**3. The B cell antigen receptor (BCR) and bone marrow B cell development**

The main function of mature immunocompetent B cells is to make antibodies upon recognition of particular new or recurrent antigens by the B cell receptor (BCR). The BCR is a membrane-bound complex of proteins, consisting of a heterodimer of identical pairs of immunoglobulin (Ig) heavy and light chains, which are responsible for the clonal diversity of the B cell repertoire and the antigen identification, but are unable to generate signals and trigger biological responses after antigen binding. This function is mediated by the disulfide-coupled heterodimer of Ig (CD79a) and Ig (CD79b), which is non-covalently associated with the Ig antigen recognition unit (Figure 4A). Ig/Ig signaling is dependent on distinct tyrosine-based activation motifs localized in the cytoplasmic tails of these proteins. It is the sequential expression and assembly of the BCR components that defines each developmental stage of the B cell pathway, and, therefore, each stage is characterized by a particular form of BCR, reflecting the progression of receptor assembly (Fuentes-Pananá et al., 2004a).

To achieve BCR clonal diversity, the Ig heavy and light chain genes are composed of constant and variable regions. The variable region is formed by a series of segments V (variable), D (diversity) and J (joining) (Figure 4B), which are brought together by a highly ordered process of VDJ recombination accomplished by the products of the recombinaseassociated genes 1 and 2 (RAG1 and RAG2) occurring first in the heavy and then in the light chain loci (Thomas et al., 2009). ProB and PreB stages are characterized by rearrangements of the Ig heavy and light chains, respectively (Figure 5) (Fuentes-Pananá et al., 2004b), and further divided according to the status of the recombination. In mice, ProB-A is the substage during which the heavy chain is in germ line state, whereas during ProB-B the heavy chain D and J fragments are recombined, and in ProB-C, V-DJ is recombined. In large PreB cells, the preBCR is already expressed in surface and the light chain V and J fragments are in germ line state, while in small PreB cells light chain V-J is recombined (Hardy et al., 1991). These stages are better known in humans as Early ProB or Pre-proB (A), ProB (B), PreB I (C), large and small PreB II (Figure 5). In the ProB stage Ig and Ig are expressed at cell surface in association with chaperon proteins such as calnexin (the proBCR). As soon as the heavy chain is successfully recombined, it is assembled with Ig and Ig and the surrogate light chains 5 and VpreB to form the preBCR. Surface expression of this receptor marks the transition to the preB stage (Figure 5) (Fuentes-Pananá et al., 2004a; 2004b).

In addition to their VDJ recombination status and pattern of surface marker expression, ProB and PreB stages can be recognized by their proliferative state (Hardy et al., 1991). RAG-1 and RAG-2 enzymes are tightly regulated during the cell cycle, being highly active in G0 and degraded before the cell enters S phase (Li et al., 1996). By assuring that proliferation and recombination are mutually exclusive mechanisms, the developing B cell guarantees that no events of non-homologous recombination will occur during DNA replication, thus avoiding an increase in the mutation rate.

#### **3.1 Self-recognition and peripheral B cell development**

Once the mature BCR is present in the surface of immature B cells, it is finally able to interact with conventional polymorphic ligands, and selection at this stage is designed to test the receptor-ligand interaction. Intimate contact between the immature B cell and the

The main function of mature immunocompetent B cells is to make antibodies upon recognition of particular new or recurrent antigens by the B cell receptor (BCR). The BCR is a membrane-bound complex of proteins, consisting of a heterodimer of identical pairs of immunoglobulin (Ig) heavy and light chains, which are responsible for the clonal diversity of the B cell repertoire and the antigen identification, but are unable to generate signals and trigger biological responses after antigen binding. This function is mediated by the disulfide-coupled heterodimer of Ig (CD79a) and Ig (CD79b), which is non-covalently associated with the Ig antigen recognition unit (Figure 4A). Ig/Ig signaling is dependent on distinct tyrosine-based activation motifs localized in the cytoplasmic tails of these proteins. It is the sequential expression and assembly of the BCR components that defines each developmental stage of the B cell pathway, and, therefore, each stage is characterized by a particular form of BCR, reflecting the progression of receptor assembly (Fuentes-

