B Cell Maturation, Activation, Proliferation, and Differentiation
B Cells: Maturation, Activation, Proliferation, and Differentiation
Learning Objectives
B lymphocytes play a crucial role in antibody production, contributing to the humoral immune response. Although their activation requires the assistance of T helper cells, this chapter aims to educate readers about:
Antigen-independent maturation of B cells within the bone marrow, where they acquire the B cell receptor (BCR) and other necessary molecules for activation.
Antigen-dependent activation of B cells in secondary lymphoid organs.
Molecular mechanisms during activation, signal transduction, and maturation of the antibody response.
Contents
Introduction
Maturation of B cells in bone marrow (antigen-independent phase)
Migration of B cells
Antigen-dependent activation of B cells in secondary lymphoid organs
Proliferation and differentiation of activated B cells
Events occurring in secondary lymphoid organs
Primary and secondary immune response
Humoral immune response to hapten:carrier conjugates
Distinction between B1 and B2 cells
Regulation of B cell development
9.1 Introduction
Both B and T lymphocytes, the major components of the acquired defense mechanism, originate from lymphoid progenitor cells in the bone marrow. Immature T cells (thymocytes) migrate from the bone marrow to the thymus for further development. In mammals, B cell development begins in the bone marrow itself and includes maturation, activation, and differentiation. Maturation occurs in the bone marrow, while activation and differentiation take place in secondary lymphoid organs.
Maturation of B cell progenitors into immunocompetent B cells is antigen-independent. A key event is the rearrangement of genes encoding heavy and light chains of immunoglobulins. IgM and IgD are expressed as surface receptors for antigens on naive B cells. Stromal cells in the bone marrow are essential for the maturation process, where each naive B cell becomes committed to respond to a unique antigenic epitope.
B cells acquire coreceptors and signaling molecules, undergo selection against self-reactivity, and then migrate to secondary lymphoid organs via blood and lymph. When B cells leave the bone marrow, they express high levels of IgM and low levels of IgD as B cell receptors (BCRs). They circulate through blood/lymph and secondary lymphoid organs until they encounter a specific antigen. Contact with the antigen in secondary lymphoid organs leads to their residence and further activation and differentiation.
After encountering a T-dependent antigen in T cell-rich areas, antigen-activated T cells move to B cell areas and activate B cells with cytokines. B cells can also directly interact with T-independent antigens. Activated B cells enter primary follicles, converting them into secondary follicles with germinal centers where proliferation, somatic hypermutations, class switching (for T-dependent antigens), and differentiation into plasma and memory cells occur. These events improve antibody quality and establish long-lasting memory. Plasma cells and antibodies exit the cortex and enter the medullary region, leaving secondary lymphoid organs through efferent lymphatic vessels (lymph node) and splenic artery to enter circulation.
9.2 Maturation of B Cells in Bone Marrow (Antigen-Independent Phase)
B cell progenitors, the first identifiable cells of B cell lineage, are known as pro-B cells. Maturation proceeds through stages such as large pro-B (or early pro-B) cells, small pro-B (or pro-B) cells, early pre-B (or large pre-B, precursor B) cells, small pre-B (or pre-B) cells, and immature B cells. Mature immunocompetent B cells express both IgM and IgD on their membrane as BCRs.
9.2.1 Role of Stromal Cells in Development of B Cells
The development of pre-B cells from pro-B cells depends on bone marrow stromal cells, which are non-lymphoid supporting cells forming a network in the bone marrow. Large pro-B cells interact with stromal cells through adhesion molecules. The initial contact is made via VLA-4 on the pro-B cell and VCAM-1 on the stromal cell, promoting attachment and interaction between c-Kit (on pro-B cells) and SCF (stem cell factor) on stromal cells. c-Kit, a tyrosine kinase, interacts with SCF, activating the IL-7 gene in stromal cells and expressing the IL-7 receptor on pro-B cells. IL-7 then binds to the IL-7 receptor on pro-B cells (also expressed on pre-B cells), signaling proliferation and Ig gene rearrangement. Table 9.1 summarizes these events.
9.2.2 Events Occurring During Transition of Pro-B Cell to Pre-B Cell
The critical event during the pro-B to pre-B transition is gene rearrangement of variable region-coding segments of immunoglobulin heavy (H) and light (L) chains. Two gene segments from light chain loci (V J) and three from heavy chain loci (V, D, J) are randomly selected for rearrangement. Gene rearrangement begins at the large pro-B stage with D and J segment joining. In small pro-B cells, a V segment joins the rearranged D-J segments, developing this cell into a large pre-B cell where heavy chain gene rearrangement stops and -chains appear in the cytoplasm. Surrogate L chains are synthesized, associate with the -chains, and are expressed on the cell membrane of pre-B cells along with Ig and Ig, which are signal transducing molecules. The expression of complete IgM (with surrogate L chain) on the small pre-B cell membrane signals for proper H chain gene rearrangement.
