b cell activation and the humoral immune response
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Each B cell after stimulation with Ag, proliferate and produces 4000 daughter cells and these cells produced 10 12 Ab molecules.
Humoral (Humoral (Antibody-Mediated) Immunity) ImmunityInvolves production of antibodies against foreign Involves production of antibodies against foreign antigens.antigens.
Antibodies are produced by a subset of lymphocytes Antibodies are produced by a subset of lymphocytes called called B cellsB cells..
B cells that are stimulated will actively secrete B cells that are stimulated will actively secrete antibodies and are called antibodies and are called plasma cellsplasma cells..
Antibodies are found in Antibodies are found in extracellular fluidsextracellular fluids (blood (blood plasma, lymph, mucus, etc.) and the surface of B cells.plasma, lymph, mucus, etc.) and the surface of B cells.
Defense against bacteria, bacterial toxins, and viruses Defense against bacteria, bacterial toxins, and viruses that circulate freely in body fluids, that circulate freely in body fluids, beforebefore they enter they enter cells.cells.Also cause certain reactions against transplanted Also cause certain reactions against transplanted tissue.tissue.
Humoral immune response properties
• Humoral immune response against non-protein Ag dose not need to helper T cells.
• Activation of B cells resulted in memory or plasma cells.
• Isotypes switching and affinity maturation are helper T cell dependent.
• The quantity and quality of primary and secondary humoral immune response is different.
Dynamics of Antibody Production
• Antibody production– Initial antibody produced in IgM– Lasts 10-12 days– Followed by production of IgG– Lasts 4-5 days– Without continued antigenic challenge antibody
levels drop off, although IgG may continue to be produced.
Comparison of Primary and
Secondary Responses
• Time course
• Antibody titer
• Antibody class
• Antibody affinity
Immunologic Memory
Virgin lymphocyte pool Virgin lymphocyte pool
PRIMARY RESPONSE PRIMARY RESPONSE
SECONDARY SECONDARY RESPONSERESPONSE
effector cells effector cells memory cell pool memory cell pool
effector cells effector cells memory cell pool memory cell pool
CR2,CD19,CD81= BCR Complex, in this model of activation using complement receptor CD19 is phosphorylated and enhance B cell activation. In
this way antigen is 1000 times more immunogen
T cell
mitogenic BcR signal
'activation' signalbut not mitogenic
mitogenesis
differentiation
present
Ag
T-independent (TI) T-cell dependent (TD)
Types of B cell Antigens
Types of antigen• T-independent (TI) antigens
– 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
B cell activation
T-independent antibody response generally have1. no memory2. no isotype switching3. no somatic mutations
Thymus-dependent because T cells are required
Thymus-independent because T cells are not needed
Some responses require T help whereas other do not
Old Paradigm:
B cell plasma cellsB cell
T-Independent (type II) Responses
Multivalent Antigen
B cell plasma cells
DC/Mø
New Model:
DC/Mø
B cell
(BAFF)
B cell plasma cells
T-Dependent Responses
DC
Antigen
T cell T cell
Dendritic Cell (DC)internalizes antigen (Ag), processes into peptides, presents peptides together with MHC molecules to T cells
B cell binds Ag via surface Ig, transmits BCR signals and presents peptides to T cells, receives T cell help (growth and differentiation factors)
Secretes Antibody (Ab)
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 ( BCR needs 104-106 lower dose)
B cell antigen presentation and the concept of linked help
protein
sugar
Polysaccharide SpecificB cell
T
Protein SpecificT cell
CD40LCytokinesBCR
MHC II
endosome
antigen-binding B cell
MHC
B7.2
TCR
CD28
IL4
FAS
IL4R
CD40
FasL
CD40L
(death)MITOSIS
antigen-specific T cell
Plasma cell
GC cell
isotype switching,
proliferation ?
additional cytokines, costimulatory signals
Cardinal features of B - T interaction
Role of CD40 in B cell activation
• TCR triggering up-regulates CD40L on T cell
• CD40 signaling promotes B cell activation
• CD40L-deficiency = 'hyper-IgM syndrome' (no isotype switching, no Germinal Centers)
• CD40 signaling also important in DC, MØ function
• Germinal center formation is CD40-CD40L interaction dependent
CD4 T cell
CD40L (TNF family)
CD40 (TNF-R family)
TRAF2 TRAF3B cell
increased expression of cell cycle molecules,
survival molecules, cytokines,promotes isotype switching
B-T interaction summary
• BCR signals induce costimulatory molecules (B7.2/CD86, ICAM1 etc), prosurvival molecules (e.g. Bclxl), entry to G1, antigen presentation
• CD40L triggers CD40 -> synergizes with BCR signals to promote mitosis
• T cell derived cytokines promote proliferation, differentiation, isotype switching:– IL2, IL6 promote differentiation
– IL4 -> IgG1, IgE
– IFN -> IgG2a, IgG3
– TGF and IL5 -> IgA
• Many other molecules involved in T-B interactions – ICOS/ICOSL (essential for GC response)
– SLAMM/SAP (promote prolonged B-T interaction essential for GC response)
– ICAM1/LFA1, CD30/CD30L, CD27L/CD27, OX40L/OX40, ...
