The study of intraclonal diversification (ID) in immunoglobulin (IG) genes offers valuable insight into the role of ongoing interactions with antigen in lymphomagenesis. We recently showed that ID in the IG heavy chain genes of patients with chronic lymphocytic leukemia (CLL) was generally limited; however, intense ID was evident in selected cases, especially those expressing stereotyped IGHV4-34 rearrangements and assigned to subset 4. Here, we report results from a large-scale subcloning study of IG light variable genes, in a total of 1008 subcloned sequences from 56 CLL cases. Multiple analogies were noted between heavy and light chains regarding the occurrence and molecular features of ID. More specifically, the impact of ID on the clonotypic light chains was generally low, with the significant exception of subset 4. Similar to the IGHV4-34 heavy chains of this subset, their partner IGKV2-30 light chains were affected by an active and precisely targeted ID process. Altogether, these findings strengthen the argument that stereotypy in subset 4 extends to stereotyped ID patterns for both heavy and light chains through persistent antigenic stimulation. Furthermore, they strongly suggest that light chains have an active role in the antigen selection process, at least for certain subsets of CLL cases.
The primary structure of the antigen-binding site in the B-cell receptor (BCR) is determined by both heavy and light chain variable domains (VH and VL, respectively). Several studies have suggested that the VH domain often has a more important role than the VL in the recognition mechanism of the immunoglobulin (IG) molecule. For instance, heavy chain dominance in antigen binding has been documented extensively for many anti-DNA antibodies.1, 2 In other cases, however, the VL domain seems to be the specificity-defining sequence, with certain light chain genes predominating in antibody responses to common pathogens (for example the IGKV2D-29 gene in infections by Haemophilus influenzae3) or serving pivotal functions in autoreactivity handling in the context of receptor editing.4, 5, 6, 7
The mature normal repertoire shows no evidence for preferential pairings of heavy/light chain genes or associations between heavy/light chain complementarity-determining region 3 (CDR3) lengths and sequences.8, 9 In contrast, the chronic lymphocytic leukemia (CLL) IG repertoire is biased10, 11 and is uniquely characterized by the existence of subsets of cases expressing quasi-identical (stereotyped) BCRs, implying the recognition of individual, discrete antigens or classes of structurally similar epitopes.12, 13, 14, 15, 16, 17, 18, 19, 20, 21 This conclusion is further supported by our recent finding of ‘CLL-biased’ somatic hypermutation (SHM) patterns in IG heavy and light variable genes of cases belonging to subsets with stereotyped BCRs.19, 20 The most distinctive SHM patterns were identified in cases expressing IGHV3-21/IGLV3-21 (subset 2) and IGHV4-34/IGKV2-30 (subset 4) BCRs.19, 20
A critical question in CLL biology is whether interaction with antigen is restricted to the progenitor cell (pre-transformation phase) or whether the putative antigen may continuously trigger the CLL clone and, perhaps, drive its evolution.22, 23, 24, 25, 26, 27 The experience from other types of B-cell malignancies suggests that useful hints for an ongoing interaction with antigen may be obtained through the study of intraclonal diversification (ID) within the IG genes expressed by the malignant clone.28 With this in mind and through a recent comprehensive analysis of ID in rearranged IGHV genes from patients with CLL, we showed that although the majority of cases showed no or low levels of ID, an intense and, likely, functionally driven ID process was evident in selected cases, especially those belonging to subset 4.29 On these grounds, we suggested that antigen involvement may affect not only the progenitor cells of such CLL clones but also the malignant cells themselves.
Given that highly distinctive SHM patterns are not limited to IGHV genes but may also extend to their partner light chain genes, at least in selected subsets of CLL cases,20 the analysis of ID in IGκ (IGK) and IGλ (IGL) light chain genes could be very relevant to understanding the interactions between the clonotypic BCRs and their cognate antigens. To this end, in this study, we applied a very stringent methodology and explored the occurrence of ID in IGK/IGL genes from 56 patients with CLL. The analysis was intentionally biased to rearrangements of the IGKV2-30 and IGLV3-21 genes since, as mentioned above, these rearrangements exhibit distinctive, CLL-biased patterns of SHM when used in the stereotyped BCRs of subsets 4 and 2, respectively,19, 20 indicative of a particular mode of interaction with the cognate antigen(s).
