development of B lymphocytes

goal of B cell development: phase 1

generate a diverse BCR through V(D)J recombination

goal of B cell development: phase 2

edit or eliminate self-reactive immature B cells (central tolerance)

goal of B cell development: phase 3

activate antigen specific B cells in secondary lymphoid tissues to differentiate into plasma and memory cells (peripheral activation)

what does bone marrow stromal cell provide

cytokines and chemokines that control B-cell differentiation and proliferation

what do mesenchymal stem cells do (MSCs)

help retain B cells in the bone marrow through CXCL12-CXCR4 interactions

what do stromal cells do

secrete IL-7, CXCL12, and other factors that guide B-cell progenitor survival and proliferation

what is IL-7 important for

early pro-B and pre-B stage development, B-cell lineage commitment

what attaches developing B cells to stromal cells

adhesion molecules (VLA-4/VCAM-1)

what happens once the pre-BCR forms

cells detach from stromal cells and progress towards independence

IL-7 signaling

drives proliferation and commitment to B-cell lineage 

Kit-SCF signaling

supports pre-pro-B and early pro-B survival

what do transcription factors do

control lineage commitment, stage progression, and gene rearrangement

what do E2A and EBF (transcription factors) do

upregulate RAG1/2, enable V(D)J

in B cell development, what is checkpoint 1 and where does it occur

• in the large pre-B cell 

• tests if H chain can pair with the surrogate L chain to form a pre-BCR 

• if this passes, cell proliferates and rearranges L chain

• if this fails, apoptosis 

in B cell development, what is checkpoint 2 and where does it occur

• occurs in immature B cell

• negative selection 1 (central tolerance)

• igM is expressed on surface

• tested for self-reactivity against self-antigens 

in B cell development, what is checkpoint 3 and where does it occur

• occurs from transitional B cell to mature B cell

• negative selection 2

• peripheral tolerance 

• begins coexpressing IgM and IgD as they mature 

what happens in the early pro-B cell

starts D to J rearrangement on H chain

what happens in the late pro-B cell

adds V to DJ rearrangement and completes H chain

what happens in the large pre-B cell

expresses a functional u H chain with surrogate L chain (forms pre-BCR)

what happens in the small pre-B cell

starts rearranging L chain (V to J)

what happens in immature B cell 

expresses IgM on cell surface 

what happens in mature B cell

co-expresses IgM and IgD through alternative splicing

what is the safest step of generating a diverse BCR chain

D-J rearrangement on both chromosomes

what is a high risk step of generating a diverse BCR chain

V-DJ rearrangement on first chromosome

what happens if V-DJ rearrangement on second chromosome fails 

50% die by apoptosis 

what happens to the functional H chains that survive in BCR chain development

signaled to survive and become pre-B cells

specifics of checkpoint 1 in large pre-B cell

• Pairs with surrogate L chain (5 or VpreB) to form pre-BCR (allows for testing of H chain functionality)
• Results in proliferation + progression to L chain rearrangement (small pre-B cell)
• Tests VDJ recombination of only 1 H chain per chromosome (allelic exclusion)
• preBCR signaling is antigen-independent

what happens once H chain passes its checkpoint

the cell moves onto rearrange L chain, first K then a, it gets four chances before apoptosis 

steps from large pre-B cell

• u H chain + surrogate L chain

• large pre-B cells stop H-chain recombination and start proliferating

• proliferation

• daughter cells become pre-B cells, ready to rearrange L chain 

steps from small pre-B cell

• rearrange K on 1st chromosome

• rearrange K on 2nd chromosome

• rearrange lambda on 1st chromosome 

• rearrange lambda on 2nd chromosome 

• apoptosis

gene rearrangement in small pre-B cell

Rearrangement starts with κ loci on both chromosomes:
•First tries κ on chromosome 1
•If nonproductive, tries κ on chromosome 2
If both fail, the cell switches to λ loci, again with two attempts.
Total = 4 chances to produce a functional light chain.
A successful light chain pairs with the μ H chain to form IgM
•IgMκ or IgMλ depending on which gene succeeded.

