Acquired Immunity

I. Humoral immunity

Chapter 8

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 Humoral vs Cellular immunity

The division of the adaptive immune system into two major components is based on
the need to recognize two distinctly different forms of foreign invaders.
Some invaders enter the body openly and grow in extracellular fluids. These
exogenous antigens are destroyed by antibodies.
Other invaders grow inside cells, where antibodies cannot reach. These are
destroyed by T cell–mediated responses.

Antibodies are produced by the lymphocytes called B cells.

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 I. Humoral Immunity via B cells

So matured B cells are mainly found in the:

• cortex of lymph nodes
• marginal zone in the spleen
• bone marrow
• throughout the small intestine (Peyer’s patches).

Few B cells circulate in the blood.

Like T cells, B cells have a large number of identical antigen-binding receptors on

their surface. Each B cell, therefore, can only bind and respond to a single antigen.
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 I. Humoral Immunity via B cells

If a B cell encounters an antigen that can bind its receptors, it will, with appropriate
co-stimulation, respond by secreting its specific B cell receptors (BCRs) into body
fluids, where they are called “antibodies”.

Each B cell thus makes antibodies of the same “binding specificity” as its receptors.

Antibodies are simply soluble BCRs secreted into body fluids; they all belong to the
family of proteins called “immunoglobulins”.

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 1. Antibodies or immunoglobulins (Igs)

When the B cells recognize a non-self surface – a virus envelope, a bacterium or a

changed body cell – the B cells turn into a so-called “plasma cells” (an activated
specialized state).

These plasma cells are real “antibody factories.” They produce very large amounts
of specialized antibodies in a short time and release them into the blood and body
fluids (extracellular fluids, lymph, milk, saliva…).

These antibodies bind to foreign antigens and “mark them” for destruction or
elimination. Antibodies are found in many body fluids but are present in highest
concentrations and are most easily obtained from blood serum.
https://www.youtube.com/watch?v=p7dEsaACm2k 5
 1. Basic structure of Igs (1)

It is not surprising, therefore, that multiple Ig classes exist. Each class is optimized
for action in a specific environment; for instance, IgA protects internal body
surfaces.
Igs may also be optimized for activity against a specific group of pathogens. For
example, IgE is important in the defense against parasitic worms.

Each Ig sub-unit is constituted of 4 polypeptide chains:

•2 identical heavy chains (H)
•2 identical light chains (L)

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 Basic structure of Igs (2)
Heavy chains:
A chemically different kind of heavy chain exists for each of the 5 classes of Igs and
is responsible for its antigenic differences. So 5 different heavy chains exist.
Each heavy chain comprises:
•The amino-terminal half of the molecule wc is the “variable” region = VH towards
the (NH2)-end
•The constant domains wc are named according to the peptide chain and numbered
according to the sequence (from amino to carboxyl terminus).

It is the amino-acid sequence of the constant region of its heavy chains wc

determines the class of an Ab whereas its variable region determines its specificity.
Both characteristics (class and specificity) are determined by genetical events in the
B-cells. 8
The hinge region is a flexible amino acid stretch in the central part of the heavy chains of the IgG
and IgA immunoglobulin classes, which links these 2 chains by disulfide bonds giving more
flexibility to the antigen binding site.

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 Basic structure of Igs (3)
Light Chains:
Only 2 types of light (L)-chains have been identified and these are:
• Kappa (κ), wc has then 5 different sub- groups
• Lambda (λ), wc has then 5 different sub-groups

Light chains have been found to possess:

• a constant region = CL, located at its carboxyl (COO-) terminal half of the
molecule
• a region of variability = VL, located at the amino (NH3+)-terminal half

https://www.youtube.com/watch?v=6A9JFaeU7Io
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Basic Igs subunit

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Basic structure of Igs

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Basic structure of Igs

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 Fragmentation of Igs

This could be done by using proteolytic enzymes wc degrade (or digest) the
immunoglobulin molecule into fragments.

The resulting products are due to a brief digestion time, as an extended time would
eventually digest the whole Ig. Thus the brief treatment cleaves only the most
susceptible bonds:

https://www.youtube.com/watch?v=8CbjogONFog

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 Fragmentation by Papain
Would split the IgG in the “hinge” region (i.e. the area of interchain disulfide bonds)
yielding 3 fragments wc can combine with an Ag but would not precipitate it and
these fragments are:

• 1 Fc (because it would crystallize during storage) fragment (COOH-end), wc

contains most of the IgG specific antigenic and biologic properties (such as a role
in complement fixation and opsonization, but no Ag binding capacity).

• 2 Fab fragments (ab, because they retain their antigen-binding activity) (NH2-end),
each of wc possesses one Ag-combining site.

