wk 8 - IMM2011 Humoral Immune Responses Notes

B Cell Biology

  • Adaptive humoral immunity is mediated by B lymphocytes.
  • B lymphocytes develop in the bone marrow and are morphologically identical to T lymphocytes.
  • Activated B cells differentiate into effector B cells called plasma cells.
  • Plasma cells secrete a soluble form of the B cell receptor, known as antibody, which has potent anti-microbial activity.

Innate vs. Adaptive Immunity

  • The innate immune system is critical early in infection.
  • If the innate immune system is overwhelmed, the adaptive immune system takes over.
  • Lymphocytes and antibodies are critical components of the adaptive immune system.
  • There are two types of lymphocytes:
    • B lymphocytes (bone marrow-dependent) for humoral immunity.
    • T lymphocytes (thymus-dependent) for cellular immunity.
  • Each lymphocyte clone expresses a unique receptor with specificity for an antigen.

Cells of the Adaptive Immune System

  • T and B cells are morphologically indistinguishable.
  • Upon activation, lymphocytes increase in size and enlarge their nucleus.
  • After repeated division and differentiation, lymphocytes become effector cells.
    • The effector cell of the B cell lineage is the plasma cell.

Clonal Selection

  • The adaptive immune system works via clonal selection.
  • A vast array of lymphocyte clones with unique specificities is generated during development, independent of foreign antigen.
  • In an immune response, antigen selects and expands clones of the appropriate specificity.

Memory and Specificity

  • The adaptive immune system remembers antigen.
  • Secondary responses are bigger and faster.

B Cell Receptor vs. T Cell Receptor

  • B cell receptor (BCR) is membrane-bound antibody (Ig).
  • T cell receptor (TCR) recognizes mainly peptides displayed by MHC molecules on APCs.
  • BCRs recognize macromolecules (proteins, polysaccharides, lipids, nucleic acids) and small chemicals through conformational and linear epitopes.
  • TCRs recognize linear epitopes.
  • Each clone has a unique specificity; potential for >10^9 distinct specificities for BCRs and >10^{11} for TCRs.
  • Antigen recognition is mediated by variable (V) regions of heavy and light chains of membrane Ig for BCRs and V regions of α and β chains of the TCR for TCRs.
  • Signaling functions are mediated by proteins (Igα and Igβ) associated with membrane Ig for BCRs and proteins (CD3 and ζ) associated with the TCR for TCRs.
  • Effector functions are mediated by secreted Ig for BCRs; TCR does not perform effector functions.

B Cell Receptor

  • The B cell receptor is surface-bound antibody.
  • The BCR of Naive B cells are IgM and IgD.
  • Antibody has two identical heavy chains and two identical light chains.
  • The BCR has an intracellular signaling domain that soluble antibodies lack.

CDRs and Antigen Binding

  • The CDRs (complementarity-determining regions) of the heavy and light chains come together to form the antigen-binding site.
  • Antibodies recognize "free" antigen.

Antibody Isotypes

  • B cells produce 5 different classes (isotypes) of antibody: IgA, IgD, IgE, IgG, and IgM.
  • IgA:
    • Subtypes: IgA1, 2 (α1 or α2).
    • Serum concentration: 3.5 mg/ml.
    • Serum half-life: 6 days.
    • Secreted form: Mainly dimer, also monomer, trimer.
    • Functions: Mucosal immunity.
  • IgD:
    • None subtype.
    • Trace serum concentration.
    • Serum half-life: 3 days.
    • Secreted form: Monomer.
    • Functions: Naive B cell antigen receptor.
  • IgE:
    • None subtype.
    • Serum concentration: 0.05 mg/ml.
    • Serum half-life: 2 days.
    • Secreted form: Monomer.
    • Functions: Defense against helminthic parasites, hypersensitivity.
  • IgG:
    • Subtypes: IgG1-4 (γ1, γ2, γ3, or γ4).
    • Serum concentration: 13.5 mg/ml.
    • Serum half-life: 23 days.
    • Secreted form: Monomer.
    • Functions: Opsonization, complement activation, antibody-dependent cell-mediated cytotoxicity, neonatal immunity, feedback inhibition of B cells.
  • IgM:
    • None subtype.
    • Serum concentration: 1.5 mg/ml.
    • Serum half-life: 5 days.
    • Secreted form: Pentamer.
    • Functions: Naive B cell antigen receptor (monomeric form), complement activation.

Monoclonal Antibodies

  • Monoclonal antibodies can be generated, producing unlimited amounts of antibody with a single defined specificity.

