Persistent polyclonal B lymphocytosis: an expansion of cells showing IgVH gene mutations and phenotypic features of normal lymphocytes from the CD27+ marginal zone B-cell compartment

Authors


José A. Brieva, Servicio de Inmunología, Hospital Universitario Puerta del Mar, Avenida Ana de Viya 21, 11009 Cádiz, Spain. E-mail: jabrieva@hpm.sas.cica.es

Abstract

Summary.  Persistent polyclonal B-cell lymphocytosis (PPBL) is an unusual and benign lymphoproliferation characterized by a polyclonal expansion of B lymphocytes, whose nature remains undetermined. The phenotypic analysis of three cases revealed that these cells were CD27+ IgMhigh CD21high CD5low and CD23low, a phenotype associated with the normal marginal zone (MZ) B-cell compartment. As MZ B cells have initiated immunoglobulin (Ig)V gene somatic mutations, PPBL IgVH genes were sequenced. An average of 73% of these sequences were mutated. The mean number of mutation per sequence was 6·9, a number similar to those observed in the MZ B-cell compartment.

Human persistent polyclonal B lymphocytosis (PPBL) is a rare condition characterized by a polyclonal expansion of peripheral blood B cells and elevated serum polyclonal immunoglobulin M (IgM). The disorder preferentially occurs in middle-aged female smokers and shows a strong association with the human leucocyte antigen (HLA) DR7 allele (Mossafa et al, 1999), suggesting that both genetic and environmental factors are involved. The expanded B-cell population contains binucleated lymphocytes and cytogenetic abnormalities, such as the presence of an isochromosome i(3q); multiple bcl2/Ig gene rearrangements are currently observed in these cells (Mossafa et al, 1999). Despite these findings, PPBL appears to be a benign entity.

The origin and nature of these B-cell expansions remains unknown. Here we describe an exhaustive phenotypic study of three cases of PPBL in which expanded lymphocytes were CD27+ and exhibited a marginal zone (MZ) B cell-like phenotype. In addition, the analysis of the IgVH gene sequences of expanded B lymphocytes revealed that a large proportion of the cells had initiated somatic mutation events. Both features strongly suggest that PPBL originates from the MZ B-cell compartment.

Patients and methods

Patients and controls.  Three women (two aged 41, one aged 47 years), who presented with PPBL were studied. The three patients had shown a moderate lymphocytosis (6·5–10·4 × 109 lymphocytes/l) for a minimum of 3 years prior to this study. B lymphocytes, all taken as CD19+ cells, represented between 40% and 60% of total lymphocytes. The κ/λ ratio in both serum Ig and B-cell surface Ig was repeatedly normal. The three patients were heavy cigarette smokers, and expressed HLA-DR7. High serum levels of polyclonal IgM were detected (8·7, 7·56 and 18·2 g/l, for cases 1, 2 and 3 respectively; normal serum IgM, range 0·4–2·3 g/l). All patients showed variable numbers of circulating binucleated lymphocytes and cytogenetical abnormalities, including additional isochromosome i(3)(q10) in the three cases, and a deletion 6q– in case 1. Fourteen healthy volunteers were included as controls.

Materials.  Fluorescein isothiocyanate (FITC)-labelled monoclonal antibody (mAb) against CD10, CD11a, CD19, CD40, CD44, CD 62-L, CD71, DR, phycoerythrin (PE)-labelled mAb against CD5, CD11c, CD20, CD21, CD22, CD23, CD24, CD25, CD27, CD50, CD54, CD69, CD79, CD80, CD95, Cy-chrome-labelled mAb against CD19 and the corresponding isotypic negative controls were provided by Becton Dickinson (San Jose, CA, USA). FITC-labelled antibody (Ab) against lambda, IgM, IgD and PE-labelled against kappa rabbit anti-human Igs were provided by Dako (Glostrup, Denmark). FITC-labelled mAb against FMC-7 was purchased from Immunotech (Luminy, France).

Lymphocyte preparation.  Peripheral blood mononuclear cells (PBMC) were obtained from the three patients and the controls by Ficoll density centrifugation, and this cell preparation was used in two- and three-colour immunofluorescent staining and flow cytometry analysis, as previously reported (Segundo et al, 1999). The expression of the different molecules was assessed on CD19+ selected cells by collecting 5000 CD19+ events/sample, and the percentage of positive cells and mean fluorescent intensity were recorded for each molecule. CD19+ cells were isolated from PBMC of the three patients by an immuno-magnetic selection procedure that used FITC-CD19 mAb followed by goat anti-FITC IgG magnetic beads, according to the manufacturers' instructions (Miltenyi Biotec, Auburn, CA, USA). These cell preparations contained more than 97% B lymphocytes, estimated as CD19+ cells, and were used for VH genes sequence studies.

