**2. The early steps in the lymphoid development**

Mature blood cells are constantly replaced from a unique cell population of hematopoietic stem cells (HSC) residing in specialized niches within the bone marrow (BM), where the hematopoietic system is organized as a hierarchy of cell types that gradually lose multiple alternate potentials while commit to lineage fates and gain specialized functions (Baba et

From HSC to B-Lymphoid Cells in Normal and Malignant Hematopoiesis 279

Fig. 1. Early lymphoid development in humans. Within bone marrow (BM), self-renewing hematopoietic stem cells (HSC) give rise to multipotent progenitors (MPP), which have the ability to differentiate into common myeloid progenitors (CMP) and into multi-lymphoid progenitors (MLP). MLP might alternately derive from HSC. NK and B-lymphoid cells are produced from B/NK-derived lineage committed precursors. Mature hematopoietic cells are exported to peripheral blood (PB). Early progenitor cells may colonize the thymus via circulation, and initiate the T-lymphoid development pathway. NKP, natural killer cell

The rigorous purification of human HSC and progenitor cell populations based on their surface phenotype has promoted the study of their biology in adult bone marrow, cord blood and G-CSF-mobilized peripheral blood (Figure 2). Importantly, some of their properties, including cell frequencies, developmental capacities, cell cycle status, transcription factors networks and growth factors production, show substantial differences between newborns and adults (Mayani, 2010). According to literature, we have found that most hematopoietic progenitors are more abundant in cord blood than in the adult tissues bone marrow and mobilized peripheral blood (Mayani, 2010). The implications of these

discrepancies during haematological neoplastic diseases are not as yet clear.

precursor; BP, B cell precursor; TP, T cell precursor.

al., 2004; Seita & Weissman, 2010). HSC possess two major characteristics: they are capable of maintaining their constant number by self-renewal and they are in charge of producing all mature blood cells through differentiation processes (Figure 1). Furthermore, HSC are mitotically inactive (quiescent) and divide very slow and intermittently under normal conditions, but are capable of proliferation and differentiation during recovery from chemotherapy or stress circumstances (Takizawa et al, 2011; Mayani, 2010; Passegue et al., 2005; Pelayo et al., 2006b). Movement into and out of a resting state might be crucial for ensuring that the correct number of new hematopoietic cells is produced.

The lymphoid pathway proceeds through critical stages of differentiation of HSC to multipotential early progenitors (MPP), which upon progressive loss of self-renewal capacity, give rise to oligopotent progenitors. Downstream, the production of lineagecommitted precursors is crucial for cell maturation. Current knowledge about development of the lymphoid system is based, in great part, on the work done in animal models, demonstrating that lymphoid specification begins in the fraction of lymphoid-primed multipotent progenitors (LMPP). A series of studies using the transgenic RAG-GFP mouse (Igarashi et al., 2002) permitted us to determine that RAG+ early lymphoid progenitors (ELP) are capable of differentiating into T, B, NK and conventional dendritic cells (cDC) (Pelayo et al., 2005a; Pelayo et al., 2006a; Welner et al., 2008a). Studies using defined cocultures and short-term reconstitution assays have shown that ELP are also good producers of plasmacytoid dendritic cells (pDC) and of interferon-producing killer dendritic cells (IKDC), both being key components of the innate immune response to infections (Pelayo et al., 2005b; Welner et al., 2007). At the same time, ELP give rise to committed oligopotent common lymphoid progenitors (CLP), which are responsible for B- and NK- precursor cells production. CLP and lineage precursors have substantially lost the possibility of differentiating into the rest of the lineages.

Due to ethical reasons and technical limitations, human hematopoietic stem cell research has been slower than it has been in mouse models. In humans, the early hematopoietic progenitors are confined in bone marrow to a cellular compartment that expresses CD34 (Blom & Spits, 2006). The fraction of multipotent stem cells is characterized by the phenotype Lin-CD34+CD38-/loCD10-CD45RA- , whereas that of probably the earliest lymphoid progenitors is Lin-CD34+CD38-/loCD45RA+CD10+ and has been recently designated as multi-lymphoid progenitor (MLP) (Doulatov et al., 2010). According to Doulatov's studies, MLP may be directly derived from HSC. However, a precise precursorproduct relationship needs to be determined (Figure 1). A description that fully matches the definition of mouse ELP is still missing, but cells with Lin-CD34+CD38+CD45RA+CD7+CD10+ phenotype seem to represent good candidates (Blom & Spits, 2006; and our unpublished observations). Lin-CD34+CD38+CD45RA+CD10+ B/NK cells, which differentiate principally into B & NK cells, are considered the counterparts of CLP in mice (Figures 1 & 2) (Doulatov et al., 2010). Of special importance is the fact that increasing levels of CD10 correspond to B-lineage specification (Ichii et al., 2010). Downstream, the differentiation of fully committed precursors gives rise to B cells that eventually are exported to peripheral lymphoid tissues (see B cell development sections below).

