Developmental stages

At the CLP stage, stem cell factor (SCF) is released from the stromal cells in the microenvironment, binding to its receptor CD117 on early B cell precursors (39). Lymphoid precursors express CD117 and IL7Rα and are capable of developing into B, T or natural killer (NK) cells, but not erythrocytes/myeloid lineages. After the CLP stage, the earliest B cell precursors express B220, along with early NK cells. However, these lineages can be separated using other surface markers such as AA4.1, which is expressed on B cells up to the immature stage (31,40).

1.4.2.1 Early B cell development

It is after Pax5 expression which promotes sequential recombination of the Ig gene segments of the heavy and light chains, forming the platform for B cell development. This functional rearrangement comprises the VDJ gene segments of the heavy chain (µ locus) and the VJ segments of the light chain (κ locus) initiated by recombination-activating genes 1 and 2 (Rag1 and Rag2). Functional rearrangement of these genes enables the cell to generate antibodies which recognise various cell specific antigens. The rearrangement pattern of the Ig heavy chain (IgH) gene segments is associated with distinct stages of B cell development – pre-proB cell, proB, preB cells (Figure 1.2, Figure 1.3). B precursors with no rearrangement are in the pre-proB cell stage. The

rearrangement of DJ segments in the H chain is correlated with the proB cell stage. Mb1 expression at the proB cell stage is vital for Ig rearrangement as Mb-1 encodes the Ig-α subunit, deficiency of which leads to BCR deficiency(41). CD19 expression is also expressed at the proB cell stage, under the regulation of Pax5. V chain addition to the DJ segment(42) marks the large preB cell stage. CD19 expression is marked by BCR dependent and independent events. CD19 interacts with the BCR to enhance BCR activity.

Figure 1.3 Diagram showing stages of early B cell development in the bone marrow along with table summarizing various stages of early B cell development including phenotype (surface markers) and events in the pre-proB cell, proB cell, preB cell and immature B cell stages.

1.4.2.2 Late B cell development

Figure 1.4Diagram showing stages of late B cell development in the spleen along with table summarizing various stages of early B cell development including phenotype (surface markers) and events in the transitional (T-3), marginal zone progenitor (MZP)/MZ, follicular 1 (fol1) and fol2 B cell and B1 B cell stages. APC – antigen-presenting cells.

After immature B cell development, the cells undergo one of three processes dependent on Ig interactions. Cells which undergo cross-linking of the BCR leading to high affinity interactions are associated with elimination via clonal deletion (negative selection). Low affinity interactions lead to non-responsive anergic cells which are short lived, or it could also lead to cells editing their BCR to become non-reactive(43). All other immature B cells, expressing surface bound IgM, migrate into the spleen where they mature into naïve, follicular (fol) or marginal zone (MZ; CD19+_CD21hi_CD23-_IgMhi_{) B cells via transitional phases (T1-}

T3). Cariappa et al., have shown there to be a transition from T1

which develop into fol2 B cell populations. Fol2 B cells give rise to MZ

progenitors (MZP; CD19+_CD21hi_CD23+_CD1dhi_{) and ultimately MZ B cells together}

with fol1 cells. Nevertheless, T1 cells also transition to T3 cells to give rise to fol1 cells (44). Association of Toll-like receptor (TLR) signalling together with pattern-associated molecular patterns (PAMPs) leads to MZ B cell development into IgM plasma cells forming the first line of innate immunity against

pathogens(45). Fol1 B cells (CD19+_AA4.1-_CD21lo_CD23+_CD1dint_IgDhi_IgMlo_{) form the}

bulk of the circulating population whereas fol2 cells (CD19+_AA4.1-

CD21lo_CD23+_CD1dint_IgDhi_IgMhi_{) form one-third of the circulating population, being}

more quiescent and considered to be more primitive(44) residing in B cell follicles (Figure 1.2, Figure 1.4). It is the interaction of T helper (Th) cells with these fol B cells which leads to the expansion of B cells comprising the germinal centres (GCs) in secondary lymphoid organs(45). CD19 expression is vital for the maintenance of peripheral B cells as CD19-null mice have shown to have

decreased survival compared to normal B cells. This maintenance is partially via the BCR/B cell lymphoma-2 (Bcl-2) axis, as increasing Bcl-2 expression in CD19- null mice rescued MZ and fol B cell generation(46). Phosphoinositide 3-kinase- delta (PI3Kδ) expression is restricted to B and T cells and has shown to play a role in late B cell development. PI3Kδ-null mice show block in B cell

development at the preB cell stage. It has been shown that PI3K expression is regulated by pre-BCR signalling, and PI3K isoforms, p110α and p110δ, modulate

Pax5 expression, absence of which arrests B cell development at the preB cell

stage(47), making PI3Kα and PI3Kδ redundant for early B cell development. PI3Kδ has shown to be vital for late B cell development, particularly for MZ cells(48,49). There is a significant reduction in MZ B cells with PI3Kδ-deficiency due to reduced CXCR5 mediated migration(50) as PI3Kδ-deficient mice have a reduction in MZ B cell homing into proliferative centres suggesting a role of PI3Kδ in MZ B cell maturation and homing capacity.