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

284 Advances in Hematopoietic Stem Cell Research

Fig. 5. Normal and leukemic B cell development. B cell stages can be divided according to

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)

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.

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.

concentration of the IL-7 is required. Homeostatic and leukemic expression of

HSC, hematopoietic stem cell.

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

#### **3.3 Regulation of B lineage commitment: The critical role of preBCR tonic signaling, IL-7R and transcription factors in context**

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

IL-7 activates the major signaling pathway JAK–STAT, with STAT5 being the essential mediator of IL-7 signals in early B cell development (Yao et al., 2006).

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 al., 2001).

From HSC to B-Lymphoid Cells in Normal and Malignant Hematopoiesis 287

proliferative expansion of clones that have successfully completed the rearrangement of their receptors, whereas late development is led by mechanisms of tolerance to self-antigens. All these processes in humans are less well understood than are their counterparts in mice. Importantly, human B cells can still be generated in severe combined immunodeficiency (SCID) patients with mutations in the IL-7R gene, suggesting that IL-7 signaling is not essential for human B cell development (Puel et al., 1998) although a recent study has demonstrated that in vitro human B cell production is dependent on IL-7 (Parrish et al., 2009). The fine regulatory mechanism separating proliferation and differentiation might explain why the proliferating ProB and PreB sub-stages are the ones generally found to be compromised in human pediatric B-cell acute lymphoblastic leukemia (B-cell ALL) and why this disease is characterized by leukemic blast cells that are often unable to progress through the differentiation pathway. This tendency to be arrested in proliferative states might result in an increased rate of mutations, leading to formation of neoplastic cells. Supporting the later, mice expressing B cell mutants in the adaptor protein BLNK are arrested in the large PreB stage and often develop B cell malignancies (Flemming et al., 2003). Proliferative stages

Acute lymphoblastic leukemia (ALL) is a disorder characterized by the monoclonal and/or oligoclonal proliferation of hematopoietic precursor cells of the lymphoid series within the bone marrow. At present, ALL is the most frequent malignancy in children worldwide and a serious problem of public health, constituting 25% of all childhood cancers and 75%-85% of the cases of childhood leukemias (Perez-Saldivar et al., 2011). Near to 80% of ALL cases have precursor B-cell immunophenotype, while approximately 15% show T-cell immunophenotype. Even when a relatively high efficiency of therapeutic agents has been demonstrated (Pieters & Carroll, 2010), there has been a slight but gradual increase in the incidence of ALL in the past 25 years, and appears to be highest in Hispanic population, which also show superior rates of high risk patients (Fajardo-Gutiérrez et al., 2007; Abdullaev et al., 2000; Perez-Saldivar et al., 2011; Mejía-Aranguré et al., 2011). Factors such as drug resistance, minimal residual disease, cell lineage switch, and the rise of mixed lineages often put the success of treatment at risk and change the prognosis of the illness. The molecular mechanism involved in these phenomena and the identities of the target hematopoietic populations have not been completely defined, due in part, to the fact that neither the precise origin of the disease, nor the susceptibility of primitive leukemic cells to

Over the last two decades, cancer stem cells (CSC) have been defined as cells within a tumor that possess the capacity to self-renew and to cause heterogeneous lineages of cancer cells that comprise the tumor (Clarke et al., 2006). According to MF Greaves, who proposed the original hypothesis for leukemogenesis, multiple consecutive carcinogenic hits in hematopoietic cells may drive the malignant transformation (Greaves, 1993; Greaves & Wiernels, 2003), where the second oncogenic event on pre-leukemic clones could be indirectly promoted by delayed infections (Greaves, 2006; Mejía-Aranguré et al., 2011). Our general current view suggests the occurring of oncogenic lesions in early development or in

occur in the early ProB, PreB-I and large PreB-II fractions (Figure 5).

**4. Acute lymphoblastic leukemia** 

extrinsic factors, is known.

**4.1 The origin of ALL** 

Once the preBCR is expressed at the end of the proB stage, it can take over many of the functions performed by the IL-7 receptor signaling. Both receptors act individually and together to allow B cell development (Figure 5). Like IL-7R, the preBCR promotes mechanisms of positive selection, survival and proliferation (Ramadani et al., 2010; Yasuda et al., 2008). The CCND3 gene, which encodes for cyclin D3, is essential for PreB cell expansion and integrates IL-7R and preBCR signals (Cooper et al., 2006).

