B Cells and Antibody Production

• Extracellular pathogens are controlled by antigen-specific antibodies which are produced by B cells
• MHC Class II processes load peptides from exogenous antigens like Streptococcus pneumoniae.
• The antigens are presented to CD4+ helper T cells (Th)

CD4+ helper T (Th) cells

• Express a CD4 co-receptor that binds to surface MHC class II molecules
• Th cells load exogenous antigenic peptide sequences
• Th cells aid B cells and their secreted antibodies
• Antibodies exist as different isotypes (subtypes) with distinct functions including, IgG, IgA, IgE, and IgM
• IgD are not mentioned in the text

Development of B Cells

• B cells develop in the bone marrow
• B cells migrate to the spleen for final maturation
• Naive B cells are mature but antigen-inexperienced
• Naive B cells express membrane-bound IgM and IgD as their B cell receptors (BCRs)
• B cells are considered pAPCs
• B cells do not have the same mobility or primary role as DCs or macrophages
• B cells comprise the B cell follicle

B Cell Receptors (BCRs): Membrane-Bound Antibodies

• B cell receptors (BCRs) are membrane-bound forms of antibodies (immunoglobulins) expressed on the surface of mature, naive B cells
• BCR's are used for antigen recognition and B Cell Activation
• BCRs are structurally identical to antibodies
• BCRs contain a hydrophobic transmembrane domain anchoring them into the plasma membrane

Structure of Antibodies (BCR/Soluble)

• Composed of a symmetrical core structure composed of 2 identical heavy chains and 2 identical light chains
• Chains are linked by disulfide bonds, forming the classic Y-shaped antibody structure
• Each chain has a constant (C) region for structural and effector function
• Each chain has a variable (V) region that determines antigen specificity

Fab Region (Fragment antigen-binding):

• Composed of the variable and constant regions of both heavy and light chains on each of the antigen-binding (Fab) arms
• Each antibody has two Fab arms
• At the tip of the Fab region is the antigen-binding site
• The antigen-binding site is formed by 3 CDRs on the heavy chain and 3 CDRs on the light chain, totalling 6 CDRs per antigen-binding site.

Complementarity-Determining Regions (CDRs) or Hypervariable Regions (HVRs):

• Short, highly variable amino acid sequences nested within the variable (V) regions of both the heavy and light chains of the Fab region
• CDRs are the contact points with their single, unique antigen's conformational epitope.
• This variability allows each individual to produce 10^11 different antibodies (repertoire)
• Antigen-specific responses can be generated against almost any pathogen
• B CELLS DO NOT GET ACTIVATED VIA MHC-PEPTIDE PRESENTATION
• B CELLS MUST RECOGNISE SINGLE NATIVE (INTACT) ANTIGENS IN THEIR FULL 3D CONFORMATION VIA A SINGLE, SPECIFIC CONFORMATIONAL EPITOPE
• Antigen is brought via lymph flow or subcapsular sinus macrophages in lymph nodes
• Avidity: Binding strength is enhanced when multiple Fab arms (antigen-binding sites) interact (avidity)

Epitopes:

• Linear: Continuous amino acid sequences
• Conformational (discontinuous):Amino acids brought together by protein folding
• Conformational epitopes recognise intact (native) antigen in its 3D conformation
• Fc Region (Fragment crystallisable region)
• Fc Region is the stem of the antibody, and is formed by the constant (C) regions of the two heavy chains
• Different Ig isotypes are represented by structurally different heavy chain constant regions
• Therefore, Fc determines the antibody (Ab) isotype
• IgG and IgE are monomers
• Isotype subclasses: IgG → IgG1, IgG2, IgG3, IgG4
• IgA are dimers
• Isotype subclasses: IgA1, IgA2
• IgM are pentamers

Functions of the Fc Region:

• Binds to Fc receptors on immune cells like macrophages, NK cells, and neutrophils
• Fc Receptors mediate effector functions like opsonisation to enhance phagocytosis
• Complement activation: IgM and IgG can activate the classical complement pathway through Fc interaction with C1q

Antibody Hinge Region:

