Lecture 6 & 7 - Immunoglobulins Flashcards
Which regions determine the specificity of Ab?
CDR: complementarity determining region
aka
Hypervariable regions
Describe the two branches of effector function of Ab
- Direct
2. Indirect
Describe direct mechanism of effector function of Ab
Neutralisation
• binds directly to toxin / virus / bacterium
Blocks:
• entry of viruses through their entry molecules
• molecules that inhibit replication etc.
• toxin can not bind to its target
Describe the indirect mechanism of effector function of Ab
Give some examples
• Ab binds to Fc receptors on immune cells
(Their function is now linked to the function of the cell)
e.g.: • Opsonisation • ADCC (Antibody dependent cell-mediated cytotoxicity) • Degranulation • C' activation
Which cells have Fc receptors?
- Macrophages
- mast cells
- eosinophils
- NK cells etc.
Which classes of Ig bind to Fc?
- IgG
* IgE
Describe opsonisation
‘enhanced phagocytosis’
• Ab coats pathogen
• Macrophage binds Fc region of Ig with its FcR
• Macrophage phagocytoses the Ig coated pathogen
• Degradation in a phagosome
Describe ADCC
(Antibody dependent cell-mediated cytotoxicity)
Used for virally infected cells (not extracellular bacteria)
- Infected cell presenting viral protein on surface of cell
- Ab binds target cell
- NK cell binds IgG via FcγR; cross-linking is important
- NK cells release toxic granules and kill the infected cell
Describe degranulation
- IgE binds FcεR on granulocyte
- IgE binds pathogen / allergen
* (steps one and two a little bit interchangeable) - Cross linking of FcR triggers degranulation
In which situations is cross linking important for activation?
- IgE cross linking pathogen when bound to Mast cells
* IgG cross linking on NK cells
Describe the various FcR’s
- FcγRI
- FcγRIIA
- FcγRIIB
- FcγRIIIA
- FcγRIIIB
• FcεR
These are each found on different cells
Which FcR’s have low affinity for Ig?
Why is this important?
FcγRII A
FcγRII B
FcγRIII A
FcγRIII B
- This is important so that not all of the Ig is bound to cells
- In addition, we don’t want an overactive response under normal conditions
If FcR’s have low affinity, how then do they bind when there is a pathogen?
When a Ig coated pathogen is encountered by the immune cell with the FcR, binding is more likely due to the increased avidity
Where does Fc bind to FcR?
Can vary
They don’t all bind in the same region
Describe IgA transcytosis
- Dimeric IgA binds to poly-Ig receptor on basolateral side of epithelial cell
- transcytosis across the cell
- Dimeric IgA released into the gut with some of the poly-Ig receptor, which is now the secretory component
What is the secretory component formed from?
The poly-Ig receptor
What are the three pathways of complement activation?
- Classical
- Alternate
- Lectin
Describe the classical pathway of C’ activation
- Ab binds to ‘multi determinant’ Ag (eg. Bacteria)
* C1q binds to IgM and IgG at Fc region
Which classes of Ig trigger the C’ pathway the best?
IgM
IgG, to a lesser degree
Which Ig isotypes are seen in secondary responses?
i.e. neutralisation
• IgG
• IgA
Which Ig isotypes are good at opsonisation?
In order of efficacy: IgG1 IgG3 IgG4 IgA
Which Ig isotypes are good at ADCC?
- IgG1
* IgG3
Which Ig isotypes are good at triggering degranulation?
IgE
Which Ig isotypes are good at C’ activation?
• IgM
• IgG1, IgG3
( • IgG2, IgA )
Which mechanisms of Ab effector function are good for extracellular bacteria?
- Neutralisation
- Opsonisation
- C’ activation
What is Idiotype?
Variation in variable regions
i.e., the different specificities
Each B cell has a different Idiotype
What is Allotype?
Different alleles of Ig genes
• Different individuals can have minor sequence differences between different individuals
• normally differences in the constant domain
(This is not particularly functionally significant)
Which regions on Ig are the most variable according to the equation?
CDRs (hyper variable regions)
• about 5 positions per Ig
• in addition, some of the CDRs are much more variable than others
What are the flanking regions on Ig that were not found to be very variable?
Framework regions
What is the genetic basis of Ab diversity?
Somatic diversification
• Rearrangement of Immunoglobulin gene segments
• This generates a unique idiotype of each B cell
What is the basic structure of a gene?
