Immuno5-6serv Adaptive Immune Response Ag-Ab
Transcript of Immuno5-6serv Adaptive Immune Response Ag-Ab
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LECOM-Pharmacy
Immunology 5 & 6
Adaptive (acquired) immune response
Antigen & AntibodyDr. Saber Hussein
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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
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Learning Objectives1. Basic structure of Abs in relation to specificity &
diversity
2. Variable & constant regions of light & heavychain
3. Biological & chemical characteristics of the 5classes of Ab
4. Compare polyclonal & monoclonal Ab
5. Three characteristics of primary Ag-Ab reaction6. The forces that foster the primary Ag-Ab reaction
7. Affinity & avidity of Abs
8. Secondary Ag-Ab reaction; lattice formation
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Objectives
9. Know the role of receptors in the recognition ofantigen in the adaptive immune system:
Antibodies as receptors of B lymphocytes
T cell receptor (TCR) for antigens10. Development of immune repertoires
1. Maturation of lymphocytes
2. Production of diverse antigen receptors3. Maturation and selection of B lymphocytes
4. Maturation and selection of T lymphocytes
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Antigenicity & Immunogenicity
Antigenicity: The ability to bind an Ab or an activated T cell
Every immunogen is an antigen, BUT not everyantigen 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
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Features of an Immunogen1. High molecular weight
2. Chemical complexity
3. Solubility or biodegradability
4. Foreignness or nonself
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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 somesimilarity with the cross-reacting Ag
Cross-reactions are related to chemical structure of Ags:
i. Chemical nature of haptens groups
ii. Position of substitutions
iii. Size of substituted groups
iv. Charge
v. Stereoisomerism
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Haptens, Antigenic Determinants (Epitopes)
Ag has 2 functional regions:
1. Hapten
2. Carrier
Epitopes are immunologically activeportions of Ag
Epitopes on an Ag are recognizedby B cells and T cells Antigenic determinants serve as fingerprint of
macromolecules
Size of an epitope is determined by the size of the Abs
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)
Ab
Carrier
Hapten
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Haptens &Antigenic Determinants
Haptens
1. Usually small molecules
2. Not immunogenic by themselves
3. Always antigenic with a specific
Ab4. Immunogenic when combined with
a carrier molecule (large)
5. Simple hapten: only 1 antigenic
determinant
6. Complex hapten: > 2 antigenic
determinants
Antigenic Determinants1. Small part of the
molecule
Few amino acids
A short carbohydratemoiety- few sugars
2. Must be accessible to
be functional
3. Charge & polarity
4. Conformation
dependent
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Ag recognized by T lymphocytes
T lymphocytes recognize only protein antigens
Proteins must be presented in the form ofshort
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
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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
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Antigens recognized by B lymphocytes
T-cell dependent route: Recognizes protein antigens
Long term immunity
IgG is produced by class switchingMemory cells
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Antigens recognized by B lymphocytes
T-cell dependent
1. Recognizes
protein antigens
2. Long term
immunity
3. IgG is produced by
class switching
4. Memory cells
T-cell independent
1. Recognizable Ags:
Lipopolysaccharides
Nucleic acids: DNA &
RNA
2. No long term
immunity results
through this route3. Only IgM is produced
4. No memory cells
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Antigen Capture and
Presentation to
Lymphocytes
A model of how a T cell receptor
(TCR) recognizes a complex of a
peptide antigen displayed by a
major histocompatibility (MHC)
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 thepeptide and some
(polymorphic) residues of the
MHC molecule
Fig. 3-1
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The capture and display of microbial antigens
Microbes enter the body
through an epitheliumand are captured byantigen-presenting cellsresident in the epithelium
enterlymphatic vessels or
blood vessels
The microbes and theirantigens are transportedto peripheral lymphoid
organs the lymph nodes,
the spleen,
where protein antigens aredisplayed for recognition
by T lymphocytes
Fig. 3-2
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The capture and
presentation of protein
antigens by dendritic cells
Immature dendritic cellsin the epithelium capturemicrobial antigens by
surface receptors and leavethe epithelium
The dendritic cells migrateto draining lymph nodes,
being attracted there by
chemokines produced in thenodes
During their migration, andin response to the microbe,the dendritic cells mature
Mature DC express highlevel MHC & costimulators
In the lymph nodes, thedendritic cells present
antigens to naive T cellsFig 3-4
Langerhans cells
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Properties of
MHC
moleculesand genes
Some of the
importantfeatures of
MHC
molecules are
listed, with their
significance forimmune
responses
Fig 3-8
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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 withtheirMHC I
Ag-presenting cells (APCs) include macrophages and
other cells
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Major Histocompatibility Complex (MHC)
m
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MHC Restriction ofT 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
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What Regions ofHLA 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]
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Antigen-MHC Class II Complex
What kind of T cell do we see here?
