Topic 5

Antigen Processing
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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 which the

The mechanism of antigen processing depends upon the compartment in 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

YY
Cross-linking of surface membrane Ig

Y Y Y Y YY YY
Proliferation and antibody production

B B B B BB B B

Y Y Y Y Y Y

No proliferation No cytokine release

Antigens must be processed in order to be recognised by T cells

Y
Soluble native Ag Cell surface native Ag Soluble peptide s of Ag Cell surface peptides of Ag

Cell surface peptides of Ag presented by cells that express MHC antigens

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
Rapid binding to cell surface

M
Internalisation

Degradation 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 Listeriaspecific T cells NO T CELLS BIND

T
NO T CELLS BIND M NO T CELLS BIND M

Listeria coated plastic

Listeria

NO T CELLS BIND M
0mins

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

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

Listeria

Antigen presentation does not require metabolically-active cells

M
Fix with paraformaldehyde or poison with sodium azide

Add Listeria specific T T CELLS BIND cells

T M

Antigen presenting cells do not need to be viable to PRESENT antigen

Where does antigen processing take place?

Listeri a M M Add Listeria specific T cells M T M
T CELLS BIND

Listeri a M

Incubate with CHLOROQUINE

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 Native Digested ovalbumin ovalbumin

Ag

APC

AP C Viabl e

AP C Fixe d
T

AP C Viabl e
T T T T T

AP C Fixe d

T cell response

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
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
CT L CT L CT L CT L CT L

CTL assay Kill No treatment + Chloroquine

Cloned antiCTL
CT CT CT CT CT LCT L CT L CT LCT L CT L L CT L L L L

Kill

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 Weak T cell response Cloned antiCTL
CT L CT CT CT CT CT LCT L CT L CT LCT L CT L L CT L L L L

CTL assay Kill No treatment + Chloroquine

No Kill

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

CT L

CT L

CTL raised with non-infectious virus

Untreate d Protein synthesis inhibitorProtein synthesis is required for treated 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
CT L

Nucleoprotei n
CT L

Peptides of nucleoprotein
CT L

Infectious virus sensitises for lysis Protein/antigen synthesis

Native antigen fails to sensitise for lysis No protein/antigen synthesis

Synthetic peptide antigens sensitise targets for lysis 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

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

Antigens generated by endogenous and exogenous antigen processing activate different effector functions

Antibodies and phagocyte activation by T helper cells that use antigens generated by EXOGENOUS PROCESSING

EXOGENOUS PATHOGENS Eliminated by:

Y
ENDOGENOUS PATHOGENS Eliminated by:
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
Y

Complement receptor mediated phagocytosis

Phagocytosis

Pinocytosis

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

Receptormediated antigen uptake Non-receptor -mediated uptake

% of max. T cell response

10 0 7 5 5 0 2 5 0

103

1010Antigen gml-1 2 1

Exogenous pathway
Cell surface Uptak e

Protein antigens In endosome

Endosome s

Increas e 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 exists Acidification of the endosome alters the in a pro-enzyme form conformation of the proenzyme to allow cleavage of the pro-region Hence: drugs that alter acidification of the endosomes disturb exogenous antigen processing

Exogenous pathway
Cell surface Uptak e

Protein antigens In endosome

Endosome s

Increas e 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

Flopp Compac y t 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 synthesised, unfolded self proteins from binding to immature MHC

Invariant chain stabilises MHC class II by non- covalently binding to the immature MHC class II molecule and 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

CLI P

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 Endosome s

Uptak e

(inv)3 complexes directed towards endosomes by invariant chain

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

HLADM

HLADR

HLA-DM: Crystallised without a peptide in the groove In space filling models the groove is very small

HLADM
Single pocket in groove insufficient to accommodate a peptide

HLADR
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-DO may also play a role in regulating DM

HLADR

HLADM Sequence in cytoplasmic tail retains HLA-DM in endosomes

MIIC compartment

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

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

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
TAP-1 TAP-1 TAP-1 Peptide Peptide TAP-2 TAP-2 TAP-2

ATP-binding Peptide cassette antigens (ABC) domain from Transporter proteasomefor >8 amino acid peptides has preference
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 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

Chemically-induced mutant antigen presenting cell line with unstable (floppy) MHC class I expressed intracellularly

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

TAP-1 TAP-1 TAP-1 Peptide

TAP-2 TAP-2 TAP-2

Endoplasmic reticulum
Calnexin binds to nascent class I chain until 2-M binds B2-M binds and stabilises floppy 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

TAP-1 TAP-1 TAP-1 Peptide

TAP-2 TAP-2 TAP-2

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