LECOM-Pharmacy

Immunology 5 & 6
Adaptive (acquired) immune response
Antigen & Antibody
Dr. Saber Hussein
 Objectives
• Define antigenicity and immunogenicity
• Four characteristics of immuogenic molecule
(requirements for immunogenicity)
• Foreignness
• High molecular weight
• Chemical complexity
• Degradability
• Define haptens and their functions
• Define antigenic determinants
• Capture of protein antigens by antigen presenting cells
• Antigen recognized by T lymphocytes
• Antigens recognized by B lymphocytes
 Learning Objectives
1. Basic structure of Abs in relation to specificity &
diversity
2. Variable & constant regions of light & heavy
chain
3. Biological & chemical characteristics of the 5
classes of Ab
4. Compare polyclonal & monoclonal Ab
5. Three characteristics of primary Ag-Ab reaction
6. The forces that foster the primary Ag-Ab reaction
7. Affinity & avidity of Abs
8. Secondary Ag-Ab reaction; lattice formation
 Objectives
9. Know the role of receptors in the recognition of
antigen in the adaptive immune system:
– Antibodies as receptors of B lymphocytes
– T cell receptor (TCR) for antigens
10. Development of immune repertoires
1. Maturation of lymphocytes
2. Production of diverse antigen receptors
3. Maturation and selection of B lymphocytes
4. Maturation and selection of T lymphocytes
 Antigenicity & Immunogenicity
• Antigenicity:
– The ability to bind an Ab or an activated T cell
– Every immunogen is an antigen, BUT not every
antigen is an immunogen
• Immunogenicity:
– Ability to elicit immune response
– Only proteins can induce cellular immunity
– Humoral immunity can be induced by:
• Proteins
• Lipopolysaccharides
• Nucleic acids
• Other substances
 Features of an Immunogen
1. High molecular weight
2. Chemical complexity
3. Solubility or biodegradability
4. Foreignness or nonself
Ab cross-reactions with different Ags
• Abs react most strongly with homologous Ag
• Sometimes they cross-react with other Ags
• Cross-reaction
– The reaction between an Ag and an Ab that was
generated against a different Ag but with some
similarity with the cross-reacting Ag
• Cross-reactions are related to chemical structure of Ags:
– i. Chemical nature of hapten’s groups
– ii. Position of substitutions
– iii. Size of substituted groups
– iv. Charge
– v. Stereoisomerism
 Haptens, Antigenic Determinants (Epitopes)
• Ag has 2 functional regions: Hapten
1. Hapten Carrier

2. Carrier Ab
• Epitopes are immunologically active portions of Ag
• Epitopes on an Ag are recognized by B cells and T cells
• Antigenic determinants serve as fingerprint of
macromolecules
• Size of an epitope is determined by the size of the Ab’s
Ag-binding site
• Size of recognizable epitope by an Ab:
– 6 sugar residues
– 15-20 amino acids (Some linear epitopes are as small as 5 aas)
 Haptens &Antigenic Determinants
Haptens Antigenic Determinants
1. Usually small molecules 1. Small part of the
2. Not immunogenic by themselves molecule
3. Always antigenic with a specific – Few amino acids
Ab – A short carbohydrate
4. Immunogenic when combined with moiety- few sugars
a carrier molecule (large)
2. Must be accessible to
5. Simple hapten: only 1 antigenic
be functional
determinant
6. Complex hapten: > 2 antigenic 3. Charge & polarity
determinants 4. Conformation
dependent
 Ag recognized by T lymphocytes