what does negative selection of immature B cells in bone marrow establish

central tolerance

checkpoint 2, negative selection 1

• Tests whether VDJ recombination of L chain pairs with established H chain to
produce a BCR that BCR that does not recognize self antigens expressed in the BM
• L chain exhibits allelic AND isotypic exclusion
• Antigen-dependent: antigens on stromal cells and soluble molecules in BM
• Central tolerance

checkpoint 2, negative selection round 1, right panel

Right panel: If IgM binds strongly to multivalent self-antigens, the cell is flagged as
autoreactive.
The B cell then gets a chance to “fix” itself via receptor editing:
•Reactivates RAG1/2 to rearrange a new L chain.
•If successful → new BCR replaces the old self-reactive one.
•If editing fails → the cell undergoes apoptosis.

checkpoint 2, negative selection round 1, left panel

Left panel: Immature B cell expresses surface
IgM after successful L-chain rearrangement.
If its receptor does not bind any self molecules in the bone marrow → it’s allowed to leave and migrate to the periphery as a transitional B cell

what is receptor editing

Additional Ig L chain rearrangements give immature B cells in BM additional
chances to replace autoreactive BCR with a non-reactive BCR.
When an immature B cell’s BCR binds a self molecule strongly:
1.The cell receives a signal through the BCR that it’s self-reactive.
2.This signal re-induces RAG1/2 expression, re-opening the light-chain locus.
3.The cell can:
1. Delete or replace the existing κ or λ light chain VJ segment with a new
rearrangement further downstream in the locus.
2. If κ is exhausted (all downstream Js used), it can still open the λ locus

replacement of L chain by receptor editing, step 1

• detection of self reactivity

• The immature B cell expresses surface IgM.

• When it binds strongly to a multivalent self-antigen, this
  causes strong cross-linking of BCRs. That signal halts development → “Wait, this BCR is self-reactive → fix it.

replacement of L chain by receptor editing, step 2

Reactivation of RAG1/2 and New L-Chain Rearrangement
•RAG genes are reactivated.
•The cell starts new V-J recombinations in the light-chain loci (usually κ first, then λ if needed).
•This process changes the antigen-binding specificity of the BCR

replacement of L chain by receptor editing, step 3

Test the New BCR
•If the new receptor no longer recognizes self, the cell survives and migrates to the periphery.
•If it still binds self strongly, it undergoes apoptosis

checkpoint 3, negative selection round 2

Tests whether BCR with central tolerance fails to recognize any new self-antigens expressed in the periphery (i.e. outside the BM)
• Antigen-dependent: expression by splenocytes and soluble molecules in spleen/circulating in the blood
• Peripheral tolerance

negative selection 2

•Transitional B cells express IgM high and IgD low at first.
•They encounter self-antigens that are only expressed outside the bone marrow: e.g.
tissue-restricted or soluble self-proteins.
•If their BCR binds these self-antigens with high affinity, they’re either:
1. get deleted by apoptosis, or
2.Rendered anergic (functionally silenced).
This ensures that any B cells that slipped past central tolerance but still recognize self don’t
activate in the periphery.

differences from negative selection in BM

1. BCRs that recognize self-molecules cannot undergo receptor editing
• Can no longer rearrange Ig L chain loci
2. Only possible outcomes are apoptosis or anergy

where do transitional B cells complete maturation

B- cell follicles in the spleen

marginal zone B cells

• Arise from weakly self-reactive B cells expressing high
levels of CD21.
• Localize to marginal zones of splenic white pulp.
• Function as first-responders to blood-borne antigens or
pathogens (rapid, T-cell-independent response)

BAFF (B cell activating factor)

• Produced by follicular DCs (FDCs).
• Binds BAFF-R on T1Bs to deliver essential survival and maturation signals.
• T1B: High IgM, no IgD; express BAFF-R
• T2B: IgM + IgD + BAFF-R + CD21 (complement receptor)

what happens to transitional T1 B cells that fail to enter follicles

• do not receive BAFF mediated survival signals

• die within 2-3 days of leaving the BM

how does IgM go to IgD

alternative splicing of primary transcript

Ig in immature B cell, and Ig in mature B cell

• immature: IgM

• mature: IgM + IgD

characteristics of IgM and IgD

•Both IgM and IgD are encoded by the same rearranged H-chain gene (VDJ region).
•The difference between them is generated after transcription, by alternative splicing of the primary RNA transcript.
•Immature B cells express only IgM on their surface.
•As B cells mature in the spleen, alternative splicing allows co- expression of IgM and IgD - both have identical antigen specificity but different constant (C) regions.