Our results indicate that, overall, the ID process affects both heavy and light chain genes of CLL malignant B cells to a similar, limited extent, with the significant exception of subset 4. Similar to what we recently reported for the IGHV4-34 heavy chains of this subset,29 their partner IGKV2-30 light chains exhibited pronounced ID with distinctive patterns of SHMs among subcloned sequences, strongly suggestive of diversification within the context of antigen-driven clonal evolution.
Patients and methods
Fifty-six patients with CLL from collaborating institutions in Scandinavia and Greece were included in the study. Patients were selected based on IGKV/IGLV repertoire with an intentional bias for cases using the IGKV2-30 and IGLV3-21 genes (12/56 and 11/56 cases, respectively). All cases were immunophenotyped as described earlier16, 18, 30 and met the recently updated diagnostic criteria of the National Cancer Institute-Working Group.31 Written informed consent was obtained according to the Helsinki declaration, and the study was approved by the local Ethics Review Committee of each institution.
Polymerase chain reaction amplification of rearranged IGK/IGL genes
Polymerase chain reaction (PCR) amplification was performed on either genomic DNA or complementary DNA, extracted from peripheral blood and bone marrow (55 cases) but also from lymph node in one case. With the exception of cases assigned to subset 4, amplification of all IGKV-J and IGLV-J rearrangements was performed using consensus primers for the KFR1/LFR1 and IGKJ/IGLJ genes.16, 17, 18, 19, 20 For the IGKV2-30 rearrangements of subset 4 cases, prompted by the high level of ID recently observed within FR4 of their partner heavy chains (IGHV4-34),29 the antisense primer (5′-IndexTermAGA CTC TCC CCT GTT GAA GCT CTT-3′) was specific for the IGKC gene, thus enabling assessment of KFR4 sequences. All amplification reactions were run using the high-fidelity Accuprime Pfx polymerase (26-fold higher accuracy compared with the Taq DNA polymerase). Purified PCR products were subjected to direct sequencing. Sequence data were analyzed using the IMGT database and tools (http://imgt.cines.fr/).
PCR amplification products were gel purified with the (QIAGEN, Hilden, Germany) DNA purification columns, ligated into the pCR2.1 vector (Invitrogen, AB, Stockholm, Sweden) and subsequently transformed into Escherichia coli/TOP10F’ competent bacteria (Invitrogen). At least 14 colonies per case (median: 17, range: 14–39) were chosen randomly, cultured and sequenced using the 20 universal primer or M13 primers.
Sequence data analysis and definitions
The obtained sequences were analyzed by the IMGT V-QUEST32 and Clustalw/EMBL tools.33 For the sequences obtained with KFR1/LFR1 consensus primers, nucleotide changes were evaluated from codon 15 in IMGT-KFR1/LFR1 down to the end of KFR3/LFR3. For the subgroup of IGKV2-30 rearrangements, which were obtained using a constant region primer, the V region was evaluated from codon 15 in IMGT-KFR1 down to KFR4. Sequence data concerning IG gene usage, percentage of identity to germline, K/LCDR3 length, SHM characteristics, and SHM hotspot targeting were evaluated and interpreted as reported earlier by our group.29
ID in sets of subcloned sequences obtained from the same sample was assessed by examination of sequence variation in the V domain. All ‘non-ubiquitous’ sequence changes from the germline were evaluated in the counts and further characterized as follows: (1) unconfirmed mutation (UCM)—a mutation observed in only one subcloned sequence from the same specimen (unique); (2) confirmed mutation (CM)—a mutation observed more than once among subcloned sequences from the same specimen (partially shared).
To compare mutation counts between the different rearrangements included in the analysis, mutations were normalized to the nucleotide length as well as the number of subcloned sequences of each rearrangement, as reported earlier by our group.29 In brief, the normalized mutation frequencies (NMF) were calculated according to the following formula: Σ (CM+UCM)/number of subcloned sequences × sequence length.
