Humoral Immunity: Antibodies and the life cycle of B cells Flashcards
Describe the molecular structure of immunoglobulins, including different isoforms
Basic Molecular Structure:
- Immunoglobulins, aka antibodies are glycoproteins produced by plasma cells (differentiated B cells)
- basic structure: two identical heavy (H) chains and two identical light (L) chains. The chains are linked by disulfide bonds. Each heavy and light chain has both a variable region (VH and VL, respectively) and a constant region (CH and CL, respectively)
- The variable regions of the light and heavy chains form the antigen-binding sites (aka paratopes). Each immunoglobulin molecule has two antigen-binding sites, making it bivalent. The antigen-binding site’s unique shape allows it to bind specifically to an epitope or a distinct portion of an antigen
Immunoglobulin Isoforms:
- IgG: Most abundant, 4 subclasses (IgG1, IgG2, IgG3, and IgG4), cross the placenta, and bind to Fc receptors on cells like macrophages and NK cells with its Fc region causing neutralisation, opsonisation, ADCC and complement activation/MAC formation
- IgA: 2 subclasses (IgA1 and IgA2). Main component of secretory antibodies found in mucosal secretions and intestinal fluids. IgA dimeric form can neutralise pathogens and toxins at the point of entry. Fc region of IgA can interact with the poly-Ig receptor on epithelial cells (basolateral surface) allowing for transcytosis of IgA from the tissue side to the lumen side of mucosal surface; acting as a defence.
- IgM: First antibody produced in a primary response to an antigen, IgM antibodies are expressed on the surface of B cells as monomers but are secreted as pentamers linked by a J-chain, which greatly increases their avidity
- IgD: found on the surface of mature naive B cells, where it functions as part of the B cell receptor (BCR) for antigen
- IgE: binds to Fc receptors on mast cells and basophils with high affinity when IgE encounters an antigen (parasite or an allergen), causing these cells to release granules containing histamine and other inflammatory mediators leading to ↑blood vessel permeability and smooth muscle contraction
Relate the different structures of immunoglobulins to their antigen-binding or effector functions
Antigen-Binding Function:
- antigen-binding of immunoglobulins is carried out by the Fab regions (fragment antigen-binding)
- Each Fab region consists of one constant domain and one variable domain from both heavy and light chains of the antibody
- That variable domains are responsible for the specificity of the antibody, as they contain a hypervariable region (aka complementarity-determining region or CDR) that can fit the specific shape of an antigen’s epitope
- Means that each specific antibody can bind to a specific antigen with high affinity, allowing for the immune system’s specificity in its response to distinct pathogens
- Diversity in antigen recognition is achieved by the recombination of the variable, diversity, and joining gene segments, V(D)J recombination along with somatic hypermutation
Effector Functions
- carried out by the Fc region, (fragment crystallizable), consists of the remaining constant domains of the heavy chain and does not bind to antigen
- Instead, it interacts with various effector molecules and cells, mediating a range of immunological responses
- Fc structure varies between isotypes of antibodies leading to different functions
- Neutralisation: variable fragment (Fv) can bind to pathogen binding site to prevent pathogen or toxin entry (mostly IgG and IgA)
- Opsonisation: variable region will bind to the pathogen while Fc region binds to Fc receptor of macrophages/neutrophils, promoting antibody-dependent cellular phagocytosis (ADCP)
- Antibody-dependent cellular cytotoxicity (ADCC): Whilst IgG’s variable is bound to a pathogen, IgG Fc region can interact with Fc receptors on NK cells triggering NK cells to release chemicals to induce apoptosis in infected/cancerous cells
- Complement activation/fixing (MAC formation): Antibodies (IgG) form immune complexes, large clumps made up of antibodies and pathogens, and can involve other molecules like c1q, c1s and c1r. Activating/fix complement system; promotes inflammation, phagocytosis and formation of membrane attack complex (MAC) which lysis cells
Explain class switching and the mechanisms of class switching
A process that occurs in activated B cells during an immune response where it enables B cells to produce different classes of antibodies (IgG, IgA or IgE) each with unique effector functions while preserving the specificity of the antibody for its antigen. The original antibody produced by B cells is IgM (sometimes IgD)
