L18 - Humoral Immunity: Antibodies & Life Cycle of B-Cells

Question 1

Describe the structure of the Antibody?

Answer 1

Made up of Constant region and Variable region.

Constant region
Same for all Ab
of same class. 
Made of heavy chain:
- Heavy: μ,δ,γ,α or ε chain
γ1, γ2, γ3, γ4
α1, α2

Made up of light chain:
Light: κ or λ chain

Variable chain contains antigen binding site and is different

Question 2

What are the two versions of Antibodies?

Answer 2

Membrane bound and Secreted antibodies

Secreted antibodies is the final fully functional version of the antibodies secreted by plasma cells

Question 3

How are the heavy and the light chains held together?

Answer 3

By disulphide bonds between the cysteine amino acid residues

Question 4

Which region of antibodies promote interaction between antibodies and other immune cells?

Answer 4

There are carbohydrate glycosylations on the CH2 regions of the heavy chain

Question 5

What are CDRs?

Answer 5

Complementarity Determining Regions

Where antibodies interacts with antigens.

Consists of Heavy/light variable regions

Question 6

What are the 4 main functions of an Antibody?

Answer 6

• variable region of antibody binds to the region where pathogen attaches to host cell. It prevents pathogen from entering the cell
• Can bind to the active sites of toxins produced by pathogens and neutralise them
• Antibodies play role in opsinisation or tagging so it becomes more visible to other immune cells
• Able to inform Immune complexes - agglutination of pathogen and antibodies which is then cleared by other cells

Question 7

What are the different classes of Antibodies?

Answer 7

IgG

IgD

IgE

IgM

IgA

Question 8

What are the differences in structure between the Antibody classes

Answer 8

IgG = has canonical Antibody structure

IgD = has a longer hinge region

IgE = Epsilon region has 5 domains

IgM and IgA are similar to IgG however they have tail peices at the end of CH3 to facilitate polymerisation and joining to J chain

Question 9

Function of IgM?

Answer 9

Main Ab of primary
response; best at
forming immune
complexes & fixing
complement;
monomer serves as
BCR

Question 10

Function of IgD?

Answer 10

BCR;
indicates
mature B
cells; only
Ab not
secreted

Question 11

Function of IgG?

Answer 11

Main Ab of
secondary
responses;
neutralize
toxins;
opsonization

Question 12

Function of IgA?

Answer 12

Secreted into
mucous, tears,
saliva,
colostrum

Question 13

Function of IgE?

Answer 13

Allergy; antiparasites

Question 14

What is Heavy Chain Class Switching?

Answer 14

 Only affects heavy chain CONSTANT region

 Different effector functions – deal with different pathogens

 Minor: Differential splicing (mRNA level)
– IgM and IgD (last lecture)

 Major: DNA recombination
– IgM to IgG, IgA, IgE
– IgG to IgA, IgE

Question 15

How do B-cells now which class to switch to?

Answer 15

They sense cytokines produced By T helper or Regulatory cells

Question 16

What are the 3 steps for class switch recombination?

Answer 16

1) Cytokine signal
2) Switch regions (switching only proceeds downstream)
3) AID and DSB repair proteins

Question 17

What is the difference in structure between Membrane bound and secreted Igs?

Answer 17

Secreted Ig has a tail peice

Membrane Ig has a hydrophobic transmembrane region and cytoplasmic tail as an anchor

Question 18

What are the 2 stages of B cells life cycle?

Answer 18

Antigen independent stage and antigen dependent stage

Question 19

Describe the Antigen independent stage of the life cycle of B-cells?

Answer 19

In the bone marrow, stem cells will differentiate into Pro-B-cell.

Pro B-cell will then undergo D-J recombination in its DNA. The DJ segment will then attach to the V segment. This is then expressed with a Mu constant region (default antibody class).

The cell becomes a Pre B-cell, when it expresses a full heavy chain with a unique variable region.
It also expresses a light chain placeholder, that forms Pseudoantibody with heavy chain.

The pre B-cell will undergo another V-J recombination on the light chain, which will determine the light chain variable and constant region

Immature B-cell expresses IgM and Mu Heavy chain

Will become Naive B-cell when it has the capacity to produce IgM and IgD, through differential splicing of mRNA

Question 20

How can additional diversity for BCRs be added

Answer 20

• Junctional Flexibility

- addition of P and N nucleotides

Question 21

How does VJ recombination of light chains occur?

Answer 21

A V and J segment is chosen and added to the constant region.

This is then transcribed into messenger RNA. Extra segments are spliced out to form mature RNA containing the L,V,J,C

RNA is translated to amino acid sequence with makes up the light chain

Question 22

How many VDJ segments are there in recombination?

Answer 22

 51 Variable (V) segments

 27 Diversity (D) segments

 6 Joining (J) segments

 Constant region (C) segments

Question 23

Where are VJ recombination genes expressed?

