The Humoral Immune Response – Antibodies and their functions

Antigen Recognition by B Cells

  • B cells recognize antigens via surface-bound immunoglobulins (Ig), known as the B cell receptor (BCR).
  • Each B cell is specific for one antigen, recognizing a particular region on a protein.
  • Immunoglobulin molecules bind to conformational shapes on proteins.

B Cell Development and Differentiation

  • B cells develop immunocompetence in primary lymphoid organs, specifically the bone marrow.
  • During B cell development, immunoglobulin gene segments rearrange through V(D)J recombination to generate a unique B Cell Receptor (BCR).
  • Self-reactive B cells are deleted during development through negative selection to prevent autoimmunity.
  • Naïve B cells require help from CD4+ T helper cells for full activation.
  • Activation induces proliferation and differentiation into antibody-secreting plasma cells and memory cells.
  • B cell presents peptide to antigen-specific CD4+ T helper cell via MHC II.
  • CD4+ T cells provide co-stimulation to the B cell.

Structure and Function of Immunoglobulins

  • An immunoglobulin molecule consists of two heavy chains and two light chains, joined by disulfide bonds.
  • The N-terminus is the variable region that binds to the antigen, while the C-terminus is the constant region that binds to Fc receptors and mediates Ig functions.

Immunoglobulin Gene Segments

  • Immunoglobulin gene segments are made from a combination of:
    • 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 enables diversity resulting in 5 \times 10^{13} possible combinations.

B Cell Development

  • Lymphocytes develop ‘immunocompetence’ in the primary lymphoid organs.
  • B-cells mature in the bone marrow.
  • 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 cell leave bone marrow ready to scan the body.

Negative Selection

  • Immune system needs to respond to foreign proteins but not self.
  • Self/non-self discrimination.
  • Important to delete any B cells that are specific for a self-protein.

Activation of B Cell/Antibody Response

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

T Cell Activation of 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

  • B & T cell activation occurs in secondary lymphoid organs such as Lymph Nodes and spleens.
  • B and T cells interact at border zones (B-cell follicle and T cell zone).

B Cell Response Refinement

  • 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 a faster and more effective response on 2nd exposure to antigen.

Germinal Centers

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

Antibody Functions

  • Antibodies perform a variety of functions, including neutralization, opsonization, and complement activation.
  • Functions are performed by the constant region on 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:
    • 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

  • Cytokines, provided by CD4+ T cells, induce switching to different isotypes.
    • IL-4 induces IgG1 and IgE, and inhibits IgM, IgG3, IgG2b
    • IL-5 induces IgA
    • IFN-γ induces IgG2a and inhibits IgG3, IgG1, IgE
    • TGF-β induces IgG2b and IgA, and inhibits IgM, IgG3, IgG1, IgG2a, IgE

Immunoglobulin Isotypes and Triggered Responses

  • Different immunoglobulin isotypes trigger different responses due to the pattern of Fc receptor expression across cell types.
  • IgM: Activates complement
  • IgG1 and IgG3: Opsonization
  • IgE: Sensitization of mast cells
  • IgA: Neutralization

Humoral Immune Response

  • The humoral immune response involves B-cell activation by antigen and helper T cells.
  • Antibodies bind to toxins, viruses, and bacteria, leading to neutralization, opsonization, and complement activation.
  • Neutralization prevents bacterial adherence.
  • Opsonization promotes phagocytosis.
  • Complement activation enhances opsonization and lyses some bacteria.

Neutralization of Bacterial/Parasitic Toxins

  • Pre-existing antibody is necessary.
  • Takes a while to initiate the adaptive immune responses – need to respond to toxins quickly.
  • Examples of diseases caused by bacterial toxins include tetanus, diphtheria, gas gangrene, cholera, anthrax, botulism, pertussis, scarlet fever, and food poisoning.

Neutralization of Viral Entry

  • Pre-existing antibody is necessary.
  • Basis for some vaccinations against viruses (e.g., COVID vaccines induce antibodies to spike protein).

Neutralization of Intracellular Bacterial Entry

  • Effective against atypical bacteria such as Chlamydia and mycoplasma species.
  • Antibodies prevent the attachment of bacteria to the cell surface.

Antibody-Dependent Cellular Cytotoxicity (ADCC)

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

IgE-Mediated Degranulation

  • Mast cells and basophils bind IgE via high-affinity Fc epsilon receptors.
  • Antigen binds to mast cell-bound IgE, resulting in crosslinking of IgE.
  • Release of mediators by mast cells.
  • Involved in anti-parasitic responses and Type I Hypersensitivity (e.g., asthma and allergy).

Opsonization

  • Antibodies bound to the pathogen bind to receptors on phagocytic cells (macrophages), triggering phagocytosis.
  • Coating of a pathogen by antibodies and/or complement proteins facilitates phagocytosis and destruction of the pathogen.
  • Opsonization can occur through IgG:Fc binding or C3b:CR1 binding.

Complement Activation

  • Complements enhances activation of phagocytes and pathogen uptake.

Complement System

  • Component of the innate immune system that complements the action of antibodies.
  • 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 System Pathways

  • The complement system has three pathways:
    • Classical pathway: C1q interacts with the pathogen surface or with antibodies bound to the surface.
    • Alternative pathway: C3 undergoes spontaneous hydrolysis to C3(H2O) to initiate eventual deposition of C3 convertase on microbial surfaces.
    • Lectin pathway: Mannose-binding lectin (MBL) and ficolins recognize and bind carbohydrates on the pathogen surface.

Complement System 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 a membrane-attack complex (MAC) that can lyse certain pathogens.
  • Complement activation is initiated when antibodies attached to the surface of a pathogen bind C1q.
  • C1q cannot bind IgM or IgG in serum.
  • IgM must undergo a conformational change to expose binding sites.
  • IgG molecules must be adjacent to create 2 binding sites and sufficient affinity for C1q.

Antibody:antigen complexes

  • Antibody:antigen complexes are 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.