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  1. MEDSCI 708 Advanced Immunology and Immunotherapy 2007 Antigen processing and presentation

  2. Antigen processing: Proteolytic cleavage of proteins into small fragments (antigen peptides) that can bind to MHC molecules on antigen presenting cells. Antigen presentation: Presentation of antigen peptides to T cell receptor on T cells

  3. MHC restriction encoded by the most polymorphic gene cluster on the human genome (many alleles) The T cell receptor will recognise a peptide only when it is bound to a particular MHC molecule. 1973: Peter Doherty and Rolf Zinkernagel (Nobel Prize in Medicine, 1996) MHC: Major Histocompatibility Complex

  4. MHC class I: found on all nucleated cells MHC class II: found only on “professional antigen presenting cells”, such as dendritic cells, macrophages, B cells

  5. exogenous pathway endogenous pathway B cell help CD8 T CD4 T CTL kill CD8 ab TcR TcR CD4 MHC I MHC II pathogen virus-infected or tumor cell Macrophage or other professional APC Antigen presenting pathways MHC I restriction MHC II restriction

  6. Class I and Class II pathways of antigen presentation From Immunology 5th ed. (Roitt et al.)

  7. MHC I peptides: 8-11 aa with 2 terminal anchor residues MHCII peptides: 8-30+ aa with anchors throughout the peptide MHCI and MHCII peptides are very different and have to be generated by different mechanisms Many different peptides have to be presented (different anchors) In human: 2x3 MHCI molecules 1245 known alleles

  8. MHC class I antigen processing Groothuis et al., Immunological Reviews Vol. 207 (2005)

  9. Endogenous (MHC class I) pathway Processing of peptide antigens Assembly of MHC and peptide loading complex Peptide loading and MHC-peptide transport

  10. Processing of peptide antigens

  11. The Proteasome - a multicatalytic protease • around 700 kDa • 7 rings of  subunits (active site) • 2 outer rings of  subunits • ATP-dependent degradation of • mostly ubiquitin-conjugated proteins • 19S regulator: • attached at both ends of 20S proteasome • binds ubiquitin-tagged proteins Kloetzel and Ossendorp, Curr. Opin. Immunol. 16 (2004)

  12. Proteasome • About 1/3 of intracellular proteolysis in mammalian cells is directed to • nascent proteins • - defective ribosomal products (DRiPs) • non-functional and potentially toxic proteins • proteins synthesised in excess (maintain protein homeostasis) • regulatory proteins • Only about 1% of the peptide pool is available to immune system

  13. Ub Ub The Ubiquitin Pathway Ub-conjugating enzyme Ub-ligase Ub E1 E2 E3 Ub-activating enzyme Target Ub Ub Ub Ub 26s proteosome degradation

  14. Binding of poly-ubiquitin chains to 19S proteasome Elongates Ub tree Groothuis et al., Immunological Reviews Vol. 207 (2005)

  15. Immunoproteasome 1i = low molecular weight protein 2 (LMP 2) 2i = multicatalytic endopeptidase complex like 1 (MECL 1) 5i = low molecular weight protein 7 (LMP 7) POMP = proteasome maturation protein PA = proteasome activator From Strehl et al., Immunological Reviews Vol.207, pp 19-30 (2005)

  16. Immunoproteasome • P28 causes N-terminal tails of the -subunits to flip upwards, thereby facilitating • substrate entry and product exit. • The immunoproteasome does not replace the constitutive proteasome completely • The immunoproteasome has a considerably shorter half-life • The immunoproteasome has an altered cleavage site preference with a strong • preference to cleave behind residues that represent correct C-terminal anchors • for MHC I presentation. • PA28 does confer new cleavage site specificities, but enhances the frequency • of the usage of minor cleavage sites to provide more peptides for MHC presentation

  17. Immunoproteasomes affect the size of the antigenic peptide pool From Strehl et al., Immunological Reviews Vol.207, pp 19-30 (2005)

  18. Trim-peptidases • Peptides produced by proteasomes are often to large for presentation (8-11 aa) • or for TAP transport (8-16 aa) • Several cytosolic and ER proteases are involved in trimming. • However, their major function is probably peptide degradation for aa recycling. Cytosolic peptidases Puromycin-sensitive aminopeptidase (PSA): - metallopeptidase - shown to both trim and destroy epitopes Thimet oligopeptidase (TOP): - metallopeptidase of the M3 family - peptides of up to 15 aa are preferred substrates - appears to be mainly involved in epitope destruction (down-regulation enhances presentation).