Importance of Antibodies

  • Antibodies protect us from infection with extracellular microbes.
  • Antibodies are important in viral infection by blocking binding to virus receptor and fusion event.
  • Antibodies protect us from helminth infestation, with IgE being the active antibody.
  • Most vaccines work by eliciting long-lived plasma cells and memory B cells.
  • Antibody immunodeficiencies lead to susceptibility to infection from pyogenic bacteria.

Clinical Significance of Antibodies

  • Monoclonal antibodies against T cell checkpoint inhibitory molecules promote cellular immunity in cancer patients.
  • Monoclonal antibodies against cancer antigens are used to kill cancers (e.g., Rituximab).
  • In some autoimmune diseases, B cells are autoreactive, and antibodies are pathogenic.
  • Monoclonal antibodies against proinflammatory cytokines are used to treat patients in some autoimmune diseases (e.g., Infliximab is anti-TNF).
  • Antibodies are used in diagnosis (e.g., ELISA to detect prostate-specific antigen in a blood test).
  • Antisera are used to neutralize venoms.
  • Antibodies can provoke allergic (hypersensitive) reactions.

Key Cellular Events in Humoral Immune Response

  • Primary antibody response: IgM is produced first, followed by isotype switching.
  • Secondary antibody response: IgG is produced, with plasma cells in bone marrow and memory B cells.

Phases of Humoral Immune Response

  • If no T cell help, IgM is produced.
  • With T cell help, IgG, IgA, and IgE are produced.
  • Needs T cell help for isotype switching to IgG, IgA, and IgE.
  • Activation of B lymphocytes leads to proliferation and differentiation into antibody-secreting plasma cells.
  • Helper T cells and other stimuli promote isotype switching and affinity maturation.

B Cell Receptor Signaling

  • Signaling through the B cell receptor is similar to TCR signaling.
    1. Membrane proximal events: tyrosine phosphorylation activates enzymes and creates docking sites for adaptor proteins.
    2. Formation of multimolecular signaling complexes.
    3. Common biochemical second messengers amplify the cell surface signal and transduce it to the nucleus.
    4. Activation of transcription factors leading to gene transcription.
  • The B cell receptor has a tiny cytoplasmic domain and associates with two signaling chains, Igα and Igβ, which have ITAM motifs in their cytoplasmic domain.
  • Src family kinases (Fyn, Lyn, Blk) phosphorylate the ITAMs of Igα and Igβ.
  • Syk is the tyrosine kinase that binds to the phosphorylated tyrosines of Igα and Igβ and is then phosphorylated by Fyn, Lyn, or Blk.
  • Syk then phosphorylates adaptor proteins, which activate 2nd messenger pathways.

Complement and B Cell Signaling

  • A B cell co-receptor complex consists of CR2 (Complement receptor, aka CD21), CD19, and CD81.
  • If complement is bound to antigen, CD21 signals via CD19 to lower the threshold of signaling required for B cell activation.

Downstream Consequences of B Cell Receptor Signaling

  • Antigen binding to and cross-linking of membrane Ig leads to changes in activated B cells:
    • Expression of proteins that promote survival and cell cycling.
    • Increased B7 expression.
    • Increased expression of cytokine receptors.
    • Increased expression of CCR7.
  • These changes result in increased survival, proliferation, antigen presentation, interaction with helper T cells, responsiveness to cytokines, migration from follicle to T cell zone, and generation of plasma cells, leading to antibody secretion (IgM).

T Cell-Dependent vs. T Cell-Independent Antibody Responses

  • T-dependent responses involve protein antigens and helper T cells, leading to isotype-switched, high-affinity antibodies, memory B cells, and long-lived plasma cells (IgG, IgA, IgE).
  • T-independent responses involve polysaccharide antigens, B-1 cells, marginal zone B cells, and other signals (e.g., complement protein, microbial product), leading mainly to IgM, low-affinity antibodies, and short-lived plasma cells.

T Cell-Independent Antibody Responses

  • Can be very important in immunity to capsulated bacteria by targeting the bacteria for phagocytosis.
  • Antigens that promote T-independent antibody responses are often polyvalent (e.g., polysaccharides).
  • They can cross-link the B cell receptor and induce B cell activation without the need for T cell help.
  • T-independent antigens may activate Pattern Recognition Receptors (PRRs) in B cells; PAMPs attached to antigens can costimulate B cells.
  • Signal 1 is the BCR, and Signal 2 is the PRR.

T Cell-Dependent Antibody Responses

  • Produce high affinity, isotype-switched antibodies, memory, and long-lived plasma cells.
  • Occur in secondary lymphoid organs: B cells in B cell follicles, T cells in T cell zones.

