Humoral Immune Response - Antibodies and Their Functions

The Humoral Immune Response

• Characterized by the action of immune complexes in the extra-cellular fluid.
• Also called “antibody-mediated immunity”.
• Involves the activation of B cells and secretion of antibodies.
• Most vaccines rely on humoral immunity to induce long-lasting antibody responses that block pathogens from establishing an infection.
• Plays a critical role in allergy (mediated by IgE) and some autoimmune diseases (mediated by IgG).
• Often deficient in patients with severe immunodeficiency, such as Common Variable Immunodeficiencies (CVID) and HIV.

B Cells

• Naïve B cells require “help” from CD4+ T helper cells for full activation.
• Present antigen as a peptide bound to MHC II (Major histocompatibility complex II).
• Activate CD4+ helper T cells, which in turn stimulate the B cell.
• Plasma cells/activated B cells secrete antibodies, leading to:
  • Neutralization
  • Opsonization
  • Complement activation
• Memory B cells provide a long-lived response.

Antigen Specificity

• Antigen: a toxin or foreign substance that induces an immune response.
• Specificity: the ability to bind one, but not another member of a family of related substances.

B Cells and Antibodies

• Each B cell is specific for one antigen, targeting a specific region on a protein.
• Antigens are recognized by Immunoglobulin (Ig) molecules.
• One B cell expresses one Ig sequence.
• Surface-bound Ig on B cells is called the B cell receptor (BCR).
• Soluble secreted Ig is called an antibody.

Structure of an Immunoglobulin Molecule

• Immunoglobulin molecule:
  • N terminus: variable region that binds to antigen.
  • C terminus: constant region that binds to Fc receptors and mediates Ig functions.
  • Composed of 2x heavy chains and 2x light chains, joined by disulfide bonds.

Antigen Binding Sites

• Immunoglobulin molecules bind regions on the protein.
• Regions can take a variety of shapes.
• Antibodies recognize conformational shapes on proteins.

Diversity in Antigen Receptors

• Made from a combination of immunoglobulin gene segments:
  • Variable (V)
  • Diversity (D)
  • Junctional (J)
  • Constant (C)
• During B cell development, these gene elements rearrange.
• The diversity region is in contact with the antigen.
• Different combinations of these genes enable diversity, with $5 \times 10^{13}$ possible combinations.

B Cell Development

• Lymphocytes develop ‘immunocompetence’ in the primary lymphoid organs.
• B-cells mature in the bone marrow.
  • Max Cooper showed in the 1960s that removal of the Bursa of Fabricius in chickens inhibited antibody production.
• Rearrangement of Ig gene elements to generate a B Cell Receptor (VDJ recombination).
• Any self-reactive cell is deleted in the development process (Negative selection).
• Immature B cells leave the bone marrow ready to scan the body.

Negative Selection

• The immune system needs to respond to foreign proteins but not self.
• Self/non-self-discrimination is important to delete any B cells that are specific for a self-protein.
• An array of self-antigens is expressed in the bone marrow.

Activation of B Cell/Antibody Response

• The B cell receptor (surface-bound immunoglobulin) binds to the antigen.
• The B cell presents a peptide to an antigen-specific CD4+ T helper cell via MHC II.
• CD4+ T cells provide co-stimulation to the B cell.
• B cell activation induces proliferation and differentiation into antibody-secreting plasma cells and memory cells.
• Microbial constituents, such as bacterial polysaccharides, can stimulate B cells independently of T cells.

T Cells Activate B Cells

• T cells activate B cells via membrane-bound and secreted molecules.
  • Membrane-bound: CD40 (co-stimulation) → B cell activation
  • Soluble: cytokines → B cell differentiation

B & T Cell Activation

• Occurs in secondary lymphoid organs (lymph nodes and spleens).
• B and T cells interact at border zones (B-cell follicle and T cell zone).

