Development of the Immune System

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B lymphocyte
Re-circulation
Bursa of fabricus (birds) Bone marrow (mammals)
B lymphocyte lineage
Lymphnodes Speen Mucosal and cutaneous Lymphoid
tissues
Blood
Blood, lymphnodes
T lymphocyte lineage
Thymus
Re-circulation
T lymphocyte
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Ordered Stages of B cell Development
Stromal Cells are critical for providing signals
to developing B cells
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Pathway of B cell Differentiation
Antigen Independent
Antigen Dependent
IgG Memory cell
Cytoplasmic m chain
Heavy chain surrogate light chain
Heavy plus light chain
Pre-Pro B
Pre-B1
Pre-B2
B
Pro B
Plasmablast
D---JH
VH--DJH
VL--JL
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Structure of the Ab Molecule
Light chains
-S-S-
-S-S-
-S-S-
-S-S-
Heavy chain
Constant regions M, D, G, A, E
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Organization of the mouse heavy chain locus on
chromosome 12
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Mechanism of VDJ recombination
DJH
VH
JH
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12
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RAG-1/RAG-2 Ku protein
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coding join
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7
VH
7
23
VH
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7
9
23
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fused heptamers
VHDJH
Cm
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m heavy chain with surrogate light chains
IL-7R (IL-7Ra and IL-2 gc chain)
Iga and Igb proteins
IL-7 and adhesive interactions between B cells
and stromal cells important for proliferation
Signal Transduction 1. clonal expansion 2.
allelic exclusion 3. light chain rearrangement
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The Stages of B cell Development
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Need to Purge Autoreactive Specificities at
KeyCheckpoints

• 5 of newly formed B cells survive to enter
  mature primary pools
• BCR signaling interaction with BAFF (BlyS)
• See Transitional B cell intermediate

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Immature B cell Microenvironments
IgM
HSAhi
HSAlo
IgD
immature
mature
New-B Fr. E
New-B Fr. E
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Characteristics of Transitional (TR) B cells

• HSAhi
• Resemble neonatal B cells in responses to BCR
  cross-linking and other signaling characteristics
• Display rapid turnover, residency in the
  transitional compartment very short (on the order
  of days)
• 2/3 or TR cells die before maturation

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• Anergic B cells enter TR pools but die before
  entering mature pools.
• Loss of autoreactive and polyreactive clonotypes
  at the TR stages
• Failure of this checkpoint in SLE patients

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Negative Selection in Developing B lymphocytes

• Mechanisms used to eliminate self-reactive B
  cells
• Deletion, anergy, and replacement of the
  self-reactive receptor with a non-reactive BCR
• Process to eliminate developing B cells that can
  interact with self-antigens in bone marrow and
  periphery
• Maintain immature B cell phenotype in periphery
• Encounter antigen in peripheral compartments
  triggers negative selection

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Default Programs

• Immature B cells
• Default is towards negative selection
• enter cell cycle but then defaults towards
  apoptosis
• Mature B cells
• Default is towards activation

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• BCR Signal Strength can Specify 3 Alternative
  fates for the TR cell.
• Differentiative failure
• Survival
• Tolerogenic elimination

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What is basis for the sensitivity of the
immature-stage B cell to negative selection?

• Differences in BCR signal transduction

IgM/Iga/Igb
PIP2
PLCg1
DAG
PKCb
IP3
NF-kB
Ca2
c-myc
Anti-apoptotic pathways
Proliferation
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Role of BAFF-BAFFr

• High levels of BAFF can rescue the maturation of
  autoreactive cells lost at the TR checkpoint
• Liklihood of rescue depends on the competing
  cells enough competitors of normal BCR
  diversity, autoreactive clonotypes fail to
  persist even with excess BAFF.

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Role of BAFF-BAFFr

• Integrates primary B cell selection and
  homeostasis
• Critical for maintaining normal peripheral B cell
  numbers
• BAFFr signaling competence dictate mature B cell
  lifespan
• Cause upregulation of Bcl-2 family members,
  prevention of PKC-??nuclear accumulation and
  activation of non-classical NF-?B pathway

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How can Immature B cells undergo receptor editing
after encountering self-antigen?
1-2 hr.
10-20 min.
10-12 hr.
apoptosis
Receptor editing
activation
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Encounter self antigen
ADD DEVD-FMK
RAG 1/2
10-12 hr.
Receptor editing
activation
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Bone Marrow
Lymph node, Spleen, Gastrointestinal tract
B7.2/CD27
CD40L
CD40
Mature, naïve B cell
MHC Class II
CD4 helper T cell
macrophage
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Cognate interactions that drive B cell
activation 1. Antigen/BCR 2. CD154/CD40
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Germinal Center Response

• B-cell follicles found in secondary lymphoid
  organs
• Spleen, lymph nodes, Peyers patches, appendix,
  tonsils, and MALT
• 1o follicles-no antigen-driven processes are
  taking place
• Network of follicular dendritic cells (FDCs),
  spaces filled w/ recirculating sIgM/sIgD small
  B cells
• Present in lymph nodes early in 2nd trimester of
  fetal life

