Humour immunity: antibodies and life cycle of B cells Flashcards
What do antibodies do?
Y-shaped molecules made by plasma cells
Fights against foreign pathogens and cancerous cells that invade our body
Work by blocking pathogens from entry or tagging them for removal by other immune cells
What is the strucutre of antibodies, what are they made up of?
Made up of two light chains and two heavy chains
Heavy chains made up of 4 domains, can be divided into mew, delta, gamma, alpha, epsilon classes
- Gamma further divided into four subclasses – gamma 1-4
- Alpha into two subclasses – alpha 1-2
Altogether 9 different heavy chains
Light chains have two domains
- Kappa and lambda chains
What region do heavy and light chains form together?
What region does rest of the antibody form?
Heavy and light chain together form variable region, this is the targeting system of the antibodies.
Different antibodies have different variable regions, binds specifically to a pathogen
- So specific that two antibodies can recognise different parts of the pathogen
Rest of antibody forms constant region, same for all antibodies of the same class
- e.g all IgM will have mew heavy chain
- All IgG have gamma chain so on
What are the two verisons of antibodies?
There are two versions of antibodies
- membrane bound version – B cell receptor
- Secreted form – final fully functional form of antibody secreted by mature plasma cells
Before it gets to the secreted form it is anchored on the membrane of B cells for weapon development.
What are the four domains of heavy chain?
What are the five classes of heavy chain?
What are the domains of light chain?
Heavy chains have four domains
- VH – variable heavy domain
- CH1-3, constant heavy domains
Heavy chain has five classes, mew, gamma, alpha, delta and epsilon
Light chains have two domains
- Light variable region, VL
- Constant light region , CL
What does the variable regions of light and heavy chain make?
Makes up the antigen binding portion of the antibody, different between antibodies secreted from different B cells.
Constant region plays a part in biological activity of an antibody, the same for all antibodies of the same class.
What are the heavy and light chains made up of?
How are they held together?
Where are the hinge regions?
Chains are a string of amino acids, start with amine NH3+ group on upstream end, ends with carboxyl COO- group.
Heavy and light chains are held together by disulphide bonds between cysteine residues in the chains, SS, and some intramolecular disulphide bonds to stabilise each of the domains.
Hinge region between CH1 and CH2 domains to provide flexibility to the molecule, not rigid. Flexes and bends.
Carbohydrate glycosylation’s on the CH2 regions near effector region active site, promotes interaction between antibodies and other immune cells it recruits.
What is the complementarity determining region?
CDR is where antibody interacts with antigens, antigens being proteins on surface of pathogens or cancer cells.
CDR of heavy and light chains are different.
L1,L2, L3 of light chains and H1, H2, H3 of heavy chain CDRs are supported by scaffolds. This is how antibody interacts with antigen.
Antibodies have four functions to combat pathogens
function 1 and 2
Variable fragment can bind to part where pathogen binds to the host cell, preventing pathogen from entering the cell
Can bind to active sites of toxins produced by pathogens and neutralises them
Antibodies have four functions in combatting pathogens
Function 3
Opsonisation, tagging pathogen so it becomes visible to other immune cells like macrophages and NK cells
Variable regions bind to pathogen
constant region will interact with FC receptor of macrophages and recruits them to perform antibody dependent cellular phagocytosis, ADCP
or recruit NK cells to perform ADCC, antibody dependent cellular cytotoxicity
Antibody has four functions to combat pathogens
Function 4
Antibodies able to form immune complexes, large complexes made up of antibody and pathogen
Agglutinates and is removed by other cells
Complex can also involve complement molecules like c1q, c1s, c1r.
When it does this antibody is set to fix complement, leads to series of events which promote inflammation, phagocytosis and formation of membrane attack complexes (MAC) which punches holes in cell membrane, causing cell lysis
What is the difference between antibody dependent cellular phagocytosis and antibody dependent cellular cytotoxicity
ADCP performed by macrophages to engulf smaller pathogens
ADCC performed by NK cells which release chemicals to induce apoptosis and generally for infected or cancerous cells.
What are the different classes of antibodies?
IgG
IgD
IgA
IgE
IgM
each one expresses different heavy chain constant region, but light and heavy chain variable region is the same for antibodies produced in same B cell
What is the role of IgM?
Heavy chain - mu
Pentamer so the largest
IgG and IgM only ones to fix complement
doesn’t cross placenta
is the main Ab of primary response; best at forming immune complexes and fixing complement
Monomer serves as BCR
What is the role of IgD?
