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Chapter 11 Antigen Processing and Presentation T cells do not recognise native antigens Y Y Y Y Y Y Y Cross-linking of surface membrane Ig YYY Y Y Y Y Y B B B B B BB B B Proliferation and antibody production T T No proliferation No cytokine release Y Y Antigens must be processed in order to be recognised by T cells T Y Soluble native Ag Cell surface native Ag Soluble peptides of Ag APC No T cell response No T cell response Cell surface peptides of Ag presented by cells that express MHC molecules Cell surface peptides of Ag ANTIGEN PROCESSING No T cell response No T cell response T cell response Chapter 11 Antigen Processing and Presentation Contents PartⅠ Introduction--concepts PartⅡ Characteristics of APCs PartⅢ Ag Processing and presentation PartⅠ Introduction-concepts Endogenous Ags: antigens synthesized within cells, including self and unself protein----class Ⅰ MHC molecules. Exogenous Ags: antigens comes outside the cells, including self and unself protein----class Ⅱ MHC molecules. Antigen processing: the conversion of native proteins to peptides which can combine with MHC molecules. Antigen presentation: the course of formation and display of peptide-MHC complexes on the surface of APCs and the course of peptide-MHC complexes recognition by T cells. Ag capturing----Endocytosis (internalization) Phagocytosis, Pinocytosis, Receptor-mediated endocytosis Production of endogenous Ags and exogenous Ags The site of pathogen replication or mechanism of antigen uptake determines the antigen processing pathway used Y EXTRACELLULAR OR ENDOSOMAL REPLICATION Vesicular Compartment Contiguous with extracellular fluid Exogenous processing (Streptococcal, tumor antigens) INTRACELLULAR REPLICATION Cytosolic compartment Endogenous processing (Viral, tumor antigens ) Antigens must be processed in order to be recognised by T cells T Y Soluble native Ag Cell surface native Ag Soluble peptides of Ag Cell surface peptides of Ag presented by cells that express MHC antigens Cell surface peptides of Ag APC No T cell response No T cell response No T cell response No T cell response T cell response Antigen-Presenting Cells (APC) APC (Accessory cells) : A group of cells play important roles in the immune response which can uptake, process antigens and present peptide-MHC complexes to T cells. Professional APC: express classⅡMHC molecules Dendritic cell Macrophage B lymphocyte Facultative APC: endothelial cells, epithelial cells, fibroblast, etc APC • • • Express classⅠ, Ⅱ MHC molecules and co-stimulatory molecules Uptake, process endogenous/exogenous antigens and present peptide-MHC to T cells Including dendritic cells, macrophages and B cells PartⅡ Characteristics of APCs Dendritic cell (DC) Macrophage B lymphocyte 1. Dendritic cell (DC) • History: DCs were first found by Steinman in 1973,named for their special spinelike projections. DCs were cultured successfully in vitro in 1993 by Inaba. • Characteristic: The most efficient APC, can present antigens to naive T cells to elicit primary immune response. Fig2-4 Mature DC suspended in media by colony( ×400) Fig2-5 scattered mature DC( ×400) Scanning electron micrograph 1. Dendritic cell (DC) (1) Identification of DC: Typical morphology—spinelike projection MLR—stimulate naïve T cells activation Surface markers : CD1a, CD11c, CD83(human) high expression of classⅡMHC co-stimulatory molecules-CD80,CD86 others—CKs, CAMs, R (2) Source of DC: pluripotent hematopoietic stem cells myeloid DCGM-CSF, IL-4 myeloid progenitor progenitor lymphoid DC lymphoid 1. Dendritic cell (DC) (3) Classification of DC : DC in lymphoid tissue: Interdigitating DC (IDC) , Folicular DC (FDC) DC in non lymphoid tissue: Langerhans cell (LC) DC in body fluid: Veiled cell, Blood DC Interdigitating DC( IDC ) Express high level of classⅠ, Ⅱ MHC molecules and B7,lack of FcR and CR, can stimulate T cells. Folicular DC(FDC) Lie in follicle of LN, no expression of class Ⅱ MHC, high level of FcR and C3bR. FDC B cell Langerhan’s cells(LC)—Birbeck particle Lie in the epithelia of the skin, gastrointestinal and respiratory tracts, express FcR and C3bR. After uptaking antigens, migrating to draining LN and becoming IDC. 1. Dendritic cell (DC) (4) Development and Maturation of DC Pre-DC phase Immature DC(iDC) phase Migration phase Mature DC(mDC) phase Blood Pre-DC Differentiation Non-lymphoid tissue Widely distributed in the body Possess ability of Ag capture and process Cytokines and Ag DC mature and move into lymphoid tissue Ability of Ag capture and processing decreases while its ability of Ag presenting increases ImmatureDC Distribute Difference between iDC and mDC • Ability of uptaking and processing antigens decreases. • Ability of antigen presentation increases. • Express high level of MHC, costimulatory molecules(CD80,CD86), CAMs(ICAM-1). • Ability to stimulate naïve T cell activation increases. 