The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is uniquely characterized by the presence of stereotyped B-cell receptors (BCRs). A major BCR stereotype in CLL is shared by immunoglobulin G-switched cases utilizing the immunoglobulin heavy-chain variable 4-34 (IGHV4-34) gene. Increased titers of IGHV4-34 antibodies are detected in selective clinical conditions, including infection by B-cell lymphotropic viruses, particularly Epstein–Barr virus (EBV) and cytomegalovirus (CMV). In this context, we sought evidence for persistent activation by EBV and CMV in CLL cases expressing the IGHV4-34 gene. The study group included 93 CLL cases with an intentional bias for the IGHV4-34 gene. On the basis of real-time PCR results for CMV/EBV DNA, cases were assigned to three groups: (1) double-negative (59/93); (2) single-positive (CMV- or EBV-positive; 25/93); (3) double-positive (9/93). The double-negative group was characterized by heterogeneous IGHV gene repertoire. In contrast, a bias for the IGHV4-34 gene was observed in the single-positive group (9/25 cases; 36%). Remarkably, all nine double-positive cases utilized the IGHV4-34 gene; seven of nine cases expressed the major BCR stereotype as described above. In conclusion, our findings indicate that the interactions of CLL progenitor cells expressing distinctive IGHV4-34 BCRs with viral antigens/superantigens might facilitate clonal expansion and, eventually, leukemic transformation. The exact type, timing and location of these interactions remain to be determined.
The immunoglobulin heavy variable 4-34 (IGHV4-34) gene encodes antibodies, which are intrinsically autoreactive by virtue of universal and largely light chain-independent recognition of the N-acetyllactosamine (NAL) antigenic determinant of the I/i blood group antigen.1, 2 The interaction with NAL epitopes, also present on various other self- and exoantigens, is largely independent of the conventional antigen-binding site and mainly involves IGHV4-34 framework regions, especially a hydrophobic patch in heavy framework region 1 (HFR1).3, 4, 5
The IGHV4-34 gene is used at a high frequency in normal individuals;6, 7 however, IGHV4-34 cells are censored at multiple checkpoints during B-cell development to alleviate their autoreactivity.7 This finding explains why IGHV4-34 antibodies are virtually undetectable in healthy sera, despite the abundance of IGHV4-34 B cells in normal individuals.7 In contrast, it has been demonstrated that IGHV4-34 antibodies are secreted in high level in patients with systemic lupus erythematosus (SLE) and closely related to tissue damage and disease activity.8, 9, 10 IGHV4-34 G-switched (IgG) antibodies have been shown to constitute a substantial fraction of SLE anti-DNA antibodies and target CD45 either on T cells or the corresponding isoform on B cells.8, 9, 10 A rise in IGHV4-34 Abs is also observed in the serum of otherwise healthy individuals in response to acute infections with herpesviruses such as Epstein–Barr virus (EBV) and cytomegalovirus (CMV) and in acute Mycoplasma pneumoniae infections.10, 11, 12, 13
The immunoglobulin gene repertoire expressed by chronic lymphocytic leukemia (CLL) malignant B cells is biased and notable for the existence of subsets with quasi-identical (stereotyped) B-cell receptors (BCRs),14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 implying the recognition of structurally similar epitopes, likely selecting the leukemic clones. However, the nature of the selecting antigens and their interactions with CLL progenitors or the malignant cells themselves remain shadowy.
In CLL, the links (if any) with common pathogens are still elusive. Of note, however, a population-based study from Denmark showed that a personal history of pneumonia mainly caused by Streptococcus pneumoniae and Haemophilus influenzae was associated with significantly increased risk for CLL development.25, 26 Furthermore, recent in vitro studies demonstrated that recombinant mAbs from patients with CLL can react with antigenic epitopes on the surface of common bacteria and, most importantly, that mAbs encoded by different immunoglobulin genes reacted with different classes of antigenic epitopes.27, 28 Along these lines, it could not be unreasonable to speculate that persistent or intermittent infections by common pathogens could stimulate CLL precursors, and possibly CLL cells themselves, and somehow contribute to malignant transformation, at least for subsets of CLL cases.
