BLymphocyte Activation and Function I Structural Basis of Antibody Diversity Lecture C409M

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B-Lymphocyte Activation and Function IStructural
Basis of Antibody DiversityLecture C4-09M

• Reading in The Immune System by Parham Chapter
  2 (pages 31-47)
• Helpful web sites
• http//www.classwire.com/Login (website for Text
  Book)
• http//www.medicine.dal.ca/micro/education/pimunit
  /home.htm
• http//www.whfreeman.com/kuby/ (has good self
  test)
• http//www-micro.msb.le.ac.uk/MBChB/ImmGloss.html
  (Glossary of terms)

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INNATE VS. ADAPTIVE IMMUNITY

• Innate immunity
• involves largely phagocytic cells (granulocytes
  and macrophages) and provides a first line of
  defense.
• Innate immunity deals with the diversity of
  pathogens that a person might encounter in their
  lifetime by having available (at all times) a few
  types of recognition molecules, each of which
  recognize a large number of pathogens.
• Adaptive immunity
• is stimulated by exposure to infectious agents
  and increases in magnitude and defensive
  capability with each successive exposure to a
  particular pathogen.
• Adaptive immunity is mediated by lymphocytes
• B-lymphocytes (also called B-cells) and
  T-lymphocytes (also called T-cells).

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ADAPTIVE IMMUNITY

• B-cells (along with T-cell help) synthesize
  antibodies that mediate humoral immunity and
  T-cells are major players in cellular immunity.
• The mechanisms of adaptive immunity are ones that
  improve recognition rather than destruction.
• Somatic gene rearrangement and somatic mutation
  in the genes for antigen receptors provide the
  lymphocyte population with a set of highly
  diverse antigen receptors, which collectively
  have the ability to recognize a vast array of
  antigens.
• An individual lymphocyte expresses receptors of a
  single and unique antigen-binding specificity.

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ADAPTIVE IMMUNITY

• B cells and T cells recognize different types of
  antigens. B-cell receptors bind whole molecules
  and intact pathogens, whereas T cell receptors
  only interact with pathogen-derived peptides
  bound to proteins of the major histocompatibility
  complex.
• IMMUNOLOGICAL MEMORY Both B cells and T cells
  of the adaptive immune system remember each
  encounter with a pathogen or foreign antigen, so
  that subsequent encounters stimulate increasingly
  more effective defense mechanisms.

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Acquired/Adaptive Immunity

• Acquired immunity develops during a hosts
  lifetime.
• Six major characteristics of acquired immunity
  are
• Specificity
• Self-nonself discrimination
• Inducible
• Diversity
• Memory
• Self-limiting

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STRUCTURAL BASIS OF ANTIBODY DIVERSITY

• Antibodies are the antigen-specific products of B
  cells, and the production of antibody in response
  to infection is the main contribution of B cells
  to adaptive immunity.
• The antibody molecule has two separate functions
  
• one is to bind specifically to molecules from the
  pathogen that elicited the immune response
• the other is to interact with various cells and
  molecules to destroy the pathogen once the
  antibody is bound to it.
• These functions are structurally separated in the
  antibody molecule, one part of which specifically
  recognizes antigen and the other engages effector
  mechanisms that dispose of it.
• The antigen-binding region varies extensively
  between antibody molecules and thus is known as
  the variable region or V region.
• The region of the antibody molecule that engages
  the effector functions of the immune system does
  not vary in the same way and is thus called the
  constant region or C region.

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Concept Antibodies are composed of polypeptides
with variable and constant regions.
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Concept The antibody molecule can readily be
cleaved in to functionally distinct fragments.
Note Papain cleaves the IgG into three pieces
two Fab (Fragment antigen binding) fragments and
one Fc fragment (Fragment crystalizable).
Note Pepsin cleaves IgG into one (Fab)2
fragment. The Fc is broken into several smaller
fragments.
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Concept All classes of antibody have the same
basic structure. Two identical heavy chains and
two identical light chains each of which
contributes to antigen binding.
The Structural Organization of the Human
Immunoglobulin Isotypes.
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HEAVY CHAINS

• All heavy chains are made up of discrete domains.
• The amino terminal domain, of VH, displays the
  greatest variability.
• The remaining domains, of CH, of the heavy chain
  form the constant region.
• There are five classes of heavy chains
  designated ?, a, µ, d, e.
• ? and a are further divided into subclasses ?1,
  ?2, ?3, ?4 and a1 a2 thus .
• There are nine isotypes of antibodies as defined
  by the heavy chains.
• All heavy chains are expressed in one of two
  molecular forms, fluid phase and membrane bound
  forms.