  19. Cytosolic peptidases (cont.) Leucine aminopeptidase (LAP): - metallopeptidase - peptides of less than 7 aa are preferred substrates - mainly for aa recycling Tripeptidyl protease II ((TPP II): - cleaves peptides larger than 15 aa - plays significant role in antigen presentation - exopeptidase activity: removes blocks of 3 N-terminal aa - endopeptidase activity: trypsin-like specificity • There are no carboxypeptidases in the cytosol • The proteasomes have to generate C-terminal anchor for MHCI binding

  20. Discovery/purification of ER peptidases AMC: aminoacyl-aminomethyl cumarin = artificial substrate that becomes fluorescent after removal of N-terminus Saveanu et al., Immunological Reviews Vol. 207 (2005)

  21. ER peptidases ER aminopeptidase associated with antigen processing (ERAAP): = ERAP1 (human) - metallopeptidase of M1 family - specific for large hydrophobic residues, such as Leu - strong preference for substrates of 10 or more aa ERAP2: - 49% identical to ERAP1 by aa sequence - specific for basic residues, such as Arg and Lys.

  22. Chaperones in antigen processing Cytosolic chaperones: • - Tailless complex polypeptide-1 (TCP-1) ring complex (TriC) • - Chaperonin-containing TCP-1 (CCT) • Exact function remains unclear • Probably involved in peptide delivery to TAP ER chaperones: • Protein disulfide isomerase (PDI) • Binding protein (BiP), hsp70 family Regulation of the translocon (lid function) Probably play a role in peptide loading of MHCI

  23. BiP PDI translocon Processing of the SIINFEKL epitope Shastri et al., Immunological Reviews Vol. 207 (2005)

  24. Transporter Associated with Antigen Presentation (TAP) 8-12 aa (up to 40aa with low efficiency) ER retention signal Transmembrane domain 2 ATP-binding cassettes Peptide-binding domain

  25. Assembly of MHC class I and peptide loading complex peptide loading complex HC: heavy chain, 3 -subunits, 45 kDa glycoprotein, polymorphic 2m: 2-microglobulin, 12 Kda, non-polymorphic CNX: calnexin, membrane-anchored chaperone, stabilises nascent HC,lectin-like CRT: calreticulin, soluble lectin-like chaperone, binds N-linked glycan on HC Erp57: oxido-reductase, binds Tapasin via S-S bonds and non-covalently to CRT Cresswell et al., Immunological Reviews V0l. 207 (2005)

  26. Peptide binding cycle Cresswell et al., Immunological Reviews V0l. 207 (2005)

  27. Tapasin • 48 kDa glycoprotein • stabilises TAP1/TAP2 which enhances peptide transport • bridges MHC class I to TAP (structural component) • facilitates peptide loading • stabilises “empty” peptide-receptive MHC complexes • optimises peptide repertoire (peptide editor)

  28. Quality control by tapasin Brocke et al. (2002)

  29. “Lost in action” or “the inefficiency of antigen presentation” • about 2 billion proteins per cell are expressed and turned over in 6h • about 100 million peptides per cell are generated in 1 minute • only a few hundred MHC I molecules are made in 1 minute • a large fraction of MHC I molecules fail to acquire a peptide • a peptide has an average in-vivo half-life of a few seconds • more than 99% of cytosolic peptides are destroyed before their • encounter TAP An antigen has to be expressed at a minimum of 10,000 copies to be presented by MHC class I

