1. Antigen processing and presentation
Endogenous antigen presentation pathway
Exogenous antigen presentation pathway
Microbial evasion strategies
Cross-presentation
Lipid presentation
Transcriptional regulation of MHC

2. (review) any nucleated cell professional APC
peptide size: aa aa
any nucleated cell
professional APC
Naive T cells can be activated only by professional APCs which can provide costimulation also. In response naive T cells can mature to effector cells, or they became anergic otherwise. * *
Janeway’s Imunolobiology 8th ed (© 2012 by Garland Science, Taylor & Francis Group, LLC)
(* Naive T cell activation is a topic of another lecture)

3. Some questions can emerge:
How could the peptide choose the appropriate MHC molecule? (MHC I   MHC II)
Where the peptides are generated?
Where and how can bind the peptides to the MHC molecules?
Is there a possibility that a more fit peptide could replace the MHC bound peptide on the cell surface?

4. General properties of the MHC-peptide interaction:
MHC molecules can be in a receptive, ”open” conformation until the appropriate peptides bind to them. (Appropriate peptide has appropriate binding motif, which allow effective binding to the MHC molecule  see last lecture)
The receptive conformation is maintained by the chaperones and the biochemical properties (e.g. pH) of the peptide loading compartment.
Appropriate peptide can induce the conformational change of the MHC molecules.
The bound peptide stabilizes the the ”closed” conformation
”Closed” MHC can detache from the chaperones and reach the cell surface

5. Simplistic overview of the antigen presentation pathways
CELLULAR AND MOLECULAR IMMUNOLOGY 8th ed. (Abbas, AK – Lichtman, AH – Pillai, S) (Elsevier, Saunders 2015)
Simplistic overview of the antigen presentation pathways

6. Endogenous antigen presentation pathway
antigen processing for MHC class I molecules

7. Synthesis and peptide binding of MHC I molecules
Freshly translated
Through the Golgí-network
ERAAP can trim the peptides to fit
calnexin, calreticulin – general chaperons for different proteins
endoplasmic reticulum aminopeptidase associated with antigen processing (ERAAP)
In the ER, the peptides are modified by the action of an enzyme known as the endoplasmic reticulum aminopeptidase associated with antigen processing, or ERAAP. ERAAP trims the aminoterminal of many peptides and allows peptides that are initially too long to bind to MHC molecules.
In all cells, proteasomes degrade cellular proteins that are poorly folded, damaged, or unwanted. When a cell becomes infected, pathogen-derived proteins in the cytosol are also degraded by the proteasome. Peptides are transported from the cytosol into the lumen of the endoplasmic reticulum by the protein called transporter associated with antigen processing (TAP), which is embedded in the endoplasmic reticulum membrane.
The peptides that bind to the MHC class I molecule are generated by a large protein complex, the proteasome, which is found in the cytoplasm. The proteasome degrades proteins within the cytosol of the cell to produce short peptides, which are then transported through the endoplasmic reticulum membrane by the TAP transporter.
MHC class I heavy chains assemble in the endoplasmic reticulum with the membrane-bound protein calnexin. When this complex binds β2-microglobulin (β2m) the partly folded MHC class I molecule is released from calnexin and then associates with the TAP, tapasin, calreticulin, ERp57 to form the peptide-loading complex. The MHC class I molecule is retained in the endoplasmic reticulum until it binds a peptide, which completes the folding of the molecule. The peptide:MHC class I molecule is then released from the other proteins and leaves the endoplasmic reticulum for transport to the cell surface.
Some peptides do not bind to the MHC molecule at all. Others can bind but are unstable. These are released from the MHC molecule—a process called peptide editing. Finally a peptide binds to the MHC molecule with high affinity to make a stable complex. This causes the final step in the folding of the class I molecule to take place, and the disassociation of the peptide loading complex.
Janeway’s Imunolobiology 8th ed © 2012 by Garland Science, Taylor & Francis Group
The chaperon complex-bound MHC molecule is ready for peptide binding
The best fit peptide wins the MHC:
PEPTIDE EDITING