• T lymphocytes recognize only protein antigens

• Proteins must be presented in the form of short
peptides
• They must be presented by an APC with the
appropriate MHC molecule:
– MHC I presents antigen to the cytotoxic T cell
– MHC II presents antigen to the helper T cell
Antigens recognized by B lymphocytes
• T-cell independent route of antigen recognition:
– B lymphocytes recognize certain antigens without
the help of the TH cell
– These include:
• Lipopolysaccharides
• Nucleic acids: DNA & RNA
– No long term immunity results through this route
– Only IgM is produced
– No memory cells
Antigens recognized by B lymphocytes
• T-cell dependent route:
– Recognizes protein antigens
– Long term immunity
– IgG is produced by class switching
– Memory cells
 Antigens recognized by B lymphocytes
T-cell dependent T-cell independent
1. Recognizes 1. Recognizable Ags:
protein antigens • Lipopolysaccharides
2. Long term • Nucleic acids: DNA &
immunity RNA
3. IgG is produced by 2. No long term
class switching immunity results
through this route
4. Memory cells
3. Only IgM is produced
4. No memory cells
 • A model of how a T cell receptor
Antigen Capture and (TCR) recognizes a complex of a
peptide antigen displayed by a
Presentation to major histocompatibility (MHC)
Lymphocytes molecule
– MHC molecules are expressed
on antigen-presenting cells
and function to display peptides
derived from protein antigens
– Peptides bind to the MHC
molecules by anchor residues,
which attach the peptides to
pockets in the MHC molecules
– The TCR of every T cell
recognizes some residues of the
peptide and some
(polymorphic) residues of the
MHC molecule

Fig. 3-1
The capture and display of microbial antigens
• Microbes enter the body
– through an epithelium
and are captured by
antigen-presenting cells
resident in the epithelium
– enter lymphatic vessels or
– blood vessels
• The microbes and their
antigens are transported
to peripheral lymphoid
organs
– the lymph nodes,
– the spleen,
• where protein antigens are
displayed for recognition Fig. 3-2
by T lymphocytes
 The capture and • Immature dendritic cells
in the epithelium capture
presentation of protein microbial antigens by
antigens by dendritic cells surface receptors and leave
the epithelium
• The dendritic cells migrate
to draining lymph nodes,
being attracted there by
chemokines produced in the
nodes
Langerhans cells
• During their migration, and
in response to the microbe,
the dendritic cells mature
• Mature DC express high
level MHC & costimulators
• In the lymph nodes, the
dendritic cells present
Fig 3-4 antigens to naive T cells
Properties of
MHC
molecules
and genes
• Some of the
important
features of
MHC
molecules are
listed, with
their
significance for
immune
responses
Fig 3-8
Role of MHC in Antigen Presentation to T Cells

• Ag processing
– The event whereby the Ag is prepared to be presented to
lymphocytes in a form they can recognize
– It includes fragmentation of the protein Ag into small
peptides in the macrophage and the presentation to T cells
• Ag-presenting cells (APCs) bind peptide Ags to their
MHC II and present it to the CD4+ helper T cells
• APCs present peptides to CD8+ cytotoxic T cells with
their MHC I
• Ag-presenting cells (APCs) include macrophages and
other cells
m

Major Histocompatibility Complex (MHC)

 MHC Restriction of T Cells
• The process by which the MHC controls
interactions between immune cells
• It involves the recognition of foreign antigens
in association with class I or II molecules
• The following reactions are MHC-restricted:
1. Antigen presentation
2. T- and B-cell cooperation
3. Cytotoxic T-cell interaction with target cells
• Malignant cells
• Viral infected cell
 What Regions of HLA Complex Encode
MHC I & MHC II?
Coding Regions:
• MHC-I coding region:
– HLA-A, HLA-B and HLA-C
• MHC-II coding region:
– HLA-D [DN, DO, DP, DQ & DR]
 Antigen-MHC Class II Complex

What kind of T cell do we see here?

 Fig 4-1: Properties of antibodies and T cell
antigen receptors (TCRs)
• Antibodies may be expressed as membrane receptors or
secreted proteins
• TCRs only function as membrane receptors
• When Ig or TCR molecules recognize antigens, signals are
delivered to the lymphocytes by proteins associated with
the antigen receptors
• The antigen receptors and attached signaling proteins
form the B cell receptor (BCR) & TCR complexes
• Single antigen receptors are shown recognizing antigens,
• Signaling requires the cross-linking of two or more
receptors by binding to adjacent antigen molecules
Fig 4-1
Fig 4-1
CD4,8

