Chapter 3 Antigen Recognition by Bcell and Tcell Receptors

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Chapter 3 Antigen Recognition by B-cell and
T-cell Receptors
Innate immunity works with few receptors or
mechanisms to recognize molecular patters
associated with microorganism (how many different
receptors?)This is sufficient to deal with
nearly all microorganisms that you encounter on a
daily basis. However, most microorganism in the
environment are not pathogenic for human because
they have not adapted to grow in or on humans
(i.e., they cannot evade innate
immunity). Pathogens generally have special
adaptations that allow them to evade some host
defenses and thus grow in or on certain hosts.
(that is why most pathogens have a narrow
host-range) The adaptive immune system can
generate many different receptors (how many?)
that can recognize molecular patterns missed by
innate immune receptors.
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Antibody (ab) Review AbIg BCR is membrane form
of Ig (mIg) found on B cells Most secreted ab
(sIg) is made by plasma cells (effector B
cell) Two functions for ab 1. antigen (ag)
binding, (i.e., ag recognition/recognition of
foreign macromolecules) 2. recruit effector
function (molecules or cells) for destruction of
ag Binding site varies with specificity (variable
region or V region).Effector function and
distribution is determined by the constant region
(C region) Antibodies generally bind antigens in
their native configuration Limited effector
functions five main forms of C regions (function
differ in the soluble and membrane forms)
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TCR Review TCR has only a membrane form TCR has
constant and variable regions homologous in
structure and function to those of
immunoglobulins TCR binds proteins that have been
processed into peptides and presented by MHC
(never ag in the native configuration)
MHC Proteins Review MHC proteins are encoded in a
large gene cluster (locus) called MHC There are
lots of MHC genetic variations in a
species/population (not in the individual) Individ
uals express several different MHC type (e.g., 6
classical MHC class I)
T cells (TCR) recognize unique structures that
result from the combination of the foreign
peptide and variable parts of MHC (not peptide,
not MHC, but peptide MHC)
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Immunoglobulins (Ig) (antibodies, BCRs) are for
antigen recognition and destruction
Two main functions for Igs1. Bind antigens
recognition (targeting)2. Recruit cells or
molecules for destruction of the antigens
(effector function)(which effector function does
not rely on 2?)
TCR has many structural similarities to Igs but
it is distinct.
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Immunoglobulins (i.e., antibodies)
Antigen binding site
Antigen binding site
Variable regions(v regions)
light chain
light chain
Constant regions(c regions)
Heavy chain
Heavy chain
Antibodies with different specificities differ in
the amino acid sequence of the variable regions
of the heavy and light chains The two heavy
chains are identical and the two light chains are
identical so the two antigen binding sites are
identical
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The Constant regions of Igs (part 1, the light
chains)
In a single Ig molecule, the C-termini amino acid
sequences of the light chains (?110 of 220 total
aa) are identical to each other. They are
similar or identical to the light chain C-termini
in all Igs, regardless of their specificity.
Thus, this part of the molecule is called the
light chain constant region.
Constant region of the heavy chain CH Constant
region of the light chain CL

• There are two major Ig light chain constant
  region types (similar but different aa
  sequences). These DO NOT determine the
  class/isotype of the Ig and are not known to link
  the molecule to specific effector functions
• The 2 major Ig light chain types are k (kappa)
  and l (lambda). k and l are encoded at different
  genetic loci.

