1. T – CELL EFFECTOR FUNCTIONS
ANTIGEN PRESENTATION
T – CELL RECOGNITION
T – CELL ACTIVATION
T – CELL EFFECTOR FUNCTIONS

2. CLP T B Th CTL PC Lymphocyte subsets Common lymphoid precursor T CELLS
B CELLS
Activate B cells
and macrophages
T HELPER CELLS Th
Kill virus-
infected cells
CYTOTOXIC T
LYMPHOCYTES
CTL
Produce antibodies
PLASMA CELLS PC

3. RECOGNITION EFFECTOR CELL
Plasma cell
Antibody production
B-lymphocyte
cytokines
BCR + antigen
cytokines
Cytotoxic T-limfocyte (Tc)
TCR + peptide + MHC-I
Cell killing
Effector cell retains specific receptor
Effector cells secrete cytokines
Helper T-lymphocyte (Th)
TCR + peptide + MHC-II
Macrophage activation
Lymphocyte activation
Inflammation

4. RECOGNITION OF EXOGENOUS AND ENDOGENOUS ANTIGENES BY T-LYMPHOCYTES
Peptides of endogenous proteins (virus, tumor) bind to class I MHC molecules Tc Th
Peptides of exogenous proteins (toxin, bacteria, allergen) bind to class II MHC molecules
Exogenous Ag
Endogenous Ag

5. The number of different T cell antigen receptors is estimated to be
1,000,000,000,000,000 ( )
How can 6 invariant molecules have the capacity to
bind to 1,000,000,000,000,000 different peptides?

6. A flexible binding site?
A binding site that is flexible enough to bind any peptide?
At the cell surface, such a binding site would be unable to
• allow a high enough binding affinity to form a trimolecular complex with the T cell antigen receptor
• prevent exchange of the peptide with others in the extracellular milieu

7. A flexible binding site?
A binding site that is flexible at an early, intracellular stage of maturation formed by folding the MHC molecules around the peptide.
Venus fly trap
Floppy
Compact
Allows a single type of MHC molecule to
• bind many different peptides
• bind peptides with high affinity
• form stable complexes at the cell surface
• Export only molecules that have captured a peptide to the cell surface

8. WHERE PEPTIDE BINDING OCCURS?
MHC molecules
Adopt a flexible “floppy” conformation until a peptide binds
Fold around the peptide to increase stability of the complex
The captured peptides contribute to the stabilization of the complex
Use a small number of anchor residues to tether the peptide
- this allows different sequences between anchors
and different lengths of peptides
WHERE PEPTIDE BINDING OCCURS?

9. INTRACELLULAR COMPARTMENTS ISOLATED BY MEMBRANSE
1) cytosol 2) vesicular system
One compartment is the cytosol, which is contiguous with the nucleus via the pores in the nuclear membrane. The other compartment is the vesicular system, which consists of the endoplasmic reticulum, the Golgi apparatus, endocytic vesicles, lysosomes, and other intracellular vesicles. The vesicular system is effectively contiguous with the extracellular fluid. Secretory vesicles bud off from the endoplasmic reticulum and, by successive fusion and budding with the Golgi membranes, move vesicular contents out of the cell. In the reverse direction, endocytic vesicles formed from infolding of the plasma membrane take up extracellular material into the vesicular system. ÜLSŐ KÖRNYEZET VAÁLTOZÁSAI EGYARÁNT VESZÉLYESEK LEHETNEK…… FIGYELNI KELLL RÁJUK
A BELSŐ ÉS A K

10. THE ENDOGENOUS ANTIGEN PROCESSING PATHWAY
Tc-cell
MHC-I +
self peptide
MHC-I +
Ag peptide
α-chain+β2m
MHC+peptide
cytoplasm
FLEXIBLE
CLOSED
MHC-I, LMP2/7, TAP
IFN induced coordinated expression
α-chain
TAP1/2
gp96 calnexin
PROTEIN
SELF
ANTIGEN
Proteasome
LMP2/LMP7

11. CYTOSOL-DERIVED PEPTIDES ARE PRESENTED BY MHC-I FOR T-CELLS
The folding and assembly of MHC class I molecules takes place in the lumen of the endoplasmic reticulum. The initial folding of the class I heavy, or α, chain is aided by the chaperone, calnexin. The partially folded chain is transferred to a second chaperone, calreticulin, which aids the further folding of the chain and the association of β-2-microglobulin. Other proteins, Erp57 and tapasin, associate with the nascent class I molecule which binds to the TAP transporter via tapasin to form a peptide loading complex. 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. In the ER, these 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. 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. The peptide loaded MHC class I molecule is now free to exit the endoplasmic reticulum and be transported, via the Golgi apparatus, to the cell surface, where it can be recognized by the antigen receptors of CD8 T cells.

12. Degradation of endogenous proteins in
(immune) proteasomes
TAP: Transporter associated
with antigen processing
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.

13. PEPTIDE-MHC INTERACTION IS SUPPORTED BY MULTIPLE PROTEINS IN THE ENDOPLASMIC RETICULUM
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, and protein disulfide isomerase (PDI) 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.

14. TRIMMING OF PEPTIDES FOR OPTIMAL SIZE BY BY ERAP
The endoplasmic reticulum aminopeptidase (ERAP) binds to MHC class I molecules in which the amino terminus of an overlong peptide hangs out of the binding site. It removes the accessible amino acid residues to leave a peptide of 8-10 amino acids with an improved fit to the heterodimer of the class I heavy chain and β2-microglobulin. It is not known whether ERAP acts on the class I molecule alone, as illustrated here, or when it is part of the peptide-loading complex, or both. Discovered by Tomo Saric and Goldberg.

