Classification of B-cells according to their differentiation status, their micro-anatomical localisation and their developmental lineage

Immunology Letters 90 (2003) 179–186

Classification of B-cells according to their differentiation status, their micro-anatomical localisation and their developmental lineage Xavier Sagaert a,∗ , Christiane De Wolf-Peeters b a

Department of Morphology and Molecular Pathology KU Leuven, Aspirant FWO Flanders, Minderbroederstraat 12, 3000 Leuven, Belgium b Department of Pathology, University Hospitals, KU Leuven, Minderbroederstraat 12, 3000 Leuven, Belgium Received 12 September 2003; accepted 15 September 2003

Abstract B-lymphocytes or B-cells form a diverse and flexible repertoire of immune cells that are reactive to almost all potential pathogens by means of the production of antigen-specific immunoglobulins. They can be divided into different populations or subsets, characterised by a distinct combination of properties. These subsets are identified on the base of their differentiation status (precursor B-cells, peripheral B-cells), their localisation in the micro-anatomical compartments of the B-cell follicle (marginal zone B-cells, lymphocytic corona B-cells, follicle centre B-cells), and the developmental lineage to which they belong (B-1 cells, and B-2 or conventional B-cells). The latter classification of B-cells into B-1 cells and B-2 cells is commonly followed by immunologists, mainly in the study of mice models, while pathologists and haematologists tend to use a terminology for B-cells which refers to their localisation in the micro-anatomical compartments of the B-cell follicle and/or differentiation status. In this review, we will discuss the various subsets of B-cells and point to the similarities between the various classification systems in use. © 2003 Elsevier B.V. All rights reserved. Keywords: B-cells; Differentiation; Micro-anatomical localisation; Developmental lineage

1. Introduction B-lymphocytes may produce a multitude of antibodies with diverse antigen-binding specificities. During B-cell ontogeny, the repertoire of B-cells secreting antibodies with unique binding specificities is produced at two stages. A primary B-cell repertoire is formed in the bone marrow through gene rearrangements, whereas a secondary B-cell repertoire is generated in the peripheral lymphoid organs (spleen, lymph nodes and mucosa associated lympoid tissue) through somatic hypermutation upon antigen encounter (Fig. 1).

2. Classification of B-cells according to their differentiation status The B-cell population can be divided into two subsets, precursor B-cells and peripheral B-cells, according to the ∗

Corresponding author. Tel.: +32-16-336610; fax: +32-16-336548. E-mail address: [email protected] (X. Sagaert).

0165-2478/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2003.09.007

differentiation status. Precursor B-cells belong to the primary B-cell repertoire and comprise pro-B-cells (early and late) pre-B-cells, immature B-cells and mature or na¨ıve B-cells (Table 1). Peripheral B-cells are part of the secondary B-cell repertoire and are classified according to their relation to the germinal centre (GC) reaction. The presence of mutations in the Ig variable region is considered to be a reliable marker for a B-cell that has been exposed to the reaction—either at GC or post-GC stage. The presence of on-going mutations—variations in the mutation pattern among GC B-cell clones—is characteristic of B-cells still at the GC stage. As such, three categories of peripheral B-cells can be recognised: pre-GC B-cells, GC B-cell, and of post-GC B-cells (Table 1). This classification system is particularly in use in medical science since it provides a very useful tool to classify B-cell lymphomas and B-cell leukemias according to their putative B-cell of origin [18]. As such, follicular lymphoma display highly mutated Ig genes and on-going mutations consistent with a GC cell origin. In contrast, mantle cell lymphomas mostly have rearranged Ig genes without somatic mutations, consistent with a derivation from naive

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Primary B-cell repertoire

Secondary B-cell repertoire

Peripheral lymphoid organs (spleen, lymph nodes, MALT)

Bone marrow

progenitor B-cell

mature B-cell

mature B-cell

rearrangement Ig heavy chain genes

memory B-cell

FOLLICLE MANTLE

antigen encounter

pre-B -cell

centrocyt -

Bblast

rearrangement Ig light chain genes

i

FOLLICLE CENTRE

mmature B-cell

IgM

centroblast

mature B-cell IgD

somatic hypermutation

short-living plasma cell

IgM

long-living plasm a cell

Fig. 1. Events in the development of the primary and the secondary B-cell repertoire.

