Download CTL - Molecular Immunology

T cells do not recognise native antigens
Y
Y
Y
Y
Y
Y
Y
Cross-linking of
surface membrane Ig
YYY Y
Y Y Y Y
B
B B
B B BB
B B
Proliferation and
antibody production
T
T
No proliferation
No cytokine release
Y
Y
Antigens must be processed in order
to be recognised by T cells
T
Y
Soluble
native Ag
Cell surface
native Ag
Soluble
peptides
of Ag
Cell surface peptides of
Ag presented by cells that
express MHC antigens
Cell surface
peptides
of Ag
ANTIGEN
PROCESSING
No T cell
response
No T cell
response
No T cell
response
No T cell
response
T cell
response
Early evidence that antigens are catabolised
M
Macrophages and radiolabelled
Listeria monocytogenes
M
Internalisation
M
Rapid binding to cell surface
Degradation
M
of bacteria
and release of
Radiolabelled
protein into
supernatant and cells
How is antigen catabolism linked to T cell proliferation?
The interaction of T cells with macrophages
requires antigen catabolism
NO T CELLS
BIND
Listeria-specific
T cells
Listeria
coated
plastic
T
Listeria NO T CELLS NO T CELLS NO T CELLS
BIND
BIND
BIND
M
0mins
M
M
T CELLS
BIND
M
60mins
T cell do not bind stably to antigen presenting cells unless the
antigen is catabolised
Only metabolically active cells can process antigen
Listeriaspecific
T cells
M
Fix with
paraformaldehyde
or poison with
sodium azide
M
Pulse with
Listeria for 60min
& wash cells
T
M
Add Listeria
specific T cells
NO T CELLS BIND
Determinants recognised by T cells are generated by catabolic activity
that is dependent upon the viability of macrophages
Antigen presenting cells must be viable to PROCESS antigen
Antigen presentation does not require
metabolically-active cells
Listeria
M
M
M
M
Fix with paraformaldehyde
or poison with sodium azide
Add Listeria
specific T cells T
T CELLS BIND
M
Antigen presenting cells do not need to be viable to PRESENT antigen
Where does antigen processing take place?
Add Listeria
specific T cells
Listeria
M
M
M
T
M
T CELLS BIND
Listeria
Incubate with CHLOROQUINE
M
M
M
M
NO T CELLS BIND
Chloroquine inhibits lysosomal function (a lysosomotrophic drug)
Antigen processing involves the lysosomal system
What form of antigen is produced by antigen processing?
T
Ovalbumin specific T cell line
Digested ovalbumin
Native ovalbumin
Ag
APC
T cell
response
APC
APC
APC
APC
Viable
Fixed
Viable
Fixed
T T T
T T
T
T
T
T T T
T T
T
T
T
Catabolism reduces antigens to peptides that can be recognised by T cells
Summary of exogenous antigen processing
• T cells can not recognise native antigens
• Antigens must be processed for recognition by T cells
• Antigens catabolism occurs inside cells
• Only metabolically active cells can process antigen
• Antigen presentation does not require metabolically-active cells
• Antigen processing involves the lysosomal system
• Catabolism reduces antigens to peptides
• Because extracellular antigens are dealt with by the lysosomal
system, lysosomal antigen processing is part of the EXOGENOUS
antigen processing pathway
Is exogenous antigen processing sufficient?
• Macrophages have welldeveloped lysosomal systems
M
• Specialised for motility,
phagocytosis and the introduction
of particles to the lysosomal system
Most cell types do not have lysosomal systems
developed as well as macrophages
BUT
Viruses can infect most cell types
A non-lysosomal mechanism to process antigens for presentation to T
cells is required
Infectious viruses raise CTL that recognise antigens
that are not generated by the exogenous pathway
Infectious
influenza
Strong T cell response
CTL
CTL
CTL
CTL
Cloned anti-
CTL assay
Kill
CTL
CTL
CTL
CTL
CTL CTL
CTL
CTL
CTL CTL CTL
CTL
CTL
No treatment
Kill
+ Chloroquine
Lysosome inhibitors do not inhibit the generation of
antigens recognised by most CTL
Most CTL do not recognise lysosomally-derived antigens
Inactive viruses raise CTL to antigens that are
generated by the exogenous pathway
Inactivated
influenza
CTL assay
CTL
Weak T cell response
Cloned anti-
Kill
CTL
No treatment
CTL
CTL
CTL CTL
CTL
CTL
CTL CTL CTL
CTL
CTL
No Kill
+ Chloroquine
Lysosomal inhibitors inhibit the generation of antigens from INACTIVE virus
Some CTL can recognise lysosomally-derived antigens
Non-lysosomal processing
The antigens of infectious & inactivated viruses are clearly generated by
different mechanisms
Infectious viruses use cellular protein synthesis machinery to replicate
Inactivated viruses do not synthesise protein
CTL raised with
infectious virus
CTL raised with
non-infectious virus
CTL
CTL
Untreated
Protein synthesis
inhibitor-treated
Protein synthesis is required for virus infected target cells to express
antigens recognised by CTL
Non-lysosomal antigen processing
Inactive virus raises a weak CTL response
The processing of antigens from inactive viruses is sensitive to
lysosomotrophic drugs
ANTIGENS FROM INACTIVE VIRUSES ARE PROCESSED VIA THE
EXOGENOUS PATHWAY
Infectious virus raises a strong CTL response
The processing of antigens from infectious viruses is NOT sensitive to
lysosomotrophic drugs
Most CTL recognise antigens generated via a non-lysosomal pathway
Protein synthesis is required for non-lysosomal antigen processing
ANTIGENS FROM INFECTIOUS VIRUSES ARE PROCESSED VIA THE
ENDOGENOUS PATHWAY
Do the two pathways generate the same type of T cell receptor ligand?
