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Antigen Processing

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Antigen Processing

Antigen Processing


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  • 1. Topic 5 Antigen Processing ©Dr. Colin R.A. Hewitt crah1@le.ac.uk
  • 2. What you should know by the end of this lecture • 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 in which the pathogen replicates • Antigen processing includes uptake, degradation, complex formation and presentation • 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
  • 3. T cells do not recognise native antigens B B B B B BB B B Y Y Y Y Y YYY Y Y Y Y Y Y Y Cross-linking of Proliferation and surface membrane Ig antibody production T T No proliferation Y No cytokine release Y
  • 4. Antigens must be processed in order to be recognised by T cells T Y Cell surface peptides of Ag presented by cells that Soluble express MHC antigens Soluble native Ag peptides Cell surface of Ag Cell surface native Ag peptides of Ag ANTIGEN PROCESSING No T cell No T cell No T cell No T cell T cell response response response response response
  • 5. Early evidence that antigens are catabolised M M Macrophages and radiolabelled Rapid binding to cell surface Listeria monocytogenes Degradation M of bacteria M and release of Radiolabelled protein into supernatant and cells Internalisation How is antigen catabolism linked to T cell proliferation?
  • 6. The interaction of T cells with macrophages requires antigen catabolism Listeria-specific NO T CELLS T cells BIND Listeria coated T plastic Listeria NO T CELLS NO T CELLS NO T CELLS T CELLS BIND BIND BIND BIND M M M M 0mins 60mins T cell do not bind stably to antigen presenting cells unless the antigen is catabolised
  • 7. Only metabolically active cells can process antigen Listeria- specific T cells T M M M Fix with Pulse with Add Listeria paraformaldehyde Listeria for 60min specific T cells or poison with & wash cells NO T CELLS BIND sodium azide Determinants recognised by T cells are generated by catabolic activity that is dependent upon the viability of macrophages Antigen presenting cells must be viable to PROCESS antigen
  • 8. Antigen presentation does not require metabolically-active cells Listeria M M M M Fix with paraformaldehyde or poison with sodium azide Add Listeria specific T cells T T CELLS BIND M Antigen presenting cells do not need to be viable to PRESENT antigen
  • 9. Where does antigen processing take place? Add Listeria specific T cells T Listeria M M M M T CELLS BIND Listeria Incubate with CHLOROQUINE M M M M NO T CELLS BIND Chloroquine inhibits lysosomal function (a lysosomotrophic drug) Antigen processing involves the lysosomal system
  • 10. What form of antigen is produced by antigen processing? Ovalbumin specific T cell line T Native ovalbumin Digested ovalbumin Ag APC APC APC APC APC Viable Fixed Viable Fixed T cell T T T T T T T T T response T T T T T T T Catabolism reduces antigens to peptides that can be recognised by T cells
  • 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 lysosomal system, lysosomal antigen processing is part of the EXOGENOUS antigen processing pathway
  • 12. Is exogenous antigen processing sufficient? • Macrophages have well- developed lysosomal systems M • Specialised for motility, phagocytosis and the introduction of particles to the lysosomal system Most cell types do not have lysosomal systems developed as well as macrophages BUT Viruses can infect most cell types A non-lysosomal mechanism to process antigens for presentation to T cells is required
  • 13. Infectious viruses raise CTL that recognise antigens that are not generated by the exogenous pathway Infectious influenza Strong T cell response CTL assay CTL CTL CTL CTL CTL Kill Cloned anti- CTL No treatment CTL CTL CTL Kill CTL CTL CTL CTL CTL CTL CTL CTL + Chloroquine Lysosome inhibitors do not inhibit the generation of antigens recognised by most CTL Most CTL do not recognise lysosomally-derived antigens
  • 14. Inactive viruses raise CTL to antigens that are generated by the exogenous pathway Inactivated influenza CTL assay CTL Weak T cell response Kill Cloned anti- CTL No treatment CTL CTL CTL CTL CTL CTL CTL CTL CTL No Kill CTL CTL + Chloroquine Lysosomal inhibitors inhibit the generation of antigens from INACTIVE virus Some CTL can recognise lysosomally-derived antigens
  • 15. Non-lysosomal processing The antigens of infectious & inactivated viruses are clearly generated by different mechanisms Infectious viruses use cellular protein synthesis machinery to replicate Inactivated viruses do not synthesise protein CTL raised with CTL raised with infectious virus non-infectious virus CTL CTL Untreated Protein synthesis inhibitor-treated Protein synthesis is required for virus infected target cells to express antigens recognised by CTL
  • 16. Non-lysosomal antigen processing 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 drugs Most 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 Do the two pathways generate the same type of T cell receptor ligand?
  • 17. Endogenous antigen processing also generates peptides Influenza virus Nucleoprotein Peptides of nucleoprotein CTL CTL CTL Infectious virus Native antigen fails Synthetic peptide antigens sensitises for lysis to sensitise for lysis sensitise targets for lysis No protein/antigen Protein/antigen No protein/antigen synthesis but peptides are synthesis synthesis pre-formed
  • 18. The site of pathogen replication or mechanism of antigen uptake determines the antigen processing pathway used EXTRACELLULAR OR ENDOSOMAL REPLICATION Vesicular Compartment Y Contiguous with extracellular fluid Exogenous processing (Streptococcal, Mycobacterial antigens) INTRACELLULAR REPLICATION Cytosolic compartment Endogenous processing (Viral antigens) Distinct mechanisms of antigen generation are used to raise T cells suited to the elimination of endogenous or exogenous pathogens
  • 19. Antigens generated by endogenous and exogenous antigen processing activate different effector functions EXOGENOUS PATHOGENS Y ENDOGENOUS PATHOGENS Eliminated by: Eliminated by: Antibodies and phagocyte Killing of infected cells by CTL activation by T helper cells that that use antigens generated by use antigens generated by ENDOGENOUS EXOGENOUS PROCESSING PROCESSING
