B Cell Activation and Germinal Centers

B Cell Activation and Germinal Centres

Deakin University, Medical School. Chapters 8 & 9 Roitt’s Essential Immunology, 12th edition. Professor John Stambas.

Outline

B cell antigen binding and activation
Germinal centres
Somatic hypermutation
Isotype switching
Plasma cells and memory B cells

Stages of B cell development

Stem cell: Initial stage.
Pro B cell: VH-DJH rearrangement occurs.
Pre B cell: VL-JL rearrangement; expresses IgM.
Immature B cell: Exit bone marrow.
Mature B cell: Expresses IgD and IgM; in search of antigen.

Pro = progenitor; Pre = precursor.

Overview of B Cell Activation and Function

Newly produced B cells: Leave bone marrow and enter circulation.
Migration: Migrate through secondary lymphoid organs and survey for antigens.
Antigen recognition: Via BCR; interaction with helper T cells; clonal expansion; isotype switching.
Low-affinity Plasma cells: Antibody production (occurs within a few days).
Germinal Center formation: Somatic mutation and affinity maturation (occurs within 1-2 weeks).
High-affinity Plasma cells: Produce antibody and memory B cells. This is an ongoing process that takes weeks.

Structure of Surface Ig on the B Cell

B cell receptor (BCR) consists of surface immunoglobulin (Ig).

Complementarity Determining Regions (CDRs)

CDRs on B cells are involved in antigen binding, similar to T cell receptor CDR regions.
Areas of high variability to allow for antigen recognition

T Dependent vs T Independent B Cell Antigens

T-dependent

Antigen Type: Protein antigens (majority of responses).
Signal Model: Requires a 2-signal model:
- Signal 1: Engagement of antigen receptor (BCR).
- Signal 2: Co-stimulatory signal via CD40-CD40L ligation and cytokine secretion.

T-independent

Antigen Type: LPS or carbohydrates.
Antibody Class: Mostly IgM.
Affinity: Modest.
Memory: No memory.
Activation: Direct BCR crosslinking.
Second Signal: Cytokines or TLRs.

T Dependent vs T Independent B Cell Responses

T-independent antigen activates B cells by direct BCR aggregation.

Recirculating B Cells

B cells normally pass through lymphoid organs (in the absence of antigen).
B cells in the blood enter through high endothelial venules (HEV).
They pass through the T cell area to get to the B cell area.

HEV and B Cell Trapping

Antigen-loaded dendritic cells migrate from the subcapsular sinus to the paracortical area of the lymph node.
T cells migrate through HEV and are trapped by antigen on DCs.
T cells proliferate.
B cells migrate through HEV; most pass through if there's no antigen.
Some B cells see antigen in the T cell area, get trapped, interact with T cells, proliferate, and migrate to the primary follicle.
Forms a germinal center

Recirculating B cells are trapped by foreign antigens in lymphoid organs.

Germinal Centre

Formation: B cells proliferate rapidly.
Structure: Transient structure of intense B cell proliferation.
Function: Releases B cells that differentiate into plasma cells.
B cells leave blood & enter lymph node via high endothelial venules.

B Cell Activation and Signaling

Ig-α and Ig-β chains: Include ITAMs that become phosphorylated on tyrosine residues.
Function of phosphorylated ITAMs: Act as docking sites for other proteins, including tyrosine kinases that initiate signaling cascades.
Signaling also requires BCR co-receptor.

B Cell Signaling Cascade

Signaling cascade downstream of antigen-driven B-cell receptor (BCR) ligation involves the BCR co-receptor:

BCR co-receptor components: CD121 (or CR2, complement receptor that binds complement-bound antigen), CD19, and CD181.
Process: Antigen binding via surface Ig and co-receptor leads to phosphorylation and activation via ITAMs, which signals to the nucleus for proliferation and survival.

