B Cell Activationand the

Humoral Immune Response

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.

Antibody Protection of the Host

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

ITAM, immuno receptor tyrosin- based activation motif

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 cells are antigen-presenting cells

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

Ig Heavy chain class (isotype) switching

neutralization

neutralization

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.

Destruction of Large Parasites by ADCC

FcgRIIIA

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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