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Blood, 1 November 2004, Vol. 104, No. 9, pp. 2879-2885.
Prepublished online as a Blood First Edition Paper on June 24, 2004; DOI 10.1182/blood-2004-01-0132.
 Previous Article | Table of Contents | Next Article 
NEOPLASIA
Subsets with restricted immunoglobulin gene rearrangement features indicate a role for antigen selection in the development of chronic lymphocytic leukemia
Gerard Tobin,
Ulf Thunberg,
Karin Karlsson,
Fiona Murray,
Anna Laurell,
Kerstin Willander,
Gunilla Enblad,
Mats Merup,
Juhani Vilpo,
Gunnar Juliusson,
Christer Sundström,
Ola Söderberg,
Göran Roos, and
Richard Rosenquist
From the Departments of Genetics and Pathology and of Oncology, Radiology, and Clinical Immunology, Uppsala University, Uppsala, Sweden; the Department of Hematology, Linköping University Hospital, Linköping, Sweden; the Department of Biomedicine and Surgery, Linköping University, Linköping, Sweden; the Department of Hematology at Karolinska University Hospital, Huddinge, Sweden; the Department of Clinical Chemistry at Tampere University Hospital, Tampere, and Helsinki University Central Hospital (HUSLAB), Helsinki, Finland; and the Department of Medical Biosciences, Pathology, Umeå University, Umeå, Sweden.
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Abstract
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We recently identified a chronic lymphocytic leukemia (CLL) subgroup using the immunoglobulin variable heavy-chain (VH) gene VH3-21 with almost identical heavy-chain complementarity determining region 3s (HCDR3s) and preferential variable light-chain (VL) gene usage, suggesting recognition of a common antigen epitope in this subset. To further explore the B-cell receptors (BCRs) in CLL, we characterized 407 VH rearrangements amplified from 346 CLLs regarding VH, diversity (D), and joining (JH) gene usage and performed multiple alignment of the HCDR3 sequences. These analyses revealed 3 small subsets (2 VH1-69 groups, 7 cases; and 1 VH1-2 group, 5 cases) with highly restricted HCDR3 features including identical VH/D/JH usage, HCDR3 lengths, and shared N-sequences, in addition to the VH3-21 group (22 cases). Furthermore, another 3 groups (9 VH1-3+ cases, 3 VH1-18+ cases, and 5 VH4-39+ cases) had essentially identical VH/D/JH use and similar HCDR3 lengths but less conserved N-regions. Analysis in all 6 of these subgroups showed restriction in VL gene use, whereas no association between VH and VL usage was found in cases without HCDR3 similarities. Altogether, structurally similar HCDR3s associated with preferential VL gene usage implies selection of BCRs, especially in subsets showing high HCDR3 similarities, thus pointing to restricted antigen recognition sites and possibly involvement of specific antigens in CLL development.
