Lecture 11: Generation of diversity

Question 1

What is the estimated number of antibody molecules that an individual is born with?

Answer 1

10^11

Antibody repertoire

More base pairs than in entire genome, how is this so? - lecture explains

Question 2

What is clonal selection theory?

Answer 2

Each and every b lymphocyte produced from bone marrow has only one antibody specificity

Question 3

What does the clonal selection theory say about antigen generation?

Answer 3

• antibody repertoire is generated randomly before birth with no antigen knowledge.
• specifications are selected by antigenic stimulation
• activated clones expand and produce antibody producing plasma cells or memory cells

Question 4

What else may the clonal selection theory apply to?

Answer 4

T cells with at TcR.

Question 5

Describe clonal selection theory

Answer 5

Naive B cells

• clonal expansion
  
  • affinity maturation
• plasma cell (AB) or memory cell

Question 6

In broad terms describe B cell origins

Answer 6

Immunoglobulin gene locus

Arise in bone marrow and mature in the spleen

Question 7

Describe in broad terms T cell development

Answer 7

T cell receptor gene locus

Arise in bone marrow and mature in the thymus

Question 8

What are the seven key elements in adaptive immunity and memory?

(In B cell development)

Answer 8

1. Repertoire Production
2. Antigen stimulation
3. Clonal expansion
4. Somatic hyper mutation
5. Affinity maturation
6. Class switching and antibody production
7. Memory

Question 9

Describe what is meant be repertoire production in B cell development?

Answer 9

Many B cells with randomly arranged Ig gene locus are produced in the bone marrow.

This is random (stochastic)

IgM is the default B cell surface antigen receptor

Question 10

Describe what is meant by antigen stimulation in B cell development;

Answer 10

Small number of B cells in lymphoid tissue encounter antigen.

Naive B cells have weak affinity toward antigen, respond and proliferate (antigen presentation is essential for this step)

Each has a unique antigen specificity

Question 11

Describe what is meant by clonal expansion in B cell development;

Answer 11

Proliferating B cells expand rapidly and form a germinal centre in a follicle.

Antigen drives this activation, expansion and later maturation

Question 12

Describe what is meant by somatic hyper mutation in B cell development;

Answer 12

During B cell development, errors occur called somatic hypermaturation, some of these will improve affinity toward and antigen.

Question 13

Describe what is meant by B cell maturation in B cell development;

Answer 13

Higher affinity B cell clones expand at a greater rate because they require less antigenic stimulation. Thus overtaking their slower growing predecessors

I.e morehigher affinity B cells are produced

Question 14

Describe what is meant by class switching and antibody production in B cell development

Answer 14

Highest affinity clones undergo a change which induces class switching from default IgM to IgG.

Majority because plasma cells producing massive amounts of IgG

A small number of IgM become memory cells. Essential for adaptive immunity

Question 15

What happens to the Ig gene loci during B cell development?

Answer 15

Undergoes somatic recombination ( same as TcR)

Question 16

Describe the Ig gene loci

Answer 16

It is segmented into germline V, D and J exons

Question 17

What happens to the germ line segments (V,D,J) during B cell development?

Answer 17

Germ line segments in the heavy and light chain loci rearrange to produce an entirely new gene.

This is completely random.

Recombination events are not inherited.

Question 18

What cells does recombination occur in!

Answer 18

Only t and B cells because of their recombinant enzymes (RAG1 & RAG2)

Question 19

What is recombination controlled by?

Answer 19

The order and process of rearrangement is strictly controlled by two important rules

Question 20

Describe the hypervariable regions and the germ line structures;

Answer 20

Ig and TcR have three hypervariable regions;

CDR/HV1 = v segment
CDR/HV2 = v segment
CDR/HV3 = VDJ segment ( junction diversity)

Question 21

What are five mechanisms that generate diversity?

Answer 21

1. Random H-L pairing
2. Segmentation of H and L gene locus
3. Somatic recombination
4. N region diversity
5. Somatic hyper mutation

Question 22

What does random HL pairing cause?

Answer 22

Random H L chain pairing provides a degree of diversity (of antibodies)

Question 23

How does H and L loci segmentation create AB diversity?

