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