B cell development
Tony DeFranco, 10/29/14
5 Themes in B cell development
+ Ig class switch and somatic mutation
Theme 1: Checkpoints in B cell development: feedback
from Ig gene rearrangements
Theme 2: Bone marrow microenvironment
Theme 3: Lineage commitment: transcription factors
Theme 4: Central and peripheral tolerance of B cells
Theme 5: 3 different types of mature B cells
Molecular mechanisms of class switch recombination and
somatic hypermutation: Activation-induced cytidine
deaminase (AID)
Lymphocyte Development
Lymphocyte development is designed
to generate functional lymphocytes
with useful antigen receptors
that are not self-reactive
Much of what happens during lymphocyte
development is designed to improve the
efficiency of adaptive immunity
B cell Development: Relevance
• Immunodeficiencies that affect B cell
development
• B cell malignancies (pre-B ALL, etc.)
• Alterations in B cell tolerance may
underlie some autoimmune diseases
• B cell development is an especially well
understood example of mammalian cell
development
Overview of B cell development
Theme 1: Ig rearrangement checkpoints for
B cell development
IgH unrearr
IgL unrearr
DJ
VDJ
unrearr unrearr
(surrogate L chain)
VDJ
VDJ
rearranging VJ
B cell development:distinctive cell surface markers
(mouse)
 chain
expressed in
cytoplasm
B220
CD43 (S7)
CD25
c-kit
+
+
+
+
+
++
+
-
+++
-
Human B cell precursors: see Blom & Spits 2006
Pro-B to pre-B checkpoint requires Pre-BCR
signaling
Surrogate light chain (VpreB + l5):
• Expressed only in proB/preB cells
• Triggers signaling (self
aggregation?Ligand present on
stromal cells?)
• Expression turned off by pre-BCR
signaling
Pre-BCR checkpoint regulates V(D)J recombination
Controlled by chromatin accessibility; epigenetic marks due to
histone modifications match those associated with transcription
Theme 2: the bone marrow
microenvironment
• Role of IL-7 in murine B cell development
• Lack of Notch ligands in the bone marrow favor B
lineage development (Notch ligands in thymus are
important for commitment to the T lineage)
Theme 2: the bone marrow
microenvironment
Based on in vitro culture experiments, the bone marrow
microenvironment has several key properties:
• Pro-B cells (-) grow indefinitely in vitro only in contact
with stromal cell layer from bone marrow
• Pre-B cells (+) will grow in vitro for a short period in
response to IL-7 and in the absence of stromal cell
contact (matches in vivo “large pre-B cell”)
Theme 2: the bone marrow
microenvironment
• Hypothesis:
– pro-B cells fill up a niche of sites bound to
the appropriate stromal cells
– cells that lose contact, stop dividing and can
attempt V(D)J recombination of IgH locus
– pre-BCR replaces the stromal signal, so
combines with IL-7R to induce burst of pre-B
proliferation
IL-7 and proliferation of B cell precursors
Clark, et al. Nat. Rev. Immunol. 14: 69-80, 2014
Pre-BCR and IL-7R: cooperation vs. antagonism?
Clark, et al. Nat. Rev. Immunol. 14: 69-80, 2014
B-ALL have lost pre-BCR signaling through Blnk
Rickert Nat. Rev.
Immunol. 13: 578591, 2013
Theme 3: B lineage commitment:
control by transcription factors
E2A-/EBF-/-
Pax5-/-
Knockouts of several transcription factors block B cell
development at discrete stages
A hierarchy of transcription factors
specifies B cell fate
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 and Commitment to B cell lineage
1. Culture Pax5-/- bone marrow in vitro to get pro-B cell
cultures
2. See if the cells can differentiate into other
hematopoietic lineages (add various growth factors)
Nutt et
al. Nature
1999
Pax5 and Commitment to B cell lineage
Nutt et
al. Nature
1999
Pax5 and commitment to the B cell lineage
• 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.)