B-cells. Finally, lymphomas with Ig gene somatic mutations but lack of on-going mutations such as approximately half of chronic lymphocytic leukemia (CLL) cases, are thought to have a post-GC cell origin. 2.1. Differentiation of precursor B-cells (primary B-cell repertoire) In adults, the primary B-cell repertoire emerges from hematopoietic stem cells located in the bone marrow [1–3]. B-cell commitment of pluripotent stem cells implies a highly

regulated series of genetic events, called immunoglobulin (Ig) heavy and light chain gene rearrangement, resulting in the formation and surface expression of a functional B-cell receptor and in the creation of a primary B-cell repertoire [4,5]. Rearrangements of Ig heavy and light chain gene segments is dependent upon the activation of the recombination activating genes RAG-1 and RAG-2 [6]. These genes are temporarily expressed in the early stages of B-cell maturation in the bone marrow. Recent evidence however, obtained in mice experiments, demonstrated that RAG-1 and RAG-2 genes can be re-activated in later stages of B-cell

Table 1 Characteristic features of B-cells, classified according to their differentiation status B-cell stage

Ig gene status

Somatic mutations

Ig production

Immunophenotype

Pro-B-cell Pre-B-cell Immature B-cell Pre-GC B-cell GC B-cells Memory B-cells Plasma cells

Germ line IgH rearrangement IgH + IgL rearrangement IgH + IgL rearrangement IgH + IgL rearrangement IgH + IgL rearrangement IgH + IgL rearrangement

None None None None Yes Yes Yes

None ␮-Chain (cytoplasm) IgM (membrane) IgM + IgD (membrane) Minimal or absent IgM IgG > IgA > IgD

CD19, CD19, CD19, CD19, CD19, CD19, CD38,

CD79a, CD10, CD34, TdT CD79a, CD10, CD34, TdT CD79a, CD10, CD20 CD79a, CD20, CD5 CD79a, CD20, CD10, BCL-6 CD79a, CD20 MUM-1, CD138

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maturation, more precisely in germinal centre cells, where they can mediate secondary rearrangements [7]. The earliest B-cell in the bone marrow is the progenitor B-cell or the pro-B-cell, which differs from the hematopoietic stem cell by the expression of B-cell antigens e.g. CD19 and CD79a, and by its ability to initiate rearrangement of the Ig gene locus. A pro-B-cell is transformed into an early pre-B-cell once that the DH and JH gene segments are recombined in the Ig heavy chain locus (with the genes for light chains still remaining in germline configuration). Further rearrangements attach one of the VH gene segments to the DH JH segment and give rise to a new B-cell stage, called the late pre-B-cell, which expresses a functionally rearranged VH DH JH C ␮-chain on the cell surface. Subsequent rearrangement of the Ig light chain gene locus leads to the surface expression of a complete IgM molecule and to a new B-cell stage, designated immature B-cell. The latter gives rise to the mature B-cell that, as a result of an alternative splicing of Ig heavy chain mRNA, expresses both IgM and IgD at the cell surface. Mature B-cells are also called na¨ıve B-cells as they did not yet encounter an antigen. 2.2. Differentiation of peripheral B-cells (secondary B-cell repertoire) Mature na¨ıve B-cells migrate into the secondary lymphoid organs where they either die or form primary B-cell follicles. Upon encounter with an antigen, the mature na¨ıve B-cells move into the extrafollicular area of the lymph node (or the periarteriolar lymphocyte sheat in the spleen). Only a small number of antigens is T-cell independent as they directly activate na¨ıve B-cells by binding to their antigen-specific B-cell receptor. Most antigens however are T-cell dependent; they indirectly activate na¨ıve B-cells by the formation of two immune synaptic interactions [8]: a first synapse between antigen-presenting cells (interdigitating dendritic cells) and T helper cells which leads to clonal expansion of those T helper cells with the required T-cell receptor, and a second synapse between the selected and antigen-primed T helper cells and mature na¨ıve B-cells. The latter interaction is a two-way process, in which B-cells present the antigen in association with MHC class II molecules to T-cells and receive signals from those T-cells for proliferation and differentiation. Antigen-activated B-cells, either with or without the interaction of antigen-presenting cells and T-cells, transform into large B-blasts that may follow two different pathways [2,3]. Some proliferate in loco and differentiate into short-living, IgM producing plasma cells, responsible for the early production of antibodies and, thus, first-line defence against the antigen. A minority of the B blasts migrates into the primary B-cell follicle, where they rapidly proliferate and differentiate into GC B-cells [9,10]. The non-antigen-triggered na¨ıve B-cells of the primary follicle are pushed aside and, thus, form the follicle mantle or mantle zone. This follicle,