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Fig 4-1: Properties of antibodies and T cell
antigen receptors (TCRs)
Antibodies may be expressed as membrane receptors orsecreted proteins
TCRs only function as membrane receptors
When Ig or TCR molecules recognize antigens, signals are
delivered to the lymphocytes by proteins associated withthe 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
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Fig 4-1
CD4,8
Fig 4-1
T cellB cell
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Fig 4-1
^ :zeta)
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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 theregions 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
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Fig 4-2
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Fig 4-3: Features of the major isotypes
(classes) of antibodies
The table summarizes some important features of the majorantibody isotypes of humans.
Isotypes are classified on the basis of their heavy chains
Each isotype may contain either O or P light chain
Each of the 5 classes differ in their locations in our bodyand how they stimulate the innate system to remove antigen
The schematic diagrams illustrate the distinct shapes of thesecreted forms of these antibodies
IgA consists oftwo subclasses: IgA1 and IgA2 IgG consists of4 subclasses: IgG1, IgG2, IgG3, & IgG4
The serum concentrations are average values in normalindividuals
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Fig 4-3: Features of the major isotypes of Abs
Breast-fedneonates get
it with the
mothers milk
Antiparasitic
activity
with mothers
milk
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Fig 4-3: Features of the major isotypes of
Abs
Diagnostic for
acute infections
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Polyclonal & Monoclonal Abs Polyclonal Abs
1. Heterogeneous mix ofAbs
2. With specificity againstthe same Ag
3. Produced by variety ofAb-producing cells
4. They are many clones ofcells
5. Polyclonal Abs recognize& react against differentepitopes on the Ag
6. Avidity
Monoclonal Abs
1. Produced by a singleclone of cells
2. Resultant Abs areidentical in all aspects
3. Same affinity
4. Same binding specificity
5. Recognize the sameepitope
6. They are produced inhybridoma betweenactivated B cells andmalignant plasma cells
(fusion)
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Three dimensional representation
of the IgG molecule
IgG molecule
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IgG
IgG digestion with papainproduces 3 fragments
2 identical Fab fragments
Fab fragments, are
capable of binding Agbecause they contain the
Ag-binding site
Fc fragment: a fragment
composed ofH chain only.
It crystallizes in the cold
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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 chaininitiates cross linking of
C3 and C4 of
five IgM monomers to
make the circulating,pentameric form of
IgM
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Dimeric structure of
IgA
Dimeric IgA held
together by
J chain and
secretory component J chain
Secretory
component
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Secretory IgA IgA represents 15-20% of serumimmunoglobulin It constitutes the majority of Ab
found in secretions Humans have 2 types of IgA:
IgA1 and IgA2
IgA1is the prominentsubclass in serum and is
found mainly as monomer IgA2 is the prominent Ig in
secretions (saliva, gut,respiratory mucus) andoccurs as a dimer with twoFc ends of the Abs boundtogether by a J chain
Secretion across the mucosa ismediated by a specific secretorycomponent which binds to a cellreceptor
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IgE
IgE is similar to IgGexcept
it has an extra constant
region domain on the H-
chain
Functions:
Type I hypersensitivity
Anti-parasitic
Degranulation of mast cells
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IgD
IgD is similar to thestructure of IgG.