8. The role of the chaperones
Retaining ”empty” MHC I molecules in the ER ER chaperones contain ER retention/targeting signals
Stabilizing empty MHC I molecules Empty MHC I molecules could become denatured and rapidly degraded without chaperones
Stabilized empty MHC I molecules will bind the best fit peptide The best fit peptide can displace the weakly bound peptides
PEPTID EDITING

9. Proteasomes degrade proteins to peptides
The function of the proteasome
A cell can contain ~30000 proteasomes (1% of the overall protein mass)
Proteasomes can be found in the cytosol and the nucleus
unfolding
~30000 proteasomes (Lodish: Mol Cell Biology, W. H. Freeman and Company 2012)
1%, nucleus, cytosol, proteolitical active center located inside (Molecular Biology of the Cell 5th 2008, 2002 by Bruce Alberts, Garland Science)
Unforlding and cleavages needs ATPs
peptides
(Molecular Biology of the Cell 5th 2008, 2002 by Bruce Alberts, Garland Science)
Proteasomes degrade proteins to peptides

10. Proteasomal peptide products could be tailored for MHC I binding
20S subunit of the constitutive proteasome
protease subunits
The Immune System 4th ed Parham,P
(© 2015 by Garland Science, Taylor & Francis Group, LLC)
20S – 20 Svedberg unit - sedimentation rate during ultracentrifugation (in connection with size, density, shape)
The two inner rings of the 20S proteasome core are composed of constitutively expressed proteolytic subunits called β1, β2, and β5, which form the catalytic chamber (see Fig. 6.4). These constitutive subunits are „sometimes” displaced by three alternative catalytic subunits, called LMP2 (PSMB9) and LMP7 (PSMB8), which are encoded within the MHC near TAPl and TAP2, and MECL-1, which is not encoded within the MHC.
Immunproteasome: There is increased cleavage of polypeptides after hydrophobic residues, and decreased cleavage after acidic residues. This produces peptides with carboxy-terminal residues that are preferred anchor residues for binding to most MHC class I molecules and are also the preferred structures for transport by TAP.
Glicine-Alanine repeats (GAr) can inhibit the proteolitic function of the proteasome (EBV EBNA1 contains GAr)
Proteasome prepares good C terinals. ER ERAAP shortens the peptide from the N terminal, leaving the C terminal unharmed
IFN-
The ”Immune proteasome”
new protease subunits replace the others (LMP2, LMP7, MECL-1)
produced peptides are more optimized for MHC I binding protein cleavage preference is changed: hydrophobic or basic amino acid on the C-terminal of the peptide

11. TAP complex transports the peptides into the ER
Prefered peptides:
8-16 aa length
hydrophobic or basic amino acids at the C-terminal
no proline in the first 3 positions (from the N-terminal)
The preferences of the TAP correspond to the cleavage specificity of the immune proteasome and the general binding preferences of MHC class I molecules
Proline is ”rigid”, can disrupt secondary protein structures inside the protein chain – so probably poorly fit into most of the MHC binding pockets
TAP complex has some specificity for the peptides it will transport. It prefers peptides of between 8 and 16 amino acids in length, with hydrophobic or basic residues at the carboxy terminus-the precise features of peptides that bind MHC class I molecules and has a bias against proline in the first three amino-terminal residues.
Janeway’s Imunolobiology 8th ed (© 2012 by Garland Science, Taylor & Francis Group, LLC)

12. Ne feledjük a végeredményt – MHC I  Tc
CELLULAR AND MOLECULAR IMMUNOLOGY 8th ed. (Abbas, AK – Lichtman, AH – Pillai, S) (Elsevier, Saunders 2015)
Ne feledjük a végeredményt – MHC I  Tc