Driven by a mechanism known as class switch recombination (CSR): genomic rearrangement in the B cell’s DNA within a region known as the immunoglobulin heavy chain locus. The heavy chain locus includes different ‘constant region’ genes for each of the isotypes (e.g., Cμ for IgM, Cγ for IgG, Cα for IgA, and Cε for IgE)
The outline of the process is as follows:
- 1) Antigen Encounter and B cell activation: A naïve B cell recognises its specific antigen through the BCR (IgM or IgD antibody on its surface). along with signals from helper T cells, triggers the B cell to proliferate and form a germinal centre within the lymph node or spleen
- 2) Cytokine signals: In the germinal centre, the activated B cell interacts with follicular helper T cells (Tfh cells), which produce cytokines that determine which isotype the B cell will switch to. I.e. Interferon-γ promotes switching to IgG3, IL-4 promotes switching to IgG4 and IgE, and TGF-β promotes switching to IgA
- 3) Activation-Induced Deaminase (AID): It deaminates cytosine residues in the DNA, converting them to uracils within the switch (S) regions that precede each constant region gene in the heavy chain locus, therefore, switching only process downstream; IgM can switch to IgG, IgA, IgE. this is major class switch and is irreversible
- 4) DNA repair: The presence of uracil in the DNA triggers a repair process that introduces double-strand breaks in the DNA at the S region, leading to the deletion of the DNA between the Sμ region (associated with the Cμ gene for IgM) and another downstream S region (associated with the gene for another isotype). The remaining DNA segments are then rejoined and cleaved after a switched DNA circle.
- 5) Transcription: Now, when the B cell transcribes the heavy chain locus, the variable region gene (antigen specificity) is joined with the new constant region gene, resulting in the production of a different isotype while retaining the original antigen specificity of the B cell.
Explain how heavy chain genes can code for membrane and secreted forms
Possible due to alternative RNA splicing:
1) Transcription: Initially, the entire immunoglobulin gene is transcribed into a primary RNA transcript or pre-mRNA by RNA polymerase II. This pre-mRNA includes the coding sequences (exons) for both the membrane-bound and secreted forms
2) RNA Processing and Alternative Splicing: The pre-mRNA is then processed and spliced. For the immunoglobulin heavy chain genes, the RNA transcript can be spliced in two different ways:
- Membrane-bound form: One splicing pattern results in mRNA encoding a hydrophobic transmembrane and cytoplasmic tail region, generating a membrane-bound form of the immunoglobulin heavy chain. This form is expressed on cell surface as part of the BCR complex
- Secreted form: The other splicing pattern results in mRNA that lack the exon for the transmembrane and cytoplasmic tail regions, thus creating a secreted form of the immunoglobulin heavy chain. This form is released from the cell and acts as an antibody for immune defences
3) Translation: The mature mRNA is then translated into the corresponding protein: either membrane-bound or secreted form of the immunoglobulin heavy chain
The specific form produced depends on the developmental stage and activation state of the B cell. Inactive or naïve B cells predominantly produce membrane-bound forms (BCRs) to detect antigens, whereas activated B cells primarily produce secreted forms (antibodies) to neutralise pathogens and activate other immune responses
Describe the important events of the antigen-independent stage of B lymphocyte life cycle
1) Early Pro-B Cell Stage: Rearrangement of the heavy chain genes of the immunoglobulin during bone marrow development. Rearrangement occurs in a specific order, with the D (Diversity) and J (Joining) segments recombining first, followed by the recombination of the V (Variable) segment with the D-J complex. The result is a rearranged VDJ segment on one chromosome, which leads to the expression of the μ heavy chain of IgM. Successful heavy chain rearrangement leads to the next stage.
2) Late Pro-B Cell Stage: The μ heavy chain pairs with a surrogate light chain (a stand-in for the actual light chain) to form the pre-BCR. This pre-BCR signals the cell to halt further heavy chain rearrangement (allelic exclusion) and initiates the rearrangement of the light chain genes.