Sequence analysis of VH genes.  mRNA was isolated from 2 to 3 × 106 purified CD19+ cells by poly dT-tailed magnetic beads (Dynal, Norway), and was reverse-transcribed into cDNA using oligo-dT as primer and avian myeloblastoid virus-reverse transcriptase (AMV-RT, Promega, Southampton, UK). VH3, VH5 and VH6 gene families were amplified from cDNA by polymerase chain reaction (PCR) using Pfu polymerase (Promega), forward family specific primers localized in the framework region 1 (FR1) (5′-GAGGTGCAGCATGGTGGAGTCT-3′ for VH3, 5′-GAGGTGCAGCTGGTGCAGTCT-3′ for VH5 and 5′-CAGGTACAGCTGCAGCAGTCA-3′ for VH6) and antisense JH gene family primer mix (5′-TGAGGAGACGGTGACCAGGGTGCC-3′ for JH1-2, 5′-TGAACAGACGGTGACCATTGTCCC-3′ for JH3, 5′-TGAGGACACGGTGACCAGGGTTCC-3′ for JH4-5 and 5′-TGAGGAGACGGTGACCGTGGTCCC-3′ for JH6). Amplified PCR products (≅350 bp) were purified and incubated with Taq polymerase (Promega) in the presence of 2 mmol/l dATP for 2 h at 72°C in order to add an adenine at both 3′ ends of the fragments. The fragments were cloned in a vector pBluescript (pBK, Stratagene, La Jolla, CA, USA) modified in the 3′ extreme with a thymidine for PCR cloning. Transformed positive clones were sequenced and the sequences were analysed and compared with the IMGT database (Lefranc, 2001). The probability that somatic mutations in the rearranged VH genes resulted from antigen selection was analysed according to Chang & Casali (1994).

Results and discussion

In an attempt to clarify the nature of the expanded B-lymphocyte subset occurring in PPBL, a broad phenotypical study was performed in three patients. B cells were identified as CD19+ cells, and additional molecules were explored in two- or three-colour FACS analysis. Figure 1A shows an example of the histogram expression of a variety of B-cell surface molecules exhibited by circulating B lymphocytes from a PPBL patient and a healthy individual.

Figure 1.

Flow cytometric analysis of the expression of several surface molecules by circulating B lymphocytes from three cases of PPBL and normal controls. (A) An example of the histogram obtained for the indicated molecule by one patient (continuous line) and one healthy individual (dotted line). (B) Summarized data obtained in the study of the three patients (cases 1, 2 and 3) and normal controls (□, mean ± SEM.).

Figure 1B summarizes the data obtained in the study of the three patients and normal controls. Only the molecules that were distinctively expressed are shown. An initial difference observed was that, although both patient and control B lymphocytes were positive for surface IgD and IgM, the expression of IgM in the former was clearly higher. An additional difference was that most PPBL cells were CD27+, while this molecule was expressed only by an average of 20% of the B lymphocytes from healthy controls. In contrast, patients' B lymphocytes exhibited lower percentages of CD23+ and CD5+ cells than controls. Moreover, PPBL cells showed higher percentages of cells expressing several molecules including the integrin CD11c, the high affinity IL-2-receptor CD25 and the death receptor CD95. Finally, PPBL cells expressed CD24, CD79, FMC7 and CD21 at levels higher than normal B lymphocytes. No difference was observed between PPBL and control B lymphocytes for the expression of CD10, CD11a, CD19, CD20, sIgD, CD22, CD40, CD44, CD49d, CD50, CD54, CD62L, CD69, CD71, CD80 and DR (data not shown).

