Humoral immunity:B cell activation, affinity maturation and class switching Flashcards
What do stem cells undergo?
• In the bone marrow, stem cells undergo DNA recombination to become immature B cell
What is there in the process of joining and what contributes to the diversity of the antibody?
• in the process of joining there is junctional flexibility and P and N nucleotide addition to contribute to the diversity of the antibody
What happens once the mature B cell encounters a pathogen and what is this for?
- Once the mature B cell encounters a pathogen, it becomes activated and undergoes affinity maturation and class switching in the germinal epithelium
* This is to code in the heavy chain constant region of the B cell
What is affinity maturation to further and what does it improve?
• Affinity maturation is to further hone the variable fragments of the antibody to that particular pathogen
-Improves affinity to the pathogen
What does the first domain of light and heavy makeup and what does the rest make up?
First domain of light and heavy makes up antigen binding variable fragment and the rest of the constant regions makes up effector function
What does the antigen binding variable fragment bind to and what does it do?
• In the antigen binding variable fragment, it binds to the docking site/host entry site of pathogen
○ Prevent the virus from entering cell
○ Or they bind to active site of toxins to neutralise them
What is opsonisation mainly for and what does it do?
• Opsonisation: mostly for bacteria as they are larger
○ Antibody tags its surface to recruit other cells e.g. macrophages that have Fc receptors – these bind to the active site of the antibody and carries out antibody dependent cellular phagocytosis
What region is the active site of the antibody?
Active site is in CH2 region of antibody ADCP
What will antibodies do to tumour cells which are large?
For tumour cells which are larger, it will opsonise them and will recruit NK cells to carry out ADCC – cellular cytotoxicity
How do antibodies form an immune complex and what does this cause?
• More and more antibodies can bind to the surface of the bacteria which forms an immune complex
○ This causes agglutination and precipitation as the complex precipitates out of the bloodstream, rendering the bacteria nonfunctional
What can you form immune complexes with other than antibodies and what does this lead to?
• Can also form immune complexes with complement known as complement fixing
○ Can lead to phagocytosis – agglutinates and precipitates and then is cleared
What can complement form in certain pathways and what does it do to tumour cells?
In certain pathways, the complement can form MAC (Membrane Attack Complex) which punches holes in the tumour cells, causing them to lyse
How many classes of antibodies are there and what are they determined by?
• 5 classes, all determined by 5 classes of heavy chain
How many heavy chain domains does IgG have and what stabilises it between the 2 chains?
IgG has 4 heavy chain domains – variable and constant 1, 2 3 – stabilised by two disulphide bonds between the heavy chains
What does IgD have a long region of and what is it stabilised by?
IgD has a long hinge region, stabilised by one disulphide bond
How many domains does IgE have?
IgE has 5 domains
What is the modification that IgA has but IgG doesn’t and what does this allow?
IgA is similar to IgG but the third CH3 domain has a modification so that it allows interaction with the J chain
What Ig is IgM similar to and what can it form and with the help of what chain?
• IgM is similar to IgD – 5 domains but it can form a pentamer with the help of the J chain
Abundances of Ig in serum
• IgG is the most abundant in serum followed by IgA and IgM
What is the only Ig to cross placenta?
• Only IgG crosses the placenta so mum can pass on antibodies to fetus
What Ig’s fix complement?
• IgM and IgG fix complement
What does IgG attract?
IgG attracts phagocytes
What does IgE attract?
IgE attracts mast cells and basophils
What is the first antibody to be produced after class switching?
• IgG is the first antibody to be produced after class switching
Life cycle of B cells(Antigen dependent)
• After VDJ recombination of B cells, they become mature circulating B cells
• Lymphoid progenitor stem cells give rise to T cells
○ T cells produced migrate to thymus where they undergo similar VDJ recombination to become TCRS
○ T cells receive signals from APCs and become activated and in turn activate B cells when there are pathogens
• B cell migrate to GC for selection
• GC is divided into dark and light zone
○ In dark zone: affinity maturation happens
• Honing of variable fragment to that particular pathogen
§ Involves clonal expansion, somatic hypermutation (enzyme creates mutation in its DNA)
○ In light zone: selection - selecting the BCR that binds to the pathogen the best
§ Undergoes class switching because by this time you know the pathogen being dealt with so need an appropriate class
• Differentiate into plasma and memory cells that will circulate in the blood and spleen
How many stages is B cell activation divided into and what are they?
• Divided into two stages
- T cell independent B cell activation
- T cell dependent B cell activation
T cell independent B cell activation
• When B cell encounters antigens expressed on the surface of the pathogens, it undergoes clonal expansion
○ Some of them will differentiate into IgM secreting plasma cells
○ This is the first line of defence
T cell dependent B cell activation
• Some of the cells will migrate into the lymph nodes
• Needs to recognize antigens, internalize them and then present it in MHC class II
• Second step: activation by TCR
○ TCR has to recognize the antigens presented on B cells and APCs
• THC produce cytokines to complete the three step verification process that the b cell activation requires
What are the 3 signals that the B cell activation requires?