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containing a GC and a mantle, is known as a secondary follicle (the histology of the secondary follicle, and its variation among different lymphoid organs, will be discussed below). Apoptosis of GC B-cells can be prevented only by survival signals delivered by T-cells and follicular dendritic cells (FDC). The latter lack the expression of class II major histocompatibility complex (MHC) molecules, but have the complement receptor CR1 (CD35) for interaction with immune complexes which they can present as such to B-cells. GC B-cells undergo a randomised introduction of mutations into the Ig gene region that encodes for the antigen binding site, a process called ‘somatic hypermutation’ [11,12]. Since the process of somatic hypermutation is at random, most of these mutations do not increase, or even decrease, the affinity of the Ig receptor or even completely prevent the expression of Ig by creating stop codons or frame shifts. GC B-cells with unfavourable mutations do not bind with high affinity to the antigen trapped by the FDC and do not appropriately interact with the GC T-cells. Therefore, they do not receive survival signals from these cells. In fact, more than 90% of the GC B-cells die as a result of apoptosis. These cells are phagocytosed and digested by the so-called tingeable body macrophages. Only those GC B-cells that synthesize high-affinity antibodies survive and may differentiate into memory B-cells or plasma cells. It has to be noted that the process of hypermutation not solely target the Ig locus as there is also evidence for the occurrence of BCL-6 mutations in GC B-cells [13,14]. The positively selected GC B-cells activate T-cells to express CD40-ligand and to secrete IL-4 and IL-10 which induces expansion and a switch of the Ig heavy chain class from IgM to IgG, IgA or less commonly IgE. After accomplishing affinity maturation and a possible isotype switch GC B-cells may either differentiate into post-GC B-cells (plasma cells or memory cells) or undergo another round (or repeated rounds) of proliferation, hypermutation and selection. The CD27 and CD40-ligand interaction with T-cells is the key-event in directing GC B-cells towards the memory B-cell pathway [15,16], while interaction with surface molecules expressed by FDC (such as CD23) appears to be important in directing the differentiation of GC B-cells into plasma cells [17]. Plasma cells are thought to reside mainly in the bone marrow and organs that are directly exposed to foreign antigens (gastro-intestinal tract, lung), whereas memory cells (which act as precursors to the recall immune response) reside in the follicle mantle or re-circulate freely to survey for secondary antigen exposure. Importantly, newly formed GC B-cells represent an oligoclonal B-cell population. On average, each mature GC is derived from only 1 to 3 B-cell clones and comprises about 104 B-cells. The GC reaction reaches its maximum by day 10–12 of the primary immune response. Without further antigenic simulation, the GC wanes by 21 days post-immunisation [8].