In summary, the population of B lymphocytes that was expanded in the blood from PPBL cases exhibited a characteristic and homogeneous phenotype expressing CD27+ IgMhigh CD21high CD5low and CD23low, a pattern of B-cell markers that associates these cells with a complex compartment of normal B lymphocytes present in the splenic marginal zone (MZ) (Tangye et al, 1998), the MZ-like area of Peyer's patches and lymph nodes (Spencer et al, 1998), and the subepithelial zone of tonsils (Dono et al, 2000). Furthermore, this phenotype is also shared by a subset of B lymphocytes occurring in the blood (Klein et al, 1998) and in the bone marrow of normal subjects (Paramithiotis & Cooper, 1997). These similarities suggest that B lymphocytes that accumulate in PPBL patients might consist of an expanded CD27+ IgD+ IgMhigh B-cell subset normally occurring in the MZ compartment. The finding that PPBL cells additionally expressed molecules such as CD11c, CD25 or FMC7 does not preclude this notion, as activated B lymphocytes have occasionally been described in normal MZ counterpart populations (Paramithiotis & Cooper, 1997; Klein et al, 1998; Spencer et al, 1998; Dono et al, 2000). In this regard, elevated expression of CD95 has also been reported in normal CD27+ B lymphocytes from the splenic MZ (Tangye et al, 1998) and BM IgMhigh B lymphocytes (Paramithiotis & Cooper, 1997). Therefore, phenotypic data strongly suggest that the B lymphocytes expanded in PPBL patients are related to the normal CD27+ MZ B-lymphocyte compartment.

Recent evidence demonstrates that B lymphocytes from normal MZ and MZ-like areas have already triggered Ig-V gene somatic mutations events (Dunn-Walters et al, 1995; Paramithiotis & Cooper, 1997; Klein et al, 1998; Tangye et al, 1998; Tierens et al, 1999; Dono et al, 2000) and, accordingly, these cells are now considered as a memory B-lymphocyte compartment. In consequence, the presence of Ig-V gene somatic mutations was examined in the B cells isolated from the three PPBL patients under study. A total of 70 Ig-VH sequences were analysed (20, 22 and 28 sequences for case 1, 2 and 3 respectively). An average of 73% of these sequences were mutated (13/20, 20/22 and 18/28, for cases 1, 2 and 3 respectively). Thus, the proportion of mutated sequences found in the PPBL cells was higher than that reported for normal blood B lymphocytes (≅30–40%).

Data of mutated Ig-VH sequences obtained from PPBL cells are summarized in Table I. As can be seen, the number of mutations per sequence ranged from 1 to 31, with a mean number of mutations per sequence of 6·9 ± 1·1 (mean ± SEM). The overall frequency of mutations per 100 bp was 2·3% ± 0·3, which is clearly higher than the figures observed for resting naive B lymphocytes (Brezinschek et al, 1995; Spencer et al, 1998; Tangye et al, 1998). The mutation frequencies detected in PPBL were in the range of those described for normal lymphocytes from the CD27+ MZ B-cell compartment (1·6–5·6) (Dunn-Walters et al, 1995; Paramithiotis & Cooper, 1997; Klein et al, 1998; Spencer et al, 1998; Tangye et al, 1998; Tierens et al, 1999; Dono et al, 2000). It is well established that Ag-selected IgVH mutations occurring in the germinal centres lead to the accumulation of replacement mutations in the complimentarity-determining regions (CDRs) while this kind of mutation are suppressed in the framework regions (FRs) (Chang & Casali, 1994). Mutations in PPBL cells were present in all three FR, as well as in CDR1 and 2, and the average number of observed R mutations was lower than that expected for FR (2·5 versus 4·1 respectively), while the same parameter was higher than expected for CDR (1·6 versus 1·05 respectively). Nevertheless, a complete pattern of Ag-selected mutations (Chang & Casali, 1994) was only observed in a few clones (Table I). The significance of the mutational process occurring in the normal MZ-like B-cell compartment is not entirely understood, and the well-defined antigen-selected mutation distribution pattern (Chang & Casali, 1994) has not been consistently demonstrated (Dunn-Walters et al, 1995; Paramithiotis & Cooper, 1997; Klein et al, 1998; Spencer et al, 1998; Tangye et al, 1998; Tierens et al, 1999; Dono et al, 2000). Therefore, PPBL cells harbour mutational events similar to those described in the normal MZ B-cell compartment. Taken together, the present results indicate that the expanded population occurring in PPBL shows phenotypic and genetic features that associate these cells with the CD27+ MZ B-cell compartment.

Table I.  Mutation analysis of the persistent polyclonal B lymphocytosis (PPBL) B cells in three cases.
 VH genenF (%)R/SExp RPCDRPFR
CDRFRCDRFR
  1. VH gene, IgH variable gene; n, total number of mutations; F, frequency of mutations (number of mutation per 100 bp sequenced); R, replacement mutations; S, silent mutations; CDR, complimentarity-determining region; FR, framework region; PCDR (PFR), probability that excess or scarcity of the R mutations in the VH gene CDRs (FR)s resulted from chance only; Ro, mean replacement mutation observed; Re, mean replacement mutation expected. Full sequences are in GenBank, accession numbers AYO33238AYO33288.

