Lecture 12

Question 1

What is the clonal selection theory?

Answer 1

This theory states that each and every B lymphocyte produced form the bone marrow has only one antibody specificity
This antibody repertoire is generated stochastically prior to birth without any prior knowledge of the antigenic universe
Specificities are selected by antigen stimulation
Activated clones expand and form either antibody producing cells or memory cells
This also holds true the T lymphocyte which expresses the T cell receptor

Question 2

What are the key elements in adaptive immunity and memory?

Answer 2

Repertoire production, antigen stimulation, clonal expansion, somatic hypermutation, affinity maturation, class switching and memory

Question 3

What is repertoire production?

Answer 3

A vast number of different B-cells each with its own randomly rearranged Ig gene locus are produced in the bone marrow, this process is completely stochastic meaning that every combination is produced randomly

Question 4

What is the role of antigen stimulation?

Answer 4

A very small number of B cells in secondary lymphoid organs encounter antigen, Naïve B cells with receptors that have weak affinity toward the antigen respond and proliferate, effective antigen presentation is essential to this step

Question 5

What is the role of clonal expansion?

Answer 5

Proliferating B-cell clone expands rapidly and froms a germinal centre in a follicle

Question 6

What is the role of somatic hypermutation?

Answer 6

During B cell clonal expansion errors occur called somatic hypermutation some of these mutations will improve affinity toward the antigen requiring less antigen to stimulate

Question 7

What is the role of affinity maturation?

Answer 7

Higher affinity B cell clones expand at a greater rate because they require less antigenic stimulation. These clones overtake their slower growing predecessors, this process is called affinity maturation. Boosting assists the process

Question 8

What is the role of class switching and antibody production?

Answer 8

The highest affinity clones undergo achange which induces class switching from the default IgM to typically IgG, the majority become plasma cells producing massive amounts of secreted IgG antibody

Question 9

What is the role of memory?

Answer 9

A small number of cells will remain IgM and become resident memory cells that are primed to expand rapidly when they next encounter small amounts of antigen, this is the basis of adaptive immunity

Question 10

What is somatic recombination?

Answer 10

The Ig and TcR loci are segmented into multiple germ line V, D and J exons
During B cell development the germ line segments in the heavy and light chain loci rearrange to produce an entirely new gene in a purely stochastic process
This occurs only in lymphocytes and is due to the activity of the recombinase enzymes (RAG1 and RAG2)
The order and process of rearrangement is strictly controlled by two rules

Question 11

What are the 5 mechanisms of lymphocytes to generate diversity?

Answer 11

Random HL chain pairing provides a degree of diversity
Segmentation of the H and L gene locus into the variable (V), diversity (D), junctional (J) and constant (C) regions and gene duplication of individual V, D, J and C segments to create a large repertoire of different segments
Somatic recombination of V, D,J and C segments to create new coding genes, D joins to J, V joins to D and J joins to C
N-region diversity which results from the imprecise joining between the V, D and J germ-line gene segments when they recombine, this allows an even greater diversity because the possible alterations at this point are almost limitless
Somatic hypermutation which is continued mutation of hotspots within CDR regions after the initial antigen stimulation and clonal expansion this process does not occur in TcR genes

Question 12

What are the important processes that create junctional diversity in N region diversity?

Answer 12

The D segments can be read in all three reading frames and sometimes in reverse
Imprecise mechanism of joining often leaves nucleotides called P nucleotides that should normally be removed during cutting and joining
N-nucleotides are added or removed through the action of terminal deoxy transferase which adds nucleotides to the ends of the V and A segments in a non-template directed fashion

Question 13

What is the temporal process of Ig Heavy chain rearrangement?

Answer 13

The Ig heavy chains are generated from four gene segments (Vh, Dh, Jh and Ch)
The first event is the joining of the D and J occurring very early in the life of the B cell while it is still in the bone marrow the second event is V-D joining which occurs much later in development
The final event is the combination of the VDJ segment with a C region segment, however this will not occur in the DNA but it an RNA splicing event allowing for the B cell to have the capability to switch classes of Ig secretion
However after affinity maturation C region switching will occur causing the intervening exons to be lost

Question 14

What is the temporal process of Ig light chain re arrangement?