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3. Classification of B-cells according to their micro-anatomical localisation B-cells of the secondary B-cell repertoire can be classified as well according to their homing in the various compartments of the B-follicle, being the follicle centre (or GC) and the follicle mantle. The latter comprises a lymphocytic corona and a marginal zone, which is particularly well developed in the white pulp of the spleen. The B-cell subsets that reside in these different compartments are referred to as follicle centre B-cells, lymphocytic corona B-cells and marginal zone B-cells [19,20]. They are characterised by a distinct morphology, immunophenotype and genotype. It has to be noted however that immunologists will refer to the lymphocytic corona as the mantle zone, hereby implying that the marginal zone is not part of the follicle mantle (or the follicle as a whole) (Fig. 2). Follicle centre B-cells are subdivided into centroblasts (large noncleaved cells) and centrocytes (small or large cleaved cells) [10]. Centroblasts are large, proliferating cells with a round, vesicular nucleus, one to three prominent nu-

cleoli located along the nuclear membrane, and a narrow rim of basophilic cytoplasm. They accumulate at one pole of the GC, forming the dark zone. They lack surface Ig expression [21] and switch off the gene that encodes the BCL-2 protein which makes them susceptible to death through apoptosis if not positively selected [22]. Centroblasts express antigens associated with activation, most of which are involved in their interaction with T-cells (such as CD23, CD71, CD40, CD86), as well as antigens associated with their adhesion to FDC (such as CD11a/18 and CD29/49d) and antigens that may promote their apoptosis (such as CD95) [23–26]. While acquiring somatic hypermutations, centroblasts differentiate into non-proliferating medium-sized centrocytes which are characterised by an irregular nucleus, inconspicuous nucleolus, and scant cytoplasm. Centrocytes accumulate at the opposite pole of the GC, known as the light zone. They re-express surface Ig, with the same VDJ rearrangement as in the parent na¨ıve B-cell and centroblast of the dark zone, but with an altered antibody binding site due to the somatic mutations in the immunoglobulin V region [27]. An important feature of follicle centre B-cells is the

Fig. 2. The micro-anatomical structure of the B-follicle in the spleen.

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expression of BCL-6, a nuclear zinc finger transcription factor that is not expressed in pre-GC B-cells or post-GC B-cells, e.g. the memory B-cells and plasma cells [13,14]. Animal experiments learn that BCL-6 is required for GC formation and T-cell dependent antibody responses. By functioning as a potent transcriptional repressor of various target genes, BCL-6 also modulates IL-4, B-cell receptor, and CD40 ligand signals essential for normal B-cell development. Alterations of the BCL-6 promoter region, including chromosomal translocation, represent a frequent genetic aberration associated with non-Hodgkin lymphoma, especially diffuse large B-cell lymphoma, a malignancy often derived from follicle centre B-cells. This suggests that deregulated expression of BCL-6 may contribute to lymphomagenesis. The inner part of the mantle zone of the B-follicle is indicated as the lymphocytic corona. It is mainly composed of mature, na¨ıve or virgin B-cells, which were pushed aside by the formation of the follicle centre. Lymphocytic corona B-cells express strongly both surface IgM and IgD and pan-B-cell antigens (CD19, CD20, CD79a) [28]. In contrast to their precursor B-cells in the bone marrow and the GC B-cells, they lack CD10 [11]. They are characterised by rearranged but unmutated Ig genes. Besides these na¨ıve mature B-cells, the lymphocytic corona also comprises memory B-cells, especially in lymphoid tissues where the marginal zone is poorly developed. The outer part of the mantle zone of the B-follicle corresponds to the marginal zone. The marginal zone is well developed in those secondary lymphoid organs where an abundant influx of antigens is known to occur [29–31]. As such, it is especially well recognisable in the white pulp of the spleen where it was originally described. It may also be easily observed in Peyer’s patches of the intestine and in tonsils. It is less obvious in lymph nodes except for the mesenteric ones. The marginal zone is populated by different kinds of cells: macrophages, some T-cells, granulocytes, plasma cells, small lymphocytes and marginal zone B-cells. The latter are characterised by an abundant clear cytoplasm and a pale, irregular, centrally located nucleus. Due to the resemblance to centrocytes, marginal zone B-cells have also been indicated as ‘centrocyte-like’ cells [30]. Marginal zone cells express pan-B-cell markers, IgM and BCL-2, and lack the expression of CD5, CD10 and CD43. Whereas the expression of IgD is generally considered to be lacking, in our and other’s experience IgD expression may vary from negative to weakly positive [30]. The negative or low expression of IgD on marginal zone B-cells is useful to distinguish them from lymphocytic corona B-cells which are strongly IgD positive. Other antigens reported to be strongly expressed by marginal zone B-cells are alkine phosphatase and complement receptors CR1 (CD35), CR2 (CD21) and CR3 (CD18/11b) [32–34]. Cells that resemble marginal zone cells but with even more nuclear indentation and abundant cytoplasm, known as monocytoid B-cells, are seen in clusters adjacent to subcapsular and cortical sinuses of some reactive

