5 Themes in B cell development Tony DeFranco, 102109

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5 Themes in B cell developmentTony DeFranco,
10/21/09

• Checkpoints in B cell development feedback from
  Ig gene rearrangements
• Stromal environment/growth factors for B cell
  development
• Lineage commitment transcription factors and
  Notch signaling
• Central and peripheral tolerance of B cells
• 3 different types of mature B cells choice of
  cell fate and relative roles

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Overview of B cell development
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Theme 1 Ig rearrangement checkpoints forB cell
development
IgH unrearr DJ VDJ
VDJ VDJ IgL unrearr unrearr
unrearr rearranging VJ
(surrogate L chain)
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B cell developmentdistinctive cell surface
markers(mouse)
? chain expressed in cytoplasm
B220 -
CD43 (S7)
- - CD25
- -
- c-kit
- -
Human B cell precursors see Blom Spits 2006
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Pre-BCR signaling

• Pre-B cell transition is blocked by
• Deletion of JH or mm exon
• Knockout of Rag1, Rag2 or Scid (DNA-PKcs)
• Knockout of signaling components Iga? Igb, Syk ,
  triple Src family KO (lyn-fyn-blk-), Btk (human,
  X-linked agammaglobulinemia), or Blnk (adapter
  molecule)
• Conclusion Need pre-BCR and signaling from
  pre-BCR

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Pre-BCR checkpoint
Cell proliferation (IL-7-dependent) Change in
cell surface markers Turn off expression of
SLCs Change in targeting of Rag1/2
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Theme 2 Stromal environment and growth
properties of B cell precursors

• B cell precursors can be cultured in vitro
  (Whitlock-Witte culture system)
• Pro-B cells (?-) grow indefinitely only in
  contact with stromal cell layer from bone marrow
• Pre-B cells (?) will grow for a short period
  in response to IL-7 and in the absence of stromal
  cell contact
• Hypothesis pro-B cells fill up a niche of sites
  bound to the appropriate stromal cells and only
  if express the pre-BCR can they proliferate
  further and proceed down developmental pathway

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Notch and lymphocyte development

• Notch promotes (or stabilizes) commitment to the
  T cell lineage and away from the B cell lineage
• Loss of function experiments B cell development
  in thymus
• Gain of function experiments T cell development
  in bone marrow
• Notch signaling also seems to participate in
  later lineage decisions in T and B cells in B
  cells Notch signaling is required for formation
  of marginal zone B cells.

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Theme 3 Control of B cell development by
transcriptional regulators
E2A-/- EBF-/-
Pax5-/-
Knockouts of several transcription factors block
B cell development at discrete stages
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A hierarchy of transcription factors specifies B
cell fate
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Pax5 and commitment to the B cell lineage

• E2A and EBF are needed to turn on B cell specific
  genes including Pax5, which turns on additional B
  cell-specific genes

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Pax5 and Commitment to B cell lineage

• Culture pax5-/- bone marrow in vitro to get pro-B
  cell cultures
• See if the cells can differentiate into other
  hematopoietic lineages (add various growth
  factors)

Nutt et al. Nature 1999
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Pax5 and Commitment to B cell lineage
Nutt et al. Nature 1999
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Pax5 and commitment to the B cell lineage

• E2A and EBF are needed to turn on B cell specific
  genes including Pax5, which turns on additional B
  cell-specific genes
• Pax5 seems to act in two ways
• It promotes progression down the B cell lineage
  (expression of Iga, Blnk)
• It shuts off genes needed to go down other
  lineages (M-CSF receptor, pre-Ta, Notch1) or
  associated with other lineages (myeloperoxidase,
  perforin, etc.)

