B Cell Development

Question 1

Describe B cell haematopoesis

Answer 1

1. The HSC first differentiates into a multipotent progenitor cell (MPP) → can produce both myeloid and lymphoid progenitors
   • Express cell surface RTK known as FTL3
   • FLT3 binds FTL3 ligand expressed on stromal cells
   • Signalling through FTL3 is needed for differentiation to the next stage
2. The cell forms the common lymphoid progenitor (CLP)
   • Accompanied by expression of the receptor for IL-7, induced by FTL3 signalling together with the TF PU-1
   • IL-7 is essential for the growth and survival of developing B Cells
   • Stem cell factor stimulates the growth of HSCs and the earliest B lineage progenitors
3. This gives rise to the earliest committed B cell lineage, the pro B cell
   • Development of the pro B cell is specified by induction of the B-lineage specific TF E2A
   • E2A then induces the expression of EBF (early B cell factor)
   • E2A and EBF work together to drive the expression of proteins that determine the pro-B cell state.
4. Next comes the pre B cell
5. Next comes the Immature B cell

• CXCL12 produced by stromal cells may help retain developing B cell precursors in the bone marrow
• TSLP may promote B cell development in the embryonic liver.
• As B cell lineages mature, they migrate within the marrow, remaining in contact with the stromal cells
• The earliest stem cells lie in the endosteum
• As they mature, they move towards the sinus
• The final stage of development, from immature to mature B cells occur in the peripheral lymphoid organs.

Question 2

What surface markers distinguish different stages of B cell development?

Answer 2

• FTL3 is expressed on HSCs and the CLP
• The earliest B lineage surface markers are CD19 – expressed throughout B cell development
• A Pro-B cell is also distinguished by Kit and the IL-7 receptor
• A late pro B cell starts to express CD24 and the IL-2 receptor alpha chain CD25
• A pre B cell is distinguished by the expression of BP-1, and Kit and IL-7R are no longer expressed

Question 3

How does gene rearrangement on the heavy chain locus proceed?

Answer 3

1. First arrangement to take place occurs in the early pro B cell and involves the locus that contains D gene segments (the Ig heavy chain) and the joining of a D segment to a J
   − Expression of E2A and EBF in the early pro-B cell induce the RAG-1 and RAG-2
   − Thus, E2A and EBF allow initiation of V(D)J recombination at the heavy chain locus, and the expression of a heavy chain
   − Another protein induced by E2A and EBF is Pax5 → targets of Pax5 include gene for the the B-cell co-receptor CD19 and the gene for Ig-alpha, a signaling cmpoment of the pre-B cell receptor and the B-cell receptor.
   − In the absence of Pax5, B cell development fails to continue, but can be induced to give T cells.
   − So Pax5 is essential for commitment of the pro-B cell to the B cell lineage

• The initial D to J rearrangements in the Ig heavy chain locus typically occur on both alleles, at which point the cell becomes a late pro –B cell.
− Most D to J joins in humans are potentially useful, because most human D gene segments can be translated in all 3 reading grames
− Thus, there is no special mechanism needed for distinguishing successful D to J joins, and no need to ensure only one allele undergoes rearrangement
− Indeed, given the likelihood of failure at the later stages, starting off with two successfully rearranged D-J sequences is an advantage. }

2. To produce a complete Ig heavy chain, the late pro-B cell now proceeds with a rearrangement of a V segment to e DJ sequence.
   − This occurs only on one chromosome
   − A successful rearrangement leads to the production of an intact u-heavy chain, after which V-DJ recombination ceases and the cell becomes a pre-B cell
   − In at least 2/3 cases, the first V-DJ rearrangement is non-productive, and rearrangement then occurs on the other chromosome
   − The chance of generating a pre-B cell is theoretically 55% (actually lower, as the V gene segment contains pseudogenes). Those pro-B cells that do not develop a u-chain are lost.
   − The diversity of the B cell antigen repertoire is enhanced at this state by TdT → adds N-nucleoides at the joints between rearranged gene segments.