al., 2004; Seita & Weissman, 2010). HSC possess two major characteristics: they are capable of maintaining their constant number by self-renewal and they are in charge of producing all mature blood cells through differentiation processes (Figure 1). Furthermore, HSC are mitotically inactive (quiescent) and divide very slow and intermittently under normal conditions, but are capable of proliferation and differentiation during recovery from chemotherapy or stress circumstances (Takizawa et al, 2011; Mayani, 2010; Passegue et al., 2005; Pelayo et al., 2006b). Movement into and out of a resting state might be crucial for ensuring that the correct number of new

The lymphoid pathway proceeds through critical stages of differentiation of HSC to multipotential early progenitors (MPP), which upon progressive loss of self-renewal capacity, give rise to oligopotent progenitors. Downstream, the production of lineagecommitted precursors is crucial for cell maturation. Current knowledge about development of the lymphoid system is based, in great part, on the work done in animal models, demonstrating that lymphoid specification begins in the fraction of lymphoid-primed multipotent progenitors (LMPP). A series of studies using the transgenic RAG-GFP mouse (Igarashi et al., 2002) permitted us to determine that RAG+ early lymphoid progenitors (ELP) are capable of differentiating into T, B, NK and conventional dendritic cells (cDC) (Pelayo et al., 2005a; Pelayo et al., 2006a; Welner et al., 2008a). Studies using defined cocultures and short-term reconstitution assays have shown that ELP are also good producers of plasmacytoid dendritic cells (pDC) and of interferon-producing killer dendritic cells (IKDC), both being key components of the innate immune response to infections (Pelayo et al., 2005b; Welner et al., 2007). At the same time, ELP give rise to committed oligopotent common lymphoid progenitors (CLP), which are responsible for B- and NK- precursor cells production. CLP and lineage precursors have substantially lost the possibility of

Due to ethical reasons and technical limitations, human hematopoietic stem cell research has been slower than it has been in mouse models. In humans, the early hematopoietic progenitors are confined in bone marrow to a cellular compartment that expresses CD34 (Blom & Spits, 2006). The fraction of multipotent stem cells is characterized by the

designated as multi-lymphoid progenitor (MLP) (Doulatov et al., 2010). According to Doulatov's studies, MLP may be directly derived from HSC. However, a precise precursorproduct relationship needs to be determined (Figure 1). A description that fully matches the definition of mouse ELP is still missing, but cells with Lin-CD34+CD38+CD45RA+CD7+CD10+ phenotype seem to represent good candidates (Blom & Spits, 2006; and our unpublished observations). Lin-CD34+CD38+CD45RA+CD10+ B/NK cells, which differentiate principally into B & NK cells, are considered the counterparts of CLP in mice (Figures 1 & 2) (Doulatov et al., 2010). Of special importance is the fact that increasing levels of CD10 correspond to B-lineage specification (Ichii et al., 2010). Downstream, the differentiation of fully committed precursors gives rise to B cells that eventually are exported to peripheral lymphoid tissues (see B cell development sections

, whereas that of probably the earliest

CD34+CD38-/loCD45RA+CD10+ and has been recently

hematopoietic cells is produced.

differentiating into the rest of the lineages.

phenotype Lin-CD34+CD38-/loCD10-CD45RA-

lymphoid progenitors is Lin-

below).

Fig. 1. Early lymphoid development in humans. Within bone marrow (BM), self-renewing hematopoietic stem cells (HSC) give rise to multipotent progenitors (MPP), which have the ability to differentiate into common myeloid progenitors (CMP) and into multi-lymphoid progenitors (MLP). MLP might alternately derive from HSC. NK and B-lymphoid cells are produced from B/NK-derived lineage committed precursors. Mature hematopoietic cells are exported to peripheral blood (PB). Early progenitor cells may colonize the thymus via circulation, and initiate the T-lymphoid development pathway. NKP, natural killer cell precursor; BP, B cell precursor; TP, T cell precursor.

The rigorous purification of human HSC and progenitor cell populations based on their surface phenotype has promoted the study of their biology in adult bone marrow, cord blood and G-CSF-mobilized peripheral blood (Figure 2). Importantly, some of their properties, including cell frequencies, developmental capacities, cell cycle status, transcription factors networks and growth factors production, show substantial differences between newborns and adults (Mayani, 2010). According to literature, we have found that most hematopoietic progenitors are more abundant in cord blood than in the adult tissues bone marrow and mobilized peripheral blood (Mayani, 2010). The implications of these discrepancies during haematological neoplastic diseases are not as yet clear.

From HSC to B-Lymphoid Cells in Normal and Malignant Hematopoiesis 281

During biological contingencies -chemotherapy, infections and transplantation procedures-, the replenishment of the innate immune system from hematopoietic stem/progenitor cells appears to be critical. Interestingly, these seminal cells can proliferate in response to stress conditions and systemic infection by using mechanisms that apparently involve interferons and tumor necrosis factors, among others (Baldridge et al., 2011). Moreover, they are capable of self/non-self discrimination through Toll-like receptors (TLR), which recognize microbial components. Mouse stem cells and early B-cell progenitors express and use TLR, a mechanism that facilitates their differentiation to the innate immune system (Nagai et al, 2006; Welner et al., 2008b; Welner et al., 2009). Recent work suggests that, as in mice, human primitive cells, including MLP, also express functional TLR (Kim et al., 2005; Sioud & Fløisand, 2007; De Luca et al, 2009; Doulatov et al, 2010). In shape with those findings, we have found that BM lymphoid progenitor-enriched fractions display TLR9 (Figure 3) and their differentiation potentials bias toward NK and DC production upon TLR9 ligation (RP & EV, unpublished observations). Thus, plasticity in primitive cells is vulnerable to extrinsic agents that can modify early cell fate decisions during infections or stress, suggesting that the stages of lineage restrictions are less abrupt than previously assumed (Welner et al.,