B Cell Response Refinement in Germinal Center

• Once activated, B cells migrate into follicles and form a germinal center where immunoglobulin modifications occur, supported by accessory immune cells.
• Somatic hypermutation (Ig gene editing) takes place to improve antibody affinity (binding strength).
• Class-switching to generate different immunoglobulin isotypes (different functional roles).
• Selection of high-affinity B cells ensures the response is faster and more effective on the 2nd exposure to the antigen.

Germinal Centers

• Generation of memory B cells.
• Generation of high-affinity plasma cells.
• Antibody-secreting plasma cells migrate and then reside in the bone marrow under homeostatic conditions.

B Cells Summary

• Each expresses a unique immunoglobulin molecule = antigen-specific.
• Composed of a combination of Ig genes (VDJ and constant region).
• Produce antibodies (secreted immunoglobulin molecules).
• APCs that express MHC II.
• Activate CD4+ T cells and receive co-stimulation.
• Need “help” from CD4+ T cells for full activation.
• Activation leads to the generation of plasma and memory cells.
• Mediate the humoral immune response.

Antibodies Functions

• Neutralize pathogens (neutralization) by binding to the surface and blocking entry into cells.
• Opsonize pathogens (opsonization) by coating pathogens with antibodies and targeting them for destruction by other immune cells (e.g., macrophages, NK cells, mast cells, and eosinophils).
• Activate the complement pathway (complement activation).
• Functions are performed by the constant region on the immunoglobulin.
• Variety of immunoglobulin isotypes that perform these functions.
• Same variable region but different constant region.

Immunoglobulin Isotypes

• Isotypes are determined by the constant region on the heavy chain.
• Types include: IgA (IgA1, IgA2), IgD, IgE, IgG (IgG1, IgG2a, IgG2b, IgG3, IgG4), IgM.
• IgD and IgM can be expressed as surface-bound receptors on naïve B cells.
• Somatic recombination (during B cell differentiation) leads to class switching and production of different Ig isotypes.

Role of Cytokines

• Different cytokines induce switching to a different isotype.
  • IL-4: Induces IgG1, IgE, inhibits IgM, IgG3, IgG2b.
  • IL-5: Augments IgA production.
  • IFN-γ: Induces IgG2a, inhibits IgG1.
  • TGF-β: Induces IgA, inhibits IgM, IgG3, IgG1, IgG2b.

Immunoglobulin Isotypes Characteristics

Tissue Tropism of Immunoglobulin Isotypes

• IgG: Systemic
• IgA: Mucosal surfaces
• IgE: Mucosal surfaces and skin

Human Immunoglobulin Class Functions and Distribution

The Humoral Immune Response: Neutralization, Opsonization, Complement Activation

• Activation of B-cells by antigen and helper T cells (CD40/CD40L interaction).
• Antibody secretion by plasma cells.
• Neutralization: Antibodies bind to toxins, viruses, and bacteria, preventing bacterial adherence.
• Opsonization: Antibodies promote phagocytosis.
• Complement activation: Antibodies activate complement, which enhances opsonization and lyses some bacteria.

Neutralization: Bacterial/Parasitic Toxins

• Pre-existing antibody is necessary as it takes a while to initiate adaptive immune responses. Need to respond to toxins quickly.

Neutralization: Gut Homeostasis

• Dimeric IgA is transported into the gut lumen through epithelial cells at the base of the crypts.
• Dimeric IgA binds to the layer of mucus overlying the gut epithelium.
• IgA in the gut neutralizes pathogens and their toxins.

Neutralization: Viral Entry

• Pre-existing antibody is necessary. Basis for some vaccinations against viruses.
• Antibody blocks binding to the virus receptor and can also block the fusion event.

Neutralization: Intracellular Bacterial Entry

• Atypical bacteria examples include Chlamydia and Mycoplasma species.
• Antibodies prevent the attachment of bacteria to the cell surface.

Common Diseases Caused by Bacterial Toxins

• Childhood vaccines are available for some of these diseases.

Antibody-Dependent Cellular Cytotoxicity (ADCC)

• IgG coated target cells activate Fc receptors on NK cells, releasing membrane- puncturing granules such as granzyme and perforin.

