L3- Antibody Structure, B cell Receptors and B cell development

Immunology is defined as understanding how the body distinguishes self from non-self.

Objectives

• Describe the detailed structure of an antibody molecule.

• Understand how binding specificity is achieved through amino acid variability.

• Appreciate the fundamental differences between the five antibody classes.

• Explain how immunocompetent B cells are produced.

• Understand how the immune system regulates against auto reactive B cells.

• Detail how the B cells are activated by antigens.

• Discuss how plasma and memory B cells are produced.

Recap: Antigens and Epitopes

The immune system recognizes 'Antigens' of the pathogen, which can be:

• Epitopes on proteins

• Peptide sequences from proteins

• Polysaccharides

• Lipids

Action as an antigen depends on:

• Foreignness

• Molecular size

• Chemical composition and complexity

• Processing pathways

B Cell Antigens

An antigen is the whole protein that elicits a response. Epitopes are the specific areas on the protein that the adaptive immune antibody or T cell receptor binds to. For antibodies, these are located on the protein surface.
Epitopes are not necessarily linear sequences.

Elucidation of Antibody Structure (1959-1960)

• Rodney Porter (1959-1960):

  • Digested immunoglobulin with papain.

  • Obtained two identical fragments of MW 45,000 and one of MW 50,000.

• Gerald Edelman (1959-1960):

  • Treated immunoglobulin with b-mercaptoethanol.

  • IgG fell apart into four chains: two identical with MW 53kDa each and two others also identical with MW 22kDa.

• Fab fragments: antigen binding

• Fc fragment: crystallised in cold storage

• Light (L) chains

• Heavy (H) chains
  An antibody molecule consists of two identical H chains and two identical L chains linked together by disulphide bonds.

• Fab consists of an L chain plus part of the H chain - ANTIGEN BINDING

• Fc contains only H chain - BIOLOGICAL FUNCTION
  *Porter and Edelman shared the Nobel Prize in 1972 for their work elucidating antibody structure.

Antibody Structure

The H and L chains are held together by interchain disulphide bonds (and reversible bonding).
The H and L chains also have intrachain disulphide bonds which creates IMMUNOGLOBULIN- FOLD domains
Abs possess a HINGE region containing mostly proline and cysteine residues. This permits flexibility between the two Fab arms
Immunoglobulins are glycoproteins. They also possess carbohydrate groups which enhance solubility and modulate their biological functions.

Light Chains

• VARIABLE (VL) REGION: first 100-110 residues

• CONSTANT (CL) REGION: two basic amino acid sequences and therefore two light chain types kappa or lambda
  A single Ab can contain either kappa or lamba chains, never both

Heavy Chains

• VARIABLE (VH) REGION: first 100-110 residues

• CONSTANT (CH) REGIONS

  • Five basic amino acid sequences and therefore five heavy chain types: gamma (\gamma), mu (\mu), alpha (\alpha), epsilon (\epsilon), delta (\delta)

  • Each of the five different heavy chains is called an ISOTYPE

Classes of Antibodies

Antiserum to the constant region of the heavy chain identifies five distinct classes of antibody called isotypes.

• IgA: alpha (\alpha) heavy chain

• IgD: delta (\delta) heavy chain

• IgE: epsilon (\epsilon) heavy chain

• IgG: gamma (\gamma) heavy chain

• IgM: mu (\mu) heavy chain

• Light-chain isotypes are:

  • kappa (\kappa)

  • lambda (\lambda)

Subclasses of IgG

Further diversity is added through the use of subclasses for IgG.
The amino acid differences between subclasses of IgG affect their biological activity, e.g. in opsonisation, complement activation, etc.

Hypervariable Regions

When the amino acid sequences of the variable regions (of both light and heavy chains) are compared, THREE regions show the greatest amount of variability. These are called HYPERVARIABLE regions.

• Light Chain Variable Domain (VL)

• Heavy Chain Variable Domain (VH)

Hypervariable Regions (2)

THREE HYPERVARIABLE REGIONS

• Participate in the binding to an antigen

• Called COMPLEMENTARITY DETERMINING REGIONs (CDRs)

• FRAMEWORK REGIONS

  • Provide the ‘scaffold’ of the immunoglobulin fold

Complementarity Determining Regions (CDRs)

The CDR regions of both the heavy and the light chains are brought together in the intact antibody molecule.
It is five or six CDRs in close proximity that form the antigen-binding site
The variability in the amino acids in the CDRs provides the diversity of shape in the antigen-binding site necessary for specificity
The forces involved in antibody-antigen binding are usually weak so it is important that there is a close fit between the two molecules to maximise the binding, i.e. shapes must match!

BCRs Require Coreceptors

Antibody molecules form a B-cell receptor (BCR) complex with molecules involved in signal transduction.

• Igα and Igβ transduce signals via ITAMs.

• CD19, CD81, and CD21 transmit and relay signals to the cell interior.

Recognition of ‘antigen’ by the B cell receptor

Crosslinking of BCR by antigen mediates activation of receptor associated src family tyrosine kinases.

Genetic Engineering of Antibodies

Have many Clinical Applications.