Answer 14

Unlike the H chain there are no D segments, this makes the first event the brining together of a V segment with a J segment which once again will only join to a C fragment via RNA splicing rather than DNA recombination
There are two L chain Loci kappa and lambda, kappa will be rearranged first and then if this is non-productive the lambda locus rearranges

Question 15

What is the most important mechanism for generation of diversity in the Ig and TcR?

Answer 15

While both recombinational and junctional diversity occur junctional diversity is what provides most of the range seen in initial Ig and TcR, the Ig diversity can be further increased by somatic hypermutation during an immune response

Question 16

What RAG 1 and RAG2?

Answer 16

These recombination activating genes which are primitive genes with a single exon in close proximity but opposite orientations to each other
They have transcription which is tightly regulated in B and T cell development which prevents more than one Ig or TcR being produced by the same cell

Question 17

Where did the process of gene recombination used in the immune system?

Answer 17

It is believed that RAG1 and RAG2 were originally transposons that integrated in a gene coding for a lectin like receptor molecule
This transposable element somehow then relocated to another part of chromosomes but left its recognition sequence behind with the receptor gene allowing them to operate in trans

Question 18

What is the recognition sequence recognised by RAG1 and RAG2?

Answer 18

There is a highly conserved heptamer/nonamer signal sequence which is the same in all species and flanks the 3’ end of all V segments, the 5’ and 3’ end of D segments and the 5’ end of J segments
These have a 23 or 12 bp intervening sequencing which is significant as it is one of two turns of a double helix

Question 19

What is the 12/23 base pair rule?

Answer 19

There is a strict rule that recombination is only allowed in a 23/12 or a 12/23 pairing
With 23/23 and 12/12 pairing are not allowed

Question 20

What are the joining rules which are defined by the 12/23 base pair rule?

Answer 20

D-regions can join in both orientation as these segments are flanked on both sides by 12bp recognition sites
H chains only allow V-D or J-D joining but not V-J joining as both V and J segments have 23 bp recognition sites
L chains allow V to J joining as there is no D segment this can occur due to Jl segments having a 12bp RS flanking their 5’ end

Question 21

What are the three joining mechanisms of somatic recombination?

Answer 21

Loop out deletion, Inversion and sister chromatid exchange

Question 22

What occurs in the loop out deletion joining mechanism?

Answer 22

This is the most common and results in the V-3’ joining the 5’-D segment causing the intervening DNA to be excised and lost forever

Question 23

What occurs in the inversion joining mechanism?

Answer 23

The V and J regions are brought together alongside each other in parallel causing the intervening DNA to end up on the 5’ region of the join rather than being excised

Question 24

What occurs in the sister chromatid exchange joining mechanism?

Answer 24

This is a rare event when the loci on one chromatid recombines with the loci on another

Question 25

What is the Stem loop structure?

Answer 25

the actual structure which forms during recombination is not known but it is presumed to be a double stranded stem between the heptamer and nonamer and two single stranded loops which intervene and are either 23 or 12 bp in size

Question 26

What is isotype switching?

Answer 26

An important function of Ig molecules it so switch there effector region
There are 5 different classes as and within the IgG there are 4 subclasses
As there are no recognition sites

Question 27

What is the role of initial splicing in creation of an mIgM molecule?

Answer 27

There are no recognitions signals 3’ of J segments or 5’ of C segments so mRNA splicing is used instead to generate a membrane bound IgM molecule
The VDJ segment can also splice to Cdelta to form a membrane bound IgD so some B cells can express both an IgM and an IgD cell surface antigen receptor
following antigenic stimulation the B cell receives signals which leads to C region class switching
This is regulated by switch signals at the 5’ end of the C segments and the 3’ end of J segments
The most common switch is to the Cgamma gene segment to produce a soluble IgG molecule it is not known exactly what regulates this switching mechanism but one important molecule CD40 and CD40L

Question 28

What is allelic exclusion with regards to antibody diversity?

Answer 28

Because there are two alleles there is a mechanism which prevents a second rearranged allele, this only occurs in IgH and TcR beta chain and not in IgL, TcR alpha loci
This prevents a B cell from having two Ig molecules or a T cell from having two functional beta chains with different specificities
However as TcR alpha and IgL do not undergo this process they can have more than one go at getting the correct recombination as if the first one is non-productive then another attempt can be had