Heavy chain delta
Doesn’t fix complement or cross placenta
function: BCR, indicates mature B cells, only Ab not secreted because it doesn’t play role in fighting off pathogens, cancer cells or parasites.
What is the role of IgG
heavy chain gamma
Fc binds to phagocytes
Fixes complement and crosses placenta
Main Ab of secondary responses, neutralises toxins, opsonisation
What is the role of IgA
Heavy chain alpha
doesn’t fix complement or cross placenta
secreted into mucous, tears, saliva, colostrum
if you have respiratory infection B cells will class switch into IgG and IgA
What is the role of IgE?
Heavy chain epsilon
doesn’t fix complement or cross placenta
Fc bidns to mast cells and basophils
Involved in allergy, anti-parasites
recruits basophils to fight off larger parasitic infections that cannot be phagocytosed by macrophages
What is heavy chain class switching?
What are the two types?
Only affects heavy chain constant region, all other regions stay the same
Class switching enables body to be more versatile in dealing with different types of pathogens
Minor: differential splicing (mRNA level)
- IgM and IgD
- Occur at RNA level, minor because it doesn’t affect DNA of B cell itself
Major: DNA recombination
- IgM to IgG, IgA, IgE
- IgG to IgA, IgE
Cytokine signalling for class switching
What do chemicals produced by T helper cells indicate?
Indicate what type of pathogen we are dealing with
increase in IL-4 favours class switch to IgG1,4 and IgE and doen’t favour IgG3
IL5- and TGFB favours switch to IgA
IFN-y favours IgG3 and doesn’t favour IgG1 and IgE
in addition to these cytokine signals, CD40L on T cell interacts with CD40 on B cells to confirm it is interacting with T cells
Major class switching uses the mechanism class switch recombination, CSR, which requires three things
- Cytokine signal
- Switch regions
- AID and DSB repair proteins
With the help of AID and other enzymes recombination will occur between switch regions, switching can only proceed downstream
Gene switching happens downstream
IgM can switch to IgG, A and E because mu gene segment is before gamma, alpha and epsilon segments
Once it has switched to IgG all the parts before it is removed, cannot revert back to IgM
How is class switching done once heavy chain gene loci has undergone V(D)J recombination and affinity maturation?
B cell with this genome will be expressing the gene in two forms, IgM and IgD through differential splicing of mRNA – minor class switch
Now it has received signals to class switch to IgA1.
With AID and other proteins, the two switch regions infront of mu segment and alpha segment are pulled together.
Cleaved and joined into a switch DNA circle whilst heavy chain loci will also be rejoined.
VDJ region + the first constant gene segment, in this case alpha1 segment, will be transcribed and translated.
B cell will now express IgA1.
What generates the secreted form of Ig?
How are secreted and membrane bound antibodies different?
Plasma cells, membrane bound BCR present on cell as B cell is an immature b cell.
Secreted and membrane bound antibody have the same heavy and light chain variable and constant regions.
Difference is the secreted version has tail piece, membrane bound has transmembrane region and cytoplasmic tail as an anchor.
Differential splicing of mu constant regions
Describe the structure of heavy chain
How is secreted and membrane bound antibodies formed
B cell loci containing leader, VDJ and constant gene segments.
C mu region is made up of mu1-4, with tailpiece at mu4.
Stop codon and poly-a tail after genes coding for the tailpiece.
Followed by M1 and M2 coding for transmembrane region and cytoplasmic tail
Another stop codon and poly-a tail
Whole region is transcribed to mRNA, these regions spliced out, forming mRNA to code for secreted antibodies.
on other hand, to make membrane bound antibodies region up to second poly-a tail will be transcribed, and 8 regions including genes encoding for tail piece and stop signal after it will be spliced out.
Two stages to B cell development, B cell independent and dependent stage
describe B cell independent stage
B cell starts life in bone marrow
Begins as stem cells
Differentiates into pro-b cell
DNA of pro-b cell undergoes D to J and V to DJ recombination to permanently code in heavy chain variable region
Variable region expressed with mu heavy chain, default first heavy chain expressed by B cells
When cell is expressing heavy chain it is called pre-B cell
Pre-B cell then undergoes another V to J recombination to permanently code in light chain variable and the constant region to become a mature B cell
These cells express IgM and will mature over time
once they can express IgM and IgD on their surface through differential splicing of the mRNA, they will become mature B cells and circulate between bloodstream, spleen and lymph nodes.