Evolutionary history of sets of subcloned sequences
The evolutionary history was inferred using the maximum parsimony method. All positions containing gaps and missing data were eliminated from the data set (Complete Deletion option). Phylogenetic analyses were conducted in Molecular Evolutionary Genetics Analysis software version 4.0 (MEGA4).34
Descriptive statistics for discrete parameters included counts and frequency distributions. For quantitative variables, statistical measures included means, medians, s.d., and min–max values. Significance of bivariate relationships between factors was assessed with the use of χ2 and Fisher's exact tests. For all comparisons, a significance level of P=0.05 was set, and all statistical analyses were performed with the use of the Statistical Package SPSS Version 12.0 (SPSS Inc., Chicago, IL, USA).
IGKV/IGLV gene repertoires and mutation status
A total of 57 productive IGKV-J and IGLV-J rearrangements from 56 CLL cases were included in the analysis: 36 cases expressed κ light chains, whereas 20 cases expressed λ light chains. One κ-expressing case carried two productive IGKV-J rearrangements. Thirteen different IGKV and seven different IGLV genes were recognized; detailed information on IG repertoires and IGKV/IGLV gene mutational status is provided in Supplementary Table 1.
Twenty-eight of 56 cases were assigned to subsets with stereotyped BCRs, as described earlier18, 19, 20 (Supplementary Table 1). Nine such cases expressed IGHV3-21/IGLV3-21 BCRs (subset 2), whereas 10 cases expressed IGHV4-34/IGKV2-30 BCRs (subset 4). The heavy chain isotype was the same among members of a subset (for cases with available data). All subsets expressed IgMD, except for subset 4, which included IgG-expressing cases, as reported earlier.15, 18, 19 The clonotypic IGHV3-21 and IGHV4-34 rearrangements of five subset 2 and nine subset 4 cases included in the present analysis have previously been evaluated for ID within IGHV genes29 (Supplementary Table 1).
ID analysis at the nucleotide level
Analysis of 651 subcloned sequences from 37 IGKV-J rearrangements revealed that 15/37 (40.5%) rearrangements carried only UCMs in certain positions of the V domain, whereas 22/37 (59.5%) carried at least one CM and, therefore, were considered as exhibiting ‘confirmed’ ID (CID). Eighteen of 22 IGKV-J rearrangements with CID had <98% germline identity; however, CID was also identified in borderline/minimally mutated IGKV sequences (Supplementary Table 2).
Overall, a total of 118 CMs and 96 UCMs were identified. The number of CMs ranged from 1 to 24/case, whereas the number of UCMs ranged from 1 to 10/case (Supplementary Table 2). Detailed information on the ‘type’ (replacement versus silent) and distribution of CMs/UCMs throughout the V region is given in Supplementary Table 3.
Eleven of 22 (50%) IGKV-J rearrangements with CID used the IGKV2-30 gene; of which, 10 were expressed by subset 4 cases. Notably, the NMF of subset 4 rearrangements were significantly higher compared with all other rearrangements included in the analysis (median NMF values: 1.8 × 10−3 versus 0.7 × 10−3, respectively; t-test, P<0.006), indicating a significantly higher impact of ID (Figure 1). Of particular importance was the observation that when IG heavy and light chains were analyzed in parallel for subset 4 and subset 2 cases, ID evidenced within the light chain was reflective of the overall level of ID observed in the corresponding partner heavy chain (Figure 2).
The high number of CMs (and UCMs) observed in certain subset 4 cases can be attributed to the existence of distinct ‘clusters’ of subcloned sequences with ‘cluster-specific’ mutational profiles. Thus, although at case level the number of CMs and UCMs was high, at cluster level the subcloned sequences were remarkably similar. In fact, four subset 4 cases (P0103, P1939, P2451, and P2920) displayed such a professed level of ID (Table 1) that subcloned sequences could be subdivided into two or more clusters exhibiting shared as well as ‘cluster-specific’ somatic mutations (Figure 3; Supplementary Figure 1).
Certain mutations appearing in single-subcloned sequences of five subset 4 IGKV2-30 rearrangements and, thus, considered as UCMs, were also present in subcloned sequences of other cases belonging to subset 4 (Supplementary Figure 2A). Given that these cases also carried many CMs, it would not be unreasonable to consider these UCMs as an imprint of the ID process. Therefore, in certain cases, ‘UCMs at the single-case level’ may be considered as ‘confirmed at subset level’ (that is confirmed by another case).
