B Cell Activation and Germinal Centres

Overview

B Cell Development:
- B cells develop in stages, starting in the bone marrow.
- Stages include stem cell, pro B cell, pre B cell, immature B cell, and mature B cell.
- Gene rearrangement events: VH-DJH and VL-JL.
- Mature B cells express IgM and IgD and exit the bone marrow in search of antigens.
B Cell Activation and Function:
- Newly produced B cells migrate through secondary lymphoid organs, surveying for antigens.
- Antigen recognition via the B cell receptor (BCR).
- Interaction with helper T cells.
- Clonal expansion and isotype switching.
- Formation of germinal centers for somatic mutation and affinity maturation.
- Differentiation into plasma cells (antibody production) and memory B cells.

B Cell Antigen Binding and Activation

Structure of Surface Ig on the B Cell: B cells express surface immunoglobulin (Ig) as their antigen receptor.
Complementarity Determining Regions (CDRs):
- CDRs are regions of high variability within the variable regions of Ig chains.
- Involved in antigen binding, similar to CDR regions on T cell receptors.
- CDR1, CDR2, and CDR3 on both heavy and light chains contribute to antigen specificity.
T-dependent vs. T-independent Antigens:
- T-dependent Antigens:
  - Typically protein antigens.
  - Require two signals for B cell activation: BCR engagement (signal 1) and co-stimulatory signal (signal 2).
  - CD40-CD40L ligation and cytokine secretion provide the co-stimulatory signal.
- T-independent Antigens:
  - Examples include LPS or carbohydrates.
  - Primarily induce IgM production.
  - Result in modest affinity antibodies and no memory B cells.
  - Activate B cells by direct BCR crosslinking.
  - Second signal from cytokines or TLRs.
Recirculation of B Cells through Lymphoid Organs:
- B cells recirculate through lymphoid organs, entering via high endothelial venules (HEV).
- Within the lymph node:
  - Antigen-loaded dendritic cells migrate to the paracortical area.
  - T cells migrate through HEV and are trapped by antigen on dendritic cells.
  - B cells migrate through HEV, and some are trapped by antigen in the T cell area.
  - Interaction between T cells and B cells leads to B cell proliferation and migration to the primary follicle.
  - Germinal centers form as a result of this process.
Germinal Centre Formation:
- Antigen enters the lymph node via afferent lymphatic vessels.
- B cells proliferate rapidly within the germinal center.
- Germinal centers are transient structures characterized by intense B cell proliferation.
- B cells differentiate into plasma cells within the germinal center.
B Cell Activation and Signaling:
- Following antigen encounter, Ig-alpha and Ig-beta chains, which include ITAMs (immunoreceptor tyrosine-based activation motifs), become phosphorylated on tyrosine residues.
- These phosphorylated ITAMs act as docking sites for tyrosine kinases that initiate a signaling cascade.
BCR Co-receptor:
- B cell signaling involves BCR co-receptors such as CD21 (or CR2, complement receptor that binds complement-bound antigen), CD19, and CD81.
- Antigen binding via surface Ig and co-receptor leads to phosphorylation and activation via ITAMs, which then signal to the nucleus for proliferation and survival.
T Cell-Dependent B Cell Response: Sequence of Events
- Antigen binding to BCR provides signal 1 to the B cell.
- Antigen is internalized, processed, and antigenic peptides are displayed on MHC II for CD4 TH cell recognition.
- TH cell recognizes antigen-MHC complex via the T cell antigen receptor (TCR), providing signal 1 to the T cell.
- B7 on the B cell binding to CD28 on the T cell provides signal 2 to the T cell.
- T cell activation leads to upregulation of CD40L, which binds to CD40, providing signal 2 to the B cell.
- Cytokine production by the activated T cell also helps to activate B cells.
- B cell proliferates and differentiates into an antibody-secreting B cell (plasma cell).