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lymph nodes, such as toxoplasma induced lymphadenitis [35]. The immunophenotype of these monocytoid B-cells is largely similar to that of marginal zone B-cells with the exception of the expression of IgM and BCL-2 which are, respectively, variable and lacking in monocytoid B-cells [36,37]. Mutation analysis of the rearranged Ig genes indicates that the marginal zone is populated by a heterogeneous B-cell population, with virgin B-cells (with unmutated Ig genes) as well as memory B-cells (with mutated Ig genes) [38–40]. A controversy on the pre-GC or post-GC origin of marginal zone B-cells remains as the presence of mutated Ig V genes in marginal zone B-cells does not preclude the possibility that these cells may not be derived from the GC (although the hypermutation mechanism is classically thought to be associated with this site) [41]. In fact, it was shown that monocytoid B-cells may acquire low-level somatic hypermutation in situ and therefore outside the GC [42]. This finding seems further more supported by observations in patients with the X-linked hyper-IgM syndrome in which IgM positive, IgD negative B-cells are seen in the absence of GC [43]. The function of the marginal zone is dual: it plays an essential role in the response to T-cell independent type 2 antigens, such as bacterial capsular polysaccharide antigens, but it is also vital in the general first-line defence against blood-borne antigens [44]. Until the age of 2 years, when the marginal zone is known to be fully developed, infants remain susceptible to infections with capsulated bacteria such as Hemophilus influenza and Neisseria meningitides. Splenectomized individuals are also notoriously susceptible to infections with these bacteria and therefore should be vaccinated.

4. Classification of B-cells according to their developmental lineage Another approach to B-cell subsetting is particularly in use in mice studies (Table 2). It is based on the surface markers and functional features of B-cells, irrespective of the cell’s homing properties, and recognizes two major subsets, the B-1 and B-2 B-cells [45,46]. Initially, the distinction was based on the presence (B-1) or absence (B-2) of CD5 on the cell surface. Later on, it was proven that not all B-1 cells express CD5 despite the presence of CD5 mRNA, which led to a further subdivision of B-1 cells into B-1a (CD5 surface positive) and B-1b (CD5 surface negative) cells. Murine B-1 cells are predominantly located in the peritoneal and pleural cavities and are virtually absent from lymph nodes, Peyer’s patches and peripheral blood [47]. They represent a small fraction of the B-cell population in adult spleen, most of which are B-2 cells. Recently, it was shown that the preferential homing of B-1 cells to the body cavities is caused by the chemokine CXCL13, which is mainly produced by cells of the omentum and the peritoneal macrophages [48]. B-1a cells (formerly known as Ly-1 positive B-cells) express surface CD5 and are derived

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Table 2 Distinctive features of murine B-cells, classified according to their developmental lineage Distinctive feature

B-1 cells

B-2 cells (conventional B-cells)