“bootcamp”, once they graduate out the B cells will start patrol, resting until they encounter pathogen
B cell dependent stage
B cells become activated when body encounters foreign protein, they go to special forces selection
B cell will further hone its ability to bind to the pathogen through affinity maturation in the germinal centre / GC, only best survive
By this time B cells receive information on what kind of enemy they are dealing with and undergo class switching to appropriate effector regions
B cell receptor can either become IgG or IgA
B cells will further develop into professional plasma cells that secrete the antibody that they code for
Whilst these processes are occurring, some of the B cells coding for IgM will differentiate into plasma cells secreting IgM as first defence
After infection, some b cells will remain as memory B cells so body will remember and be ready to fight off the same infection should it encounter it again
Antibdy independent stage
When does the pro-b cell become a pre-b cell?
What does VJ recombination by pre-B cell determine?
How can additional diversity be generated?
- When it can fully express heavy chain with a unique variable region.
Pre-B cell also expresses light chain place holder, forms pseudo antibody with heavy chain.
- determines light chain variable and constant region
- During VDJ and VJ recombination on heavy and light chains, junctional flexibility and P and N nucleotide addition can generate additional diversity.
Mechanisms which are random in nature is enough to generate more than a billion different unique b cells.
Immature B cell will express full IgM with mu heavy chain and either kappa or lambda light chain, will become mature B cell which has capacity to produce IgM and IgD through differential splicing of mRNA.
Mature B cells also called resting or naïve B cells.
Light chain and heavy chain gene loci is made up of different gene segments
How does recombination occur?
V, J, C for light chain
V, D , J, C for heavy chain
Heavy chain simplified to Cmu
Gene segments undergo recombination, one v segment will be selected at random to recombine with one j segment.
in heavy chain, random v, d, j segments recombine.
DNA will now encodes complete antibody with the VJ or VDJ segments encoding for the light or heavy chain variable fragments and C segments coding for constant domain’s.
DJ portion of antibody genes codes for the CDR3, finger protrusions that interact with the antigen.
What is infront of each V segment?
How does VJ recombination occur?
How does VJ recombination form the kappa light chain?
Leader segment, so cell knows where protein is going to end up.
V and J segments are randomly chosen e.g V23/J4, to form a leader V+J segment, extra J and constant region.
Then transcribed into mRNA.
Extra J segment and other bits and pieces are spliced out to form a mature mRNA, containing only leader, V, J, C segments as a whole.
mRNA is translated into amino acid sequences of the light chain.
Amino acid is then folded and their leader sequence cleaves off when the protein reaches where it needs to go.
Results in a unique kappa light chain.
Lambda chain mechanism similar but slightly more complex.
VDJ recombination of gammay heavy chain genes (chr14)
- 51 Variable segments
- 27 diversity segments
- 6 Joining segments
- Constant region segments
First recombination to take place is the D to J joining.
D7/J3 randomly selected to join.
Then V segment e.g V20, joins to D7/J3 segment that has formed.
Recombined hardcoded DNA then transcribed into mRNA transcripts.
In population of mRNA transcripts, some of them will express lead sequence, followed by VDJ and C mu sequence.
Eventually translated into IgM heavy chain.
Other transcripts in population will express leader, VDJ C delta sequence.
Extra J segment and Cmu is spliced out.
Eventually translated into IgD heavy chain to signal maturity of b cells.
Where are the turns in the light chain
For the light chain we have V,J, C segments.
Red and green triangles are one and two turns.
Two turns are downstream of V segments of the heavy chain and lambda light chain
and upstream of heavy chain and kappa chain J segments
One turns on both sides of the heavy chain J segment, upstream of lambda J segments and downstream of kappaV segments.
Where are the turns on the heavy chain
In the first VJ recombination to join DJ segments there is first one turn two turn join
to join V segment there is another one-two turn join
Therefore the one-two turn rule prevents D fragments or VJ fragments from accidentally combining
Combinatorial diversity
How many antibody genes can you get when using all V,D,J segments of heavy chain and V, J segments of light chain?
Heavy chain:
V (51), D(27), J(6) = 8262 (94 segments inherited)
Light chain kappa:
V (40), J(5) = 200 (45 segments inherited)
Lamda:
V(30), J (4) = 120 (30 segments inherited)
8262 x 200 x 120 = 1.98 x10^8
but you need 1 billion B cells
Mechanism of joining gene segments
What enzymes bind to the turns?