9.2.3 Selection of B Cells in the Bone Marrow
Since BCR gene rearrangement is random, B cells with BCRs recognizing self-antigens are present. Immature B cells with strong reactivity for self-antigens undergo apoptosis to prevent autoimmune diseases. Some self-reacting B cells are rescued by receptor editing. If gene rearrangement creates receptors recognizing self-antigens, cells are held at G0 phase, RAG proteins are re-expressed, and light chain gene rearrangement from the other homologue is initiated. B cells successfully producing alternative receptors without strong self-antigen reactivity survive.
9.3 Migration of B Cells
Mature but naive B cells express high levels of surface IgM and low levels of IgD, produced from differential splicing of a common primary transcript, maintaining the same antigen specificity.
Released from the bone marrow, mature naive B cells circulate in blood, secondary lymphoid tissues, and lymph. They enter peripheral lymphoid organs from the blood through endothelial venules via extravasation. Stromal cells in the cortex of lymphoid organs secrete chemokine CCL-21, which interacts with the CCR27 receptor on B cells. Guided by the chemokine gradient, B cells migrate to the primary follicle and are activated with the help of T cells if they encounter a specific antigen.
Passage through primary follicles is necessary for mature B lymphocyte survival; otherwise, they undergo apoptosis. If they do not encounter an antigen within a few days, they exit secondary lymphoid organs and re-enter circulation. Upon encountering an antigen, they are activated, enter the germinal center, and differentiate into plasma or memory cells.
9.4 Antigen-Dependent Activation of B Cells in Secondary Lymphoid Organs
Essential components for B cell activation include antigens, T cells, and cytokines secreted by T cells. Additionally, membrane proteins Ig/Ig, part of the B cell receptor (BCR) complex, and B cell coreceptors are crucial for signal transduction.
9.4.1 Activation of B Cells by Thymus-Dependent and Thymus-Independent Antigens
B cells are activated by two types of antigens: T-dependent (TD) and T-independent (TI). TD antigens require direct contact between B cells and T cells, while TI antigens do not. T cell-dependent antigens are typically soluble proteins requiring both direct contact between B and T cells and cytokines secreted by T cells. Activation by TD antigens results in a strong humoral response with affinity maturation, class switching, and generation of antigen-specific memory B cells.
Activation with TI antigens has been studied extensively in mice. Two classes of TI antigens exist: TI-1 and TI-2. Neither class requires direct contact with T cells for activation; however, TI-2 antigens require cytokines secreted by T cells. Bacterial cell wall lipopolysaccharide (LPS) is a TI-1 antigen example, while capsular polysaccharides with repetitive sequences are common TI-2 antigens. Macromolecules with repetitive structures, such as polymeric proteins, can also act as TI-2 antigens. Cross-linking of membrane BCRs is essential for stimulation with TI-2 antigens.
9.4.2 B Cell Activating Signals
Naive B cells are in a resting (G0) phase. Driving them to the S phase requires competence signals to move them from G0 to G1 phase and progression signals to push them from G1 to S phase.
B cell activation by TI-1 antigens
At high concentrations, bacterial cell wall lipopolysaccharides act as mitogens, binding to LPS-binding protein on B cell membranes, causing polyclonal activation irrespective of antigen specificity. At low concentrations, LPS reacts with B cells with BCRs specific for LPS, inducing activation of only antigen-specific B cells.
B cell activation by TI-2 antigens
TI-2 antigens have repetitive structures, leading to cross-linking of membrane BCRs. However, this signal alone is insufficient; cytokines from T cells provide the second signal for B cell activation.
9.4.3 Activation Signals by TD Antigens
Activated T cells secrete cytokines that can interact with any cell type with specific cytokine receptors. Two signals are needed for antigen-specific stimulation of T cells: MHC:antigenic peptide: TCR interaction and interaction between B7 (on APC) and CD28 (on T cell). B cells also act as APCs.
Interaction between B cell and T cell
Binding with the antigen is insufficient for B cell activation interacting with soluble protein antigens. Direct contact between B cell and T cell is essential. In this interaction, the B cell acts as an antigen-presenting cell. The B:TÍ interaction requires another set of second signals like CD40 on B cells and CD40L (ligand) on T cells.
