Lecture 18 - B cells - Development Flashcards
Where do B cells mature?
Give some features of this location
Where did they get their name?
Bone marrow
• Secure (encased in bone)
• Anatomically distinct compartment
Bursa of Fabricius:
• birds generate their immature B cells in this organ
What are so called ‘effector B cells’?
Plasma cells
Why is the bone marrow a ‘dangerous place’?
Prone to disease
e.g. irradiation affecting the bone marrow could lead to leukaemia
Until when can new B cells be produced?
Throughout life
NB T cells: up to 15 years
What is the BCR?
Antibodies bound to the surface of B cells
Antibody: secreted soluble BCR
Describe the events after antigen engagement of the BCR
1. Engagement of native antigen w/ BCR (NB do not require presentation from MHC or other molecules 2. Proliferation 3. Differentiation into plasma cells 4. Secretion of antibodies
Which sort of antigen are B cells really good at recognising?
Why is this good?
- Higher molecular weight
- Folded proteins in native form:
- toxins
- Do not require co-stimulation
This is good because it means that the B cells can recognise the antigen much more quickly; doesn’t need time for processing and presentation
What is the reason for the name of plasma cells?
Often found in the plasma
What are the functions of Ab?
- Direct neutralisation
- Opsonisation
- ADCC
- Complement activation
What is the life span of B cells?
Where do memory B cells reside?
Around 10 years
However, the memory response against a given antigen will last up to 60 years
Reside in the BM, LNs, spleen
Describe development of B cells
In BM:
- Pre-pro B cell
- Pro B cell
- Pre B cell
- Immature B cell
Periphery:
- Mature B cell (naïve)
6a. Plasma cell
6b. Memory B cell
At what stage is B cell development complete?
Compare the following terms:
• Naïve B cell
• Mature B cell
• Immature B cell
After B cells leave the BM, they are considered mature = ‘naïve’
A naïve B cell is mature
The order is:
- Immature (still in BM)
- Mature = naïve (secondary lymphoid organs, yet to experience antigen)
Compare pre and pro B cells
‘Pro’ comes before ‘pre’
Pro: progenitor
Pre: precursor
Describe McFarlane Burnet’s Clonal Selection Theory
- Pool of naïve B cells in lymph nodes
- Antigen drains into lymphatics, and into lymph nodes
- B cells come into contact w/ the antigen
- The single B cell that is specific for the Ag binds it with its BCR
- BCRs cluster, binding Ag, cross linking
- Intracellular activation signal in B cell
- Migration, proliferation of millions of clones (all with the same specificity)
- Differentiation into plasma cells, which release the Ab into the blood & tissues
What is the estimated number of antibodies?
1 x 10^6
What is the lineage of B cell development?
- Pluripotent Haematopoeitic stem cell (HSC)
- Multipotent progenitor (MPP)
- Common lymphoid progenitor (CLP)
- Pre-B cell
- B cell
What are MPPs?
Multipotent progenitors
- Can not self-renew
- Can form CLP & CMP
What is CLP?
What can it form?
Common lymphoid progenitor
Forms:
• Pre-NK cells
• Pre-B cells
• Thymocyte (goes on to form T cells)
Compare committed and uncommitted cells in the BM
Uncommited:
• HSC
• CLP
• CMP
Committed:
• B cells etc.
What is the default of a developing cell?
Why is this important?
To die
To survive, it has to receive a positive signal
It must assemble a functional B cell receptor to receive this positive signal
The B cells are only useful to the body if they have a functional B cell receptor
What are stromal cells?
Where are they found?
Describe their function
Critical for B cell development
• Often provide the critical positive signals
Located:
• Bone Marrow
• Lymph nodes
They are not white blood cells, rather tissue cells
Function: Release of essential factors for bone development: • IL-7 • CXCL12 • FLT3 ligand • SCF
Describe the location inside the BM where B cells develop
Endosteum: inner lining of central sinus in long bone
Cells first develop here, then move along the stromal cell into the centre of the sinus
Endosteum → Middle of stromal cells → Central sinus → into vasculature, & circulation
Describe the ordered events in B cell development in the BM
- MPP attracted to BM stromal cell w/ chemokines (CXCL12)
- Stromal cell releases factors (IL-7, FLT3 ligand)
- Development into CLP; migration along stromal cells into the centre of the sinus
- Exposed to the next set of signals; IL-7 released by stromal cells, IL-7R expressed by CLP
- Developing B cells migrate further into the centre along stromal cells
NB Integrins are important for attaching the developing B cells to the stromal cells
What is the role of IL-7R?
When is it expressed
What happens w/o its expression?
Receptor for IL-7
IL-7R expressed at CLP stage
IL-7 signalling through the receptor helps CLP → pro B cell
B cells don’t develop without it
Compare:
• pro-B cells
• Large pre-B cells
• Immature B cells
Pro-B cell:
• No functional protein expressed
• DJ has rearranged
Large Pre-B cell:
• Expression of:
• Heavy chain + SLC
• Have pre-B cell receptor
Immature B cell:
• Expression of:
• Heavy chain + Light chain
What is allelic exclusion?