The IGHV4-34 gene is also very frequent in CLL.14, 22 We recently reported two major subsets of CLL cases expressing stereotyped IGHV4-34 BCRs who also shared unique biological and clinical features.15, 16 Both subsets were characterized by distinctive antigen-binding sites, reminiscent of pathogenic anti-DNA antibodies.14, 22 They also exhibited distinctive somatic hypermutation patterns, with shared (‘stereotyped’) amino-acid changes, leading us to suggest that the leukemic progenitor cells may have responded in a similar fashion to the selecting antigens.24 Interestingly, though mutated, stereotyped IGHV4-34 BCRs showed nonmutated HFR1 motifs and, in theory, retained the ability to engage in superantigenic-like interactions with various auto- and exoantigens.24
Along these lines, in this study we explored possible links with latent or persistent activation by EBV and CMV in CLL cases expressing the IGHV4-34 gene. Remarkably, in a cohort of CLL cases expressing various IGHV genes, we identified a strong and statistically significant association between persistence of both EBV and CMV and usage of the IGHV4-34 gene, especially in stereotyped IGHV4-34 BCRs.
Materials and methods
Ninety-three patients with CLL were included in the study. Patients were selected based on IGHV gene usage as well as the availability of serial samples collected over an extended time period, ranging from 2 to 5 years. In particular, a median of 4 (2–10) peripheral blood samples were analyzed per case (overall 356 samples). All cases were immunophenotyped as described previously22 and met the recently updated criteria of the National Cancer Institute-Working Group.29 Written informed consent was obtained at study entry. The study was approved by the local ethics review committee of each institution.
The study also included as a control group 61 hematopoietic cell donors who were subjected to molecular testing for CMV and EBV before hematopoietic cell donation.
PCR amplification of IG rearrangements and sequence analysis
Amplification and sequence analysis of IGH/IGK/IGL rearrangements was performed as previously described.15, 16, 17, 18, 19, 20, 21, 22 Sequence data were analyzed using the ImMunoGeneTics database (http://imgt.cines.fr).30, 31 Sequences with a germline identity ⩾98% were considered as unmutated, and those with an identity <98% were considered as mutated.32 The repertoire and mutational status of all sequences were published previously.22
Detection of CMV and EBV genome copies by real-time PCR
Total genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) using the QIAmp DNA Blood Mini kit (Qiagen, Hilden, Germany). Twelve million cells PBMCs were used per extraction. Quantitative real-time PCR analysis for CMV and EBV genome copies was performed by RealArt CMV RG PCR kit (Qiagen) and RealArt EBV PCR kit (Qiagen), respectively, on Rotor-Gene 6000 real-time analyzer (Corbett Research, St Neots, Cambridgeshire, UK). The protocol is based on the amplification and simultaneous detection of sequences of (1) the CMV major intermediate early gene and (2) the EBV nuclear antigen 1 gene with hydrolysis probes and two fluorochromes; succinimidyl ester of carboxyfluorescein (for sample reaction) and 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE; for internal control (IC) reaction). Each Kit is equipped with a series of different dilutions of an EBV/CMV quantitation standard (QS), which allows plotting a standard curve for the precise quantitation of the pathogen load.33 The included IC distinguishes between false-negative results and true-negative results by simultaneous amplification of the IC and the specific sample in one reaction tube. Data analysis was performed with Rotor-Gene software according to the manufacturer's manual.
All samples, QS standards and nontemplate controls were analyzed in duplicate. Samples were evaluable for analysis only if tested positive for IC amplification and exhibited a normal exponential curve in JOE analysis. Cases were considered as CMV/EBV-positive only if two or more samples from different time points exceeded the real-time PCR threshold. In each case, the final result was expressed as CMV or EBV genomes per 106 PBMCs. A threshold of 1000 viral genome copies per 106 PÂMCs was utilized for assessment of positive/negative samples. Given that the EBV and CMV viral load varies over a wide range between healthy individuals,34 this rather high real-time PCR cutoff value for positivity was adopted to define only chronic, persistent high viral load carriers.
Descriptive statistics for discrete parameters included counts and frequency distributions. For quantitative variables, statistical measures included means, medians, standard deviation 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).
IGHV gene repertoire
Twenty-four functional IGHV genes were identified in the present series (Table 1). For the reasons detailed in the Introduction, our study was intentionally biased for cases utilizing the IGHV4-34 gene (23/93 rearrangements, 24.73%). That notwithstanding, the present series also included subgroups comprised of at least six cases each who utilized other IGHV genes frequent in CLL (for example, IGHV1-69, IGHV3-23, IGHV3-7). Following the 98% identity cutoff value, 67/93 sequences (72%) were defined as ‘mutated,’ whereas the remainder (26/93 sequences, 28%) had ‘unmutated’ IGHV genes. The asymmetric distribution of mutated/unmutated cases compared to previous studies is likely attributed to the overrepresentation of IGHV4-34-expressing cases. In fact, all IGHV4-34 rearrangements of this study had less than 98% identity to germline (ranging from 90 to 97.4%).
