MEDSCI 708

Antigen processing and presentation

Advanced Immunology and Immunotherapy

2007

Antigen processing:

Proteolytic cleavage of proteins into small fragments(antigen peptides) that can bind to MHC moleculeson antigen presenting cells.

Antigen presentation:

Presentation of antigen peptides to T cell receptoron T cells

MHC restriction

encoded by the most polymorphic gene cluster on the human genome (many alleles)

1973: Peter Doherty and Rolf Zinkernagel (Nobel Prize in Medicine, 1996)

The T cell receptor will recognise a peptide only when it is bound to a particular MHC molecule.

MHC: Major Histocompatibility Complex

MHC class I:

found on all nucleated cells

MHC class II:

found only on “professional antigen presenting

cells”, such as dendritic cells, macrophages, B cells

endogenous pathway

CD8 T

TcR CD8

virus-infected or tumor cell

MHC I

CTLkill

exogenous pathway

Macrophage or other professional APC

pathogen

MHC II

CD4 T

TcR CD4

B cell help

ab

MHC I restriction MHC II restriction

Antigen presenting pathways

Class I and Class IIpathways of antigenpresentation

FromImmunology 5th ed. (Roitt et al.)

MHC I peptides: 8-11 aawith 2 terminal anchor residues

MHCII peptides: 8-30+ aa with anchors throughout the peptide

In human:

2x3 MHCI molecules

1245 known alleles

MHCI and MHCII peptides are verydifferent and have to be generatedby different mechanisms

Many different peptides haveto be presented (different anchors)

Two distinct patways exist to provide antigen for T cells

Class I MHC Class II MHC

Peptide source Inside the cell Outside the cell

Peptide size 8-10 amino acids Any length but usually 8-30 aa

T cell response Cytotoxic CD8 T cells Helper CD4 T cells

Outcome Infected cell is killed T cell, B-cell and macrophage are activated

Ig isotype switching occurs

Type of infection

predominantly viral predominantly bacterial, parasitic and toxins

Pathway Endogenous Exogenous

Groothuis et al., Immunological Reviews Vol. 207 (2005)

MHC class I antigen processing

Endogenous (MHC class I) pathway

1. Processing of peptide antigens

2. Assembly of MHC and peptide loading complex

3. Peptide loading and MHC-peptide transport

Processing of peptide antigens

Kloetzel and Ossendorp, Curr. Opin. Immunol. 16 (2004)

The Proteasome - a multicatalytic protease

- around 700 kDa

- 7 rings of subunits (active site)

- 2 outer rings of subunits

- ATP-dependent degradation of mostly ubiquitin-conjugated proteins

19S regulator:

- attached at both ends of 20S proteasome

- binds ubiquitin-tagged proteins

Proteasome

About 1/3 of intracellular proteolysis in mammalian cells is directed to

nascent proteins

- defective ribosomal products (DRiPs)

- non-functional and potentially toxic proteins

- proteins synthesised in excess (maintain protein homeostasis)

- regulatory proteins

- Only about 1% of the peptide pool is available to immune system

UbUb

E1E1

UbUb

E2E2

UbUb

E3E3

UbUb

26s proteosome degradation26s proteosome degradation

TargetTarget

The Ubiquitin PathwayThe Ubiquitin Pathway

UbUbUbUbUbUb

Ub-activating enzyme

Ub-conjugating enzyme Ub-ligase

Groothuis et al., Immunological Reviews Vol. 207 (2005)

Binding of poly-ubiquitin chains to 19S proteasome

Elongates Ub tree

From Strehl et al., Immunological Reviews Vol.207, pp 19-30 (2005)

1i = low molecular weight protein 2 (LMP 2)

2i = multicatalytic endopeptidase complex like 1 (MECL 1)

5i = low molecular weight protein 7 (LMP 7)

POMP = proteasome maturation protein

PA = proteasome activator

Immunoproteasome

• P28 causes N-terminal tails of the -subunits to flip upwards, thereby facilitating

substrate entry and product exit.

• The immunoproteasome does not replace the constitutive proteasome completely

• The immunoproteasome has a considerably shorter half-life

• The immunoproteasome has an altered cleavage site preference with a strong

preference to cleave behind residues that represent correct C-terminal anchors

for MHC I presentation.

• PA28 does confer new cleavage site specificities, but enhances the frequency

of the usage of minor cleavage sites to provide more peptides for MHC presentation

Immunoproteasome

From Strehl et al., Immunological Reviews Vol.207, pp 19-30 (2005)

Immunoproteasomes affect the size of the antigenic peptide pool

Trim-peptidases• Peptides produced by proteasomes are often to large for presentation (8-11 aa) or for TAP transport (8-16 aa)

• Several cytosolic and ER proteases are involved in trimming.

• However, their major function is probably peptide degradation for aa recycling.

Cytosolic peptidases

Puromycin-sensitive aminopeptidase (PSA):- metallopeptidase- shown to both trim and destroy epitopes

Thimet oligopeptidase (TOP):- metallopeptidase of the M3 family- peptides of up to 15 aa are preferred substrates- appears to be mainly involved in epitope destruction (down-regulation enhances presentation).