Localisation Reconstitution Antibody production CD5 CD5 mRNA IgM IgD CD11b (MAC1) CD23 (FcεR)

Body cavities By division of fully mature B-1 cells Polyreactive + on B-1a − on B-1b + on B-1a + on B-1b +++ + + (− on splenic B-1 cells) –

Blood, spleen, lymph nodes, Peyer’s patches By differentiation of progenitors located in the bone marrow Monoreactive – – + (+++ on marginal zone B-2 cells) +++ (− to + on marginal zone B-2 cells) – + (− on marginal zone B-2 cells)

from progenitors that are present in fetal omentum and fetal liver but are largely absent from adult bone marrow; in contrary, B-1b cells (formerly known as Ly-1 negative B-cells) do not express surface CD5 and are derived from progenitors that are present in fetal omentum, fetal liver, and also in adult bone marrow [45]. The number of B-1a and B-1b cells varies in different mouse strains, e.g. B-1b cells represent 20–25% of the B-1 cells in BALB/c congenics and 40–50% of the B-1 cells in CBA congenics [49,50]. Both B-1a and B-1b cells are self-renewing cells, which means that they are reconstituted by division of fully mature B-1 cells. B-1 cells preferentially produce IgM (especially in spleen), IgA (especially in the intestinal lamina propria) and IgG3 of low affinity and with a broad affinity for polysaccharides, lipids and proteins of micro-organism coat antigens and auto-antigens. These antibodies are generally encoded by unmutated Ig V genes. Importantly, despite the fact that B-1 cells constitute only a minor fraction of all B-cells in the mouse, they appear to produce much of the Ig and probably most of the natural antibodies in serum and mucosal sites. It has been suggested that B-1 cells mainly participate in natural immunity (by preventing dissemination of pathogens and by enhanced antigen-trapping in lymphoid organs in a T-cell independent manner) and auto-immunity (increased number of B-1 cells have been found in mice that spontaneously develop auto-immune haemolytic anaemia–NZB mice, or murine systemic lupus erythematosus–NZB/NZW F1 mice) [51,52]. Murine B-2 cells or conventional B-cells develop later in ontogeny compared to B-1 cells and they emerge from progenitors which are found in the fetal liver and the adult bone marrow but which are missing from the fetal omentum [45]. The mature B-2 cell population is itself heterogeneous: recirculating B-2 cells locate in the B-follicles of the spleen and lymph nodes, whereas a particular population of mostly non-recirculating B-2 cells is located in the marginal zone of the spleen [53,54]. In contrast to B-1 cells, which maintain their numbers in adult mice by self-replenishment, B-2 cells are generated throughout life by differentiation of progenitors in the bone marrow. With the exception of marginal zone cells which are only slightly IgD positive, B-2 cells are characterised by low IgM expression and high IgD expression. This immunophenotype is in contrast to that of B-1

