1. ANTIGEN RECOGNITION BY T-CELLS REQUIRES PEPTIDE ANTIGENS AND ANTIGEN PRESENTING CELLS THAT EXPRESS MHC MOLECULES Y T No T-cell response soluble Ag Native membrane Ag Peptide antigen Cell surface MHC- peptide complex T-cell response Cell surface peptides APC

2. REQUIREMENTS FOR ANTIGEN PRESENTATION 1.Expression of MHC molecules 2.ANTIGEN a) Synthesis of viral antigens - intracellular b) Uptake of protein antigens – extracellular 3. „Processing” of antigen generation of peptides suitable for T-cell recognition 4. Presentation of peptides in complex with MHC molecules on the cell surface T-cells with  TCR are specialized for recognizing protein – derived fragments

3. THE STRUCTURE OF MHC

4. MEMBERS OF THE IMMUNOGLOBULIN SUPERGENE FAMILY FUNCTION RECOGNITION Ig, TCR, MHC-I, MHC-II ADHESION ICAM-1, ICAM-2, VCAM-1, NCAM BINDING CD4, CD8, CD28, B7, IL-1RI, PDGFR, Fc  RII, poly-IgR MOLECULES CONTAINING ONE OR MORE Ig DOMAIN(S) V or C domain related

5. There are two types of MHC molecule, MHC class I and MHC class II.

6. 3,838,986 bp 224 gene 6 kromoszóma http://webace.sanger.ac.uk/cgi-bin/ace/pic/6ace?name=MHC&class=Map&click=400-1 MHC sequencing consortium Nature 401, 1999 The full sequence and the map of the human MHC locus HUMAN GENOME PROJECT

7. Genes and proteins of MHC

8. AZ MHC restriction TCR/ MHC + peptid complex recognized A single TCR recognize a single MHC-peptid komplex The same peptide presented on a different MHC is not recognized. The same MHC molecule with a different peptide is not recognized by a given TCR (other TCRs may recognize)

9. MHC class I and MHC class II molecules bind to different T-cell co-receptors.

10. The structure of MHC proteins

11. THE PEPTIDE BINDING SITE OF MHC CLASS I MOLECULES

12. THE PEPTIDE BINDING SITE OF MHC CLASS II MOLECULES PEPTIDE

13. MHC class I accommodate peptides of 8-10 amino acids Cleft geometry MHC class II accommodate peptides of >13 amino acids  -M  -chain Peptide  -chain  -chain Peptide

15. 11 33 22 2m2m 22 11 22 11 Allelic polymorphism is concentrated in the peptide antigen binding site Polymorphism in the MHC affects peptide antigen binding Allelic variants may differ by 20 amino acids Class II (HLA-DR) Class I

17. DPB1*01011 TAC GCG CGC TTC GAC AGC GAC GTG GGG GAG TTC CGG GCG GTG ACG GAG CTG GGG CGG CCT GCT GCG GAG TAC TGG AAC AGC CAG AAG GAC ATC CTG GAG GAG DPB1*01012 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*02012 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*02013 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AC -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0202 CT- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0301 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*0401 -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0402 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0501 CT- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0601 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*0801 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*0901 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- --- --- --- --- DPB1*1001 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*11011 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- DPB1*11012 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- DPB1*1301 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*1401 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*1501 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- C-- --- --- --- DPB1*1601 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*1701 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- --- --- --- --- DPB1*1801 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*1901 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*20011 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*20012 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --C --- --- --- --- --- --- --- C-- --- --- --- DPB1*2101 CT- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*2201 CT- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*2301 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*2401 -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -AG --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*2501 -T- -T- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- -A- -A- --- --- --- --- --- --- --- --- C-- --- --- --- DPB1*26011 --- --- --- --- --- --- --- --- --A --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- DPB1*26012 --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- 30 HLA-DP  allele sequences between Nucleotides 204 and 290 (amino acids 35-68) Most polymorphisms are point mutations Y-F A-V Silent A-D A-E E-A I-L Polymorphic nucleotides encode amino acids associated with the peptide binding site

18. Klasszikus MHC gének POLIMORPHIC HLA – Human Leukocyte Antigen system HLA –A,B, C I osztály ALL NUCLEATED CELLLS HLA – DR, DP, DQClass II ON PROFESSIONAL APC Non- classical MHC genes E, G, F 6 kromoszóma rövid karjaMHC 15 kromoszóma  2m STRUCTURE OF THE MHC Class III