Question 4

What is the importance of the pre-B cell receptor produced after heavy chain rearrangement?

Answer 4

The pre-B cell receptor produced after V-DJ recombination tests for successful production of a heavy chain and signals transition into the pre-B cell stage.
− The imprecise nature of V(D)J recombination produces increased diversity, but also results in many unsuccessful rearrangements.
− Pro-B cells synthesise two invariant ‘surrogate’ light chains (because the light chain gene segments have not yet been re-arranged), and pair these with the u- heavy chain to form the pre-B cell receptor.
− The surrogate chains are encoded by nonrearranging genes separate from the antigen receptor loci
− Their expression is induced by E2A and EBF
− One is called lambda-5 and the other is the VpreB
− Ig-alpha and Ig-beta are also required (these are expressed from the pro-B cell stage until the death of the cell)
− The assembly of the pre-B cell receptor signals to B cells that a productive V-DJ gene rearrangement has been made.
− Pre-B cell receptors are thought to interact with each other, forming dimers that generate signals → these signals halt gene rearrangement of the heavy chain locus and allow the cell to become sensitive to IL-7 (induces proliferation and transition to large Pre-B Cel

• As well as inhibiting further heavy chain locus rearrangement, the pre-B cell receptor enforces allelic exclusion:
− Successful rearrangements at both heavy chain alleles could result in a B cell producing two receptors of different antigen specificities
− To prevent this, pre-B cell receptor signaling enforces allelic exclusion – only one of the two alleles is expressed
1. Reduces V(D)J recombinase activity by reducing expression of RAG genes
2. Causes the degredation of the RAG genes
3. Reduces the access of the heavy chain locus to the recombinase machinery
− Rag activity will be switched on later on to carry out light chain rearrangement.

Question 5

Describe rearrangement at the light chain locus

Answer 5

Transition from pro to large pre B cell accompanied by several rounds of cell division
− A large pre-B cell therefore gives rise to several small pre-B cells
4. RAGs are produced again, and rearrangement at the light chain locus begins
− Light chain rearrangement also exhibits allelic exclusion, with rearrangement occurring only one allele at a time.
− The light chain loci lack D segments, and rearrangement occurs by V-J joining
− Repeated rearrangements can occur using unused segements on one chromosome, before initiating rearrangements on the second chromosome (if no functional light chain is made)
− This greatly increases the chance of light chain produciton, especially as there are two different light chain loci (kappa and lambda) on each chromosome.
− As well as allelic exclusion, light chains also display isotype exclusion → only one type of chain, kappa or lambda, is expressed.
− Kappa tends to be rearranged first

Question 6

Explain B cell central tolerance

Answer 6

No self reaction:
• Immature B cells with no strong reactivity to self antigens are allowed to mature

Clonal Deletion/Receptor Editing
• Clonal deletion (removal of particular antigen specificity from the repertoire) seems to predominate when the interacting self antigen is multi-valent and strongly cross-linking.
• Not all lymphocytes undergo clonal deletion, some undergo receptor editing
• Interval before cell death during which the B cell can be resuced by further gene rearrangement
• Strong cross-linking of the IgM ensures RAG gene expression continues
• light chain rearrangement can continue → these secondary rearrangements can resuce immature self-reactive B cells by deleting the self reactive light chain and replacing it with a new one.
• If the new light chain is non-autoreactive, B cell continues normal development
• If it is autoreactive, rearrangement continues until a non-autoreactive one is produced, or segments are exhausted and the B cell undergoes apoptosis
• Not clear whether receptor editing occurs at the heavy chain
• No available D segments at a rearranged heavy chain locus, so a new rearrangement cant occur by the normal mechanism
• Instead, a process of Vh replacement may occur

• It has always been thought that RAG gene turn-off results in allelic exclusion. The ability of cells to keep them turned on to undergo receptor editing suggests that it may not be the sole method.

