Humoral Immunity &Immunoglobulin Structure and

Function

Dr. Adel Almogren

Humoral (Humoral (Antibody-Mediated) Immunity) Immunity + +Involves production of antibodies against foreign Involves production of antibodies against foreign

antigensantigens..

+ +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 cellsplasma, lymph, mucus, etc.) and the surface of B cells..

+ +Defense against bacteria, bacterial toxins, and Defense against bacteria, bacterial toxins, and viruses that circulate freely in body fluids, viruses that circulate freely in body fluids, beforebefore they they enter cellsenter cells..

+ +Also cause certain reactions against transplanted Also cause certain reactions against transplanted tissuetissue..

How Do B Cells Produce Antibodies?How Do B Cells Produce Antibodies? B cells develop from B cells develop from stem cellsstem cells in the bone marrow in the bone marrow

of adults (liver of fetuses).of adults (liver of fetuses).

After maturation B cells migrate to lymphoid organs After maturation B cells migrate to lymphoid organs (lymph node or spleen). (lymph node or spleen).

Clonal SelectionClonal Selection: When a B cell encounters an : When a B cell encounters an

antigen it recognizes, it is stimulated and divides into antigen it recognizes, it is stimulated and divides into

many clones called many clones called plasma cellsplasma cells, which actively , which actively

secrete antibodies, and secrete antibodies, and memory B cellsmemory B cells

Each B cell produces antibodies that will recognize Each B cell produces antibodies that will recognize only one antigenic determinant.only one antigenic determinant.

Clonal Selection of B Cells is Caused by Antigenic Stimulation

Humoral Immunity (Continued)Humoral Immunity (Continued)

Clonal SelectionClonal Selection Clonal SelectionClonal Selection: B cells that encounter : B cells that encounter

stimulating antigen will proliferate into a large stimulating antigen will proliferate into a large group of cells (also apply to T cells).group of cells (also apply to T cells).

Why donWhy don’’t we produce antibodies against our t we produce antibodies against our own antigens? We have developed own antigens? We have developed tolerancetolerance to to them.them.

Clonal DeletionClonal Deletion: B (and T) cells that react against : B (and T) cells that react against selfself antigens appear to be destroyed during fetal antigens appear to be destroyed during fetal development. Process is poorly understood.development. Process is poorly understood.

Antibody ProductionAntibody ProductionTT--Dependent Antigens:Dependent Antigens:

Antibody production Antibody production requiresrequires assistance from T helper cells.assistance from T helper cells. A macrophage cells ingest antigen and presents it to TA macrophage cells ingest antigen and presents it to THH cell.cell. TTHH cell stimulates B cells specific for antigen to become cell stimulates B cells specific for antigen to become

plasma cells.plasma cells. Antigens are mainly proteins on viruses, bacteria, foreign red Antigens are mainly proteins on viruses, bacteria, foreign red

blood cells, and haptenblood cells, and hapten--carrier molecules.carrier molecules.TT--Independent Antigens:Independent Antigens:

Antibody production does not require assistance from T cells.Antibody production does not require assistance from T cells. Antigens are mainly polysaccharides or lipopolysaccharides Antigens are mainly polysaccharides or lipopolysaccharides

with repeating subunits (bacterial capsules).with repeating subunits (bacterial capsules). Weaker immune response than for TWeaker immune response than for T--dependent antigens.dependent antigens.

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

CL VL

S

S

S

S

SS

SS

CH3

CH2 CH1

VH

Fc Fab

F(ab)2

Domains are folded, compact, protease resistant structures

Domain Structure of Immunoglobulins

Pepsin cleavage sites - 1 x (Fab)2 & 1 x FcPapain cleavage sites - 2 x Fab 1 x Fc

Light chain Cdomains or

Heavy chain Cdomains

or

CH3

CH3

CH2

CH3

CH2

CH1

CH3

CH2

CH1VH1

CH3

CH2

CH1VH1

CL

CH3

CH2

CH1VH1

CL

VL

CH3

CH2

CH1VH1

CLVL

Hinge

CH3

CH2

CH1VH

CL VL

Elbow

FbFv

CH3

CH2

Fb

Fv

Fv

FvHinge

Elbow

CH3

CH2

Fb

Fv

Flexibility andmotion of immunoglobulins

Hypervariable regions

FR1 FR2 FR3 FR4CDR2 CDR3CDR1

Amino acid No.

