Topic 5 Antigen Processing ©Dr. Colin R.A. Hewitt crah1@le.ac.uk

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

T cells do not recognise native antigens Y B Y B Y B Y B Y B Y B Y B Y B Y B Y Y Y Y Y Y Cross-linking of surface membrane Ig Proliferation and antibody production Y T Y T No proliferation No cytokine release

Antigens must be processed in order to be recognised by T cells Soluble native Ag Cell surface native Ag Soluble peptides of Ag Cell surface peptides of Ag Cell surface peptides of Ag presented by cells that express MHC antigens ANTIGEN PROCESSING No T cell response No T cell response No T cell response No T cell response T cell response

Early evidence that antigens are catabolised M M Macrophages and radiolabelled Listeria monocytogenes Rapid binding to cell surface M Degradation of bacteria and release of Radiolabelled protein into supernatant and cells M Internalisation How is antigen catabolism linked to T cell proliferation?

T The interaction of T cells with macrophages requires antigen catabolism NO T CELLS BIND T Listeria-specific T cells Listeria coated plastic NO T CELLS BIND NO T CELLS BIND T CELLS BIND NO T CELLS BIND Listeria M M M M 0mins 60mins T cell do not bind stably to antigen presenting cells unless the antigen is catabolised

Only metabolically active cells can process antigen Listeria-specific T cells M M M Fix with paraformaldehyde or poison with sodium azide Pulse with Listeria for 60min & wash cells Add Listeria specific T cells NO T CELLS BIND 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

Antigen presentation does not require metabolically-active cells Listeria M M M M Fix with paraformaldehyde or poison with sodium azide T Add Listeria specific T cells M T CELLS BIND Antigen presenting cells do not need to be viable to PRESENT antigen

Where does antigen processing take place? Add Listeria specific T cells Listeria M M M M T CELLS BIND Incubate with CHLOROQUINE M M M M NO T CELLS BIND Chloroquine inhibits lysosomal function (a lysosomotrophic drug) Antigen processing involves the lysosomal system

What form of antigen is produced by antigen processing? Ovalbumin specific T cell line Native ovalbumin Digested ovalbumin Ag APC T cell response Fixed APC Fixed APC APC Viable APC Viable T T T T Catabolism reduces antigens to peptides that can be recognised by T cells

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

Is exogenous antigen processing sufficient? MF • Macrophages have well- developed lysosomal systems • 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

Infectious viruses raise CTL that recognise antigens that are not generated by the exogenous pathway Infectious influenza No treatment CTL assay Strong T cell response Kill CTL Cloned anti- CTL Kill + Chloroquine CTL Lysosome inhibitors do not inhibit the generation of antigens recognised by most CTL Most CTL do not recognise lysosomally-derived antigens

Inactive viruses raise CTL to antigens that are generated by the exogenous pathway Inactivated influenza No treatment CTL assay CTL Weak T cell response Kill Cloned anti- CTL CTL + Chloroquine No Kill Lysosomal inhibitors inhibit the generation of antigens from INACTIVE virus Some CTL can recognise lysosomally-derived antigens

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 infectious virus CTL CTL raised with non-infectious virus CTL Untreated Protein synthesis inhibitor-treated Protein synthesis is required for virus infected target cells to express antigens recognised by CTL

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?

Endogenous antigen processing also generates peptides Influenza virus Nucleoprotein Peptides of nucleoprotein CTL CTL CTL Native antigen fails to sensitise for lysis No protein/antigen synthesis Infectious virus sensitises for lysis Protein/antigen synthesis Synthetic peptide antigens sensitise targets for lysis No protein/antigen synthesis but peptides are pre-formed

The site of pathogen replication or mechanism of antigen uptake determines the antigen processing pathway used EXTRACELLULAR OR ENDOSOMAL REPLICATION Y Y Vesicular Compartment 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

Antigens generated by endogenous and exogenous antigen processing activate different effector functions Y EXOGENOUS PATHOGENS Y ENDOGENOUS PATHOGENS Eliminated by: Antibodies and phagocyte activation by T helper cells that use antigens generated by EXOGENOUS PROCESSING Eliminated by: Killing of infected cells by CTL that use antigens generated by ENDOGENOUS PROCESSING

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

Uptake of exogenous antigens Membrane Ig receptor mediated uptake Y Y Phagocytosis Complement receptor mediated phagocytosis Y Pinocytosis Y Fc receptor mediated phagocytosis Uptake mechanisms direct antigen into intracellular vesicles for exogenous antigen processing

Receptor-mediated uptake enhances the efficiency of the T cell response Non-receptor -mediated uptake Receptor-mediated antigen uptake 100 50 75 25 % of max. T cell response 10-1 10-2 10-3 Antigen gml-1

Exogenous pathway Cell surface Protein antigens Uptake In endosome Increase in acidity Protein antigens In endosome Endosomes 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

Activation of Cathepsin B at low pH Loss of the pro-region exposes the catalytic site of the protease 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

Exogenous pathway Cell surface Protein antigens Uptake In endosome Increase in acidity Protein antigens In endosome Endosomes 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

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

MHC class II maturation and invariant chain In the endoplasmic reticulum Invariant chain stabilises MHC class II by non- covalently binding to the immature MHC class II molecule and forming a nonomeric complex Need to prevent newly synthesised, unfolded self proteins from binding to immature MHC

Invariant chain structure Three extended peptides each bind into the grooves of three MHC class II molecules to form the nonomeric complex

Invariant chain CLIP peptide and b 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)

Class II associated invariant chain peptide (CLIP) Cell surface Uptake Endosomes (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

Removal of CLIP ? How can the peptide stably bind to a floppy binding site? Competition between large number of peptides

HLA-DM assists in the removal of CLIP HLA-DR HLA-DM: Crystallised without a peptide in the groove In space filling models the groove is very small

HLA-DM HLA-DR Single pocket in “groove” insufficient to accommodate a peptide HLA-DR Multiple pockets in groove sufficient to accommodate a peptide

HLA-DM catalyses the removal of CLIP 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 MIIC compartment Sequence in cytoplasmic tail retains HLA-DM in endosomes HLA-DM HLA-DR

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

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

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

Crystal Structure Of The 20s Proteasome From Yeast View End on

ENDOPLASMIC RETICULUM 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 access to the ER in order to be loaded onto MHC class I molecules CYTOSOL

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

Discovery of the role of TAP1 & TAP2 in antigen processing Normal antigen presenting cell line with stable surface MHC class I expression Analysis of genes in the MHC of the mutant cell line showed mutations in a pair of ABC transporter genes X √ Chemically-induced mutant antigen presenting cell line with unstable (floppy) MHC class I expressed intracellularly 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

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 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

Fate of MHC class I Exported to the cell surface Sent to lysosomes for degradation

Evasion of immunity by interference with endogenous antigen processing Endoplasmic reticulum Sent to lysosomes for degradation 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

Evasion of immunity by interference with endogenous antigen processing Normally exported to the cell surface Sent to lysosomes for degradation Adenoviral protein retains MHC class I in the ER

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