes for either the bcl-1 or bcl-2 loci, as detailed in Materials and Methods. The hybridization pattern was consistent with that of both proto-oncogenes being in germline configuration (Fig. 6Go).



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  		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.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies show that IgM+CLL B cells can express Ig V(D)J gene segments containing variable numbers of somatic point mutations. They also show that these mutations do not display any preferential enrichment of R changes within the CDRs, and, in each CLL patient, they are absolutely conserved not only in multiple Ig cDNAs from the same time point, but also throughout multiple time points over a 2-yr period. The scarcity of R mutations in the CDRs, and absolute conservation of the sequences of the Ig V(D)J gene cDNA transcripts among different B cells of the same clonotype, are consistent with an absence of intraclonal diversification. These findings were derived from the analysis of three patients diagnosed with CD5+IgM+CLL, each of whom fulfilled the established clinical and morphologic diagnostic criteria for the disease, including B cell surface expression of both CD5 and CD23 at high density. The CLL nature and the Ag-independent emergence of these neoplastic clones were strengthened further by the germline configuration of the bcl-1 and bcl-2 proto-oncogenes, which translocate (to chromosome 14) in neoplastic B cells undergoing a high degree of intraclonal somatic diversification and selection by Ag, such as in mantle cell lymphoma and follicular lymphoma (56, 57).

The first issue addressed by these studies was whether (IgM+) CLL B cells can express somatically mutated Ig V genes. In each of the three B cell clones, the expressed VH gene segment sequences displayed a number of differences as compared with those of the closest reported germline genes V6-1, V4-59, and DP-88. The probability that these differences stemmed from the utilization of heretofore unreported germline gene was estimated to be negligible in each of the three CLL VH gene segments due to the following considerations. V6-1 has been shown to be among the most conserved Ig VH genes throughout the human population. V4-59 has been reported to be polymorphic, but the allelic variants in this gene have been shown to consist of only few nucleotide differences (38, 39). DP-88 is one of the 13 related variants of the V1-69 gene. Each of these variants has been sequenced, thereby producing a complete profile of this gene’s polymorphism (41, 42). The contention that the expressed Ig VH genes constitute somatically mutated forms of V6-1, V4-59, and DP-88 templates is supported further by the presence of somatic mutations in the juxtaposed D and JH genes, and by the isolation of a VH gene template identical to the germline V6-1 gene from the genomic PMN DNA of patient 1.19. In each B cell clone, the load of somatic mutations in the expressed Ig VHDJH gene segment was accompanied by a comparable load of putative somatic mutations in the paired L chain variable segments. For instance, the CLL 1.19 B cell V{kappa} gene sequence displayed 19 nucleotide differences when compared with that of the closest reported {kappa}-chain germline gene, 02/012; the CLL 1.32 B cell V{lambda} gene sequence displayed five nucleotide differences when compared with that of the closest reported {lambda}-chain germline gene, lv318; and the CLL 1.69 B cell V{kappa} gene sequence displayed two nucleotide differences when compared with that of the closest reported {kappa}-chain germline gene, A23. Like their H chain counterparts, the J{kappa} and J{lambda} genes were also mutated.

The next question asked by our experiments was whether the nature and the distribution of the R mutations in the CLLs Ig V segment were consistent with a selection by Ag. Ag-selected Abs have been shown to include a higher frequency of R mutations in the Ig V gene CDRs than in the FRs, in which the proportion of S mutations may be greater. The recent findings by Chang and Casali (26) suggest that when assessing the Ag-selected nature of somatic point mutations in an expressed Ig V gene, the inherent susceptibility to amino acid replacement or replacement frequency, Rf, needs to be calculated for the progenitor germline gene sequence. The Rf is then used to calculate the theoretically expected number of R mutations in the CDRs or FRs of that particular gene given a random distribution of R mutations. This principle was applied to the analysis of the Ig V genes of the three CLL subjects. For each expressed H or L chain V gene and in each CLL patient, the number of expected CDR and FR R mutations was calculated, and used to determine, on the basis of a binomial distribution model, the probability that any excess and scarcity of R mutations in the CDRs and FRs were due to chance only. The results of these calculations clearly indicated that in none of the three CLL B cell H and L chain V segments was there evidence for positive selection of R mutations in the CDRs, a result, in general, of a process of Ag-driven clonal selection. In some cases, it could be hypothesized that a negative, rather than a positive, selective pressure applies to R mutations, that is, the unmutated product rather than a somatically mutated form of the germline gene is selected by Ag. This is a distinct possibility (58), but has never been experimentally substantiated in vivo. Even in such a case, however, negative pressure on R mutations in CDRs should be accompanied by a similarly negative pressure in the FRs to preserve a structurally sound Ab molecule. Thus, a significant scarcity of FR R mutations should be an important feature of Ag-selected Ab-producing cell clones. The present findings show, with the exception of the CLL 1.19 VH gene segment, no significant negative selection of R mutations in the FRs of the Ig H and L chain V segments expressed by the three CLL B cell clones.