15. Transporters associated with antigen processing (TAP1 & 2)
ER membrane
Lumen of ER
Cytosol
ER membrane
Lumen of ER
Cytosol
ATP-binding cassette
(ABC) domain
Hydrophobic
transmembrane
domain
Peptide antigens
from proteasome
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
Transporter has preference for longer than 8 amino acid peptides
with hydrophobic C termini.

16. Maturation and loading of MHC class I
Endoplasmic reticulum
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
Calnexin binds
to nascent
class I chain
until 2-M binds
B2-M binds and stabilises floppy MHC
Tapasin, calreticulin, TAP 1 & 2 form a complex with the floppy MHC
Cytoplasmic peptides are loaded onto the MHC molecule and the structure becomes compact

17. THE EXOGENOUS ANTIGEN PROCESSING PATHWAY
Th-cell
MHC-II +
Ag peptide
self peptide
INVARIANT CHAIN (Ii)
Chaperone – conformation
Inhibition of peptide binding
Transport/retention
CLOSED
FLEXIBLE
Ii+αβ
CLIP
DMA/B
DMA/DMB
1. Support the peptide receptive conformation
2. Exchange of CLIP for exogenous peptides

19. THE INVARIANT CHAIN PROTECTS THE MHC CLASS II BINDING SITE UNTIL REACHING THE APPROPRIATE COMPARTMENT
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, 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.
INVARIANT CHAIN LÁNC (Ii)
Chaperon – Conformation
Inhibition of the peptide binding site
Transport and retention
DMA/DMB
1. Stabilization of peptide accessable conformation
2. Exchange of CLIP to peptides derived from exogenous proteins

20. GENERATION OF MHC – I EPITOPES
Viral protein
GENERATION OF MHC – II EPITOPES
HLA-DR1/HLA-DR4
B27 A2
B35
C42
HLA-A,B,C binding
Overlapping peptides
HLA-DQ2/HLA-DQ7
The Tc response is focused to few epitopes
ENSURE RECOGNITION OF ANY PATHOGENIC PROTEIN
The Th response is directed to overlapping epitopes
ENSURE RECOGNITION OF ALL PROTEINS

21. TARGETS OF EPSTEIN-BARR VIRUS-SPECIFIC Tc (CTL) RESPONSES
LATENT ANTIGENS +
± (?) ++ -
EBNA3
EBNA4
EBNA6
LMP2
EBNA5
EBNA2
EBNA1
LMP1 W W W W W W N h e t C H F Q U P O M S L E Z R K B G D T X V I A N h e t W W W W W Y
LYTIC ANTIGENS
BHRF1
BMLF1
BMRF1
BZLF1
BARF0
A poliklonális CTL válasz elsősorban a litikus antigének és az
EBNA3,4,6 nukleáris fehérjék ellen irányul
Erősen fókuszált egy adott MHC - peptid kombinációra
Az endogén EBNA1 nem processzálódik és így nem ismerhető fel

22. MHC class I MHC class II Bound peptide Generation of peptide
source
self or foreign proteins
size
8-10 amino acids
13-21 amino acids
heterogenity
limited
overlapping set of peptides
natural
cytoplasmic and nuclear proteins
~70% MHC derived,
membrane and extracellular proteins
Generation of peptide
site
cytoplasm
vesicles, endo/lysosomes
enzyme
proteasome
LMP-2, LMP-7 regulatory subunits
vesicular acidic proteases
cathepsins
transport
TAP - size and C-terminal dependent
cytoplasm  ER no
MHC transport
Ii - target, retention
ER  vesicular system
special compartment
MHC - peptide interaction ER
vesicles, CIIV
chaperons
calnexin, toposin
Ii - CLIP, DMA/B
MHC - peptide complexes In the cell surface
stable complexes reflecting the endogenous environment of the cell
few instable empty molecules
stable complexes reflecting the exogenous/endogenous environment of the cell
few re-circulating molecules complexed with CLIP

23. REGULATION OF CLASS I AND II MHC MOLECULES
IFNγ
IFNγR
Type II immune IFNγ increases MHC expression
Inflammatory cytokines and IFNγ induces MHC class II expression in certain tissue cells (endothelial, astrocyte, microglia)
Co-ordinated upregulation of MHC-I, TAP, LMP and MHC-II, DM, Ii

24. B-cell T-cell Appearance of antigen Nature of the antigén Ligand
Soluble, particles
Any cell surface molecule
Cells carrying self MHC-peptide complexes
Nature of the antigén
Natíve proteins,
carbohydrate, lipids, metals, any structure
Processed protein fragments = peptides
Ligand
Conformational determinant
Ssequential determinant
MHC-peptide complex
Antigen recognizing receptor on the cell surface
Variable BCR
ligand (antigen) – spcific
bivalent
Variable TCR
MHC + peptide pecific
monovalent
Soluble antigen recognizing receptor
antibody -
Collaboration of other cells
Antigen processing and presenting cells
APC – interaction of two cells
Antigen processing, presentation
Intracellular enzymatic degradation, peptide or MHC transportation
Result of full activation
Production of effector molecule antibody = soluble BCR
Activation of new genes
Activation molecules, production of lymphokines, TCR on the cell surface
Possibililties of cell activation
FULL
plasma cell, antibody
PARTIAL
funcional anergy
APOPTOSIS
Various lymphokines
functional anergy
certain lymphokines
Co-receptors
CD19, CD21,
CD4, CD8, CD28/CTLA4, CD2, CD38