Answer 23

Gene duplication of individual V,d,j,c segments creates a large repertoire of different segments.

Question 24

How does somatic recombination contribute to antigen diversity?

Answer 24

Somatic recombination of VJDC segments to create new coding genes.

D joins V

V joints D

J joints C

Question 25

Describe what n region diversity is and how it contributes to antigen diversity

Answer 25

N region diversity results from the imprecise joint between V,D,J germ line gene segments when they combine.

Thus allowing greater diversity as the possible alterations are limitless

Question 26

What creates the junction all diversity of the n region?

Answer 26

• d segment can be read in all three reading frames and sometimes in reverse.
• imprecise joining leaves p nucleotides that would otherwise be removed
• n nucleotides are added or removed to the ends of D or V segments (TgT)

Question 27

Describe how somatic hypermutation contributes to antibody diversity

Answer 27

Continued mutations at hot spots within CD-R regions of Ig gene during clonal expansion.

Key for affinity maturation

Question 28

What gene does somatic hypermaturation not occur in?

Answer 28

TcR genes

Question 29

Describe the temporal process of Ig heavy chain rearrangement (look at book page 88)

Answer 29

In the genome, somatic recombination occurs twice. Firstly it reorganises genome so that d and j segments join. Then this occurs again that d and v join.

VDJ (now one segment, no other random DNA between them) joins the c region (constant) via mRNA splicing not recombination.

C(m) region is typical at this stage. During affinity maturation isotope switching can occur and c regions can be switched.

Question 30

Describe the temporal process of Ig light chain rearrangement

Answer 30

No D segments!

Somatic rearrangement v and j.

mRNA splicing joins VJ and C(kappa) (

Question 31

What is essential for recombination?

Answer 31

RAG 1 & 2. These are recombination genes and are tightly regulated to ensure that one cell produces one antibody

Question 32

Describe the RAG origin.

Answer 32

RAg 1 & 2 originally transposing inter grated with PRR gene,

Transposable elements genes relocated to different part of genome but left recognition sequence behind, (Rs) could operate in trans

Trans recombination events - gene duplication and segmentation

Question 33

What recognises the RS sequence left behind?

Answer 33

RAG 1 and 2

Question 34

What is the key to recombination events?

Answer 34

The preserved signal sequence at the flank

3’ end of all v segments

5’ and 3’ of d segments

5’ end of j segments

With 12 or 23 base pair intervening sequence (2 or 1 helix turns)

Question 35

What is recombination regulated by?

Answer 35

12/23 base pair joining rule.

I.e 12/12 and 23/23 are not allowed.

Question 36

Considering the 12/23 rule, what happens in segment binding?

Answer 36

1. D regions can be joined in both orientations (by j) as 12bp either end, j is 23
2. H chains only allow v-d or d-j. But no v-j as j and v both 23 bp for RS
3. L chains allow v j binding as no d segment. J(l) unlike j(h) has 12 bo at 5 end instead

Question 37

What are the three joining mechanisms of recombination?

Answer 37

1. Loop out deletion(I,e v-3’ joins 5’-D and intervening DNA is excised)
2. Inversion. V and j brought into parallel and same result, but intervening DNA ends up 5’ end of bind, not excised
3. Sister chromatid exchange of loci

Question 38

What Ig class has four sub classes?

Answer 38

IgG

Question 39

Describe how the initial splicing of mRNA creates and IgM molecule

Answer 39

VDJ is spliced (joined) to c(m) region segment. (Default) though IgD can occur too

Question 40

How does c class switching occur?

Answer 40

Following antigenic stimulation signals are received by the B cell which initiates c region class switching.

Exact mechanism of how VDJ is spliced or moved to C(g) is unknown but CD40 is required…

Question 41

What is allelic exclusion?

Answer 41

It’s believed that one allele is methylated preventing its rearrangement.

This occurs if Ig (heavy) and TcR beta. (Not IgL or TcR a)

Thus preventing two IgG molecules.

Question 42

How does affinity maturation occur?

Answer 42

Results from somatic hyper mutation of h and l chains post B cell selection.

Mutations in variable regions

As antigen depletes in germinal centre, higher affinity B cells are selected (boosting)

A very high affinity antibody is obtained eventually and this is the memory cell.