1. Antigen Processing

2.

That T and B cells recognise antigen differently

The experimental evidence that antigen catabolism takes place

Antigen processing generates antigenic peptides

That antigen processing can take place in lysosomes

That there is a non-lysosomal mechanism of antigen processing

The mechanism of antigen processing depends upon the compartment inwhich the pathogen replicates

Antigen processing includes uptake, degradation, complex formation andpresentation

The role of invariant chain HLA-DM and CLIP in antigen processing

The role of the proteasome and transporters in antigen processing

How pathogens evade immunity by disrupting antigen processing

What you should know by the end of this lecture 3. T cells do not recognise native antigens Y Y Y Y Y Y Proliferation and antibody production No proliferation No cytokine release Cross-linking of surface membrane Ig Y Y B Y B Y T Y T Y B Y B Y B Y B Y B Y B Y B 4. Antigens must be processed in order to be recognised by T cells T cell response No T cell response No T cell response No T cell response No T cell response ANTIGEN PROCESSING Cell surface peptides of Ag Y T Soluble native Ag Cell surface native Ag Solublepeptides of Ag Cell surface peptides of Ag presented by cells that express MHC antigens 5. Early evidence that antigens are catabolised Macrophages and radiolabelled Listeria monocytogenes Internalisation Rapid binding to cell surface Degradation of bacteria and release of Radiolabelled protein into supernatant and cells How is antigen catabolism linked to T cell proliferation? M M M M 6. The interaction of T cells with macrophages requires antigen catabolism Listeria T cell do not bind stably to antigen presenting cells unless the antigen is catabolised M M M M 0mins 60mins T Listeria -specific T cells NO T CELLS BIND NO T CELLS BIND T CELLS BIND Listeria coated plastic NO T CELLS BIND NO T CELLS BIND 7. Only metabolically active cells can process antigen Determinants recognised by T cells are generated by catabolic activity that is dependent upon the viability of macrophages Fix with paraformaldehyde or poison with sodium azide Pulse with Listeriafor 60min & wash cells AddListeria specific T cells NO T CELLS BIND Antigen presenting cells must be viable toPROCESSantigen M M M T Listeria -specific T cells 8. Fix with paraformaldehyde or poison with sodium azide Listeria T CELLS BIND Antigen presenting cells do not need to be viable toPRESENTantigen Antigen presentation does not require metabolically-active cells M M M M T AddListeria specific T cells M 9. Where does antigen processing take place? Incubate withCHLOROQUINE T CELLS BIND NO T CELLS BIND Chloroquine inhibits lysosomal function (a lysosomotrophic drug) Antigen processing involves the lysosomal system M M M M M M Listeria Listeria M M T AddListeria specific T cells 10. What form of antigen is produced by antigen processing? Catabolism reduces antigens to peptides that can be recognised by T cells T Ovalbumin specific T cell line APC Viable APC Viable T T T T T T T T T T T T T T T T Digested ovalbumin Fixed APC Fixed APC Native ovalbumin Ag APC T cell response 11. Summary of exogenous antigen processing T cells can not recognise native antigens Antigens must be processed for recognition by T cells Antigens catabolism occurs inside cells Only metabolically active cells can process antigen Antigen presentation does not require metabolically-active cells Antigen processing involves the lysosomal system Catabolism reduces antigens to peptides Because extracellular antigens are dealt with by the lysosomalsystem, lysosomal antigen processing is part of theEXOGENOUS antigen processing pathway 12. Is exogenous antigen processing sufficient? Most cell types do not have lysosomal systems developed as well as macrophages BUT Viruses can infect most cell types Specialised for motility,phagocytosis and the introductionof particles to the lysosomal system A non-lysosomal mechanism to process antigens for presentation to T cells is required M Macrophages have well- developed lysosomal systems 13. Infectious viruses raise CTL that recognise antigens thatare notgenerated by the exogenous pathway Most CTLdo notrecognise lysosomally-derived antigensLysosome inhibitors do not inhibit the generation of antigens recognised by most CTL Strong T cell response + Chloroquine Infectiousinfluenza CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL Clonedanti-CTL No treatment CTL assay Kill Kill 14. Lysosomal inhibitors inhibit the generation of antigens from INACTIVE virus SomeCTL can recognise lysosomally-derived antigens Inactive viruses raise CTL to antigens that are generated by the exogenous pathway No Kill Inactivatedinfluenza Clonedanti-CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL Weak T cell response + Chloroquine No treatment CTL assay Kill 15. Non-lysosomal processing The antigens of infectious & inactivated viruses are clearly generated by different mechanisms Protein synthesis is required for virus infected