Among 23 IGHV4-34-expressing cases in our series, two subsets of collectively 11 mutated sequences with different, restricted HCDR3s were identified. Both subsets have been reported previously (subsets 4 and 16; subset numbering follows our previous publications22, 24). The main subset (subset 4) included nine IgG-switched cases expressing stereotyped IGKV2-30 light chains. The minor subset (subset 16) included two IgG-switched cases expressing stereotyped IGKV3-20 light chains.
Detection of CMV and EBV genome copies by real-time PCR
Molecular analysis for CMV and EBV genome copies was performed in serial samples over a period of 2–5 years. Given the wide range of reported rates for EBV/CMV DNA positivity of donor blood,34 we adopted a high real-time PCR cutoff value for positivity (>1000 viral genome copies). Cases were considered as ‘CMV-positive’ or ‘EBV-positive’ only if exceeding the real-time PCR cut off in at least two different samples from different time points. Following this definition, 13/93 cases (14%) and 30/93 cases (32.3%) of the present series were considered as ‘CMV-positive’ or ‘EBV-positive,’ respectively.
In the group of healthy individuals who served as controls, the frequency of CMV or EBV positivity was significantly lower compared to the CLL cohort (with the caveat that healthy individuals were tested only once). In particular, CMV genome copies were detected in only 1 of 61 healthy donors (1.6 vs 14% among CLL cases); the frequency of EBV-positive cases was higher (11/61 cases; 18%) but still significantly below the corresponding frequency in the CLL cohort (32.3%).
On the basis of the molecular testing for CMV and EBV, cases were subdivided in three groups: (1) Group A, double-negative patients: 59/93 cases (63.4%) proved to be negative either for CMV or EBV. (2) Group B, single-positive cases: 25/93 cases (27%) tested positive for only one viral genome (CMV or EBV). In particular, 4/25 group B cases were CMV+ whereas 21/25 cases were EBV+. (3) Group C, double-positive patients: 9/93 cases (9.7%) tested positive for both CMV and EBV in at least two (median 4, range 2–9) samples from different time points. Although the patients considered as ‘positive’ had quite different CMV and EBV loads at different time points, each retained a unique characteristic load at a roughly stable level over the period of study.
Given the retrospective character of our study, serological data for anti-CMV or anti-EBV IgM/IgG antibodies were available only in a minority of cases. All cases with available information (including six of nine group C cases) carried IgG antibodies against both CMV and EBV, whereas no case tested positive for IgM anti-CMV or -EBV antibodies.
Clinical and biological features of CLL groups defined by patterns of viral positivity
The three groups of CLL patients defined according to patterns of molecular positivity for CMV and EBV differed in terms of clinical presentation and outcome as well as immunophenotypic profile, cytogenetic aberrations, IG gene repertoire and BCR stereotypy (Table 2). In particular, with comparable median follow-up times, disease progression requiring treatment was significantly more frequent in groups A+B vs C (P=0.04; Figure 1). All but one group C case were characterized by sole expression of surface IgG (sIgG), whereas sIgG expression was infrequent in group A or B (P<0.001). Group C cases were uniformly negative for cluster of differentiation 38 (CD38); in contrast, with a 7% cut off for positivity,35 CD38 expression was frequent among group A and, especially, group B cases (P<0.02 for comparison between groups B vs C).
Groups A and B displayed heterogeneous IGHV gene usage. However, the frequency of the IGHV4-34 gene was significantly higher in group B (9/25 cases vs 5/59 group A cases; χ2-test: P=0.002). Group C was comprised by cases expressing exclusively the IGHV4-34 gene (χ2-test: P<0.001 for comparison to group A or B; Figure 2). Strikingly, seven of nine group C cases were found to express stereotyped IGHV4-34/IGKV2-30 BCRs (subset 422, 24, 36); of the remaining two group C cases, one belonged to subset 1622, 24 with stereotyped IGHV4-34/IGKV3-20 BCRs. Therefore, it would not be unreasonable to consider group C as broadly corresponding to subset 4.