  • 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. Uptake of exogenous antigens Membrane Ig receptor mediated uptake Phagocytosis Y Complement receptor mediated phagocytosis Pinocytosis Y Fc receptor mediated phagocytosis Uptake mechanisms direct antigen into intracellular vesicles for exogenous antigen processing
  • 22. Receptor-mediated uptake enhances the efficiency of the T cell response Receptor-mediated Non-receptor antigen uptake -mediated uptake 100 % of max. T cell response 75 50 25 0 10-3 10-2 10-1 Antigen gml-1
  • 23. Exogenous pathway Cell surface Protein antigens Uptake In endosome Endosomes Increase in acidity To lysosomes Cathepsin B, D and L proteases are activated by the decrease in pH Proteases produce ~24 amino acid long peptides from antigens Drugs that raise the pH of endosomes inhibit antigen processing
  • 24. Activation of Cathepsin B at low pH Loss of the pro- region exposes the catalytic site of the protease At higher pH cathepsin B Acidification of the Hence: drugs that alter exists in a pro-enzyme form endosome alters the acidification of the conformation of the endosomes disturb proenzyme to allow exogenous antigen cleavage of the pro-region processing
  • 25. Exogenous pathway Cell surface Protein antigens Uptake In endosome Endosomes Increase in acidity To lysosomes Cathepsin B, D and L proteases are activated by the decrease in pH Proteases produce ~24 amino acid long peptides from antigens Drugs that raise the pH of endosomes inhibit antigen processing
  • 26. Flexibility of the peptide binding site in MHC molecules MHC molecules possess binding sites that are flexible at an early, intracellular stage of maturation Floppy Compact 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
  • 27. MHC class II maturation and invariant chain In the endoplasmic reticulum Need to prevent newly Invariant chain stabilises MHC class synthesised, unfolded II by non- covalently binding to the self proteins from binding immature MHC class II molecule and to immature MHC forming a nonomeric complex
  • 28. Invariant chain structure Three extended peptides each bind into the grooves of three MHC class II molecules to form the nonomeric complex
  • 29. Invariant chain CLIP peptide and chains of MHC class II molecules CLIP A peptide of the invariant chain blocks the MHC molecule binding site. This peptide is called the CLass II associated Invariant chain Peptide (CLIP)
  • 30. Class II associated invariant chain peptide (CLIP) Cell surface Endosomes Uptake ( inv)3 complexes Cathepsin L degrades MHC Class II directed towards Invariant chain containing vesicles endosomes by CLIP blocks groove in MHC fuse with antigen invariant chain molecule containing vesicles
  • 31. Removal of CLIP ? How can the peptide stably bind to a floppy binding site? Competition between large number of peptides
  • 32. HLA-DM assists in the removal of CLIP HLA-DM HLA-DR HLA-DM: Crystallised without a peptide in the groove In space filling models the groove is very small
  • 33. HLA-DM Single pocket in “groove” insufficient to accommodate a peptide HLA-DR Multiple pockets in groove sufficient to accommodate a peptide
  • 34. HLA-DM catalyses the removal of CLIP 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-DR HLA-DM HLA-DO may also play a role in regulating DM Sequence in cytoplasmic tail retains HLA-DM in MIIC compartment endosomes
  • 35. Surface expression of MHC class II- peptide complexes Exported to the cell surface (t1/2 = 50hr) Sent to lysosomes for degradation MIIC compartment sorts peptide-MHC complexes for surface expression or lysosomal degradation
  • 36. Endogenous antigen processing 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
  • 37. Degradation in the proteasome Cytoplasmic cellular proteins, including non-self proteins are degraded continuously by a multicatalytic protease of 28 subunits 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
  • 38. Crystal Structure Of The 20s Proteasome From Yeast View End on
  • 39. Peptide antigens produced in the cytoplasm are physically separated from newly formed MHC class I ENDOPLASMIC RETICULUM Newly synthesised MHC class I molecules Peptides need CYTOSOL access to the ER in order to be loaded onto MHC class I molecules
  • 40. Transporters associated with antigen processing (TAP1 & 2) Hydrophobic transmembrane Lumen of ER domain Peptide ER membrane Cytosol Peptide Peptide Peptide antigens ATP-binding cassette from proteasome (ABC) domain Transporter has preference for >8 amino acid peptides with hydrophobic C termini.
  • 41. Discovery of the role of TAP1 & TAP2 in antigen processing Analysis of genes Normal antigen in the MHC of the presenting cell line with stable mutant cell line showed mutations X surface MHC class I expression in a pair of ABC transporter genes √ Chemically-induced Transfection of mutant antigen normal TAP genes presenting cell line into mutant APC with unstable (floppy) restored stable MHC class I surface MHC expressed intracellularly 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
  • 42. Maturation and loading of MHC class I Peptide Peptide Peptide Endoplasmic reticulum Calnexin binds B2-M Tapasin, calreticulin, TAP Cytoplasmic peptides to nascent binds and 1 & 2 form a complex with are loaded onto the class I chain stabilises the floppy MHC MHC molecule and the until 2-M binds floppy structure becomes MHC compact
  • 43. Fate of MHC class I Exported to the cell surface Sent to lysosomes for degradation
  • 44. Evasion of immunity by interference with endogenous antigen processing Peptide Peptide Endoplasmic reticulum Sent to lysosomes for degradation HSV protein blocks transport of viral peptides into ER
  • 45. Evasion of immunity by interference with endogenous antigen processing Normally exported to the cell surface Adenoviral protein retains MHC class I in the ER Sent to lysosomes for degradation
  • 46. Summary • 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, complex formation 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