Endogenous antigen processing also generates peptides
Influenza virus
CTL
Nucleoprotein
CTL
Peptides of nucleoprotein
CTL
Infectious virus
sensitises for lysis
Native antigen fails
to sensitise for lysis
Synthetic peptide antigens
sensitise targets for lysis
Protein/antigen
synthesis
No protein/antigen
synthesis
No protein/antigen
synthesis but peptides are
pre-formed
The site of pathogen replication or mechanism of antigen
uptake determines the antigen processing pathway used
Y
EXTRACELLULAR OR
ENDOSOMAL REPLICATION
Vesicular Compartment
Contiguous with extracellular fluid
Exogenous processing
(Streptococcal, Mycobacterial antigens)
INTRACELLULAR REPLICATION
Cytosolic compartment
Endogenous processing
(Viral antigens)
Distinct mechanisms of antigen generation are used to raise
T cells suited to the elimination of endogenous or exogenous pathogens
Y
Antigens generated by endogenous and exogenous
antigen processing activate different effector functions
EXOGENOUS
PATHOGENS
Eliminated by:
ENDOGENOUS
PATHOGENS
Eliminated by:
Antibodies and phagocyte
activation by T helper cells that
use antigens generated by
EXOGENOUS PROCESSING
Killing of infected cells by CTL
that use antigens generated by
ENDOGENOUS
PROCESSING
Stages of endogenous and exogenous
antigen processing
UPTAKE
Access of native antigens and pathogens to intracellular
pathways of degradation
DEGRADATION
Limited proteolysis of antigens to peptides
ANTIGEN-MHC COMPLEX FORMATION
Loading of peptides onto MHC molecules
ANTIGEN PRESENTATION
Transport and expression of peptide-MHC complexes on the
surface of cells for recognition by T cells
Uptake of exogenous antigens
Membrane Ig
receptor mediated
uptake
Y
Phagocytosis
Complement receptor
mediated phagocytosis
Pinocytosis
Y
Fc receptor mediated phagocytosis
Uptake mechanisms direct antigen into intracellular vesicles
for exogenous antigen processing
Receptor-mediated uptake enhances the
efficiency of the T cell response
Receptor-mediated
antigen uptake
Non-receptor
-mediated uptake
100
% of max.
T cell
response
75
50
25
0
10-3
10-2
10-1
Antigen gml-1
Exogenous pathway
Cell surface
Uptake
Protein antigens
In endosome
Endosomes
Increase
in acidity
To lysosomes
Cathepsin B, D and L proteases are activated by the decrease in pH
Proteases produce ~24 amino acid long peptides from antigens
Drugs that raise the pH of endosomes inhibit antigen processing
MHC class II maturation and invariant chain
In the endoplasmic reticulum
Invariant chain stabilises MHC class
Need to prevent newly
II by non- covalently binding to the
synthesised, unfolded
self proteins from binding immature MHC class II molecule and
forming a nonomeric complex
to immature MHC
Invariant chain CLIP peptide
 and b chains of MHC
class II molecules
CLIP
A peptide of the invariant chain blocks the MHC molecule binding site.
This peptide is called the CLass II associated Invariant chain Peptide
(CLIP)
Class II associated invariant chain peptide (CLIP)
Cell surface
Uptake
(binv)3 complexes
directed towards
endosomes by
invariant chain
Endosomes
Cathepsin L degrades
Invariant chain
CLIP blocks groove in MHC
molecule
MHC Class II
containing vesicles
fuse with antigen
containing vesicles
Removal of CLIP
?
