Transcript B cells - Altervista

B CELL DEVELOPMENT
AND ACTIVATION
• In healthy people, there are mature B cells with the
capacity to make antibodies to virtually any antigen.
• Bone marrow is the primary lymphoid organ in which
B cell development occurs.
• Bone marrow is the
primary lymphoid organ
in which B cell
development occurs.
• Following initial
development in bone
marrow, mature B cells
migrate to various
secondary lymphoid
tissues, including lymph
nodes, spleen, gutassociated lymphoid
tissue and blood.
• There, mature B cells can
interact with antigen,
become activated, and
further differentiate into
antibody-secreting cells
• B and T cells undergo distinct differentiation pathways.
• B cells are generated in the bone marrow, with mature B
cells, which are ready to respond to antigen, then exiting
and migrating to lymph nodes and spleen. T cells are
generated in the thymus.
• The development of B cells, starting from
hematopoietic stem cells and ending with cells that
produce antibodies, can be divided into four phases:
Phase 1 – development of B cells in bone
marrow
• This first phase of B cell development is the generation
of B cells in bone marrow.
• There, stem cells develop into pro-B cells, then pre-B
cells, and finally mature B cells, which exit the bone
marrow and migrate to secondary lymphoid organs.
• This phase of B cell development is not driven by
contact with antigen: antigen independent.
• The DNA rearrangements that result in a functional
cell-surface immunoglobulin molecule occur during this
phase.
Phase 1 – development of B cells in bone marrow
• Stem cells have both their H-chain and L-chain genes in
germ-line, un-rearranged configuration
• The earliest cell that has made the commitment to the B
cell lineage is the pro-B cell: pro-B cells have begun to
rearrange their H-chain gene.
• Once a B lineage cell expresses cell surface H-chain (m)
it is defined as a pre-B cell.
• However, the early pre-B cell receptor is not the final
form of surface immunoglobulin: H-chain + surrogate
light chain (molecule that mimics L-chain)
• Further DNA rearrangements result in the formation of a
functional L-chain, then IgM (H-chain + L-chain) is
expressed on the cell surface.
• When a cell has productive rearrangements of both H- and
L-chains) it becomes an immature B cell:
• This first phase of B cell development in bone marrow is
dependent on association with stromal cells.
• Stromal cells are non-lymphoid cells that provide an
appropriate microenvironment for B cell development.
• Bone marrow stromal cells produce both cell surfacestimulatory molecules, as well as growth factors and
cytokines, which help drive B cell development.
• For a B cell to survive
this phase of
development, it must
have productive
rearrangements of
both H-chain and Lchain.
• Failure to do this results
in cell death - cells that
have unproductive
rearrangements (such
as rearrangements that
are not in a correct
reading frame) are
eliminated.
• A given B cell can
undergo repeated
rearrangements.
• The rearrangements that result in functional H- and
L-chains occur in a specific order:
• Expression of a
functional B cell
receptor protein on the
cell surface stops
further rearrangement
of the gene encoding
that product:
Phase 2 – elimination of self-reactive cells
• Once B cells express a functional cell surface receptor
for antigen (immature/mature B cell stage) they have
the potential to be stimulated by contact with antigen,
and to become antibody-secreting cells.
• Since the DNA rearrangements that result in functional
H-chain and L-chain are not antigen-driven, a fraction of
immature B cells will have a BCR that, by chance,
reacts with some component of self - self antigen
reactive cells
• These immature B cells are removed by clonal
deletion, either in the bone marrow, or shortly after
leaving the bone marrow.
• Encounter with
self-antigen results
in apoptosis
(death),
or anergy
(unresponsiveness)
• B cells that survive
this step express
surface IgD as well
as IgM:
mature B cells
Goodnow experiments
• After elimination of self-reactive B cells, mature cells,
which express both cell surface IgM and IgD, are ready
to leave the bone marrow, can interact with antigen in
secondary lymphoid organs.