19. CELL SURFACE EXPRESSION OF MHC ON VARIOUS CELL TYPES Szövet MHC I MHC II T cells+++ +/- B cells+++ +++ Makrophages+++ ++ Dendritic cells +++ +++ Epithelial cells + +++ Neutrophyls+++ - Hepatocytes + - Kidney + - Brain + - Eritrocytes -- Cell surface expression of MHC is influenced by activation MHC class I molecules are important in immune responses agains viruses and tumour cells MHC class II plays a role in the activation of immunocytes and in the regulation of cell cell cooperation

20. INHERITENCE OF CLASS I AND CLASS II MHC GENES HUMAN LEUKOCYTE ANTIGEN HLA EVERY CELL α1β1α1β1α2β2α2β2 PROFESSIONAL APC I osztály II osztály Ko-domináns öröklésmenet

21. Populations need to express variants of each type of MHC molecule The rate of replication by pathogenic microorganisms is faster than human reproduction In a given time a pathogen can mutate genes more frequently than humans and can easily evade changes in MHC molecules The number of types of MHC molecules are limited To counteract the flexibility of pathogens The MHC has developed many variants of each type of MHC molecule These variants may not necessarily protect all individuals from every pathogen, but will protect the population from extinction

22. Classical MHC genes are inherited as haplotypes BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR X Parents DP-1,2 DQ-3,4 DR-5,6 B-7,8 C-9,10 A-11,12 DP-9,8 DQ-7,6 DR-5,4 B-3,2 C-1,8 A-9,10 DP-1,8 DQ-3,6 DR-5,4 B-7,2 C-9,8 A-11,10 DP-1,9 DQ-3,7 DR-5,5 B-7,3 C-9,1 A-11,9 DP-2,8 DQ-4,6 DR-6,4 B-8,2 C-10,8 A-12,10 DP-2,9 DQ-4,7 DR-6,5 B-8,3 C-10,10 A-12,9 BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR BCADPDQDR Offspring

23. MHC MOLECULES ARE EXPRESSED WITH BOUND PEPTIDES DERIVED FROM SELF OR NON-SELF PROTEINS B-cell, macrophage, dendritic cell Kidney epithelial cell Liver cell Present intracellular environment Present intra- and extracellular environment Class I MHC Peptides of restricted size, which derive from cytosolic or nuclear proteins Class II MHC Overlapping peptides of various sizes, which derive from membrane proteins 70% derives from MHC molecules

24. THE OUTCOME OF INFECTION IN A POPULATION WITH POLYMORPHIC MHC GENESMHC-Gen v v v v v v v v v v v v v vvv v v v v v v v v v Example: If MHC X was the only type of MHC molecule Population threatened with extinction V – virus infection Pathogen that evades MHC X MHC XX Population is protected

25. Molecular basis of MHC types and variants POLYMORPHISM Variation >1% at a single genetic locus in a population of individuals MHC genes are the most polymorphic known The type and variant MHC molecules do not vary in the lifetime of the individual The diversity in MHC molecules exists at the population level This sharply contrast diversity in T and B cell antigen receptors which exists within the individual POLYGENISM Several MHC class I and class II genes encoding different types of MHC molecule with a range of peptide-binding specificities.

26. AZ MHC FUNCTIONS –Presentation of peptides– self/altered self/foeign peptides –Continous expression of self-peptidesto monitor cellular health –Determination of immunological self Self MHC + self peptide – individual MHC pluss és saját peptid Allogeneic response to fotreign MHC (transplantation) Self MHC– autolgous foreign MHC allogeneic activation. Az The ratio of alloreactive T-cells is very high: 1-10% –A differentiation and selection of developing thymocytes (in the thymus) –promotion of T-limphocyte survival in the priphery week” tonic” signals induced by MHC / TCR interactions provide survival signals –Inhibitory ligands for NK cells, maintainance of host cell integrity.

27. ANTIGEN PRESENTATION

28. How can 6 invariant molecules have the capacity to bind to 1,000,000,000,000,000 different peptides? The number of different T cell antigen receptors is estimated to be 1,000,000,000,000,000 (10 15 )

29. A flexible binding site? At the cell surface, such a binding site would be unable to with the T cell antigen receptor 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 binding site that is flexible enough to bind any peptide?

30. 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. FloppyCompact 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 Venus fly trap

31. 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?