Case 1
1IGHV1-02*02 4 1·40/13/00·52·50·560·36
2IGHV3-11*01 8 2·83/14/01·14·90·070·22
3IGHV3-11*01 4 1·41/00/30·52·50·350·02
4IGHV3-30*10 3 1·041/01/10·41·80·300·27
5IGHV5-51*01 6 2·10/15/00·83·70·420·20
6IGHV5-51*01 2 0·71/00/10·31·20·230·15
7IGHV6-01*01 4 1·31/02/10·62·40·370·35
8IGHV6-01*01 2 0·71/01/00·31·20·260·48
9IGHV1-69*04 5 1·72/03/00·63·10·120·35
10IGHV3-07*02 8 2·83/02/31·14·90·070·03
11IGHV6-01*01 5 1·71/03/10·83·00·390·35
12IGHV6-01*01 2 0·701/10·31·20·710·48
13IGHV5-51*01 4 1·41/10/20·52·50·350·02
Mean 4·07 1·41Ro = 1·1Ro = 1·9Re = 0·6Re = 2·50·30·23
Case 2
1IGHV1-18*01 1 0·30/01/00·10·60·870·62
2IGHV3-23*01 5 1·73/02/00·73·10·020·07
3IGHV6-01*0129 9·769/29/94·517·30·020·001
4IGHV3-21*01 9 3·12/13/31·25·50·240·06
5IGHV3-23*01 3 1·040/02/10·41·80·650·44
6IGHV3-7*0210 3·52/15/21·36·20·260·19
7IGHV4-34*01 1 0·30/01/00·10·60·870·62
8IGHV4-34*0122 7·74/18/92·813·70·170·01
9IGHV6-1*01 2 0·70/11/00·30·50·720·48
10IGHV6-1*01 5 1·70/04/10·80·30·430·26
11IGHV6-1*01 3 1·010/02/10·51·80·600·43
12IGHV1-03*01 2 0·70/02/00·31·20·750·38
13IGHV1-18*01 3 1·042/01/00·41·80·050·27
14IGHV1-18*0113 4·56/43/01·780.0040·005
15IGHV3-30–3*01 1 0·31/000·10·60·130·38
16IGHV3-30–3*01 1 0·301/00·10·60·870·62
17IGHV3-33*05 9 3·11/01/71·25·50·270·02
18IGHV1-03*0128 9·74/410/103·817·20·210·003
19IGHV6-1*01 3 1·010/12/00·51·70·600·43
20IGHV3-23*0131 10·766/314/84·219·10·120·026
Mean 8·6 3·0Ro = 2Ro = 3·6Re = 1·2Re = 5·10.3740·253
Case 3
1IGHV1-46*03 2 0·702/00·71·20·750·38
2IGHV1-46*03 1 0·31/000·10·60·130·38
3IGHV3-7*01 6 2·11/02/30·83·70·420·26
4IGHV5-51*01 8 2·82/04/21·14·90·210·22
5IGHV5-a*03 1 0·31/000·10·60·130·38
6IGHV5-51*01 4 1·41/02/10·52·50·350·34
7IGHV6-01*01 2 0·71/01/00·31·90·260·48
8IGHV6-01*01 9 31/23/31·45·40·360·07
9IGHV1-02*02 4 1·41/02/10·52·50·350·33
10IGHV1-02*02 6 2·11/04/10·83·70·390·32
11IGHV5-51*01 3 1·041/01/10·41·90·300·27
12IGHV5-51*01 3 1·041/11/00·41·90·300·27
13IGHV3-01*01 2 0·71/100·31·20·230·15
14IGHV1-f*0113 4·509/41·78·00·150·20
15IGHV3-21*0110 3·54/01/51·36·20·030·001
16IGHV5-51*01 4 1·41/02/10·52·50·350·34
17IGHV6-01*0129 9·7610/110/84·4817·320.0060·004
18IGHV5-51*01 7 2·42/21/20·94·30·180·01
Mean 6·0 2·1Ro = 1·3Ro = 1·9Re = 0·9Re = 3·702580·232

Acknowledgments

This study has been supported by grants 96/2116 and 01/1590 from Fondo de Investigaciones Sanitarias of Spain. We would like to thank Daniel Armenta for cytogenetic studies and Olga Caballer for excellent technical help.

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