cells which are IgMhi IgDlo cells. Two other immunophenotypic markers deserve special attention as well. Complement receptor CR3 (CD11b) is expressed on peritoneal and pleural cavity B-1 cells but is missing on conventional B-cells and splenic B-1 cells [45,50]. FcεR (CD23) is present on all conventional (IgDhi ) B-cells in the peritoneal cavity and on the predominant conventional B-cell population in the spleen; however, it is not expressed on either marginal zone (IgDlo ) B-cells in the spleen or B-1 cells from any location [55]. Thus, in the peritoneal cavity (but not in the spleen), these markers alone can be used to distinguish B-1 cells from conventional B-cells, as B-1 cells are CD11b positive and CD23 negative whereas conventional B-cells are CD11b negative and CD23 positive. Conventional B-cells are mainly involved in the acquired immunity and in the immune response to T-cell dependent antigens. The human adult B-cell repertoire comprises B-cell subsets similar to those seen in the mouse: B-1a, B-1b and B-2 cells. B-1a and B-1b cells express comparable levels of CD5 mRNA, and B-1a cells are surface CD5 positive whereas B-1b cells are surface CD5 negative. It is generally believed that the CD5 molecule, by inhibiting nuclear translocation of NF-␬B, can function as a negative regulator of the B-cell receptor signalling pathway that may help prevent inappropriate activation of autoreactive B-1a cells [52]. B-1a cells account for 15 tot 20% of the circulating, splenic and tonsillar B lymphocytes in human adults and constitute the major B-cell population in the foetus and the neonate. B-1b cells account for 2 tot 6% of the circulating adult B lymphocytes; however, their occurrence in the secondary lymphoid organs is not clear. B-2 or conventional B-cells are CD5 surface and CD5 mRNA negative and constitute the major population of the adult B-cell repertoire. B-2 cells mainly produce antigen-induced monoreactive IgG, in contrast to B-1 cells that produce IgM antibodies, the majority of which are polyreactive. Some issues surrounding the classification of B-cells into B-1 and B-2 cells are still unresolved an debated, and need to be further clarified. To begin with, there is a striking difference between the polyreactive antibodies produced by the murine and the human B-1 cells, hampering the use of the same B-1/B-2 cell classification system in both murine and human B-cell studies: in contrast to the polyreactive

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antibodies in the mouse, the polyreactive antibodies in the human are somatically diversified as they display a somatic mutation pattern indicative of antigen-driven selection [56]. Secondly, the gene selection and junctional pattern of the VH DH JH of the human polyreactive antibodies do not reflect those thought to be characteristic of the foetal and/or neonatal B-cell repertoire where B-1 cells predominate [56,57]. The characteristic features reminiscent of the foetal B-cell repertoire, e.g. a preferential use of VH 3 family genes, short complementarity determining regions 3 due to limited N nucleotide additions in the DH JH junctions and a biased use of short DH gene segments, absence of somatic mutations in the VH genes, have not been observed in the adult B-1 cell repertoire. Whether human adult B-1 cells are derived through antigen-driven selection of a restricted set of the B-1 developmental waves of colonisation from the foetal and neonatal B-cell repertoire or emerge from mature virgin B-cells in the bone marrow is still enigmatic. In support of the latter is the observation that adult bone marrow B-cells can progress along two differentiation pathways, B-1 and B-2, in response to different stimuli [58]. B-cells induced to proliferate by cross-linking of surface IgM by soluble anti-␮ F(ab )2 fragments express surface CD5, whereas B-cells induced to proliferate in response to thymus-dependent signals, e.g. CD40-ligand, display high density CD23 but no CD5. Cross-linking of surface IgM in the absence of engagement of CD40 by CD40-ligand on T helper cells is characteristic of T-cell independent type 2 antigens, including ubiquitous self-antigens or bacterial antigens. A third debated issue in the B-1/B-2 cell classification system is whether human marginal zone B-cells belong to the B-1 or the B-2 developmental lineage. Marginal zone B-cells (as well as their putative neoplastic counterpart called the marginal zone lymphoma) display a somatic mutation pattern indicative of antigen-driven selection, a genetic fingerprint similar to that of B-1 cells. Moreover, marginal zone B-cells (and marginal zone lymphomas) predominantly rearrange VH 3 family and to a lesser extent VH 4 and VH 1 family genes, similar to what was is observed in the foetal B-1 cell repertoire. However, human marginal zone B-cells are generally accepted to be part of the B-2 cell subset, as they lack surface expression of the B-1 cell marker CD5. The observation that marginal zone B-cells share a similar VH gene rearrangement and a similar range of somatic mutations in their rearranged VH genes with B-1 cells, makes some authors wondering whether marginal zone B-cells do not represent the CD5 surface negative, CD5 mRNA positive, naturally antibody producing B-1b cell subset, a question yet to be resolved in the future [40].

5. Summary The B-cell population in mouse and human, is heterogeneous and can be subdivided into distinct subsets, by their stage of differentiation, their micro-anatomical localisation

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