B Cells: Maturation, Activation, Proliferation, and Differentiation Notes

B Cells: Maturation, Activation, Proliferation, and Differentiation

Learning Objectives

• Role of B lymphocytes in antibody production (humoral immune response).

• Antigen-independent maturation in bone marrow.

• Acquisition of B cell receptor (BCR) and accessory molecules.

• Antigen-dependent activation in secondary lymphoid organs.

• Molecular events during activation, signal transduction, and antibody response maturation.

Contents

• Introduction to B cells.

• Maturation in bone marrow (antigen-independent phase).

• Migration of B cells.

• Antigen-dependent activation in secondary lymphoid organs.

• Proliferation and differentiation of activated B cells.

• Events 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

• B and T lymphocytes originate from lymphoid progenitor cells in bone marrow.

• T cells mature in the thymus.

• B cell development: maturation, activation, and differentiation.

• Maturation occurs in bone marrow while activation and differentiation occur in secondary lymphoid organs.

• Maturation is antigen-independent.

• Rearrangement of immunoglobulin heavy and light chain genes occurs in bone marrow.

• IgM and IgD act as surface receptors on naive B cells.

• Stromal cells in bone marrow support maturation.

• Each naive B cell is committed to one antigenic epitope.

• B cells acquire coreceptors and signaling molecules.

• Selection against autoreactive cells occurs.

• Mature B cells express high IgM and low IgD levels.

• B cells circulate through blood/lymph until encountering a specific antigen.

• Interaction with T-dependent antigens occurs in T cell-rich areas.

• T cells activate B cells via cytokines.

• B cells interact directly with T-independent antigens.

• Activated B cells enter primary follicles, forming secondary follicles with germinal centers.

• Proliferation, somatic hypermutations, class switch, and differentiation occur in germinal centers.

• These events lead to higher quality antibodies and long-lasting memory.

• Plasma cells and antibodies leave through efferent lymphatic vessels and splenic artery.

9.2 Maturation of B Cells in Bone Marrow (Antigen-Independent Phase)

• B cell progenitors are called pro-B cells.

• Maturation stages: large pro-B, small pro-B, early pre-B, small pre-B, and immature B cells.

• Mature B cells express both IgM and IgD as BCR, becoming immunocompetent.

9.2.1 Role of Stromal Cells

• Development of pre-B cells from pro-B cells depends on bone marrow stromal cells.

• Stromal cells are non-lymphoid supporting cells.

• Large pro-B cells interact with stromal cells through adhesion molecules.

• VLA-4 on pro-B cells binds to VCAM-1 on stromal cells, initiating contact.

• This interaction promotes expression and interaction of c-Kit on pro-B cells and SCF (stem cell factor) on stromal cells.

• c-Kit is a tyrosine kinase involved in signal transduction.

• c-Kit and SCF interaction activates the IL-7 gene in stromal cells and IL-7 receptor on pro-B cells.

• IL-7 binds to IL-7 receptor on pro-B and pre-B cells, signaling proliferation and Ig gene rearrangement.

9.2.2 Events During Pro-B to Pre-B Transition

• Gene rearrangement of variable portions of heavy (H) and light (L) chains of immunoglobulins is the most important event.

• Two gene segments (V and J) rearrange for the light chain.

• Three gene segments (V, D, and J) rearrange for the heavy chain.

• Rearrangement starts with $D<em>H$ and $J</em>H$ segments joining in large pro-B stage.

• A V segment joins the rearranged D-J segments in small pro-B cells, developing into large pre-B cells.

• Heavy chain gene rearrangement stops, and $μ$-chains appear in the cytoplasm.

• Surrogate L chains are synthesized, associate with $μ$-chains, and are expressed on the pre-B cell membrane along with Ig$α$ and Ig$β$, which are signal transducing molecules.

• Expression of complete IgM (with surrogate L chain) on the small pre-B cell membrane signals for proper H chain gene rearrangement.

• Proper light chain is synthesized, and proper IgM is expressed on the B cell membrane as B cell receptor.

• Naive B cells express IgD along with IgM, ready to enter circulation.

• Proliferation occurs at each stage of B cell development, enhanced by IL-7 binding to IL-7 receptors.