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Introduction
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The recombination of the immunoglobulin (Ig) genes during B-cell development is a key step in generating the repertoire of antibody diversity in the immune system,1 and the large number of combinatorial possibilities of the different heavy-chain variable (VH), diversity (D), and joining (JH) gene segments in the Ig heavy-chain locus and light-chain variable (VL) and joining (JL) gene segments in the 2 light-chain loci constitute the base for this diversity.2,3 During V(D)J recombination, additional diversity is provided by random insertion/deletion of nucleotides in the D-JH, VH-DJH, and VL-JL junctions and by the usage of 3 different reading frames for the D gene, hence creating the most hypervariable regions for antigen binding (ie, the heavy- and light-chain complementarity determining region 3 [CDR3]). Furthermore, the final affinity maturation of the Ig molecules occurs in the germinal center during an antigen response, which can generate high-affinity antibodies by introduction of somatic hypermutation in the Ig gene rearrangements. Thus, the rearrangements of the Ig genes will provide each B-cell with a highly unique B-cell receptor (BCR) and can therefore serve as tumor markers in B-cell malignancies.4
B-cell chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries and is characterized by a continuous expansion of CD5+/CD19+/CD23+ monoclonal B cells. In this clinically heterogeneous disease, analysis of the somatic hypermutation status of the VH genes has proven to be a powerful prognostic predictor with mutated cases showing almost twice as long overall survival compared with patients with tumor cells lacking somatic hypermutation.5-10 Analysis of VH gene usage has revealed a biased repertoire with particular VH genes preferentially used compared with normal B cells, where the most prevalent are the VH1-69, VH3-07, VH3-21, and VH4-34 genes.6,11-18 The VH1-69+ rearrangements in CLL were previously reported to display molecular peculiarities such as longer than average heavy-chain CDR3s (HCDR3s;  19 amino acids) with skewed D3-3 and JH6 gene usage compared with normal B cells.13,14,17,18 This finding of preferential VH, D, and JH gene usage as well as similar amino acid motifs in the HCDR3s in this subset of CLL led to the speculation of a possible antigen component involved in the pathogenesis of CLL with the hypothesis that BCRs encoded by specific VHDJH combinations could generate Ig molecules with affinity to similar antigenic epitope(s).13-15
Recently, we demonstrated that CLL cases with tumor cells rearranging the VH3 family gene, VH3-21, showed poor overall survival,16,18 which has also lately been confirmed by others.19,20 The VH3-21 subset (11% of our CLL cohort) displayed predominantly mutated VH genes but still had an inferior clinical outcome and therefore did not fit into the prognostic classification scheme of mutated or unmutated CLL.18 In addition, the VH3-21 cases showed certain molecular characteristics with shorter than average HCDR3s (10 amino acids) and almost identical HCDR3 sequences with a conserved motif (ala-arg-asp-ala-asn-gly-met-asp-val) and a highly restricted usage of the V 2-14 gene in most cases.16,18 Considering that the CDRs of both the heavy- and light-chain V region comprise the antigen-binding site of the Ig molecule and that the HCDR3 is believed to contribute the most diversity in this respect,21 the finding of similar HCDR3s coupled with V2-14 gene usage indicated a common antigen-binding site in the BCRs of VH3-21+ CLL and suggested stimulatory influence of an unknown antigen in the disease development.
To further investigate signs of antigen selection in CLL, we studied the BCRs by analyzing the VH, D, and JH gene usage as well as performing multiple sequence alignment of the HCDR3 amino acid sequences obtained from 368 functional VH gene rearrangements amplified in 346 CLL cases. Furthermore, we carried out VL gene analysis in selected groups. Our data reveals further subsets of CLL with restricted HCDR3 features combined with preferential VL gene usage, thus indicating that BCRs with similar structures of the antigen-binding site were selected, which further corroborates the possibility of antigen involvement in CLL development.
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Patients, materials, and methods
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Patients and materials
Tumor samples were collected from 346 patients with CLL that had been identified from the archives of frozen tissue specimens at the University Hospitals of Uppsala (n = 164), Linköping (n = 79), Umeå (n = 51), and Huddinge (n = 19) in Sweden and at Tampere University Hospital (n = 33) in Finland, between 1981 and 2001. Frozen tumor material was obtained mainly from peripheral blood and bone marrow but in a few cases also from lymph nodes, spleen, and other sources. Morphologic classification was performed according to the World Health Organization (WHO) classification, and tumor cells typically expressed CD5 and CD23 and had weak expression of Ig.22 The median age at diagnosis was 65 years (range, 36-88 years) for the entire cohort, with a male-to-female ratio of 2:1.