IgE-Mediated Degranulation

• Mast cells and basophils bind IgE via high affinity Fc epsilon receptor.
• Antigen binds and crosslinks IgE, leading to:
  • Anti-parasitic response
  • Type I Hypersensitivity (e.g., asthma and allergy)

Opsonization

• Antibodies bound to the pathogen are recognized by phagocytic cells, such as macrophages.

Opsonisation Definition

• Coating of a pathogen by antibodies and/or complement proteins to facilitate phagocytosis and destruction of the pathogen.

Opsonisation Process

• Opsonisation can occur through IgG:Fc binding or C3b:CR1 binding.
• Bacterium is coated with complement and IgG antibody.
• When C3b binds to CR1 and antibody binds to the Fc receptor, bacteria are phagocytosed.
• Macrophage membranes fuse, creating a membrane-enclosed vesicle, the phagosome.
• Lysosomes fuse with these vesicles, delivering enzymes that degrade the bacteria.

Complement Activation

• Complements enhance antibody and phagocytes to clear pathogens (antigen) from an organism.

Complement System

• Component of the innate immune system that “complements the action of antibodies”.
• A collection of plasma proteins (>30).
• Many are pro-enzymes (Zymogens) that are activated by proteolysis.
• Activation causes a cascade of reactions leading to the destruction of pathogens.
• Needs to be tightly regulated to prevent host damage.

Complement Pathways

• Classical pathway: Activated by antibodies coating the target cell.
• Lectin pathway: Activated by lectins binding to specific sugars on the microorganism's surface.
• Alternative pathway: Activated spontaneously; a lack of inhibitors on the microorganism's surface allows the process to proceed.
• All pathways lead to C3 convertase formation and C3 cleavage.

Complement System Activation

• Complement proteins recognize features of microbial surfaces and mark them for destruction.
  • Lectin-pathway: Recognizes carbohydrates.
  • Classical pathway: C1q interacts with the pathogen surface or binds to surface-bound antibody.
  • Alternative pathway: Spontaneous C3 hydrolysis.
• All pathways lead to cleavage of C3.
  • C3b bound to the microbial surface (covalent).
  • C3a is soluble and helps induce inflammation.

Complement System: Three Functions

• Some complement proteins initiate inflammation (e.g., C3a, C5a).
• Phagocytes have complement receptors (CR) and can bind “flagged” pathogens (i.e., opsonized).
  • C3b can bind to CR1.
• Formation of the membrane-attack complex (MAC) that can lyse certain pathogens.

Antibody:Antigen Complexes Activate Complement

• Complement activation is initiated when antibodies are attached to the surface of a pathogen via C1q.
• C1q cannot bind IgM or IgG in serum.
• IgM must have a conformational change to expose binding sites.
• IgG molecules must be adjacent to create 2 binding sites and sufficient affinity for C1q.

Antigen:Antibody Complexes

• Too few molecules of IgG to efficiently bind to Fc receptors.
• Antigen can be coated with complement and transported to relevant organs (e.g., spleen) for destruction.
• Can lead to insoluble immune complex deposition and complement activation, leading to chronic inflammation in autoimmune diseases and serum sickness (Type III hypersensitivity).
• Complement receptors are important in the removal of immune complexes from the circulation.

Summary: Complement

• Complement proteins recognize features of microbial surfaces and mark them for destruction.
• All pathways lead to cleavage of C3.
• Complement function:
  • Initiate inflammation (e.g., C3a, C5a).
  • Facilitate phagocytosis.
  • Formation of the membrane-attack complex (MAC).

Summary

• B cells present antigen to CD4+ Helper T cells to ensure full activation.
• Proliferation and differentiation into plasma or memory B cell.
• Cytokines released by CD4+ T cells promote isotype class switching.
• Germinal center reactions ensure maturation of the B cell response (higher affinity antibodies and isotype class switching).
• Activated B cells secrete antibodies to mediate effector functions, including:
  • Neutralization
  • Opsonization
  • Activation of the complement pathway