B lymphocytes (B cells) are part of the adaptive immune system and are essential for the humoral immune response against foreign antigens. They generate high affinity antibodies that are secreted to effectively inactivate the specific antigen. Antibodies are composed of four immunoglobulins (Ig): two identical heavy chain immunoglobulins (IgH) and two identical light chain immunoglobulins (IgL) linked by disulfide bonds. Each of these polypeptides consists of a carboxyterminal constant region (C) and an aminoterminal variable (V) region. Each mature B cell expresses a membrane bound antibody on its surface that is associated with cofactors (Igα and Igβ). Together they form the B cell receptor (BCR). This receptor gains its high specificity during B cell maturation where complex genomic modifications to the immunoglobulin genes are introduced.
Early development of B cells is initiated in the fetal liver and subsequently relocated into the bone marrow of the mammalian embryo (reviewed in (Melchers, 2015)). A multipotent hematopoietic stem cell in the bone marrow gives rise to a lymphoid progenitor, which devel-ops into a precursor B cell that subsequently undergoes complex rearrangements of the im-munoglobulin heavy and light chain variable region genes (reviewed in (Seifertet al., 2013)).
This process is called V(D)J recombination, because the variable region of the heavy and light chain immunoglobulin gene is encoded on different gene segments: variable (V), diversity (D) and joining (J) segments. From each of these segments one is randomly selected and step wise joined together with the other segments. This process leads to a high variability in newly formed immunoglobulins. Cells that express a functional, but non-autoreactive B-cell receptor survive the selection process and are released as naive B cells into the blood.
Upon encountering of a T cell dependent antigen these cells are activated, migrate into sec-ondary lymphatic organs like the lymph nodes, tonsils, spleen, mucosa associated lymphoid tissue (MALT) or Peyer’s patches, where they maturate their B-cell receptor, undergo clonal expansion and further differentiate into antibody secreting plasma cells or long lived memory cells (reviewed in (De Silva and Klein, 2015)).
One site of B cell maturation are the lymph nodes. They are characterized by follicles that are build up from naive B cells which are separated from each other by an interfollicular re-gion that is surrounded by a T cell zone. Within the follicular centers a network of follicular dendritic cells (FDC) can be found. When a naive B cell encounters an antigen, it migrates to the border of T cell and B cell zone and forms long-lived interactions with antigen-specific T cells leading to full activation of the B cell. A subset of these B cells differentiate into short-lived plasmablasts, which secrete low affinity antibodies. Another subset of these cells enter the germinal center (GC) pathway, where the BCR is further refined. Activated T and B cells migrate to the follicular center and interact with the dendritic cells. The T cells become T
follic-1.1. B CELL DEVELOPMENT, MATURATION AND MALIGNANT TRANSFORMATION 3
ular helper cells (TF H) thus they upregulate B cell lymphoma 6 (BCL6), the master regulator of TF H and GC B cell development (Baumjohannet al., 2011). The B cells start to divide rapidly and to populate the follicle displacing the naive B cells, which thereby form a so called “mantle zone” around the newly formed germinal center. Within the germinal center two different zones develop: a densely packed “dark zone” containing proliferating B cells (centroblasts) and retic-ular cells and a “light zone” containing non-proliferating B cells (centrocytes),TF H cells, FDCs and macrophages. The centroblasts in the dark zone undergo somatic hypermutation (SHM), a process that further diversifies the rearranged IgV genes. SHM results in different B cell clones with a broad range of affinities against the antigen. The clones expressing a high-affinity antigen receptor are positively selected within the light zone. Effective antigen binding leads to enriched antigen capture followed by strong BCR signaling (see section 1.2.1) and longer interactions with FDCs andTF H cells (fig. 1.1). The bystander cells (FDCs andTF H cells) provide CD40L and secrete interleukine-4 and -21 (IL-4 and Il-21) (Liuet al., 2015; Shul-man et al., 2014). In sum, these stimulations provide a survival signal, promoting positive selection. Cells expressing an auto-reactive or defective BCR undergo apoptosis. Induction of MYC expression during the selection process induces recircularization of the positive se-lected cells between dark and light zone resulting in further refinement of the antigen specificity (Dominguez-Sola et al., 2012). CD40 stimulation leads to nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) mediated upregulation of Interferon Regulatory Factor 4 (IRF4) expression (Saitoet al., 2007), which represses BCL6 thereby terminating the dark zone program. Within the light zone the cells undergo class switch recombination (CSR) or finally differentiate into plasmablasts or memory cells and leave the germinal center (reviewed in (De Silva and Klein, 2015)). CSR is a process were the isotype of the immunoglobulin is switched (from IgM or IgD) by a new combination of the variable hypermutated VDJ gene ele-ments with genes encoding for a different heavy chain (IgA, IgE or IgG), rendering their effector function.
Notably, most aggressive B cell lymphoma resemble germinal center B cells expressing mark-ers that reflect their origin. The cells seem to be frozen at a particular differentiation step during germinal center reaction (reviewed in (Küppers, 2005)). Notably, some GC derived lymphoma still undergo SHM. Indeed, aberrant SHM and CSR can promote lymphomagensis (Lenzet al., 2007; Pasqualucciet al., 2008). If errors occur during CSR, free DNA ends are produced that can cause chromosomal translocations, a genetic hallmark of lymphoma. The translocations of proto-oncogeneMYCandBCL6to immunoglobulin promoters are characteristic for aggres-sive B cell lymphoma. Moreover, constitutive expression of BCL6 maintains a pro-proliferative and DNA-damage tolerant phenotype leading to additional mutations which might further pro-mote lymphomagensis (Cattorettiet al., 2005). Enhanced expression ofMYCresults from the translocation into the immunoglobulin heavy chain or light chain loci and is characteristic for Burkitt lymphoma (BL) cells (section 1.3). Aberrant SHM acting in the 5´ regulatory or coding
4 1| Introduction
Figure 1.1.: Germinal center reaction
During germinal center reaction activated B cells generate due to genomic modification of the immunoglobulin genes high affinity antibodies and differentiate into antibody secreting plasmablasts or memory cells. Upon antigen binding, activated B cells differentiate into centroblasts that undergo clonal expansion and somatic hypermutation (SHM). SHM introduces point mutations into the V(D)J region of already rearranged immunoglobulin variable region (IgV) genes (red dots). These cells move into the light zone, were T follicular helper cells (TF H cells) and follicular dendritic cells (FDCs) help to elect these cells, that generated an B cell receptor (BCR) with improved binding specificity. Higher affinity leads to increased antigen capture and promotes TF Hcell binding and CD40L signaling resulting in a survival signal. Cells with low binding capacity to the antigen undergo apoptosis. A subset of positive selected B cells recirculates into the dark zone to further refine the BCR, whereas another subgroup undergoes class switch recombination (CSR). CD40L = CD40 ligand, TCR = T cell receptor, MHC = major histocompatibility complex, BL = Burkitt Lymphoma, FL = Follicular Lymphoma, DLBCL = Diffuse Large B cell Lymphoma. (modified from de Silva and Klein, 2015)