Lecture 12 - Development of B Lymphocytes

the goal of B cell development phase 1

generate a diverse BCR through V(D)J recombination

the goal of B cell development phase 2

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

the 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 is involved in phase 1 of B cell development

B-cell precursor rearranges its immunoglobulin genes

what is involved in phase 2 of B cell development

immature B cell bound to self cell-surface antigen is removed from the repertoire by receptor editing or apoptosis

what is involved in phase 3 of B cell development

mature B cell bound to foreign antigen is activated

activated B cells give rise to plasma cells and memory cells

what supports early B-cell survival and differentiation

bone marrow and stromal cells

what do stromal cells secrete and what do they do

stromal cells secrete IL-7, CXCL12, and other factors that guide B-cell progenitor survival and proliferatoin

what is IL-7 especially important for

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

what do mesenchymal cells do

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

early B cell development requires signals from what

bone marrow stromal cells

in the earliest stages (up to pre-B cell), developing B cells are physically attached to stromal cells through ______

adhesion molecules (VLA-4/VCAM-1)

stromal cells provide survival and differentiation signals through _____

IL-7 and stem cell factor (SCF)

once the pre-BCR forms, what happens

B cells detach from stromal cells and progress toward independence

IL-7 signaling

drives proliferation and commitment to the B-cell lineage

Kit-SCF signaling

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

transcription factors do what

control lineage commitment, stage progression, and gene rearrangement

• E2A and EBF → upregulate Rag1/2 → enable V(D)J

stages in B-cell development

checkpoint #1

at large pre-B cell stage

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

• pass→ proliferate and rearrange L chain

• fail → apoptosis

checkpoint #2

at immature B cell stage

negative selection 1 - central tolerance

• expresses IgM on surface

• Tested for self-reactivity against self-antigens

checkpoint #3

negative selection 2

• peripheral tolerance

• begin co-expressing IgM and IgD and they mature

where does antigen-independent B cell development occur

a) thymus

b) spleen

c) bone marrow

d) lymph node

c) bone marrow

development of a B-lineage cell proceeds through several stages marked by what

rearrangement and expression of the Ig genes

early pro-B cell

starts D→ J rearrangement on H chain

late pro-B cell:

adds V→ DJ rearrangement (completes H chain)

large pre-B cell:

expresses a functional u H chain with surrogate L chain (VpreB +λ5) → forms pre-BCR

small pre-B cell

starts rearranging L chain (V→ J)

immature B cell

expresses IgM on surface

mature B cell

co-expresses IgM + IgD through alternative splicing

checkpoint 1:

Pre-BCR test (H chain works)

checkpoint 2

L chain works, IgM on surface → test for self-reactivity

checkpoint 3

IgM + IgD expression → mature and leave bone marrow

Ig L and H chains undergo recombination at distinct phases of B cell development

generation of a diverse BCR begins with the rearrangement of the H chain

checkpoint #1

Pre-BCR testing

• pairs with surrogate : chain (λ5 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

Pre-B cell receptor

• composed of μ H chain + surrogate L chain (λ5 + VpreB), with Iga/IgB for signaling

• signals without antigen - ligand-independent activation

• outcomes of signaling:

i. stops heavy-chain rearrangement (allelic exclusion)

ii. induces proliferation of pre-B cells

iii. triggers light chain (k then λ) gene rearrangements

iv. turns off surrogate-chain transcription

Pre-TCR

• composed of B chain + pTa (surrogate a chain) and CD3 complex for signaling

• also ligand-independent - tests whether B chain works

• outcomes:

i. stops further B rearrangement (allelic exclusion)

ii. induces proliferation of pre-T cells

iii. stimulates a-chain rearrangement

iv. turns on CD4 and and CD8 expression (→ DP stage)

v. shuts off pTa transcription after checkpoint

which of the following correctly pairs a developmental stage with its key event?

