Antigendependent B cell development

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Antigen-dependent B cell development Prof.
Sylvie Fournier
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Traditional view of B lymphocyte development
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The development of B lymphocytes from early
precursors to the naïve mature B cell stage has
been historically considered to be
antigen-independent while the differentiation of
naïve mature B cells to plasmocytes and memory B
cells in response to activation by foreign Ags
has been referred to as the antigen-dependent
phase of B cell development.
Antigen-independent
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In this lecture we will see that this classical
distinction between antigen-dependent and
-independent stages of B cell development is no
longer so clear cut. We will examine whether
signals through the BCR are required for the
development of immature B cells to naive and
naïve like mature B cell populations
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B-1
Fetal Liver
IgMhi IgDlo CD21hi CD23- HSAhi
?
IgMhi IgDhi CD21hi
IgMlo IgDhi CD21int CD23 HSAlo
T2
IgMhi IgDlo CD211o CD23- HSAhi Transitional of
type 1
T1
IgMhi IgD -
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T1 cells are recent emigrants from the bone
marrow. Their phenotype is closely related to
that of immature B cells in the bone marrow
T1 IgMhigh , IgDlow/neg, CD21low/neg, CD23
low/neg. T1 cells that successfully enter
splenic B-cell follicles become T2 cells. T2
cells have a mixed phenotype between that of T1
cells and mature follicular (FO) B
cells T2 IgMhigh , IgDhigh, CD21high, CD23
. FO IgMlow , IgDhigh, CD21int, CD23 . T2 B
cells are considered to be precursors for FO and
MZ B cells, and some B-1 cells. MZ IgMhigh ,
IgDlow, CD21high, CD23low/neg.
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Relationship of T1, T2 and mature B cells
Loder , F. et al. 1999. B cell development in the
spleen takes place in discrete steps and is
determined by the quality of B cell receptor
derived signals. J. Exp. Med. 190 75-89.
Transfer of T1 cells into Rag -/- mice.
Figure 2. T1 B cells are the precursors of T2
and mature B cells. (A) Splenocytes of 1- (left)
and 3- (center) wk-old and adult (right) C57BL/6
mice were stained with Abs to CD21 and IgM and
analyzed by flow cytometry. (B) Splenic cells
from recipient RAG-2-/- mice were analyzed at the
indicated times after transfer of 2 x 106 splenic
B cells from a pool of 1-wk-old mice. Cells were
stained with Abs to IgM, IgD, and CD21. Top
panels, IgM vs. IgD staining. Bottom panels, the
CD21 vs. IgM profile of IgDIgM donor B cells.
200,000 events were collected. Data is shown as
dot plots to highlight the few transferred cells
that home to the spleen. In the dot plots
corresponding to the control (adult) spleen, only
5 of the collected events are shown.
48 h after transfer, T2 B cells were 52 of all
splenic B cells, and mature B cells were 36.
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Figure 3. T2 B cells develop into mature B cells
in the spleen. (A) Spleen cells of adult mice
were stained with Abs to HSA and CD21, and T1,
T2, and mature (M) B cells were identified
(before sort). CD21HSAbright T2 B cells were
sorted (after sort) and transferred into adult
RAG-2-/- recipient mice. (B) The spleens of
recipient RAG-2-/- mice were analyzed 24 h after
transfer and compared with the spleen of an adult
control mouse. T1, T2, and mature B cells were
identified on the basis of the expression of HSA,
B220, IgM, and IgD. The plots show the IgD and
IgM staining of cells that were positive for B220
and either bright or dull for HSA.
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Emigration and loss of recently generated B cells

• What supports massive cell loss at or soon after
  the immature B-cell
• Stage in the bone marrow?
• The high rate of B cell production Immature B
  cells 15-20 x 106/day
• (Osmond, D. G. 1986. Population dynamics of bone
  marrow B lymphocytes.
• Immunol. Rev. 93103.)
• Half-life of mature follicular B cells 4.5
  months
• (Hao, Z. and Rajewsky, K. 2001. Homeostasis of
  peripheral B cells in the absence of B cell
• influx from the bone marrow. J. Exp. Med. 194
  1151-1163.)
• One way the relatively long lifespan of
  follicular B cells can be reconciled with the
  high
• rate of production is to assume that a large
  proportion of recently generated B cells are lost