• Located in the constant region of the heavy chain
• Hinge region is specifically located between the CH1 domain (in the Fab region) and the CH2 domain (in the Fc region)
• A short, flexible segment of amino acids
• Typically consists of 10–60 amino acids
• Depends on the antibody isotype such as IgG and IgA
• Contains cysteine residues that form inter-heavy-chain disulfide bonds
• Provides structural stability while allowing flexibility between the two Fab arms for rotation and spatial movement
• Allows the antibody to bind antigens at various angles and distances
• Increases the ability to neutralise pathogens
• B cells have evolved to detect external, surface-exposed structures such as viral spikes and bacterial capsules to generate effective, neutralising antibodies
• MHC-peptide presentation cannot provide external defense.
• MHC presentation breaks down antigens into peptides, destroying the conformational epitopes B cells need to bind.
• This presentation is fine for CD4+ T cells, which rely on MHC-II to detect processed fragments of what an APC has ingested
• B cells must detect external, surface-exposed structures (e.g. viral spikes, bacterial capsules) to generate effective, neutralising antibodies.
• Antigens are brought via lymph flow or subcapsular sinus macrophages in lymph nodes.

B Cell Antigen-Specific Internalisation

• Before encountering an antigen, each naïve B cell expresses a unique B cell receptor (BCR)
• The BCR is a membrane-bound immunoglobulin (IgM and IgD) whose antigen specificity is determined by complementarity-determining regions (CDRs) in the Fab region, generated during V(D)J recombination in the bone marrow.
• The BCR recognizes and internalizes a single, specific conformational epitope on an intact (native) antigen.

Conformational Determinants (Epitopes)

• Defined by the 3D structure of the antigen (tertiary/quaternary folding)
• The binding site is formed by amino acids that are distant in the primary sequence, but brought together by protein folding.
• Denaturation: Loss of 3D structure which destroys the epitope
• An antibody cannot bind anymore
• Conformational epitopes are lost upon denaturation
• No Ab:Ag binding

Linear Determinants (Epitopes)

• Composed of a continuous sequence of amino acids in the primary structure
• Binding depends on sequence, not structure
• If a linear epitope is exposed in native form: An antibody binds both native and denatured forms
• If the epitope is buried in native form: Denaturation may expose the epitope, allowing Ab binding only in denatured form
• Some Abs are designed to bind only denatured forms of a protein
• Occurs when denaturation reveals otherwise hidden epitopes
• Linear epitopes may still bind Abs after denaturation, depending on accessibility

Specificity and Cross-Reactivity of the Antibody Fab Region

• Specificity: the ability of the Fab region to bind one unique epitope with high precision
• The CDRs are within the variable regions of the heavy and light chains
• Cross-Reactivity: When an antibody binds to more than one antigen epitope when a similar epitope resembles the original in shape or charge but is not identical.
• Weaker binding which may result in partial activation or non-protective binding
• Cross-reactivity can be beneficial or problematic

Why B Cells Are Unique Professional APCs:

• Antigens are generally presented as 'intact' thus in native conformation in B cells compared to the TCR which requires processing and presentation of a linear peptide by APCs
• B cells specifically internalise the native antigen via receptor-mediated endocytosis through their B cell receptor (BCR) binding to its cognate conformational epitope, enabling highly selective antigen uptake.

BCR

• Membrane-bound immunoglobulin (IgM or Igd) binds to the antigen
• Iga and Igß (Cd79a/CD79b) ITAM motifs become phosphorylated, initiating intracellular signalling cascades that lead to B cell activation
• The signal induces antigen internalisation, upregulation of co-stimulatory molecules (CD80/86) and migration to the T-B border for T cell help
• B cells remain confined to the B cell follicle (in lymph nodes) and do not migrate to peripheral tissues like other APCs
• Instead, the antigen is delivered to them via lymphatic flow or by APCs
• For lymphatic flow, small antigens from peripheral tissues can enter secondary lymphoid organs via afferent lymphatic vessels
• The antigens are delivered directly to the B cell follicle through the lymph node conduit system
• For APC delivery, antigens such as large microbes or Ag:Ab complexes are captured by subcapsular sinus macrophages or resident DCs
• The antigens are then delivered to B cells in lymph node follicles
• Antigens can remain intact on the cell surface of phagocytes and follicular dendritic cells
• Internalise and process the entire antigen for T Cell-Dependent Activation of B Cells
• Once internalised, the entire antigen is processed into multiple linear peptide fragments, even though the B cell binds one specific conformational epitope of a native antigen through it's unique BCR
• Multiple peptides are loaded onto MHC class II molecules and presented on the SAME B cell surface