• Productive rearrangement of $μ$-chain allows development into pre-B cells.

• Non-productive rearrangement leads to apoptosis.

• Proper $κ$ or $λ$ gene rearrangement allows association with the $μ$-chain dimer and survival.

9.2.3 Selection of B Cells

• BCR gene rearrangement is random, leading to B cells recognizing self-antigens.

• Immature B cells with high reactivity to self-antigens undergo apoptosis.

• Receptor editing can save some self-reacting B cells.

• Cells are held at G0 phase, RAG proteins are re-expressed, and light chain gene rearrangement is initiated from the other homologue.

• Cells producing alternative receptors without strong reactivity for self-antigens survive.

9.3 Migration of B Cells

• Mature naive B cells express high levels of surface IgM and low levels of IgD.

• IgM and IgD are produced from differential splicing of a common primary transcript, sharing antigen specificity.

• Mature naive B cells circulate in blood, secondary lymphoid tissues, and lymph.

• B cells enter peripheral lymphoid organs from blood through endothelial venules via extravasation.

• Stromal cells in the cortex of lymphoid organs secrete chemokine CCL-21, interacting with receptor CCR7 on B cells.

• Under chemokine gradient, B cells migrate towards primary follicles and activate with T$_{H}$ cells upon encountering the antigen.

• Passage through primary follicles is essential for survival; otherwise, apoptosis occurs.

• If antigen is encountered, B cells activate, 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: antigens, T$_{H}$ cells, cytokines, Iga/Ig$β$ (BCR complex), and B cell coreceptors.

9.4.1 Activation by Thymus-Dependent (TD) and Thymus-Independent (TI) Antigens

• T-dependent antigens require direct contact between B and T cells.

• T-independent antigens do not require direct contact.

• TD antigens are generally soluble protein antigens and require T cell cytokines.

• TD antigens result in a strong humoral response, affinity maturation, class switch, and memory B cell generation.

• TI antigens: TI-1 and TI-2.

• TI-2 antigens require T cell cytokines but no direct T cell contact.

• Lipopolysaccharide (LPS) is a typical TI-1 antigen.

• Capsular polysaccharides with repetitive sequences are common TI-2 antigens; 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 Receptor Activating Signals

• Naive B cells are in G0 phase.

• Competence signals drive them from G0 to G1, and progression signals drive them from G1 to S phase.

• B cell activation by TI-1 antigens:

  • At high concentrations, LPS acts as a mitogen, causing polyclonal activation irrespective of antigen specificity.

  • At low concentrations, LPS reacts with B cells with BCRs specific for LPS.

• B cell activation by TI-2 antigens:

  • Repetitive structure results in cross-linking of membrane BCRs.

  • T cell cytokines provide the second signal for activation.

9.4.3 Activation Signals by TD Antigens

• Antigen-specific stimulation of T$_{H}$ cells requires two signals: MHC:antigenic peptide:TCR interaction and B7 (on APC):CD28 (on T cell) interaction.

• When the B cell acts as an APC, another signalling interaction set is required.

• Direct contact between B cell and T$_{H}$ cell is essential for B cell activation with soluble protein antigens.

• The B cell acts as an antigen-presenting cell (APC).

• The B:T$_{H}$ interaction requires a second signal comprising CD40 (on B cells) and CD40L (ligand) on T cells.

• After binding antigen to BCR, the BCR internalizes the antigen via receptor-mediated endocytosis.

• B cells increase expression of class II MHC molecules and costimulatory B7 molecules.

• Antigenic peptides displayed on MHC class II are recognized by T cells, forming a T$_{H}$:B cell conjugate.

• B and T cells recognize different epitopes of the same antigen.

• The B7 molecule on B cells interacts with CD28 on T cells, activating T cells to express CD40L.

• CD40 on B cells interacts with CD40L, resulting in activation of signal transduction pathways and cytokine receptor expression on B cells.

• IL-4 secreted by T cells binds to receptors on B cells, driving them into S phase and proliferation.

• Transduction of activating signals

  • BCRs have short cytoplasmic tails.

  • Membrane-bound antibodies (IgM, IgD, IgA, IgG, IgE) cannot interact with intracellular signalling molecules.