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The Constant regions of Igs (part 2, the heavy
chains)
In a single Ig molecule, the C-termini amino acid
sequences of the 2 heavy chains (?330 of 440
total aa) are identical to each other. They are
similar or identical to the heavy chain C-termini
in all Igs, regardless of their specificity.
Thus, this part of the molecule is called the
heavy chain constant region.
Constant region of the heavy chain CH Constant
region of the light chain CL
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The Structure of Immunoglobulins
2 heavy (H) chains (50kD) 2 light (L) chains
(25kD) Total mw gt150 kD
Effector function is determined by the constant
regions of the H chains. The 5 classes of
immunoglobulins are determined by the H chain
constant region.BCRs (mIg) and soluble Igs
(sIg) of the same class have somewhat different C
termini
There are gt109 different antigen binding sites.
How can you make millions of different binding
sites (how can you have millions of different
amino acid sequences) with hundreds of genes or
gene segments?
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The five major Ig classes are IgM IgG (Human
IgG1, IgG2, IgG3, IgG4) (mouse IgG1, IgG2a,
IgG2b, IgG3) IgA (Human IgA1, IgA2) IgD IgE
There are also sub classes (4 IgG subclasses and
2 IgA subclasses in humans.
Isotype means the same thing as class and
subclass thus, in humans, there are 9 isotype
IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgE and
IgD. For mice, IgG1, IgG2a, IgG2b IgG3, IgA,
IgM, IgE and IgD.
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Greek letters are used to name the heavy and
light chain constant regions g, m, a, d, e for
the heavy chainsk, l for the light chains (kl
21 human 201mice 120 bovine)
IgG
IgG
IgD
The heavy chain determines the class of the Ig,
thus If the heavy chain is g the class is IgG
(and g1 for IgG1, etc.) If the heavy chain is m
the class is IgM If the heavy chain is a the
class is IgA If the heavy chain is d the class is
IgD If the heavy chain is e the class is IgE
A IgA
Different heavy chains provide different
functions and distribution there is no known
difference in function for k and l
A a and l
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Each immunoglobulin chain comprises domains
Domains are discrete, compactly folded regions of
a protein. Each light chain comprises 2 domains
and each heavy chain comprises 4 or 5 domains
In immunoglobulins, the domains are structurally
similar but not identical (similar amino acid
sequences and folding) and thus referred to as
immunoglobulin domains. Many other proteins fold
similarly and are said to belong to the
immunoglobulin super family. Domains, by
themselves or via interactions with other domain,
form functional units.
When comparing many antibodies, amino terminal
domains are highly variable. Within an isotype,
the remaining domains are constant (i.e., all IgM
CH1 are essentially identical, all CH2 are
essentially identical CH1 and CH2 are similar
but not identical)
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Heavy and light chains are disulfide linked
(interchain disulfide bond)
Heavy chains are disulfide linked (interchain
disulfide bond)
Each immunoglobulin domain has an intrachain
disulfide bond
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Y
Different way to depict an Ig molecule
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Fragment antigen binding Fab Fragment
crystallizable Fc
The ag binding specificity of the Fab and F(ab)2
is exactly like that of the whole molecule.
However, other properties of the Ig molecule are
lost. For example?
Note error in book book say papain
Prime () because F(ab)2 is more than just 2 Fab
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Flexibility in the hinge region allows binding to
surfaces or antigens with various shapes or
orientations.
Multiple binding sites allows for stronger
binding (higher avidity) and for cross-linking
antigens into complex matrices
Although not shown, antigens with three (or more)
sites where three (or more) antibodies can bind
simultaneously, allow for three-dimensional
antigen-antibody complex of enormous size.
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Ig domains have similar structures (i.e.,
immunoglobulin folds)
Here, the light chain constant region (CL) has
the Ig fold.
Although the variable domains of the H and L
chains are highly variable (determining the
specificity of the antibody) they are still
recognizable as Ig domains
Many other molecules have a similar structure
(Ig-like domains) and constitute the
immunoglobulin (Ig) super family.
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This is a comparison of the amino acid sequences
from many different heavy chains (same isotype).
The variability relates to the chances that, at a
given amino acid position, different amino acids
will be used.
Wu-Kabat hypervariablity plot
These plots show that, within a species, the
constant region does not vary from Ig to Ig of
the same isotype (this is what we expect in
almost all proteins).
In the variable (V) region , there is variability
at every amino acid position. There are three
areas where there is hypervariability i.e., the
hypervariable regions (HV). The other variable
parts of the V regions are called framework (FR).
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In both the heavy and light chain variable
regions there is variability at every position
and there are hypervariable regions
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X-ray crystallography has shown which amino
acids form the antigen-contact residues. The
crystallographers called the contact residues the
complementarity determining regions (CDR). They
showed there are three CDRs per variable region
Variability vs. 3-D Structure
It turns out that the CDRs and the HV regions
comprise the same amino acids.
Framework regions (FR) play a crucial part in
shaping the antigen-binding site and thus, along
with the HV regions, are key in determining
specificity of the antibody
The term hypervariable comes from protein
sequencing studies (pre-DNA-sequencing era)
whereas complementarity determining comes from
x-ray crystallography (protein structure) studies.
x-ray crystallography is a technique to
determine the three dimensional structure of a
protein
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The antigen binding site is made-up of both heavy
and light chain CDRs
Fig. 3.8
Antigenonly the part of the antigen that fills
the binding site is shown. The antigen can be
much much bigger such that only a small part of
the whole antigen is in contact with the antigen
binding site (see next slides)
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Epitope antigenic determinant
Antibodies contact epitopes or antigenic
determinants
The epitope is small (6 amino acids or 6
sugars) or a small part of a larger antigen
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Epitope antigenic determinant
Antibodies contact epitopes or antigenic
determinants
Peptides, and other small molecules, will usually
not induce an immune response. Thus, although
the antibody recognizes and binds a small part of
a big molecule, the isolated small part of the
molecule cannot induce an immune response.
An antigen is the whole molecule (or cell) bound
by the antibody. Also, the antigen could be an
isolated epitope. Immunogens induce immune
response and are the target of the induced
response (i.e., bound by antibody). Immunogens
are antigens but not all antigens are immunogens.
The epitope is small (6 amino acids or 6
sugars) or a small part of a larger antigen.
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For antibodies, epitopes are conformational (aka
discontinuous) or continuous (aka linear)
TCRs bind only continuous epitopes. Why?
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Antigen-antibody complexes are held together by
non-covalent forces (therefore, antigen binding
by antibody is reversible)
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Antigen recognition by the TCR
BCR ormembrane Igorsurface Ig
(soluble)
Note the transmembrane and cytoplasmic amino
acids here but they are absent on the soluble
forms of Igs
B cell
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The TCR
There is also a TCR made of a g and d chain (the
gd TCR)
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Antibodies, unlike TCRs, bind continuous (linear)
or conformational epitopes