Answer 23

Chromosome 2

Question 24

Where are VDJ recombination genes expressed?

Answer 24

Chromosome 14

Question 25

How does VDJ recombination of Heavy chains occur?

Answer 25

A D and J segment are chosen to join Then a V segment will join to the D and J segment together. This hardcoded DNA will then be transcribed into the MRNA transcripts. Differential splicing of mRNA occurs to form mature RNA. Sequence includes L,V,D,J,CMu This will then be translated into the IgM heavy chain

Question 26

How many VJ segments are there in recombination?

Answer 26

 40 Variable (V) segments  5 Joining (J) segments  Constant region (C) segment

Question 27

What are Recombination signal sequences (RSS)?

Answer 27

It is a recombination mechanism They are conserved sequences upstream or downstream of gene segments Thye are made up of turns consisting of a conserved Heptamer (7 bp DNA sequence), that is the same in all turns. Followed by 12/23bp spacer, and then a nonamer (9bp)

Question 28

What is the difference between a 1 and 2 turn in RSS and what is the 1 turn/2 turn rule?

Answer 28

2 turn = 23bp spacer 1 turn = 12bp spacer There are 2 types of turns, as DNA recombination only occurs between a 12 and 23bp spacer. So only a 2 turn can be combined with 1 turn.

Question 29

How many Heavy chain combinations can be made from the segments?

Answer 29

8262

Question 30

How many Light chain kappa and Lambda combinations can be made from the segments?

Answer 30

``` Kappa = 200 Lambda = 120 ```

Question 31

How many BCR combinations can be made?

Answer 31

1.98 x 10^8

Question 32

How are major and minor Hairpins formed?

Answer 32

MAJOR: The whole DNA folded in half. The enzymes RAG1 and RAG2 will bind to the turns, pulling them closer together, to form a hairpin. MINOR: Between the 2 strands of DNA. DNA is nicked and automatically forms a hairpin at the ends of gene segments

Question 33

What enzyme is involved in Hairpin opening?

Answer 33

Artemis

Question 34

What enzyme is involved in Hairpin processing?

Answer 34

exonucleases TdT

Question 35

What is Junctional Flexibility?

Answer 35

Removal of nucleotides between gene segments during VDJ/VJ recombination

Question 36

Describe the process when B-cells are activated?

Answer 36

The activated B-cells migrate to the germinal centre (GC) where it will undergo Affinity Maturation. This proccess is to improve their affinity for attacking pathogens. Affinity Maturation involves clonal expansion and somatic hypermutation which takes place in the Dark zone. Cells then migrate to the light zone to undergo selection. Then the antibody receives signals that indicate which pathogens they will encounter, and they will undergo class switching. The B-cells will differentiate into plasma cells, secreting antibodies and at the same time, maintaining some receptors on their surface. A few will differentiate into memory cells which also circulate the bloodstream with plasma cells

Question 37

Describe the T-cell independent stage of B-cell activation

Answer 37

When pathogens invade, they partially activate the B-cell when they process antigen. Then the B-cells will make clones of itself, which becomes the 1st defence army secreting IgM to hold off pathogen. The other clones will migrate to the lymph node to await T-cell activation

Question 38

Describe the T-cell dependent stage of B-cell activation

Answer 38

some clones will migrate to the lymph node to await T-cell activation. T-cell dependant phase requires a triple verification process: - First the B-cell need to encounter the pathogen and internalise the antigens. Antigen is then presented on the surface of the B-cell, via MHC class 2 receptor. - Then it has to be activated by a T-cell which has in turn been activated by the same pathogen (pathogen was engulfed by the dendritic cells and presented on the surface, T-helper cells are activated). T-helper cell then activated the B-cell - T-helper cell produces cytokines: - Fully activated B-cells will undergo affinity maturation plus switching and differentiate into plasma cells which secrete antibodies (IgG secreted)

Question 39

Describe the process of Affinity Maturation and where it occurs?

Answer 39

Takes place in the germinal centre of the lymph node (circular cell clusters at the periphery After antibody binds to antigen for the 1st time, it binds at low affinity. Affinity maturation process generates mutations in the variable region genes, and selects the antibody with the highest affinity: Activated B-cells undergo clonal expansion in dark zone. Then they undergo somatic hypermutation via Actiavtion-induced-deaminase (AID) which are point mutation in DNA. All B-cells are now slightly different to one another. The hyper-mutated B-cells go into light zone and undergo selection. Follicular dendritic cells (FDC) will present antigens on the surface, and B-cells compete for limited number of antigens on the surface, and remove them. B-cells then present the antogens to TFH (T follicular cells) cells, which will gove it a survival signal B-cells that survive the process migrate back to the dark zone where process starts again.