Topic 5

Antigen Processing

Dr. Colin R.A. Hewitt

crah1@le.ac.uk
What you should know by the end of this lecture

That T and B cells recognise antigen differently

The experimental evidence that antigen catabolism takes place
Antigen processing generates antigenic peptides
That antigen processing can take place in lysosomes
That there is a non-lysosomal mechanism of antigen processing
The mechanism of antigen processing depends upon the compartment in
which the pathogen replicates
Antigen processing includes uptake, degradation, complex formation and
presentation
The role of invariant chain HLA-DM and CLIP in antigen processing
The role of the proteasome and transporters in antigen processing
How pathogens evade immunity by disrupting antigen processing
 T cells do not recognise native antigens

B B
B B B BB
B B
Y Y Y Y
Y YYY Y

Y
Y
Y

Y
Y
Y
Cross-linking of
Proliferation and
surface membrane Ig
antibody production

T T No proliferation
Y No cytokine release
Y
 Antigens must be processed in order
to be recognised by T cells

T
Y
Cell surface peptides of
Ag presented by cells that
Soluble express MHC antigens
Soluble
native Ag peptides
Cell surface of Ag Cell surface
native Ag peptides
of Ag

ANTIGEN
PROCESSING

No T cell No T cell No T cell No T cell T cell

response response response response response
 Early evidence that antigens are catabolised

M
M

Macrophages and radiolabelled Rapid binding to cell surface

Listeria monocytogenes

Degradation
M of bacteria
M
and release of
Radiolabelled
protein into
supernatant and cells
Internalisation

How is antigen catabolism linked to T cell proliferation?

 The interaction of T cells with macrophages
requires antigen catabolism

Listeria-specific NO T CELLS
T cells BIND
Listeria
coated
T plastic

Listeria NO T CELLS NO T CELLS NO T CELLS T CELLS

BIND BIND BIND BIND

M M M M

0mins 60mins
T cell do not bind stably to antigen presenting cells unless the
antigen is catabolised
Only metabolically active cells can process antigen

Listeria-
specific
T cells
T

M M
M

Fix with
Pulse with Add Listeria
paraformaldehyde
Listeria for 60min specific T cells
or poison with
& wash cells NO T CELLS BIND
sodium azide

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 T
Listeria

M M M 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?

Ovalbumin specific T cell line T

Native ovalbumin Digested ovalbumin

Ag

APC APC APC APC APC

Viable Fixed Viable Fixed

T cell T T T T T T T
T T
response 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 well-

developed 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 assay

CTL CTL CTL CTL CTL Kill

Cloned anti- CTL No treatment

CTL CTL Kill

CTL CTL CTL
CTL CTL CTL CTL
CTL CTL + 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
Kill
Cloned anti- CTL
No treatment
CTL CTL
CTL CTL CTL
CTL CTL CTL CTL No Kill
CTL CTL
+ 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 CTL raised with

infectious virus 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 Nucleoprotein Peptides of nucleoprotein

CTL CTL CTL

Infectious virus Native antigen fails Synthetic peptide antigens

sensitises for lysis to sensitise for lysis sensitise targets for lysis
No protein/antigen
Protein/antigen No protein/antigen
synthesis but peptides are
synthesis synthesis
pre-formed
The site of pathogen replication or mechanism of antigen
uptake determines the antigen processing pathway used

EXTRACELLULAR OR
ENDOSOMAL REPLICATION
Vesicular Compartment
Y 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
 Antigens generated by endogenous and exogenous
antigen processing activate different effector functions

EXOGENOUS
PATHOGENS
Y ENDOGENOUS
PATHOGENS
Eliminated by: Eliminated by:
Antibodies and phagocyte Killing of infected cells by CTL
activation by T helper cells that that use antigens generated by
use antigens generated by ENDOGENOUS
EXOGENOUS PROCESSING 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
Phagocytosis
Y
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 Non-receptor
antigen uptake -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

Protein antigens
Uptake
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
 Activation of Cathepsin B at low pH

Loss of the pro-

region exposes the
catalytic site of the
protease

At higher pH cathepsin B Acidification of the Hence: drugs that alter

exists in a pro-enzyme form endosome alters the acidification of the
conformation of the endosomes disturb
proenzyme to allow exogenous antigen
cleavage of the pro-region processing
 Exogenous pathway
Cell surface

Protein antigens
Uptake
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
 Flexibility of the peptide binding
site in MHC molecules
MHC molecules possess binding sites that are flexible at an early,
intracellular stage of maturation

Floppy Compact

Although this example shows MHC class I molecules, the flexibility in the
peptide binding site of MHC class II molecules also occurs at an early
stage of maturation in the endoplasmic reticulum
MHC class II maturation and invariant chain
In the endoplasmic reticulum

Need to prevent newly Invariant chain stabilises MHC class

synthesised, unfolded II by non- covalently binding to the
self proteins from binding immature MHC class II molecule and
to immature MHC forming a nonomeric complex
 Invariant chain structure

Three extended peptides each bind into the grooves of three MHC class II
molecules to form the nonomeric complex
 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

Endosomes
Uptake

(binv)3 complexes Cathepsin L degrades MHC Class II

directed towards Invariant chain containing vesicles
endosomes by CLIP blocks groove in MHC fuse with antigen
invariant chain molecule 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 sub-
stoichiometric levels)
Discovered using mutant
cell lines that failed to
present antigen
HLA-DR HLA-DM HLA-DO may also play a
role in regulating DM

Sequence in cytoplasmic
tail retains HLA-DM in
MIIC compartment endosomes
 Surface expression of MHC class II-
peptide 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
Crystal Structure Of The 20s Proteasome
From Yeast

View
End on
 Peptide antigens produced in the cytoplasm are
physically separated from newly formed MHC class I

ENDOPLASMIC RETICULUM

Newly synthesised
MHC class I molecules

Peptides need
CYTOSOL access to the ER in
order to be loaded onto
MHC class I molecules
 Transporters associated with
antigen processing (TAP1 & 2)
Hydrophobic
transmembrane
Lumen of ER domain
Peptide

ER membrane

Cytosol Peptide
Peptide

Peptide antigens ATP-binding cassette

from proteasome (ABC) domain

Transporter has preference for >8 amino acid peptides

with hydrophobic C termini.
Discovery of the role of TAP1 & TAP2 in antigen processing

Analysis of genes
Normal antigen in the MHC of the
presenting cell
line with stable
mutant cell line
showed mutations X
surface MHC
class I expression
in a pair of ABC
transporter genes

Chemically-induced Transfection of
mutant antigen normal TAP genes
presenting cell line into mutant APC
with unstable (floppy) restored stable
MHC class I surface MHC
expressed intracellularly 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

Calnexin binds B2-M Tapasin, calreticulin, TAP Cytoplasmic peptides

to nascent binds and 1 & 2 form a complex with are loaded onto the
class I chain stabilises the floppy MHC MHC molecule and the
until b2-M binds floppy structure becomes
MHC 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
Sent to lysosomes
for degradation

HSV protein blocks transport

of viral peptides into ER
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