B Cell Peptide-MHC II Presentation For T Cell-Dependent Activation

• Present multiple linear peptide fragments derived from the processed protein on MHC class II
• This allows for interaction with multiple, already differentiated CD4+ Helper T cells (e.g., especially Tfh subset) with different peptide specificities
• Broad T Cell Help is enabled
• B cells require 2 key signals for full activation
• For both T Cell-Dependent and T Cell-Independent B Cell Activation, BCR-native antigen acts as Signal 1

T cell-dependent (TD) B cell activation

• Occurs when B cells recognise protein antigens and receive help from T follicular helper (Tfh) cells via CD40–CD40L interaction and cytokine signals (e.g., IL-4, IL-21)
• Leads to germinal centre formation, class switching, somatic hypermutation, and memory B cell generation

T cell-independent (TI) B cell activation

• Triggered by non-protein antigens (e.g., polysaccharides, lipids)
• Induces strong BCR cross-linking or TLR engagement which leads to a rapid, short-lived IgM response with lower-affinity and broader specificity
• Occurs without germinal centre formation (mostly extrafollicular), class switching, or memory.

T Cell-Dependent Activation of B Cells

• B cells act as pAPCs only in the context of T cell-dependent activation
• B cells act specifically to cognate CD4+ T follicular helper (Tfh) cells
• Different CD4+ T cells, each with a unique TCR, can recognise distinct peptide:MHC-II complexes derived from the same internalised antigen as presented on the same B cell
• This allows MULTIPLE (already activated & differentiated) cognate CD4+ helper T cells to provide help, especially Tfh cells
• After B cell engagement with its cognate antigen, the B cell migrates to the T-B border of the lymphoid follicle between the follicular B zone and T cell zone
• It scans for an already-differentiated T follicular helper (Tfh) cell whose TCR specifically recognises the presented peptide:MHC-II complex
• Therefore, B Cells interact with ALREADY differentiated COGNATE Tfh cells instead of naive CD4+ T cells.

Helper Synapse - Tfh–B Cell Interaction

• Initial Pre-Germinal Center formation occurs at the T–B border
• Occurs in the in secondary lymphoid organ, between an effector CD4* helper T cell such as Tfh and an antigen-captured B cell
• TCR on Tfh recognises MHC-II:peptide on a B cell
• CD4 co-receptor stabilises interaction and initiates signal transduction
• Adhesion molecules LFA-1 (Tfh cell) binds ICAM-1 (B cell) stabilising the synapse
• Co-stimulation (CD40–CD40L)
• CD40 (B cell) binds CD40L (Tfh cell)
• Delivers essential activation signal to B cell promoting class-switch recombination and survival
• Tfh secretes polarised cytokines such as IL-4, IL-6, and IL-21 into the synaptic cleft
• B cells cannot provide co-stimulation or the full set of polarising cytokines required to activate naive CD4+ T cells

T cell-dependent B cell (activation)

• Process can follow two distinct pathways
• Extrafollicular T-dependent B cell activation: Prolonged T-B interactions outside the follicle leading to short-lived plasma cells, producing lower-affinity IgM (no affinity maturation) with limited isotype switching.
• Germinal center (GC) response: After initial T-B contact, B cells migrate into follicles to form germinal centers where they interact with Tfh cells, promoting long-lived plasma cell differentiation, isotype switching, and somatic hypermutation/affinity maturation.
• T cell-independent and extrafollicular T cell-dependent (NO GC formation) responses BOTH result in the rapid production of short-lived plasma cells that secrete moderate- to low-affinity antibodies, primarily IgM
• T-cell-dependent responses can ALSO include some class-switched IgG or IgA