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FDCs

• Antigen is taken up by FDCs in the form of immune
  complexes and held in a non-degraded form for
  months
• Important in maintaining T-cell-dependent
  responses
• Molecules important in B cell-FDC interactions
• ICAM-I-LFA I, Ig, CD44 (adhesion molecule)

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Organization of the Germinal Center
FDC network Zone Fine processes
Follicular mantle
Follicle
Loose network Outer zone Dense network
Apical light zone Dense network Basal light
zone Fine processes Dark zone
T cell zone outside B cell follicles
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The Germinal Center Reaction
B cells being tested for antibody affinity
Light zone
Antigen-retaining FDCs
Macrophage capturing dead B cells
Dark zone
Rapidly dividing and mutating B cells deep in the
center
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The Germinal Center Reaction
Selection
Light zone
Retention
Diversification
Dark zone
Recruitment
Expansion
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Immunological Memory

• Four distinct phases
• 1. Induction of B cell memory
• GC reaction
• 2. Maintenance of a B cell memory compartment
• Non-secreting precursor to the memory response
• 3. Expression of B cell memory on rechallenge
• 4. Replenishment of the memory compartment

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Induction of Memory

• Ag-activated B cells migrate to follicular
  regions 4 days after immunization
• Regionalize to light zone and dark zone
• Centroblasts somatically diversify their B cell
  receptors (BCR)
• Improved fit?--re-enter GC cycle or exit

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Hypermutation and selection

• Affinity of Serum Ig increases during an immune
  response
• Somatic mutation in Ig V region genes, in
  response to protein, in B cells in mouse spleen
  does not start until after the onset of Ab
  production
• 2. Mutational process occurs in
  centroblasts--centrocytes are selected on basis
  of their capacity for activation by antigen held
  on FDCs

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Somatic Hypermutation (SHM) and selection

• Somatic Hypermutation
• Experiments with different model haptens, e.g.
  2-phenyl-5-oxazolone
• Immunize and analyze sequences from hybridomas
• two types of changes
• 1. Highly mutated germline VH gene (first used
  VHOx1/VkOx1--
• 2. Changed to other VH/VK combinations at
  later times
• CALLED repertoire Shift

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The mutation Domain

• Mutations largely confined to variable regions
• Occur over a 1-2 kB region around the rearranged
  VJ gene segment
• Found predominantly in CDRs but due to selection

CDRs
Light chains
-S-S-
-S-S-
-S-S-
-S-S-
Constant regions M, D, G, A, E
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Nucleotide Substitution Preferences

• Individual substitutions, transitions are more
  common than transversion
• Gs more often than Cs and As more than Ts
• strand polarity
• Hotspots for mutations
• defined by local DNA sequence
• Pu-G-Py-A/T (where Pu A or G and Py C or
  T)
• Not all consensus sequences define a hotspot

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Requirements for recruiting SHM

• In kappa locus both intronic and 3 enhancer
  elements are required
• Transcription is required, mutations decrease
  with distance from the promoter
• Expression of AID (activation-induced cytidine
  deaminase) essential enzyme for CSR, gene
  conversion and SHM

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VDJ
E
P
Initiating event asymmetric DSB into one of the
two sister Chromatids. DNA synthesis involves an
Error-prone polymerase Introduction of point
mutations
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Differentiation Signals

• Physiological signals that can extend the
  survival of GC cells--differentiation
• 1. Signaling through CD40
• A. Induces cells to leave cell cycle, acquire
  phenotype of small lymphocytes
• B. express bcl-2 protein
• C. Express sIg
• 2. Stimulation with CD23 and IL-1Ra prevents GC
  cells from entering apoptosis
• A. induces some features of plasmablasts, bcl-2
  and cytoplasmic Ig
• B. development of the ER

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Differentiation Signals, contd.

• Physiological signals that can extend the
  survival of GC cells--differentiation
• Expression of IL-2 allows GC cells to remain in
  cell cycle
• A. cells dont express bcl-2 protein

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Differentiation Signals, contd.
-Burketts lymphoma-translocation of bcl-2 gene
on Chromosome 18 and IgH on chromosome
14. -constitutive expression of bcl-2
protein -Possible reason why these cells
survive without the need for interaction with
antigen
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Memory cell
plasmablast
CD40L
CD23 IL-1a
Apical light zone
IL-2
plasmablast
Interaction w/Ag.
?
Basal light zone
Outer zone
No interact. W/Ag.
Death by apoptosis
?
Dark zone
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Organization of the human heavy chain locus
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Germline Transcription
C?2 C?4 ? C?2
C? C? C?3 C?1 ?? C?1
VHDJH
EH
3?1E
3?2E
Germline I? transcript
Germline ? transcript
VDJ
S?
C?
C?
I?
S?
C?
5
3

• Germline transcription is required and precedes
  switch recombination

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Switch Recombination Induced by T-cell Signals
(IL-4, CD154)
Mechanism of Switch Recombination
VDJ
S?
C?
C?
I?
S?
C?
5
3
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Patient B cells can undergo Class Switch
Recombination (CSR)
CD154IL-4
CD154IL-4
CD154
CD154
PBMC
IL-4
PBMC
IL-4


3
VD J
m
d
g
3
VD J
g
Patient
Control
d
m
Switched Circle
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Activation of NF-kB and other signaling pathways
are required for GL transcription and switch
recombination