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How T cells recognize antigen
Notion of immunological “SELF”
Skin graft transplantation compatibility
• Rejection by adaptive immune system T cell response
• Graft compatibility genetically determined
Extremely polymorphic trait -many alleles
Governed by genes of major histocompatibility
complex (MHC) that encode MHC molecules, the
principal targets of rejection
Differences in MHC molecules between individuals are
central to determining “SELF”
Clonal recognition of antigen by T lymphocytes
• Each clonal T cell receptor (TCR) is specific for a
particular sequence of amino acids in a peptide
• The peptide is generated from proteins in the peptidepresenting cell
• The peptide is not directly identified by the TCR, but
recognized in a form bound to a MHC molecule
Clonal recognition of antigen by T lymphocytes
The huge diversity of pathogen peptides to be recognized mandates a
large number of different T cell clones, necessitating:
• A somatic recombination mechanism to generate the large
number of clones
• A clonal selection process to identify the appropriate clones
for the clonal repertoire
The selection process (Thymic “education”) has two stages
• First stage selects clones capable of recognizing self peptide in an
individual’s own MHC molecules - positive selection
• Second stage eliminates
overtly self reactive clones
with high affinity for self
peptide:MHC- negative
selection
*** (Self-peptides are used as a
surrogate for foreign peptides
since there are few non-self
peptides)
The TCR repertoire differs from individual to individual
• The specificity of self/non-self peptide binding to MHC molecules
determined by pockets that only bind certain amino acid side chains
• MHC genes are extremely polymorphic and alleles encode pockets
with specificities for different amino acid side chains
The TCR is specific
for both peptide
and MHC- A
complex ligand
Polymorphic
residues of
MHC
The definition of immunologic self is made by selecting
the clonal T cell repertoire on self-peptides bound to the
individual’s particular allelic forms of MHC molecules
Immunologic self is both the collective
recognition specificity of an individual’s T cell
repertoire for peptides presented by the MHC
and the set of self-peptides and self-MHC
molecules that generated the repertoire
The immune response to pathogens
Many of the events in the clonal selection process are
recapitulated during the clone’s response to peptide in an
immune response
Non-self peptides from pathogens, etc., are bound to MHC
molecules and recognized by T cells because their peptides
are partial mimics of self
Polymorphic
residues of MHC
Because of MHC polymorphisms we each likely
recognize non-self antigens and pathogens
differently from the next individual
Major selective advantages to the species
However because the adaptive immune system is patterned on
self, it sets the stage for the development of autoimmune disease
T cells recognize peptide antigens from two main
types of pathogens
Bacteria or components of
Virus - or Pathogen - infected cell
Any nucleated cell
an extracellular pathogen
that have been internalized
Macrophage/DC
B cell
A viral peptide on a cell signifies it is infected and should
be killed, while a pathogen’s peptide on a phagocytic cell
signifies the cell has ingested a foreign substance and must
be helped to eliminate the pathogen
The adaptive immune system must make this distinction
The immune system makes this distinction by loading and
recognizing peptides in either class I or class II MHC
Challenge:
Cytosolic Virus
or Pathogen
Presenting cell:
Any cell
Peptide degraded in: Cytosol
Ingested Bacteria or
Endocytic Pathogen
Macrophage/DC
Extracellular Pathogen
or Toxin
B cell
Endocytic vesicles
Endocytic vesicles
Peptides bind to:
MHC class I
MHC class II (or I)
MHC class II
Presented to:
CD8 T cells
CD4 T cells (or CD8)
CD4 T cells
Activation of cell to
enhance pathogen
killing
Provision of help to B
cell for production of
antibodies
Effect on presenting Death of cell
presenting the
cell of T cell
viral antigen
recognition:
Structure of MHC molecules
What are the structural features of the MHC that
determine peptide binding?
Organization of the MHC
Two classes of MHC molecules
0
1
2
3
Class II loci
Class I loci
HLA-DR
HLA-DQ
HLA-DP
HLA-A
HLA-B
HLA-C
Ch 6 Human Leukocyte Antigens (HLA)
4m bp
a1
N
a2
Structure of peptide-binding class I MHC domain
The ligand for the CD8 T cell TCR
b 2m
MHC Class I Domains
Peptidebinding
domains
The overall structure of class I and class II MHC is rather similar
Class I
Class II
MHC class I and II molecules have homologous domain
organization, but different chain structure
Peptide
a1
a2
a1
b1
b 2m
a3
a2
b2
MHC class I molecule
MHC class II molecule
The Structure of MHC Molecules: MHC Class I
• The a chain is~ 350 AA long
• Three globular domains, a1,
a2 and a3, each ~90AA
• a1 and a2 form the antigenbinding cleft
• b2 microglobulin ~100AA,
associates with the a3 domain,
not MHC encoded
• Transmembrane and
cytoplasmic portion
The Structure of MHC Molecules: MHC Class II
• Composed of two similar
membrane spanning
proteins, the a-chain and
b-chain both encoded
within the MHC
• Each chain is made of two
globular domains, each
~90AA, e.g. a1 and a2
• a1 and b1 domains form
the antigen-binding cleft
At the genetic level the MHC class I and II system is
complex!