Phase 2 – elimination of self-reactive B cells,
generation of mature IgM+, IgD+ cells:
Phase 3 – activation of B cells on
contact with antigen
• Following the generation
of a functional B cell
receptor for Ag, and the
removal of self-reactive
cells, mature Agresponsive B cells
(IgM+, IgD+) emigrate
from bone marrow.
• These mature B cells go
to secondary lymphoid
organs.
• In the lymph node, B cells gather in primary lymphoid
follicles, where they receive viability-promoting signals,
interacting with follicular dendritic cells, and wait for
antigen
• B cells enter
lymph nodes
via high
endothelial
venules (HEV)
to reach
these primary
follicles.
• B cells can
recirculate out
via the lymphatic
circulation, and
back into blood.
What is meant by ‘B cell activation’?
• Go -> G1 of cell cycle (increase in size)
• upregulate
–
–
–
–
MHC class II
costimulatory molecules (B7-2)
adhesion molecules (ICAM1)
cytokine receptors (IL-2R)
• migrate to outer T zone
–
•
altered response to chemokines
become receptive to T cell help
–
protected from fas
• enter mitosis if provided with submitogenic doses of
other stimuli (LPS, CD40L, IL-4)
Types of antigen
• T-independent (TI) antigens - Type I
– induce division/differentiation independently of BCR (polyclonal
mitogens)
• LPS, bacterial (CpG) DNA
• T-independent (TI) antigens - Type II
– induce division/differentiation by BCR signaling alone
• bacterial polysaccharides, repeating surface molecules on viruses
• T-dependent (TD) antigens
– activate via BCR but depend on additional signals from helper T cells
to cause division/differentiation
• any antigen containing protein
•
•
Most pathogens contain both T-I and T-D antigens
Only TD antigens can induce Germinal Center responses
Types of B cell Antigens
T-independent (TI)
T-cell dependent (TD)
T cell
present
Ag
mitogenic
BcR signal
'activation' signal
but not mitogenic
mitogenesis
differentiation
-> most pathogens contain both T-independent and T-dependent antigens
Innate features of pathogens act
as B cell costimulators
• pathogen multivalency
– provides a level of BCR crosslinking optimal for activation
• many pathogens activate TLRs
– TLR signaling synergizes with BCR signal
• many pathogens activate the complement cascade
and become C3d coated
– complement receptor (CR) crosslinking synergizes with BCR
signal
Antigen-C3d complexes cross-link BCR and CR2CD19 complex - increase sensitivity to antigen
T-Independent (type II) Responses
Current Paradigm :
Multivalent Antigen
B cell
B cell
plasma cells
Emerging Model:
B cell
B cell
plasma cells
T-Dependent Responses
Antigen
DC
T cell
Dendritic Cell (DC)
internalizes antigen (Ag),
processes into peptides,
presents peptides
together with MHC
molecules to T cells
T cell
B cell
B cell binds Ag via surface
Ig, transmits BCR signals
and presents peptides to T
cells, receives
T cell help (growth and
differentiation factors)
plasma cells
Secretes
Antibody (Ab)
• Antigen-specific B cells are detained in the T cell-areas,
where they interact with antigen, and with antigen-specific
activated helper T cells. Stimulated antigen-specific B
cells then proliferate and differentiate, eventually forming
plasma cells and germinal centers:
B cells are antigen-presenting cells
• BCR cross-linking induces antigen
internalization to endosomes
• antigen is proteolysed to peptides
• peptides associate with MHC class II
• MHC class II-peptide complexes traffic to
surface of B cell\
• B cells present antigen recognized by their BCR
~105 x more efficiently than other antigens
B cell antigen presentation and
the concept of linked help
protein
sugar
T
Sugar Specific
B cell
Protein Specific
Antigen internalization, proteolysis
T cell
-> presentation of peptides
Interactions between antigen-specific B and T cells
1 day after HEL antigen injection
HEL-specific (Ig-tg) B cells
HEL-specific (TCR7 tg) T cells
Onset of B-T interaction
HEL-specific B cells
HEL-specific T cells
B cells can interact with multiple T cells
HEL-specific B cells
HEL-specific T cells
Cardinal features of B - T interaction
• However, signaling via the BCR alone is not sufficient to
activate the B cell -
• Second signals (co-stimulatory signals) are necessary
for activation.