32. INTRACELLULAR COMPARTMENTS ISOLATED BY MEMBRANSE 1) cytosol 2) vesicular system

33. THE ENDOGENOUS ANTIGEN PROCESSING PATHWAY Tc-cell Proteasome LMP2/LMP7 PROTEIN SELF ANTIGEN TAP1/2 gp96 calnexin α-chain α-chain+β2mMHC+peptide MHC-I + Ag peptide MHC-I + self peptide CLOSEDFLEXIBLE cytoplasm MHC-I, LMP2/7, TAP IFN  induced coordinated expression

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

35. ER membrane Lumen of ER Cytosol Transporters associated with antigen processing (TAP1 & 2) Transporter has preference for longer than 8 amino acid peptides with hydrophobic C termini. 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 ER membrane Lumen of ER Cytosol TAP-1 TAP-2 Peptide ATP-binding cassette (ABC) domain Hydrophobic transmembrane domain Peptide antigens from proteasome

36. PEPTIDE-MHC INTERACTION IS SUPPORTED BY MULTIPLE PROTEINS IN THE ENDOPLASMIC RETICULUM

37. TRIMMING OF PEPTIDES FOR OPTIMAL SIZE BY ERAP

38. CYTOSOL-DERIVED PEPTIDES ARE PRESENTED BY MHC-I FOR T-CELLS

39. Th-cell INVARIANT CHAIN (Ii) 1.Chaperone – conformation 2.Inhibition of peptide binding 3.Transport/retention CLOSEDFLEXIBLE Ii+αβ CLIP DMA/B DMA/DMB 1. Support the peptide receptive conformation 2. Exchange of CLIP for exogenous peptides MHC-II + Ag peptide MHC-II + self peptide THE EXOGENOUS ANTIGEN PROCESSING PATHWAY

40. THE INVARIANT CHAIN PROTECTS THE MHC CLASS II BINDING SITE UNTIL REACHING THE APPROPRIATE COMPARTMENT INVARIANT CHAIN LÁNC (Ii) 1.Chaperon – Conformation 2.Inhibition of the peptide binding site 3.Transport and retention DMA/DMB 1. Stabilization of peptide accessable conformation 2. Exchange of CLIP to peptides derived from exogenous proteins

41. EXTRACELLULAR PROTEIN-DERIVED PEPTIDES ARE PRESENTED BY MHC-II FOR T-CELLS

43. MHC class IMHC class II Bound peptide sourceself or foreign proteins size8-10 amino acids13-21 amino acids heterogenitylimitedoverlapping set of peptides naturalcytoplasmic and nuclear proteins~70% MHC derived, membrane and extracellular proteins Generation of peptide sitecytoplasmvesicles, endo/lysosomes enzymeproteasome LMP-2, LMP-7 regulatory subunits vesicular acidic proteases cathepsins transportTAP - size and C-terminal dependent cytoplasm  ER no MHC transport noIi - target, retention ER  vesicular system special compartment MHC - peptide interaction siteERvesicles, CIIV chaperonscalnexin, toposinIi - 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

44. Classical MHC genes POLYMORPHIC HLA – Human Leukocyte Antigen HLA –A,B, C class I EXPRESSED IN ALL NUCLEATED CELLS HLA – DR, DP, DQclass II EXPRESSED IN PROFESSIONAL APC Non- classical MHC genes E, G, F Chromosome 6 short armMHC Chromosome 15  2m LOCALIZATION OF MHC GENES Class III genes

45. OTHER GENES IN THE MHC – not polymorphic MHC Class 1b genes Encoding MHC class I-like proteins that associate with  -2 microglobulin Restricted tissue expression HLA-G trophoblast, interacts CD94/NKG2 (NK-cell receptor). Inhibits NK cell attack of foetus/ tumours HLA-E in the membrane of certain cells, binds conserved leader peptides from HLA-A, B, C. Interacts with CD94/NKG2 HLA-F fetal liver, eosinophil surface, function unknown MHC Class II genes Encoding several antigen processing genes HLA-DM   and  in professional APC, proteasome components ( LMP-2 & 7 ), peptide transporters ( TAP-1 & 2 ) Many pseudogenes MHC Class III genes Encoding complement proteins C4A and C4B, C2 and FACTOR B TUMOUR NECROSIS FACTORS-  Immunologically irrelevant genes Genes encoding 21-hydroxylase, RNA Helicase, Caesin kinase Heat shock protein 70, Sialidase