13. Exogenous antigen presentation pathway
antigen processing for MHC class II molecules

14. Newly translated MHC II αβ dimers bind to Ii (invariant chain, CD74) chaperon
nonameric
complex
α-β-Ii complex
A part of the Ii chain can fit into the peptide binding site of various MHC II molecules
The bound Ii is blocking the binding site and prevents the binding of ER resident peptides
Ii have endosomal localisation signal sequence
ER chaperones help in assembling {MHCII alpha-beta + Ii} trimeric complexes (e.g. calnexin)
3 timeric complex complexed together (nonameric complex) is stable enough to detach from calnexins and leave the ER
Triple trimeric structure could help to stabilize the weak Ii-MHCII bonds (remember the pentameric IgM, and its overall avidity or the binding mechanism of FcReceptors). After the degradation of the Ii chains , the weakly bound CLIP peptides can easily detached.
MHC II – Ii complex travels through the Golgi-apparatus into the endosome
Janeway’s Imunolobiology 8th ed © 2012 by Garland Science, Taylor & Francis Group

15. The assembly of the [MHC class II molecule – exogenous peptide] complex
CLIP peptide
peptide editing
Janeway’s Imunolobiology 8th ed © 2012 by Garland Science, Taylor & Francis Group
CLIP - Class II-associated invariant chain peptide
The invariant chain prevents peptides from binding to an MHC class II molecule until it reaches the site of extracellular protein breakdown.
In the endoplasmic reticulum (ER), MHC class II α and β chains are assembled with an invariant chain that fills the peptide-binding groove; this complex is transported to the acidified vesicles of the endocytic system. The invariant chain is broken down (cathepsins), leaving a small fragment called class II-associated invariant-chain peptide (CLIP) attached in the peptide-binding site. The vesicle membrane protein HLA-DM catalyzes the release of the CLIP fragment and its replacement by a peptide derived from endocytosed antigen that has been degraded within the acidic interior of the vesicles.
HLA-DM  MHC class II region encoded monomorphic  structural (and genetic) homology with classical polymorphic MHC II proteins
proteases: e.g. cathepsins
CLIP - Class II-associated invariant chain peptide
HLA-DM helps in the peptide editing

16. The peptide binding (editing) of the MHC II molecules could take place in a multivesicular/multilamellar endo-lysosomal vesicle: named MIIC or CIIV compartment
CIIV – Class II Vesicle or
MIIC – MHC class II Compartment
Immunity, Vol. 22, 221–233, February, 2005, Copyright ©2005 by Elsevier Inc. DOI /j.immuni
multivesicular and multilamellar compartment (MHC class II-containing
Compartments (MIIC or CIIV))
DM – 10nm nanogold, DR3 – 15nm nanogold
Almost every endocytic compartment, including the two novel compartments CIIV and MIIC, is a candidate for the denomination of “MHC class II containing compartment”. In itself, the intracellular location where class II molecules accumulate may not be too relevant. The site where class II molecules are loaded with peptide, however, is. This perhaps can be best approached by establishing the localization of HLA-DM, an essential mediator in class II peptide loading
(Eur. J. Immunol : 1421–1425 )
Immuno-electron-microscopy, double immunogold labeling
HLA-DM : 10nm nanogold
HLA-DR: 15nm nanogold
The compartment of the peptide loading is indicated by the presence of HLA-DM

17. The peptid editing of HLA-DR by the help of HLA-DM in the endo-lysosomal compartment
(1)
best fit
antigenic
peptide
CLIP
HLA-DRβ
antigen derived
peptide
CLIP detachment starts in the acidic endo-lysosomal compartment
CLIP detachment can start in the acidic endo-lysosomal compartment. The empty MHC II molecules (eg. HLA-DR on the picture) rapidly denatured without the stabilizing effect of HLA-DM.
DM interacts with the alpha chain of the MHC II – one reason why the alpha chains have limited polimorphy
MHC I and MHC II peptide editings are analogous:
MHC I: A calreticulin-tapasin-Erp57 complex binds/stabilizes MHC I during peptide editing (The chaperon complex binds the N-terminal part of the MHC I α2 domain  binds to antigenic peptide C-terminal part).
MHC II: A DM binds the DRα  binds the N-terminal part of the peptide (P1 binding pocket)
DM stabilise the ”empty” DR
Wouter Pos et al.: Cell 2012, 151, 1557–1568