Only one of the heavy chain alleles is rearranged
NB we have two alleles; one on each chromosome
The other allele remains in germ line configuration
What is the heavy μ-chain?
Successfully rearranged heavy chain for IgM (i.e. the first heavy chain made)
Describe the pre-B cell checkpoint
It is important to check that the rearrangement of the heavy chain is successful before we progress on to rearrangement of the light chain
Surrogate light chain binds to the heavy chain
If the heavy chain can form a complex with the SLC, a success signal will be transmitted (with Igα and Igβ)
This also finalises allelic exclusion.
If the pre-B cell receptor is not signalling, we still have the other allele, which will then undergo arrangement
Which genes are important for the expression of the surrogate light chain?
E2A+EBF
What is the important thing about pre-BCR engagement?
It is antigen independent
Do not need antigen for the receptor to be engaged
When do we call it a pre-B cell?
Once it has passed the pre-B cell checkpoint
How many potential alleles of the light chain before rearrangement?
4; two of each κ and Lambda
Why does it take 3 months for Bruton’s XLA to emerge?
IgG from the mother cross the placenta and provide protection for the first three months
What is isotopic exclusion?
Only one of κ and lambda is expressed
Describe the expression pattern of RAG
Two peaks:
• Pre-pro B cell & Pro B cell: heavy chain rearrangement
• Small Pre B cell: light chain rearrangement
What is PAX5?
When is it important?
Transcription factor, vital for B cell development
Expressed around the pre B cell, immature B cell stage
Elimination of PAX5 from B cell, development of B cells from pro- to pre- can be reversed
Who is Sir Gustav Nossal?
PhD student of McFarlane Burnet
• Confirmed clonal selection theory, through proving allelic exclusion, i.e.
One cell produces only one antibody
His experiment:
• Two different rabbits
• Made a chimera mouse by irradiating it, then giving it a bone marrow transplant from two different types of rabbits.
• Two different types of B cells in mouse
• However, there was only ever one type of Ab produced; either Ab from one of the donors, or from the other
• This proves that one B cell only makes one antibody
Describe the process of selection
Negative selection:
• If the B cell encounters a strongly cross-linking antigen in the BM
→ Clonal deletion
→ Receptor editing
List the checkpoints of B cell deveopment
- V-DJ rearrangement (heavy chain)
* V-J rearrangement (light chain)
What is µ:κ?
Ig that has used the kappa chain
What is the result of the spurt of proliferation that occurs after successful rearrangement of the heavy chain?
Multiple clones have the same heavy chain.
Each clone then goes on to rearrange the light chain differently.
They do not end up with the same iodiotype
How does allelic exclusion occur?
Not known at the moment
What is E2A important for?
At what stage is it expressed?
Surrogate light chain expression
Expressed at CLP stage
Forms a complex with EBF, which is vital at the pre B cell stage
When is FLT3 expressed?
What is its role?
Expressed at the MPP stage of B cell development
Induces expression IL-7R at the CLP phase
Compare the Immunoglobulin expressed on the surface of immature and mature B cells
Immature: IgM+
Mature: IgD+, IgM+
What releases IL-7?
Bone marrow stromal cells
What effect does IL-7 bring about?
Growth factor for all cells of the lymphoid lineage (B cells, T cells, NK cells)
What is the B cell equivalent of naïve T cells?
What are the features of these?
Naïve T cells = Immature B cells
They have both completed their development and moved out into the periphery.
Neither have yet experienced their antigen
At which stages of development do B cells express IL-7R?
Up until the immature B cell stage
At which stage does the following occur:
• V(L)-J(L)
• V(H) - DJ(H)
• D(H) - J(H)
V(L)-J(L): small pre B cell
V(H) - DJ(H): pro B cell
D(H) - J(H): Pre-pro B cell
At which stage is the heavy chain rearrangement complete?
Large pre B cell
At which stage is the light chain rearrangement complete?
Immature B cell
At which stage do B cells leave the BM?
Once they are immature (i.e. heavy and light chains rearranged)
When is IgD expressed?
Describe the process
Mature B cells (i.e. naïve B cells)
- Light chain is rearranged and expressed as IgM
- No strongly binding antigen encountered
- Delta heavy chain expressed
- IgD expressed on cell surface
Which factors do BM stromal cells release that are vital for B cell development?
What are some other factors vital for B cell development?
- IL-7
- CXCL12
- FLT3 ligand
- SCF
- IL-7R expression
- RAG
- E2A+EBF (SLC expression)
Describe Receptor editing
- RAG is still active after light chain rearrangement
- It can rescue cells form clonal deletion:
- Continuing light chain rearrangement
- New receptor specificity is now expressed
- If receptor is still self-reactive, the B cell is deleted