  • Iga/Ig$β$ act as signal transducers.

• Role of Iga/Ig$β$ in signal transduction

  • Long cytoplasmic tails contain immunoreceptor tyrosine-based activation motifs (ITAMs).

  • Antigen binding and membrane antibody cross-linking activate membrane-associated protein tyrosine kinases from the Src family.

  • Tyrosine kinase phosphorylates tyrosine residues in ITAMs, providing docking sites for signalling molecules like Syk.

  • Syk activates various signal transduction pathways, leading to gene activation for proliferation, differentiation, and antibody synthesis.

• Role of B cell coreceptor complex in signal transduction

  • Amplifiers of signal transduction: CD19, CD21 (CR2), and TAPA-1.

  • CD19 is a transmembrane protein with Ig-like domains and tyrosine residues; CD21 binds to C3d (complement breakdown product); TAPA-1 is a transmembrane protein.

  • CD21 binds to C3d-coated microbes cross-linked by BCRs.

  • The coreceptor complex, BCR, and Iga/Ig$β$ come together to transduce the signal.

  • Activated protein kinases phosphorylate tyrosine residues of CD19, recruiting more signalling molecules and 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.

• IL-4 binding to receptors on B cells signals progression from G0 to G1 and S phase.

• IFN-$γ$ and TGF-$β$ are essential for differentiation into plasma and memory cells.

• CD40 and CD40L interaction is responsible for class switching.

• Differential splicing of RNA transcript results in 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 to Primary Follicles, Formation of Secondary Follicles and Germinal Centres

• Activation occurs in T cell-rich areas of secondary lymphoid organs.

• Activated B cells migrate toward the peripheral zone of the paracortex with T cells, forming small foci in primary follicles.

• These develop into secondary follicles, and further activation takes place in germinal centers (GCs) in 7-10 days.

• Germinal centers contain follicular dendritic cells (FDCs) with Fc receptors and complement receptors.

• FDCs capture ag.ab complexes and retain them for a long period.

9.6.2 Role of Follicular Dendritic Cells (FDCs) in Selection of B Cells with High Affinity

• Proliferation occurs in germinal centers of secondary follicles; activated B cells interact with FDCs.

• FDCs are non-hematopoietic, possibly from stromal cells in secondary lymphoid organs.

• They express abundant Fc receptors and complement receptor CR1 but do not express class II MHC molecules.

• Long processes of FDCs have iccosomes coated with immune complexes via FcR or CR1.

• Activated B cells attach to iccosomes via BCRs.

• Iccosomes shed from FDCs interact with B cells, promoting B cell proliferation.

• Activated B cells entering the germinal centers are called centroblasts.

• Centroblasts undergo extensive proliferation in the dark zone and undergo somatic hypermutations in their hypervariable regions.

• Random hypermutations lead to B cells with high, low, or unchanged affinities for the antigen.

• Hypermutated centroblasts, now called centrocytes, move to the light zone containing FDCs.

• Affinity maturation and class switch occur in the light zone.

• Centrocytes with high affinity bind firmly to the antigen from ag.ab complexes on FDCs, selecting for high-affinity cells (affinity maturation).

• Centrocytes with low or unchanged affinity undergo apoptosis.

• B cells having high affinity show class switch and differentiate into plasmablasts and 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.

• Mutations are substitutions restricted to a single base within CDRs in the variable regions of heavy and light chains; no other B cell genes are affected.

• Hypermutation rate is 10-³/bp/division, much higher than normal somatic cell mutation rate (10-9/bp/division).

• Each centroblast acquires a mutation after two cell divisions.

• Mutations accumulate, and since they occur in CDRs, they change the coding sequence, generating variability in antigen affinity.

• Activation-induced cytidine deaminase (AID) causes mutations.

• During transcription, single-stranded DNA is temporarily formed; AID deaminates cytosine to uracil.

• Uracil DNA glycosylase cleaves off the unwanted uracil, leaving 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.

• Eisen and Siskind observed that the affinity of antibodies increases about a hundred-fold during the course of the humoral response obtained after successive exposures of the host to the same antigen.

• High-affinity hypermutated B cells are selected on iccosomes, differentiate into plasma cells, and produce antibodies with high affinity.

