Lecture 23: Antibodies and gene rearrangement

Question 1

What are the three key points of adaptive immunity?

Answer 1

1) Has memory. Secondary response is stronger and more rapid than the primary.
2) Affinity of B cells towards antigen increases with time and persistence of antigen.
3) You are born with a massive repertoire of B and T lymphocytes. Each lymphocyte represents a different antigen specificity randomly produced by rearrangement of the genes coding for the antigen receptors.

Question 2

How did adaptive immunity come about in humans?

Answer 2

• Relied on gene rearrangement or recombination (only genetic locus capable of this)
• Transposon inserted into primordial receptor 500m years ago… transposase the enzyme that operates the transposon moved away from the receptor gene allowing it to rearrange the genes without affecting its own position in the gene.
• Ancient transposes in our genome are RAG1 and RAG2 (recombinant activation genes), the recognition sequence in genes are the substrate for RAG-1 and RAG-2.
• RAG-1 and RAG-2 and recognition sequences are identical in all species that possess adaptive immunity

i.e any gene that re-arranges has a recognition sequence which RAG-1 or RAG-2 can bind to and start to recombine that gene. RS and RAG-1/2 are conserved amongst all species that have adaptive immunity. It facilitates the creation of the large repertoire of adaptive immune cells to recognise nearly all pathogens.

Question 3

Write some notes on what the immunoglobulin protein fold is:

Answer 3

Antibodies are formed from repeated protein units called Ig domains. Found in hundreds of other proteins and is evolutionary very successful.

The Ig domain fold is called a Beta-barrel of ~110 amino acids.

Question 4

Describe the antibody protein structure:

Answer 4

• 4 protein chains (repeating Ig domains); 2 light chains and 2 heavy chains
• Light chains contain 2Ig domains, Heavy chains contain 4Ig domains.
• 1 Heavy chain is disulphide linked to 1 Light chain.
• The 2 heavy chains are also disulphide linked.

L-s-H-s-H-s-L

Forming a Y shape! (Effector Fc region and Antigen binding region (Fab)

Question 5

Why do antibodys form a Y shape?

Answer 5

• Allows for two flexible arms and the antigen binding sites are located at the tip of the two arms.
• Formed from the N terminal domains of the L and H chains
• Effector region is invariant and is bound by Fc receptors and compliment component C1.

Question 6

What determines the function fo the five Ig classes?

Answer 6

Different functions depending on which H chain gene is used.

IgM, IgG, IgD, IgE and IgA

Question 7

Describe where each of the five Ig classes are found:

Answer 7

IgM = Default, serum and membrane 
IgG = Serum 
IgD = Serum and membrane 
IgE = Serum 
IgA = Serum and mucosa 

pneumonic: IgGAMED

Question 8

Write some notes on IgM:

Answer 8

• Default antibody made by naive B cells
• Comes membrane bound (monomer) and soluble (pentamer) forms.

1) Membrane form is the B-cell antigen receptor
2) Soluble form has 10 antigen binding sites.

• Avidity binding to microbes
• Good at fixing complement with 5 Fc regions that bind complement component C1.

Question 9

What is affinity?

Answer 9

When the sum of the attractive molecular forces at the two surfaces exceeds the repulsive forces, there is affinity.

The higher the affinity, the fewer the molecules it takes per unit volume to associate and to dissociate slowly.

Question 10

Describe avidity:

Answer 10

Avidity results from multiple affinity contacts. Like velcro, the strength of the binding can be orders of magnitude higher than the individual affinities.

Avidity is a very important concept in immunology.

Question 11

What does complementarity mean when it comes to antibodies?

Answer 11

• An antibody can form complementarity to virtually anything because of potential amino acid diversity at the antigen binding site is vast….
• Affinity arises when the sum of attractive forces exceeds the sum of repulsive molecular forces.

Question 12

What gives the diversity of antibodies to bind to many different antigens?

Answer 12

Antigen receptor repertoire.

• Few genes, but, amino acid variation is found in 3 discrete regions of the antibody. Complemetarity Determining Regions (CDR). These are the loops that connect the strands of the 1st domains of the H and L chains.
• 3 loops from Vh and Vl juxtapose in the folded protein.
• There are two identical antigen binding sites.

i.e the loops are what form the antigen binding site..

Question 13

Describe the recombination of the Ig locus:

Ig = immunoglobulin

Answer 13

• Germ line genes are segmented into clusters called Variable, Diversity, Joining and Constant regions.
  (light chain locus has no D segements)
• RAG1 and RAG2 are responsible for the rearrangement and are only active in B and T lymphocytes.
• D to J then V to D. Intervening DNA is lost.
• Joining is very imprecise so base pairs are changed during the repair. This leads to huge variation at the VDJ join.
• VDJ join region codes for CD3.

Question 14

What is the role of clonal selection in immune repertoire formation?

Answer 14

The immune system randomly makes as many receptor combinations as possible then allows the pathogen to select those clones that are useful.

Question 15

Describe the formation of high affinity B cell clones:

Answer 15

• Naive low affinity B cells with IgM receptors in the B-cell follicles of the lymph nodes….
• Under the influence of persistent antigen and over time form:
• Memory B cells and High affinity B cell clones that produce high affinity soluble IgG.

One B cell = One antigen specificity.

= Clonal selection by antigen.

Question 16

Describe the affinity maturation of B cells:

Answer 16

• Massive naive B cell repertoire generated stochastically before birth, each with a unique B cell receptor.
• B cells encounter antigen in lymph bodes. Expanding a small number clonal b cells with receptors that weakly bind the antigen.

Maturation process:

• Random SOMATIC HYPERMUTATION of the Ig gene results in some clones with HIGHER antigen receptor AFFINITY.
• After SUCCESSIVE rounds of this, the mature B cells becomes a plasma cell secreting soluble Ig.
• Some B cells reside in lymph nodes as long-term memory cells.
• This is the fundamental mechanism behind adaptive immunity, memory and immunisation.

AKA: Somatic hypermutation means the highest antigen receptor affinity is selected through successive rounds of replication. It goes on to produce mature high affinity producing IgG cells and long term memory B cells.

Question 17

Write some notes on clostridium tetani:

Answer 17

• Produces tetanus toxin
• Neurotoxin that binds to the presynaptic membrane of the neuromuscular junction and causes spastic paralysis. Known as lockjaw because the pts jaw often freezes open. Incurable. causes flow and painful death.

Question 18

Write some notes on immunity to tetanus:

Answer 18

• Every baby has IgM antibodies that bind to TT weakly but not enough to neutralise its activity
• Vaccination with a non-toxic version called TETANUS TOXOID drives affinity maturation of those IgM antibodies into high affinity IgG antibodies that block TT receptor binding.
• Single boost required ~ every ten years.

Affinity maturation is the reason why vaccination works