College 21 mei

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The Development of B lymphocytes
Chapter 6 Parham
Hans de Haard
21th of May 2010
Agenda
•  Stages in development of B cells
•  B cell development in bone marrow
•  Heavy chain rearrangement
•  Light chain rearrangement more efficient process
•  Checkpoints
•  B1 versus B2 lymphocytes
•  Removal self reactivity
•  B cell development in secondary lymphoid tissues
•  Differentiation into mature B lymphocytes
•  Maturation into plasma cells and memory cells
•  Various B cell tumors represent different stages in B cell development
Agenda
•  Stages in development of B cells
•  B cell development in bone marrow
•  Heavy chain rearrangement
•  Light chain rearrangement more efficient process
•  Checkpoints
•  B1 versus B2 lymphocytes
•  Removal self reactivity
•  B cell development in secondary lymphoid tissues
•  Differentiation into mature B lymphocytes
•  Maturation into plasma cells and memory cells
•  Various B cell tumors represent different stages in B cell development
Stages in development B cells
•  Phase 1: B cell precursors in bone marrow acquire
functional antigen receptors via gene rearrangements
•  Phase 2: elimination anti-self (tolerance) in bone
marrow and 2nd lymphoid tissues
•  Phase 3: positive selection for B cells expression of
pathogen specific Ig in 2nd lymphoid tissues
•  Phase 4: mature B cells circulate via lymph and blood
to lymphoid tissues patrolling for infections
•  Phase 5: proliferation and clonal expansion pathogen
specific B-cells in 2nd lymphoid tissues
•  Phase 6: maturation into:
•  plasma cells =>remain in 2nd lymphoid tissues, express large quantities
pathogen specific Ab
•  memory B cells (circulate) for rapid Ab generation in case of future
infections with same pathogen
Agenda
•  Stages in development of B cells
•  B cell development in bone marrow
•  Heavy chain rearrangement
•  Light chain rearrangement more efficient process
•  Checkpoints
•  B1 versus B2 lymphocytes
•  Removal self reactivity
•  B cell development in secondary lymphoid tissues
•  Differentiation into mature B lymphocytes
•  Maturation into plasma cells and memory cells
•  Various B cell tumors represent different stages in B cell development
Development Immature B cells in
bone marrow
•  B and T cells originate from common lymphoid progenitor cell (CD34+
pluripotent hematopoietic stem cell)
•  Earliest B cell lineage is early pro-B cell with starting DH – JH rearrangements
•  Late pro-B cell VH rearranges with DJ, in pre-B cell stage VL with JL
•  In immature B cell stage IgM expressed on surface
Heavy chain rearrangement inefficient
process
•  Due to imprecise joining and addition of N / P nucleotides during VDJ
recombination (3 vs 2 segments for light chain) high probability for incorrect
reading frame yielding non-functional Ab => non-productive rearrangement
•  Check for correctly expressed heavy chain; if not, then rearrangement of
other chromosome
•  If again non-productive arrangement, then cell dies by apoptosis
•  Just over half of total number of B cells make functional heavy chain
Quality check (pre-) B cell receptor
•  Correctly rearranged m heavy chain capable to
combine with light chain
•  Check performed in ER with surrogate light chain
consisting of VpreB and l5 in pre-B cell receptor
•  IgM incorporated in cell membrane via MC, but
associated Iga / Igb responsible for signaling
•  Final check with rearranged light chain in
B cell receptor expressed on cell membrane
•  After successful VDJ recombination on first chromosome assembled pre-B
cell receptor signals to stop recombination of 2nd chromosome (allelic
exclusion)
Light chain rearrangement more
efficient process
• 
Large pre-B cell with rearranged heavy
chain divides yielding approx. 100 cells
per B cell clone (i.e. with identical VDJ)
• 
Kappa light chain rearranges first, if
unsuccessful followed by lambda
• 
Only V and J need to recombine, therefore
higher success rate then with additional D
• 
If first recombination fails to give correct
fusion, then other V region in upstream position will recombine with downstream J
• 
If again non-productive, then recombination of kappa locus on other chromosome
• 
If kappa fails, then lambda locus rearranges
• 
85% of pre-B cells yield productive light chain rearrangement
Checkpoints during B cell development in
bone marrow
•  Check (1): pre-B cell receptor with correct VDJ fused to
m heavy chain and
associated with surrogate light chain (in vesicle)
•  Check (2): B cell receptor with correctly rearranged light chain and
heavy chain (on cell membrane)
m
Regulated and time dependent expression
of proteins involved in Ig rearrangement
•  Expression of signaling proteins
important for differentiation and
development of B cell:
•  FLT3 => receptor kinase, receives
signal stromal cells for differentiation
of common lymphoid progenitors
•  CD19 => co-receptor participating in
Ag – BCR complex
•  CD45 => receptor phosphatase
modulating signals from Ag – BCR
complex
•  RAGs are expressed two times,
during rearrangement of heavy
and later during light chain locus
Pax-5 opens up chromatin in heavy chain