18. Antigen presentation pathways of exogenous and endogenous proteins
short draft summary:
(exogenous p.)
(endogenous p.) DM
CD4+
Helper
(by TAP) T Ii
Processing of antigens for presentation by MHC class II or MHC class I molecules occurs in different cellular compartments.
The left half of the figure shows the fate of peptides derived from extracellular antigens and pathogens. Extracellular material is taken up by endocytosis and phagocytosis into the vesicular system of the cell, in this case a macrophage. Proteases in these vesicles break down proteins to produce peptides that are bound by MHC class II molecules, which have been transported to the vesicles via the endoplasmic reticulum (ER) and the Golgi apparatus. The peptide:MHC class II complex is transported to the cell surface in outgoing vesicles. The right half of the figure shows the fate of peptides generated in the cytosol as a result of infection with viruses or intracytosolic bacteria. Proteins from such pathogens are broken down in the cytosol by the proteasome to peptides, which enter the ER. There the peptides are bound by MHC class I molecules. The peptide:MHC class I complex is transported to the cell surface via the Golgi apparatus. T
CD8+
CTL

19. An evasion strategy of the Epstein-Barr virus (EBV)
The schematic gene map of the EBV and the targets of the EBV specific CTL response - +
± (?) ++ ++ ++ +
LMP2
EBNA-LP
EBNA2
EBNA3A
EBNA3B
EBNA3C
EBNA1
LMP1 W W W W W W
Jhet C H F Q U P O M S L E Z R K B G D T X V I A N
Nhet W W W W W Y
BHRF1
BMLF1
BMRF1
BZLF1
BARF0 ++
Lytic antigens and EBNA3 nuclear proteins are the main targets of the polyclonal CTL response
The processing of endogenous EBNA1 is ineffective EBNA1 contains repeated Glycine-Alanine sequences which inhibits the proteasomal degradation  ineffective antigen presentation EBNA1 expression is enough to maintaining the latent viral phase  the virus is invisible for the CTL immune response in the latent phase
„Litic antigens” are labeled with red
EBNA1 stabilize the viral genom in dividing memory B cell
Letters from A to Z represents BamH1 restriction endonuclease cleavage fragments (A-largest, Z-smallest, W-lots of similar small genome fragments)
New and old EBNA nomenclature:
EBNA1, EBNA2, EBNA3A (old EBNA3), EBNA3B (old EBNA4), EBNA3C (old EBNA6), EBNA-LP (old EBNA5)

20. Microbes can use different immune evasion strategies
Janeway’s Imunolobiology 8th ed © 2012 by Garland Science, Taylor & Francis Group

21. Viral strategies against antigen presentation
(Immonoevasins)
Janeway’s Imunolobiology 8th ed © 2012 by Garland Science, Taylor & Francis Group
Pathogens try to use various mechanisms to evade the presentation of their antigens

22. ”Missing self” theory (It was mentioned on a previous lecture)
Pathogens try to evade the immune response by disabling the MHC I expression of the host cells
”Missing self” theory (It was mentioned on a previous lecture)
In the case of decreased or missing MHC I molecule expression, the cells can be targeted by NK cells
Directly
HLA-C alleles are potent NK inhibitors (ligands of KIR2DL1, KIR2DL2/3)
various HLA-A and HLA-B alleles inhibit the NK cell activation with different efficiency (ligands of KIR3DL1, KIR3DL2, KIR2DL1)
Indirectly HLA-E is a potent NK inhibitor (by NKG2A:CD94) . HLA-E cannot leave the ER without binding signal peptides from the classical polymorphic MHC I. Weak MHC I translation  weak HLA-E expression.
NK cell activation  killing the cells with ”missing self”