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Light CHAINS

• All antibody light chains fall into two classes
  
• ? (kappa) and ? (lambda).
• Each light chain, whether ? or ?, is folded into
  separate V and C domains.
• There is a single gene for the kappa constant
  region, thus there is a single isotype of kappa
  chains.
• There are six separate genes for the constant
  region of lambda chains, thus, there are six
  isotypes of lambda chain.

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Immunoglobulin Domains

• Both H and L chains contain several homologous
  units of about 110 amino acids termed a DOMAIN.
• Each domain contains an intrachain disulfide bond
  which forms a loop of approximately 60 amino
  acids.
• L chains contain one variable domain (VL), and
  one constant domain (CL)
• H chains contain one variable domain (VH) and
  either three or four constant domains (CH1,
  CH2, CH3, and CH4) depending on the antibody
  class.

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General Structures of the Five Classes of
Immunoglobulins in Serum
IgE
IgG
IgD
IgA (dimer)
IgM (pentamer)
Note The heavy and light chains of an
immunoglobulin molecule are
made up of similar protein domains.
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Immunoglobulin Isotypes are Selectively
Distributed in the Body

• The principal immunoglobulins in plasma are IgG,
  IgA and IgM (the concentration is IgGgtIgAgtIgM.
  In a healthy host IgM is found only in the
  plasma, whereas IgG and IgA are found in other
  compartments.
• IgG and monomeric IgA are the major isotypes in
  the extracellular fluid.
• IgG is the only isotype to cross the placenta.
• Dimeric IgA is the predominate isotype in
  secretions across epithelia.
• IgE is associated mainly with mast cells and
  basophiles (subject of a later lecture).
• IgD is predominately found as a receptor on B
  cells.
• In healthy individuals the Ig concentration in
  the CNS is low.

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Concept An antigen-binding site is formed from
the hypervariable regions of a H-chain and
L-chain V domain.

• The amino-terminal domain of both H and L chains
  is termed a variable (V) region due to the
  discovery of extensive seequence divergence
  between different antibody proteins in this part
  of the molecule. These are designated VH and VL
  for H and L chains, respectively.
• V regions have been demonstrated to be
  responsible for antigen specificity of the
  immunoglobulin.
• Sequence analysis of many VH and VL domains
  revealed that variability is concentrated in
  several hypervariable (HV) regions.
• There are three hypervariable regions in each VH
  and VL domain.
• The stretches of amino acid between the HVs are
  termed framework regions and these exhibit far
  less variability.
• The interaction between the HVs of the H and L
  chains form the antigen binding site of the
  antibody molecule.
• Since these HVs fold into a structure that is
  complementary to the antigen epitope, the HVs are
  also called complementarity-determining regions
  (CDRs).

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Antigen Combining Site
A
L-chain HVs

• Schematic of and IgG molecule showing the
  approximate location of the hypervariable regions
  (CDRs).
• Schematic showing how the CDRs in each heavy- and
  light-chain pair for an antigen binding site.

H-chain HVs

• 1. Hypervariable Regions (HV) areas of extreme
  variability in the V domain of both H and L
  chains.
• Hypervariable regions are also called
  complementarity determining regions (CDRs).
• Framework Regions stretches of amino acids
  between the hypervariable regions that show
  relatively less variability.

B
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Antigen Receptor Diversity

• The human genome is estimated to contain 34
  thousand genes (give or take a few thousand)
• The estimated number of antibody specificities is
  estimated to be 30 million or more the number of
  T cell receptor specificities is about the same.
• Is there a problem with these numbers?????
• How can fewer than 105 genes encode for nearly
  108 antigen receptor specificities plus all of
  the other proteins needed by the body????????
• The answer
• Each receptor chain is encoded by different gene
  segments

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CONCEPT The DNA sequence encoding a complete V
region is generated by the somatic recombination
of separate gene segments.

• Three separate multigene families (kappa, lambda,
  and heavy chain loci) located on different
  chromosomes encode immunoglobulin light chains
  and heavy chains.
• In germline DNA, each multigene family contains
  numerous gene segments
• Ig genes undergo a process of somatic DNA
  recombination or rearrangement during B cell
  development to form the variable regions of light
  chains and heavy chains.
• Functional light-chain and heavy-chain genes are
  generated by random rearrangement of the variable
  region gene segments (two for light chains and
  three for heavy chains) in developing B lineage
  cells.