After antigen binding to a specific BCR, the BCR internalizes the antigen by receptor-mediated endocytosis and is processed in the endocytic vesicle. BCR engagement increases the expression of class II MHC molecules and costimulatory B7 molecules on B cells. At low antigen concentrations, B cells are better antigen-presenting cells than macrophages and DCs. Antigenic peptides displayed with class II MHC molecules on the B cell surface are recognized by T cells, forming a T:B cell conjugate. B and T cells recognize different epitopes of the same antigen. Presentation of the antigen constitutes the first signal. The costimulatory molecule B7 on the B cell membrane interacts with CD28 on the T cell, activating the T cell to express CD40L on its membrane.
CD40, a glycoprotein on the B cell membrane, interacts with CD40L, acting as the second signal, leading to activation of signal transduction pathways, resulting in cytokine receptor expression on the B cell membrane. IL-4 from the T cell binds to its receptors on the B cell, driving the B cell into S phase and proliferation.
Transduction of activating signals
BCRs on B cells have short cytoplasmic tails. Membrane-bound IgM and IgD have cytoplasmic tails with only three amino acids, IgA has 14, while IgG and IgE have 28. Consequently, membrane-bound antibodies cannot interact with intracellular signaling molecules. The membrane proteins associated with mIg, namely Ig/Ig, act as signal transducers.
Role of Igα and Igβ in signal transduction
Membrane-bound Ig and Ig molecules have long cytoplasmic tails with 61 and 48 amino acids, respectively, acting as intracellular signaling molecules. The cytoplasmic regions themselves do not have tyrosine kinase activity but contain immunoreceptor tyrosine-based activation motifs (ITAMs). Antigen binding and cross-linking of membrane antibodies activate membrane-associated protein tyrosine kinases from the Src family. The activated tyrosine kinase phosphorylates tyrosine residues in ITAMs, providing docking sites for other signaling molecules like Syk, which activate various signal transduction pathways, ultimately activating specific genes for proliferation, differentiation, and antibody synthesis.
Role of B cell coreceptor complex in signal transduction
B cell activation is enhanced by a group of proteins on the B cell membrane, amplifiers of signal transduction, called the B cell coreceptor complex. This complex consists of CD19, CD21 (CR2), and TAPA-1. CD19 is a transmembrane protein with three Ig-like domains on the extracellular side, and its long cytoplasmic tail contains six tyrosine residues. CD21 has a binding site for C3d, the breakdown product of the C3 component of the complement system. TAPA-1 is a transmembrane protein traversing the membrane four times. CD21, through its C3d binding site, binds to the C3d-coated microbe cross-linked by the BCRs, causing the B cell coreceptor complex, BCR, and Ig/Ig to come together and transduce the signal. Activated protein kinases of the Src family phosphorylate the six tyrosine residues of the cytoplasmic tail of CD19, recruiting more signaling molecules in the BCR region, amplifying the activating signal.
9.5 Proliferation and Differentiation of Activated B Cells
Activated T cells secrete cytokines IL-2, IL-4, and IL-5 sequentially. IL-4 binding to receptors on B cells signals the progression from G0 to G1 and S phase. IFN- and TGF- are essential for differentiation into plasma and memory cells. CD40 and CD40L interaction causes class switching for TD antigens. IL-2, IL-4, and IL-5 induce B cell proliferation, while IL-4, IFN- , and TGF- induce class switching. Differential splicing of the RNA transcript produces secretory and membrane-bound antibodies. Membrane-bound IgM is monomeric, while secretory IgM is pentameric.
9.6 Events Occurring in Secondary Lymphoid Organs
All antigen-specific B cell activation events occur in secondary lymphoid organs.
9.6.1 Migration of Activated B Cells to Primary Follicles, Formation of Secondary Follicles and Germinal Centers
The activation processes take place in the T cell-rich areas of the secondary lymphoid organs (e.g., lymph node). Activated B cells migrate towards the peripheral zone of the paracortex along with a few T cells, undergoing proliferation to form small foci in the primary follicles. Proliferating B cells convert primary follicles into secondary follicles, with subsequent activation steps occurring in the germinal centers (GCs) in about 7-10 days. GCs contain FDCs with long processes rich in Fc receptors and complement receptors, capturing ag.ab complexes and retaining them on FDCs for extended periods.