Overall, 357 subcloned sequences from 20 IGLV-J rearrangements were evaluated (Supplementary Table 4). Identical sets of subcloned sequences were obtained in 6/20 (30%) analyzed IGLV-J rearrangements. Among the remaining rearrangements, 8/20 (40%) carried only UCMs, whereas 6/20 (30%) carried at least one CM and, were therefore, considered to exhibit CID (Supplementary Table 4). Overall, the number of ‘non-ubiquitous’ mutations was low: in fact, only 8 CMs and 30 UCMs were identified (Supplementary Table 4).
Four of six IGLV-J sequences with CID had <98% germline identity; the two remaining cases used the IGLV3-21 gene and both belonged to subset 2 with stereotyped IGHV3-21/IGLV3-21 BCRs. Three additional subset 2 IGLV3-21 rearrangements were found to carry UCMs, which were also present in subcloned sequences of other cases belonging to subset 2 and, thus, could be considered as ‘confirmed by another case’ (Supplementary Figure 2B).
Nucleotide substitution analysis and targeting of the ID process
Nucleotide substitution analysis of the mutations introduced as part of the ID process among subcloned sequences revealed that transitions predominated over transversions both for CMs and UCMs, in keeping with a canonical SHM process35; furthermore, purines were targeted more frequently than pyrimidines (Supplementary Table 5).
The analysis for targeting to SHM hotspots, that is the tetranucleotide (4-NTP) motifs RGYW/WRCY (R=A/G, Y=C/T, and W=A/T)36 and DGYW/WRCH (D=A/G/T, H=T/C/A)37 as well as the dinucleotide (2-NTP) motifs WA and TW38 revealed that only 82/214 (38.3%) mutations identified among sets of IGKV-J subcloned sequences occurred within a hotspot motif. A similar percentage was found for IGLV-J rearrangements (13/38 mutations, 34.2%). No significant difference was noted between CMs and UCMs regarding targeting to SHM hotspots motifs.
ID patterns among cases expressing stereotyped BCRs: analysis at the amino acid level
A total of 135 amino acid (AA) changes were identified among sets of subcloned IGKV-J rearrangements (Supplementary Tables 2 and 6). Seventy AA changes (52%) were classified as confirmed (CAA), whereas the remainder (65/135; 48%) were classified as unconfirmed (UAA) (Supplementary Table 2). Two confirmed changes concerned a single AA deletion within the KCDR3 (IMGT codon 115; both deletions were identified in subset 4 cases). The frequency of CAA changes was significantly higher among subset 4 versus all other IGKV-J rearrangements analyzed (10/10 versus 9/27 rearrangements, P<0.001).
The IGKV2-30/IGKJ2 rearrangements of subset 4 cases were also exceptional due to the fact that many CAA changes were ‘stereotyped,’ in the sense that different cases could exhibit recurrent AA changes among subcloned sequences (Figure 4). An illustrative example is provided by position 66 in the KFR3 of IGKV2-30 genes expressed by subset 4 cases, which, as previously shown by our group,20 is targeted for a recurrent N-to-D AA change. Indeed, 5/10 (50%) subset 4 cases of this study carried this AA change in all subcloned sequences; of note, the same change was identified as a CAA in two additional cases. Taken together, 7/10 (70%) subset 4 cases carried an N-to-D replacement in the KFR3, even in a minority of subcloned sequences. A comprehensive list of ‘stereotyped’ AA changes attributed to ID among IGKV2-30/IGKJ2 rearrangements of subset 4 cases is provided in Supplementary Table 7.
Among analyzed IGLV-J rearrangements, CAAs were found in only three cases and at a low level. Two cases with CAAs carried borderline/minimally mutated IGLV3-21 gene sequences and belonged to subset 2 with stereotyped IGHV3-21/IGLV3-21 BCRs (Supplementary Table 8).
