lecture outline mhc i ag presentation mhc ii ag ...€¦ · what cells express mhc class ii...
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Advanced Course in Basic & Clinical Immunology
February 19, 2018
1
Antigen presentation
Kenneth L. Rock, M.D.
Professor & Chair
Department of Pathology
UMass Medical School
Lecture outline
MHC I Ag presentation
MHC II Ag presentation
Antigen presenting cells (brief)
Advanced Course in Basic & Clinical Immunology
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CD8 T lymphocyte
How do theyrecognize thathese cells ar
abnormal?
The principal adaptive immune defense against cancersvirally infected cells
MHC class I molecule
Advanced Course in Basic & Clinical Immunology
February 19, 2018
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Genes
Virus
ERMHC I
Nucleus
MHC class I antigen presentation
TAP
Parham
The MHC I antigen binding receptor
Parham
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From Kuby MHC class I genes
MHC class I genes are highly polymorphic
Now >10,000 alleles of MHC class I genes identified!!
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Nomenclature
(defined by serology)
(defined by DNA sequence)
From Parham – The Immune System
Where are the polymorphic residues?
Class I & II genes are highly polymorphic
What will the polymorphisms affect?
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Clinical importance of MHC polymorphism
Transplant rejection
Susceptibility to infectious disease (e.g. HIV elite controller)
Susceptibility to autoimmune disease
Responses to vaccines & immunotherapy
GWAS HIV elite controlers
The International HIV Controllers StudyScience 2010
Vol. 330 pp. 1551-1557
MHC I alleles
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How many different MHC I molecules can cells express?
H2N C C N C COOH
R R’
HH O
Common
Unique
Peptide Structure
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Peptides bound to MHC molecules
1 2 3 4 5 6 7 8 9N COOH
Side chains not bound
side chains bound by MHC
moleculeFixed (few) amino acids
Can be any of the 20 amino acids
Therefore, a single MHC I molecule can bind huge numbers of peptides (but not all)
With 6 different MHC I molecules, can “cover” much of the antigenic universe
So, how do we get from proteins to
presented peptides
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Parham
MHC class I pathway utilizes the peptides generated from the normal catabolism of
cytosolic & nuclear proteins
ProteinProteolysis by Proteasomes
Active sites
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0
50
100
150
CP
M X
10-
3
0.3 0.6 1.25 2.5 5 10
Vaccinia - OVA
Inhibitor0
50
100
150
0.3 0.6 1.25 2.5 5 10
Vaccinia - SIINFEKL
APC X 10-4
Inhibitor
Effect of proteasome inhibitors on antigen presentation
MH
C I
antig
en p
rese
ntat
ion
Rock et. Al. 1994. Cell 78: 761-771
Immune system modificationof proteolysis
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ImmunoproteasomesThe picture can't be displayed.
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Antigen
Immunoproteasome
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IFNMHC
1i
immunoproteasome ß-subunits
5i
Non-MHC
2i
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Proteasome (constitutive)
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5
2
Different cleavages result in different peptides
1
[Also a 5t subunit expressed only in the thymus]
Proc. Natl. Acad. Sci. USA 91: 9212-9217
How important are immunoproteasomes?
1i/2i/5i-/-
Immunoproteasome knock out
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H-2Db H-2Kb
Peptides eluded from MHC I
Wt Immuno-Proteasome-/-
Wt Immuno-Proteasome
50% qualitatively different!
Conclusion
The proteasome is required to generate the majority of presented peptides
The immune system evolved modifications of proteasomes to optimize antigen
presentation.
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How do class I molecules access cytosolic peptides?
ass I molecules access cytosolic peptides? Cytosol
ER
ER membrane
Peptide
1i TAP1 5i TAP2
MHC CHROMOSOME 6
MHC I
TAPCytosol-to-ER peptide transporter
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Other events in the ER
MHC I
Chaperones-calnexin-ERP57
Tapasin
TAP
PeptideRibosomes
To cell surfaceEndoplasmic reticulum
MHC I
Tapasin
Encoded in MHC
Thought to play a role of retaining “empty” MHC I in the ER
Also promotes peptide loading of MHC I molecules
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H2-Kb 8mers (some 9mers)
HLA-A,B, C 9-10mers
Size of peptides bound by MHC I molecules
Does the proteasome generate presented peptides in their final
form?
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What is the size of proteasome products?
Cascio et al., EMBO J. 20:2357-66 (2001)Kisselev et al., J Biol Chem 274:3363-71 (1999)
Too short Too longJust right
MHC binding
Are long peptide fragments precursors for epitopes?
epitopeN COOH
Presented
epitope
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Cleavages to generate MHC I peptides
epitopeN COOH
PresentedOnly
ProteasomeOther
peptidase
Summary of key points:• To generate peptides from expressed genes the
immune system has utilized the normal cellular pathway that degrades proteins
• The initial cleavages are made by proteasomes and especially immunoproteasomes, often making long antigenic precursors.
• Most peptides are generated in the cytoplasm
• The correct C-terminal cleavage is ~only made by proteasomes
• Other peptidases can trim the N-terminus of peptides
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N-terminal trimming of peptides
Much of the trimming occurs in the ER
By the aminopeptidase = ERAP1/ERAAP
(note humans but not mice also have an ERAP2)
Importance of ERAP1 in vivo
ERAP1 “knock out”
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*
**
*
*
LCMV immunodominance hierarchy in ERAP1-/- mice
0
1
2
3
4
5
6
7
8
Per
cent
of T
cel
l res
pons
eERAP1 -/-Wild type
Proc. Natl. Acad. Sci. USA. 103: 9202-7
Humans
ERAP1 polymorphisms linked to autoimmune diseases and immune
responses.