9.6.2 Role of Follicular Dendritic Cells (FDCs) in the Selection of B Cells with High Affinity
Activated B cell proliferation occurs in germinal centers of the secondary follicles, interacting with follicular dendritic cells (FDCs) in the secondary follicles. FDCs are non-hematopoietic in origin, possibly from stromal cells of secondary lymphoid organs, expressing abundant Fc receptors and complement receptor CR1, but not class II MHC molecules. The long processes of FDCs have tiny nodules called iccosomes, heavily coated with immune complexes via FcR or CR1. Activated B cells attach to iccosomes on FDCs via BCRs, with some iccosomes being shed and interacting with B cells. Extensive B cell proliferation in the germinal center is thought to result from interaction with antigens on free-floating iccosomes.
Activated B cells entering germinal centers are called centroblasts. Centroblasts proliferate extensively in the dark zone, undergoing somatic hypermutations in their hypervariable regions. Random hypermutations produce B cells with high, low, or unchanged affinities for the antigen. Hypermutated centroblasts, now called centrocytes, move towards the peripheral light zone containing FDCs. Affinity maturation and class switching of activated B cells occur in this light zone. Centrocytes with high affinity for the specific antigen bind firmly to the antigen from ag.ab complexes on FDCs, selecting B cells with high affinity, called affinity maturation. Centrocytes with low or unchanged affinity undergo apoptosis because they cannot compete with B cells with high-affinity receptors in binding iccosomes. B cells having high affinity class switch and are differentiated into large plasmablasts and small memory cells in the presence of T cells. Plasmablasts migrate towards the medullary zone and develop into antibody-producing plasma cells.
9.6.3 Somatic Hypermutations
Point mutations occur in antibody genes of proliferating centroblasts in the dark zone of the germinal center. These mutations are substitutions, restricted to a single base, occurring in CDRs in the variable regions of both heavy and light chains. No other genes are affected. The hypermutation rate is a million-fold higher than the normal somatic cell mutation rate (10/bp/division vs. 10/bp/division). Each centroblast acquires a mutation after two cell divisions, accumulating in CDRs, changing the coding sequence and antigen affinity. Activation-induced cytidine deaminase (AID) causes the mutations. During transcription, single-stranded DNA is temporarily formed. AID deaminates cytosine to uracil. The unwanted uracil is cleaved off by uracil DNA glycosylase, leaving behind only the sugar-phosphate backbone. During the next round of replication, any one of the four bases of DNA will be incorporated against this gap in the ssDNA. Depending on the base incorporated, the codon is changed, and the original amino acid is replaced by a different amino acid, causing a change in affinity.
9.6.4 Affinity Maturation
As the acquired immune response to infection proceeds, antibodies of progressively higher affinity for the infecting pathogen are produced. This phenomenon of producing antibodies of higher affinity with the progression of the immune response is called affinity maturation. Eisen and Siskind first observed that the affinity of antibodies increases by about a hundred-fold during the course of the humoral response obtained after successive exposures of the host to the same antigen. When high-affinity hypermutated B cells get selected on iccosomes, they differentiate into plasma cells and produce high-affinity antibodies.
The process of affinity maturation is repeated in the germinal center in each round of infection/immunization. Since only the B cells with high affinity are selected and allowed to undergo differentiation, an increase in the affinity of antibodies occurs each time.
9.6.5 Antibody Class Switch
Changing the heavy chain of an antibody without changing its antigen specificity is called class switching or isotype switching.
IgM is the first class of antibodies produced in the primary immune response (first exposure to antigen). Although IgM has strong antigen binding due to ten antigen-binding sites, it is not efficient at antigen removal. Different antibody classes are needed for different effector functions. Class switching replaces the -chain with another heavy chain, maintaining the same variable region. The resulting antibodies have the same antigen specificity but different effector functions due to the isotype change.
Isotype switching, like somatic hypermutation, depends on the enzyme AID. It occurs when activated B cells are in the germinal center. Isotype switching is due to recombination at the switch region within the cluster of C genes. In each C gene, the flanking 5' region (except -gene) has highly repetitive nucleotide sequences called switch regions. Recombination occurs in these regions with AID. For example, in a switch between and -3, AID deaminates the cytidine residues from both switch regions of -gene and -3 gene to uracil. Uracil is removed by the uracil DNA glycosylase enzyme. The sequence then lacks a base. A specific nuclease then produces a nick in both the DNA strands at both switch regions at 5' end of and 5' end of -3, which facilitates joining of rearranged V region genes directly to -3, removing the DNA sequences in between the two nicks by forming a DNA loop. The C gene of the -3 chain is now placed in juxtaposition with the V region gene and is joined. After transcription and translation, the IgG3 antibody is produced instead of IgM, without changing the antibody specificity.