To achieve BCR clonal diversity, the Ig heavy and light chain genes are composed of constant and variable regions. The variable region is formed by a series of segments V (variable), D (diversity) and J (joining) (Figure 4B), which are brought together by a highly ordered process of VDJ recombination accomplished by the products of the recombinaseassociated genes 1 and 2 (RAG1 and RAG2) occurring first in the heavy and then in the light chain loci (Thomas et al., 2009). ProB and PreB stages are characterized by rearrangements of the Ig heavy and light chains, respectively (Figure 5) (Fuentes-Pananá et al., 2004b), and further divided according to the status of the recombination. In mice, ProB-A is the substage during which the heavy chain is in germ line state, whereas during ProB-B the heavy chain D and J fragments are recombined, and in ProB-C, V-DJ is recombined. In large PreB cells, the preBCR is already expressed in surface and the light chain V and J fragments are in germ line state, while in small PreB cells light chain V-J is recombined (Hardy et al., 1991). These stages are better known in humans as Early ProB or Pre-proB (A), ProB (B), PreB I (C), large and small PreB II (Figure 5). In the ProB stage Ig and Ig are expressed at cell surface in association with chaperon proteins such as calnexin (the proBCR). As soon as the heavy chain is successfully recombined, it is assembled with Ig and Ig and the surrogate light chains 5 and VpreB to form the preBCR. Surface expression of this receptor marks the

transition to the preB stage (Figure 5) (Fuentes-Pananá et al., 2004a; 2004b).

avoiding an increase in the mutation rate.

**3.1 Self-recognition and peripheral B cell development** 

In addition to their VDJ recombination status and pattern of surface marker expression, ProB and PreB stages can be recognized by their proliferative state (Hardy et al., 1991). RAG-1 and RAG-2 enzymes are tightly regulated during the cell cycle, being highly active in G0 and degraded before the cell enters S phase (Li et al., 1996). By assuring that proliferation and recombination are mutually exclusive mechanisms, the developing B cell guarantees that no events of non-homologous recombination will occur during DNA replication, thus

Once the mature BCR is present in the surface of immature B cells, it is finally able to interact with conventional polymorphic ligands, and selection at this stage is designed to test the receptor-ligand interaction. Intimate contact between the immature B cell and the

**3. The B cell antigen receptor (BCR) and bone marrow B cell development** 

Pananá et al., 2004a).

stromal cells of the bone marrow allows those receptors capable of recognizing self-antigens to be identified and eliminated through a variety of mechanisms collectively termed "tolerance". Non-self-reactive B cells exit to the periphery and reach the spleen where they are again tested for reactivity against self-antigens before they transition to the mature stage (Figure 5) (von Boehmer & Melchers, 2010). Three main mechanisms of B-cell tolerance are known: receptor editing, deletion of auto-reactive clones (negative selection) and anergy. Only those B cells that carry receptors without self-specificity are allowed to exit the bone marrow and become mature B cells in peripheral lymphoid organs.

Fig. 4. The B cell antigen receptor (BCR). A) Heavy and light chains are comprised of variable regions where VDJ recombination occurs (shown in dark blue) and constant regions (green). The signaling domains are present in the cytoplasmic leaflet of Ig and Ig. B) Variable regions are formed by a number of segments termed V (variable), D (diversity) and J (joining) within the heavy chain, and by segments V and J within the light chain, which are brought together by a VDJ recombination process. Randomly, D and J segments recombine at first, followed by V segments joining the DJ fragment (shown in dark blue squares is an example of segment choice). This mechanism is responsible for the extensive repertoire of BCR specificities.

From HSC to B-Lymphoid Cells in Normal and Malignant Hematopoiesis 285

On the basis of their cell-surface phenotype, peripheral immature B cells are further divided

cells inhabit the spleen's red pulp and give rise to T2 cells (Allman et al., 2001). There is an additional population designated T3, but it is controversial whether this is a population in line in the progression to the mature stage or whether it represents a population of anergic