What is the difference between minor and major hairpin?
Enzymes are RAG1 and RAG2, bind to turn, pulling them together to form hairpin
DNA will be nicked and automatically forms a hairpin at the ends of the gene segments
Minor hairpin
- Between two strands of DNA
Major hairpin
- Whole DNA folded in half
A whole host of enzymes will come in to repair and process the ends, eventually forming coding joint of V17 and J3, and a signal joint containing turns and other DNA between two joining gene segments
Hairpin opening and joining
Hairpin opening by artemis/DNA PK
DNA ends processed by other enzymes like exonucleases, TdT
These enzymes mess around with free DNA ends by adding or removing nucleotides
Ends then joined together by other series of enzymes
What happens once the hairpin is formed?
Once hairpins are formed, enzyme artemis randomly knocks one end of double stranded DNA.
Nicked ends linearise, forming overhanging ends.
Repair enzyme in some cases will fill in the gaps, p nucleotide additions.
Terminal deoxynucleotidyl transferase (Tdt) will add the N nucleotides into the two ends before the ends are ligased together again.
N nucleotide addition by Tdt occurs almost exclusively in heavy chain.
Addition of p and N nucleotides before joining the segments together will cause addition of extra amino acids or even shifting the whole reading frame.
Even when two B cells end up selecting the same VDJ or VJ segments, they will end up with totally different antibodies because of P and/or N nucleotide additions
What is junctional FLEXIBILITY?
Removal of nucleotides between gene segments during V(D)J recombination
Ends will overlap if they have enough complimentary sequences.
There will be mismatched nucleotides which have to be removed by exonucleases before repair enzyme can work.
Sometimes exonucleases can be too enthusiastic and overtrim the ends.
For example, recombination between V kappa 21 and J kappa 1 segments
Before B cells managed to pick the same segments to join the exonuclease can remove different lengths of DNA from the gene segments.
- so in B cell 1, the DNA has lost TCC on V end, GT on J end
- B cell two has lost even more on V segment and only G in J segment and so on
Joining of signal joints on the other hand is always precise
Like p and n nucleotide additions, the deletions by exonucleases will cause deletions of amino acid sequences and frame shifts generate completely different antibody variable regions by different B cell lines
Life cycle of B cells, antigen dependent
Lymphoid progenitor stem cells give rise to B cells and to T cells which migrate to the thymus
T cells have their own TCR generated through similar VDJ recombination process
T helper cells are a subset of T cells involved in activation of B cells during infection
Activated B cells migrate to the GC, where it undergoes affinity maturation
Process is to improve affinity for attacking antigens
Affinity maturation process involves
- Clonal expansion
- Somatic hypermutation, which occurs in dark zone
Cells migrate to light zone to undergo selection
Process is repeated several times
Antibody will receive signals to tell them what pathogens they’re fighting, and they undergo class switching so that they have appropriate effector functions
After that B cell differentiates into plasma cells, secreting antibodies whilst still maintaining some BCR
Few will become memory B cells
Memory B cells and plasma cells circulate in the bloodstream
What happens in T cell independent and dependent B cell activation?
When pathogen invades, B cells partially activated, binds to and processes antigens
B cells then make clones of itself through clonal selection
Some of the clones will become first defence army, secreting IgM
Other clones migrate to lymph node to wait for T cell activation
T cell dependent B cell activation requires a triple verification process to ensure B cells don’t get activated by mistake
- First B cell encounters pathogen and internalise antigens, antigen then presented on surface of B cell via MHC class 2 receptor
- Then has to be activated by Th cell which has been activated by the same pathogen
- Pathogen will be engulfed by dendritic cells presented on the surface and T helper cells will detect and be activated
- Then activates B cell in turn
CD40/CD40L confirms that the cell is a t helper cell and not anything else
Third signal it requires is cytokines produced by t helper cell
Fully activated B cells then undergo affinity maturation, class switching and differentiate into plasma cells which secrete antibodies
What is affinity maturation?
Process to improve affinity of antibody to antigen. Antibody has low affinity for antigen, binding takes longer, binds loosely and can quickly fall away after binding.
After VDJ and VJ recombination body has produced 1 bil naive B cells that haven’t been exposed to antigen.
Each B cell has unqie BCR
When pathogen attacks, B cell binds to pathogen
Makes clones of itself - clonal expansion
Clone undergoes affinity maturation