Kinetics of Humoral T-Dependent Immune Response

  • Isotype switching: First antibody produced is IgM, peaking around day 7, then diminishes as B cells switch to other isotypes (e.g., IgG).
  • Immunological memory: Primary response peaks around day 14; secondary response is greater and quicker upon re-exposure.
  • Affinity maturation: Early in the immune response, the affinity of antibody produced is low but improves with time; the affinity of antibody produced in a secondary immune response is generally higher than that produced in the primary immune response.

Secondary Responses

  • Quicker, larger, and higher affinity compared to primary responses.
  • Lag after immunization is shorter (1-3 days vs. 5-10 days).
  • Larger response.
  • Relative increase in IgG and, under certain situations, in IgA or IgE (heavy-chain isotype switching).
  • Higher average affinity (affinity maturation).

Germinal Center Reaction

  • Site of intense B cell proliferation and differentiation within the B cell follicles of secondary lymphoid organs during an adaptive immune response.
  • A feature of T cell-dependent antibody responses.
  • Follicular Dendritic cells (FDC) and T Follicular Helper cells (TFH) play a role within germinal centers.

Follicular Dendritic Cells (FDC)

  • Not conventional Dendritic cells; they are mesenchymal and not bone marrow-derived.
  • They don’t process antigen and present it on MHC.
  • Act as an antigen depot; intact antigen is stuck on the FDC surface via Fc and complement receptors.
  • Ag-specific B cells acquire antigen from FDC-displayed Ag via the BCR.

T Follicular Helper Cells

  • Another type of T helper cell found within B cell follicles.
  • Specialized in driving B cell proliferation, isotype switching, and affinity maturation.

B Cells Present Antigen to T Follicular Helper Cells

  • B cells acquire antigen via their B cell receptor (from FDC).
  • They endocytose, digest, and process the antigen.
  • Peptide fragments are presented on class II MHC to TFH.
  • TFH cell/B cell interactions are critical in T cell-dependent antibody responses.

T Cell Help Drives B Cell Proliferation and Differentiation

  • B cells present antigen to T cells on MHC II.
  • T cells stimulate B cells with CD40L and cytokines, driving B cell survival, proliferation, and differentiation.

Germinal Center Zones

  • Dark zone: B cells proliferate
  • Light zone: interactions with T helper cells and follicular dendritic cells
  • Affinity maturation and isotype switching take place within germinal centers.
  • The germinal center reaction produces long-lived plasma cells and memory B cells.

Biological Affinity

  • A thermodynamic expression of the strength of interaction between two molecules.
  • A dissociation constant is often used to describe the affinity between a ligand and a protein; the smaller the dissociation constant, the tighter the binding.

Affinity Maturation

  • Low affinity antibody is produced early in an immune response.
  • During the germinal center reaction, the enzyme AICD (Activation Induced Cytidine Deaminase) introduces somatic point mutations in the Ig V genes through a process known as somatic hypermutation.
  • B cell clones that display high affinity mutations are selected in the germinal center reaction.

Selection of High Affinity B Cells

  • Antigen is limited in the germinal center and is presented on the surface of follicular dendritic cells.
  • High affinity B cells outcompete low affinity B cells for antigen from FDC.
  • They endocytose this antigen and present it (on MHC II) to T follicular helper cells (TfH).
  • TfH provide germinal center B cells with survival and proliferation signals.
  • Low affinity B cells can't acquire antigen and therefore receive no T cell help; they die by apoptosis.

Molecular and Cellular Basis of Affinity Maturation

  • In the dark zone of the germinal center reaction, AICD introduces point mutations in the V genes of B cells, creating clones with variable affinities.
  • In the light zone, high affinity B cells outcompete low affinity B cells for antigen (from FDC) and help from TFH.
  • High Affinity B cells proliferate and differentiate into plasma cells and memory B cells.
  • Low affinity B cells die by apoptosis.

T Cell Help Drives Isotype Switching

  • B cells present antigen to T cells on MHC II.
  • T cells drive B cell isotype switching via CD40L and cytokines.

Cytokines and Isotype Switching

  • IgM is the default isotype; without T cell help (and cytokines), B cells will produce IgM.
  • IFNγ drives (some subclasses of) IgG.
  • IL-4 drives IgE.
  • TGFβ drives IgA.

Mechanism of Isotype Switching

  • Adjacent to each constant region gene is a switch region (S).
  • Upon CD40L and cytokine signaling, AICD alters nucleotides in the switch region.
  • The switch regions are then cleaved by other enzymes and joined to downstream switch regions.
  • DNA is “looped out” and lost from the genome.

Antibody Feedback

  • B cells express an inhibitory Fc receptor (FcγRIIB).
  • Late in an immune response, when there is excess antibody, FcγRIIB will recognize immune complexes and transduce signals that inhibit B cell activation.