Following antigen binding, mature B cells activate pathways that lead to proliferation and further differentiation into antibody-producing B cells (plasma cells) or memory B cells. In the spleen, mature B cells are sub-divided into follicular (FO, AA4.1-CD21intCD23high) and marginal zone (MZ, AA4.1-CD21highCD23-) B cells according to both their location and their cell-surface phenotype. A distinct subset of mature B cells is preferentially present in the peritoneal cavity; these are known as B1 cells [B220+CD11b+CD5+ (B1a) or CD5- (B1b)]. Among them, FO B cells are responsible for adaptive antibody responses, whereas MZ and B1 mature populations respond rapidly to antigenic stimulus but do not go through germinal-center reactions and thus their response can be independent of T cell help (Martin et al., 2001). Therefore, MZ and B1 B cells are thought to be part of an innate–like response. The origin of both of these populations is not well understood. While MZ B cells share part of FO pathway, the fetal liver was thought to originate a large fraction of the adult B1 B cells (Tung et al., 2006). Recently, a novel developmental model suggests that some B1 cell

**3.3 Regulation of B lineage commitment: The critical role of preBCR tonic signaling,** 

Limitation of lineage choice during development is regulated by a combination of signaling pathways and transcription factors (TF). In mice, the main receptor controlling the ProB stage is the IL-7R, which is composed of a chain (IL-7R) and the common cytokine receptor chain (c). Deletion of IL-7R or c leads to developmental arrest at the early ProB

IL-7 activates the major signaling pathway JAK–STAT, with STAT5 being the essential

By the other hand, an important characteristic of the developmental process that distinguishes B and T lymphocytes from other cell lineages is the continuous selection of these lymphoid cells for their ability to express a competent, non-self receptor. B cells that fail to express a receptor are eliminated. Thus, BCR and BCR-like receptors must generate active permissive signals that allow differentiation through the different developmental stages. Because the preBCR lacks of the light chain and therefore of the capacity to bind polymorphic ligands, it has been proposed that this receptor is able to signal constitutively and independently on ligand, an activity also known as tonic signaling. Although there is little understanding of how tonic signals are generated, the view is supported by receptor-less B cells able to differentiate into mature B cells by expression of a chimeric construct of Ig and Ig positioned in the cell surface membrane (Bannish et

) and transitional 2 (T2, AA4+IgMhighCD23+). T1

into transitional 1 (T1, AA4+IgMhighCD23-

**3.2 Innate and adaptive mature B cell populations** 

progenitors can be produced in bone marrow (Esplin et al., 2009).

mediator of IL-7 signals in early B cell development (Yao et al., 2006).

**IL-7R and transcription factors in context** 

al., 2001).

stage (von Freeden-Jeffrey et al., 1995; Cao et al., 1995).

cells (Merrell et al., 2006).

Fig. 5. Normal and leukemic B cell development. B cell stages can be divided according to the main processes guiding development: receptor assembly, self-recognition and activation (top panel). Receptor assembly occurs in bone marrow (light blue box) by VDJ recombination in the Pro-B and Pre-B stages, whereas self-recognition starts in bone marrow and ends in periphery, and activation takes place at peripheral level. Nomenclature for each sub-stage in mice is shown in black letters while the most common nomenclature for their counterparts in humans is shown in red letters. The dashed lines separating all stages indicate checkpoints at which signaling from the preBCR and BCR is required for positive selection and progression along the B-cell maturation pathway. The proBCR, preBCR, and mature receptor are also illustrated in their respective stages. Replication and recombination processes are mutually exclusive as denoted by the circular arrows and VDJ signs inside the cell. The replication stages are also frequently compromised in pediatric B cell acute leukemia. Black lines under IL-7R and preBCR indicate the stages where these receptors are most required. The differential thickness in the IL-7R line shows the sub-stages where a higher (nanograms) or lower (picograms) concentration of the IL-7 is required. Homeostatic and leukemic expression of transcription factors along the B cell pathway are shown in the middle and bottom panels. Blue bars mark normal gene expression, and the most common modified forms of the transcription factors associated with B cell acute lymphoblastic leukemia are revealed. HSC, hematopoietic stem cell.

On the basis of their cell-surface phenotype, peripheral immature B cells are further divided into transitional 1 (T1, AA4+IgMhighCD23- ) and transitional 2 (T2, AA4+IgMhighCD23+). T1 cells inhabit the spleen's red pulp and give rise to T2 cells (Allman et al., 2001). There is an additional population designated T3, but it is controversial whether this is a population in line in the progression to the mature stage or whether it represents a population of anergic cells (Merrell et al., 2006).
