lecture 3 Flashcards
Why make IgM and then change to IgG?
- Secreted IgM is pentameric
- IgM provides 10 binding sites thus greatly increasing avidity despite potentially low affinity
- Secreted IgG is dimeric
- Provides 2 binding sites, so binding reflects affinity rather than avidity (somatic mutation increases the affinity of the binding site over time)
- changes effector functions: IgM is very limited in its effector function
- Pentameric IgM allows low affinity antibody to bind antigen
- As affinity increases, IgM can be replaced with IgG or IgA with their specialised effector functions
What are the different effector functions of human Igs?
- Complement activation (G1/2/3, M)
- Placental transfer (G1/2/3/4)
- Mucosa (A1/2) (M - poorly)
- Extravascular transfer (G1/2/3/4, A1/2)
- Sensitising mast cells (E)
How does Class Switch Recombination occur?
- Alters Ig class without affecting specificity
- Each has a switch (S) region upstream that is homologous to other S regions
- CSR is deletional recombination mediated by S-S recognition requiring double stranded breaks in the DNA
- Enzyme AID introduces nicks into S region DNA, providing a substrate for recombination
- VDJ segment is unaffected by CSR
- CSR occurs at the Heavy chain locus only and requires the enzyme AID
What is the role of activation induced cytidine deaminase (AID) in CSR?
- discovered about 14 years ago
- Recognises a target sequence in DNA and it finds cytosines within that sequence and deaminates them which converts them into uracil
- Uracil should not be in DNA and there are various repair mechanisms in cells that will remove it
- this creates gaps/nicks in the DNA
- these gaps are close enough together that they actually represent a double stranded break in the DNA
- AID also does the same at another point in the DNA (before whichever constant region is required)
- the two staggered cuts are joined together, removing the DNA in between
- shifts the variable region in front of a new constant region
What is the relationship between the antigen and the immunoglobulin isotype?
e.g. you have a worm
this worm has specific antigens your body has learnt to recognise as being part of a worm.
When those bits of worm are detected by antigen presenting cells e.g. Dendritic cells they are activated and go on to stimulate T cells. It will do it through cell surface markers and cytokines (soluble molecules) that programme the T cell in a particular way.
The immune system has evolved so that , in response to a worm, the T cell will produce IL4, IL5, IL13, IL25 (i.e. a set programme: worms always induce cytokines of this type in an immune response).
Simultaneously parts of the worm will be recognised by the B cell as antigens. It will recognise this as foreign and migrate to a point where it will meet up with the T helper cell that has the appropriate antigen from the same worm. The B cell receives the specific set of cytokines from the T cell and these tell it what to do. The B cell undergoes CSR specific for that antigen. –> IgE is the best isotype to make in response to worms because it invokes mast cells.
Mast cells degranulate on exposure and release toxins and compounds capable of expelling worms.
This process is not perfect but it works pretty well to induce the correct CSR differentiation.
How and why are mutations introduced into GC B cell Ig V genes?
- mutations are introduced at ‘random’ = somatic
- this disversifies the binding properties of the expressed immunoglobulin
- amino acid replacements that improve affinity for Ag are selected for
- when B cells start they are exactly as they were in the bone marrow - germline
- if you take a B cell undergoing a response out you will notice a small number of changes at random - these are unique to that V gene undergoing the response i.e. somatic (e.g. day 7; low mut. freq.)
- introduced as random nucleotide changes along the gene
- as the response progresses, the frequency of these mutations, in any V gene, increases
- these mutations tend to be clustered into the regions of the antibody that interact with the antigen
- day 10; increasing
- day 21; plateau
- only a small number of these mutations will increase the binding affinity, but those that do will be very strongly selected for and will dominate the whole response
- the affinity can increase by up to 10000 fold just by changing a few nucleotides and selection for improved binding
Which enzyme is used to create somatic mutations in the Ig V gene?