Fig. 3. Lymphoid progenitors from human bone marrow express TLR9. Adult bone marrow is fractionated according to cell surface expression of lineage markers, CD34, CD45RA and CD7/CD10 (A). Lin-CD34+CD45RA- HSC/MPP, Lin-CD34+CD45RA+CD7/CD10- myeloid progenitors (MP) and Lin-CD34+CD45RA+CD7/CD10+ lymphoid progenitors (LP) were tested for their intracellular expression of TLR9 by flow cytometry using a specific anti-TLR9

CD34+CD38-

identification of T-cell progenitors (TP), B/NK progenitors and ELP-like cells (C). Cell frequencies for each population from the different sources are shown (B and D panels). CMP, common myeloid progenitor; GMP, granulocyte & monocyte progenitor; MEP,

megakaryocyte & erythrocyte progenitor. The identity and functions of Lin-

CD45RA+CD123hi cells still need more investigation.

2008a).

antibody (B).

Fig. 2. Prospective identification of human myeloid and lymphoid progenitor cells by flow cytometry. HSC and early progenitor cells reside in the Lin- CD34+ fraction of adult normal bone marrow (NBM), as well as in umbilical cord blood (UCB) and mobilized peripheral blood (MPB). Based on the surface expression of CD38, CD123 and CD45RA, multilymphoid progenitor cells (MLP) and most of the myeloid progenitors can be recognized (A). Further fractionation of Lin-CD34+CD45RA+ cells into CD7 & CD10-expressing cells allows the

Fig. 2. Prospective identification of human myeloid and lymphoid progenitor cells by flow

bone marrow (NBM), as well as in umbilical cord blood (UCB) and mobilized peripheral blood (MPB). Based on the surface expression of CD38, CD123 and CD45RA, multilymphoid progenitor cells (MLP) and most of the myeloid progenitors can be recognized (A). Further fractionation of Lin-CD34+CD45RA+ cells into CD7 & CD10-expressing cells allows the

CD34+ fraction of adult normal

cytometry. HSC and early progenitor cells reside in the Lin-

identification of T-cell progenitors (TP), B/NK progenitors and ELP-like cells (C). Cell frequencies for each population from the different sources are shown (B and D panels). CMP, common myeloid progenitor; GMP, granulocyte & monocyte progenitor; MEP, megakaryocyte & erythrocyte progenitor. The identity and functions of Lin-CD34+CD38- CD45RA+CD123hi cells still need more investigation.

During biological contingencies -chemotherapy, infections and transplantation procedures-, the replenishment of the innate immune system from hematopoietic stem/progenitor cells appears to be critical. Interestingly, these seminal cells can proliferate in response to stress conditions and systemic infection by using mechanisms that apparently involve interferons and tumor necrosis factors, among others (Baldridge et al., 2011). Moreover, they are capable of self/non-self discrimination through Toll-like receptors (TLR), which recognize microbial components. Mouse stem cells and early B-cell progenitors express and use TLR, a mechanism that facilitates their differentiation to the innate immune system (Nagai et al, 2006; Welner et al., 2008b; Welner et al., 2009). Recent work suggests that, as in mice, human primitive cells, including MLP, also express functional TLR (Kim et al., 2005; Sioud & Fløisand, 2007; De Luca et al, 2009; Doulatov et al, 2010). In shape with those findings, we have found that BM lymphoid progenitor-enriched fractions display TLR9 (Figure 3) and their differentiation potentials bias toward NK and DC production upon TLR9 ligation (RP & EV, unpublished observations). Thus, plasticity in primitive cells is vulnerable to extrinsic agents that can modify early cell fate decisions during infections or stress, suggesting that the stages of lineage restrictions are less abrupt than previously assumed (Welner et al., 2008a).

Fig. 3. Lymphoid progenitors from human bone marrow express TLR9. Adult bone marrow is fractionated according to cell surface expression of lineage markers, CD34, CD45RA and CD7/CD10 (A). Lin-CD34+CD45RA- HSC/MPP, Lin-CD34+CD45RA+CD7/CD10- myeloid progenitors (MP) and Lin-CD34+CD45RA+CD7/CD10+ lymphoid progenitors (LP) were tested for their intracellular expression of TLR9 by flow cytometry using a specific anti-TLR9 antibody (B).

From HSC to B-Lymphoid Cells in Normal and Malignant Hematopoiesis 283

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

Fig. 4. The B cell antigen receptor (BCR). A) Heavy and light chains are comprised of

BCR specificities.

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

marrow and become mature B cells in peripheral lymphoid organs.
