Multipotent progenitors Can not self-renew Can form CLP & CMP

Lecture 18 - B cells - Development Flashcards by Eleanor Nicklason

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

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What are so called ‘effector B cells’?

Plasma cells

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Why is the bone marrow a ‘dangerous place’?

Prone to disease

e.g. irradiation affecting the bone marrow could lead to leukaemia

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Until when can new B cells be produced?

Throughout life

NB T cells: up to 15 years

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What is the BCR?

Antibodies bound to the surface of B cells

Antibody: secreted soluble BCR

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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

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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

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What is the reason for the name of plasma cells?

Often found in the plasma

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What are the functions of Ab?

Direct neutralisation
Opsonisation
ADCC
Complement activation

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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

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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

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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)

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Compare pre and pro B cells

‘Pro’ comes before ‘pre’

Pro: progenitor
Pre: precursor

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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

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What is the estimated number of antibodies?

1 x 10^6

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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

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What are MPPs?

Multipotent progenitors

Can not self-renew
Can form CLP & CMP

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What is CLP?

What can it form?

Common lymphoid progenitor

Forms:
• Pre-NK cells
• Pre-B cells
• Thymocyte (goes on to form T cells)

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Compare committed and uncommitted cells in the BM

Uncommited:
• HSC
• CLP
• CMP

Committed:
• B cells etc.

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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

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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

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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

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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) 1. Light chain is rearranged and expressed as IgM 2. No strongly binding antigen encountered 3. Delta heavy chain expressed 4. 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