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ERAP1 unique-trims with a molecular ruler
EPITOPE
11 10 9 8
EPITOPE
ER
ERAP1
Nature Immunology, 3: 1169-1176
Leu-AMC
Active site
ERAP1 shows allosteric activationAMC
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Leu-AMC
+Active site
ERAP1 shows allosteric activation
Peptides ≤7 AA AMC
Leu-AMC
+Active site
ERAP1 shows allosteric activation
Peptides ≤7 AA
Leu-AMC
+Active site
Peptides ≥8 AA
AMC
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Crystal Structure
Reorientation of Tyr438
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Crystal structure
trigger
Cytosol trim
cleave
Crystal structure
trigger
Cytosol trim
cleave
X
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Summary
Influences responses
ER Trimming has specificity
N-extended peptides are trimmed in the ER by ERAP1
ERAP1 trims with a molecular ruler
1/2
Source proteins for MHC I presentation
Immunoribosomes?
Turn over of the proteome
Products of protein splicing
Defective proteins (DRIPs)?
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Antigen presentation is a “bell & whistle”
MHC I molecules, tapasin, TAP transporter, immunoproteasomes, ERAP1
are not required for cell viability
Microbial immune evasion
PeptideCMV
CMVHSV
EBVAntigen
Endoplasmic reticulum
TAP
Proteasome
MHC I
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Cancer immune evasion
This will be a barrier to cancer immunotherapy
Many cancers are genetically unstable
Can lose expression of cancer antigens, MHC I, Tapasin, TAP
Variable for precision medicine?
E
Summary of Pathway
ERAP
TAPASIN
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MHC II antigen presentation
Macrophage
Microbe
Phagocytosis
Destroy microbe
Infection
Extracellular microbe
Outcomes of bacterial infection
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The major immune defense against cells infected with bacteria are CD4 T
cells
CD4 Tcell
MHC II sample peptides in endocytic compartments
MHC II molecule
ExtracellularAntigens
Microbe
Endocytic uptake
CD4+TH
Vacuoles
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MHC II peptide-binding receptors
Very similar tertiary structure to MHC I
3 molecules = HLA-DR, DP, DQ
Same regions highly polymorphic
Parham
MHC II monitors peptides from phagosomes (& endocytic compartments)
How are these generated?
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February 19, 2018
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The endocytic compartment is also catabolic.
H+ H+
Lysosome
Inactiveprotease
Antigen
Phagosome
Activeprotease
Proton Pump
H+
Peptides
How do class II molecules get to phagosoms/endosomes to sample
these compartments
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Synthesis & assembly of MHC class II molecules
MHC IIInvariant chain
Ribosomes
Endoplasmic reticulum
Clip region
How is class II prevented from being saturated with peptides in the ER & then get to the right compartments?
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How is class II prevented from being saturated with peptides in the ER & then
get to the right compartments?
Invariant chain
Clip region blocks the groove
Sorting sequence to endosomes
Key points• Invariant chain binds newly synthesized class II in the ER
• Invariant chain blocks peptide binding to class II
• Invariant chain directs class II molecules to endocytic compartments
• Peptides are generated in endosomalcompartments by acid optimal proteases
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How are class II molecules activated in endosomal vesicles?
HLA-DM
H+
Endosomal Vacuole (MIIC)
CLIP
Antigen
To cell surface
DM promotes loading/editing of antigenic peptide within antigen presenting cells
DM MHC II
Affinity
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DM
DM
DR DRpeptide
DM-MHCII interaction(from crystal structure of a trapped complex)
Model for DM-dependent peptide exchange
2
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Key Points• Invariant chain is hydrolyzed by proteases in the endocytic compartment
• A fragment of invariant chain (CLIP) is left in the peptide binding groove
• CLIP is removed by HLA-DM
• Peptides bind to class II molecules (facilitated by HLA-DM)
What cells express MHC class II molecules?
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What cells express MHC class II molecules?
All nucleated cells
EffectorCD8+CTL
Dendritic cell Macrophage B cell
MHC I
EffectorCD4+CTL
MHC II
These are precisely the cells that CD4 T cells need to monitor and activate
B cells & macrophages, DCsAll can take up antigen through endocytosis and
present via the mechanisms we’ve discussed so far.
In addition, some have specializations:
MØ & immature DCs can acquire Ag through phagocytosis (important for microbes & cell Ags)
B cells can capture Ag through their surface antibody.This is important for antigen specific B cells to acquire antigen, process it & present peptides to T helper cells
DCs can transfer eaten antigens into the MHC I pathway (called cross presentation)
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Dendritic cells are key antigen presenting cells (APCs) for initiating T cell responses
Why?
The initial frequency of any anti-viral T cell is very low
AntigenX
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The “where’s Waldo” problem
The “where’s Waldo” problem
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Huge territory
How can this work?
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CCR7CCR7
scoutsFort
dendritic cells Lymph node
“Wild west” strategy
Dendritic Cells- Key APCsAnatomic location – located where the antigens are
(mucosa & all tissues)
Have receptors that sense danger (infection & injury) & stimulate the DC
(pattern recognition & cytokine receptors)
When stimulated, DCs express all the signals needed to activate naïve T cells (Ag, costim., cytokines)
Trafficking: Tissues(CCR7) L. NodesT cells(naïve T cells don’t traffic through tissues)
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Implications for cancer
The cells that initially present cancer antigens (dendritic cells) have immunoproteasomes
In the absence of inflammation, cancer cells may lack immunoproteasomes
Therefore, the T cells that are elicited might not optimally recognize the cancer
END