All IGHV4-34 monoclonal antibodies recognize the NAL carbohydrate epitope on the surface of red blood cells6, 7, 37 and, furthermore, many such antibodies cross-react with other nonprotein antigens present in self- and microbial antigens.6, 9, 10, 11 Increased titers of IGHV4-34 antibodies have been found in individuals infected by various pathogens, especially CMV, EBV and M. pneumoniae,10, 11, 12, 13, 38, 39, 40 which engage in superantigenic-like interactions with a conserved motif in the HFR1 of IGHV4-34 Abs.3, 4, 5 Indeed, it has been proposed that the IGHV4-34 gene may have been selected during evolution for its ability to encode protective antibodies, as indicated by the fact that the immune response to pathogens carrying NAL epitopes selectively targets IGHV4-34 B cells.11, 40, 41
In this study, we investigated whether patients with CLL-expressing IGHV4-34 BCRs might somehow be linked with chronic activation by common herpesviruses, in particular EBV and CMV. The ideal approach would be that of testing directly the CLL cell capacity of being stimulated by viral antigens in vitro. However, given that this approach can be very difficult, we followed an alternative, admittedly indirect, approach and sought for molecular evidence of CMV and EBV persistence in a large collection of samples from 93 CLL cases, with an intentional bias for the usage of the IGHV4-34 gene. Overall, 34/93 patients tested positive for the presence of EBV and/or CMV genomic DNA in at least two blood samples over the period of study. Importantly, EBV and/or CMV-positive cases had a characteristic viral genome load at a roughly stable level, consistent with the view that these patients are maintaining their CMV/EBV-positive status through persistence of a long-term infection rather than through a series of transient infections.42
Striking differences with regard to IGHV gene repertoire were observed in EBV- and/or CMV-positive vs negative CLL cases. In particular, positive cases were notable for overrepresentation of the IGHV4-34 gene. Furthermore, all nine EBV/CMV double-positive cases utilized exclusively the IGHV4-34 gene. Seven of nine EBV/CMV double-positive cases belonged to subset 4 and expressed mutated BCRs characterized by distinctive antigen-binding sites, with HCDR3s enriched in aromatic and positively charged amino acids, especially arginine.22, 24 In the past, similar HCDR3 sequence motifs have been shown to correlate strongly with reactivity of IGHV4-34 antibodies against both apoptotic cells and DNA.40, 43, 44 On the basis of these features, we have previously postulated that the progenitors of CLL cases with stereotyped IGHV4-34 BCRs may have originated as cells with reactivity against DNA or apoptotic bodies.22, 24 Sequences of this subset were also recently reported by our group to display distinctive somatic hypermutation patterns, in particular recurrent (‘stereotyped’) amino-acid changes, typified by the consistent introduction of aspartic acid residues in HCDR1.24 On the basis of analogous findings in transgenic mice with edited anti-DNA BCRs,45, 46 we proposed that this type of modification may have represented a means to diminish responsiveness of the CLL progenitors against DNA. That notwithstanding, these same cells could retain the ability to receive stimulation signals by superantigenic-like interactions as they retained the NAL-binding motif in HFR1 in germline conformation.24
Given that all nine EBV/CMV double-positive cases from our series utilized the IGHV4-34 gene, mostly in stereotyped rearrangements, it can be hypothesized that EBV and CMV may be involved in the pathogenesis of a subset of CLL cases expressing IGHV4-34 BCRs with distinctive molecular features. In this scenario, the IGHV4-34 CLL precursor might be triggered by viral antigens through recognition of NAL epitopes mediated by their intact HFR1 motif.3, 5, 47 This contention is also supported by studies on IGHV4-34 antibodies from patients with SLE, which have been shown to possess a NAL determinant reactivity apart from the well-known anti-DNA reactivity.2, 38, 39, 48
In conclusion, our study demonstrates for the first time a possible link between latent or persistent infection by EBV and CMV and a subset of CLL cases expressing IGHV4-34 BCRs. However, several important issues remain to be answered. (1) Did viral infection precede or follow malignant transformation? The ideal, though admittedly difficult, approach to explore links between EBV/CMV and IGHV4-34 CLL would be to test directly the CLL cell capacity of being stimulated by viral antigens in vitro, as previously carried out with antigens from other pathogens in other lymphoma subtypes. (2) Connected with this issue, what could be the exact type and location of interactions between CLL progenitors (or even the CLL cells themselves) and CMV or EBV? (3) Why was viral persistence observed preferentially in cases with IgG-switched stereotyped rather than heterogeneous IGHV4-34 BCRs? (4) Could activation by viral antigens/superantigens be important in promoting clonal expansion of IGHV4-34-expressing B cells (in analogy to monoclonal T-cell expansions specific for latent or persistent EBV and CMV infections49, 50) and, perhaps, eventually malignant transformation? How could this proposed activation be reconciled with the indolent disease course of EBV/CMV (+) cases (group C)? The answers to these questions may be hard to obtain, but the reported association is too striking to ignore.
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