Two levels of diversity:
Within individual: duplicated loci
Class I: HLA-A, HLA-B, HLA-C
Class II: HLA-DR, HLA-DQ, HLA-DP
Between individuals: hundreds of alleles for most of these
loci
The MHC is by far the most genetically polymorphic region in
the genome
The diversity of MHC class I and II genes
An evolutionary response to the structural diversity
and mutation potential of microorganisms
Arises from two mechanisms:
Duplication of a gene locus in an individual resulting in multiple
loci, polygeny
A
B
A
(isoforms or isomorphs)
Development of multiple alleles at a locus among individuals in the
species, polyallelism
A1
A2
A3
A4
A1
A2
A3
A4
B1
B2 Alleles
B3 (Allotypes)
B4 In different
individuals
MHC alleles have a dominant mode of action because the
molecules they encode have the ability to bind particular peptides
Corollary: if your MHC molecules cannot bind a
peptide, you cannot make a T cell response against it
Duplicating a locus allows it to mutate and develop new peptide
presenting specificities that operate in parallel with older ones
However each duplication increases “self” and mandates more
negative clonal selection during repertoire formation, reducing the
size of the repertoire
Practical maximum is ~ three loci each for class I and class II
HLA-DR
HLA-DQ
HLA-DP
No limit on the number of alleles
HLA-A
HLA-B
HLA-C
How T cells recognize antigen
Notion of immunological “SELF”
Skin graft transplantation compatibility
• Rejection by adaptive immune system T cell response
• Graft compatibility genetically determined
Extremely polymorphic trait -many alleles
Governed by genes of major histocompatibility
complex (MHC) that encode MHC molecules, the
principal targets of rejection
Differences in MHC molecules between individuals are
central to determining “SELF”
Clonal recognition of antigen by T lymphocytes
• Each clonal T cell receptor (TCR) is specific for a
particular sequence of amino acids in a peptide
• The peptide is generated from proteins in the peptidepresenting cell
• The peptide is not directly identified by the TCR, but
recognized in a form bound to a MHC molecule
Clonal recognition of antigen by T lymphocytes
The huge diversity of pathogen peptides to be recognized mandates a
large number of different T cell clones, necessitating:
• A somatic recombination mechanism to generate the large
number of clones
• A clonal selection process to identify the appropriate clones
for the clonal repertoire
The selection process (Thymic “education”) has two stages
• First stage selects clones capable of recognizing self peptide in an
individual’s own MHC molecules - positive selection
• Second stage eliminates
overtly self reactive clones
with high affinity for self
peptide:MHC- negative
selection
*** (Self-peptides are used as a
surrogate for foreign peptides
since there are few non-self
peptides)
The TCR repertoire differs from individual to individual
• The specificity of self/non-self peptide binding to MHC molecules
determined by pockets that only bind certain amino acid side chains
• MHC genes are extremely polymorphic and alleles encode pockets
with specificities for different amino acid side chains
The TCR is specific
for both peptide
and MHC- A
complex ligand
Polymorphic
residues of
MHC
The definition of immunologic self is made by selecting
the clonal T cell repertoire on self-peptides bound to the
individual’s particular allelic forms of MHC molecules
How peptides bind to class I MHC
molecules
How does polymorphism influence binding of different
peptides and what is its immunologic significance?
Cluster of
tyrosines
recognize NH2
NH2
How peptides bind
Class I MHC
COOH
+ charged
amino acids
interact with
negative COOH
Specific bound peptide always oriented in the same
direction in the MHC
Length of peptide is constrained to 8 or 9 amino acids
TCR
3
N
Anchor
2
5
How peptides bind
7
MHC class I
C
peptide
9
Anchor
Rules for binding to MHC class I molecules
• Usually peptides are 8-9 amino acids in length
• Always oriented with NH2 terminus to the left
• Most often anchored by interactions of the side
chains of their 2nd (P2) and 9th (P9) amino acids to
MHC pockets that confers specificity for amino
acids with similar physical properties, e.g. size,
charge, hydrophobicity, etc.