T Cell Dependent B Cell Response

Signal 1 to B cell: Antigen binding to BCR.
Antigen processing: Antigen is internalized, processed, and antigenic peptides are displayed on MHC II for CD4 TH cell recognition.
Signal 1 to T cell: TH (helper T cell) recognizes antigen-MHC complex via the T cell antigen receptor (TCR).
Signal 2 to T cell: B7 on B cell binds to CD28 on T cell.
Signal 2 to B cell: T cell activation leads to up-regulation of CD40L which binds to CD40.
Activation: Cytokine production by activated T cell also helps to activate B cells.
Outcome: B cell proliferates and differentiates into antibody-secreting B cell (plasma cell).

Lymph Node and Germinal Center

Germinal centers are B cell areas within secondary lymphoid organs.
T cell areas are also present.
Recirculating lymphocytes are looking for antigen.

Germinal Centers Overview

Function: Germinal centers are sites within secondary lymphoid organs where mature B lymphocytes proliferate, differentiate, increase affinity of 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.

Germinal Centre Differentiation

Differentiation of B cells in the germinal center leads to increasing maturation.

Stages of B Cell Activation in the Germinal Centre

Events occurring in lymphoid germinal centers.

What Happens in the Germinal Center?

Affinity Maturation.

Affinity Maturation

Affinity maturation: the increase in the average affinity (binding) of 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.

Affinity Maturation: Mutations and Binding

Mutations are targeted to antigen-binding region of antibody CDR.
CDR = complementarity-determining region, also known as the hypervariable region (part of the V domain that binds the antigen).
Affinity ("strength of binding") maturation improves the ‘fit’ of the antibody for the antigen - increasing the binding affinity

Somatic Hypermutation

Somatic Hypermutation in variable region CDRs increases over time and following re-exposure to antigen.

Somatic Hypermutation via AID

AID (Activation-induced cytidine deaminase) dependent mutator complex causes DNA replication error.
AID deaminates Cytosine turning it into uracil (which is recognized as a thymine).
Repair enzymes recognize Uracil as Thymine, come in and this leads to error prone repair.

Somatic Hypermutation: Altered Antigen Binding

Now encodes antibody molecule with slightly altered antigen binding site. Sometimes, by chance, this site will have an improved ability to bind the inducing antigen (i.e. a higher affinity).

Somatic Hypermutation Increases Affinity

Darwinian selection by antigen of B-cells with antibody mutants of high affinity

Affinity Maturation and Selection

Occurs in the germinal centre.

Somatic Hypermutation Overview

Occurs within germinal centers of secondary lymphoid organs.
Hypermutation mechanism generates point mutants in variable regions especially CDRs.
B cells undergoing rapid cell division.
B cells with best affinity survive, divide and differentiate.
B cells that can’t compete die by apoptosis.

What Else Happens in the Germinal Center?

Isotype Switching

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.

Isotype Switching Details

IgM is the first Ab that is 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 switch sequence (except δ).
Regulated by cytokines secreted by T cells.

Class-Switch Recombination (CSR)

Other Ig isotypes (IgG, IgA, IgE) are generated by a second type of somatic recombination called Class-Switch Recombination (CSR).

A “Switch site” is located 5’ to each CH segment and targets the recombination machinery.
Once a B cell has switched to make IgG, it can no longer make IgM (remember IgM found on surface of mature naïve B cell).
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.

Isotype Switching Visualized

Switching from IgM to IgA: Variable region stays the same, constant region changes.

What Drives Class Switching?

T cells can determine the type of Ig produced by a B cell by the type of cytokines they secrete.

Plasma Cell Development

After appropriate activation and differentiation, the B cell leaves the GC and secretes antibody (or Plasma Cell).

Plasma cells develop in secondary lymphoid organs then home to the bone marrow or mucosal surfaces continually secreting antibody

Mature B Cell vs Plasma Cells

Displays the differences between mature B cells and plasma cells.

Memory B Cells

Generated in germinal centers – therefore we have 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.