L18 - Humoral Immunity: Antibodies & Life Cycle of B-Cells Flashcards
Describe the structure of the Antibody?
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
What are the two versions of Antibodies?
Membrane bound and Secreted antibodies
Secreted antibodies is the final fully functional version of the antibodies secreted by plasma cells
How are the heavy and the light chains held together?
By disulphide bonds between the cysteine amino acid residues
Which region of antibodies promote interaction between antibodies and other immune cells?
There are carbohydrate glycosylations on the CH2 regions of the heavy chain
What are CDRs?
Complementarity Determining Regions
Where antibodies interacts with antigens.
Consists of Heavy/light variable regions
What are the 4 main functions of an Antibody?
- 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
What are the different classes of Antibodies?
IgG
IgD
IgE
IgM
IgA
What are the differences in structure between the Antibody classes
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
Function of IgM?
Main Ab of primary response; best at forming immune complexes & fixing complement; monomer serves as BCR
Function of IgD?
BCR; indicates mature B cells; only Ab not secreted
Function of IgG?
Main Ab of secondary responses; neutralize toxins; opsonization
Function of IgA?
Secreted into
mucous, tears,
saliva,
colostrum
Function of IgE?
Allergy; antiparasites
What is Heavy Chain Class Switching?
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
How do B-cells now which class to switch to?
They sense cytokines produced By T helper or Regulatory cells
What are the 3 steps for class switch recombination?
1) Cytokine signal
2) Switch regions (switching only proceeds downstream)
3) AID and DSB repair proteins
What is the difference in structure between Membrane bound and secreted Igs?
Secreted Ig has a tail peice
Membrane Ig has a hydrophobic transmembrane region and cytoplasmic tail as an anchor
What are the 2 stages of B cells life cycle?
Antigen independent stage and antigen dependent stage
Describe the Antigen independent stage of the life cycle of B-cells?
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
How can additional diversity for BCRs be added
- Junctional Flexibility
- addition of P and N nucleotides
How does VJ recombination of light chains occur?
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
How many VDJ segments are there in recombination?
51 Variable (V) segments
27 Diversity (D) segments
6 Joining (J) segments
Constant region (C) segments
Where are VJ recombination genes expressed?
Chromosome 2
Where are VDJ recombination genes expressed?
Chromosome 14
How does VDJ recombination of Heavy chains occur?
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
How many VJ segments are there in recombination?
40 Variable (V) segments
5 Joining (J) segments
Constant region (C) segment
What are Recombination signal sequences (RSS)?
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)
What is the difference between a 1 and 2 turn in RSS and what is the 1 turn/2 turn rule?
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.
How many Heavy chain combinations can be made from the segments?
8262
How many Light chain kappa and Lambda combinations can be made from the segments?
Kappa = 200 Lambda = 120
How many BCR combinations can be made?
1.98 x 10^8
How are major and minor Hairpins formed?
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
What enzyme is involved in Hairpin opening?
Artemis
What enzyme is involved in Hairpin processing?
exonucleases
TdT
What is Junctional Flexibility?
Removal of nucleotides between gene segments during VDJ/VJ recombination
Describe the process when B-cells are activated?
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
Describe the T-cell independent stage of B-cell activation
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
Describe the T-cell dependent stage of B-cell activation
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)
Describe the process of Affinity Maturation and where it occurs?
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.