23. Cross-presentation:
exogenous antigen(!)  MHC I molecule(!)
Naive, viral antigen specific CD8+ T cells need activation by dendritic cells to mature CTL.
Lots of viruses are not able to infect DC, so direct MHC I presentation cannot be achieved
Specialised DC are able to present exogenous antigens by MHC I molecules
The underlying mechanisms are not fully unravelled.
FcRn can protect immuncomplexes from the complete endo-lysosomal degradation.
The engulfed antigen possibly should reach the ER by reverse transport.
ER contains lots of transport mechanisms which can transport out unsuccessfully translated/unfolded proteins into the cytosol for proteasomal degradation.
There is no direct proof that such transporters take part also in the cross-presentation steps.
Effector CTL
CELLULAR AND MOLECULAR IMMUNOLOGY 8th ed. (Abbas, AK – Lichtman, AH – Pillai, S) (Elsevier, Saunders 2015)
Endocytosed viral antigens should reach the cytosol to enter the conventional endogenous antigen presentation pathway

24. Lipid presentation by CD1 molecules (1
Lipid presentation by CD1 molecules (1.) synthesis, cell surface expression
CD1: Non MHC encoded MHC I –like molecules (”class Ib”) Their synthesis and folding is similar to conventional MHC I molecules
Lipid transfer proteins (LTP) fill the lipid binding site of the CD1 molecules in the ER
They can reach the cell surface with bound endogenous lipids.
NATURE REVIEWS IMMUNOLOGY VOLUME 5 | JUNE 2005 | 485-
Figure 4 | Intracellular trafficking of CD1 molecules. Newly synthesized CD1 molecules are transported through the Golgi
and trans-Golgi network (TGN) to reach the cell surface, with the exception of CD1e, which remains in the cell. Cell surface CD1
molecules are internalized in clathrin-coated pits and are sorted as follows. a | CD1a routes to early recycling endosomes and
back to the plasma membrane in an ADP-ribosylation factor 6 (ARF6)-dependent manner. b | CD1b, after interacting with
adaptor protein 2 (AP2), is transported to the late endosomes and, after binding to AP3, traffics to the lysosomes. c | CD1c, after
reaching the sorting endosomes, preferentially routes to the early endosomes, and to a lesser extent to the late endosomes and
lysosomes, and then recycles to the plasma membrane. d | CD1d is transported mainly to the late endosomes and only partially
to the lysosomes. Mouse, but not human, CD1d associates with AP3. A fraction of the mouse CD1d binds to the invariant chain
in the endoplasmic reticulum (ER), which escorts this molecule to the late endosomes (not shown). e | CD1e accumulates in the
Golgi stacks of immature dendritic cells, and routes to the late endosomes and the multivesicular body-enriched compartment
in mature dendritic cells, where it is cleaved and remains in a soluble form. CD1e never reaches the plasma membrane.
β2m, β2-microglobulin; MIIC, MHC class II compartment.
NATURE REVIEWS IMMUNOLOGY
VOLUME 5 | JUNE 2005 | 485-