locus: start recombination
•  Pax-5 is B cell specific transcription
factor
•  Binds enhancer elements downstream
(i.e. 3’ direction) of constant region
•  Thereby opens up chromatin resulting
in low level transcription from
promoters upstream (5’) of D and J
•  Transcription allows access of RAGs to
“open” HC locus to start
recombination of D – J
•  Brings enhancer closer to V region
leading to transcription from
promoters upstream of V
•  Induces V recombination with DJ
B1 versus B2 lymphocytes
•  Minority human B cells are of B1 class
=> CD5 marker typical for T cells
=> little or no surface IgD •  Arise from stem cell active in
prenatal period and therefore less
developed
=> use V genes closely positioned to D
=> no or limited TdT activity (i.e. few
N nucleotides introduced)
•  Low affinity antibodies and polyspecific (i.e. reactive against multiple
antigens), often carbohydrates recognized
•  When generated in postnatal period these are better developed and matured;
no longer produced by bone marrow, but are self-renewal at sites in
peripheral circulation (IL-10 dependent)
•  Chronic lymphocytic leukemia caused by B1 cells
Summary B cell development in bone
marrow
Removal self-reactive antibodies
•  Self-reactive immature B cells
removed
=> 75% total population
•  Potential multivalent self antigens
are glycoproteins, proteoglycans
and glycolipids on cells
•  Negative selection starts in bone
marrow on self antigens
presented by stromal and
hematopoietic cells
•  Immature B cells with self
reactivity are maintained within
bone marrow, others will go into
circulation
Receptor editing for changing self reactivity
•  Self reactivity detected in bone
marrow
=> binding to mutivalent
antigen
=> signaling to reduce
surface expression IgM
•  Maintaining RAG expression
=> excision old light chain
=> replaced by newly recombined
light chain
(receptor editing)
•  If self reactivity has been removed,
then B cell development continues
•  If not, B cell in apoptosis
(clonal deletion)
Self reactivity against monovalent antigen
results in anergy
•  Self reactivity detected in bone marrow
=> binding to monovalent antigen
=> B cells inactivated and unresponsive
against antigen
(Anergy)
•  Anergic cells:
=> no production cell surface exposed IgM
(remains intracellular)
=> only IgD presented (not activating)
=> go into circulation, short T½ (1 – 5 days vs 40
days for other B cells)
•  Central tolerance: check within bone marrow
leading to removal of anti-self or anergy
•  Peripheral tolerance: removal of anti-self outside
bone marrow (i.e. by apoptosis or anergy)
Agenda
•  Stages in development of B cells
•  B cell development in bone marrow
•  Heavy chain rearrangement
•  Light chain rearrangement more efficient process
•  Checkpoints
•  B1 versus B2 lymphocytes
•  Removal self reactivity
•  B cell development in secondary lymphoid tissues
•  Differentiation into mature B lymphocytes
•  Maturation into plasma cells and memory cells
•  Various B cell tumors represent different stages in B cell development
Maturation B cells in secondary lymphoid
tissues
•  Immature B cells developed in bone
marrow recirculate between blood and
secondary lymphoid tissues
•  Development from immature (sIgM, IgD)
into mature B cells (sIgM, IgD) occurs
within secondary lymphoid tissues:
=> enter lymph nodes through walls
High Endothelial Venules (HEV) driven
by chemotaxis (gradient of chemokines)
=> guided by other chemokines to primary lymphoid follicles, i.e. network of
Follicular Dendritic Cells (FDC)
=> contact FDC and B cells; if no encounter with antigen B cell detaches and
goes back in circulation via lymph
•  Now developed into mature naive B cell; survival dependent on regular passage
through primary follicles of secondary lymphoid tissues; long T½ (~100 days)
Differentiation of activated B cells into
plasma cells
•  Encounter of mature naive B cell
with antigen leads to interaction
with antigen specific TH:
activation leads to proliferation and
differentiation into plasma cells
•  Change of phenotype:
=> expression of secreted IgM ( sIgM-)
=> not sensitive for signaling, not dividing
•  Migration to primary follicle thereby
becoming secondary lymphoid follicle with germinal centers
•  B cells become large proliferating centroblasts, subsequent
maturation into more slowly dividing centrocytes (isotype
switch and affinity matured)
•  As primary response subsides differentiation in memory cell
(picture lymph node see p 210)
Various B cell tumors reflect different
development stages of B cells
•  Mistake in rearrangement can lead
to activation of oncogenes
(controling cell growth, division and
differentiation)
•  B cell tumors are clonal, i.e. derived
from single recombined B cell
•  B cell tumors represent different
stages of B cell development
•  Tumors derived from naive B cells
grow in follicles of lymph nodes
=> follicular center cell lymphoma
•  Tumors derived from plasma cell
tumors grow in bone marrow
=> myeloma
Summary B cell development
The Development of B lymphocytes
Chapter 6 Parham
Hans de Haard
21th of May 2010
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