Lecture 3: Diversity

Question 1

Causes of VARIETY/DIVERSITY in B-cell VDJ gene recombination (4)

Answer 1

There are multiple copies of the V, D and J genes that can be joined together in different combinations.

B-cell progenitors in thebone marrow randomly rearrange theirVDJgenes.

Further diversity is added as the junctions between rearranged gene segments contain small insertions and deletions
- junctional diversity

Finally, pairing between the chains can also increase variation.

Question 2

What determines the specificity of the antibody

Answer 2

Specificity of an antibody is determined by the shape of its variable region(made of variable(V),diversity (D), andjoining (J)genes on the heavy chain and VJ on the light chain).

Question 3

Recombination process for light chain (terms of V, D and or J gene combination sequence)

Answer 3

1 V segment fuses with 1 J segement vy recombination

Question 4

Recombination process for Heavy chain (terms of V, D and or J gene combination sequence)

Answer 4

1 D segment fuses with 1 J segment to form a recombined DJ segment. DJ segment fuses with 1 V segment
This recombined gene fuses with the constant part of the antibody forming a functional antibody.

Question 5

What directs recombination and where is it found and what is it made up of

What proteins cooridinate the recombination

Answer 5

Recombination signal sequence (RSS)

found next to every V, D, and J segment.

made of highly conserved heptamer sequences (7 base pairs), spacer sequences, nonamer (9 base pairs) sequences. They are the RSS sequences

Coordinated by recombinase-activating genesRAG-1andRAG-2
Rag 1 and Rag 2 recombinase make dsDNA breaks at the RSS

Enzyme terminal
deoxynucleotidyl transferase(TdT) adds extra nucleotides between the V, D and J regions (junctional diversity)

Question 6

When does VDJ recombination occur?

Answer 6

In bone marrow pro-immature B-cell

Question 7

Purpose of BCR Somatic Hypermutation

Answer 7

increase antibody affinity

- to maximise antibody response

Question 8

BCR recombination: Process of BCR Somatic Hypermutation

Answer 8

Random point mutations of variable (VDJ) region of BCR

Performed by the enzymeactivation-induced cytidine deaminase(AID) randomly insertions point mutations in B cells changing the affinity of antibody

Results in many B cell with their BCRs sightly different from each other
Some will have higher affinity, some will not (apoptosis)

B cells with increased affinity to the antigen will proliferate and survive preferentially

Question 9

General process of T cell from bone marrow to blood (alpha and beta chains)

Answer 9

1. Bone marrow the common lymphoid progenitor cell is formed
2. immature T cell enters blood stream towards thymus for maturation
3. Thymocyte enters thymus and undergoes maturation and POS/NEG selection (98% apoptosis) to form gamma/delta T cells, CD4+ and CD8+ (alpha and beta)
4. Leave thmyus and enter bloodstream as peripherall Tcell repertiore.

Question 10

TCR is made from…

Most common chain types.

Answer 10

TCR are made up of two polypeptide chains:
~95% alpha (α) and a beta (β) chain
~5% gamma (γ) and delta (δ )chain

Question 11

Recombination processes of TCR

Answer 11

recombination of different V, D, and J gene segments.
- use of RSS, RAG-1 and RAG-2, TdT.

Recombination signal sequence (RSS) found next to every V, D, and J segment.
RSS is made of highly conserved heptamer sequences (7 base pairs), spacer sequences, nonamer (9 base pairs) sequences. They are the RSS sequences

Coordinated by recombinase-activating genesRAG-1andRAG-2
Rag 1 and Rag 2 recombinase make dsDNA breaks at the RSS

Enzyme terminal
deoxynucleotidyl transferase(TdT) adds extra nucleotides between the V, D and J regions (junctional diversity)

TCR genes do not undergo somatic hypermutations.

Question 12

What gene segments does alpha and gamma chains have?

Answer 12

V and J

Question 13

What gene segments does beta and delta chains have?

Answer 13

V, D and J

Question 14

Why does a TCR have more diversity than antibody despite similar structure?

Answer 14

• increased α J segments
• additional junctional regions
• increased nucleotide addition/deletion during recombination

Question 15

Beta chain recombination process for TCR (in terms of V, D and J sequence)

Answer 15

D to J, then DJ to V

Question 16

Alpha chain recombination proces for TCR (in terms of V, D and J sequence)

Answer 16

V to J

Question 17

What is SCID

Answer 17

Severe combined immunodefiency (cant make T and B cells and thus cant fight infections)

Question 18

What are the immunophenotypes of SCID and what lack/presence of T and B and NKC cells do they have?

Answer 18

Lecture slides

gamma C/JAK3 SCID
IL-7RALPHA defiency

ADA SCID
RAG1/2 defiency SCID

Question 19

MCH class 2 defiency

Answer 19

has normal amounts of dysfunctional T and B cells

Question 20

MCH class 1 defiency

Answer 20

have normal humoral response, low NK, Tc cell response. Very rare

Question 21

RAG 1/2 Defiency effect

Answer 21

defects in the RAG1 or RAG2 genes

- T and B cell receptor complexes cannot be assembled.

Question 22

SCID Treatment Options

Answer 22

Depends on underlying genetic defect

Most infants treated with bone marrow transplant/stem cell transplant for permanent cure

Gene therapy currently undergoing clinical trials

Enzyme replacement therapy for ADA-SCID to boost ADA enzyme

Before the treatments above..
Antibiotics, antivirals, antifungals etc
Immunoglobulin therapy

Question 23

Describe Multiple Myeloma and effects

Answer 23

Develops in B cells after they have left the BM (not during VDJ recombination)
During class switch recombination process causes MM
defective plasma cell preferentially proliferates in the BM

Uncontrolled secretion of monoclonal antibody, cytokines and chemokines