25. Lipid presentation by CD1 molecules (2.) - recycling
They are internalized from the cell surface into the endo-lysosomal system. Endo-lysosomal LTPs mediate the exchange of the bound lipids - ”lipid editing”
Different type of CD1 (a, b, c, d) can reach different compartments of the endo-lysosomal system. There they are loaded with different lipids for presentation accordingly.
NATURE REVIEWS IMMUNOLOGY VOLUME 5 | JUNE 2005 | 485-
Figure 4 | Intracellular trafficking of CD1 molecules. Newly synthesized CD1 molecules are transported through the Golgi
and trans-Golgi network (TGN) to reach the cell surface, with the exception of CD1e, which remains in the cell. Cell surface CD1
molecules are internalized in clathrin-coated pits and are sorted as follows. a | CD1a routes to early recycling endosomes and
back to the plasma membrane in an ADP-ribosylation factor 6 (ARF6)-dependent manner. b | CD1b, after interacting with
adaptor protein 2 (AP2), is transported to the late endosomes and, after binding to AP3, traffics to the lysosomes. c | CD1c, after
reaching the sorting endosomes, preferentially routes to the early endosomes, and to a lesser extent to the late endosomes and
lysosomes, and then recycles to the plasma membrane. d | CD1d is transported mainly to the late endosomes and only partially
to the lysosomes. Mouse, but not human, CD1d associates with AP3. A fraction of the mouse CD1d binds to the invariant chain
in the endoplasmic reticulum (ER), which escorts this molecule to the late endosomes (not shown). e | CD1e accumulates in the
Golgi stacks of immature dendritic cells, and routes to the late endosomes and the multivesicular body-enriched compartment
in mature dendritic cells, where it is cleaved and remains in a soluble form. CD1e never reaches the plasma membrane.
β2m, β2-microglobulin; MIIC, MHC class II compartment.
CD1e probably takes part in the lipid editing or as chaperone for the CD1b, c, d
NATURE REVIEWS IMMUNOLOGY
VOLUME 5 | JUNE 2005 | 485-
- CD1 molecules can present both endogenous,
- and engulfed exogenous lipids also

26. Transcriptional regulation of MHC Class I and Class II molecules
IFNγ increases the expression of MHC molecules
CIITA coactivator play central role in the coordinated regulation of MHC expression (MHC I, TAP, LMP, MHC II, HLA-DM) – increasing both the expression of the antigen presenting-, and the antigen processing molecules
Proinflammatory cytokines, TLRs can have additional effect (NFκB pathway)

27. MHC II expression of human monocyte derived dendritic cells
- before the activation
- after the activation
with TLR7/8 ligands
The histograms below show the results of the flow cytometric measurements
~10 fold
difference
activation: 1μg/ml CL075 (TLR7/8 ligand) overnight
the vertical axis indicates the cell numbers
the horizontal axis indicate each measured cells’ the fluorescence intensities corresponding the relative quantity of surface MHC II (HLA-DR) molecules detected by fluorescent dye (FITC) coupled mouse monoclonal antibodies recognizing the human HLA-DR molecules
non-activated
activated

28. ANTIGEN PROCESSING AND PRESENTATION
short summary
(endogenous Ag.)
(exogenous Ag.)
8-10aa
10-20aa
(CTL)
(helper)
(CIIV/MIIC)
CELLULAR AND MOLECULAR IMMUNOLOGY 8th ed. (Abbas, AK – Lichtman, AH – Pillai, S) (Elsevier, Saunders 2015)

29. Themes and topics (you should know): various terms (you should know):
Endogenous antigen presentation pathway
Exogenous antigen presentation pathway
Cross-presentation
Lipid presentation
Transcriptional regulation of MHC
protein, peptide
cellular compartments
MHC I, MHC II molekulák
proteasome, immunoproteasome
TAP (1, 2)
chaperon
tapasin
signal sequence/peptide
protein targeting/sorting signals
Ii (invariant chain), CLIP
HLA-DM
endosome, MIIC/CIIV
peptide editing
cross-presentation
”missing-self” theory
lipid transport proteins (LTP)
lipid editing
CIITA
The Immune System (P. Parham, 4th ed): chapter 5-10 – 5-17 (p ), – (p ), 5-20 (p138)
Basic Immunology (Abbas, 4th ed): chapter 3: p58-68, , p289 (missing: lipid presentation, IFN-γ – MHC expression signalling)