3) Pre-B Cell Stage: The light chain gene rearrangement begins, involving the recombination of V and J segments. Successful light chain rearrangement results in the expression of a complete IgM molecule, which can be transported to the cell surface.
4) Immature B Cell Stage: Immature B cells express IgM BCR on their surface. These cells undergo a check for auto-reactivity. B cells that bind too strongly to self-antigens undergo either receptor editing (further light chain rearrangement to change the specificity of the BCR), anergy (become unresponsive), or apoptosis
5) Mature B Cell Stage: Surviving B cells begin to express IgD in addition to IgM and migrate from the bone marrow into circulation, marking the end of the antigen-independent phase of B cell development.
Compare the differences between somatic recombination and differential splicing, using examples from the B lymphocyte life cycle
Somatic Recombination:
- Responsible for the assembly of the variable regions of immunoglobulin genes during B cell development in the bone marrow.
- It is the process through which V (variable), D (diversity), and J (Joining) gene segments are combined in various permutations to generate a functional and diverse repertoire of BCRs
- 1) In the early Pro-B Cell Stage, the D and J segments of the heavy chain locus recombine
- 2) In the late Pro-B Cell Stage, the V segment is combined with the DJ complex to generate a complete VDJ segment, which forms the variable region of the heavy chain
- 3) In the Pre-B Cell Stage, a similar process occurs for the light chain genes, but only V and J segments
- Mediated by RAG1 and RAG2 enzymes (recombination activating genes), which make cuts in the DNA at specific recombination signal sequences (RSSs)
- The junctions are then processed and ligated together by other enzymes (DNA-PK, Artemis and Ligase IV). This process introduces junctional diversity
Differential Splicing:
- Post-transcriptional process that allows for the generation of multiple proteins from a single gene
- After transcription of a gene, the pre-mRNA is processed; exons are joined together in various combinations to form different mRNAs
- 1) In the immature B Cell stage, the primary RNA transcript from the rearranged immunoglobulin gene includes the sequence for both membrane-bound and secreted forms of the antibody
- 2) Differential splicing of this transcript allows the cell to produce either form of the antibody. If the sequence encoding the transmembrane domain is included; a membrane-bound antibody is produced (for BCR). If this sequence is spliced out; secreted form
So, while somatic recombination provides the antigen-binding diversity in the B cell receptor, differential splicing allows the B cell to express its receptor in different forms to serve different roles.
Explain how an enormous diversity of antigen-binding sites was produced during B lymphocyte development
1) Somatic Recombination:
- Firstly the random recombination of variable (V), diversity (D), and joining (J) segments in immunoglobulin heavy and light chain loci, happens during the early stages of B cell development in the bone marrow
- The V, D and J segments contain the genetic information that codes for the antigen-binding site of the antibody
- There are many different V, D and J segments in the genome, and the random selection and joining of these segments during somatic recombination create a large number of unique combinations
2) Junctional Diversity:
- During the process of joining the V, D and J segments together, the enzymes involved (RAG 1, RAG2, Artemis, and DNA ligase IV) can add, remove or modify a few nucleotides at the junction between these segments
- Introduces additional diversity at the most variable part of the antigen-binding site
3) Pairing of Heavy and Light Chains:
- Each B cell produces a unique heavy chain and a unique light chain, which pair together to form the final antibody molecule
- The combination of a specific heavy chain with a specific light chain adds another level of diversity
4) Somatic Hypermutation:
- Once a B cell has been activated by an antigen, it undergoes rapid proliferation in germinal centres within lymph nodes
- During this process, the activated B cell’s immunoglobulin gene is subject to a high rate of mutation, driven by AID (activation-induced cytidine deaminase) enzyme.
- These mutations primarily affect the variable regions of the antibody, further diversifying the antigen-binding sites
5) Isotype/Class Switching:
- Activated B cells can also change the isotype of the antibody they produce by a process called class switching
- This process doesn’t change the antigen specificity of the antibody but it does alter its effector function, allowing the immune response to adapt to different types of pathogens
Describe the mechanisms for genetic diversity during the development of B lymphocytes, including V(D)J recombination, P/N nucleotide addition, and junctional flexibility
1) V(D)J Recombination:
- The immunoglobulin gene segments are encoded in the genome as separate gene segments – multiple Variable (V), Diversity (D), and Joining (J) segments.