• The process of affinity maturation is repeated in the germinal center in each round of infection/immunization.

9.6.5 Antibody Class Switch

• Change in the heavy chain of antibody without changing its antigen specificity.

• IgM is the first class of antibodies produced in the primary immune response.

• Antibodies from different classes are needed to perform different effector functions.

• The $μ$-chain is replaced by another heavy chain, keeping the same variable region.

• Isotype switch depends on the enzyme AID and occurs when activated B cells are in the germinal center.

• Isotype switch is due to recombination at the switch region within the cluster of C genes.

• In each C gene (except $δ$-gene), the flanking 5' region has highly repetitive nucleotide sequences called switch regions.

• Recombination occurs in these regions with the help of the AID enzyme.

• As an example of switch between $μ$ and $γ$-3, AID deaminates the cytidine residues from both switch regions of $μ$-gene and $γ$-3 gene to uracil.

• Uracil is then removed by the uracil DNA glycosylase enzyme.

• A specific nuclease then produces a nick in both DNA strands at both switch regions at 5' end of $μ$ and 5' end of $γ$-3, which facilitates joining of rearranged V region genes directly to $γ$-3, removing the DNA sequences in between the two nicks by forming a DNA loop.

• The C gene of the $γ$-3 chain is now placed in juxtaposition with the V region gene and is joined.

• Cytokines IL-4, TGF-$β$, and IFN-$γ$ act as switch signals.

• High-affinity BCR-bearing B cells must interact with CD4 T cells in the germinal center, which produces the switch signalling 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 with affinity maturation and class switch differentiate into plasmablasts and memory B cells.

• Physical interaction with T cells and cytokines produced by T cells are required for differentiation of centrocytes into plasma cells and memory cells.

• Plasma cells do not express membrane-bound antibodies; they synthesize large amounts of secretory antibodies.

• Plasma cells acquire a capacity to process the primary RNA transcript in such a way that the membrane-anchoring segment is deleted and only a secretory form of the antibody is synthesized.

• Memory B cells can produce the membrane-bound form of the antibody molecule from the same primary transcript.

• 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 result in the production of plasma cells and secretion of antigen-specific antibodies.

• The immune response that occurs after one exposure to the antigen (primary immune response) gets amplified on the second or repeated exposures of the host to the same antigen (secondary immune response).

• Antibodies produced in primary and secondary immune response differ in their nature, kinetics, magnitude, and biological functions.

• In the primary response, the host is exposed to the antigen for the first time, and naive B cells generate an IgM response.

• The heightened response in subsequent exposures is due to the activation of memory cells.

• Generation of memory cells is characteristic of TD antigens and, to some extent, TI type 2 antigens.

• Memory B cells undergo rapid proliferation after the second stimulus and are more easily activated than naive B cells.

• The population of antigen-specific memory B cells is higher than the population of antigen-specific naive B cells, resulting in the amplification of the secondary response.

• IgG isotypes are the predominant contributors to the secondary immune response because memory B cells have already undergone class switch.

• Antibodies produced in the secondary immune response have higher affinity for the antigen, and the response lasts longer compared to the primary response.

9.8 Humoral Immune Response to Hapten:Carrier Conjugates

• Hapten is a small organic compound that, in itself, does not have antigenicity but can interact with specific antibodies.

• When conjugated with a carrier protein, it gains the capacity to induce antibody response.

• Commonly used haptens belong to molecules containing nitrophenyl groups; carrier proteins are high-molecular weight proteins (bovine serum albumin, bovine gamma globulin, human gamma globulin, tetanus toxoid, and diphtheria toxoid).

• Conjugation of hapten:carrier protein induces production of antibodies to hapten, epitopes on carrier proteins, as well as new antigenic epitopes created by conjugation.

• If a host is initially immunized with a hapten linked to a certain carrier, the same carrier is used to enhance the secondary immune response to the hapten.

• Changing the carrier protein does not enhance the stimulation of the carrier in getting the secondary immune response expected.

• The carrier molecules induce a helper T cell response and if the first carrier protein that enhanced the T response is altered no heightened hapten-specific antibody response will be produced even though the same hapten is introduced for the second time.

• The requirement for the presence of both B cell and T cell epitopes on the same carrier molecule is particularly important.