- Promoter
- Leading exon; axons
- Introns
- Enhancer
- Poly-A addition signal
What is the difference between the DNA and mRNA?
- T replaced with U
* Introns spliced out
What is meant by tandem arrays of genes segments at the Heavy and Light chain loci?
• Many V, D & J segments
Draw the Kappa locus in germ line configuration
S33
Draw the Lambda locus in germ line configuration
S33
Draw the heavy chain locus in germ line configuration
S33
How many V segments are there on the K, L and Heavy chain loci?
K: 40
L: 30
H: 45
How many D segments are there on the K, L and Heavy chain loci?
K: 0
L: 0
H: 25
How many J segments are there on the K, L and Heavy chain loci?
K: 5
L: 4
H: 6
How is individual segment selection in gene rearrangement determined?
It’s random
What are the variability plots called?
Kabat-Wu plots
On a Katat-Wu plot, where do the variable regions correspond to on the light chain?
The 3 loops on the variable domain
where the complimentary epitope binds
Describe the process of gene rearrangement of the heavy chain
- D-J rearrangement
- V-DJ rearrangement
- mRNA transcription
- Translation
Describe the process of gene rearrangement of the light chain
- V-J rearrangement
- mRNA transcription
- Translation
On a Katat-Wu plot, where do the variable regions correspond to on the heavy chain?
Two in variable region
One at D-J junction
What are the flanking sequences of DNA of the segments, and why are they important?
RSS: recognition signal sequences
These form loops which are recognised by the recombinase enzyme complex
Describe how intervening sequences are excised in recombination
The RSS loop (containing all the intervening segments) binds the REC, which makes cuts at the V and J junctions.
These junctions then join up again with the help of more enzymes
What is the 12/23 rule?
How does this arise?
Prevents V → J rearrangement at the heavy chain locus
The Recom. enzyme complex only recognises 12-23 nucleotide loops, not 23-23.
When V-J forms a loop, you get 23-23 loops, which won’t be recognised.
What is the V(D)J recombinase complex?
Comprised of:
• RAG-1, RAG-2
• Exonucleases
• TdTs
What is the function of RAG?
Breaking & joining of DNA
What is the function of exonucleases?
Removal of nucleotides at the ends of broken chains
What is the function of TdT?
Addition of random nucleotides on the ends
What is the name for these random nucleotides added by TdT called?
N-regions / N-nucleotides
Why have N-regions?
Contribute to diversity
Downside: can lead to failure of rearrangement
How is diversity of Ig specificity generated?
Ig gene rearrangement
• Combinatorial diversity
• Junctional diversity
• Somatic mutation
Describe combinatorial diversity
Two aspects: 1. Combinatorial joining (the different segments) 2. Combinatorial association (heavy chain & light chain)
Describe Junctional diversity
Determined by N-regions generated by TdT
Describe somatic mutation.
Which regions accumulate mutation?
Further diversity as the immune response progresses
Mutations in:
• V regions (not C) of both chains
• Predominantly in CDRs, but not exclusively
Where are the different aspects of diversity occurring?
Combinatorial & junctional diversity: 1° lymphoid organ
Somatic mutation: 2° lymphoid organ
Describe the factors of maturation of antibody function
- CSR (class switch recombination)
* Affinity maturation
What is required for CSR?
T cell help
Once T cells are activated by DCs, they express CD40L.
They can now interact with B cells and their CD40 molecules on the surface.
When does somatic diversification occur?
During the development of B cells
When talking about Ig gene rearrangement, why do we talk about ‘deletion’ of DNA instead of splicing?
Splicing is what’s going on at the RNA transcript.
In Ig gene rearrangement, the DNA is actually being deleted from that cell.
What does the massive peak in the Kabat-Wu plot correspond to?
Why?
To the junction between the V and the J segments.
The two smaller peaks are part of the exons, and they are thus germ line encoded.
The junction represents so much diversity, because this combination is cell specific.
What is the difference in the genome between a mature lymphocyte and any other cell in the body?
Deletion of most Ig gene segments in mature lymphocytes
Describe the structure of the RSSs and why this is important
Made up of heptamers and nonamers
The two heptamers anneal, as do the two no namers.
This forms a loop which can be recognised by the recombinase enzyme complex
What does RAG stand for?