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CONCEPT Light chain variable regions are
assembled from to gene segments V and J (for
joining).
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REARRANGEMENT OF THE LIGHT CHAIN LOCUS
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CONCEPT Heavy chain variable regions are
assembled from three gene segments V
(variable), D (for diversity) and J (for joining).
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REARRANGEMENT OF THE HEAVY CHAIN LOCUS
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CONCEPT The reaction that recombines V,D, and J
gene segments involves both lymphocyte-specific
and ubiquitous DNA-modifying enzymes that are
collectively call the recombinase complex.

• Rearrangement of Ig genes occurs principally by a
  mechanism of excision (looping out) of the DNA
  between the various gene segments followed by
  ligation of these segments.
• Ig gene rearrangement is a special kind of
  recombination involving non-homologous gene
  segments that is mediated by a system of enzymes.
• The enzymes involved include
• lymphocyte-specific enzymes, which are products
  of the recombinase activating genes (Rag1 and
  Rag2)
• and, other nuclear DNA-modifying enzymes (DNA
  repair) that are not tissue specific.

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RECOMBINASES

• Recombinases (RAG-1 and RAG-2) recognize specific
  DNA sequences called recombination signal
  sequences (RSS) that flank each V, D, and J gene
  segment.
• Recombinases are cell type-specific. They are
  active in lymphocytes but not in non-lymphoid
  cells.
• Recombinases function in the early developmental
  stages of B and T lymphocytes and are absolutely
  required for recombination to occur.
• Recombinase activating genes 1 and 2 (Rag-1 and
  Rag-2) are present in all cells but are only
  expressed in B and T cells.

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CONCEPT Gene segments encoding the variable
region are joined by recombination at
recombination signal sequences (RSS).
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CONCEPT The diversity of the immunoglobulin
repertoire is generated by four main processes.

• Multiple inherited germline gene segments used in
  different combinations.
• Junctional diversity antibody molecules show
  the greatest variability at the junctions of the
  variable gene segments which form the third
  hypervariable region, or CDR3. Junctional
  diversity arises by two mechanisms
• Introduction of P-nucleotides by a feature of the
  recombination reaction that generates short
  palidromic sequences at the cut ends of the DNA
  strands.
• N-nucleotides formed from the untemplated
  addition of nucleotides by the enzyme terminal
  deoxynucleotidyl transferase (TdT).
• Combination of any H-chain with any L-chain.
• Somatic Hypermutation (occurs after encounter
  with antigen).

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CONCEPT Junctional diversity is generated
during recombination of variable gene segments.
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CONCEPT The development of B cells proceeds
through stages defined by the rearrangement and
expression of the immunoglobulin genes.
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The Other Side of the Coin

• Antigens/Immunogens
• Antigen antibody generators
• Immunogen any substance that can elicit an
  immune response in either B cells or T cells

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ANTIGENS/Immunogens

• Substances capable of inducing an immune response
  are called antigens or immunogens.
• To protect against infectious disease, the immune
  system must be able to recognize bacteria,
  bacterial products (e.g., toxins), fungi,
  parasites, and viruses as immunogens.
• The immune system actually recognizes particular
  macromolecules of an infectious agent, generally
  either proteins or polysaccharides.
• Proteins are the most potent immunogens with
  polysaccharides ranking second.
• In contrast, lipids and nucleic acids of an
  infectious agent generally do not serve as
  immunogens unless they are complexed to proteins
  or polysaccharides.

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Contribution of the Immunogen to Immunogenicity

• FOREIGNNESS
• In order to elicit an immune response, a molecule
  must be recognized as nonself by the biological
  system (i.e., the host).
• MOLECULAR SIZE
• There is a correlation between the size of a
  macromolecule and its immunogenicity. Generally,
  a molecule must have a molecular mass gt10,000
  daltons.
• CHEMICAL COMPOSITION AND COMPLEXITY
• Size and foreignness are not, by themselves,
  sufficient to make a molecule immunogenic other
  properties are needed. The macromolecules must
  have internal complexity. Simple polymers are
  not immunogenic.
• SUSCEPTIBILITY TO ANTIGEN PROCESSING AND
  PRESENTATION
• The development of most humoral and all
  cell-mediated immune responses requires
  interaction of T cells with antigen that has been
  processed and presented together with MHC
  molecules. Thus, if a macromolecule can not be
  degraded by the biological system it is not
  immunogenic.

