Humoral immunity

B Cell Development

• B lymphocytes, a type of white blood cell, are responsible for humoral immunity, which involves the production of antibodies.
• B cell development, also known as B cell maturation, is the process by which these cells become mature and capable of mounting an immune response.
• This process occurs primarily in the bone marrow, a spongy tissue found within bones, particularly in humans and mice.
• The bone marrow provides a unique microenvironment conducive to B cell development.
• Immature B cells undergo a series of developmental stages within the bone marrow, resulting in mature B cells that are capable of recognizing and responding to specific antigens.

B Cell Activation and Differentiation

• Mature B cells reside in the lymph nodes and spleen, where they encounter antigens.
• Activation of B cells occurs when a B cell receptor (BCR) on the surface of the B cell binds to a specific antigen.
• This binding event triggers a cascade of intracellular signaling events.
• Activated B cells differentiate into plasma cells and memory B cells.
• Plasma cells, which are antibody-producing factories, are responsible for producing and secreting antibodies that target the specific antigen encountered by the B cell.
• Memory B cells are long-lived cells that remain in the body after an infection, providing immunological memory.
• These memory B cells quickly differentiate into antibody-producing plasma cells if the body encounters the same antigen again, providing a faster and more robust immune response upon re-exposure.

B Cell Activation

• B cell activation is triggered by antigen presence.
• Activated B cells proliferate and differentiate into plasma cells and memory B cells.
• Naive B cells that don't encounter antigens die within a few weeks.
• B cell activation can occur via two pathways: thymus-dependent (TD) antigens and thymus-independent (TI) antigens.
• Activation by TD antigens requires the presence of helper T cells (CD4+).

T-dependent vs T-independent Antigens

• T-independent antigens (TI) can directly activate B cells without the help of T cells.

T-independent Antigen Types

• TI-1 antigens: Polyclonal activators of B cells. Act as mitogens.
  • Recognized by B cell receptors (BCRs) or Toll-like receptors (TLRs).
• TI-2 antigens: Activate B cells by extensively cross-linking to multiple membrane immunoglobulin receptors.

T-independent Response Characteristics

• Weak immune response: Not as strong as T-dependent responses.
• No memory cell differentiation: No long-term memory immune cells formed.
• Dominant immunoglobulin: IgM is the primary antibody produced.

B Cell Receptor Structure and Signaling

• B cell receptors (BCRs) are composed of immunoglobulin molecules, primarily IgM and IgD, which bind antigens.
• BCRs have a short cytoplasmic tail, insufficient for signal transduction.
• Signal transduction is accomplished by Igα and Igβ chains, which are associated with the BCR.
• Igα and Igβ chains have longer cytoplasmic tails containing Immunoreceptor Tyrosine-based Activation Motifs (ITAMs).
• ITAMs are crucial for initiating intracellular signaling cascades upon antigen binding.

Humoral Immune Response

• Initial exposure to an antigen triggers the primary immune response
• The primary immune response involves antibody production by plasma cells and the generation of memory B cells
• The kinetics of the primary immune response are influenced by several factors:
  • Nature of the antigen
  • Administration route (vaccine or infection route)
  • Presence or absence of an adjuvant (vaccines)
  • Species of infected or immunized individuals
• The primary immune response is characterized by a lag time
• During the lag time, the following events occur:
  • Clonal selection: B cells with specificity for foreign antigens are selected
  • Clonal expansion: The number of antigen-specific B cells increases
  • Differentiation: Plasma cells and memory cells develop
  • Antibody production: Mostly IgM is produced, with lesser amounts of IgG

Primary Immune Response

• Characterized by low antibody levels.

Secondary Humoral Immune Response

• Occurs upon subsequent exposure to the same antigen.
• Dependent on memory B and T helper cells.
• Generated rapidly compared to the primary response.
• Stronger than the primary response due to memory B and T cells.
• Lasts longer than the primary response.
• Antibodies secreted have a higher affinity to antigens.
• Characterized by higher levels of IgG than IgM.