- Antigen binding to BCRs
- Co-stimulation by activated Th cell specific to same antigen 3. Th cell-derived cytokines to verify that there is an active infection going on
What does BCR binding cause the activation of?
Causes the activation of tyrosine kinase
What does tyrosine kinase phosphorylate and activate?
• Tyrosine kinase phosphorylates other proteins and activates pathways involved in cell proliferation as B cell needs to make copies of itself
What does VDJ recombination in the body produce?
• VDJ recombination in the body produce 1 billion B cells with unique BCRs
What can BCR do when pathogen invades?
• When pathogen invades, the BCR that can bind to it and makes clones of itself
Where do clones of BCR migrate to?
• Clones migrate to GC for affinity maturation
Where does affinity maturation happen and what 2 cells is it helped by?
• Affinity maturation happens in the GC and is helped by two types of cells
○ T follicular helper cells (Tfh): subset of T cells that can enter the GC
○ Follicular dendritic cells (FDC): not your normal DCs
Where do follicular dendritic cells stay in and present?
• FDC stay in GC and present antigens for selection process
Steps involved in affinity maturation and class switching in GC
• B cells will be activated by Th and migrate to GC while undergoing clonal expansion
• Enzyme causing mutation in variable region
• Migrate to light zone and with the help of FDC and TFH it will undergo selection
○ This will repeat until affinity is high enough
• B cell will undergo class switching to select for the right antibody class before differentiating into plasma cells and memory cells
What zones does affinity maturation happen in?
• Happens in dark and light zone
Detailed steps involved in affinity matuation
• Activated T cell makes clones of itself
• Activation induced cytidine enzyme (AID) makes point mutations in the variable regions of the DNA
○ Before this, the B cells are clones
○ After AID, they are all unique B cells with unique mutations
• These migrate to the light zone where the Tfh present antigens on its surface
• B cells with high affinity BCR on surface will try to grab the antigens on its surface and then present to Tfh
○ Tfh will give a survival signal
• The rest also have point mutations in the DNA and these can cause improved affinity, reduced affinity or no improvement in affinity
○ Those with reduced or no improvement will undergo apoptosis because there is no survival signal from Tfh
• Have to repeat this process multiple times until affinity is high enough
What happens to B cells once the affinity is high enough?
○ Once affinity is high enough, it progresses to the next stage – class switching
What does class switching only affect and not affect?
• Class switching only affects heavy chain constant regions
○ Doesn’t affect variable regions of the heavy and light chain
○ Constant region of light chain stays the same
What are the 2 different types of heavy class switching?
- Minor:Differential splicing(mRNA level)
2. Major:DNA recombination(Irreversible)
What is minor class switching between?
○ Between IgM and IgD
Reversibility of minor class switching
This is reversible
What does major class swithich occur between?
○ Class switching can only occur from IgM to IgG, IgA, IgE ANDIgG to IgA, IgE
Why is major class switching only between the specific Ig?
○ This is because of the locations of the constant regions
What does class switch recomination(CSR) require?
○ Cytokine signal
○ Switch regions – similar to RSS but is in front of heavy chain constant region gene segments
○ AID and DSB (double stranded break) repair proteins
What is recombination between in class switch recombination?
• Recombination between switch regions
How does switching proceed in class switch recombination?
• Switching only proceeds downstream
○ IgM to IgG, IgA, IgE
○ IgG to IgA, IgE
○ E..g you cannot switch from IgE to IgA
What does AID help in?
• AID helps in somatic hyper mutation
What is the default after affinity maturation and before class switching?
• After affinity maturation and before class switching, B cell still makes IgM and IgD as this is the default
What is the final form of Ig?
• Secreted form is final form
When is Ig membrane bound?
• Membrane bound is during antibody development
What piece does secreted Ig have?
• Secreted has tail piece
What is the Ig anchor on the membrane made of?
• Membrane has an anchor made of hydrophobic transmembrane region and cytoplasmic tail
What is true differential splicing?
• True differential splicing is how the B cell knows how to make both of these forms of antibodies
What remains unchanged in DIFFERENTIAL SPLICING OF μ CONSTANT REGIONS?
• Leader VDJ remains unchanged
How does differential splicing of μ constant regions for secreted heavy chains?
• For secreted heavy chains, the DNA region is transcribed into MRNA and spliced at first polyA site
§ This generates mRNA coding for the new secreted heavy chain
What do you need to splice up first if you want to encode membrane bound new chain?
• If you want to encode membrane bound new chain, you need to splice up first polyA site
What does differential splicing of μ constant regions?q
• Prevents antibody from secreting out of B cell