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Fragmentation by Papain

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Fragmentation by Papain

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 Fragmentation by pepsin

Is another proteolytic enzyme that acts on IgG by degrading the H-chain beginning
at the COOH-terminal end and resulting in:

•one F(ab’)2-fragment having 2 Ag-binding sites remaining linked and therefore

could still visibly precipitate Ags while the

• COOH-end is lost with most of its specific antigenic properties located on it.

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Fragmentation by pepsin

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Fragmentation by pepsin vs papain

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 Fragmentation by other proteolytic enzymes?

• Other proteolytic enzymes can also fragment the other Igs (but the fragments
produced are not necessarily comparable to those derived from IgG digestion).
However, in all Ig-molecules, the regions are named according to the system
described for the IgG i.e. Fab, Fc and F(ab’)2.

• Enzymes produced by some bacteria e.g. protease by the enteric Streptococci or

Pneumococcus, would fragment the IgA1 molecule at the hinge region (J-chain)
producing Fab and Fc (papain-like) fragments having a shortened half-life in
mucous secretions and are not able to agglutinate microorganisms.

• However, the dominant form of IgA in the human intestinal tract is IgA2 wc is
insensitive to protease and this was attributed to the evolution of the human
immunity system to resist such bacteria. 23
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 IgA1 vs IgA2
The major difference between IgA1 and IgA2 resides in the hinge region that lies
between the two Fab arms and the Fc region.

IgA1 features a very extended hinge due to the insertion of a duplicated stretch of
amino acids, which is lacking in IgA2. The longer hinge in IgA1 may have evolved
to offer advantages in antigen recognition by allowing higher avidity bivalent
interactions with distantly spaced antigens. However, this extended reach is
accompanied by an increased vulnerability to proteolytic attack.

Indeed, a number of important pathogenic bacteria, such as Streptococcus

pneumonaie, Neisseria meningitidis and Haemophilus influenzae, have evolved
enzymes independently that cleave specifically in the IgA1 hinge. The ability of
these IgA1 proteases to compromise IgA function is thought to facilitate the
initiation of infection at mucosal surfaces by these microorganisms.
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 Igs classes

Antibody molecules are glycoproteins called immunoglobulins (Igs). There are five
different classes (or isotypes) of Igs:
• The class found in highest concentrations in serum is called immunoglobulin G
(IgG) ().
• The class with the second highest serum concentration (in most mammals) is IgM
().
• The third highest concentration in most mammals is IgA(). IgA is, however, the
predominant immunoglobulin in secretions such as saliva, milk, and intestinal
fluid.
• IgD () is rarely encountered in body fluids.
• IgE () is found in very low concentrations in serum (so mainly in mucosa such
as respiratory and intestinal mucosal cells) and mediates allergic reactions.
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 The characteristics of each of these Ig classes
PROPERTY IMMUNOGLOBULIN CLASS

IgM IgG IgA IgE IgD

Molecular weight 900,000 180,000 360,000 200,000 180,000

Subunits 5 1 2 1 1

Heavy chain µ γ α ε δ

Largely Spleen and lymph Spleen and lymph Intestinal and Intestinal and Spleen and lymph
synthesized in: nodes nodes respiratory tracts respiratory tracts nodes

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 Igs classes: IgG (monomer)

IgG is produced by plasma cells in the spleen, lymph nodes, and bone marrow. It is
the immunoglobulin found in highest concentration in the blood and plays the major
role in antibody-mediated defenses.
It has two identical light chains and two identical γ heavy chains. Its light chains
may be of the κ or λ type.

Because it is the smallest of the immunoglobulin molecules, IgG can escape from
blood vessels more easily than can the others. This is especially important in
inflammation, in which increased vascular permeability allows IgG to participate in
the defense of tissues and body surfaces.

IgG binds to specific antigens such as those found on bacterial surfaces. Binding of
these antibody molecules to bacterial surfaces can cause clumping (agglutination)
and opsonization. 28
The structure of IgG, the prototypical immunoglobulin molecule

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 Igs Classes: IgM (pentamer)
IgM is also produced by plasma cells in the secondary lymphoid organs.

It occurs in the second highest concentration after IgG in most mammalian serum.
The secreted form of IgM consists of five units linked by disulfide bonds in a
circular fashion. A small polypeptide called the “J” chain joins two of the units to
complete the circle.

Each IgM monomer is of conventional Ig structure and consists of two κ or λ light

chains and two µ heavy chains.