- Each B cell randomly selects and joins one of each type of gene segment (V, D, and J for the heavy chain, and V and J for the light chain) to construct the gene that encodes its unique BCR.
- This process, catalysed by enzymes called recombination activating genes (RAG1 and RAG2), results in a recombined VDJ (or VJ) sequence that encodes the variable region of an immunoglobulin heavy (or light) chain
2) P/N Nucleotide Addition:
- After the initial cleavage events, the cut ends of the gene segments form hairpin loops
- When these loops are opened to generate coding ends for joining, palindromic (P) nucleotides can be created
- P-nucleotides extend the coding sequence of the gene segments, and their generation increases the variability of the junctions between the gene segments
- In addition to P-nucleotide addition, the enzyme terminal deoxynucleotidyl transferase (TdT) can randomly add non-template (N) nucleotides to the ends of the gene segments before they are joined
- The addition of these N nucleotides is another major contributor to diversity, as it introduces additional random sequences at the junctions of the V, D, and J segments
3) Junctional Flexibility:
- Refers to the imprecision that occurs during the joining of the V, D, and J segments.
- The RAG complex introduces a degree of flexibility at the junctions by allowing nucleotides to be randomly deleted from the ends of the segments before they are joined together
- This results in a greater variability of the length and sequence of the junctions, which further increases the diversity of the antigen binding sites
Explain deductions from protein sequence and classical genetics: allelic exclusion, separate chromosomal locations for lambda, kappa, and heavy chain
1) Allelic Exclusion:
- Each B cell produces an immunoglobulin that has one unique type of antigen specificity ensuring monospecificity
- This is despite the fact that each B cell, being diploid, has two sets of immunoglobulin genes (one from each parent)
- The principle of allelic exclusion states that only one allele (either maternal or paternal) is actively transcribed and translated into a protein product
- This ensures that each B cell produces a BCR of a single specificity
- Evidence for allelic exclusion comes from the observation that B cells produce immunoglobulin of only one light chain type (typer kappa or lambda, but not both) and one heavy chain type.
- Mechanism involves a feedback mechanism where the successful production of a heavy or light chain prevents rearrangement of the other allele
2) Separate Chromosomal Locations
- The genes encoding the immunoglobulin chains are located on separate chromosomes.
- In humans, the heavy chain genes are located on chromosome 14, the kappa light chain genes on chromosome 2, and the lambda light chain genes on chromosome 22
- This separation allows for independent recombination events to occur at each locus, increasing the diversity of potential antigen receptors
- Also allows for sequential recombination events where heavy chain recombination occurs first, followed by kappa and then lambda
- This sequence is maintained by tightly regulated process called “checkpoints” where successful heavy chain production is required before light chain recombination can begin
Describe the important events of the antigen-dependent stage of the B lymphocyte life cycle
Occurs in secondary lymphoid tissues such as lymph nodes or spleen, where B cells interact with antigens
1) Antigen Recognition and Binding:
- Begins when a mature B cell, encounters antigen specific to its unique BCR
- The unique aspect of BCRs is that they can bind to native, unprocessed antigens, unlike TCRs
2) B Cell Activation:
- The binding of the antigen to the BCR serves as the initial activation signal for the B cell. If the antigen is a thymus-dependent antigen (protein), a second signal is required for full activation, provided by helper T cells called linked recognition
- The antigen-bound B cell processes the antigen and presents fragments on its surface using major histocompatibility complex II (MHC II) molecules. These fragments are recognised by a helper T cell, which, in turn, releases stimulatory cytokines that provide the second signal.
3) Proliferation and Differentiation:
- After receiving both signals, rapid proliferation begins, creating a lone of identical B cells.