Primary vs. Secondary Humoral Responses

• Responding B Cells:
  • Primary Response: Naive B Cells
  • Secondary Response: Memory B Cells
• Lag Time: Time between antigen administration and the start of the immune response
  • Primary Response: 4-7 days
  • Secondary Response: 3-4 days
• Peak of Response: Time when the immune response is at its highest
  • Primary Response: 7-10 days
  • Secondary Response: 3-5 days
• Magnitude of Response:
  • Primary Response: Varies depending on the antigen
  • Secondary Response: 100 to 1000 times stronger than the primary response
• Isotype and Antibody Production:
  • Primary Response: IgM is the dominant antibody
  • Secondary Response: IgG is the dominant antibody
• Type of Antigen Inducing Response:
  • Primary Response: Both T-dependent and T-independent antigens
  • Secondary Response: Primarily T-dependent antigens
• Antibody Affinity:
  • Primary Response: Low affinity
  • Secondary Response: High affinity

Sites of humoral immunity induction

• Antigens from external surfaces concentrate in lymph nodes.
• Bloodborne antigens concentrate in the spleen.

Sequence of events in humoral immune responses to T cell– dependent protein antigens

• B cells and CD4+ T cells initiate immune responses by recognizing antigens.
• Activated lymphocytes migrate towards B cells.
• B cell proliferation and differentiation occur through interactions between activated lymphocytes.

Humoral Immune Responses to T Cell–Dependent Protein Antigens

• The immune system is initiated by the recognition of antigens by B cells and CD4+ T cells.
• Activated lymphocytes migrate towards B cells, leading to B cell proliferation and differentiation.
• Helper T cell stimulation in extrafollicular sites produces short-lived plasma cells and early isotype switching.
• Activation of T cells by B cells induces follicular helper T cells.
• Late events occur in germinal centers, including:
  • Selection of high-affinity cells (affinity maturation)
  • Additional isotype switching
  • Memory B cell generation
  • Generation of long-lived plasma cells
• B cell activation requires two signals:
  • Signal 1: Antigen engagement (TCR + Ag on MHC II of B cells)
  • Signal 2: Binding of CD40 on the B cell and CD40L on the activated Th cell, inducing B7 which interacts with CD28 (co-stimulatory signals)

Germinal Centers

• Germinal centers (GC) form in secondary lymphoid tissues, like lymph nodes and spleen.
• B cells in GC are activated by specific antigens, and undergo rapid proliferation.
• GCs are essential for the production of long-lived antibody-secreting plasma cells and memory B cells.
• Plasma cells produce antibodies, which are proteins that can bind to and neutralize specific antigens.
• Memory B cells can quickly respond to reinfection with the same antigen, providing protection against reinfection.

Germinal Centers

• Germinal centers are located within the follicles of secondary lymphoid organs.
• Each germinal center has two distinct zones: a dark zone and a light zone.
• The dark zone is characterized by proliferating B cells.
• The light zone contains follicular dendritic cells which display antigens for B cell selection.

Germinal Center Reaction

• B cells activated by antigen migrate to the germinal center.
• Activated B cells undergo rapid proliferation in the dark zone.
• During proliferation, B cells undergo somatic hypermutation, allowing for affinity maturation.
• The B cells also switch their immunoglobulin isotype.
• High affinity antibody-secreting cells and memory cells exit the germinal center.

Ig class switching

• B cells undergo heavy chain isotype switching in response to different types of microbes by changing the class of antibody produced
• This occurs by recombining the VH DH JH unit with C regions of any heavy chain
• The result of this is that B cells can produce different classes of antibody with different functions
  • For example, switching from IgM to IgG leads to the production of antibodies that can activate complement or bind to Fc receptors on other cells
• Isotype switching is regulated by cytokines produced by helper T cells
  • Interleukin-4 can induce isotype switching to IgG1
  • Interferon gamma can induce isotype switching to IgG3 or IgG2a
  • TGF-beta can induce isotype switching to IgG2b
  • Interferon alpha can induce isotype switching to IgG2a
• LPS induces IL-4

Clonal Expansion

• Proliferation of T cells originating from a single T cell with a specific antigen recognition site is known as clonal expansion.
• This expansion occurs after the T cell encounters its specific antigen.

Clonal Selection

• The clonal selection hypothesis explains how the immune system generates a diverse repertoire of B cells, each with unique antigen specificity.
• B cells express receptors specific for a particular antigen before encountering it.
• Binding of the antigen to the B cell's receptor activates the cell.
• This activation triggers proliferation of the B cell, creating a clone of daughter cells with the same antigen specificity.
• These daughter cells are responsible for producing antibodies against the specific antigen that triggered their expansion.