Although produced in small amounts, IgM is more efficient than IgG at complement
activation, opsonization, neutralization of viruses, and agglutination. Because of
their very large size, IgM molecules rarely enter tissue fluids even at sites of acute
inflammation. 30
The structure of IgM. This diagram shows a pentameric structure with 5 subunits
which is the most abundant form of the molecule (linked by a “J” chain
which joins two of the units to complete the circle)

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The relative amounts of each immunoglobulin class produced during the primary
and secondary immune responses. Note that IgM predominates in a primary
immune response, whereas IgG predominates in a later response.

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 Igs Classes: IgA (1)

Immunoglobulin A (IgA, also referred to as sIgA in its secretory form) is

an antibody that plays a crucial role in the immune function of mucous membranes.
IgA has 2 forms: serum IgA and secretory IgA (sIgA) and with two subclasses of
IgA, named IgA1 and IgA2.
The predominant serum IgA form is monomeric, i.e., comprises 2HC and 2LC, with
a subclass distribution of about 90% IgA1.
In contrast, the main molecular form found at mucosal surfaces, known as secretory
IgA (SIgA), is dimeric; an average distribution being about 40% IgA1 and 60%
IgA2.

The amount of IgA produced in association with mucosal membranes (SIgA) is

greater than all other types of antibody combined (up to 15% of total
immunoglobulins produced throughout the body).
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Shorter hinge region in IgA2 as compared to that of IgA1, making it less
accessible to proteolytic enzymes secreted by some resistant pathogens

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 IgA (2)

IgA does not activate the classical complement pathway, nor can it act as an
opsonin.
It can, however, agglutinate particulate antigens and neutralize viruses.
IgA prevents the adherence of invading microbes to body surfaces.

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The structure of IgA and secretory IgA. The secretory component is found on
the surface of certain epithelial cells.

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 SIgA
SIgA produced in body surfaces is transported through epithelial cells into external
secretions. Thus most of the IgA made in the intestinal wall is carried into the
intestinal fluid. This IgA is transported through intestinal epithelial cells bound to
the secretory component (which is synthesized by epithelial cells).
Secretory component binds IgA dimers to form a complex molecule called secretory
IgA (SIgA). It protects the IgA from digestion by intestinal proteases.
https://www.youtube.com/watch?v=rQHSn9HePGE

Secretory IgA is of critical importance in protecting the intestinal, respiratory, and

urogenital tracts, the mammary gland, and the eyes against microbial invasion.

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 SIgA
Secretory IgA is present in saliva,
nasal mucus, bronchial secretions,
mucus of small intestines, tears etc.

It is secreted by mucosal plasma

cells as monomers and dimers.

The secretory piece is synthesized

by epithelial cells and it is added to
the dimer which is then transported
to the surface of the epithelium
where they are free to react with
the appropriate antigens of
microbes. 39
IgA is the principal class of antibody in secretions (saliva, milk, tears, and respiratory and intestinal
secretions). Whereas IgA is a 4-chain monomer in blood, it is an 8-chain dimer in secretions.

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 Igs classes: IgE (monomer)
IgE occurs in serum at the lowest concentration.
The Fc-molecule of the IgE molecule has a unique characteristic. It has an extremely
high affinity for special cell surface-receptors on tissue mast cells (are also
basophilic leukocytes but found in tissues thus the name “mast cells”) and on the
basophilic leukocytes (are granulated WBCs).

Therefore mast cells and basophils may carry on their surface Ab-molecules of the
IgE-class.
https://www.youtube.com/watch?v=SW9iSnxTcqQ
The IgE molecules are held by the Fc-portion and therefore their Ag-binding sites
are freely extended into the microenvironment. Cross-linking of IgE-molecules by
Ag, or by anti-IgE Abs, triggers the mast cells or basophils to release potent
pharmacological active factors which are responsible for symptoms of many acute
allergic conditions and hay fever. 41
IgE: allergy reaction to help eliminating the invader

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 Igs classes: IgD (monomer)

IgD is found in serum but in very low concentration, is very sensitive to enzymes
and to heat and its role is still unclear.

In contrast to the other Igs classes, IgD:

-shows many variations in structure (concerning MW, number of heavy or constant
domains..) between animal species.
-Is not found in some animal species (rabbits, cats, chicken..).

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 Ig Variants (1)

All Ig molecules are made of:

- 2 heavy chains and
- 2 light chains.

The type of heavy chain (between bracket) determines the type of Ig. For example:
• IgG ( chain)
• IgM ( chain)
• IgA ( chain)
• IgD ( chain)
• IgE ( chain)
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 Ig Variants (2)

These different classes of Igs may have each subclasses to in some animal species.

The subclasses are the result of minor variation in amino acid sequences in heavy
chains during the course of evolution. These variations, among species, are not of
major biological significance.

All animals of a species will possess then these subclasses.

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