How can the peptide stably bind to a floppy binding site?
Competition between large number of peptides
HLA-DM assists in the removal of CLIP
HLA-DM
HLA-DR
HLA-DM: Crystallised without a peptide in the groove
In space filling models the groove is very small
HLA-DM
Single pocket in “groove”
insufficient to accommodate
a peptide
HLA-DR
Multiple pockets
in groove sufficient to
accommodate a peptide
HLA-DM catalyses the removal of CLIP
HLA-DM
Replaces CLIP with a
peptide antigen using a
catalytic mechanism (i.e.
efficient at substoichiometric levels)
Discovered using mutant
cell lines that failed to
present antigen
HLA-DR
HLA-DM
MIIC compartment
HLA-DO may also play a
role in regulating DM
Sequence in cytoplasmic
tail retains HLA-DM in
endosomes
Surface expression of MHC class IIpeptide complexes
Exported to the cell surface (t1/2 = 50hr)
Sent to lysosomes for degradation
MIIC compartment sorts peptide-MHC complexes for surface expression or
lysosomal degradation
Endogenous antigen processing
UPTAKE
Antigens/pathogens already present in cell
DEGRADATION
Antigens synthesised in the cytoplasm undergo limited
proteolytic degradation in the cytoplasm
ANTIGEN-MHC COMPLEX FORMATION
Loading of peptide antigens onto MHC class I molecules
is different to the loading of MHC class II molecules
PRESENTATION
Transport and expression of antigen-MHC complexes on
the surface of cells for recognition by T cells
Degradation in the proteasome
Cytoplasmic cellular proteins, including non-self proteins
are degraded continuously by a multicatalytic protease of 28 subunits
The components of the proteasome include MECL-1, LMP2, LMP7
These components are induced by IFN- and replace constitutive
components to confer proteolytic properties.
LMP2 & 7 encoded in the MHC
Proteasome cleaves proteins after hydrophobic and basic amino acids
and releases peptides into the cytoplasm
Peptide antigens produced in the cytoplasm are
physically separated from newly formed MHC class I
ENDOPLASMIC RETICULUM
Newly synthesised
MHC class I molecules
CYTOSOL
Peptides need
access to the ER in
order to be loaded onto
MHC class I molecules
Transporters associated with
antigen processing (TAP1 & 2)
Hydrophobic
transmembrane
domain
Lumen of ER
Peptide
ER membrane
Cytosol
Peptide
Peptide
Peptide antigens
from proteasome
ATP-binding cassette
(ABC) domain
Transporter has preference for >8 amino acid peptides
with hydrophobic C termini.
Discovery of the role of TAP1 & TAP2 in antigen processing
Normal antigen
presenting cell
line with stable
surface MHC
class I expression
Chemically-induced
mutant antigen
presenting cell line
with unstable (floppy)
MHC class I
expressed intracellularly
Analysis of genes
in the MHC of the
mutant cell line
showed mutations
in a pair of ABC
transporter genes
X
√
Transfection of
normal TAP genes
into mutant APC
restored stable
surface MHC
class I
expression
Mutations in TAP genes affect the supply of peptides to the ER
MHC class I stability is dependent upon a supply of peptides
Maturation and loading of MHC class I
Peptide
Peptide
Peptide
Endoplasmic reticulum
B2-M
Calnexin binds
binds and
to nascent
stabilises
class I chain
floppy
until b2-M binds
MHC
Tapasin, calreticulin, TAP
1 & 2 form a complex with
the floppy MHC
Cytoplasmic peptides
are loaded onto the
MHC molecule and the
structure becomes
compact
Fate of MHC class I
Exported to the cell surface
Sent to lysosomes for degradation
Evasion of immunity by interference with endogenous
antigen processing
Peptide
Peptide
Endoplasmic reticulum
HSV protein blocks transport
of viral peptides into ER
Sent to lysosomes
for degradation
Evasion of immunity by interference with
endogenous antigen processing
Normally exported to the cell surface
Adenoviral
protein
retains MHC
class I in the ER
Sent to lysosomes for degradation
Summary
• T and B cells recognise antigen differently
• Antigen must be catabolised before T cells can recognise it
• Antigen processing generates antigenic peptides
• Exogenous antigen processing takes place in lysosomes
• Endogenous processing is non-lysosomal
• The mechanism of antigen processing depends upon the
compartment in which the pathogen replicates
• Endogenous and exogenous antigen processing both involve
uptake, degradation, complex formation and presentation
• Exogenous antigen processing uses invariant chain and HLA-DM
• Endogenous antigen processing uses proteasomes and peptide
transporters in antigen processing
• Pathogens can evade immunity by disrupting antigen processing