Immunoglobulin Structure and Function III Effector Mechanisms of Humoral Immunity Lecture 12 August 10, 2004 Lecturer: Wolcott Reading for Lectures 11-13: Kuby 5e, 87-104: 57-75: and 137-160

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Understanding Immune Recognition
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Antigen Recognition

• B cells can recognise antigens via their surface
  Ig molecules
• T cells can only recognise antigen in association
  with a Major Histocompatibility Complex (MHC)
  molecule.

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Antigen Recognition

• B cells can recognise antigens via their surface
  Ig molecules
• T cells can only recognise antigen in association
  with a Major Histocompatibility Complex (MHC)
  molecule.

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The Immunoglobin Fold
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Immunoglobin Fold

• V and C domains share the basic Ig fold
• Differences between the two domains
• C domain is built of seven b-strands arranged so
  that four strands form one sheet and three
  strands form a second sheet.
• The strands are closely packed together and
  joined by a single disulphide bond
• Most of the invariant residues of the constant
  domain are in the sheets
• Overall structure of the V domain very similar
  but there are nine strands instead of seven. The
  two additional strands harbour CDR2

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Structure of antibody
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Complementarity Determining Regions in Ig
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The Complementarity Determining Regions

• Six loops of the VH (H1, H2 and H3) and VL (L1,
  L2 and L3) domains create a great variety of
  surfaces
• Deep binding cavities such as those seen in some
  antibody-hapten complexes
• Wide pockets seen in certain antibody-peptide
  complexes
• Flat surfaces seen in antibody-protein
  interactions
• H3 is the most variable of the loops and in all
  crystallographically solved antibody-antigen
  complexes makes several contacts with antigen

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What Do Antibodies Recognize?

• Proteins (conformational determinants, denatured
  or proteolyzed determinants)
• Nucleic acids
• Polysaccharides
• Some lipids
• Small chemicals (haptens)

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AntigenAntibody complex

• Antibodies bind to antigens by recognizing a
  large surface, and through surface
  complementarity.
• Thus, these complexes have a very high affinity
  for each other.

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Weak forces vs high affinity

• The interaction between an antigen and antibody
  can be very strong, and yet all of the forces
  involved are considered to be relatively weak.
  How can weak hydrogen bonds, electrostatic
  attractions, hydrophobic forces, and van der
  Waals contacts lead to a high affinity?
• Contact between antigen and antibody occurs over
  a wide surface area, allowing multiple weak
  interactions that give a strong affinity
• Hydrogen bonds join the antibody and antigen over
  a wide surface area. Other weak forces, including
  van der Waals forces, electrostatic attractions
  and hydrophobic forces, add to the strength and
  specificity of antibody/antigen binding

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Antibody-Hapten Complex

• Haptens, having a limited total surface area,
  deeply embed themselves into the VL/VH dimer
  interface
• Hapten binding antibodies frequently show a deep
  central cavity, long CDR L1 loops and a CDR H3
  loop with an "open" conformation, allowing the
  hapten to bind as much as 80 of its total
  surface in the interaction.

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Intimate interaction between Ab and Hapten
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Peptide Antibody Complex
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Protein Antibody Complex

• In contrast, proteins preferentially to a
  relatively flat binding surface
• In a "closed" CDR H3 conformation, the CDR H3
  loop packs down onto the central cavity, and the
  protein antigen binds on top of it.

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Effector response is mediated via Ig-FcR complex
formation

• Antibodies not only must recognize antigen, but
  also must invoke responses effector functions
  that will remove the antigen and kill the
  pathogen.
• Variable regions of antibody are the sole agents
  of binding to antigen.
• The heavy chain constant region (CH) is
  responsible for interactions with other proteins
  (e.g. complement), cells (elements of innate
  immune system), and tissues that result in the
  effector functions of the humoral response.
• FcR recognize the Fc portion of antibodies not
  antigens

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The Fc-Fc Receptor complex

• FcR plays important role in antibody mediated
  immune responses
• Ig and FcR binding activates effector functions
• Fc Receptor interacts with the CH2 and CH3
  domains of Immunoglobulins

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Mode of interaction of FcR with difference Ig
molecules
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Immune Recognition MHC and TCR interactions
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Antigen Recognition

• B cells can recognise antigens via their surface
  Ig molecules
• T cells can only recognise antigen in association
  with a Major Histocompatibility Complex (MHC)
  molecule.

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T cells

• T cells display TCR as their antigen recognition
  protein
• When stimulated they become Cytotoxic or Helper T
  cells
• Secrete cytokines that recruit other cells of the
  IS
• TCRs only recognise short peptides.

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MHC T cells

• T cells have a requirement to recognise both the
  ANTIGEN and the MHC molecule. This is because
  the molecular structure of the MHC-Antigen
  complex is arranged so that some of the
  polymorphic amino acids of the MHC molecule are
  in direct contact with the TCR
• Therefore T cell recognition of antigen is said
  to be MHC restricted.