Cytokines IL-4, TGF- and IFN- act as switch signals. For the class selected, high-affinity BCR bearing B cells must interact with CD4 T cells in the germinal center, which produce the switch signaling cytokines. Interaction of CD40 on B cells with CD40L on CD4 T cells is required for class switch.
9.6.6 Differentiation into Plasma Cells and Memory B Cells
Differentiation of centrocytes occurs in the light zone of germinal centers. Centrocytes that have undergone affinity maturation and class switching differentiate into plasmablasts and memory B cells. T cell interaction and cytokines are required. Plasma cells do not express membrane-bound antibodies but synthesize large amounts of secretory antibodies. Plasma cells acquire the capacity to process the primary RNA transcript, such that the membrane anchoring segment is deleted and only a secretory form of antibody is synthesized. On the other hand, memory B cells can produce the membrane-bound form of the antibody molecule from the same primary transcript.
Memory B cells differ from naive B cells. Naive B cells express only IgM and IgD antibodies as membrane receptors. Memory B cells express IgG, IgA, and IgE as BCRs.
9.7 Primary and Secondary Immune Response
Antigen-dependent activation and differentiation of B cells produce plasma cells and secrete antigen-specific antibodies. The immune response after one exposure to an antigen (primary immune response) is amplified upon subsequent exposures (secondary immune response). Antibodies produced in primary and secondary immune responses differ in nature, kinetics, magnitude, and biological functions.
In the primary response, the host is exposed to the antigen for the first time. Naive B cells respond differently to TI and TD antigens, generating an IgM response. The heightened response in subsequent exposures to the antigen is due to the activation of memory cells. Memory cell generation is characteristic of TD antigens and, to some extent, TI type 2 antigens.
Memory B cells undergo rapid proliferation after a second stimulus and are more easily activated than naive B cells, resulting in a shorter lag period in the secondary immune response. The population of antigen-specific memory B cells is also much higher, amplifying the secondary response. IgG isotypes are the predominant contributors as memory B cells have already undergone class switching. Furthermore, antibodies in the secondary response have higher affinity and a longer-lasting response.
9.8 Humoral Immune Response to Hapten:Carrier Conjugates
Hapten is a small organic compound that lacks antigenicity but can interact with specific antibodies. When conjugated with a carrier protein, it induces an antibody response. Common haptens contain nitrophenyl groups, while carrier proteins are high-molecular weight proteins such as bovine serum albumin, bovine gamma globulin, human gamma globulin, tetanus toxoid, and diphtheria toxoid.
Hapten:carrier protein conjugation induces antibodies against the hapten, carrier protein epitopes, and altered antigenic epitopes created by the conjugation. To obtain a secondary immune response to the hapten, the same hapten linked to the same carrier should be used. Changing the carrier protein is considered a primary stimulant producing no heightened response to either the hapten or the carrier protein, known as the carrier effect. Helper T cell responses are induced by carrier molecules.
Another property is the requirement of both B cell and T cell epitopes on the same carrier molecule.
9.9 Distinction Between B1 and B2 Cells
Two types of B cells exist: B1 and B2. B2 is the predominant cell type involved in the adaptive immune response. B1 cells are present in fetal life and continue to exist in small numbers in adults in peritoneal and pleural cavities. Self-renewal is a characteristic property. B1 cells are polyspecific, producing antibodies against bacterial lipopolysaccharides and carbohydrates, but not against protein antigens. The antibodies have low affinity for the antigen. B1 cells express CD5 molecules on their membrane, distinguishing them from B2 cells.
9.10 Regulation of B Cell Development
B cell development is regulated by various transcription factors, DNA-binding proteins interacting with promoter or enhancer DNA sequences, stimulating or inhibiting specific gene expression. The B cell-specific activator protein (BSAP) is critical. It is expressed only by B lineage cells, influencing all B cell maturation stages. BSAP also affects differentiation into plasma and memory cells. Genes stimulated by BSAP include Vpre-B and 25 genes for surrogate chain synthesis, the J chain gene of polymeric IgM, Ig heavy chain switch sites, many genes involved in B cell activation, and transcription factors such as NF-B, Ets-1, c-Jun, Ikaros, and Oct-2.
Ikaros controls the expression of genes responsible for stem cell to pro-B cell development. BSAP, E2A, and Oct-2 control the transition of pro-B cells to immature B cells.
The heavy chain -gene has an enhancer region called E3'. BSAP inhibits the binding of NF-P factor, thereby blocking the expression of the -chain and memory B cell differentiation. BSAP, at low levels, allows normal differentiation of activated B cells into plasma cells, as NF-P can bind to the E3' region.