Pr 1
Pr 2
Pr 3
Different
Proteins yield
different
peptides that can
bind to the same
MHC molecule
because they
share anchor
residues
Y
Y
Y
L
L
I
Y=Tyr
L=Leu
I=Ile
Each different MHC Class I
allotype has pockets that
preferentially bind different
amino acid side chains at
positions P2 and P9
Only a few peptides from each protein will bind to
a given MHC class I allotype
Peptide binding to class II MHC molecules
2
5
7 8
peptide
1
4
6
9
Protein 1
Protein 2
Protein 3
Similar pockets for peptide side chains, but peptides binding class II
molecules vary in length 12-24 AA and are anchored in the middle
For both class I and class II molecules
• Binding is highly stable with no peptide interchange on cell
surface
• Specificity for original loaded peptide is maintained because
empty MHC molecule is very unstable
How are proteins metabolized within the cell
to yield peptides that bind to the MHC?
How do cytosolic peptides from protein synthesized in virally
infected cells get loaded on class I and not class II molecules to
trigger killing by CD8T cells?
How do peptides from ingested proteins get loaded on class II and
not class I molecules to elicit macrophage activation and B cell help?
Class I peptide loading
Synthetic
endocytic
Cytosolic
peptides
synthesized by
virally infected
cells get loaded
on class I MHC
The endocytic and synthetic pathways are usually quite separate
Peptides generated in the cytosol by the proteasome bind to MHC
class I molecules while they are being synthesized within the ER
Top
Side
The 26S proteasome is composed of the 20S proteasome core
containing the catalytic chamber and two 19S regulatory
complexes that bind and unfold ubiquitylated proteins
3 of the 7 subunits in each b ring of
the 20s core confer the proteolytic
activity, b1, b2 and b5
Responsible for the proteolysis
of polyubiquitylated proteins in
basal protein turnover
Proteasome core catalytic chamber cross section showing a ring of
7 b subunits
Three have proteolytic activity; b1, b2 and b5
Proteins are unfolded and pass through the active proteolytic sites
IFN-g greatly enhances antigen processing and
presentation
• An immunomodulatory cytokine produced by activated T
helper lymphocytes, CD8 CTLs and natural killer (NK)
cells
• Upregulates classic MHC I and II and TAP gene
products
• Upregulates the synthesis of three new proteasome
subunits with different proteolytic activity that replace the
three constitutively active proteolytic subunits, changing
specificity of proteasome towards production of
hydrophobic peptides that have greater binding affinity
for MHC pockets
Generation of the immunoproteosome
3 immunosubunits LMP2 (ib1), LMP7 (ib5) and MECL-1 (ib2)
replace the constitutive b1, b5 and b2 subunits in the 20S core and
change specificity to more hydrophobic peptides
Generation of peptides for class I MHC requires precise proteolysis
because the overall peptide length is restricted to 8 or 9 residues
The 26S immunoproteasome system is used to get
precise cuts at the C termini of the peptides
Other peptidases nibble back the N termini until the
peptide fits exactly, e.g. the IFN-g-inducible cytosolic
leucine aminopeptidase (LAP) cleaves a single residue
from the N- terminus
The process has a relatively low efficiency, especially since the
peptide production system is not coordinated with the peptide
binding specificity of the individual’s MHC class I molecules
Almost no peptides presented by class I molecules are derived from
“old” proteins
A proportion of newly synthesised proteins are defective ribosomal
products (DRiPs) that are rapidly ubiquitylated and degraded by
proteasomes
DRIPS are the major source for the generation of antigenic peptides
synthetic
Class II peptide
loading pathway
Endocytic
Proteins endocytized
by DC, B cell or
macrophage are
contained in
endocytic vesicles
Class II presentation is centered in
the vesicular system
Acidic endosomal proteases digest
ingested proteins into peptides that
will load MHC class II molecules
This process does not require the precise proteolysis needed in the class
I system, since the peptide terminii are not constrained by MHC class II
Invariant chain (Ii)
A chaperone that complexes with MHC class II molecules
during their synthesis in the endoplasmic reticulum has two
critical actions
• Ii Blocks the class II peptide binding groove and prevents
loading by peptides destined for class I molecules
• The recognition sequence on the Ii transmembrane portion
targets the nascent MHC II molecule from the synthetic
compartment to the acidic endosomal compartment to join with
the degraded ingested exogenous peptides
Ii is degraded to CLIP (Class II-associated invariant chain peptide) by
specific endosomal acidic cysteine proteases (cathepsins)
CLIP is only bound to the MHC groove by its peptide
backbone and its side chains do not engage the pockets
• HLA-DM, an ancient but non-classical class II molecule catalyzes the
release of CLIP and the binding of high affinity peptides via
interaction of peptide amino acid side-chains with MHC pockets
• Without Ii the MHC class II molecule traffics to the cell membrane