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Ikaros
Repression of genes needed to become myeloid cell
Repression of genes needed to become T cell or
myeloid cell
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Putting it all together growth factors
transcription factors in B cell development
Harinder Singh et al. 2005 Solid lines more
solid data Dashed lines less well established
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Putting it all together growth factors
transcription factors in B cell development
Singh et al. 2005 C/EBPa transcription factor
that directs macrophage development Notch1
directs T cell development GATA-1 directs
erythroid development
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Theme 4 Fate of self-reactive B cells
Periphery
Bone Marrow
Antigen Independent
Antigen Dependent
IgMµ
IgM
IgM
Pre BCR
IgM
IgD
Plasma Cell
pre-B
Immature
T2
T1
Mature
antigen encounter proliferation
autoreactive
autoreactive
autoreactive
deletion or editing
deletion or anergy
anergy (w/o T cell help)
Positive Selection
Negative Selection
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Receptor Editing Mechanisms
1. Upstream V? can rearrange to downstream J?
Vk
Jk
Ck
2. Upstream V? can rearrange to KDE (?deleting
element) deleting C? this would be followed
by a rearrangement of another light chain allele
KDE
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Receptor editing vs. clonal deletion

• Contact with antigen in bone marrow leads to
  maturational arrest (no exit from bone marrow)
  and receptor editing
• Contact with antigen in periphery leads to
  deletion
• The difference appears to be that bone marrow
  stromal cells promote survival to allow editing
  to occur. If apoptosis is blocked with caspase
  inhibitors, editing can occur in vitro. Also in
  vitro culture of immature B cells stromal cells
  can allow editing (Monroe et al.)

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Clonal deletion vs. clonal anergy

• Anti-lysosyme transgenic mice with high affinity
  antibody presence of soluble lysozyme either as
  transgenic or injected leads to anergy (Goodnow
  et al.)
• In contrast, membrane-bound form of lysozyme
  induces deletion
• Anti-DNA transgenics (autoantigen of lupus) mIg
  with high affinity for dsDNA results in strong
  editing and deletion but mIg with lower affinity
  leads to anergy (Weigert, Erikson)

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Characteristics of Anergic B cells

• Anergic B cells exhibit chronic low grade BCR
  signaling, further stimulation of BCR gives weak
  response (contrast to acute stimulation of BCR of
  naïve B cells).
• Anergic B cells have decreased survival in vivo,
  especially in competition with naïve B cells.
  This is due to decreased ability to respond to
  the survival factor BAFF.
• Anergic B cells localize to the edge of the T
  cell zone next to B cell follicles, same as
  acutely stimulated naïve B cells.
• Anergy in the presence of competent helper T
  cells is enforced by Fas killing. BCR signaling
  is too low to rescue B cells from FasL-induced
  killing. Autoantibody production in MRL/lpr mice
  (Fas-deficient) may result from defect here.
• NOTE B cell anergy is best thought of as a range
  of phenotypes from deep anergy to light anergy

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Mechanisms of B cell tolerance (summary)

• Immature B cells in bone marrow are very
  sensitive to antigen receptor editing and
  deletion
• Immature B cells in periphery (transitional B
  cells) can be deleted by antigen (strong signal)
• Immature or mature B cells in periphery
  moderate BCR signal move to T cell zone in
  absence of T cell help they become anergic.
• Anergic B cells can receive T cell help
  normally, but are killed by Fas unless the BCR
  can signal adequately to protect (anergic B cells
  have weak BCR signaling induced by the anergizing
  form of the antigen)

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Theme 5 3 types of mature B cells
B1, marginal zone, and follicular B cells
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Three types of mature B cells

• Recirculating follicular B cells (aka
  conventional B cells, B2 cells) circulate
  between LN follicles and blood size of
  population determined by BAFF levels
• Marginal zone B cells reside in marginal zone
  of spleen where they can respond to particulate
  antigen in blood (bacteria, etc.) also dependent
  on BAFF for survival
• B1 B cells prominent in peritoneal and pleural
  cavities, present in spleen, absent in lymph
  node. Produce natural antibody and also
  respond to T-independent antigens. (less
  dependent on BAFF)

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(No Transcript)
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Biological roles of three types of B cells
follicular
MZ FO
MZ B1
B1
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Marginal zone of the spleen
The spleen filters the blood, marginal zone B
cells are exposed to particles in the blood
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Signaling and maturation of B cells
The simplest interpretation moderate to strong
BCR signaling is needed to become mature B1 less
to become mature Follicular B cell and still less
to become MZ B cell (Note also need Notch2
signaling become MZ B cell)
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SM610 transgenic B cells develop and produce
autoantibody only in animals expressing Thy1
suggests positive selection by antigen
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BCR signaling and B cell fate
(maturation B1)
(folliculargtMZ?)