PCR amplification and nucleotide sequence analysis
DNA was prepared from fresh-frozen tumor material using standard protocols including proteinase K treatment. VH and VL gene family-specific polymerase chain reaction (PCR) amplification was performed using consensus VH/JH primers and VL/JL primers as previously described.23,24 The sequence reactions were performed using either the BigDye Terminator Cycle Sequencing Reaction kit (Applied Biosystems, Foster City, CA) or the DYEnamic ET Dye Terminator Kit (Amersham Biosciences, Piscataway, NJ) and analyzed with an automated DNA sequencer (ABI 377 or ABI3700, Applied Biosystems; and MegaBACE 500 DNA Analysis System, Amersham Biosciences). The sequences were aligned to Ig sequences from the GenBank,25 V-BASE,26 and IMGT27 databases. VH sequences deviating more than 2% from the corresponding germ line gene were defined as mutated. For D gene determination, a requirement of a minimum of 7 matching nucleotides was used. The length of the HCDR3 was calculated between codons 95 and 102 as previously described by Kabat.28 All in-frame VH rearrangements were converted into amino acid sequences and the HCDR3 sequences were aligned in the multiple sequence alignment software Clustal X (1.83) for Windows (UBC Bioinformatics Centre, Vancouver, BC, Canada). Selection of VH groups with restricted HCDR3s was performed using the following criteria: (1) the group members had same VH gene usage, (2) the VH gene rearrangements showed at least 60% mean homology between the HCDR3s, and (3) the group contained at least 3 CLL cases. The numbering of cases in the figures and Table 3 refers to the order in which the VL gene PCR was performed.
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Results
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VH gene usage and somatic hypermutation status
A total of 407 VH gene rearrangements were PCR amplified and sequenced from 346 CLL cases, of which 294 cases displayed 1 rearrangement, 52 cases displayed 2 rearrangements, and 3 cases displayed 3 rearrangements. In total, 39 VHDJH sequences were either out of frame or had a stop codon introduced in the HCDR3. The VH family gene usage was as follows: VH1 (28%); VH2 (2%); VH3 (47%); VH4 (19.5%); and VH5, VH6, and VH7 (< 2% each). In this CLL cohort, 7 VH genes accounted for approximately 50% of the total VH genes rearranged: VH1-69 (14%), VH3-21 (9%), VH4-34 (7%), VH3-30 (6%), VH3-23 (5%), VH4-39 (4%), and VH1-2 (4%) (Table 1). Of the 51 functional VH genes, 49 were represented in this material. Using the 2% mutation border, 146 (42%) cases were considered mutated with a mean mutation frequency of 5.8% (range, 2.1%-13.2%), and 200 (58%) cases were unmutated. In terms of mutated CLL cases, the most frequent VH gene rearranged was VH3-21 (24 cases), whereas VH1-69 (54 cases) represented the most frequent VH gene in unmutated cases (Table 1).
D and JH gene usage
The D gene segment rearranged was identified in 341 of 407 VH gene rearrangements with 5 genes representing 57% of the rearranged D genes: D3-3, D6-19, D2-2, D3-22, and D3-10 (Figure 1). The identification of a D gene segment was possible in 94% of the unmutated rearrangements but in only 64% of the mutated rearrangements. The D genes D3-3, D2-2, and D6-19 represented 49% of the rearrangements in the unmutated VH gene rearrangements, whereas a more diverse spread was seen in the mutated rearrangements. JH4 and JH6 were the most frequently rearranged JH genes (71%, 287 of 365 identified JH genes; Figure 1) in both the mutated and unmutated subsets.
Identification of groups with restricted HCDR3 features and analysis of their VL gene usage
In this material, there were 13 different VH/D/JH combinations that were present in 3 or more cases (data not shown). However, considering that the preferential usage of certain VH/D/JH combinations does not necessarily indicate that the HCDR3s will show similarities at the amino acid level, since the D segment can be used in all 3 reading frames and as the rearrangements can display different N-regions, we decided to perform cluster analysis of the HCDR3 amino acid sequences only to identify subsets with restricted HCDR3 structures using a particular VH gene, as have been shown for the VH3-21 group. Thus, the translated amino acid sequences of the HCDR3s from 368 functional VH gene rearrangements were collected into one file and multiple sequence alignment analysis was carried out. From this analysis, we could identify 4 groups using the VH3-21 (22 cases), VH1-69 (2 groups, 4 and 3 cases, respectively), and VH1-2 (5 cases) genes with high similarities (75%-95% mean homology) in their HCDR3s (designated as "Groups with highly restricted HCDR3s"; see Table 2). In addition, another 3 groups with VH1-3 (9 cases), VH1-18 (3 cases), and VH4-39 (5 cases) gene usage showed restricted features but with less HCDR3 homology (60%-75%; designated as "Groups with moderate HCDR3 homology"; see Table 2). The largest group contained 22 VH3-21 cases with highly restricted HCDR3s, 19 of which have been published previously and will therefore not be shown further.18 All amino acid sequences are illustrated in Figure 2 in their respective groups. None of the groups showed evidence of somatic hypermutation in their VH regions. The nucleotide sequences from the VH gene rearrangements are available in GenBank25 at accession numbers AY486162
[GenBank]
-AY486231.