Its only known
function is as part of
the signalingcomplex of B cells
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Primary Ag-Ab reaction1. 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
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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 solublemacromolecules in their native (folded) conformation.
The heavy chains (H) of the antibody are red
L chains:yellow
Ag is blue
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Chemical forces foster Ab-AgFour 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
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Secondary Ag-Ab reaction &
Secondary response Secondary Ag-Ab reaction:
The conversion of the invisible primary
reactions macroscopically visible ones as inthe case of precipitation and agglutination
Secondary response:
The immune response which follows asecond encounterwith a particular Ag
It is usually stronger (affinity maturity)
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Lattice formation
Occurs wheni. Ag-Ab complexes
aggregate in formofprecipitation in
liquidmedium -ii. Agglutination,
includingparticulate
components, otherthan Ag and Ab,such as cells
AgAb
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Affinity & Avidity Affinity is the strength of Ag-Ab bonds between a single
epitope and an individualAbs bindingsite
Avidity: The binding strength between a multivalentAb
(polyclonal Ab) and a multivalentAg
Ag + Ab Ag..AbK = [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
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Affinity maturity
Ag + Ab Ag..AbKD = [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
M l l Ab d ti
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Monoclonal Ab production Immunize animals, rats or mice, with Ag
When the animals start to make a good Ab response remove theirspleens and prepare a cell suspension
Fuse spleen cells with a myeloma cell line by the addition ofpolyethylene 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 metabolicpathway)
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 metabolicdefect 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 immortalityof myeloma and HAT-resistance of the spleen cells
Some produce antibody
Any wells containing growing cells are tested forthe production of the desired Ab (often by solid
phase immunoassay)
Positive ones are cloned by plating out so thatthere is only one cell in each well
This produces a clone derived from a singleprogenitor, which is both:
Immortal Producer of monoclonal Ab
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Humoral Immune Response
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B cells produce Abs
B cells are specialized white blood cells produced
in the bone marrow.
Each B cell contains multiple copies of one kindof antibody as a surface receptor for antigen.
The entire population of B cells has the abilityto specifically bind to millions of differentantigens
When the antibody on the surface of a B cell
binds to an antigen, the cell can be stimulated toundergo proliferation and differentiation.
This process is called clonal selection.
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Clonal selection The cells produced make the same Ab, but become
memory cells and plasma cells
Memory cells insure that subsequent infections by thepathogen receive a more rapid response.
Plasma cells secrete large amounts of the Ag-specificAb
T helper cells are required for the clonal selection of Bcells
Ab secreted by plasma cells forms complexes with freepathogens and their toxic products
The complexes can:
inactivate pathogens &
stimulate otherinnate systems including
phagocytes and complement to eliminate thedanger from our extracellular fluids
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Abs and their diversity
An Ab immunoglobulin is a "Y" shapedmolecule made up of two identical "light" and
"heavy" chains of amino acids.
The variable region includes the N-terminal110-130 amino acids of the light and heavy
chains, and is responsible for binding to antigen.
The constant region is the C-terminal end andcontains similar amino acids for each class of
Ab.
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b i i ( )
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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 foran 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.
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The immune system creates billions of different Abs
with a limited number of genes by rearrangingDNA segments during B cell development, prior to
Ag exposure.
Mutation can also increase genetic variation in Abs
Heavy chain
Light
chain
Abs Diversity
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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 rearrangementchanging the heavy chain constant gene in
memory cells.
Loss of coding regions for the constant part of theheavy chain causes IgG, IgA, and IgE to be
produced.
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Ab Class switching to produce IgA