target cells to express antigens recognised by CTL Infectious viruses use cellular protein synthesis machinery to replicate Inactivated viruses do not synthesise protein Protein synthesis inhibitor-treated CTL raised with infectious virus CTL CTL raised with non-infectious virus CTL Untreated 16. Inactive virus raises a weak CTL response The processing of antigens from inactive viruses is sensitive to lysosomotrophic drugs ANTIGENS FROM INACTIVE VIRUSES ARE PROCESSED VIA THE EXOGENOUS PATHWAY Infectious virus raises a strong CTL response The processing of antigens from infectious viruses is NOT sensitive to lysosomotrophic drugsMost CTL recognise antigens generated via a non-lysosomal pathway Protein synthesis is required for non-lysosomal antigen processing ANTIGENS FROM INFECTIOUS VIRUSES ARE PROCESSED VIA THE ENDOGENOUS PATHWAY Non-lysosomal antigen processing Do the two pathways generate the same type of T cell receptor ligand? 17. Endogenous antigen processing also generates peptides Infectious virus sensitises for lysis Protein/antigen synthesis Synthetic peptide antigens sensitise targets for lysis No protein/antigen synthesis but peptides are pre-formed Peptides of nucleoprotein Native antigen fails to sensitise for lysis No protein/antigen synthesis CTL Influenza virus Nucleoprotein CTL CTL 18. Y The site of pathogen replication or mechanism of antigen uptake determines the antigen processing pathway used Cytosolic compartment Endogenous processing (Viral antigens) Vesicular Compartment Contiguous with extracellular fluid Exogenous processing (Streptococcal, Mycobacterial antigens) Distinct mechanisms of antigen generation are used to raise T cells suited to the elimination of endogenous or exogenous pathogens INTRACELLULAR REPLICATION EXTRACELLULAR OR ENDOSOMAL REPLICATION Y 19. Y Eliminated by: Killing of infected cells by CTL that use antigens generated byENDOGENOUS PROCESSING Eliminated by: Antibodies and phagocyte activation by T helper cells that use antigens generated by EXOGENOUS PROCESSING Antigens generated by endogenous and exogenous antigen processing activate different effector functions ENDOGENOUS PATHOGENS EXOGENOUS PATHOGENS Y 20. Stages of endogenous and exogenous antigen processing UPTAKE Access of native antigens and pathogens to intracellular pathways of degradation DEGRADATION Limited proteolysis of antigens to peptides ANTIGEN-MHC COMPLEX FORMATION Loading of peptides onto MHC molecules ANTIGEN PRESENTATION Transport and expression of peptide-MHC complexes on the surface of cells for recognition by T cells 21. Y Pinocytosis Phagocytosis Membrane Ig receptor mediated uptake Uptake of exogenous antigens Complement receptor mediated phagocytosis Fc receptor mediated phagocytosis Uptake mechanisms direct antigen into intracellular vesicles for exogenous antigen processing Y Y Y 22. % of max. T cell response Antigen gml -1 Receptor-mediated uptake enhances the efficiency of the T cell response 100 50 75 25 0 10 -1 10 -2 10 -3 Receptor-mediated antigen uptake Non-receptor -mediated uptake 23. Proteases produce ~24 amino acid long peptides from antigens Drugs that raise the pH of endosomes inhibit antigen processing Exogenous pathway Protein antigens In endosome Cathepsin B, D and L proteases are activated by the decrease in pH Endosomes Increase in acidity Cell surface To lysosomes Uptake 24. Activation of Cathepsin B at low pH At higher pH cathepsin B exists in a pro-enzyme form Acidification of the endosome alters the conformation of the proenzyme to allow cleavage of the pro-region Hence: drugs that alter acidification of the endosomes disturb exogenous antigen processing Loss of the pro-region exposes the catalytic site of the protease 25. Proteases produce ~24 amino acid long peptides from antigens Drugs that raise the pH of endosomes inhibit antigen processing Exogenous pathway Protein antigens In endosome Cathepsin B, D and L proteases are activated by the decrease in pH Endosomes Increase in acidity Cell surface To lysosomes Uptake 26. MHC molecules possess binding sites that are flexible at an early, intracellular stage of maturation Flexibility of the peptide binding site in MHC molecules Although this example shows MHC class I molecules, the flexibility in the peptide binding site of MHC class II molecules also occurs at an early stage of maturation in the endoplasmic reticulum Floppy Compact 27. Need to prevent newly synthesised, unfolded self proteins from binding to immature MHCInvariant chain stabilises MHC class II by non- covalently binding to the immature MHC class II molecule and forming a nonomeric complex In the endoplasmic reticulum MHC class II maturation and invariant chain 28. Invariant chain structure Three extended peptides each bind into the grooves of three MHC class II molecules to form the nonomeric complex 29. A peptide of the invariant chain blocks the MHC molecule binding site. This peptide is called theCL ass II associatedI nvariant chainP eptide ( CLIP )Invariant chain CLIP peptide