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Figure 2.. Alignment of amino acid sequences in groups with restricted HCDR3s. Alignment of amino acid sequences from the VH gene and VL gene rearrangements in the 3 groups with highly restricted HCDR3s (the VH3-21 alignment has been published elsewhere18; A) and the 3 groups with moderate HCDR3 restriction (B). The VH gene rearrangements sequences are shown to the left and the VL sequences to the right. A dot indicates homology with the top sequence. The CDR1, CDR2, and CDR3 are indicated. The D gene segment and the start of the JH/JL genes are underlined. In CLL27, the third amino acid in the HCDR3 is underlined, as it is part of the D3-16 gene. ND indicates that the rearrangement was not determined; and *, position with allelic variants.
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To investigate any association between VL gene usage and HCDR3 composition as in the VH3-21 group, VL gene analysis was performed in these groups with high or moderate HCDR3s similarities. The nucleotide sequences from the VL gene rearrangements are available at GenBank25 accession numbers AY490826
[GenBank]
-AY490888. Interestingly, the same rearranged VL gene was found in the majority of cases when coupled to a similar HCDR3 in the VH gene rearrangement (Figure 2).
Groups displaying highly restricted HCDR3s
VH1-69 (2 groups). The first VH1-69 group (4 cases) used a D3-16 and a JH3 gene (Table 2) and displayed only 1 to 2 amino acid differences in the 18-amino acid-long HCDR3 sequence (Figure 2A). The D gene was used in the same reading frame and the HCDR3 showed identical lengths in all rearrangements. The N-segment in the VH/D junction consisted of 3 amino acids (except CLL27) and 2 amino acids were identical in all rearrangements, whereas 4 of 5 amino acids were the same in the D/JH junction. In one position, 1 amino acid difference was found between all rearrangements, which contained either an Asn, Tyr, His, or Asp amino acid, 3 of which have polar/hydrophilic properties. All 4 cases rearranged a V A27 gene combined with a J1 or J3 gene (Figure 2A; Table 3). In addition to these cases, we identified from the public databases 4 other VH1-69+ CLL cases and 1 anticardiolipin antibody with highly similar HCDR3s. The differences between the HCDR3s in the 8 CLL cases were restricted to 2 positions in this 18-amino acid-long region (Figure 2A).
The second VH1-69 group (3 cases) rearranged a D3-3 and a JH6 gene. The HCDR3 was 21 amino acids long and the same D gene reading frame was demonstrated. The D/JH junction contained 1 amino acid that was identical in all sequences (Figure 2A), whereas the VH/D junction displayed higher variability with varying amino acid properties between the sequences. Two of the 3 cases rearranged a V2-6 gene combined with a J2 gene (Figure 2A; Table 3), whereas the VL sequence was not determined in the remaining case (CLL72).
VH1-2. The VH1-2 group consisted of 5 cases using the D1-26 and JH6 genes. These rearrangements displayed an identical D gene reading frame and identical HCDR3s lengths (14 amino acids; Figure 2A). The VH/D and D/JH junctions consisted of only 1 amino acid each; 1 position was identical between the rearrangements, whereas the other position contained the same amino acid in 3 cases and different amino acids in the remaining 2 cases, all differences resulting in a hydrophobic amino acid. Three cases rearranged a VB3 gene joined with a J2 or J4 gene, whereas 1 case rearranged a VA17 gene, and in 1 case the VL usage was not determined (Figure 2A; Table 3).