- or
-deficient mice and human do not
undergo isotype switching
They only have IgM: hyper IgM syndrom
Phase 4 – differentiation to
antibody-secreting cells
• Some of the progeny of these antigen-activated B cells
differentiate into IgM-secreting plasma cells (antibodysecreting cells).
• Plasma cells:
– terminally-differentiated cells
– derived from activated B cells or
memory cells
– loaded with endoplasmic
reticulum
– devoted to protein (antibody)
synthesis
– no longer express surface
immunoglobulin or MHC class II
– no longer responsive to antigen
contact
– live for several weeks
– migrate away from the site of
initial contact with helper T cells,
either to the medullary cords of
the lymph nodes or to the bone
marrow
Plasma cells
Surface Surface High rate Growth Somatic Isotype
Ig
MHC II Ig secretion
hypermut’n switch
B
High
Yes
No
Yes
Yes
Yes
Low
No
Yes
No
No
No
Mature B cell
B
Plasma cell
• Other antigen activated B cells give rise to germinal
centers (GC), zones of proliferating activated B cells:
• These germinal centers (GC) contains:
– proliferating (D - centroblasts) B cells express low
levels of Ig, especially IgD
– differentiating (L - centrocytes) B cells express high
levels of Ig
• B cells can interact with an
antigen that is bound to the
surface of follicular dendritic
cells in the lymph nodes.
• These cells trap and
concentrate antigen,
maximizing the interaction of
antigen with B cells
Follicular Dendritic cells (stained blue)in the Germinal Centre
Retention of Antigens on Follicular Dendritic Cells
Radiolabelled antigen localises on the surface of Follicular Dendritic cells
and persists there, without internalisation, for very long periods
Maturation of Follicular Dendritic cells
Club-shaped tips of developing dendrites
Filiform dendrites
Bead formation on dendrites
Bead formation on dendrites
Iccosome formation and release
DC veils
The veils of antigen-bearing dendritic
cell surround the beads and the layer of
immune complexes is thickened by
transfer from the dendritic cell.
These beads are then released and are
then called ICCOSOMES
Iccosomes (black coated particles)
bind to and are taken up by B cell
surface immunoglobulin
Uptake of Iccosomes/Antigen by B cells
Anti-
Y
Iccosomes bearing
different antigens
Surface Ig captures antigen
Cross-linking of antigen receptor activates B cell
Activated B cell expresses CD40
B cell
B
CD40
Fate of Antigens Internalised by B cells
B
1. Capture by antigen
specific Ig maximises
uptake of a single antigen
2. Binding and internalisation via cell
surface Ig induces the expression
of CD40
3. Antigen enters exogenous antigen
processing pathway and is degraded
4. Peptide fragments of antigen are loaded
onto MHC molecules intracellularly.
MHC/peptide complexes are then
expressed at the cell surface
B
• This selection process involves competition for both
antigen and for helper T cells.
• Antigen is trapped on the surface of follicular dendritic
cells in the form of immune complexes (antigen +
antibody complexes).
• B cells that bind to Ag with high affinity live, others die by
apoptosis.
• Centrocytes interact with T cells by presenting
processed antigen to them via their MHC class II
molecules.
• Centrocytes that receive co-signaling (via CD40, MHC
class II and cytokines), as well as signaling via their
antigen receptor survive
• Centrocytes that do not bind antigen and T cells with
sufficient affinity die by apoptosis.
• Germinal centers are where isotype switching and
somatic hypermutation occur.
