Antigen processing involves two main pathways: 1. Exogenous antigens are processed in the lysosomes by proteases and presented on MHC class II molecules. This involves uptake, degradation, complex formation and presentation. 2. Endogenous antigens from cytosolic pathogens are processed by the proteasome and transported into the ER by TAP for loading onto MHC class I. This non-lysosomal pathway generates peptides for CTL response. Pathogens can evade immunity by interfering with these antigen processing pathways, such as by blocking transporter function or retaining MHC in the ER. Proper antigen processing is essential for generating effective T cell immunity.

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 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 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 Soluble peptides 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 Listeria for 60min & wash cells Add Listeria specific T cells NO T CELLS BIND Antigen presenting cells must be viable to PROCESS antigen 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 to PRESENT antigen Antigen presentation does not require metabolically-active cells M  M  M  M  T Add Listeria specific T cells M 

9. Where does antigen processing take place? Incubate with CHLOROQUINE 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 Add Listeria 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 lysosomal system, lysosomal antigen processing is part of the EXOGENOUS 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 introduction of 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 that are not generated by the exogenous pathway Most CTL do not recognise lysosomally-derived antigens Lysosome inhibitors do not inhibit the generation of antigens recognised by most CTL Strong T cell response + Chloroquine Infectious influenza CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL CTL Cloned anti- CTL No treatment CTL assay Kill Kill

14. Lysosomal inhibitors inhibit the generation of antigens from INACTIVE virus Some CTL can recognise lysosomally-derived antigens Inactive viruses raise CTL to antigens that are generated by the exogenous pathway No Kill Inactivated influenza Cloned anti- 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 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 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 by ENDOGENOUS 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 MHC Invariant 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 the CL ass II associated I nvariant chain P eptide ( CLIP ) Invariant chain CLIP peptide and  chains 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 of normal 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 antigen presenting 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 degradation Exported 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 degradation Normally 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, 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 Summary