A network of transcription factors
specifies B cell fate
Ikaros
PU.1
Repression of genes needed to
become myeloid cell
Repression of genes needed to
become T cell or myeloid cell
Theme 4: Tolerance of B cells
Method for studying the self-reactivity of human B cells
Sorted based on
phenotype and/or
specificity, etc.
Test antibodies for
properties, self
reactivity, etc.
The primary repertoire of B cells
includes many self-reactive cells
Meffre and Wardemann, COI
20:632, 2008
Theme 4: Fate of self-reactive B cells
Bone Marrow
Antigen
Independent
Pre BCR
Periphery
Antigen Dependent
IgM
μ
IgM
IgM
IgD
IgM
Plasma
Cell
pre-B
Immature
autoreactive
T1
autoreactive
deletion
or editing
deletion
or anergy
Negative Selection
T2
Mature
autoreactive
antigen
encounter &
proliferation
anergy
(w/o T cell help)
Positive Selection
Receptor Editing Mechanisms
1. Upstream V can rearrange to downstream J
V
J
C
2. Upstream V can rearrange to KDE (deleting
element) deleting C; this would be followed by a
rearrangement of another light chain allele
KDE
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
• Bone marrow stromal cells promote survival
to allow editing to occur
Clonal deletion vs. clonal anergy
• Anti-lysozyme transgenic mice with high affinity
antibody:
-presence of soluble lysozyme either as
transgenic or injected leads to anergy (Goodnow
et al.)
-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
-mIg with lower affinity leads to anergy
(Weigert, Erikson)
Characteristics of Anergic B cells
• Anergic B cells exhibit chronic low grade BCR signaling and
attenuated response to further stimulation
• Anergic B cells localize to the edge of the T cell zone next to B
cell follicles, same as acutely stimulated naïve B cells.
• Anergic B cells have decreased survival in vivo due to decreased
ability to respond to the survival factor BAFF (BLyS).
• Anergy in the presence of competent helper T cells is enforced
by Fas killing.
NOTE: B cell anergy is best thought of as a range of phenotypes
from deep anergy to light anergy
Theme 5: 3 types of mature B cells
B1, marginal zone, and follicular B cells
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.
Also dependent on Notch signaling
• 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)
Biological roles of three types of B cells
follicular
MZ + FO
MZ + B1
B1
BCR signaling and B cell fate
(maturation B1)
(follicular>MZ?)
Changes to rearranged Ig genes
during immune response
Ig Heavy chain class (isotype) switching
Affinity maturation and antibody responses
Ig mutations are localized near
transcription start site
from Longacre and Storb Cell 102: 541, 2000.
Activation-induced cytidine deaminase (AID)
• Discovered as an induced gene in a cell line with
inducible class-switch recombination (subtractive
hybridization)
• Transfection into B cell lines induces class switch
recombination
• AID KO mice have strong defect in class switch
recombination AND in somatic hypermutation
• Hyper-IgM syndrome type 2 (autosomal) is due to
mutation in AID; very similar phenotype to mice (no
IgG, IgA, IgE; very much reduced somatic mutation)
AID: How does it work?
• AID is highly related to APOBEC-1, a cytidine
deaminase that edits mRNA for Apolipoprotein B
(via a targeting subunit) and APOBEC-3, which
mutates retroviral genomes
• indirect action or direct action in class switch and
hypermutation?
AID could edit mRNAs for factors that act in
class switch and factors that act in class switch
OR
it could act directly in both processes
AID as a mutator of DNA
• AID is mutagenic in bacteria and mutations
are increased by deficiency in Uracil-DNA
glycosylase (enzyme that removes U from
DNA and triggers DNA repair)
• Class switch is inhibited and hypermutation
perturbed in UNG-deficient mice
• These results favor the hypothesis that
AID directly acts on C residues in DNA to
promote class switch and hypermutation
Model for direct actions of AID in
somatic mutation and class switch
In hypermutation:
U in DNA could lead to direct mutations and
secondary mutations via mismatch repair
and/or error-prone DNA polymerases
In class switch recombination:
U in DNA could lead to nick formation by repair
enzymes:
nicks on both strands-->ds breaks-->recombination