Anergy
• Immature B cells that encounter more weakly cross-linking antigen of low valence respond differently
• They tend to be inactivated, entering a state of permanent un-respinsiveness (anergy) but do not immediately die
• Anergic B cells cannot be active by their specific antigen.
• Migration of anergic B cells to the LN is impaired
• They will eventually die as they cannot get survival signals from T cells

Clonal Ignorance
• Remain in as state of immunological tolerance
• Have affinity for self-antigen, but do not sense and respond to it
• Antigen may not be accessible to the B cell, it may be in low concentration, or it may bind too weakly
• They are not however, inert → may be activated in times of inflammation or if the self antigen reaches an unsually high concentration.

Question 7

Explain B cell peripheral tolerance

Answer 7

• Not all potential self antigens are expressed in the lymphoid organs → some are highly tissue specific, or compartmentalized
• Newly emigrated self-reacitve lymphocytes that encounter their specifc autoantigen in the periphery must be eliminated or inactivated
• In the absence of infection, newly mature B cells that encounter a strongly cross-linking antigen in the periphery will undergo clonal deletion
• There is no receptor editing – they are mature and cant rearrange gene loci any more
• As with immature B cells, mature B cells that encounter and bind abundant soluble antigen become anergised
• The question arises: if the encounter of a mature naïve lymphocyte with a self antigen leads to death or anergy, why does this not happen to a lymphocyte that recognizes a pathogen antigen?
• Infection sets up inflammation, which induces expression of co-stimulatory molecules and cytokine production that promote lymphocyte activation

B Cell Maturation
Immature B cells arriving in the spleen turn over rapidly and require cytokines and positive signals through the B-cell receptor for maturation:
• Immature B cells express high levels of IgM but little IgD, mature B cells express low IgM and high IgD
• the daily output of new B cells form te bone marrow is 5-10% the pool of lymphocytes in the steady state peripheral pool
• The size of this pool remains constant, so the stream of new B cells needs to be balanced by the removal of an equal number of B cells
• However, the majority of B cells in the periphery are long-lived, only 1-2% die each day
• Most B cells that die are in the rapidly turning-over immature B cells, of which more than 50% die each day.
• Seems to be due to competition between peripheral B cells to access the follicles in peripheral lymphoid tissues
• If they don’t enter a follicle, their passage through the periphery is altered and they die
• Peripheral B cells also include memory B cells → competition for follicular entry favours these, as mature B cells have undergone phenotypic changes that make their access to the follcile easier, eg, they express CXCR5, the receptor for CXCL13 expressed by FDCs

Question 8

What other types of B cell are there?

Answer 8

B1 Cells
• 5% of all B cells
• High IgM and low IgD
• Called B1 because first to appear during fetal development
• Found primarily in the peritoneal and pleural cavity fluid
• Innate-like lymphoid cell - don’t require T cell help
• certain environemtnal antigens and autoantigens are thought to drive their expansion and maintenance, eg) phosphocholine found on the surface of bacteria that colonise the gut.
• Not clear whether they arise from a unique precursor cell, or the same as B2 cells → evidence favours a selection step, rather than their being distinct lineages.

Marginal Zone B Cells
• Reside in the marginal sinus of the white pulp in the spleen
• Seem to be resting mature B cells, yet have a different set of surface proteins:
• Lower CD3 (receptor for IgE)
• High levels of the MHC-I like molecule CD1, and CR2 and 3
• Restricted antigen specificities – bias towards self antigen and common bacterial antigen
• May be adapted to provide a quick response if the bacterial antigen enters the bloodstream
• Functionally and phenotypically resemble B1 cells

Functions of these cells:
• Still being clarified
• Locations suggests a role for B1 in defending the body cavity and marginal zone in defense against bacteria that enter the blood stream
• Restricted repertoire seems to equip them for function in the innate immune response
• They seem to have evolved to recognize common bacterial antigens → allowing them to contribute to the very early phase of the adaptive immune response
• IN practice, found that B1 cells make little contribution to the adaptive immune response, but contribute strongly to some antibody responses against carbohydrate antigens..
• A large proportion of IgM normally circulating in the blood derives from B1 cells → natural antibodies