Cytosolic peptidases (cont.)

Leucine aminopeptidase (LAP):

- metallopeptidase

- peptides of less than 7 aa are preferred substrates

- mainly for aa recycling

Tripeptidyl protease II ((TPP II):

- cleaves peptides larger than 15 aa

- plays significant role in antigen presentation

- exopeptidase activity: removes blocks of 3 N-terminal aa

- endopeptidase activity: trypsin-like specificity

• There are no carboxypeptidases in the cytosol

• The proteasomes have to generate C-terminal anchor for MHCI binding

Discovery/purification of ER peptidases

Saveanu et al., Immunological Reviews Vol. 207 (2005)

AMC: aminoacyl-aminomethyl cumarin = artificial substrate that becomesfluorescent after removal of N-terminus

ER peptidases

ER aminopeptidase associated with antigen processing (ERAAP):

= ERAP1 (human)

- metallopeptidase of M1 family

- specific for large hydrophobic residues, such as Leu

- strong preference for substrates of 10 or more aa

ERAP2:

- 49% identical to ERAP1 by aa sequence

- specific for basic residues, such as Arg and Lys.

Chaperones in antigen processing

- Tailless complex polypeptide-1 (TCP-1) ring complex (TriC)- Chaperonin-containing TCP-1 (CCT)

• Exact function remains unclear

• Probably involved in peptide delivery to TAP

Cytosolic chaperones:

ER chaperones:

- Protein disulfide isomerase (PDI)- Binding protein (BiP), hsp70 family

Regulation of the translocon (lid function)Probably play a role in peptide loading of MHCI

Shastri et al., Immunological Reviews Vol. 207 (2005)

Processing of the SIINFEKL epitope

translocon

BiPPDI

http://www.cryst.bbk.ac.uk/pps97/assignments/projects/coadwell/003.htm

Transporter Associated with Antigen Presentation (TAP)

Peptide-binding domain

Transmembrane domain

2 ATP-binding cassettes

ER retention signal

8-12 aa (up to 40aa with low efficiency)

Cresswell et al., Immunological Reviews V0l. 207 (2005)

Assembly of MHC class I and peptide loading complex

HC: heavy chain, 3 -subunits, 45 kDa glycoprotein, polymorphic2m: 2-microglobulin, 12 Kda, non-polymorphicCNX: calnexin, membrane-anchored chaperone, stabilises nascent HC,lectin-likeCRT: calreticulin, soluble lectin-like chaperone, binds N-linked glycan on HCErp57: oxido-reductase, binds Tapasin via S-S bonds and non-covalently to CRT

peptide loading complex

Cresswell et al., Immunological Reviews V0l. 207 (2005)

Peptide binding cycle

Tapasin

• 48 kDa glycoprotein

• stabilises TAP1/TAP2 which enhances peptide transport

• bridges MHC class I to TAP (structural component)

• facilitates peptide loading

• stabilises “empty” peptide-receptive MHC complexes

• optimises peptide repertoire (peptide editor)

Brocke et al. (2002)

Quality control by tapasin

“Lost in action” or “the inefficiency of antigen presentation”

• about 2 billion proteins per cell are expressed and turned over in 6h

• about 100 million peptides per cell are generated in 1 minute

• only a few hundred MHC I molecules are made in 1 minute

• a large fraction of MHC I molecules fail to acquire a peptide

• a peptide has an average in-vivo half-life of a few seconds

• more than 99% of cytosolic peptides are destroyed before their

encounter TAP

An antigen has to be expressed at a minimum of 10,000 copies to be presented by MHC class I

The exogenous (MHC class II) pathway

Right here after the break ….

Exogenous (MHC class II) pathway

Villadangos et al., Immunological Reviews Vol. 207 (2005)

Exogenous (MHC class II) pathway

1. MHC assembly and transport to peptide loading compartment

2. Uptake and processing of exogenous antigen

3. Peptide loading (CLIP exchange)

Assembly of MHC class II requires the invariant chain

MHC II: HLA-DR, -DQ, -DP (human), glycosylated heterodimers,

- generic chain (30-34 kDa)

- highly polymorphic -chain (26-29 kDa)

Invariant Chain (Ii)

• scaffold to facilitate proper folding and assembly of MHC II

• blocking premature class II peptide association

• direct trafficking of MHCII-invariant chain to endosomal pathway

• modulating the proteolytic environment within endosome.

4 domains:

4 functions:

• short N-terminal cytosolic domain (sorting motif)

• single TM domain

• class II-associated invariant chain peptide (CLIP)

• C-terminal trimerisation motif and protease inhibitor motif (only

some isoforms).

Uptake of exogenous antigen

Endocytosis: Uptake of material into the cell by the formation of a membrane-bound vesicle.

Endosome: endocytotic vesicle derived from the plasma membrane.