Germinal Center

• A specialized microenvironment within B cell follicles of secondary lymphoid organs
• Germinal centre formation is triggered by the initial interaction between an antigen-activated B cell and its cognate T follicular helper (Tfh) cell at the T–B border
• Every major event in the Germinal Centre such as clonal expansion, class switching, affinity maturation, and differentiation is a direct result of the earlier Tfh–B interaction (cytokine signals + CD40-CD40L)
• The signals received from the Tfh cell direct the fate of the B cell within the GC

The B cell responses include:

• B Cell Proliferation: The B cell undergoes rapid clonal expansion
• Somatic Hypermutation (SMH)/Affinity Maturation: The B cell undergoes Somatic hypermutation + selection for high-affinity BCRs (in the GC).
• Class-switch Recombination: Switches antibody isotype (e.g., to IgG, IgA, or IgE) based on cytokine signals.
• Differentiation: The B cells become either high affinity Plasma cells or memory B cells.

Somatic Hypermutation (SHM) / Affinity Maturation, Positive Selection

• Somatic Hypermutation (SHM) / Affinity Maturation occurs during rapid B cell proliferation in the dark zone of the germinal center (GC)
• The enzyme AID introduces random point mutations in the V-region genes specifically in the complementarity-determining regions (CDRs) or hypervariable regions (HVRs) of both heavy and light chains within the Fab region of the antibody
• This generates a diverse pool of BCR variants with different affinities for the same antigen
• Positive Selection for High-Affinity B Cells IN the Germinal Center Light Zone

Mutated B cells migrate to the light zone of the GC

• Follicular dendritic cells (FDCs) display native (intact) antigen on their surface
• Low-affinity clones fail to compete and undergo apoptosis
• Only B cells with High-affinity BCRs can effectively bind native antigen displayed on follicular DCs and receive survival/proliferation signals from T follicular helper (Tfh) cells. The Tfh/B-Cell Survival signals require CD40L and cytokines like IL-21
• Second, Post-selection Tfh-B Cell Interaction occurs in the Light Zone of the Germinal Center which is a critical post-selection checkpoint
• Positively selected B cells that have high-affinity BCRs that have successfully bound native antigen on follicular DCs present processed peptide:MHC-II to differentiated Tfh cells
• Tfh cells provide essential survival and maturation signals through CD40L-CD40 interaction and cytokines such as IL-21 and IL-4

Class Switch Recombination (CSR): Diversification of Antibody Isotypes

• Cytokines that drive Class Switch Recombination (CSR) are primarily secreted during this second interaction between high-affinity B cells and T follicular helper (Tfh) cells in the light zone of the germinal center, following somatic hypermutation (SHM) and positive selection.
• Tfh cells in this contact provide CD40L and cytokines that signal B cells to initiate CSR, switching antibody isotype
• Downstream of this, B cells can switch to downstream isotypes (e.g., IgG → IgA) but cannot revert to upstream isotypes.

Plasma Cell Differentiation

• BCRs are not cleaved off the membrane
• Instead, secreted and membrane-bound forms of antibodies are the result of alternative mRNA splicing of the same immunoglobulin heavy chain gene.
• Naive B cells produce membrane-bound IgM and IgD by including exons that encode a hydrophobic transmembrane region
• These antibodies become the B cell receptor (BCR)
• Upon activation and differentiation into plasma cells, the B cell undergoes alternative mRNA splicing, excluding the exons encoding the transmembrane and cytoplasmic tail domains
• Instead, exons that code for a soluble tail are included, so that the antibody is secreted instead of membrane-bound This results in secreted antibodies which retain the same antigen specificity as the original BCR because the variable (V(D)J) region is unchanged, but without the hydrophobic transmembrane anchor, so they can diffuse and act systemically
• In soluble form, effector functions such as pathogen neutralisation, opsonisation, and complement activation are performed; no longer as receptors, but as antibodies in the humoral immune response.
• B cells must undergo two critical interactions with already-differentiated T follicular helper (Tfh) cells
• With the first interaction at the T–B border pre-germinal center via presenting peptide:MHC-II to a Tfh cell with a matching TCR
• Receives early help via CD40–CD40L and cytokines, initiating B cell activation, proliferation, and entry into the germinal centre
• The second interaction occurs in the light zone of the GC post-selection
• If high-affinity B cells re-engage with Tfh cells to receive further CD40L and polarised cytokines
• Drives class switch recombination and differentiation into plasma cells or memory B cells

