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

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

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

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

The number of different T cell antigen receptors is estimated to be 1,000,000,000,000,000 (1015 - 17) How can 6 invariant molecules have the capacity to bind to 1,000,000,000,000,000 different peptides?

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

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

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?

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

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

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.

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.

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.

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.

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.

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

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

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

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

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

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

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

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