9.9 Distinction Between B1 and B2 Cells

• Two types of B cells exist: B1 and B2, with B2 being the predominant type; B2 cells are usually simply referred to as B cells.

• B1 cells exist in fetal life and continue to exist in adult life in small numbers (peritoneal and pleural cavities).

• Self-renewal is a characteristic property of B1 cells.

• B1 cells are polyspecific in nature and produce antibodies against bacterial lipopolysaccharides and carbohydrates but not against protein antigens.

• The antibodies have low affinity for the antigen.

• The characteristic feature of B1 cells is the expression of CD5 molecule on their membrane.

9.10 Regulation of B Cell Development

• Development of B cells is regulated by various transcription factors (DNA-binding proteins).

• The most critical transcription factor is the B cell-specific activator protein (BSAP), which is expressed only by B lineage cells and influences all stages of B cell maturation and affects differentiation of B cells into plasma cells and memory B cells.

Genes stimulated by BSAP include the following:

• Vpre-B and 25, genes for the synthesis of surrogate chain, J chain gene of polymeric IgM, Ig heavy chain switch sites in B cell development

• Many genes involved in B cell activation

• Transcription factors involved in B cell regulation such as NF-$κ$B, Ets-1, c-Jun, Ikaros, Oct-2

• Pu-1, EBF, BCF and E2A

Summary

• B cell development happens in two steps, the first is independent of antigens in bone marrow, the second is an antigen dependent which occurs in the germinal centers of the secondary lymphoid organs.

• Precursor B cells pass through Pro-B, Pre-B and immature B-cell stages in bone marrow, crucially aided by bone marrow stromal cells. Interaction between c-Kit and SCF encourages secretion of cytokine IL-7 which promotes proliferation.

• The immunoglobulin heavy and light chains are mainly modified at the lg gene rearrangement stage, which prepares each cell to respond to one singular antigenic epitope.

• Regarding heavy chain locus, they get rearranged first, with the pro-B cell membrane then expressing $μ$-chain with a surrogate light chain (Vpre-$β$ and 25) accompanied by signaling molecules Iga and Ig$β$. Signals are set to appropriately rearrange the light chain gene locus, making the naïve B cell express its proper IgM.

• The expression of IgM and IgD happens on the naïve B cell membrane making the B cell receptor for antigens (BCR). B cells also express a co-receptor molecular complex consisting of CD19, CD21 and TAPA-1 to help amplify the signal between the already mentioned Iga, and Ig$β$. B cells undergo elimination based apoptosis during negative selection when the cells bear a BCR that can recognize its own antigens.

• Receptor editing occurs occasionally in autoreactive cells held in the G0 phase of the cell cycle, and the light chain gene rearranges the homologus chromosome to make a new BCR from there.

• Circulating immature B-cells enter the follicles of the outer cortical region of the secondary lymphoid organs, where there is the full development in the encounter of a proper antigen. B-cells take diverse pathways based on TD (thymus-dependent), or TI (thymus-independent), in which there are bacterial lipopolysaccharides (TI-1), or polysaccharide components(TI-2).

• As signals get relayed through the Iga/Ig$β$ heterodimer, in addition to the co-receptor a chain of reaction gets induced through the phosphorylation of tyrosine residues to assist in activating proliferation genes and encouraging cell proliferation and divergence.

• On the first contact of the B-Cell with the antigen the initial stage of responses sets known as the primary immune, while subsequence exposure leads to the response from memory-cells produces in this stage. Thus it's rapid, a quick and strong robust from memory cells.

• Mostly the antigen-driven gathering happens on the T-cell regions located in the secondary lymphoid regions, and B-cells then navigate to the primary follicles. Secondary lymphoid also occur from activated B-Cells(centroblasts) during prolifertion, and dark-zones and hypermutation in those region also occur.

• The hypervariable V region heavy and light chains undergo hypermutations during dark zone B region division. Follicular dentritic cell encourage B cell selection as they help present ag.ab complexes with the help of hypermutated affinity receptors. Interaction between The cells and the subsequent cytokines results lead to an induced switch of isotypes while the antigen triggers better affinity through germinal divisions, creating plasma cells and B cells.