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• At the immature stage, about 90 of the immature
  B cells produced are lost. A large part of this
• loss can probably be explained by the deletion
  of autoreactive B cells in the bone marrow.
• However, signaling through the BCR appears to
  play a role in the emigration of immature
• B cells from the bone marrow to the spleen.
• Mice with an engineered mutation in the
  cytoplasmic tail of Iga B cell development up to
  the
• immature B cell stage in the bone marrow is
  minimally affected, but emigration to the
  periphery
• appears to be markedly reduced. (Torres, R. M.,
  et al. 1996. Aberrant B cell development and
  immune
• response in mice with a compromised BCR complex.
  Science 2721804).
• BCR Tg mice with a deletion of Syk B cells at
  the immature stage are detected in the bone
• marrow but very few are present in the spleen.
  (Turner, M. et al. (1997). Syk tyrosine kinase is
  required
• for the positive selection of immature B cells
  into the recirculating B cell pool. J. Exp. Med.
  1862013-2021.)
• CD45-/- and Xid (Btk defective) mice Marked
  increase of immature B cells in the spleen that
• may be consistent with an accelerated emergence
  of immature B cells from the bone marrow.
• According to these findings, it is therefore
  assumed that emigration from the bone marrow

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Now if there is a BCR-driven checkpoint exits at
this stage of B cell development in the
bone marrow, the question is whether this BCR
signal is constitutive or generated by
self-antigens. The most commonly held view is
that it is probably a constitutive signal. If
the BCR- generated signal is constitutive it may
be ligand independent (tonic signaling the
mere process of receptor assembly), or it may
depend on some extracellular ligand that
recognizes a nonvariable portion of every
BCR. Perhaps not not every possible
immunoglobulin heavy-light chain is capable of
associating well enough to generate appropriate
levels of properly assembled receptor to initiate
a tonic signal. It is possible that what is being
selected at the immature B cell stage in the bone
marrow is an appropriate fit between pairs of
heavy and light chains
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Signaling through the B cell receptor
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Signals required for the T1 to T2 transition
Only a small percentage of T1 cells proceed to
the T2 stage. Most T1 undergo clonal deletion or
anergy. These represent B cells that cross-react
with self-antigens expressed only in peripheral
tissues. However, basal BCR signals are required
for the transition from T1 to T2 B cell stage.
Syk -/- Ig? ?c/?c Ig? ?c/?c
Baff -/- A/WySnJ (mutation in BaffR)
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Signals required for the T2 to FO transition
Btk -/- CD45 -/- PLCg2 -/- Dominant negative of
MEK
Syk -/- Ig? ?c/?c Ig? ?c/?c
T1
T2
FO
Baff -/- A/WySnJ (mutation in BaffR)
As disruption of BCR signaling components further
downstream of Ig?/ Ig? or Syk seems to affect
the transition from T2 to FO B cells. Thus, it
is suggested that a higher level of BCR signal
might be required for this transition compared
with the T1 to T2 transition. In vitro, BCR
crosslinking of T2 cells induces
their differentiation to FO B cells. So, basal
BCR signaling might not be sufficient for this
process, and interaction of the BCR with unknown
ligands, possibly self-Ags, might be required.
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If the differentiation of mature B cells is
driven by the selection of specific B cells by
self-antigen(s), it should be reflected by a
difference in the BCR repertoire between immature
B cells in the spleen and mature B cells in the
spleen. In a recent report, researchers have
examined the BCR repertoire between immature B
cells in the spleen and mature B cells in the
spleen. (Levine, M.H. et al. PNAS 972743-2748,
2000.)
They used IgH ?transgenic mice to fix one half of
the BCR. This allowed them to focus on the light
chain repertoire as an indicator of the
specificity of the BCR. They compared the
in-frame ? light chain repertoires of peripheral
immature and mature B cells within individual
mouse.
Fig. 1.   FACS sorting of immature (population E)
and mature (population F) B cells. B220/IgMa
splenocytes from Meg mice were gated to separate
immature and mature B cells from other cells
(a), and then sorting of immature B cells (E,
HSAhi) from mature B cells (F, HSAint) was
performed by using the indicated gates (b). The
bold line shows unstained cells. FACS reanalysis
of immature (thin line) and mature (bold line) B
cells from a and b was performed. (c) Reanalysis
of the separation of population E and F cells by
surface HSA levels as shown in b. (d) Reanalysis
of surface IgM levels in population E and
F.  Note the uniformly high IgM levels in
population E