T cell-independent (TI) B cell Activation

• Triggered by non-protein antigens that induce strong BCR cross-linking and TLR/Complement enhanced engagement
• Leading to a rapid, short-lived IgM response with lower-affinity and broader specificity without germinal centre formation, class switching or memory
• Antiboby binding alone is not enough to make a B cell differentiate, therefore a second signal is required In T-independent B cell activation, the second signal is provided by the microbe itself or by a microbe-associated complement protein

TLR-Mediated Activation

• The BCR binds the antigen as SIGNAL 1
• Simultaneously, PAMPs on the same microbe engage Toll-like receptors (TLRs) on the B cell as SIGNAL 2
• Both BCR and TLR co-stimulation activates the B cell
• Result: Induces proliferation and differentiation, yielding mostly IgM, short-lived, and no memory

Complement-Enhanced Activation

• Microbial antigen such as bacterial polysaccharide is opsonised by complement protein as a TI-2 - Antigen
• The BCR binds the antigen as SIGNAL 1 Since these are highly reptitive structures, they can cross-link multiple BCRs
• Simultaneously, C3d binds to CR2-CD21 as SIGNAL 2
• This co-ligation of BCR and CR2 amplifies BCR signalling

T cell-independent

• The activation is triggered by non-protein antigens that induce strong BCR cross-linking and TLR/Complement enhanced engagement
• Then it triggers a rapid, short-lived IgM response with lower affinity and broder specificity without germinal center formation, class switching ormemory

Sequence of Events

1. B Cell Activation, where the, Antigen binds the BCR via CD40-CD40L interaction with Tfh cell and the signal travels through Th cytokines
2. Clonal Expansion rapid proliferation that occurs in the dark zone of the germinal center
3. The goal of Somatic Hypermutation(SHM) is to improve antigen affinity thanks to the AID enzyme introduces mutations in variable regions
4. Positive Selection in Light Zone, where high-affinity BCRs binds antigen on FDCs through the present peptide:MHC-II to the Tfh which recieves the survival signals to move on
5. Class-Switch Recombination is AID mediated and there is switching of constant via (C) region
6. B cells still membrane-bound and they move into plasma cell differentiation and after that alternative mRNA splicing removes exons for transmembrane region

Effector Mechanisms of Antibodies

• Allow for the effector functions of B cells at distal sites from the cell, thus enabling various antibody-mediated responses

Major Activities

• Neutralisation, opsonisation such as antibody-dependent phagocytosis, complement-dependent cytotoxicity, and antibody-dependent Cell Cytotoxicity. The effector function of each antibody isotype depends on its tissue distribuiton and location Antibodies (as well as complement) can opsonise pathogens Antibody-mediated opsonisation causes antibodies (specifically IgG and IgA) to be considered opsonins

Neutralisation

• When antibodies bind to microbial surface antigens from binding and prevent these ligands from interacting with their specific host cell receptors
• Depends on the antibodies and their ability to bind and recognize antigens

Additional Notes

• It can be noted that it does not necessarily require antibodies to bind to immune cells or components
• The neutralizing antibodies can be any Ab Isotype
• Opsonization is a process in which antibodies bind to the surface that they need to via their FAB region and expose their FC regions.
• This process is performed via FC receptors that have recognized or bound to the region
• Neutrophils, DC's, and Macrophages participate in this section of the immune response as innate immune cells
• In order to have greater specificity, efficiency, selectivity, speed and microbial recognition they will engage in killing.

Antibody-Dependent Cell Cytotoxicity (ADCC)

• This is performed by immune cytotoxic effect cells that kill or are coded with antibodies from another microbe region
• FCYRILL which CDIG on NK cells binds the FC region to deliver an activating signal