Groups displaying moderate HCDR3 homology
VH1-3. Nine cases using the VH1-3/D6-19/JH4 genes displayed a mean of 65% homology between their HCDR3s, with 5 of 9 showing 70% to 80% homology (Table 2; Figure 2A). The same D gene reading frame was used in all rearrangements and the HCDR3 had the same length in 7 of 9 cases. The VH/D junction (1 amino acid) was identical in 7 of 9 cases; however, more variability was indicated in the D/JH junction. Difference in the HCDR3s included amino acids with similar properties in 3 positions (1 in the VH/D junction and 2 in the D/JH junction), such as glu/asp/gln (all polar/hydrophilic), val/pro/gly/ala or ile/leu/met/phe/ala/pro (all non polar/hydrophobic), and 2 positions (both in the D/JH junction) with varying properties. All VL rearrangements used a VO2 gene together with a J1 (4 cases), J2 (4 cases), or J4 (1 case) gene (Figure 2A; Table 3).
VH1-18. Three cases were found to rearrange either a D6-19 (CLL57 and CLL56) or a D3-22 (CLL59) gene, all of which used a JH4 gene (Table 2). Even though 2 different D gene segments were used, they resulted in a conserved 3-amino acid segment, Gln, Trp, and Leu, and the HCDR3s were of the same length in all 3 sequences (Figure 2B). The CDR3s showed 60% homology, but the N regions were less conserved and the different amino acids displayed varying properties. All 3 cases rearranged a functional VO2 with either a J1, J2, or J3 gene (Figure 2B; Table 3).
VH4-39. Homology in the VH4-39/D6-13-D6-19/JH5 group varied between 62% and 75% in 5 cases (Table 2). The D gene displayed the same reading frame, and the rearrangements had similar lengths of the HCDR3s despite differences in the VH/D and D/JH junctions (Figure 2B). In 3 positions (1 in the VH/D and 2 in the D/JH junction), 4 different amino acids were used in the separate sequences, with 2 positions (amino acid differences of Asp, Gly, Ser, or Ala and Thr, Asn, Gly, or Leu) showing differing properties and the third position (Arg, Ser, Thr, or Glu) having similar properties (polar/hydrophilic). Four of 5 cases rearranged a VO2 gene with either a J2 or J4 gene and the remaining case (CLL8) had a VA3/J2 rearrangement (Figure 2B; Table 3).
Ig rearrangements with restricted HCDR3 features with low frequency
In addition to the above groups, a number of HCDR3s were found in sets of 2 sequences, which, because of their low frequency, are detailed separately. Two VH1-69/D3-16/JH4, 2 VH1-2/D3-9, D3-3/JH4, and 2 VH1-46/D3-22/JH4 rearrangements as well as 2 VH4-34/D-/JH6 and 2 VH4-34/D2-15/JH6 rearrangements showed restricted HCDR3s with more than 60% homology (data not shown). The 2 VH4-34 groups and 1 of the VH1-2 sequences displayed evidence of somatic hypermutation. The VH4-34/D-/JH6 cases both rearranged aVA27 gene, whereas the 2 VH4-34/D2-15/JH6 cases rearranged a VA17 gene (Table 3). The VL gene rearrangement was determined in 1 of the VH1-69/D3-16/JH4 cases, which rearranged a VO8 gene, and in 1 of the VH1-46/D3-22/JH4 cases rearranging a VL2 but in none of the 2 VH1-2/D3-9, D3-3/JH4 cases (Table 3).