1. Receptor-mediated endocytosis: mannose and lectin-like receptors

2. Macropinocytosis: uptake of fluid-filled vesicles (mainly DC)

3. Phagocytosis: uptake of complete cells

Phagocytosis

Desjardins et al., Immunological Reviews Vol 207 (2005)

Processing of exogenous antigen and invariant chain

Cathepsins: endosomal proteases involved in Ii degradation and antigen processing

- exact role for each cathepsin not clear

- some might have redundant functions

- some were shown to be cell specific

- best studied are cathepsin L and cathepsin S

- both are papain-like cysteine endoproteases and are

required for invariant chain degradation

Other endosomal proteases

Asparaginyl cysteine endoprotease (AEP):

- initiates first cuts in protein

-interferon-induced lysosomal thiol reductase (GILT):

- reduces disulfide bridges in proteins (S-S to -SH)

Invariant chain degradation in endosome

Note: within the same compartment, proteases also generate peptide fragments derived from endocytosed antigens

Hsing & Rudensky, Immunological Reviews Vol 207 (2005)

Regulation of endosomal proteases

• N-terminal propiece that blocks substrate binding. Propiece stabilises

protease during traffick through ER and dissociates

upon maturation and acidification of the endosome.

• Cystatins: naturally occuring lysosomal protease inhibitors. Wedge-

shaped binding region fills and obstructs active site.

• Ii isoform p41: Highly specific for cathepsin L. Enhances presentation

of certain antigens.

Peptide loading and editing by HLA-DM

Brocke et al., Curr. Opin. Immunol. Vol. 14 (2002)

Peptide loading and editing by HLA-DM

Peptide loading and editing by HLA-DM

Brocke et al., Curr. Opin. Immunol. Vol. 14 (2002)

Possible role for HLA-DO in B cells

Now it seems to be all so clear and logical ….but is it ?

Problem 1:

The stimulation of a naïve CD8 T cell requires co-stimulatory molecules, such asCD86, but these are absent from the majority of cell types!!

A professional APC has to acquire viral antigens from infected cell, e.g by phagocytosis

Problem 2:

DC has to present same peptide antigen as infected cell, but exogenous pathway produces different peptides than endogenous pathway.

Cross-presentation of exogenous antigens

How does endocytosed protein get into the cytosol for endogenous pathway?

Hypothesis:

Retro-transport of endocytosed protein:

TGN - ER - cytosol (ERAD pathway?)

This pathway is used by certain toxins, such as cholera toxin, ricin

ERAD: ER Associated Degradation

Possible routes that phagocytosed antigens take to reach proteasomes in the cytosol.

Vol. 19,Feb. 2007

Lilley & Ploegh, Immunological Reviews Vol. 207 (2005)

Strategies of immune evasion

Manipulation of endocytic pathway

Interference with T cell receptor recognition

e.g. m04/gp34 (murine cytomegalovirus)

Protein binds to MHCI in the ER, sterical inhibition

of TcR binding

Increasing the endocytotic rate of MHC I

e.g. K3, K5 (Kaposi’s sarcoma-associated herpesvirus)

Proteins act as E3 ubiquitin ligases that ubiquinate Lys

in cytosolic parts of their target. Sorting into lysosome

for degradation

Re-routing of MHC I complex from TGN to lysosomes

e.g. Nef (HIV), U21 (Herpesvirus 7), m06/gp48 (MCMV)

Proteins interact with adaptor proteins, which are

important for localizing cargo to distinct compartments

Prevention of peptide transport by TAP

e.g. ICP47 (Herpes simplex virus), US6 (human

cytomegalovirus), UL49.5 (Varicelloviruses)

Proteins bind to TAP1/2 at different sites.

Lilley & Ploegh, Immunological Reviews Vol. 207 (2005)

Strategies of immune evasion

Manipulation of endocytic pathway

Retention of MHC I complexes in the ER

e.g. E19 (Adenovirus), US3 (human cytomegalovirus)

E19 interacts with MHC I complex in ER and retains

them via a KKXX ER-retention motif

MHC I dislocation

e.g. US2, US11 (human cytomegalovirus)

Proteins block MHC I biosynthesis by catalysing the

transport of new MHCI HC to the cytosol for

proteasomal degradation

Retention of MHC I complexes in the ER-Golgi

intermediate compartment (ERGIC)

e.g. m152 (murine cytomegalovirus)

Exact mechanism is unknown

Lilley & Ploegh, Immunological Reviews Vol. 207 (2005)

Strategies of immune evasion

Manipulation of exocytotic pathway

Blocking recognition of MHC class II productse.g. gp42 (Epstein-Barr virus)Secretion of gp42, binding to HLA-DR (co-

receptor for EBV infection)

Degradation of MHC II proteinse.g. US2, US3 (human cytomegalovirus)probably catalyse the destruction of HLA-DR

and HLA-DM

Vpu targets newly synthesised CD4 in ER for proteasomal degradation.

Nef routes CD4 to lysosomes using a di-leucine-based sorting motif.

Downregulation of CD4e.g. Nef, Vpu (HIV)