1. INTRODUCTION
1.3 B-CELL LYMPHOID NEOPLASMS
1.3.3 GENETIC CHANGES IN B-CELLS WITHIN GERMINAL CENTERS
During T‑cell-dependent antibody responses to exogenous antigen, germinal centers (GCs) are formed by proliferating B cells in the follicles of peripheral lymphoid tissues, including the spleen, lymph nodes, Peyer’s patches and tonsils.
Naïve B cells first migrate to the T-cell-rich area (known as the T-cell zone) of lymphoid tissues due to their upregulation of specific chemokine receptors in response to their encounter with antigen presented by dendritic cells. In the T-cell zones, B T-cells become fully activated as a result of their interaction with CD4+ T cells and antigen presenting cells 47. The initiation of the GC response requires the interaction of co-stimulatory B-cell-surface receptors with ligands expressed by T cells and/or antigen-presenting cells, of which the most important is that between the tumor-necrosis factor (TNF)-receptor family member CD40, which is expressed by all B cells, and its ligand CD154 expressed by helper T cells. Activated B cells can then either develop directly into antibody-secreting cells in specialized extra follicular sites of plasmablast growth and differentiation, such as the medullary cords of lymph nodes, or mature into GC‑precursor B cells and move to the primary follicle, a structure made of recirculating IgM+IgD+ B cells within a network of follicular dendritic cells (FDCs).
Here, B cells start to proliferate rapidly and push the IgM+IgD+ B cells aside to form the mantle zone around the GC, yielding a structure known as the secondary follicle. After a few days of vigorous proliferation, the characteristic structure of the GC becomes apparent with a dark zone consisting almost exclusively of densely packed proliferating B cells known as centroblasts, and a light zone comprised of smaller, non-dividing centrocytes situated within a mesh
of FDCs, T cells and macrophages. Centroblasts diversify their IgV genes by somatic hyper mutation (SHM), and those cells that express newly generated modified antibodies are selected for improved antigen binding in the light zone.
Some centrocytes eventually differentiate into memory B cells or plasma cells (figure 1.8).
The main characteristic of centroblasts is the high proliferation rate that is required for the generation of large number of modified antibodies, from which the few B cells that display antibodies with improved antigen-binding are selected. Accordingly, gene expression profile analysis has shown that the differentiation of an antigen-activated B cell into a centroblast is accompanied by a dramatic up-regulation of genes associated with cell proliferation and the down-regulation of genes encoding negative regulators of clonal expansion 48. Moreover, centroblasts express several pro-apoptotic molecules 49, 50, which allow the rapid execution of cell death by default or in response to exogenous signals. The major benefit of this process is the rapid elimination of GC B cells with newly generated immunoglobulin mutations that produce a non-functional or non-binding antibody. This feature and the spatial concentration of proliferation, mutation and selection of antigen-activated B cells may have the driving force for the evolution of the highly specialized GC structure 47.
Figure 1.8. Biology of B-cell differentiation (Modified from Klein, U, 2008 47)
The low level of NF-kB activation may be due to the fact that the dark zone is largely avoided of T cells that express CD154 50. These observations suggest that centroblasts are not subject to active CD40-signalling. However, the established GCs can be dissolved by inhibiting the CD40-CD154 interaction 51, suggesting that CD40 stimulation may specifically occur in the light zone, leading to the delivery of survival signals necessary for cell exit from the GC or their recirculation within GC. Very little is known about how the centroblast-specific transcriptional programme is established but some candidate factors are known, include several transcription factors like B-cell lymphoma 6 (BCL6), the ETS family member SPI-B and interferon-regulatory factor 8 (IRF8) 47.
1.3.3.1 SOMATIC HYPERMUTATION
The GC, dark zone in particular, is the main structure where SHM occur. SHM is a process that modifies the Ig variable region (IgV) of the rearranged antibody genes in B cells during an immune response (figure 1.9). SHM is associated with DNA strands breaks and introduces mostly single nucleotide exchanges, but also small deletion and duplications (about 6% of all mutation events) 52 into the rearranged IgV region. The base changes are distributed throughout V region, but not completely randomly: there are some ‘hotspots’ of mutation that indicate a preference for characteristic short motifs of four or five nucleotides, and perhaps also for secondary structural features. Some of the mutant immunoglobulin molecules bind antigen better than the original B-cell receptors, and they are preferentially selected to mature into antibody-secreting cells 53.
The SHM requires the activity of activation-induced cytidine deaminase (AID), which is an enzyme that catalyses the initiation event by deaminating cytidines directly on DNA 54. Then, error-prone repair that involve different DNA repair factors leads to the introduction of somatic mutations 55.
SHM, beside acting on the IgH, κ and λ loci, also affects some non-immunoglobulin genes, most notably the BCL6 proto-oncogene, the master transcriptional regulator of centroblasts. BCL6 is a nuclear phosphoprotein that belongs to a large BTB/POZ family of nuclear factors that contain zinc-finger motif and acts as transcriptional repressor 56. Centroblasts have the unique ability to tolerate this state of proliferation and genomic instability due in part to BCL6-mediated repression of the ATR (ataxia telangiectasia and Rad3 related), TP53 and p21 genes 57 Thus, BCL6 may allow GC B cells to sustain the physiological genotoxic stress that is associated with high proliferation, and sustain the DNA breaks that are induced by SHM without eliciting the p53-dependent and p53–inp53-dependent growth arrest 47.
Figure 1.9. Molecular Processes Modifying the Genes Encoding Antibody Molecule (Modified from Kuppers R, 2003 53).
1.3.3.2 CLASS SWITCH RECOMBINATION
The light zone of the GC is the site for three main B-cell development processes:
the selection of centroblast that produced high affinity antibodies, the process of class switch recombination (CSR) and the initiation of centrocytes into plasma cells or memory B cells (figure 1.9).
Compared with centroblasts, centrocytes are very heterogeneous, because they can undergo diverse developmental stages, including the differentiation back into centroblasts and into post-GC cells with the acquisition of a high-affinity B-cell receptor 42.
CSR is an irreversible somatic recombination mechanism by which B cells can switch their immunoglobulin class expression from IgM and IgD to either IgG, IgA and IgE. This process alters the effector functions of an antibody that can be specifically required to clear a particular antigen by leaving the antibody specificity defined by the rearranged V(D)J region of the heavy-chain unchanged, but allowing this same region to associate with different constant regions. CSR occurs by DNA recombination involving non-homologous end-joining processes between specific repetitive regions of several hundred base pairs (known as switch regions) that precede the immunoglobulin constant region genes 47. This process needs expression of activation-induced cytidine deaminase (AID) and can occur in both T-dependent and T-independent manner, and it is the combination of specific cytokines and co-stimulatory signals that determine the nature of resulting immunoglobulin class. For CSR, CD40-CD154 interaction is critical, and recent studies demonstrated an important role for IRF4 that seem to regulate AID expression through intermediate molecules rather than through a direct mechanism. IRF4 may be up-regulated by the CD40-NF-kB signalling pathway that is activated in centrocytes47.
Thus, SHM and CSR are not temporally linked. Also, the processes are spatially separated, with SHM occurring in the dark zone and CSR in the light zone of GCs (figure 1.7).