B cell T cell
Fig 4-1

 :zeta)
Fig 4-2: The structure of antibodies

• Schematic diagrams of a secreted IgG (A) and a

membrane form of IgM (B) illustrate the
domains of the heavy and light chains and the
regions of the proteins that participate in antigen
recognition and effector functions
• N and C refer to the amino-terminal and carboxy-
terminal ends of the polypeptide chains,
respectively
Fig 4-2
 Fig 4-3: Features of the major isotypes
(classes) of antibodies
• The table summarizes some important features of the major
antibody isotypes of humans.
• Isotypes are classified on the basis of their heavy chains
• Each isotype may contain either  or  light chain
• Each of the 5 classes differ in their locations in our body
and how they stimulate the innate system to remove antigen
• The schematic diagrams illustrate the distinct shapes of the
secreted forms of these antibodies
• IgA consists of two subclasses: IgA1 and IgA2
• IgG consists of 4 subclasses: IgG1, IgG2, IgG3, & IgG4
• The serum concentrations are average values in normal
individuals
Fig 4-3: Features of the major isotypes of Abs

with mother’s
milk
Breast-fed
neonates get
it with the
mother’s milk

Antiparasitic
activity
Fig 4-3: Features of the major isotypes of
Abs

Diagnostic for
acute infections
                                                                   

       
            
 Polyclonal & Monoclonal Abs
• Polyclonal Abs • Monoclonal Abs
1. Heterogeneous mix of 1. Produced by a single
Abs clone of cells
2. With specificity against 2. Resultant Abs are
the same Ag identical in all aspects
3. Produced by variety of 3. Same affinity
Ab-producing cells 4. Same binding specificity
4. They are many clones of 5. Recognize the same
cells epitope
5. Polyclonal Abs recognize 6. They are produced in
& react against different hybridoma between
epitopes on the Ag activated B cells and
6. Avidity malignant plasma cells
(fusion)
Three dimensional representation
of the IgG molecule

IgG molecule
 IgG
• IgG digestion with papain
produces 3 fragments
– 2 identical Fab fragments
• Fab fragments, are
capable of binding Ag
because they contain the
Ag-binding site
– Fc fragment: a fragment
composed of H chain only.
• It crystallizes in the cold
 Pentameric structure of IgM
• The structure of IgM is
similar to that of IgG
except the IgM heavy chain
has an extra domain.
• A small, cysteine-rich
protein called J chain
initiates cross linking of
C3 and C4 of
– five IgM monomers to
make the circulating,
pentameric form of
IgM
Dimeric structure of
IgA
• Dimeric IgA held
together by
– J chain and
– secretory component J chain
Secretory
component
Secretory IgA • IgA represents 15-20% of serum
immunoglobulin
• It constitutes the majority of Ab
found in secretions
• Humans have 2 types of IgA:
– IgA1 and IgA2
– IgA1is the prominent
subclass in serum and is
found mainly as monomer
– IgA2 is the prominent Ig in
secretions (saliva, gut,
respiratory mucus) and
occurs as a dimer with two
Fc ends of the Abs bound
together by a J chain
• Secretion across the mucosa is
mediated by a specific secretory
component which binds to a cell
receptor
 IgE
• IgE is similar to IgG
except
– it has an extra constant
region domain on the H-
chain
• Functions:
– Type I hypersensitivity
– Anti-parasitic
– Degranulation of mast cells
 IgD

• IgD is similar to the

structure of IgG.
• Its only known
function is as part of
the signaling
complex of B cells
 Primary Ag-Ab reaction
1. The first interaction between Ag & Ab
2. Key-lock principle
3. Ag-Ab interaction is precise = specific
4. Characteristics of Ag-Ab reaction:
i. Rapid, in seconds
ii. Independent of electrolytes, salt, buffer
iii. Not visible
 Fig 4-4: Binding of an Ag by an Ab
• This model of a protein antigen bound to an antibody molecule shows how the antigen-
binding site can accommodate soluble macromolecules in their native (folded)
conformation.
• The heavy chains (H) of the antibody are red
• L chains: yellow
• Ag is blue
 Chemical forces foster Ab-Ag
Four noncovalent interactions hold antigenic
determinants w/in Ab-binding site:
1. Coulombic (electrostatic, ionic)
interactions
2. Van der Waals forces
3. Hydrogen bonds
4. Hydrophobic interactions
 Secondary Ag-Ab reaction &
Secondary response

• Secondary Ag-Ab reaction:

The conversion of the invisible primary
reactions macroscopically visible ones as in
the case of precipitation and agglutination
• Secondary response:
The immune response which follows a
second encounter with a particular Ag
It is usually stronger (affinity maturity)
 Lattice formation
Ab Ag
• Occurs when
i. Ag-Ab complexes
aggregate in form
of precipitation in
liquid medium -
ii. Agglutination,
including
particulate
components, other
than Ag and Ab,
such as cells
 Affinity & Avidity
• Affinity is the strength of Ag-Ab bonds between a single
epitope and an individual Ab’s binding site
• Avidity: The binding strength between a multivalent Ab
(polyclonal Ab) and a multivalent Ag
• Ag + Ab  Ag..Ab
K = [Ag..Ab]/[Ag][Ab]
• The higher [Ag..Ab], the larger is K (the associated Ab
and Ag), the higher is affinity of the Ab to the Ag.
• K = Equilibrium constant
= Association constant
= Ab affinity
 Affinity maturity
• Ag + Ab  Ag..Ab
KD = [Ag][Ab] / [Ag..Ab]
• The lower the KD (dissociation constant) the
higher the affinity
• Affinity maturity: after repeated exposure to
the Ag the affinity increases
 Monoclonal Ab production
• Immunize animals, rats or mice, with Ag
• When the animals start to make a good Ab response remove their
spleens and prepare a cell suspension
• Fuse spleen cells with a myeloma cell line by the addition of
polyethylene glycol (PEG), which promotes membrane fusion
– Only a small proportion of the cells fuse successfully
• The fusion mixture is then set up in culture with medium containing
“HAT”
– HAT = Mixture of
• Hypoxanthine
• Aminopterin (powerful toxin that blocks a metabolic
pathway)
• Thymidine (H & T intermediate metabolites help the cell
bypass the pathway when added)
– Spleen cells can grow/survive in HAT
– Myeloma cells are sensitive to HAT because of metabolic
defect that prevents them from using the bypass
– HAT culture contains:
• Spleen cells: die naturally in 1-2 weeks
• Myeloma cells: Killed by HAT
• Fused cells (hybridoma): Survive because of immortality
of myeloma and HAT-resistance of the spleen cells
– Some produce antibody
– Any wells containing growing cells are tested for
the production of the desired Ab (often by solid
phase immunoassay)
– Positive ones are cloned by plating out so that
there is only one cell in each well
– This produces a clone derived from a single
progenitor, which is both:
» Immortal
» Producer of monoclonal Ab
Humoral Immune Response
 B cells produce Abs
• B cells are specialized white blood cells produced
in the bone marrow.
• Each B cell contains multiple copies of one kind
of antibody as a surface receptor for antigen.
• The entire population of B cells has the ability
to specifically bind to millions of different
antigens
• When the antibody on the surface of a B cell
binds to an antigen, the cell can be stimulated to
undergo proliferation and differentiation.
• This process is called clonal selection.
 Clonal selection
• The cells produced make the same Ab, but become
memory cells and plasma cells
– Memory cells insure that subsequent infections by the
pathogen receive a more rapid response.
– Plasma cells secrete large amounts of the Ag-specific
Ab
• T helper cells are required for the clonal selection of B
cells
• Ab secreted by plasma cells forms complexes with free
pathogens and their toxic products
• The complexes can:
– inactivate pathogens &
– stimulate other innate systems including
• phagocytes and complement to eliminate the
danger from our extracellular fluids
 Abs and their diversity
• An Ab immunoglobulin is a "Y" shaped
molecule made up of two identical "light" and
"heavy" chains of amino acids.
• The variable region includes the N-terminal
110-130 amino acids of the light and heavy
chains, and is responsible for binding to antigen.
• The constant region is the C-terminal end and
contains similar amino acids for each class of
Ab.
 Abs Diversity (con)
• When a stem cell changes to become a B cell,
DNA segments for both heavy (VDJ) and light
(VJ) chains are randomly combined.
• Each B cell ends up with functional genes for
making one light and one heavy chain coding for
an Ab as a membrane-bound receptor.
• Ab specificity depends on the gene fragments
used.
• Abs are produced that can react with almost any
chemical structure in nature.
 Abs Diversity
The immune system creates billions of different Abs
with a limited number of genes by rearranging
DNA segments during B cell development, prior to
Ag exposure.
Mutation can also increase genetic variation in Abs

Light
chain

Heavy chain
 Ab Class switching
• At first, B cells contain IgM molecules only.
• Class switching occurs after Ag binding, when
plasma cells are produced.
• Class switching refers to a DNA rearrangement
changing the heavy chain constant gene in
memory cells.
• Loss of coding regions for the constant part of the
heavy chain causes IgG, IgA, and IgE to be
produced.
Ab Class switching to produce IgA