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SUMMARY OF THE
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Contribution of the Biological System to
Immunogenicity

• Even if a macromolecule has the properties that
  contribute to immunogenicity, its ability to
  produce an immune response will depend on
  certain properties of the biological system that
  the antigen encounters.
• GENOTYPE OF THE RECIPIENT HOST
• The genetic composition of the immunized host
  influences the type and degree of the manifested
  response.
• HEALTH AND WELL BEING OF THE HOST
• sickness, stress, malnutrition, and age (too old
  or too young) of the host can affect the type and
  degree of an immune response.
• IMMUNOGEN DOSE AND ROUTE OF ADMINISTRATION
• Too high or too low of a dose can result in a
  poor immune response (tolerance to the antigen).
  The administration route strongly influences
  which immune organs and cell populations will be
  involved in the response.

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EPITOPES

• The molecular sites that stimulate immune
  responses are termed epitopes or antigenic
  determinants
• B-cell Epitopes may be sequential or
  conformational
• B cell epitopes can be found in proteins,
  carbohydrates, lipids, chemicals, drugs, etc.
• Haptens --Non-immunogenic substances (small
  drugs, lipids,peptides, etc) can be coupled to
  carrier proteins and then can serve as B cell
  epitopes.
• B cells recognize antigen that is free in the
  fluid phase.
• T-cell Epitopes are made up of sequential
  residues in a protein immunogen.
• T cells only recognize antigen after it has been
  processed by APCs and presented in the context of
  MHC

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CONCEPT Epitopes of a protein can be linear or
discontinuous (conformational)
A linear epitope of a protein antigen is formed
from contiguous amino acids. A discontinuous
epitope is formed from amino acids from different
parts of the polypeptide that are brought
together when the chain folds, i.e., are
dependent on the conformation of the protein.
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Concept Epitopes can bind in pockets or grooves
or on extended surfaces in the binding site of
antibodies.
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Forces Binding Antigen to Antibody

• The combination of antigen with antibody is a
  non-covalent interaction.
• Ag Ab Ag-Ab (immune complex)
• The strength of the association is a summation of
  several weak intermolecular forces
• Van der Waals Forces
• Hydrogen Bonds
• Hydrophobic interactions
• Electrostatic (Ionic) interactions

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Cross-Reactivity of Antibodies

• ENIGMA If antibodies are so highly specific why
  is it often observed that they will cross-react
  with unrelated antigens?
• Same molecular structure (structurally similar
  epitope)
• Structural mimicry
• Examples
• ABO blood group antigens
• Rheumatic heart disease
• Post-streptoccocal glomerulonephritis

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Summary of Antibodies

• are glycoproteins
• are built of subunits containing
• two identical light chains (L chains), each
  containing about 200 amino acids
• two identical heavy chains (H chains), which are
  at least twice as long as L chains
• The first 100 or so amino acids at the N-terminus
  of both H and L chains vary greatly from antibody
  to antibody the are termed the variable (V)
  regions
• unless members of the same clone (often not even
  then!), no two B cells are likely to secrete
  antibodies with the same V region
• the amino acid sequence variability in the V
  regions is especially pronounced in 3
  hypervariable regions also called CDRs
• the tertiary structure of the antibodies brings
  the 3 CDRs of both the H and L chains together
  to form the antigen binding site which binds the
  antigen epitope
• Only a few different amino acids sequences are
  found in the C-terminus of the H and L chains and
  these are called constant (C) regions
• two different kinds of C regions for L chains --
  kappa (?) and lambda (?)
• five different kinds of C regions for H chains
• mu (µ) chains, IgM
• gamma (?) chains, IgG
• alpha (a) chains, IgA
• epsilon (e) chains, IgE
• delta (d) chains, IgD
• each of these 5 kinds of H chains can pair with
  either lambda or kappa L chains

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Summary of Antigen Receptor Diversity

• Each receptor chain is encoded by several
  different gene segments
• the gene segments are V, D, and J for heavy
  chains and V and J for light chains
• Each gene segment has an adjacent recombination
  signal sequenc (RSS)
• RSSs are recognized by two recombinase proteins
  encoded by Rag-1 and Rag-2 genes
• During the differentiation of the B cell the DNA
  is cut by the recombinase enzymes and recombined
  to make an intact gene coding for a receptor
  chain.
• DJ joining occurs first in a developing B cells
  followed by VDJ
• VJ recombination of light chain genes occurs
  after the successful completion of VDJ
  recombination of the heavy chain genes.
• Random assortment of gene segments contributes to
  diversity
• Junctional diversity, P-nucleotides and
  N-nucleotides contribute to additional receptor
  diversity.
• Somatic hypermutation after the B cell encounters
  antigen adds additional diversity
• Ergo little bit of DNA a whole bunch of
  antibodies