Variability80

100

60

40

20

20 40 60 80 100 120

•Most hypervariable regions coincided with antigen contact points -

the COMPLEMENTARITY DETERMINING REGIONS (CDRs)

Hypervariable CDRs are locatedon loops at the end of the Fv regions

•The sequences of the hypervariable loops are highly variable

amongst antibodies of different specificities

•Variable amino acid sequence in the

hypervariable loops accounts for the diversity of

antigens that can be recognised by a repertoire of

antibodies

Hypervariable loops and framework: Summary

Antibody + complement- mediated damage to E. coli

Healthy E. coli

Electron micrographs of the effect of antibodies and complement upon bacteria

Structure and function of the Fc region

CH3

CH2

IgA IgD IgG

CH4

CH3

CH2

IgE IgMThe hinge region is replaced by an additional Ig domain

Fc structure is common to all specificities of antibody within an ISOTYPE(although there are allotypes)

The structure acts as a receptor for complement proteins and a ligand for cellular binding sites

Monomeric IgM

IgM only exists as a monomer on the surface of B cells

Monomeric IgM has a very low affinity for antigen

C4

C3C2 C1

N.B. Only constant heavy chain

domains are shown

IgM forms pentamers and hexamers

CC

C

C

C C

Multimerisation of IgM

C4 C

3

C2

C

C

C4

C3

C2

C CC4

C3

C2

C

C

C4

C3C2

C

C

C4

C3

C2

C

C

s s

ss

ss

C

Css

IgM facts and figures

Heavy chain: - 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

??IgD & IgM ??

Heavy chain: - 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

IgA dimerisation and secretion

IgA is the major isotype of antibody secreted at mucosal surfaces

Exists in serum as a monomer, but more usually as a J chain-

linked dimer, that is formed in a similar manner to IgM pentamers.

JC C

SS

SS

C

C

SS

SS

C

C

s s

IgA exists in two subclasses

IgA1 is mostly found in serum and made by bone marrow B cells

IgA2 is found in higher concentration in mucosal secretions, colostrum

Epithelialcell

JC C

SS

SS

C

C

SS

SS

CC

ss

Secretory IgA and transcytosis

B

JC C

SS

SS

CC

SS

SS

CCss

JC C

SS

SS

C

C

SS

SS

CC

ss

JC C

SS

SS

C

C

SS

SS

CC

ss

pIgR & IgA areinternalised

‘Stalk’ of the pIgR is degraded to release IgA containing part of the pIgR - the secretory component

JC C

SS

SS

C

C

SS

SS

CC

ss

IgA and pIgR are transported to the apical surface in vesicles

B cells located in the submucosaproduce 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: 1or2 - Alpha 1 or 2

Half-life: IgA1 5 - 7 daysIgA2 4 - 6 days

Serum levels (mgml-1): IgA1 1.4 - 4.2IgA2 0.2 - 0.5

% of Ig in serum: IgA1 11 - 14

IgA2 1 - 4

Complement activation: IgA1 - by alternative and lectin pathwayIgA2 - No

Interactions with cells: Epithelial cells by pIgRPhagocytes by IgA receptor

Transplacental transfer: No

IgE facts and figures

its role in protecting against parasitic infectionsIgE is also closely linked with allergic diseases

Heavy chain: - 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 cellsVia low affinity IgE receptor on B cells and monocytes

Transplacental transfer: No

IgG facts and figures

Heavy chains: 123 4 - Gamma 1 - 4

Half-life: IgG1 21 - 24 days IgG2 21 - 24 days

IgG3 7 - 8 days IgG4 21 - 24 days

Serum level (mgml-1): IgG1 5 - 12 IgG2 2 - 6IgG3 0.5 - 1 IgG4 0.2 - 1

% of Ig in serum: IgG1 45 - 53 IgG2 11 - 15IgG3 3 - 6 IgG4 1 - 4

Complement activation: IgG1 +++ IgG2 + IgG3 ++++ IgG4 No

Interactions with cells: All subclasses via IgG receptors on macrophages and phagocytes

Transplacental transfer: IgG1 ++ IgG2 +IgG3 ++ IgG4 ++

The neonatal Fc receptor may be responsible!

Carbohydrate is essential for complement activation

Subltly different hinge regions between subclasses accounts for differing abilities to activate complement

C1q binding motif is located on the C2 domain

Fc receptors

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)

Hinge

Fv

Fb

Fab

CH3

CH2

CH1VH1

CL VL

Fc

Elbow

Carbohydrate

Antibody Dependent Cell Mediated Cytotoxicity (ADCC)

Target cell is covered with antibodies, Target cell is covered with antibodies, leaving Fc portion sticking outwardsleaving 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 cellsstimulated to kill targeted cells..

Target organism is lysed by substances Target organism is lysed by substances secreted by attacking cellssecreted by attacking cells..

Used to destroy large organisms that Used to destroy large organisms that cannot be phagocytosedcannot be phagocytosed..

Destruction of Large Parasites by ADCC