Much insight into the development and progression of CLL has been gained through molecular analysis of the IG genes expressed by the malignant B cells. This analysis has provided significant prognostic information39, 40 and, furthermore, has played a central role in the evolution of our ideas about the role of antigen in the ontogenesis of CLL. The skewed IG gene repertoire characteristic of CLL,10, 11 the existence of quasi-identical (stereotyped) Ag-binding sites12, 13, 14, 15, 16, 17, 18, 19, 20, 21 and the very precise targeting and distinctive features of SHM in selected subgroups of CLL patients19, 20 imply the recognition of individual, discrete Ags or classes of structurally similar epitopes, likely selecting the leukemic clones.
Although BCR-related studies put a step forward in clarifying the antigen-driven model of CLL ontogeny, further studies are necessary to specify the timing of interactions with antigen(s). Sequence-based insight into the aforementioned issues may be gleaned from the analysis of ID of IG genes. In this context, our recent, extensive study of ID in IGHV genes expressed by a large cohort of CLL cases provided evidence suggestive of an ongoing interaction with antigen in certain cases carrying distinctive IGs, especially those belonging to subset 4 with stereotyped IGHV4-34/IGKV2-30 IGs.29 Prompted by these findings, as well as earlier reports from our group about the important role of light chains in recognition of and selection by antigen in CLL,20 we extended our studies by exploring the occurrence of ID in the IG light chains expressed by CLL malignant B cells so as to gain a more comprehensive view of the interactions between the clonotypic IGs and the selecting antigens. On the basis of cumulating published data, most notably the distinctive SHM patterns observed in the light chains of subset 2 and subset 4 cases20 and the more recent finding of ID in the stereotyped partner IGHV4-34 receptors,29 we selected cases in a non-random manner specifically focusing on subsets 2 and 4. Admittedly, our analysis was biased in this regard; however, we also included cases assigned to various other subsets, those classed as non-subset, and also unmutated cases, to obtain a more comprehensive and rounded view on the nature and timing of SHM in the development and, perhaps, evolution of CLL.
The analysis of IGKV-J rearrangements at nucleotide level documented the existence of ID in a significant proportion of cases; a lower level of ID was observed in IGL genes. Although the breakdown of the present series with regard to the level of ID revealed that most cases were not significantly affected by the ID process, the striking exception concerned IGKV2-30 rearrangements of subset 4 cases, which were distinguishable due to a professed level of ID and very precise patterns of mutational activity, similar to what we recently reported for their partner IGHV4-34 heavy chains.29 On these grounds, we propose that subset 4 IGs are ‘fine-tuned’ and perhaps evolve by an intense ongoing ID process involving both heavy and light chains in a concerted manner. It is perhaps relevant to note that the patients assigned to this subset are also characterized by distinctive clinicobiological features. In keeping with our earlier reports,18, 19, 29 subset 4 cases included in the analysis had a young median age at diagnosis (45 years, range: 43–69 years) and were diagnosed in the early clinical stages; the leukemic clones were uniformly CD38 negative and IgG switched; finally, 5/7 cases with available data carried del(13q) as the sole cytogenetic abnormality (Supplementary Table 9).
The findings of this study provide evidence that certain residues of the VK domains of subset 4 cases may be critically implicated in shaping the binding affinity of the clonotypic BCRs. A prime example is offered by the mutational pattern at codon 66 in the KFR3 of the IGKV2-30 gene. As we have recently shown, this codon is often targeted for a ‘subset–biased,’ recurrent N-to-D AA replacement.20 In accordance with this observation, this change was shared by all subcloned sequences of 5/10 (50%) cases of this study; intriguingly, two additional cases exhibited the same change in a minority of subcloned sequences (Figure 3). These stereotyped patterns of ID are analogous to those recently reported by our group for the IGHV4-34 rearrangements of subset 429 and may be considered as additional evidence for the continuous operation of antigen-driven SHM even after the transforming event. Furthermore, the remarkable molecular features of subset 4 heavy and light chains strongly indicate that the conformational changes introduced by SHM are not stochastic but very likely directed by selection for functionality.