Downstream the IL-7 and preBCR receptors, a handful of transcription factors (TF) are critical for commitment to the B cell lineage and early development; these include E2A/TCF3 (immunoglobulin enhancer binding factors E12/E47/transcription factor 3), EBF1 (Early B cell Factor 1) and PAX5 (Paired box 5) (Figure 5). Loss of E2A and EBF1 blocks entry into the B cell lineage, while loss of PAX5 redirects B cells into other lineages (Nutt et al., 1999; O'Riordan & Grosschedl, 1999). Acting together with E2A, EBF1 and STAT5, one of the main molecular functions of PAX5 is to allow VDJ recombination (Hsu et al., 2004). Also, E2A, PAX5, IKZF1 and RUNX1, among other TF, are responsible for RAG expression (Kuo & Schlissel, 2009). Moreover, IL-7R signaling fulfills an essential role in early B cell development, with STAT5 participating in the activation of the B cell regulatory genes E2A, EBF1 and PAX5. E2A encodes two TF via alternative splicing, E12 and E47. In mice lacking the E2A gene, the B cell lineage is lost, there is no heavy chain recombination, and the expression of the B cell-restricted genes EBF1, PAX5, CD79A/B and VPREB1 (CD179A) is also affected.

Enforced expression of EBF1 and PAX5 is sufficient to overcome the developmental block in mice deficient in E2A, IL-7 or IL-7R, further illustrating the transcriptional hierarchy of the B cell-specific program triggered by IL-7 receptor signaling (Nutt & Kee, 2007). EBF1 acting together with PAX5 drives the expression of many genes critical for early B cell development and B cell function, including FOXO1, MYCN, LEF1, BLNK, CD79A (MB-1), RAG2, CD19 and CR2 (CD21) (Nutt & Kee, 2007; Smith & Sigvardsson, 2004).

Although PAX5 is a positive regulator of B-cell specific genes, also functions as a repressor of non B-lineage genes such as M-CSFR, NOTCH1 and FLT3 (Cobaleda et al., 2007) so B cell development is unidirectional and mostly irreversible in homeostatic conditions.

Also important for lymphoid development are members of the Ikaros family of TFs, mainly IKZF1 (which encodes Ikaros) and IKZF3 (which encodes Aiolos). Ikaros activates B cell genes and represses genes that are unrelated to the B lineage. Expression of IKZF1 and IKZF3 is regulated by alternative splicing, which produces long isoforms (Ik-1, Ik-2, Ik-3, Aio-1, Aio-3, Aio-4 and Aio-6) that efficiently bind to DNA, and short isoforms (Ik-4, Ik-5/7, Ik-6, Ik8, Aio-2, Aio-5) that are unable to bind DNA with high affinity and do not activate transcription (Liippo et al., 2001). Ikaros is activated in early stages of lymphopoiesis and is required for both early and late events in lymphocyte differentiation. Aiolos is not required during the early specification of the B and T lineages but is essential during further B cell maturation. They also act in concert to promote preB cell cycle exit and transition to small PreB stage (Ma et al., 2010).

#### **3.4 Human B cell development**

Selection processes operating on developing B cells are similar in all mammals. Thus, early B cell development in humans is also mainly guided by VDJ recombination and by the proliferative expansion of clones that have successfully completed the rearrangement of their receptors, whereas late development is led by mechanisms of tolerance to self-antigens. All these processes in humans are less well understood than are their counterparts in mice. Importantly, human B cells can still be generated in severe combined immunodeficiency (SCID) patients with mutations in the IL-7R gene, suggesting that IL-7 signaling is not essential for human B cell development (Puel et al., 1998) although a recent study has demonstrated that in vitro human B cell production is dependent on IL-7 (Parrish et al., 2009). The fine regulatory mechanism separating proliferation and differentiation might explain why the proliferating ProB and PreB sub-stages are the ones generally found to be compromised in human pediatric B-cell acute lymphoblastic leukemia (B-cell ALL) and why this disease is characterized by leukemic blast cells that are often unable to progress through the differentiation pathway. This tendency to be arrested in proliferative states might result in an increased rate of mutations, leading to formation of neoplastic cells. Supporting the later, mice expressing B cell mutants in the adaptor protein BLNK are arrested in the large PreB stage and often develop B cell malignancies (Flemming et al., 2003). Proliferative stages occur in the early ProB, PreB-I and large PreB-II fractions (Figure 5).