Germinal Centres

Definition: Germinal centers are sites within secondary lymphoid organs where mature B lymphocytes proliferate, differentiate, increase the affinity of their antibodies (through somatic hypermutation), and switch the class of their antibodies during a normal immune response.
Development: They develop dynamically after the activation of B-cells by T-dependent antigen.
Events occurring in lymphoid germinal centers: Differentiation and increasing maturation of B cells.

Affinity Maturation

Affinity maturation is the increase in the average affinity (binding strength) of an antibody for its antigen.
Occurs in germinal centers and is the result of:
- Somatic hypermutation of Ig-genes in dividing B cells.
- Selection of B cells for their ability to bind more strongly (with higher affinity) to the inducing antigen via follicular dendritic cells.
The high-affinity B cells emerging in germinal centers give rise to long-lived plasma cells and memory B cells.
Mutations are targeted to the antigen-binding region of the antibody.
CDR (complementarity-determining region) is also known as the hypervariable region (part of the V domain that binds the antigen).
Affinity improves the 'fit' of the antibody for the antigen, increasing the binding affinity.
Somatic hypermutation in variable region CDRs increases over time and following re-exposure to antigen.

Somatic Hypermutation

Somatic hypermutation is AID-dependent.
AID (Activation-induced cytidine deaminase) deaminates Cytosine, turning it into uracil (which is recognized as thymine).
Repair enzymes are recruited, leading to error-prone repair.
Somatic hypermutation increases affinity. Darwinian selection by antigen of B-cells with antibody mutants of high affinity.
Occurs within germinal centers of secondary lymphoid organs.
The hypermutation mechanism generates point mutants in variable regions, especially CDRs.
B cells undergo rapid cell division.
B cells with the best affinity survive, divide, and differentiate.
B cells that can't compete die by apoptosis.

Isotype Switching

Isotype switching is the mechanism by which antibodies are generated with the same specificity but of a different isotype (class of antibody), e.g., IgM, IgG, IgA, IgE. This enables antibodies to perform different functions.
Constant region encodes antibody class.
Variable region binds antigen.
IgM is typically the first antibody secreted.
IgM is pentameric, and each H chain can bind complement proteins.
Isotypes with other effector functions are produced by activated B cells (IgG, IgA, IgE, IgD).
Rearrangement of DNA using SWITCH regions - all C genes preceded by the switch sequence (except d).
Regulated by cytokines secreted by T cells.
Other Ig isotypes (IgG, IgA, IgE) are generated by a second type of somatic recombination called Class-Switch Recombination (CSR).
A Switch site located 5' to each CH segment targets the recombination machinery.
Once a B cell has switched to make IgG, it can no longer make IgM (remember IgM found on the surface of mature naïve B cells).
DNA is nicked and broken at two selected Switch-regions by the activity of a series of enzymes, including Activation-Induced (Cytidine) Deaminase (AID), uracil DNA glycosylase, and apyrimidic/apurinic (AP)-endonucleases.
Variable region stays the same, and the constant region changes during switching from IgM to IgA.
T cells can determine the type of Ig produced by B cells by the type of cytokines they secrete.

Plasma Cells

After appropriate activation and differentiation, the B cell leaves the GC and secretes antibody (or becomes a Plasma Cell).
Plasma cells develop in secondary lymphoid organs then home to the bone marrow or mucosal surfaces, continually secreting antibodies.
Plasma cells contain prominent rough ER.

Memory B Cells

Generated in germinal centers (strong humoral memory to T-dependent antigens).
Small, recirculating cells.
Often isotype switched (e.g., IgG+ or IgA+).
Typically have higher affinity for the inducing Ag.
Longer-lived – Persistence of memory B cells after antigen exposure ensures that we have increased numbers of B cells specific for the antigen and ready to respond on re-encounter.
e.g., memory B cells found circulating >50 years after smallpox vaccination.

Learning Objectives

Recall the structure of immunoglobulin.
Describe the process of B cell activation and the signalling events involved.
Understand T-dependent activation and function of B cells.
Explain germinal centre development and the processes of affinity maturation, somatic hypermutation, and class switching that occur within the germinal centre following T-dependent B cell activation.
Describe the function of plasma cells and memory cells.