• Somatic hypermutation:
– rapid mutation (hypermutation)
of immunoglobulin genes
– results in antigen-binding
affinity that is higher, or lower,
than its original binding affinity
– selection by antigen results in
the generation of BCR with
increased affinity for antigen
• Only those B cells that have
enhanced their antigen receptor’s
binding affinity survive.
• Isotype switching - change in the type of H-chain that
is used by a given B cell, also occurs in germinal
centers:
• Isotype switching involves DNA rearrangements replacement of one H-chain class gene with another
• Isotype switching also occurs in germinal centers
• Isotype switching is guided by the pattern of cytokines
that are produced by helper T cells:
• These somatically mutated and
isotype switched B cells can then
continue to differentiate into
memory cells or plasma cells,
producing:
– IgG or other switched isotypes
– much higher affinity Ab, due to
somatic hypermutation
increasing the antigen binding
affinity
– migrate from the secondary
lymphoid organs to the bone
marrow, where cytokines (IL-6,
IL-11) produced by bone
marrow stromal cells keep
these cells viable and
producing Abs
Together, these processes result in a 1-2 log increase in the
number of antigen-specific B cells (clonal selection and
expansion), an increase in antibody binding affinity for
antigen (somatic hypermutation), and the expression of new
Ig subclasses (Ig isotype switching):
• Other germinal center B cells develop into memory B cells:
– quiescent, long-lived
– increased in frequency following primary responses
– found in secondary lymphoid organs (spleen, lymph nodes,
Peyer’s patch)
– posses high-affinity, isotype-switched (IgG, IgA, IgE)
antigen receptors
– form a pool of cells ready to mount a rapid secondary
antibody response, on subsequent exposure to antigen:
• The combined result of clonal
selection (expansion of pool
size of Ag-reactive cells),
somatic hypermutation,
isotype switching, and the
generation of memory cells,
is the creation of a pool of cells
that can respond rapidly and
vigorously to subsequent
contact with antigen, with highaffinity, IgG and IgA
antibodies.
Immunoglobulins:Structure and Function
• Definition: Glycoprotein molecules that are produced by
plasma cells in response to an immunogen and which
function as antibodies
Amount of protein
+
albumin
globulins
a1
a2
e
g
Immune serum
Ag adsorbed serum
Mobility
General Functions of
Immunoglobulins
• Ag binding
– Can result in protection
• Effector functions (Usually require Ag binding)
– Fixation of complement
– Binding to various cells
Immunoglobulin Structure
• Heavy & Light
Chains
• Disulfide bonds
– Inter-chain
– Intra-chain
Disulfide bond
Carbohydrate
CL
VL
CH2
CH1
VH
Hinge Region
CH3
Immunoglobulin Structure
Disulfide bond
• Variable &
Constant Regions
– VL & CL
– VH & CH
Carbohydrate
CL
VL
• Hinge Region
CH2
CH1
VH
Hinge Region
CH3
Structure of the Variable Region
• Hypervariable (HVR) or complimentarity determining
regions (CDR)
Variability Index
HVR3
150
100
HVR2
HVR1
50
FR2
FR1
0
25
FR3
75
50
Amino acid residue
• Framework regions
FR4
100
Immunoglobulin Fragments:
Structure/Function Relationships
• Fab
Papain
– Ag binding
– Valence = 1
– Specificty
determined by VH
and VL
• Fc
– Effector functions
Fc
Fab
Immunoglobulin Fragments:
Structure/Function Relationships
Ag Binding
Complement Binding Site