VL gene usage is restricted to HCDR3 composition
In order to study whether the restricted VL gene usage was linked to the HCDR3 composition or to the particular VH gene used, an additional 33 cases were analyzed by VL gene amplification within these groups using the same VH gene (VH1-69, VH4-39, VH1-2, VH1-3, and VH1-18; Table 3). As illustrated in Figure 3, a diverse usage of VL genes was found in 20 VH1-69 using cases, and VL gene restriction was only demonstrated in cases with similar HCDR3s; the 4 VH1-69/D3-16/JH2 cases and the 2 VH1-69/D3-3/JH6 cases used a VA27 gene or a V2-6 gene, respectively. These 2 VL genes were found in only 2 of the 14 other VH1-69 cases without restricted HCDR3s and instead 7 different V and 3 different V genes were used. In the VH4-39 cases, 12 of 16 were analyzed for VL gene usage, 4 of 5 cases with similar HCDR3s rearranged the VO2, whereas 7 other cases without HCDR3 homology rearranged 6 different VL genes (Table 3). In the VH1-2 cases, the 5 with restricted HCDR3s showed preferential VL gene use, whereas the 4 nonrestricted cases rearranged different VL genes: VO2, VL2, VL10, and V1-17 (Table 3). In the VH1-3-using cases, 11 were analyzed with 9 showing restricted HCDR3s and a VO2 rearrangement, whereas the remaining 2 cases without HCDR3 homology used a VB3 and a VL12 gene (Table 3). Six VH1-18 cases were analyzed and 3 of these had restricted VO2 usage and similar HCDR3s, whereas the remaining 3 cases showed no bias in VL gene use (Table 3).
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Figure 3.. Alignment of HCDR3 amino acid sequences obtained from 20 VH1-69 using rearrangements. The VL gene usage in each case is indicated to the right. A dot indicates homology with the top sequence. The 4 VH1-69/D3-16/JH3 and the 2 VH1-69/D3-3/JH6 rearrangements are aligned to the top sequence in respective group, whereas the amino acid sequences from the remaining VH 1-69+ sequences are presented without alignment.
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Discussion
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Alignment analyses of the HCDR3s from 368 functional VH rearrangements revealed 3 subsets with highly similar HCDR3s (> 75% homology) as well as 3 subsets with moderate HCDR3 homology (60%-75%) in addition to the previously described VH3-21+ group. The first 3 groups (2 VH1-69+ groups and 1 VH1-2+ group) displayed identical VH, D, and JH gene use; identical CDR3s lengths; and shared N-regions; whereas the latter 3 groups (VH1-3, VH1-18, and VH4-39) showed restricted VH, D, and JH gene usage; similar lengths of the CDR3s; and similar D reading frame but less conserved N-region composition. In parallel with the VH3-21+ subset, VL gene rearrangement analysis showed a strikingly biased VL gene usage for both groups showing high and moderate HCDR3 homology (Figure 2). For instance, in the VH1-69/D3-16/JH3 group, all 4 cases with similar HCDR3s had the same VA27 gene usage, whereas 14 other cases without HCDR3 restriction used 10 different VL genes (Figure 3). Additionally, the VH4-39 group with HCDR3 restriction showed preferential VL gene use, whereas the remaining VH4-39 cases used a variety of VL genes. Notably, despite having lower homology in the N-regions in the VH1-3, VH1-18, and VH4-39 groups, they all displayed VL gene rearrangements with almost identical VL use (V02), which is in contrast to the non-HCDR3-restricted cases using the same VH genes. This latter finding strengthens the association between restricted HCDR3 features and biased VL use, although the N-regions do not show identical amino acid sequences.