  30. The exogenous (MHC class II) pathway Right here after the break ….

  31. Exogenous (MHC class II) pathway Villadangos et al., Immunological Reviews Vol. 207 (2005)

  32. Exogenous (MHC class II) pathway MHC assembly and transport to peptide loading compartment 2. Uptake and processing of exogenous antigen 3. Peptide loading (CLIP exchange)

  33. Assembly of MHC class II requires the invariant chain MHC II: HLA-DR, -DQ, -DP (human), glycosylated  heterodimers, - generic  chain (30-34 kDa) - highly polymorphic -chain (26-29 kDa)

  34. Invariant Chain (Ii) 4 domains: • short N-terminal cytosolic domain (sorting motif) • single TM domain • class II-associated invariant chain peptide (CLIP) • C-terminal trimerisation motif and protease inhibitor motif (only • some isoforms). 4 functions: • scaffold to facilitate proper folding and assembly of MHC II • blocking premature class II peptide association • direct trafficking of MHCII-invariant chain to endosomal pathway • modulating the proteolytic environment within endosome.

  35. Uptake of exogenous antigen Endocytosis: Uptake of material into the cell by the formation of a membrane-bound vesicle. Endosome: endocytotic vesicle derived from the plasma membrane. Receptor-mediated endocytosis: mannose and lectin-like receptors Macropinocytosis: uptake of fluid-filled vesicles (mainly DC) Phagocytosis: uptake of complete cells

  36. Phagocytosis Desjardins et al., Immunological Reviews Vol 207 (2005)

  37. Processing of exogenous antigen and invariant chain Cathepsins: endosomal proteases involved in Ii degradation and antigen processing • exact role for each cathepsin not clear • some might have redundant functions • some were shown to be cell specific • best studied are cathepsin L and cathepsin S • both are papain-like cysteine endoproteases and are • required for invariant chain degradation

  38. Other endosomal proteases Asparaginyl cysteine endoprotease (AEP): - initiates first cuts in protein -interferon-induced lysosomal thiol reductase (GILT): - reduces disulfide bridges in proteins (S-S to -SH)

  39. Invariant chain degradation in endosome Hsing & Rudensky, Immunological Reviews Vol 207 (2005) Note: within the same compartment, proteases also generate peptide fragments derived from endocytosed antigens

  40. Regulation of endosomal proteases • N-terminal propiece that blocks substrate binding. Propiece stabilises • protease during traffick through ER and dissociates • upon maturation and acidification of the endosome. • Cystatins: naturally occuring lysosomal protease inhibitors. Wedge- • shaped binding region fills and obstructs active site. • Ii isoform p41: Highly specific for cathepsin L. Enhances presentation • of certain antigens.

  41. Peptide loading and editing by HLA-DM Brocke et al., Curr. Opin. Immunol. Vol. 14 (2002)

  42. Peptide loading and editing by HLA-DM

  43. Peptide loading and editing by HLA-DM

  44. Possible role for HLA-DO in B cells Brocke et al., Curr. Opin. Immunol. Vol. 14 (2002)

  45. Now it seems to be all so clear and logical …. but is it ? Problem 1: The stimulation of a naïve CD8 T cell requires co-stimulatory molecules, such as CD86, but these are absent from the majority of cell types!! A professional APC has to acquire viral antigens from infected cell, e.g by phagocytosis Problem 2: DC has to present same peptide antigen as infected cell, but exogenous pathway produces different peptides than endogenous pathway.

  46. Cross-presentation of exogenous antigens

  47. How does endocytosed protein get into the cytosol for endogenous pathway? Hypothesis: Retro-transport of endocytosed protein: TGN - ER - cytosol (ERAD pathway?) This pathway is used by certain toxins, such as cholera toxin, ricin ERAD: ER Associated Degradation

  48. Possible routes that phagocytosed antigens take to reach proteasomes in the cytosol. Vol. 19,Feb. 2007