Transcript Document

MEDSCI 708
Advanced Immunology and Immunotherapy
2007
Antigen processing and presentation
Antigen processing:
Proteolytic cleavage of proteins into small fragments
(antigen peptides) that can bind to MHC molecules
on antigen presenting cells.
Antigen presentation:
Presentation of antigen peptides to T cell receptor
on T cells
MHC restriction
The T cell receptor will recognise a peptide only
when it is bound to a particular MHC molecule.
1973: Peter Doherty and Rolf Zinkernagel (Nobel Prize in Medicine, 1996)
MHC: Major Histocompatibility Complex
encoded by the most polymorphic gene cluster on the
human genome (many alleles)
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
Antigen presenting pathways
exogenous pathway
endogenous pathway
B cell help
CD4 T
CD8 T
TcR
MHC I
CD8
CTL
kill
ab
TcR
CD4
MHC II
pathogen
virus-infected or
tumor cell
MHC I restriction
Macrophage or other
professional APC
MHC II restriction
Class I and Class II
pathways of antigen
presentation
From
Immunology 5th ed. (Roitt et al.)
MHC I peptides: 8-11 aa
with 2 terminal anchor residues
MHCII peptides: 8-30+ aa with
anchors throughout the peptide
MHCI and MHCII peptides are very
different and have to be generated
by different mechanisms
Many different peptides have
to be presented (different anchors)
In human:
2x3 MHCI molecules
1245 known alleles
Two dis tinct 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 -1 0 amino acids
Any length but usually 8 -3 0 aa
T cell respo nse
Cyt otox ic CD8 T cells
Helper CD4 T cells
Out come
Inf ected cell is killed
T cell, B-cell and macro phage are activ ated
Ig isot y pe swit ching occ urs
Ty pe of
inf ection
predominant ly v iral
predominant ly bacterial, parasit ic and t oxins
Pathway
Endogenous
Exogenous
MHC class I antigen processing
Groothuis et al., Immunological Reviews Vol. 207 (2005)
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
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
Kloetzel and Ossendorp, Curr. Opin. Immunol. 16 (2004)
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
The Ubiquitin Pathway
Ub-conjugating enzyme
Ub
E1
Ub-ligase
Ub
Ub
E2
E3
Ub-activating enzyme
Target
Ub
Ub
Ub
Ub
26s proteosome degradation
Binding of poly-ubiquitin chains to 19S proteasome
Elongates
Ub tree
Groothuis et al., Immunological Reviews Vol. 207 (2005)
Immunoproteasome
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
From Strehl et al., Immunological Reviews Vol.207, pp 19-30 (2005)
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
Immunoproteasomes affect the size of the antigenic peptide pool
From Strehl et al., Immunological Reviews Vol.207, pp 19-30 (2005)
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
AMC: aminoacyl-aminomethyl cumarin
= artificial substrate that becomes
fluorescent after removal of N-terminus
Saveanu et al., Immunological Reviews Vol. 207 (2005)
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
Cytosolic chaperones:
- 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
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
Processing of the SIINFEKL epitope
BiP
PDI
translocon
Shastri et al., Immunological Reviews Vol. 207 (2005)
Transporter Associated with Antigen Presentation (TAP)
8-12 aa
(up to 40aa with low efficiency)
ER retention signal
Transmembrane domain
2 ATP-binding cassettes
Peptide-binding domain
http://www.cryst.bbk.ac.uk/pps97/assignments/projects/coadwell/003.htm
Assembly of MHC class I and peptide loading complex
peptide loading complex
HC: heavy chain, 3 -subunits, 45 kDa glycoprotein, polymorphic
2m: 2-microglobulin, 12 Kda, non-polymorphic
CNX: calnexin, membrane-anchored chaperone, stabilises nascent HC,lectin-like
CRT: calreticulin, soluble lectin-like chaperone, binds N-linked glycan on HC
Erp57: oxido-reductase, binds Tapasin via S-S bonds and non-covalently to CRT
Cresswell et al., Immunological Reviews V0l. 207 (2005)
Peptide binding cycle
Cresswell et al., Immunological Reviews V0l. 207 (2005)
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)
Quality control by tapasin
Brocke et al. (2002)
“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)
4 domains:
• 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).
4 functions:
• 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.
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
Hsing & Rudensky, Immunological Reviews Vol 207 (2005)
Note: within the same compartment, proteases also generate peptide
fragments derived from endocytosed antigens
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. Wedgeshaped 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
Possible role for HLA-DO in B cells
Brocke et al., Curr. Opin. Immunol. Vol. 14 (2002)
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 as
CD86, 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
Strategies of immune evasion
Manipulation of endocytic pathway
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
Interference with T cell receptor recognition
e.g. m04/gp34 (murine cytomegalovirus)
Protein binds to MHCI in the ER, sterical inhibition
of TcR binding
Prevention of peptide transport by TAP
Lilley & Ploegh, Immunological Reviews Vol. 207 (2005)
e.g. ICP47 (Herpes simplex virus), US6 (human
cytomegalovirus), UL49.5 (Varicelloviruses)
Proteins bind to TAP1/2 at different sites.
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
Retention of MHC I complexes in the ER-Golgi
intermediate compartment (ERGIC)
e.g. m152 (murine cytomegalovirus)
Exact mechanism is unknown
MHC I dislocation
e.g. US2, US11 (human cytomegalovirus)
Proteins block MHC I biosynthesis by catalysing the
Lilley & Ploegh, Immunological Reviews Vol. 207 (2005)
transport of new MHCI HC to the cytosol for
proteasomal degradation
Strategies of immune evasion
Manipulation of exocytotic pathway
Blocking recognition of MHC class II products
e.g. gp42 (Epstein-Barr virus)
Secretion of gp42, binding to HLA-DR (coreceptor for EBV infection)
Degradation of MHC II proteins
e.g. US2, US3 (human cytomegalovirus)
probably catalyse the destruction of HLA-DR
and HLA-DM
Downregulation of CD4
e.g. Nef, Vpu (HIV)
Nef routes CD4 to lysosomes using a dileucine-based sorting motif.
Vpu targets newly synthesised CD4 in ER
for proteasomal degradation.
Lilley & Ploegh, Immunological Reviews Vol. 207 (2005)