Our novel data are indicative of antigen selection in subsets of CLL, especially in the groups with highly similar HCDR3 features, but we also consider this possibility for the subgroups showing moderate HCDR3 homology. This interpretation is drawn from the following facts. Considering the probability that 2 VHDJH rearrangements occur by chance is low (1/51 x 1/27 x 1/6 = 1/8262), and in combination with a particular VL gene rearrangement very low (in combination with 1 VJ, 1/8262 x (1/40 x 1/5) = 1/1.6 million), these findings are unlikely to be a random phenomenon. Also, if the junctional diversity and the use of D reading frame is taken into account, the probability will diminish to less than 10-12. Furthermore, using a similar approach to analyze 227 HCDR3s from normal CD5+ B cells available from the public databases (Genbank25), similar rearrangements were demonstrated between only 2 sequences, indicating that our findings in CLL seem not to be a reflection of the normal B-cell repertoire (data not shown).29,30 This is in parallel with the finding of no duplicate Ig rearrangements in about 10 000 Ig sequences obtained from normal tonsils.31 Further support to our data are also given by the previous reports showing CLL cases using the VH1-69/D3-3/JH6 genes or VH4-39/D6-13/JH5 combinations with similar restriction of amino acid motifs within the HCDR3 as in our groups.13,32 We could also identify CLL rearrangements in the GenBank database25 with similar sequences such as the 4 VH1-69/D3-16/JH3 rearrangements (db1-4; Figure 2A), thus strengthening the finding of this particular subgroup. In parallel with our report, a recent abstract also showed combined VH/VL gene usage in VH1-69, VH3-21, VH4-34, VH1-2, and VH1-3-using CLL with high HCDR3 homology.33 Our study thus revealed indirect signs of antigen selection in subsets of CLL, but we cannot exclude that other CLL subgroups could exist that we have not been able to detect due to low frequencies in this material. Indeed, a number of small groups occurring in low frequency were identified using the VH1-69, VH4-34, and the VH1-2 genes with restricted HCDR3 structures and also with similar light-chain rearrangements. A large-scale worldwide study of VH/VL gene rearrangements in CLL is now necessary in order to elucidate all different subgroups in detail and possible geographical differences. For instance, the VH3-21 group has been shown in a higher frequency in Sweden compared with other large VH gene studies, which may indicate that regional difference between populations may be of importance.34-36
Our data suggests a limited number of antigens involved in the selection of these BCRs; however, the identity of such antigens is currently unknown. These antigens could either be self- or non-self-antigens that may trigger and promote proliferation of B cells expressing certain BCRs, leading to clonal expansion and increased risk of transformation. The groups with high HCDR3 restriction, such as the VH1-69 and VH1-2 subgroups, showed limited variation between their HCDR3s with a higher likelihood that they could bind similar antigens, whereas this is less obvious in the moderate-homology groups that had further amino acid differences, although their restricted VH/D/JH and VL rearrangement may indicate antigen selection also within these groups. Interestingly, the recently described VH4-39/VO2-expressing IgG+ CLL subset, with BCR structures similar to our VH4-39 group, displayed comparable antigen-binding sites as monoclonal antibodies reacting toward bacterial carbohydrates as well as different autoantigens.32 Furthermore, a VH1-69/VA27 antibody obtained from a CLL patient showed low-affinity RF activity and bound to myoglobulin, thyroglobulin, actin, and ssDNA.37 An anticardiolipin antibody encoded by a rearrangement similar to the VH1-69/D3-16/JH3 group in this study has also been indicated (GenBank25 accession no. AAL67508
[GenBank]
, but no data were available concerning the VL gene usage. Moreover, the restriction of the HCDR3s found in our subgroups was mainly confined to the unmutated cases with poor prognosis. Recent data has shown that unmutated cases display a higher capacity to signal through their BCRs as measured by syk phosphorylation.38 Signaling through the BCR may therefore play a vital role in the development and, perhaps, progression of the malignant clone in unmutated CLL. The restricted BCRs shown in groups with unmutated VH genes would hence indicate that a selected number of antigens could play an important role in the progression of the clonal expansion in the unmutated CLL subsets. Further studies of the binding sites of the restricted BCRs and putative antigens are now necessary to elucidate the possibility of antigen involvement in CLL development.
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Acknowledgements
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The authors are grateful to Inger Eriksson and Anita Lindström for skilful technical assistance and to Elisabeth Grönlund and Magnus Hultdin for assistance with the diagnostics of CLL.
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Footnotes
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Submitted January 13, 2004;
accepted June 7, 2004.
Prepublished online as Blood First Edition Paper, June 24, 2004; DOI 10.1182/blood-2004-01-0132.
Supported by grants from the Swedish Cancer Society; Lion's Cancer Research Foundation, Umeå and Uppsala; and the research foundation of the Department of Oncology at Uppsala University.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Richard Rosenquist, Dept of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden; e-mail: richard.rosenquist{at}genpat.uu.se.
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Abstract
Introduction