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In all three mice of this line, the immature B
cell  light chain sequences were diverse (Fig. 2
a-c). However, in the mature B cells of all
three mice, the frequency of a single member of
the V24/25 family, sequence 80 as denoted by
Strohal et al. (15) was significantly enhanced
(Fig. 2 a-c). The frequency of the sequence
80 light chain was 3- to 7-fold higher in the
mature B cell population compared with the
immature B cell population within the same mouse.
The magnitude of difference in all mice analyzed
was similar, and in all cases the sequence
80 light chain was the most prevalent light
chain isolated from the mature B cells.
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Fig. 2.   In-frame  light chain repertoire is
more restricted in mature naive B cells than in
immature B cells from three Meg (VH186.2
transgenic   JH/) mice.  light chain joins to J2
from immature (population E,white bars) and
mature (population F, black bars) B cell
populations were amplified, cloned, and
sequenced. Each panel compares the in-frame light
chain repertoire of immature and mature splenic B
cells from an individual mouse. The horizontal
axis is labeled with V families, and chain
numbers as assigned by Strohal et al. (15).
Chains not identified by Strohal are labeled
N1-N9. The vertical axis depicts standardized
 light chain expression frequency as a percentage
of total  chains sequenced.(a) V24/25 family
member number 80 (marked with an asterisk) was
found to be at a three-fold greater percentage in
mature (black bars) than immature (white bars) B
cells within the first mouse (E, n  37 F,
n  42). This V24/25 family member also
represents the most common  light chain in the
mature B cell population. (b) In the second
mouse, the same V24/25 family member number
80 was found to be enhanced at the E to F
transition, representing 12 of total in-frame V
chains in the mature B cell population and only
2 in the immature population (E, n  48 F,
n  59). (c) In a third mouse analyzed, the same
V24/25 family member number 80 once again
appeared at a much greater frequency in the
mature B cell population (12) than the immature
B cell population (2.5) (E, n  40 F, n  50).
(d) Tabulation of all V-J2 sequences from the Meg
line for immature (E) and mature (F) cells is
shown. The horizontal axis is labeled with as
above whereas the vertical axis depicts total
number of sequences with the immature numbers
normalized to be equivalent to the mature numbers
(n  151 for both populations). V24/25 sequence
80 (marked with an asterisk) represents just
under 5 sequences of 151 in immature B cells
(3.2) and represents 19 of 151 sequences (12.6)
in mature B cells. This enhancement is highly
significant (P lt 0.003)
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The fundamental significance of these data is
that they demonstrate that the loss of the
majority of B cells at the peripheral immature
to mature follicular transition is selective
rather than stochastic. Although not proved
directly by these studies, the authors favor the
interpretation that certain cells are positively
selected. If positive selection is occurring at
this transition, the question is raised Why
must B cells be positively selected, and what is
the nature of the selecting ligand? It is
possible that environmental antigens and
nonpathogenic flora may be "preselecting" a naive
recirculating B cell repertoire that is
predisposed to recognize pathogenic antigens it
may later encounter. Regardless of the purpose
of receptor-specific selection of immature B
cells into the mature compartment, the present
work adds another dimension to the mounting
evidence that receptor-ligand interactions signal
for the ongoing survival of lymphocytes in the
periphery.
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• Does the specificity of the BCR dictate the
  developmental pathway
• of a B cell clone?
• VH81X transgenic mice (this BCR recognizes
  phosphorylcholine with low affinity)
• B cells differentiate into marginal zone B cells
• Anti-HEL transgenic mice
• B cells differentiate into follicular B cells
• A restricted BCR repertoire that is weakly
  self-reactive is selected preferentially in MZ
  and
• B1 B cells.
• Microbial products, recognized specifically
  through TLRs, are sufficient to drive the
• differentiation of T2 cells to MZ B cells in
  vitro.
• In mice that lack the normal commensal
  intestinal flora, the generation of MZ and B1
  cells is
• impaired.

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Does the strength of the signal generated by the
BCR influence the developmental pathway of a B
cell clone?
Btk -/-
T2
FO
Btk -/-
MZ

• VH81X transgenic mice (this BCR recognizes
  phosphorylcholine with low affinity)
• B cells differentiate into marginal zone B cells
• T15 transgenic mice (this BCR recognizes
  phosphorylcholine with high affinity)
• B cells differentiate into B-1 cells