andchains of MHC class II molecules

CLIP 30. Class II associated invariant chain peptide (CLIP) ( inv)3 complexes directed towards endosomes by invariant chain Cathepsin L degrades Invariant chain CLIP blocks groove in MHC molecule MHC Class II containing vesicles fuse with antigen containing vesicles Endosomes Cell surface Uptake 31. Removal of CLIP ? How can the peptide stably bind to a floppy binding site? Competition between large number of peptides 32. HLA-DM HLA-DR HLA-DM assists in the removal of CLIP HLA-DM: Crystallised without a peptide in the groove In space filling models the groove is very small 33. HLA-DM HLA-DR Single pocket in groove insufficient to accommodate a peptide Multiple pockets in groove sufficient to accommodate a peptide 34. HLA-DM catalyses the removal of CLIP MIIC compartment HLA-DM Replaces CLIP with a peptide antigen using a catalytic mechanism (i.e. efficient at sub-stoichiometric levels) Discovered using mutant cell lines that failed to present antigen HLA-DO may also play a role in regulating DM Sequence in cytoplasmic tail retains HLA-DM in endosomes HLA-DM HLA-DR 35. MIIC compartment sorts peptide-MHC complexes for surface expression or lysosomal degradation Surface expression of MHC class II- peptide complexes Exported to the cell surface (t1/2 = 50hr) Sent to lysosomes for degradation 36. UPTAKE Antigens/pathogens already present in cell DEGRADATION Antigens synthesised in the cytoplasm undergo limited proteolytic degradation in the cytoplasm ANTIGEN-MHC COMPLEX FORMATION Loading of peptide antigens onto MHC class I molecules is different to the loading of MHC class II molecules PRESENTATION Transport and expression of antigen-MHC complexes on the surface of cells for recognition by T cells Endogenous antigen processing 37. Degradation in the proteasome The components of the proteasome include MECL-1, LMP2, LMP7 These components are induced by IFN- and replace constitutive components to confer proteolytic properties. LMP2 & 7 encoded in the MHC Proteasome cleaves proteins after hydrophobic and basic amino acids and releases peptides into the cytoplasm Cytoplasmic cellular proteins, including non-self proteins are degraded continuously by a multicatalytic protease of 28 subunits 38. Crystal Structure Of The 20s Proteasome From Yeast View End on 39. ENDOPLASMIC RETICULUM CYTOSOL Peptide antigens produced in the cytoplasm are physically separated from newly formed MHC class I Newly synthesised MHC class I molecules Peptides need access to the ER in order to be loaded onto MHC class I molecules 40. Transporters associated with antigen processing (TAP1 & 2) Transporter has preference for >8 amino acid peptides with hydrophobic C termini. ER membrane Lumen of ER Cytosol 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 41. Discovery of the role of TAP1 & TAP2 in antigen processing Transfection ofnormal TAP genes into mutant APC restored stable surface MHC class I expression Mutations in TAP genes affect the supply of peptides to the ER MHC class I stability is dependent upon a supply of peptides Analysis of genes in the MHC of the mutant cell line showed mutations in a pair of ABC transporter genes Normal antigenpresenting cell line with stable surface MHC class I expression Chemically-induced mutant antigen presenting cell line with unstable (floppy) MHC class I expressed intracellularly X 42. 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 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 43. Fate of MHC class I Sent to lysosomes for degradationExported to the cell surface 44. Evasion of immunity by interference with endogenous antigen processing 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 HSV protein blocks transport of viral peptides into ER Sent to lysosomes for degradation 45. Evasion of immunity by interference with endogenous antigen processing Sent to lysosomes for degradationNormally exported to the cell surface Adenoviral protein retains MHC class I in the ER 46.

T and B cells recognise antigen differently

Antigen must be catabolised before T cells can recognise it

Antigen processing generates antigenic peptides

Exogenous antigen processing takes place in lysosomes

Endogenous processing is non-lysosomal

The mechanism of antigen processing depends upon the compartment in which the pathogen replicates

Endogenous and exogenous antigen processing both involve uptake, degradation, complexformation and presentation

Exogenous antigen processing uses invariant chain and HLA-DM

Endogenous antigen processing uses proteasomes and peptide transporters in antigen processing

Pathogens can evade immunity by disrupting antigen processing

Summary