As shown by the study of a variety of specific experimental Ab responses, Ag-driven selection and expansion of a B cell clone not only result in positive selection of R mutations in the Ig V segment CDRs, but also lead to a significantly high degree of intraclonal diversification, revealed at the DNA transcription level by the appearance of colinear Ig V(D)J DNA sequences sharing and differing in various numbers of somatic point mutations. Intraclonal diversification has been shown in neoplastic equivalent of GC B cells, such as follicular lymphoma (57, 59, 60), and one patient with CD5-CLL (61). In these cells, as in normal cells undergoing specific expansion and selection by Ag, somatic diversification has been found to be associated with positive selection of CDR R mutations and negative selection of FR R mutations. To better verify whether selective forces were influencing the biologic behavior of the CLL clones in vivo, we addressed the issue of intraclonal diversification by not only analyzing multiple independent bacterial isolates from a given time point, but by also examining multiple time points of interest during a 11/2- to a 2-yr period. By following these clones individually through their natural history in vivo, we failed to detect any nucleotide variation throughout our time point analysis study, therefore concluding that these CLL B cells lacked traces of intraclonal diversification. Lack of intraclonal diversification in CLL B cells is supported further by Fais et al. (62, 63), who demonstrated that clonally related IgG- and IgA-switched progeny of IgM+CLL B cells fail to accumulate appreciable numbers of new somatic mutations in their Ig V genes. However, IgM+ progenitors of IgG+CLL B cells retain the ability to accumulate somatic mutations (64), presumably because they have yet to receive the final "hit" in the transformation process. The occurrence of switching without the accumulation of V gene mutations suggests that the processes of differentiation and diversification are not necessarily linked, and that in CLL B cells, clonal differentiation can occur in the absence of V gene mutation.

Taken together, our findings suggest that antigenic stimulation is unlikely involved in the clonal diversification of our panel of CD5+IgM+CLL B cells. They differ from those by Hashimoto and coworkers (65), who detected a high number of somatic mutations in the expressed Ig H chain genes in two (CLLs 055 and 030) of seven IgG+CD5+CLLs, as compared with their respective germline genes (V4-34 and H11, respectively). The distribution of R mutations in both of these CLL IgG+ clones was consistent with selection by Ag. Thus, different selective forces are present in IgG-producing CLL cell, as discussed by Hashimoto et al. (65), as compared with IgM-producing CLL cells, as shown by this study.

The different selective pressures that are applied to IgM+ and IgG+ CLLs need to be further investigated. The majority of studies on CLL suggest that the B cells are clonally expanded, and use a biased set of Ig V genes, which are generally unmutated, to encode for low affinity, polyreactive autoantibodies (8). These findings are consistent with the general unmutated nature of the putative non-neoplastic CLL progenitors, B-1a cells. Recent findings by us and others (16, 17, 18, 19, 20), however, have demonstrated that normal human B-1a lymphocytes can produce somatically mutated and Ag-selected Abs, indicating that these cells do indeed possess the machinery for somatic hypermutation, and can undergo an affinity maturation process, and therefore suggesting that CLL B-1a cells may also be able to mutate the expressed Ig V(D)J genes. By indicating that mutations can occur in the V genes of both H and L chains, and that the numbers and locations of these mutations are closely paralleled in individual patients, our findings in IgM+CLLs extend those by Hashimoto et al. in IgG+CLLs (65). However, unlike these previous studies in IgG+CLL, they did not show evidence of a pattern of Ag selection of R mutations, nor did they show any evidence of intraclonal diversification over a significant period of the disease. It is thus unlikely that the reported somatic mutations are inherent to the clonal evolution of CLL, but rather represent a pre-existing feature of the normal B-1a cell clones "hit" by the transforming event(s).


   Acknowledgments
 
We are grateful to Drs. E. Newcomb and M. Potmesil for providing the CLL samples. We thank Dr. A. Matolscy for his help with the proto-oncogene analysis, and Ms. N. Pacheco for providing her skillful technical assistance.


   Footnotes
 
1 This work was supported by U.S. Public Health Service Grants CA 68541 and AR 40908 to P.C., and AI 10811 to N.C. Back

2 Address correspondence and reprint requests to Dr. Paolo Casali, Division of Molecular Immunology, Department of Pathology, Cornell University Medical College, 1300 York Avenue, New York, NY 10021-4896. Back

3 Abbreviations used in this paper: CLL, chronic lymphocytic leukemia; CDR, complementarity-determining region; FR, framework region; H chain, heavy chain; L chain, light chain; PMN, polymorphonuclear cell; R, replacement (mutation); S, silent (mutation). Back

Received for publication June 10, 1997. Accepted for publication September 26, 1997.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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