Topic 5 Antigen Processing

8/2/2019 Topic 5 Antigen Processing

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Topic 5

Antigen Processing

Dr. Colin R.A. Hewitt

[email protected]


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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 thepathogen replicates Antigen processing includes uptake, degradation, complex formation andpresentation 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

What you should know by the end of this lecture


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T cells do not recognise native antigens

YY

B

YY Y YY

Y

Y

B

Y

T

Y

T

Proliferation and antibodyproduction

No proliferationNo cytokine release

Cross-linking of surfacemembrane Ig

Y

B

Y

BY

B

Y

B

Y

BY

B

Y

B


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Cellsurface

peptidesof Ag

Antigens must be processed in orderto be recognised by T cells

Y

T

T cellresponse

No T cellresponse

No T cellresponse

No T cellresponse

No T cellresponse

Soluble

nativeAg Cellsurfac

enative Ag

Soluble

peptidesof Ag

Cell surface peptides ofAg presented by cellsthat express MHC

antigen

s

ANTIGENPROCESSING


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M

M

Early evidence that antigens are catabolised

M

Macrophages and radiolabelledListeria monocytogenes

Internalisation

Rapid binding to cell surface

M

Degradationof bacteria

and release of

Radiolabelledprotein into supernatant andcells

How is antigen catabolism linked to T cell proliferation?


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MM

M

M

0mins 60mins

T

Listeria-specificT cells

The interaction of T cells with macrophagesrequires antigen catabolism

Listeria

NO TCELLSBIND

NO TCELLSBIND

TCELLSBIND

Listeriacoatedplastic

NO TCELLSBIND

NO TCELLSBIND

T cell do not bind stably to antigen presenting cells unless the antigen is

catabolised


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Only metabolically active cells can process antigen

Determinants recognised by T cells are generated by catabolic activity that is dependentupon the viability of macrophages

Fix withparaformaldehydeor poison withsodium azide

M

Pulse withListeria for 60min& wash cells

M

AddListeriaspecific T cells

M

T

Listeria-

specificT cells

NO T CELLS BIND

Antigen presenting cells must be viable to PROCESS antigen


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Fix with paraformaldehydeor poison with sodium azide

M

M

M

M

Listeria

TAddListeriaspecific TcellsT CELLS BIND M

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

Antigen presentation does not requiremetabolically-active cells


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M

M

M

M

M

M

Listeria

Listeria

Where does antigen processing take place?

M

M

Incubate with CHLOROQUINE

TAddListeriaspecific Tcells

T CELLS BIND

NO T CELLS BIND

Chloroquine inhibits lysosomal function (a lysosomotrophic drug)

Antigen processing involves the lysosomal system


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What form of antigen is produced by antigen processing?

TOvalbumin specific T cell

line

Catabolism reduces antigens to peptides that can be recognised by T cells

APC

Viable

APC

ViableT T

T T

TT

T T

T TT

T T

TT

T

Digestedovalbumin

Fixed

APC

Fixed

APC

Nativeovalbumin

Ag

APC

T cellresponse


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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 antigenprocessing pathway


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Is exogenous antigen processing sufficient?

Most cell types do not have lysosomal systems developed as well asmacrophages

BUT

Viruses can infect most cell types

M Macrophages have well-developed lysosomal systems Specialised for motility, phagocytosisand the introduction of particles to thelysosomal system

A non-lysosomal mechanism to process antigens for presentation to T cells is required


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+Chloroquine

Infectiousinfluenza

CTL CT

L

CTL

CTL

CTL

CTL

CT

LCTL

CT

LCTL

CTL

CTL

CTL

CTL

CTL

CTL

Infectious viruses raise CTL that recognise antigensthat are notgenerated by the exogenous pathway

Most CTL do notrecognise lysosomally-derived antigens

Cloned anti-CTL

Notreatment

CTLassay

Lysosome inhibitors do not inhibit the generation ofantigens recognised by most CTL

Kill

Kill

Strong T cell response


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Lysosomal inhibitors inhibit the generation of antigens from INACTIVE virus

Some CTL can recognise lysosomally-derived antigens

Inactivatedinfluenza

Cloned anti-CTL

Inactive viruses raise CTL to antigens that aregenerated by the exogenous pathway

CT

LCTL

CT

L

CT

L

CT

L

CTL

CTL

CT

L

CTL CT

LCTL

CT

L

Weak T cell

response

+Chloroquine

Notreatment

CTLassay

Kill

No Kill


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Non-lysosomal processing

The antigens of infectious & inactivated viruses are clearly generated by different mechanisms

Proteinsynthesisinhibitor-treatedProtein synthesis is required for virus infected target cells to express

antigens recognised by CTL

CTL raisedwith

infectiousvirus

CTL

CTL raised withnon-infectiousvirusCT

L

Untreated

Infectious viruses use cellular protein synthesis machinery to replicateInactivated viruses do not synthesise protein


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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 EXOGENOUSPATHWAY

Infectious virus raises a strong CTL response

The processing of antigens from infectious viruses is NOT sensitive to lysosomotrophicdrugs

Most CTL recognise antigens generated via a non-lysosomal pathway

Protein synthesis is required for non-lysosomal antigen processingANTIGENS FROM INFECTIOUS VIRUSES ARE PROCESSED VIA THEENDOGENOUS PATHWAY

Non-lysosomal antigen processing

Do the two pathways generate the same type of T cell receptor ligand?