Affinity Maturation

• Somatic mutations (point mutations, deletions, and insertions) of Ig genes occur in germinal centers leading to increased antibody affinity
• B cells with high affinity for foreign antigens are selected to survive, while those with low affinity are eliminated through clonal deletion
• Follicular dendritic cells (FDCs) play a crucial role in clonal selection by presenting antigens on their cell surface for B cell interaction
• T CD4+ cells are essential for the selection process, recognizing antigens presented by B cells (centrocytes) in the context of MHC class II
• After selection, B cells differentiate into plasma cells, which lose their BCR and produce antibodies, and memory B cells

Generation of Memory B Cells

• Memory B cells are generated during germinal center reactions, enabling rapid responses to subsequent antigen encounters
• They can survive for extended periods without continuous antigenic stimulation
• Memory B cells can reside in lymphoid organs or circulate between the blood and lymphoid organs
• Effective vaccines against microbes and their toxins must induce both affinity maturation and memory B cell formation

Antibodies

• Antibodies are proteins produced by B cells in response to antigens.
• Found in serum, milk, tears, saliva, mucus, and bile.
• Composed of four chains: two light (L) and two heavy (H) chains.
• Light chains have a molecular weight of 22,000 daltons each.
• Heavy chains have a molecular weight of 55,000 daltons each.
• L and H chains are associated by disulfide bonds and non-covalent interactions.
• Two L-H dimers associate via disulfide bonds to form a heterodimer (antibody).
• The first 110 amino acids of the L and H chains are highly variable, called the variable region (V_L and V_H).
• The region below the variable region is constant, called the constant region (C_L and C_H).
• Papain digestion of IgG produces three fragments: two Fab fragments and one Fc fragment.
• Fab fragments bind antigens.
• Fc fragment does not bind antigens but crystallizes at low temperatures.
• Two types of L chains: kappa (κ) and lambda (λ).
• Antibodies have only one type of L chain (κ or λ).
• Five types of H chains: alpha (α), gamma (γ), delta (δ), epsilon (ε), and mu (μ).
• The type of H chain determines the immunoglobulin class (IgA, IgG, IgD, IgE, or IgM).
• Immunoglobulins with different heavy chains are called isotypes.

Immunoglobulin Subclasses

• Immunoglobulin subclasses are determined by small differences in the amino acid sequences of heavy chains.
• IgG is the most abundant immunoglobulin in serum, accounting for approximately 80%.
• IgG molecules consist of two γ heavy chains and two light chains, which can be either κ or λ.
• The number of IgG subclasses varies across different animal species.
• In humans, IgG1, IgG3, and IgG4 can cross the placenta to protect the developing fetus.
• IgG3 activates the complement system, which enhances the immune response.
• IgG3 binds to Fc receptors on macrophages, promoting opsonization and phagocytosis.

What is IgA?

• IgA makes up 10-15% of total immunoglobulins in serum.
• It is the dominant immunoglobulin in body secretions like milk, tears, and mucous in mucosal linings of the respiratory, digestive, and reproductive tracts.
• IgA exists mostly as a monomer but can be found as a dimer, trimer, or tetramer.

Secretory IgA (sIgA)

• The IgA found in external secretions is a dimer called secretory IgA.
• Two units of this IgA dimer are connected by Fc, a joining chain, and a secretory component.
• Plasma cells producing secretory IgA are found in the submucosal of the mucous membranes.
• Secretory IgA is formed during translocation through epithelial cells of the mucosal.

How is Secretory IgA Formed?

• IgA dimer binds to the poly-Ig receptor on the epithelial cell surface.
• This receptor is present on the basolateral surface of many mucosal epithelia, including the digestive tract and mammary, salivary, and lacrimal glands.
• IgA is transported through endocytosis with the poly-Ig receptor across the cell to the opposite side of the cell surface (luminal side, e.g., intestines).
• The poly-Ig receptor is cleaved enzymatically and becomes part of the secretory IgA dimer.

Function of Secretory IgA

• The secretory component protects the hinge regions of IgA from digestion by proteolytic enzymes.
• High IgA levels are maintained at surfaces rich in proteolytic enzymes.
• IgA protects against pathogens (bacteria, viruses) entering the body through mucosal surfaces.
• Antigen-antibody complexes formed at these surfaces are removed by local mechanisms like ciliated epithelial cells in the respiratory tract or peristalsis in the digestive tract.