- Some of these cells differentiate into plasma cells, which are large cells producing and secreting antibodies specific for the antigen
- Others differentiate into memory B cells, which will respond more rapidly and robustly upon subsequent exposure to the same antigen
4) Affinity Maturation:
- During the proliferation phase, the B cells undergo a process called somatic hypermutation, which introduces random mutations into the genes coding for the BCR
- B cells with mutations that increase the affinity of the BCR for the antigen are selected for survival, positive selection
5) Class Switching:
- Activated B cells can undergo class switch recombination (CSR), a process that changes the class or isotype of the antibody that B cell produces white maintaining the specificity for the antigen
- CSR is influenced by cytokines released by helper T cells
6) Memory Response:
- After the initial immune response, most of the activated B cells undergo apoptosis and are removed from the system
- However, memory B cells persist in the body
- Upon subsequent exposure to the same antigen, these cells mount a faster and more potent immune response, providing long-term immunity
Differentiate between naïve and activated B lymphocytes
Naive B lymphocytes:
- Matured in the bone marrow but has not yet encountered an antigen
- These cells express BCRs, membrane-bound form of IgM and IgD antibodies
- Naive B cells circulate in the blood and lymph and reside in lymphoid organs, such as lymph nodes
- When naive B cell encounters its specific antigen, it becomes activated, mediated by T cells (T-dependent activation)
- Alternatively, some antigens (polysaccharides and lipids) can activate B cells without T cell help (T-independent activation)
Activated B lymphocytes:
- Encountered its specific antigen and received additional activation signals from helper T cells
- Causing the B cells to proliferate rapidly, aka clonal expansion
- Some of these cells differentiate into plasma cells; produces and secretes specific antibody to neutralise the antigen
- Other cells differentiate into memory B cells
- Also can undergo class-switch recombination (changing the class of the antibody they produce) and affinity maturation (improving the affinity of their antibodies for the antigen)
Describe the events during B lymphocyte activation and differentiate between T-cell independent and T-cell dependent B cell activation
T-cell Dependent B cell Activation:
- Antigen recognition: B cell recognises and binds its specific antigen through its BCR, the BCR-antigen complex is then internalised and processed into peptide fragments
- Presentation to T cells: These peptide fragments are displayed on the B cell surface, bound to MHC II molecules
- Interaction with helper T cells: The antigen-MHC II complex is recognised by a helper T cell with a complementary TCR. This interaction is stabilised by CD40 ligand on the T cell, binding to CD40 on the B cell
- Cytokine signalling: The helper T cell, once activated by this interaction, releases cytokines like IL-4, IL-5 and IL-6 which provide signals that lead to the B cell’s proliferation and differentiation into plasma and memory cells, as well as class-switch recombination and affinity maturation.
T-cell independent B cell activation:
- Involves antigens with repeating subunits, like polysaccharides or lipids
- Multivalent binding: These antigens can cross-link multiple BCRs on the B cell surface, providing a strong enough signal for activation
- Alternative signals: Signals from pattern recognition receptors, i.e. Toll-like receptors, or activation of the complement system, can contribute to T-cell independent B cell activation
- Limitations: While this pathway can lead to the production of antibodies, the resulting response is usually of lower affinity and does not result in memory B cell formation or class-switching
Describe the affinity maturation process and how it contributes to immunoglobulin affinity
- Generate high-affinity antibodies in response to an infection, primarily occurs in the germinal centres of secondary lymphoid organs such as lymph nodes or the spleen
- B cell activation and germinal centre formation: Presentation of antigen-MHC II to Th cells leads to the formation of a germinal centre, a site where activated B cells (centroblast) begin to proliferate rapidly
- Somatic hypermutation: Within the germinal centre, the BCR genes (especially the variable regions) undergo a high rate of point mutations (somatic hypermutation or SHM) and are mediated by an enzyme called activation-induced cytidine deaminase (AID). SHM results in a collection of B cells with varied BCRs, and consequently varied affinities to the antigen
- Selection of high-affinity B cells: The mutated B cells then express their mutated BCRs and capture the antigen from follicular dendritic cells (FDCs). B cells with higher affinity BCRs for the antigen capture more antigen and thus more presentation to follicular helper T cells (Tfh) = more survival and proliferation signals
- Clonal expansion and differentiation: The high-affinity B cells undergo clonal expansion and differentiate into either memory B cells or plasma cells
- Affinity maturation explains why the antibodies produced in a secondary immune response are of higher affinity than those produced in a primary immune response