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Endogenous antigen processing also generates peptides

Peptides ofnucleoprotein

Native antigen fails tosensitise for lysis

No protein/antigensynthesis

Infectious virussensitises for lysis

Protein/antigensynthesis

CTL

Influenzavirus

Nucleoprotein

CTL

Synthetic peptide antigenssensitise targets for lysis

No protein/antigensynthesis but peptides arepre-formed

CTL


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Y

The site of pathogen replication or mechanism of antigen uptakedetermines the antigen processing pathway used

Y

Cytosolic compartmentEndogenous processing(Viral antigens)

Vesicular CompartmentContiguous with extracellular fluidExogenous processing(Streptococcal, Mycobacterial antigens)

Distinct mechanisms of antigen generation are used to raiseT cells suited to the elimination of endogenous or exogenous pathogens

INTRACELLULAR REPLICATION

EXTRACELLULAR ORENDOSOMAL REPLICATION


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Y

Eliminated by:Killing of infected cells byCTL thatuse antigens generated byENDOGENOUS PROCESSING

Y

Eliminated by:Antibodies and phagocyteactivation by T helper cellsthat useantigens generated byEXOGENOUS PROCESSING

Antigens generated by endogenous and exogenous antigenprocessing activate different effector functions

ENDOGENOUSPATHOGENS

EXOGENOUSPATHOGENS


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Stages of endogenous and exogenousantigen processing

UPTAKEAccess of native antigens and pathogens to intracellular pathways ofdegradation

DEGRADATIONLimited proteolysis of antigens to peptides

ANTIGEN-MHC COMPLEX FORMATIONLoading of peptides onto MHC molecules

ANTIGEN PRESENTATIONTransport and expression of peptide-MHC complexes on the surfaceof cells for recognition by T cells


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Y Y

Pinocytosis

Phagocytosis

Membrane Ig

receptor mediateduptake

Y

Uptake of exogenous antigens

Complement receptormediated phagocytosis Y

Fc receptor mediated phagocytosis

Uptake mechanisms direct antigen into intracellular vesicles

for exogenous antigen processing


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100

50

75

25

0

% of max.T cellresponse

10-

1

10-

2

10-

3 Antigen gml-1

Receptor-mediatedantigen uptake

Non-receptor-mediateduptake

Receptor-mediated uptake enhances theefficiency of the T cell response


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Proteases produce ~24 amino acid long peptides from antigens

Drugs that raise the pH of endosomes inhibit antigen processing

Endosomes

Exogenous pathway

Increaseinacidity

Cell

surface

Tolysosomes

Uptake

Protein antigensIn endosome

Cathepsin B, D and L proteases are activated by the decrease in pH


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Activation of Cathepsin B at low pH

At higher pH cathepsin B existsin a pro-enzyme form

Acidification of theendosome alters theconformation of theproenzyme to allow cleavageof the pro-region

Loss of the pro-regionexposes the catalyticsite of the protease

Hence: drugs that alteracidification of theendosomes disturbexogenous antigenprocessing


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Proteases produce ~24 amino acid long peptides from antigens

Drugs that raise the pH of endosomes inhibit antigen processing

Endosomes

Exogenous pathway

Increaseinacidity

Cell

surface

Tolysosomes

Uptake

Protein antigensIn endosome

Cathepsin B, D and L proteases are activated by the decrease in pH


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MHC molecules possess binding sites that are flexible at an early, intracellular stage ofmaturation

Floppy

Compact

Flexibility of the peptide bindingsite in MHC molecules

Although this example shows MHC class I molecules, the flexibility in the peptide bindingsite of MHC class II molecules also occurs at an early stage of maturation in the endoplasmicreticulum


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Need to prevent newlysynthesised, unfolded selfproteins from binding toimmature MHC

Invariant chain stabilises MHC class II bynon- covalently binding to the immature MHCclass II molecule and forming a nonomericcomplex

In the endoplasmic reticulum

MHC class II maturation and invariant chain


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Invariant chain structure

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


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A peptide of the invariant chain blocks the MHC molecule binding site.This peptide is called the CLass II associated Invariant chain Peptide (CLIP)

Invariant chain CLIP peptide

and

CLIP

Cl II i d i i h i id (CLIP)


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Endosomes

Cell

surfaceUptake

Class II associated invariant chain peptide (CLIP)

(inv)3 complexesdirected towardsendosomes byinvariant chain

Cathepsin L degrades InvariantchainCLIP blocks groove in MHC

molecule

MHC Class IIcontaining vesiclesfuse with antigen

containing vesicles


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Removal of CLIP

?