Recombination activating genes
Describe the molecular mechanism of isotope switching
- Switch regions come into contact, forming a loop
2. Loop excised from DNA
Draw the constant regions with the switch regions
S53
Is class switching reversible?
No, because once the Constant region genes have been excised they cannot be gotten back.
Why is the tail piece important?
Needs to be there for J-chain association to make a multimer
Where is the constant region cut when there should be no tail piece?
At the “occult” splicing site
Which forms of Ig are made when there isn’t an immune response?
Contrast this after the immune response is activated
Normally: membrane bound Ig
Immune response activated: secreted Ig
How much more diversity does junctional diversity give?
10^4
What does alternative splicing determine?
What is really important to remember about this?
Which isotope is produced
NB: this is occurring at the RNA level, not DNA (as in Ig gene rearrangement)
Describe the result of affinity maturation
As the immune response progresses, the affinity of Ab and Ag increases.
This occurs through the process of somatic hypermutation.
Describe the process of SHM
(Somatic hypermutation)
• de novo Mutations accumulate in variable regions of both chains
• in the exons
• mostly in CDR; but not exclusively
Which enzymes mediate SHM?
AID
Activation induced cytidine deaminase
In which cells is AID expressed?
Compare this with RAG
AID: Germinal centre B cells
RAG: developing B cells in bone marrow
Describe the evolution of mutation in Ab over primary, secondary and tertiary immune response
S63
What sometimes happens when Ag binds IgM?
Conformational change in IgM
This allows C1q to bind better
In the classical pathway of C’ activation, why must the Ag be multi determinant?
Multiple copies of the antibody must bind to the antigen in order to activate C1q
Which cells have FcγRI?
What is the effector function?
What is the affinity?
Cells:
• Macrophages
• Neutrophils
Effector function:
• Phagocytosis
Affinity:
• High
Which cells have FcγRII A?
What is the effector function?
What is the affinity?
Cells:
• Macrophages
• Neutrophils
Effector function:
• Phagocytosis
Affinity:
• Low
Which cells have FcγRII B?
What is the effector function?
What is the affinity?
Cells:
• B cells
Effector function:
• Inhibition of cell activity
• NB no phagocytosis
Affinity:
• Low
Which cells have FcγRIII A?
What is the effector function?
What is the affinity?
Cells:
• NK cells
Effector function:
• ADCC
Affinity:
• Low
Which cells have FcγRIII B?
What is the effector function?
What is the affinity?
Cells:
• Neutrophils
Effector function:
• Phagocytosis
Affinity:
• Low
Which cells have FcεR?
What is the effector function?
What is the affinity?
Cells:
• Mast cells
• Eosinophils
• Basophils
Effector function:
• Degranulation
Affinity:
• High
Describe what is required in terms of antigen for cross linking to occur?
Pathogens with many determinants
Many antibodies coat it
Where does C1q bind?
What does this stimulate?
What is required for C1q binding to Ab?
To the Fc region of Ab
This triggers the classical pathway of complement activation
Free Ab will not bind C1q, the Ab must be bound to a determinant
How many idiotypes can one individual have present in their body?
Up to 10^7
i.e. how many different Ab specificities
How can N-regions lead to failure of rearrangement?
- Frameshift insertions / deletions
* Insertion of STOP codons
What determines whether secreted or transmembrane Ig is made?
Selection of the membrane anchor in alternate splicing of mRNA:
• pAm
Affinity maturation coincides with an decrease in …
valency
eg. IgM –> IgG
At which level does selection IgM / IgD in B cell development occur?
Characterise this process
Occurs at mRNA level
‘Alternate RNA splicing’
Describe alternate mRNA splicing
At what stage is this occurring?
When:
• The B cell is developing in the bone marrow
• There is IgM and IgD being produced at the same time, within the one B cell
- Both the Cδ or the Cμ regions are transcribed into RNA
- Alternate splicing removes either the Cδ or the Cμ region
- Either IgM or IgD is made
What does alternate splicing control?
- IgM / IgD selection
2. Transmembrane / Secretory form of the Ig
What is required for a protein to be membrane bound?
A membrane anchor
Where is the occult splice site?
Describe what goes on here
The occult splice site is at the junction of the Cμ regions and the ‘Tail piece’ region.
If the Ig is to be secreted:
• tail piece is maintained in the RNA transcript
If the Ig is to be membrane bound:
• Membrane anchor region is spliced into the occult splice site
• Tail piece region spliced out
