Antibody Diversity

Question 1

Before clonal expansion, in what two ways can we tweak the antibodies to give a better immune response?

Answer 1

1. Switching constant region - that’s associated with variable exon on the heavy chain.
2. Somatic hypermutation - mutate variable exon

Question 2

What immunoglobulins are expressed in naive B cells?

Answer 2

IgM and IgD

Question 3

How are IgD and IgM expressed?

Answer 3

Alternative splicing

Question 4

How are constant domains switched? (2 points)

Answer 4

• T cells signal to activate transcription of the switch regions.
• Enzymes create breaks, DNA is kicked out and then is joined - DNA is permanently changed unless T cells signal to change again

Question 5

What does AID do? (4 points)

Answer 5

• enzyme that deaminates cytosine to uracil - creating G/U mismatch - AID mutates DNA
• Works in Class Switch Recombination and Somatic hypermutation
• Only works on single-stranger DNA - so needs transcription
• Works in germinal centres - lymph nodes

Question 6

What size regions of DNA do somatic hypermutation and class switch recombination remove?

Answer 6

• Somatic hypermutation only removes a few bases - small amount
• Class switch recombination removes lots of bases - creating breaks all along DNA

Question 7

Does Class Switch recombination create variability?

Answer 7

• It does not change the antigen binding site

- It only changes the heavy chains associated with the variable exon

Question 8

How does Somatic Hypermutation create variability?

3 points

Answer 8

After AID has created G/U mismatches - they are processed by:

1. Cell replicates and half of daughter cells carry mutation - A/U - (not G/C) - Then U is replaced = A/T
2. U is removed and replaced with any random base - error prone polymerase
3. DNA is blocked - around 6 bases - mutation is repaired with random bases - error prone polymerase

Question 9

How can somatic recombination cause cancer?

Answer 9

Mis-targeting of AID - leads to oncogene activation

e.g., tumour suppressor genes: ARF; INK4B
Oncogenes: MYC; BCL6

Question 10

How can class switch recombination (CSR) cause cancer?

Answer 10

Translocation of oncogene MYC: C-MYC gene to IgH switch - appears error in CSR - creates aberrant MYC activation and Burkitt’s lymphoma

Question 11

What is cryptic recombination?

Answer 11

Recombination with a cryptic recombination signal sequence outside of the antigen receptor loci

• Leads to over expression of oncogenes - contributes to lymphoid cancers
• e.g., LM02, SIL/SCL, TCR/IgH inversion

Question 12

What is End Donation?

Answer 12

RAGs cuts DNA but then drops it

• When section is reintergrated - can lead to over expression of oncogene
  e. g., BCL-2/IgH - incorrect translocation of gene leads to over expression of gene preventing apoptosis - so cancer cells won’t die - mantle lymphoma

Question 13

Structure of antibody (3 points)

Answer 13

• Two identical heavy chains - with different constant regions - 1 locus - chromosome 14 - variable region + 3/4 constant regions
• Two identical light chains - one variable region, one constant region - (k or lambda )
• Linked with disulphide bridges

Question 14

How does the V(D)J recombination reaction create antibody diversity? (1 point)

Answer 14

• Mixes/matches gene segments to create more different exons

Question 15

What does V(D)J stand for and which regions does each occur in?

Answer 15

V - Variable
D - Diversity
J - Joining

Light chain - V & J
Heavy chain - V, D, J

Question 16

What does the 12-23 rule ensure? (1 point)

Answer 16

Ensures V-J are always joined - to give properly functioning exon

Question 17

What does V(D)J recombination of gene segments rely on? (1 point)

Answer 17

Recombination signal sequence (RSS)

Question 18

What does RAG1 do?

Answer 18

• Cuts/cleaves DNA - is active at C-terminus core

Question 19

What does RAG2 do?

Answer 19

• Regulates RAG1 and the joining of segments afterwards

- Active at N-terminus core

Question 20

Mechanism of RAGS - (hairpin etc) (3 points)

Answer 20

• RAGS bind RSS into a synaptic complex
• RAG1 nicks/breaks strand - releases free OH group which attacks opposite strand - direct transesterfication
• Creates hairpin structure at coding ends and blunt double strand break at signal end
• Broken ends are processed - build the coding exon of immunoglobulin (V-J)
• Hairpin is kicked out and V-J is sealed in a circle
• All occurs in hyper variable region 3

Question 21

How are the ends rejoined after RAGs?

Answer 21

• Uses Nono-homologous enjoining machinery
• Ku70/80 proteins and Artemis protein - open hairpin end - alter and process DNA
• DNA ligase binds region together

Question 22

Where does Junctional Diversity take place?

Answer 22

• During joining of V-J - DNA is processed

- AAs are added/deleted - adding Junctional Diversity

Question 23

Which immunoglobulins require class switch recombination?

Answer 23

IgG, IgA, IgE

Question 24

Where does CSR and Somatic hypermutation occur?

Answer 24

• Germinal centre of lymph nodes

Question 25

Why are recombination reactions dangerous?

Answer 25

• Body usually protects DNA very well
• B-cells are careless and create breaks/mutations from CSR and Somatic hypermutation
• gives chance for recombination events to lead to an over expression of oncogenes - and develop cancer