IgM Properties

• 5-10% of total immunoglobulin in serum
• Free IgM is a pentamer
• Monomeric IgM is found as a receptor on B cells
• Molecular weight: 900,000 daltons
• Five subunits arranged in a star-like fashion, connected through the Fc region and a joining chain
• 10 antigen-binding regions (Fab fragments)
• First immunoglobulin synthesized by newborns
• First immunoglobulin produced during a primary humoral immune response

IgM Function

• Can only bind 5 large antigens due to its structure
• Effective in binding antigens with repetitive epitopes (e.g., viruses, red blood cells)
• More effective in activating complement than IgG
• Two Fc fragments in close proximity are required for complement activation

IgE

• Found in small amounts in serum
• Plays a critical role in triggering allergic reactions
• Binds to Fc receptors on basophils and mast cells
• When an antigen binds to two IgE molecules, mast cell granules move to the cell surface and release their contents
• These granules contain mediators of inflammation, contributing to allergic responses

IgD

• Accounts for approximately 0.2% of total serum immunoglobulins
• Alongside IgM, IgD acts as a B cell receptor (BCR) on the surface of naive B lymphocytes
• No known biological effector function has been identified for IgD

Antigenic Determinants on Immunoglobulins

• Immunoglobulins (Ig) are themselves antigens, meaning they can induce an immune response when introduced to other species.
• This immune response produces anti-Ig antibodies which are useful for studying B lymphocyte development and for diagnostic purposes.

Categories of Antigenic Determinants

• Isotypic determinants distinguish the different isotypes of immunoglobulins.
  • These determinants are found in the constant regions of heavy and light chains.
  • Each species has different isotypes, making antibodies from one species foreign antigens to another, triggering an immune reaction.
• Allotypic determinants distinguish between different alleles of isotype genes within a single species.
  • These determinants are encoded by alleles that cause small amino acid sequence variations within isotypes.
  • Allotypic antibodies are produced by injecting immunoglobulin from one member of a species into another member of the same species.
• Idiotypic determinants are found in the variable regions of both light and heavy chains of a given antibody.
  • They determine the unique specificity of an individual antibody.

Antibody Functions

• Antibodies do not directly kill or remove pathogens.
• Antibodies bind antigens using their variable region, and interact with other cells via their constant region (Fc).
• Antibody binding to an antigen triggers effector functions in other immune cells, leading to pathogen removal.

Antibody Functions

• Antibodies bind to foreign antigens recognized within the host.
• Antibodies mediate effector functions that lead to the elimination of foreign antigens, such as infectious agents.

Opsonization

• Antibodies coat antigens, facilitating phagocytosis by macrophages and neutrophils.
• Macrophages and neutrophils have Fc receptors on their cell surfaces.
• Phagocytosis is initiated when Fc receptors engage with Fc fragments on antibodies.

Antibody-Dependent Cellular Cytotoxicity (ADCC)

• Target cells are recognized through CD16 only when antibodies are present.

Fc Receptors

• Glycoproteins found on the surface of many cells.
• Responsible for various biological functions of antibodies:
  • Induce regulatory signals that affect cell activation or inhibition.
  • Transcytosis.
  • Mediation of phagocytosis.
  • Antibody-dependent cell cytotoxicity.
• Antibodies bound to Fc receptors on cell surfaces can:
  • Induce regulatory signals that affect activation or inhibition of cells bearing the Fc receptors.
  • Participate in transcytosis (transport).
  • Mediate phagocytosis.
  • Initiate Antibody-Dependent Cell Cytotoxicity (ADCC).

Types of Fc Receptors

• Poly-Ig receptors transport IgA and IgM through epithelial cells.
• FcRn (neonatal Fc receptor) transports IgG through the placenta to the blood circulation of the fetus and regulates serum IgG levels.
• FcαR binds IgA.
• FcεR binds IgE.
• FcγR exists in multiple forms (RI, II-A, RII-B1, RII-B2, RIII) and binds IgG and its subclasses.

Monoclonal Antibodies

• Most antigens have multiple antigenic epitopes, inducing the proliferation and differentiation of distinct B cell clones producing different antibodies.
• This creates heterogeneous serum containing a mixture of antibodies specific to different antigens.
• These antibodies are called polyclonal antibodies.
• Polyclonal antibodies are suitable for various effector functions, such as mediating phagocytosis and activating complement.
• Polyclonal antibodies are not suitable for diagnostic or therapeutic procedures.
• Monoclonal antibodies are produced by a single clone and are specific for a single antigenic epitope.
• Köhler and Milstein developed a method to produce monoclonal antibodies by fusing normal plasma cells with myeloma cells.
• The hybrid cells are immortal and continuously produce antibodies of single specificity.