• Anti-tumor antibody

• Anti-T-cell receptor

• Toxin

• Chimeric immunotoxin

• Heteroconjugate

B Cell Development Introduction

• Explain how immunocompetent B cells produced

• Understand how the immune system regulates against auto reactive B cells

• Detail how the B cells are activated by antigens

• Discuss how are plasma and memory B cells produced

The Humoral Branch of Adaptive Immunity: B cells

B cell lineage: Stem Cell -> Pro-B-cell -> Immature B-cell -> Mature Naïve B-cell -> Memory B-cell, Plasma B-cell

Developing B cells undergo a selection process

B-Cell Development Overview

B-cell development begins in the bone marrow and is completed in the periphery.
Overall selection of cells not expressing antibody that recognises ‘self’must occur.
This is known as negative selection

B cell development:- Antigen independent phase in the bone marrow

• Generate 5 \times 10^7 cells/day

• Only 5 \times 10^6 leave the bone marrow

• Once mature circulate in blood through secondary lymphoid organs

• Unless encounter antigen die by apoptosis within a few weeks

B cell development:- Antigen independent phase

In the bone marrow: Pro-B Cell -> Pre-B-cell -> Immature B-cell -> Mature naïve B-cell Expresses IgM and IgD with SAME antigen specifivity

B cell development:- Antigen independent phase Expression of surface markers.

B cell development:- Antigen Independent phase. Gene Rearrangement and expression of antibody.

B cell development in the Bone Marrow:- Negative selection

• Interaction with Self antigen -> IgM on surface -> Cell Death

• No interaction with Self antigen -> ‘Non-self interacting’ B-cell -> Mature naïve B-cell (IgM and IgD on surface)

Tolerance Induction

Immature B cells in the bone marrow are exquisitely sensitive to tolerance induction.
They bear membrane IgM, B220, CD25, IL-7R, and CD19.
Surface receptors are tested against self-antigens; there are three possible outcomes:

• clonal deletion of strongly autoreactive cells by apoptosis

• receptor editing via reactivation of light-chain recombination machinery

• induction of anergy, a state of nonresponsiveness to further stimuli (even self-antigen stimuli)
  Clonal deletion and receptor editing are termed central tolerance as they occur in bone marrow.

Deletion of Autoreactive B cells by Negative Selection

Self Antigen Binds to IgM on Immature B-cell -> Crosslinking of IgM -> Apoptosis is activated -> Development is arrested
No binding to IgM -> Maturation continues
Some light chain editing -> Still self reactive -> No longer self reactive

Maturation of Transitional B Cells

B cells that are exported from the bone marrow are functionally immature.
There are two subsets – T1 and T2 – that ultimately differentiate into conventional B-2 cells.
Note that these are NOT T cells!
The subsets differ in gene expression as they progress through the spleen for further maturation.

Subsets of B cells: Transitional 1 and 2 (T1 and T2 Cells)

Two subsets of transitional B cells are known as T1 and T2.
The T1 subset is still undergoing screening and can undergo negative selection.
The possibility of negative selection is lost as the cells transition to the T2 stage.
T2 B cells express the BAFF-receptor (BAFF-R) for the B-cell survival factor BAFF.
Progressions to T2 and mature B cells are supported by a variety of responses to strong BCR signaling in each subset.

B cell development:- Antigen dependent phase in the periphery

Mature B-cell -> Plasma B-cell, Memory B-cell

B cell development:- Antigen dependent phase in the periphery

Two types of B cell response:-

1. Activated by Thymus-dependent (TD) antigen

   • requires CD4+ T cells (helper T cells)

   • linked recognition

   • T-cell dependent differentiation of B cell results in memory

   • Mediated through follicular B2 B cells

2. Activated by Thymus independent (TI) antigen

   • large polymeric molecules with repeating units (PAMP)

   • can be considered part of innate immunity

   • able to cross link B-cell antibody leads to primarily IgM production

   • do not result in memory

   • Mediated through B1 subset of B cells

Thymus Dependent Response: Mature Follicular B-2 B Cells

Mature, primary B-2 B cells migrate to lymphoid follicles.
They express high levels of IgM/IgD on their surfaces.
They recirculate between blood and lymphoid organs.
They are often called follicular B cells because they enter the follicles in lymph nodes and the spleen.
They help to respond to antigens with T-cell help by producing antibodies.
They have a half-life of approximately 4.5 months in the periphery.

Activation of B-cells

• TI pathway

  • B-cell

• TD pathway

  • B-cell - T helper-cell

  • CD40-CD40L

  • CD4 T cell receptor

  • MHC

Activation of B-cells: The B cell receptor

Components: IgM, Igβ chain, Igα chain, B cell plasma membrane

Recognition of ‘antigen’ by the B cell receptor (2)

Crosslinking of BCR by antigen mediates activation of receptor associated src family tyrosine kinases.

Activation of proliferation and further development of B-cells

B7 -> CD28 between B-cell and T helper-cell
Release of cytokines to further activate B-cell resulting in proliferation and differentiation

Sites for proliferation and further development of B-cells: Lymph nodes and Germinal centres

Components: Primary Follicle, Secondary Follicle, Germinal Centre, Medulla, Cortex, Paracortex, Lymphatic vessels

Affinity Maturation of Antibodies through Somatic Hypermutation

Naive B cell -> Clonal expansion B cell -> Germinal centre;
Somatic hypermutation occurs. Improved affinity selected, disadvantageous mutations lead to apoptotic B cell;
Differentiation via FDC and T cell -> Plasma cell (Class switching) or Memory B cell
Dark zone, Light zone, Mantle zone (shown in figure)

Primary and Secondary Responses