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
• Some induce type I IFNs- IFNR signaling synergizes with BCR
• Many activate the complement cascade and become C3d coated- complement receptor (CR) crosslinking synergizes with BCR
signal
membrane Ig
secretory Ig
B cell Plasma Cell
After appropriate activation the B cell differentiates into an antibody secreting cell, also known as a Plasma Cell
blimp1
XBP1
Production of membrane vs secreted Ig
membrane Ig
CH tm cy
CH tmcyAAAA
CH
AAAA
secretory Ig
splice, use poly A site 2
1 2
no splice, use poly A site 1
CH tm cy1 2
naive B cell activated B cells
3 days 12 divisions
plasma cells
1 day differentiation
1 day104 Ab/cell/sec
antibodies
1 212 = 4,096 4,096 >1012
B cell antibody response - clonal replication enters into a higher order upon plasma cell differentation
bacteria - dividing every ~60 min5 days = 2120 divisions = 1.3x1036
Plasma Cells are antibody secreting cells
Two types:
1. Plasma cells generated early in the primary response- short-lived (~ few days)
- typically low affinity
- form in T-independent and T-dependent responses
- home to red pulp of spleen, medullary cords of lymph nodes
- IgM but also IgG and other isotypes
2. Plasma cells generated later in the primary response and that predominate in secondary responses- arise predominantly from germinal centers (in primary) or from memory B
cells (in secondary)
- long-lived (possibly several months)
- often home to bone marrow, gut, lactating mammary gland
- predominantly isotype switched
BCR isotype switching
VDJ b a
55 kb
(IL-4)T cell help
antigen
IgM/D+ naive B cell
IgG1+ memory cell
IgG1 secreting plasma cell
Affinity Maturation
• Affinity maturation occurs in germinal centers and is the result of somatic hypermutation of Ig-genes in dividing B cells followed by selection of high affinity B cells by antigen displayed by FDCs
• The high affinity B cells emerging in germinal centers give rise to long-lived plasma cells and memory B cells
Antibody Affinity Maturation
Low affinity B High affinity B
mutation selection
Germinal Center
VH VL
Ag VH VL
VH VL
VH VL VH VL
AgVH VL
FDCFDC
FDC
Light Zone
Dark Zone
FDC
FDC
FDC
CBCB
CBCB
CBCB
CB
CB
T T
Tmemory B cell
plasma cell
CB
CB
MØ
T – T cellFDC – follicular dendritic cellMØ – macrophageCC - centrocyteCB - centroblast
CC
CCCC
CCCC
MØ
CB
Proliferation, somatic hypermutation
Selection, proliferation, differentiation
CB
CBCB CB
Germinal Center Organization
B
High Affinity
Low Affinity
CC
CC, doubling time 6-12 hr , within 5 days each B cell produces 5000 cells, FDC:CR-1,2,3+, MHCII+
Germinal CentersFunction: to generate B cells that produce antibodies with increased
affinity for the inducing antigen
=> affinity maturation
Germinal Center Reaction:
Activated B cells give rise to Centroblasts
- localize in follicle, undergo rapid cell division and turn on machinery that causes somatic mutation in V-regions
Centroblasts give rise to Centrocytes
- migrate to the FDC-rich region of the Germinal Center
- survival is dependent on interaction with FDC-bound Ag and presentation of Ag to T cells
- centrocytes that successfully compete to bind antigen (e.g. by having higher affinity BCR) and to receive T cell help are selected and may differentiate into long-lived plasma cells or memory B cells
mantle zone
GC light zone (CD23++ FDCand centrocytes)
GC dark zone
T zone
(Ki67 cell cycle antigen+ centroblasts)
(CD23+ naive B cells)
Germinal Center
from Liu et al., Immunology Today 13, 17-21 (1992)
Affinity maturation is T-dependent
• T cells are enriched in the light zone
• Represent ~10% of GC cells
LZ
DZ
CD3
IgD
Somatic hyper mutation occurred 10 3 in V H and VL gene and the neucleotide sequences of mutated IgG is 50% more than germinal gene and resulted in change of 10 a.a. in variable area.
Selection Model
• GC B cells that have improved affinity capture more antigen in a given amount of time, receiving a stronger BCR signal and presenting more MHC-peptide complexes to GC T cells, outcompeting surrounding B cells for T cell help
Memory B cells
• Generated during the primary response– best characterized for T-dependent responses involving GC but some
evidence exists for memory to TI antigens
• Small, recirculating cells• Typically isotype switched (e.g. IgG+ or IgA+)• Typically have higher affinity for the inducing Ag• Longer lived than naïve B cells
– Persistence of memory B cells after an immune response ensures that we have increased numbers of B cells specific for the antigen and ready to respond on re-encounter
• May have intrinsic differences that promote greater clonal expansion and more rapid differentiation to plasma cells- differences in cytoplasmic domains of IgG vs IgM/D
- upregulation of TLRs
Down-regulation of B cell response by pre-formed IgG
syk
lyn
IgM
FcRII
Y
calcium and lipid dependent signaling pathways
antigen
I T I M
SHIP
IgG
Antibody Dependent Cell Mediated Cytotoxicity (ADCC)
Target cell is covered with antibodies, Target cell is covered with antibodies, leaving Fc portion sticking outwards.leaving Fc portion sticking outwards.
Natural killer and other nonspecific cells Natural killer and other nonspecific cells that have receptors for Fc region are that have receptors for Fc region are stimulated to kill targeted cells.stimulated to kill targeted cells.
Target organism is lysed by substances Target organism is lysed by substances secreted by attacking cells.secreted by attacking cells.
Used to destroy large organisms that Used to destroy large organisms that cannot be phagocytosed.cannot be phagocytosed.
Receptor Cell type Effect of ligation
FcRI Macrophages Neutrophils,
Eosinophils, Dendritic cells Uptake, Respiratory burst
FcRIIA Macrophages Neutrophils,
Eosinophils, Platelets
Langerhans cells Uptake, Granule release
FcRIIB1 B cells, Mast Cells No Uptake, Inhibition of
stimulation
FcRIIB2 Macrophages Neutrophils,
Eosinophils Uptake, Inhibition of stimulation
FcRIII NK cells, Eosinophils,
Macrophages, Neutrophils
Mast cells Induction of killing (NK cells)
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