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We will examine more closely data that supports
the notion that the strength of the BCR signal
that B cells receive during the late phases of
their differentiation drives their
differentiation into different mature B cell
subsets.
Cariappa A. et al. Immunity, 14603-615,
2001. The follicular versus marginal zone B
lymphocyte fate decision is regulated by Aiolos,
Btk and CD21.
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Aiolos -/- BCR signaling is increased. Xid
(Btk is not functional) BCR signaling is
decreased.
T1 IgMhigh IgDlow T2 IgMhigh IgDhigh FO IgMlow
IgDhigh
The ratio of mature B cells to newly formed B
cells is increased in Aiolos -/- mice but
decreased in Xid mice.
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MZ IgMhigh IgDlow like T1, but in contrast to
T1 they are CD21high and CD1 high.
T2 IgMhigh, IgDhigh, CD21 high, CD43-
Aiolos -/- absence of MZ B cells and their
putative precursors, but presence of FO B cells.
Xid absence of FO B cells but presence of MZ
B cells and their putative precursors.
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Cr2 (CD21) enhances BCR signaling.
Aiolos -/- decreased MZ B cells increased FO B
cells Cr2 (CD21) -/- increased MZ B
cells decreased FO B cells
These findings further support the view that an
impairment in BCR signaling may negatively
influence the generation of FO B cells
but simultaneously favor the generation of MZ B
cells.
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It is known that B cells bearing high-affinity
receptors for PC (bearing the T15 idiotype for
instance) develop into B-1 cells whereas
antigen-receptors with low avidity for PC acquire
a MZ B-cell phenotype. Although specific BCRs
can clearly favor B-1 differentiation, reducing
the level of surface expression of a B-1
committed BCR can induce a cell to acquire a
follicular or B-2 fate. B-1-cell numbers are
enhanced in mice that lack the immunoreceptor
tyrosine-based inhibition motif (ITIM)-containing
negative regulator of B-cell signaling, CD72,
which is known to recruit SHP-1, as well as in
viable motheaten mice, which harbor a defect in
SHP-1. These data argue that very strong BCR
signals favor B-1-cell generation.
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The BCR signal-strength model for lineage
commitment in peripheral B cells

• An emerging theme for peripheral B-cell
  development is that the strength of signal
  delivered
• via self-antigen/BCR interactions contributes in
  a significant way to the differentiative fate of
  an emerging
• B-cell clone. This model is based on the study of
  B cell development in mice that lack various
  components
• of the BCR signaling machinery.
• Disruption of the proximal and pivotal kinase
  Syk leads to the absence of the three subsets of
  mature B cells
• (MZ, FO, B-1).
• In the absence of Btk alone (Weak BCR signals
  only) or of its major substrate, PLCg2,
  follicular B cells
• and B-1 B cells are lost. However, MZ B-cell
  development is by large normal.
• In the absence of PKC alone, which is downstream
  of PLCg2 in the signaling cascade of the BCR,
• (Weak' and Intermediate BCR signals' only),
  follicular B cells are not lost and MZ B cells
  presumably
• remain unaffected, but B-1 cells are markedly
  deficient.
• Weak BCR signals are therefore permissive for MZ
  B-cell development, but not for follicular and
  B-1
• cells,
• intermediate signals are required for follicular
  B-cell development, and
• strong BCR signals are required for the positive
  selection of B-1 cells.

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Figure 3   Development of transitional and
mature B cells.  Basal (weak) B-cell receptor
(BCR) signals are required for the transition
from transitional type 1 (T1) to T2 B-cell
stages. Disruption of proximal BCR signalling
components the cytoplasmic tails of
immunoglobulin-associated protein- (Ig) or Ig,
and SYK blocks this transition. BAFF
(B-cell-activating factor of the
tumour-necrosis-factor family) is also required
for the maintenance of T2 cells. For the
transition from T2 to follicular (FO) B-cell
stages, intermediate levels of BCR signals are
required. Disruption of some BCR signalling
components leads to a block at this stage. Again,
BAFF might be required for the maintenance of
follicular B cells. A weak BCR signal and a
stronger BCR signal are required for the
generation of marginal-zone (MZ) B cells and
peritoneal B cells, respectively (see also Table
1). BAFF might be required for the maintenance of
MZ B cells, but not peritoneal B cells. The
Toll-like receptor (TLR) signals that are
provided by blood-borne and commensal pathogens
might regulate the survival of MZ B cells also.
BAFFR BAFF receptor BCAP, B-cell adaptor for
PI3K BLNK, B-cell linker IKK, inhibitor of NF-B
(IB) kinase NF-B, nuclear factor-B PB cell,
peritoneal B cell PI3K, phosphatidylinositol
3-kinase PLC, phospholipase C.