a) early pro-B cell - expression of IgM

b) late pro-B cell - heavy chain VDJ arrangement

c) large pre-B cell - light chain rearrangement

d) immature B cell - pre-BCR expression

b) late pro-B cell - heavy chain VDJ rearrangment

at the large pre-B cell stage, what does the μ heavy chain pair with to form the pre-B cell receptor?

a) λ light chain

b) k light chain

c) surrogate light chain (VpreB + λ5)

d) Iga/IgB complex only

c) surrogate light chain (VpreB + λ5)

gene rearrangement in small pre-B cell

once H chain passes its checkpoint, the cell now moves on to rearrange L chain - first k, then λ. It gets FOUR chances before apoptosis

how many chances does the L chain have to rearrange before apoptosis

FOUR

large pre-B cells stop H chain recombination and start proliferating, what do these daughter cells become

small pre-B cells, ready to rearrange L-chain

steps of L chain rearrangement

rearrangement starts with k loci on both chromosomes:

• first tries k on chromosome 1

• if nonproductive, tries k on chromosome 2

if both fail the cell switches to the 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

• IgMk or IgMλ depending on which gene succeeded

what establishes central tolerance

negative selection of immature B cells in bone marrow establishes 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 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 B<

• central tolerance

binding to self molecules in bone marrow can lead to what

receptor editing or to death of immature B cells

right panel shows what

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

left panel shows

immature B cell expresses surface IgM after successful L chain rearrangement

if its receptor does not bind any self molecules in the bone marrow → its allowed to leave and migrate to the periphery as a transitional B cell

receptor editing

additional Ig L chain rearrangements give immature B cells in BM additional chances to replace autoreactive BCR with a non-reactive BCR

what happens when an immature B cell’s BCR binds a self molecule strongly

1. the cell receives a signal through the BCR that it is 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 k or λ light chain VJ segment with a new rearrangement further downstream in the locus

2. if k is exhausted (all downstream J’s used), it can still open the λ locus

what can rescue some self-reactive B cells by changing their antigen specificity

replacement of L chains by receptor editing

steps of receptor editing

1. detection of self-reactivity

2. reactivation of RAG1/2 and New L-Chain rearrangement

3. test the new BCR

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”

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 k first, then λ if needed)

• this process changes the antigen binding specificity of the BCR

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

receptor editing does what

gives an autoreactive B cell a second chance to fix its receptor

what establishes peripheral tolerance

negative selection of transitional B cells in spleen establishes peripheral tolerance

what happen to immature B cells that survive central tolerance in the bone marrow (no strong self reactivity)

they arent fully safe yet.

once they leave the marrow, they enter the spleen as transitional B cells; this is where they face peripheral tolerance

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 splees/circulating in the blood

• peripheral tolerance

transitional B cells express what at first

IgM high and IgD low at first

what happens to transitional B cells

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

in what ways does peripheral tolerance differ from central tolerance

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

what is the fate of immature B cells in the bone marrow that strongly bind multivalent self-antigen?

a) proliferation and class switching

b) receptor editing or apoptosis

c) anergy

d) migration to spleen for positive selection

b) receptor editing or apoptosis

where do transitional B cells complete their maturation

in 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

what is the role of BAFF in B cell maturation?

a) induces heavy chain recombination

b) provides survival signals to transitional B cells entering follicles

c) mediates receptor editing in immature B cells

d) promotes class switching to IgG

b) provides survival signals to transitional B cells entering follicles

alternative splicing of primary transcript to generate what

IgM + IgD

immature B cell -

Mature B cell -

immature B cell - IgM

mature B cell - IgM + IgD

both IgM and IgD are encoded by what

the same rearranged H-chain gene (VDJ region)

what is the difference between IgM and IgD

the differenc between them ia generated after transcription, by alternative splicing of the primary RNA transcrip

what do immature B cells express on their surface

only IgM

as B cells mature in the spleen, what allows co-expression of IgM and IgD

alternative splicing allows co-expression of IgM and IgD - both have identical antigen specificity but different constant regions