- Activation-Induced Deaminase (AID)
- initiates somatic mutation by converting cytosines to uracils (U=T) in Ig genes
- This induces error-prone DNA repair -> these constitute the mutations
- doesn’t result in DNA ds breaks because the number of nicks is much smaller
- many of the mutations will be deleterious, leading to a defective antibody or no improvement with affinity
- many cells in the GC will die in the process because the mutation leads to poorer binding
- very few selected but this is okay because we make so many lymphocytes in such a short amount of time that wasting 90% of them is irrelevant
- the 10% you are left with do a fantastic job - they have the high affinity antibody to fight of the infection
What is affinity maturation? How does it occur?
The selective survival of Ag-binding variants, generated by V gene SHM, with improved affinity for antigen.
- In the early GC there is T cell driven proliferation CSR from IgM to IgG/A - no SHM
- Onset of SHM diversifies V genes by mutation leading to a range of antigen binding affinities including improvement, diminution or complete loss of Ig expression
- Selective expansion of B cell clones with improved binding to antigen and death of remainder increases affinity of the population. Repeat process.
What are the interactions between B and T cells when responding to an antigen?
- The B cell and T cell meet at the edge of the B/T areas and decide they need to do something about the antigen
- B cells migrate in and start proliferating to form the GC
- T cell enters in as well
- B cell and T cell communicate with each other through cell surface protein interactions and cytokines (soluble molecules)
- Whole series of ligands and receptors on the B cells and the T cells that are communicating with each other throughout the response: intermittently but repetitively
- this is to ensure that only those cells involved in the response are initiate proliferation and differentiation.
- B cells have to be activated and in that activation they will express several of these molecules
- T cells also have to be activated, and in that activation they express the counter molecules
- So you need to have two activated cells that express the right molecules on the surface in order to tell the other that they are the activated cell, therefore stopping the activation of bystander cells
- Surrounding B cells are not expressing the correct molecules and are therefore ignored by the T cells
How do TH cells induce CSR and SHM of B cells?
- activated T cells express CD40L and ICOS
- B cells express the receptors for these: CD40 and ICOSL
- It is signalling through both of these which is what driving proliferation and SHM and CSR
- there is a signalling cascade downstream of both of these receptors
- CD40 runs through a relatively complex pathway involving NF-kappaB - these are crucial for activating the AID genes
- AID will then activate CSR and SHM
- CSR targeted by the cytokines secreted by the T cell
- mutations in each of these steps are causative of immune deficiency
What is the enzyme that removes Uracil from DNA?
UNG (Uracil DNA deglycosylase)
What deficiencies are associated with the induction in B cells of CSR and SHM by T cells?
- XL-CD40L deficiency
- AR-CD40 deficiency
- XL-HIGM-ED (x linked hyper IgM with anhydrotic ectodermal displasia) (ass. with NF-kappaB)
- AR-AID deficiency
- AR-HIGM
- AR-UNG
- mutations in the cascade from CD40 onwards result in the same phenotype: people who are able to make IgM but unable to activate class switch recombination or affinity maturation
What is the molecular basis of Hyper-IgM?
- CD40L – X-linked, combined immune deficiency with cellular, humoral and innate defects. Defective B cell proliferation, no GC, no memory, defective DC activation
- CD40 – humoral immune deficiency, no GC, no memory (autosomal recessive)
- AID - GC form but no CSR, no switched memory, no SHM (autosomal recessive)
- AID-Cterm - GC form, no CSR, but SHM normal (autosomal dominant)
- UNG - Acts downstream of AID, GC, no CSR, no SHM (AR)
- NFkappaB signalling - abrogates signals from CD40, no GC, no CSR, no SHM (X-linked and AD)
Therapy for people with HIgM?
- IVIg (intragram)
- or for XL-HIGM bone marrow transplantation: often used for people who have the combined immune deficiencies, they get someone else’s hematopoietic stem cells and that reconstitutes their immune system with now fully functional immune cells - has a lot of its own issues but is a relatively effective immune therapy
What is the largest class of primary antibody deficiency?
CVID: common variable immunodeficiency