• Usually antigens recognized by antibodies are in
  their native configuration. Antibodies bind on
  the surface of the antigen (e.g., amino acids
  that are buried in the center of a globular
  protein are not accessible by antibodies)..
  Antibodies bind linear and conformational
  epitopes.

• Antibodies can bind proteins, carbohydrates,
  lipids and nucleic acids (essentially any
  macromolecule). (most antibodies bind proteins or
  carbohydrates)

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Unlike antibodies, the TCR binds only continuous
(linear) epitopes

• The TCR binds peptides from processed protein
  (proteins that have been chopped-up).
  Accordingly, TCRs can bind peptides that are
  derived from the surface of a proteins or
  peptides from the interior of the protein.

• Because of processing and presentation of
  peptides, the TCR binds only linear epitopes.
  TCRs can bind non-surface epitopes buried in the
  center of a big protein and can bind peptides
  from proteins from inside of bacteria and
  viruses. TCRs bind only peptides not nucleic
  acids, not lipids and not carbohydrates (rare
  exceptions) because MHC presents only peptides
  (not nucleic acids, not lipids, not
  carbohydrates).

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Two classes of T cells are distinguished by
surface proteins CD4 and CD8 (surface molecules
used to ID cells are called markers)
CD4 cells are assumed to be T cells with TCRs
that bind antigen (peptide) plus MHC class II
CD4 binds invariant parts of MHC class II
CD8 cells are T cells that bind antigen
(peptide) plus MHC class I
CD8 binds invariant parts of MHC class I
Since either CD4 or CD8 binding to MHC is
required for the TCR to effectively bind to MHC
plus peptide, CD4 and CD8 are TCR co-receptors
There are CD4 cells that are not T cells, so
be careful
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TCR plus CD8 interacting with MHC class I plus
peptide
T cell (CTL)
membrane
membrane
Any cell
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TCR
peptide
peptide
TCR
peptide
CD8a CD8b
This is another depiction of the same thing shown
on the previous slide
MHC a
b2M
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TCR plus CD4 interacting with MHC class II plus
peptide
( TH1 or TH2 )
membrane
membrane
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Expression of MHC class I and class II proteins
in humans, but not mice, activated T cell
express low levels of MHC class II
Dendritic cells
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MHC class I
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MHC Class II
MHC Class I
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MHC class I peptide
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Various Peptides isolated from two different MHC
Class I proteins.The peptides isolated from
identical MHC proteins have similar motifs
Single letter code for amino acids
Ytyrosine Fphenylalanine Llucine Iisolucine V
valine
aromatic AA
hydrophobic AA
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• MHC class I peptide-binding motifs are
  characterized as having
• 8-10 amino acids
• anchors at termini
• anchors in the middle

Since there are hundreds of different MHC class I
proteins in a species (polymorphic and
polygenic)(only a few in each individual) there
are hundreds of MHC class I binding motif in a
species (but few in an individual)
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MHC class II
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Peptides isolated from from mouse and human MHC
class II
Same core sequence but different lengths
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• MHC class II peptide-binding motifs are
  characterized as having
• As few as 9 amino acids in the cleft but usually
  13-17 amino acids total
• anchors in the middle but not necessarily at the
  ends

Since there are hundreds of different MHC class
II proteins in a species (polymorphic and
polygenic) (only a few in each individual) there
are hundreds of MHC class II binding motif in a
species
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Looking down on the TCR binding site
TCR
CDRs
peptide
MHC class I
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