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Antigen Processing and Presentation

• Fragmentation of protein into peptides
• Association of peptide with an MHC molecule
• Transport to cell surface for expression
• Different cellular pathways for association of
  peptide with MHC class I and class II molecules

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MHC Antigens

• MHC Class I
• present endogenously derived peptides.
• these can be either self or derived from viruses
• because MHC Class I is present on all cells any
  cell can interact with T cells if infected by a
  virus

• MHC Class II
• present exogenous antigen which has been
  phagocytosed and processed.eg. Bacteria
• This is performed by professional antigen
  presenting cells eg macrophages

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MHC

• MHC Class II
• seen only on the professional antigen processing
  cells e.g macrophage
• slightly less polymorphic
• accepts peptides of up to 15 aa acids

• MHC Class I
• detected on all nucleated cells
• very highly polymorphic
• Tight fit for peptides of only about 9 aa
• consists of an a-chain of 3 domains associated
  with b-2 microglobulin

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MOLECULES OF T LYMPHOCYTE RECOGNITION

• Major histocompatibility complex (MHC)
  humanHuman Leukocyte Antigen (HLA) mouseH-2
• Gorer and Snell identified a genetic basis for
  graft rejection and Snell named it
  histocompatibility 2 (H-2). Nobel prize awarded
  to Snell.
• Highly polymorphic genes organized in a complex
  on chromosome 6 (human) and 17 (mouse).
• Glycoproteins expressed on the surface of cells.
  MHC class I is composed of one polypeptide,
  non-covalently associated with b2microglobulin.
  MHC class II is composed of two polypeptides,
  referred to as a and b.

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MHC Class I and Class II Proteins

• Class I
• Alpha Chain
• 3 External domains
• 1 Transmembrane
• 1 Cytoplasmic tail
• Encoded in MHC
• Beta-2 Microglobulin
• 1 External domain
• No transmembrane
• No Cytoplasmic tail
• Not encoded in MHC

• Class II
• Alpha Chain
• 2 External domains
• 1 Transmembrane
• 1 Cytoplasmic Tail
• Encoded in MHC
• Beta Chain
• 2 External domains
• 1 Transmembrane
• 1 Cytoplasmic Tail
• Encoded in MHC

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MHC Class I and Class II Proteins
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Peptides bind to MHC molecules in a polyproline
II conformation
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Class IPeptide Binding
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MHC-II Structure
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Peptide Binding by Major Histocompatibility
Complex (MHC) Antigen-presenting Proteins
MHC I
MHC II

• Peptides of intracellular origin
• Peptides 9-10 residues long
• Deep pockets bind peptide sidechains
• Deep pockets bind peptide N- and C-termini

• Peptides of extracellular origin
• Peptides 15 residues or longer
• Shallow pockets bind peptide sidechains
• Peptide termini free
• H-bonds to peptide backbone

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MHC Polymorphism

• Both Class I and Class II genes are highly
  polymorphic
• Most polymorphic residues of Class I are in the
  alpha 1 and alpha 2 domains
• Most polymorphic residues of Class II are in the
  alpha 1 and beta 1 domains

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Location of Polymorphic Residues
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Location of Polymorphic Residues
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Allelic variation in MHC occurs at the peptide
binding site and on the top/sides of the binding
cleft
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T-cell Receptor
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The T cell receptor (TCR) is a complex of
integral membrane proteins that participates in
the activation of T cells in response to the
presentation of antigen. Specific recognition
and binding by the clonotype-specific a/b
heterodimer leads to activation of transcription
and commitment of the T cell to CD4 or CD8
fate. This activation involves other subunits of
the receptor complex as well as other
membrane-associated molecules that couple the
extracellular liganding event to downstream
signaling pathways such as protein
phosphorylation, the release of inositol
phosphates and the elevation of intracellular
calcium levels.
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TCR binds peptide/MHC with a restricted (but
variable) orientation
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peptide binding interface 21-34 proportion of
TCR contacts with the peptide26-47 contact are
different between TCR-MHC complex -the
contribution to the binding energy is still
uncleared!
Bandovich and Garcia. 2003. Immunity 18,7-11
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TRI-MOLECULAR COMPLEX CHARACTERISTICS
-CDR1 and CDR2 interact with MHC molecules (a
helices)
-CDR3 interacts with the peptide
-interaction always in the same orientation -45
to 70 degrees angle related to peptide -Va see
N-ter of the peptide -Vb see C-ter of the peptide
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TRI-MOLECULAR COMPLEX CHARACTERISTICS
- most of the binding interface is between the
TCR and MHC helices - conformational change
in the TCR CDR loops enhances TCR
crossreactivity - no conformational change in
the TCR constant region (except in one complex
out of ten)
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Recognition of the Super Antigens
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Antigen Recognition by Antibodies (Ab) and T-cell
Receptors (TCR)
Ab - Ag
TCR MHC/peptide

• Surface area 2x750 Å2
• Epitope discontinuous in antigen (Ag) sequence

• Surface area 2x1000 Å2
• Ag peptide contributes only 40 of surface area
• Epitope continuous in Ag sequence
• Otherwise similar to Ab - Ag recognition

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PARADOX
-TCR-MHC interaction has a weak
affinity -affinity 10 mM -half-life 10s
-restricted numbers of ligands (100) are
displayed at the surface of antigen presenting
cells
-T cell activation requires a long interaction
with antigen presenting cells (gt2h)