How can the peptide stably bind to a floppy binding site?

Competition between large number of peptides


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HLA-DM

HLA-DR

HLA-DM assists in the removal of CLIP

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


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HLA-DM

HLA-DR

Single pocket in grooveinsufficient to accommodatea peptide

Multiple pocketsin groove sufficient toaccommodate a peptide


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HLA-DM catalyses the removal of CLIP

MIIC compartment

HLA-DM

Replaces CLIP with a peptide

antigen using a catalyticmechanism (i.e. efficient atsub-stoichiometric levels)

Discovered using mutant celllines that failed to presentantigen

HLA-DO may also play a rolein regulating DM

Sequence incytoplasmic tail retainsHLA-DM in endosomes

HLA-DM

HLA-DR


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MIIC compartment sorts peptide-MHC complexes for surface expression orlysosomal degradation

Surface expression of MHC class II-peptide complexes

Exported to the cell surface (t1/2 =50hr)

Sent to lysosomes fordegradation


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UPTAKEAntigens/pathogens already present in cell

DEGRADATIONAntigens synthesised in the cytoplasm undergo limited

proteolytic degradation in the cytoplasm

ANTIGEN-MHC COMPLEX FORMATIONLoading of peptide antigens onto MHC class I molecules isdifferent to the loading of MHC class II molecules

PRESENTATIONTransport and expression of antigen-MHC complexes on thesurface of cells for recognition by T cells

Endogenous antigen processing


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Degradation in the proteasome

The components of the proteasome include MECL-1, LMP2, LMP7

These components are induced by IFN-and replace constitutive components to conferproteolytic properties.

LMP2 & 7 encoded in the MHC

Proteasome cleaves proteins after hydrophobic and basic amino acids and releases

peptides into the cytoplasm

Cytoplasmic cellular proteins, including non-self proteinsare degraded continuously by a multicatalytic protease of 28 subunits


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Crystal Structure Of The 20s ProteasomeFrom Yeast

View

Endon


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ENDOPLASMIC RETICULUM

CYTOSOL

Peptide antigens produced in the cytoplasm are physicallyseparated from newly formed MHC class I

Newly synthesisedMHC class I molecules

Peptides needaccess to the ER in

order to be loaded ontoMHC class I molecules

i i


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ER membrane

Lumen of ER

Cytosol

Transporters associated withantigen processing (TAP1 & 2)

Transporter has preference for >8 amino acid peptideswith hydrophobic C termini.

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

ER membrane

Lumen of ER

Cytosol

TAP-1 TAP-2

Peptide

ATP-bindingcassette(ABC) domain

Hydrophobictransmembranedomain

Peptideantigensfrom

proteasome


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Discovery of the role of TAP1 & TAP2 in antigen processing

Transfection ofnormal TAP genesinto mutant APCrestored stable

surface MHC class Iexpression

Mutations in TAP genes affect the supply of peptides to the ER

MHC class I stability is dependent upon a supply of peptides

Analysis of genes inthe MHC of the

mutant cell lineshowed mutationsin a pair of ABCtransporter genes

Normal antigen

presenting cellline with stablesurface MHCclass I expression

Chemically-inducedmutant antigenpresenting cell linewith unstable (floppy)

MHC class Iexpressed intracellularly

X


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Endoplasmicreticulum

Calnexin bindsto nascentclass I chainuntil 2-M binds

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

B2-Mbinds andstabilisesfloppyMHC

Tapasin, calreticulin, TAP 1& 2 form a complex with thefloppy MHC

Cytoplasmic peptides areloaded onto the MHCmolecule and the structurebecomes compact

Maturation and loading of MHC class I


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Fate of MHC class I


Exported to the cellsurface


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Endoplasmicreticulum

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

Peptide

TAP-1 TAP-2

HSV protein blockstransport

of viral peptides into ER

Sent tolysosomesfor degradation

Evasion of immunity by interference with endogenous antigenprocessing

E i f i it b i t f ith


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Normally exported to the cellsurface

Adenoviral

proteinretains MHCclass I in theER

Evasion of immunity by interference withendogenous antigen processing

S


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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 inantigen processing

Summary

Topic 5 Antigen Processing

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