The IGHV4-34 gene, paired with the IGKV2-30 gene in subset 4 BCRs, is inherently autoreactive, as it recognizes the N-acetyllactosamine determinant on the I/i red blood cell antigen, DNA, cytoskeletal proteins, the Fc fragment of IgG and apoptotic bodies.41, 42 IGHV4-34 B cells are censored at multiple checkpoints during B-cell development and evade clonal deletion by several strategies.43 Light chains could have a pivotal role in handling the autoreactive potential of IGHV4-34 heavy chains, in analogy to earlier reports in transgenic mouse models of autoimmunity.44, 45, 46, 47 For instance, in mice transgenic for the heavy chain of antibodies that bind DNA, it has been shown that just a few strategically located acidic residues within the light chains may eliminate or even abrogate the anti-DNA specificity.47 Along these lines, the results of this study strengthen our earlier assumptions regarding the potential editing function of somatically introduced acidic residues in the IGKV2-30 light chains of subset 4 BCRs.19, 20
Several positions identified as ‘hotspots’ for ID among subset 4 IGKV2-30 rearrangements were characterized by either silent mutations or remarkably conservative AA changes. The foremost examples, perhaps unexpectedly, were KFR4 codons 124 (L) and 125 (E), which exhibited ID in all subset 4 cases. However, the introduced changes concerned AA replacements of a very conservative nature (L-to-V, E-to-D). This limited range of permissible AAs could reflect selection events governed by structural constraints for optimal antigen recognition.
The striking impact of the ID process on the KFR4 of subset 4 light chains again mirrors our earlier findings for their partner heavy chains,29 which show a clustering of CMs and UCMs in HFR4, and may be considered as evidence for the active role of regions outside the conventional antigen-binding site in the antibody–antigen interface, perhaps through non-conventional, superantigenic or superantigenic-like interactions. This notion also goes along with our recent report linking subset 4 with persistent infection by CMV and EBV,48 which are known to interact with IGHV4-34 IGs in a superantigenic-like manner.
Non-ubiquitous mutations among subcloned IG gene sequences could reflect imprints (relics) or representatives of intermediate stages in the affinity maturation process. In this context, the precise, ‘stereotyped’ ID patterns in subset 4 heavy29 and light chain genes are highly indicative of antigen-driven clonal evolution. This argument is also supported by the finding of distinct ‘clusters’ of subcloned sequences likely corresponding to different tumor subpopulations deriving from a common ancestor that were perhaps selected by classes of similar, yet subtly different epitopes and subsequently evolved along related yet distinct pathways. That notwithstanding, several critical issues remain unresolved, especially with regard to the type and range of antigenic stimuli as well as the microenvironment fostering ongoing interactions of the clonotypic BCRs with their cognate antigen(s). Bearing to these questions is whether the CLL clone was stimulated by the same antigen(s), which had originally selected the clonal precursor cell, or, alternatively, by related epitopes present on both exogenous and self-antigens? This is particularly relevant, in view of accumulating recent evidence that CLL mAbs may recognize and be triggered by antigens of common pathogens as well as autoantigens.48, 49, 50, 51
Whatever the answers to these questions, this study offers ample evidence for the critical role of light chains in antigen recognition by the leukemic BCRs. Furthermore, it provides further evidence that subset #4 is characterized by a highly distinctive BCR with SHM patterns in both heavy and light chain genes indicative of a ‘stereotyped response’ to an active, ongoing interaction with antigen(s).
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We thank Professor Marie-Paule Lefranc and Dr Veronique Giudicelli, Laboratoire d’Immunogenetique Moleculaire, LIGM, Universite Montpellier II, Montpellier, France, for their long-standing support and guidance with the large-scale immunoglobulin sequence analysis throughout this project. We also acknowledge the contribution of Christer Sundström, Karin Karlsson and Juhani Vilpo for providing samples and associated data, and Andreas Agathagelidis, Vasilis Bikos, Nikos Papakonstantinou, Gerard Tobin, Ulf Thunberg and Mia Thorsélius to the sequence analysis. This work was supported by the Swedish Cancer Society, the Swedish Medical Research Council, the Medical Faculty of Uppsala University, Uppsala University Hospital, and the Lion's Cancer Research Foundation, Uppsala, Sweden; the BioSapiens Network of Excellence (contract number LSHG-CT-2003-503265); and, the General Secretariat for Research and Technology of Greece (Program INA-GENOME). EK is a recipient of a fellowship from the Propondis Foundation, Athens, Greece.
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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