Binding to Fc
Receptors
Placental Transfer
Immunoglobulin Fragments:
Structure/Function Relationships
Pepsin
• Fab
– Ag binding
• Fc
– Effector functions
• F(ab’)2
Fc
Peptides
F(ab’)2
Human Immunoglobulin Classes
•
•
•
•
•
IgG - Gamma heavy chains
IgM - Mu heavy chains
IgA - Alpha heavy chains
IgD - Delta heavy chains
IgE - Epsilon heavy chains
Human Immunoglobulin Subclasses
• IgG Subclasses
–
–
–
–
IgG1 - Gamma 1 heavy chains
IgG2 - Gamma 2 heavy chains
IgG3 - Gamma 3 heavy chains
IgG4 - Gamma 4 heavy chains
• IgA subclasses
– IgA1 - Alpha 1 heavy chains
– IgA2 - Alpha 2 heavy chains
Human Immunoglobulin
Light Chain Types
• Kappa ()
• Lambda ()
Human Immunoglobulin
Light Chain Subtypes
• Lambda light chains
–
–
–
–
Lambda 1 (1)
Lambda 2 (2)
Lambda 3 (3)
Lambda 4 (4)
Immunoglobulins
• Nomenclature
– IgM (kappa)
– IgA1(lambda 2)
– IgG
• Heterogeneity
Monomeric IgM
IgM only exists as a monomer on the surface of B cells
Monomeric IgM has a very low affinity for antigen
Cm2
N.B. Only constant
heavy chain
domains are shown
Cm4 contains the transmembrane and cytoplasmic regions. These are
removed by RNA splicing to produce secreted IgM
Polymeric IgM
IgM forms pentamers and hexamers
Cm2
N.B. Only constant
heavy chain
domains are shown
Cm3 binds C1q to initiate activation of the classical
complement pathway
Cm1 binds C3b to facilitate uptake of opsonised antigens by
macrophages
Cm4 mediates multimerisation (Cm3 may also be involved)
Multimerisation of IgM
Cm2
1. Two IgM monomers in the ER
(Fc regions only shown)
C
3. A J chain detaches
leaving the dimer
disulphide bonded.
C
2. Cysteines in the J chain
form disulphide bonds
with cysteines from each
monomer to form a dimer
4. A J chain captures another
IgM monomer and joins it
to the dimer.
6. The J chain remains
attached to the IgM
pentamer.
Cm4
5. The cycle is repeated
twice more
ss
Cm4
Antigen-induced conformational changes in IgM
Planar or ‘Starfish’ conformation found in
solution.
Does not fix complement
Staple or ‘crab’ conformation of IgM
Conformation change induced by
binding to antigen.
Efficient at fixing complement
IgM facts and figures
Heavy chain:
m - Mu
Half-life:
5 to 10 days
% of Ig in serum:
10
Serum level (mgml-1):
0.25 - 3.1
Complement activation:
++++ by classical pathway
Interactions with cells:
Phagocytes via C3b receptors
Epithelial cells via polymeric Ig receptor
Transplacental transfer:
No
Affinity for antigen:
Monomeric IgM - low affinity - valency of 2
Pentameric IgM - high avidity - valency of 10
IgD facts and figures
Heavy chain:
d - Delta
Half-life:
2 to 8 days
% of Ig in serum:
0.2
Serum level (mgml-1):
0.03 - 0.4
Complement activation: No
Interactions with cells:
T cells via lectin like IgD receptor
Transplacental transfer: No
IgD is co-expressed with IgM on B cells due to differential RNA splicing
Level of expression exceeds IgM on naïve B cells
IgD plasma cells are found in the nasal mucosa - however the function of IgD in
host defence is unknown - knockout mice inconclusive
Ligation of IgD with antigen can activate, delete or anergise B cells
Extended hinge region confers susceptibility to proteolytic degradation
IgA dimerisation and secretion
IgA is the major isotype of antibody secreted at mucosal sufaces
Exists in serum as a monomer, but more usually as a J chainlinked dimer, that is formed in a similar manner to IgM pentamers.
