Themes in B cell development Tony DeFranco, 101708

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Themes in B cell developmentTony DeFranco,
10/17/08

• 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 tolerance of B cells
• Peripheral tolerance of B cells
• 3 different types of mature B cells choice of
  cell fate and relative roles

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Order of Gene Rearrangements to Create a
Functional IgH gene in a B cell
VH1
VH2
VH3
VH4
DH1
DH3
JH4
JH1
IgH gene in germ line
Rearrangement 1
DJH
Rearrangement 2
VDJH
IgH gene in B cell
Transcription, RNA splicing, Translation
Ig H chain
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B cell development as defined byIg gene
rearrangement status
Early lymphoid progenitor
Hematopoietic Stem cell
B Cell
Pre-B Cell
Pro-B Cell
VDJH ( surrogate L)
H L
DJH
Ig gene rearrangement
none
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B cell development Distinctive cell surface
markers
Hematopoietic Stem Cell
B Cell
Pro-B Cell
Pre-B Cell
B220 -
CD43 (S7)
-
- CD25 -
-
- c-kit
- -
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Checkpoint for Success of FirstGene Rearrangement
VpreB
l5
m
Iga/Igb
SIGNAL

• Pre-BCR m heavy chain surrogate light chains
  (l5 and VpreB) Iga/Igb

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Requirements for pro-B cell to pre-B cell
transition

• This 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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BCR signaling mechanism
adaptor
adaptor
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B cell development in human
Blom Spits 2006
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B cell development in human
Blom Spits 2006
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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 (m-) grow indefinitely only in
  contact with stromal cell layer from bone marrow
• Pre-B cells (m) 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 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 seems to be 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-
Hematopoietic Stem cell
Pro-B Cell
Pre-B Cell
B Cell
Rag1 Rag2 Iga, Igb l5, VpreB
Genes expressed
E2A binds to sites in Ig enhancers EBF binds to
site in Iga gene Pax5 binds to site in Iga gene
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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
Multivalent antigen in periphery only
Soluble antigen
Multivalent antigen in bone marrow
Maturational arrest Receptor editing
Anergy
Deletion
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Checkpoints in B cell tolerance induction
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 Vk can rearrange to downstream Jk
Vk
Jk
Ck
2. Upstream Vk can rearrange to KDE (k deleting
element) deleting Ck 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 MRLlpr mice
  (Fas-deficient) may result from defect here.

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Mechanisms of B cell tolerance

• 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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Theme 5 Three types of mature B cells

• Recirculating follicular B cells (aka
  conventional B cells, B2 cells) circulate
  between LN follicles and blood
• Marginal zone B cells reside in marginal zone
  of spleen where they can respond to particulate
  antigen in blood (bacteria, etc.)
• 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.

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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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Biological roles of three types of B cells
follicular
MZ FO
MZ B1
B1
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Effect of BCR signaling mutations on development
of mature B cell types
Presence of mature B cell type
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Signaling events required for B1 development
Mutations of components in orange boxes no B1
cells
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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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Antigen-dependence for B cell maturation

• Immature B cells that dont experience a strong
  BCR signal in bone marrow exit to the periphery.
• These cells have an average lifespan of about 7
  days, but about 20 enter the long-lived
  recirculating B cell population
• Entry into the long-lived follicular B cell pool
  is competitive and requires BCR signaling (like
  positive selection of thymocytes)

IgMhi IgDlo HSAhi
IgMint IgDhi HSAlo
BCR signal
No BCR signal
Immature B cell in bone marrow
Immature B cell in periphery (transitional B
cell)
Mature follicular B cell in periphery
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Tolerance of B1 B cells

• To study autoimmune hemolytic anemia, an Ig
  transgenic mouse was made with anti-RBC
  specificity (Honjo et al.)
• There are no transgene-positive B cells in blood,
  spleen or lymph node, but there are in the
  peritoneum and they are CD5
• About 50 of the animals develop autoimmune
  hemolytic anemia and the rest are healthy.
  Apparently, infections can activate these B cells
  to produce the autoantibody
• Injection of RBC into the peritoneum causes rapid
  apoptosis of the transgenic B cells and cures the
  mice
• In vitro, B1 cells do not proliferate to anti-IgM
  stimulation, but instead undergo apoptosis
  Injection of anti-IgM into the peritoneum induces
  apoptosis of the B1 cells there (How are B1
  cells activated?)

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Level of signaling and fate of immature B cell
maturation arrest receptor editing
deletion
anergy
maturation (B1)
Strength of BCR signal
maturation (follicular)
lack of maturation (or marginal zone?)