S
S
S
J
S
ss
S
S
S
S
IgA exists in two subclasses
IgA1 is mostly found in serum and made by bone marrow B cells
IgA2 is mostly found in mucosal secretions, colostrum and milk and is made
by B cells located in the mucosae
Secretory IgA and transcytosis
S
S
SS
SS
SS
SS
ss
ss
S
S
J
S
S
S
S
S
S
J
ss
S
S
S
S
SS
S
S
B
J
J
Epithelial
cell
pIgR & IgA are
internalised
ss
SS
S
S
SS
J
SS
S
S
ss
IgA and pIgR
are transported
to the apical
surface in
vesicles
SS
‘Stalk’ of the pIgR is degraded to release IgA
containing part of the pIgR - the secretory
component
SS
B cells located in the submucosa
produce dimeric IgA
Polymeric Ig
receptors are
expressed on the
basolateral surface of
epithelial cells to
capture IgA produced
in the mucosa
IgA facts and figures
Heavy chains:
a1 or a2 - Alpha 1 or 2
Half-life:
IgA1 5 - 7 days
IgA2 4 - 6 days
Serum levels (mgml-1):
IgA1 1.4 - 4.2
IgA2 0.2 - 0.5
% of Ig in serum:
IgA1 11 - 14
IgA2 1 - 4
Complement activation: IgA1 - by alternative and lectin pathway
IgA2 - No
Interactions with cells:
Epithelial cells by pIgR
Phagocytes by IgA receptor
Transplacental transfer: No
To reduce vulnerability to microbial proteases the hinge region of IgA2 is truncated,
and in IgA1 the hinge is heavily glycosylated.
IgA is inefficient at causing inflammation and elicits protection by excluding, binding,
cross-linking microorganisms and facilitating phagocytosis
IgE facts and figures
Heavy chain:
e - Epsilon
Half-life:
1 - 5 days
Serum level (mgml-1):
0.0001 - 0.0002
% of Ig in serum:
0.004
Complement activation: No
Interactions with cells:
Via high affinity IgE receptors expressed
by mast cells, eosinophils, basophils
and Langerhans cells
Via low affinity IgE receptor on B cells
and monocytes
Transplacental transfer: No
IgE appears late in evolution in accordance with its role in protecting against
parasite infections
Most IgE is absorbed onto the high affinity IgE receptors of effector cells
IgE is also closely linked with allergic diseases
The high affinity IgE receptor (FceRI)
The IgE - FceRI interaction
is the highest affinity of any
Fc receptor with an
extremely low dissociation
rate.
Binding of IgE to FceRI
increases the half life of IgE
a chain
S
g2
S
S
b chain
S
S
S
Ce3 of IgE interacts with the
a chain of FceRI causing a
conformational change.
IgG facts and figures
Heavy chains:
g 1 g 2 g3 g4 - Gamma 1 - 4
Half-life:
IgG1
IgG3
21 - 24 days
7 - 8 days
IgG2
IgG4
21 - 24 days
21 - 24 days
Serum level (mgml-1):
IgG1
IgG3
5 - 12
0.5 - 1
IgG2
IgG4
2-6
0.2 - 1
% of Ig in serum:
IgG1
IgG3
45 - 53
3-6
IgG2
IgG4
11 - 15
1-4
+++
++++
IgG2
IgG4
+
No
Complement activation: IgG1
IgG3
Interactions with cells:
All subclasses via IgG receptors on macrophages
and phagocytes
Transplacental transfer: IgG1
IgG3
++
++
IgG2
IgG4
+
++
C1q binding motif is
located on the Cg2
domain
Carbohydrate is essential for
complement activation
Subltly different hinge regions
between subclasses accounts
for differing abilities to activate
complement
Fcg receptors
High affinity Fcg receptors from the Ig superfamily:
Receptor
FcgRI
FcgRIIA
FcgRIIB1
FcgRIIB2
FcgRIII
Cell type
Effect of ligation
Macrophages Neutrophils,
Eosinophils, Dendritic cells
Uptake, Respiratory burst
Macrophages Neutrophils,
Eosinophils, Platelets
Langerhans cells
Uptake, Granule release
B cells, Mast Cells
No Uptake, Inhibition of stimulation
Macrophages Neutrophils,
Eosinophils
Uptake, Inhibition of stimulation
NK cells, Eosinophils,
Macrophages, Neutrophils
Mast cells
Induction of killing (NK cells)