1. Dendritic cell (DC) (5) Antigens capturing: • Phagocytosis—cell, bacteria • Pinocytosis—soluble antigen • Receptor-mediated endocytosis FcγRⅡ, C3bR, mannose receptor 1. Dendritic cell (DC) (6) Function of DC : • Capture, process, present antigens—APC • Stimulate T or B lymphocytes—mature DC • Induce immune tolerance—immature DC 2. Macrophage ( MФ ) Stem from monocytes in blood • • Have strong phagocytosis (big phagocyte) • Can not stimulate naïve T cells • Capture antigens by phagocytosis, pinocytosis, receptor-mediated endocytosis 2. Macrophage( MФ) Function : • • Phagocytosis Presentation of antigens Unactivated macrophage Activated macrophage: Class Ⅱ MHC molecules and co-stimulatory molecules 3. B cells Functions • Mediate humoral immune response • Immunological regulation • Present antigens to T cell Soluble Ag--pinocytosis Specific receptor-mediated endocytosis The three kinds of professional APC Antigens must be processed in order to be recognised by T cells T Y Soluble native Ag Cell surface native Ag Soluble peptides of Ag Cell surface peptides of Ag presented by cells that express MHC antigens Cell surface peptides of Ag APC No T cell response No T cell response No T cell response No T cell response T cell response PartⅢ Ag Processing and Presentation Class Ⅱ MHC pathway ------exogenous antigens Class Ⅰ MHC pathway ------endogenous antigens Cross – presentation of antigen SectionⅠ Class Ⅱ MHC pathway 1. Capture of exogenous Ag 2. Processing of Ag 3. Synthesis and transportation of class Ⅱ MHC molecules 4. Peptide loading of classⅡ MHC molecules 5. Presenting to CD4+T cells 1. Capture of exogenous Ag • Endocytosis: Phagocytosis: particles or granules Pinocytosis: soluble antigens Receptor-mediated endocytosis: • Form endosome Uptake of exogenous antigens Membrane Ig receptor mediated uptake Y Phagocytosis Complement receptor mediated phagocytosis Pinocytosis Y Fc receptor mediated phagocytosis 2. Processing of Ag endosome + lysosome Cathepsin Ag antigen peptides(10-30aa) Exogenous pathway Cell surface Uptake Protein antigens In endosome Endosomes To lysosomes Cathepsin B, D and L proteases are activated by the decrease in pH 3. Synthesis and transportation of class Ⅱ MHC molecules Synthesis of class Ⅱ MHC molecules in ER Ii chain --- class Ⅱ MHC molecule (Ii3α3β3 ) ①Promote formation of class Ⅱ MHC dimer ②Preventing endogenous peptide from combining with classⅡMHC molecules within ER ③Leading classⅡMHC molecules into endosome from ER Endosome (MIIC) *Ii chain: Ia-associated invariant chain 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) MHC class II maturation and invariant chain In the endoplasmic reticulum Ii chain CLIP Invariant chain stabilises MHC class Need to prevent newly II by non- covalently binding to the synthesised, unfolded self proteins from binding immature MHC class II molecule and forming a nonomeric complex to immature MHC 4. Peptide loading of class Ⅱ MHC molecules Ii - class Ⅱ MHC molecules protease Ii chain cleaving CLIP - class Ⅱ MHC molecules HLA-DM CLIP releasing Antigen peptide - class Ⅱ MHC complexes Class II associated invariant chain peptide (CLIP) Cell surface Uptake (binv)3 complexes directed towards endosomes by invariant chain Endosomes Cathepsin L degrades Invariant chain CLIP blocks groove in MHC molecule MHC Class II containing vesicles fuse with antigen containing vesicles Removal of CLIP ? How can the peptide stably bind to a floppy binding site? Competition between large number of peptides HLA-DM catalyses the removal of CLIP HLA-DM Replaces CLIP with a peptide antigen using a catalytic mechanism (i.e. efficient at substoichiometric levels) Discovered using mutant cell lines that failed to present antigen HLA-DR HLA-DM MIIC compartment HLA-DO may also play a role in peptide exchange Sequence in cytoplasmic tail retains HLA-DM in endosomes 5.Presenting to CD4+T cells Antigen peptide-class Ⅱ MHC molecuels presented on cell membrane by exocytosis Surface expression of class II MHC 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 CD4+T cells sectionⅡ class Ⅰ MHC pathway 1. Processing of endogenous Ag 2. Transporting of antigen peptide into ER 3. Peptide loading of class Ⅰ MHC molecules 4. Presenting to CD8+T cells 1. Processing of endogenous Ag • Proteosome : 20S, 26S • Low molecular weight polypeptide (LMP) : LMP2, LMP7,LMP10 • Ag antigen peptides (6-30aa) Degradation in the proteasome Cytoplasmic cellular proteins, including non-self proteins are degraded continuously by a multicatalytic protease Binding ubiquitin The components of the proteasome include LMP2, LMP7, MECL-1(LMP10) *MECL-1:Multicatalytic endopeptidase complex subunit 2. Transporting of antigen peptide into ER TAP(transporter associated with antigen processing): Consisting of TAP1 and TAP2 ATP dependent transporter Selective transporting (8-15aa) Peptide antigens produced in the cytoplasm are physically separated from newly formed class I MHC ENDOPLASMIC RETICULUM Newly synthesised class I MHC molecules CYTOSOL Peptides need access to the ER in order to be loaded onto class I MHC molecules Transporters associated with antigen processing (TAP1 & 2) Hydrophobic transmembrane domain Lumen of ER Peptide ER membrane Cytosol Peptide Peptide Peptide antigens from proteasome ATP-binding cassette (ABC) domain Transporter has preference for >8 amino acid peptides with hydrophobic C termini. 3. Peptide loading of class Ⅰ MHC molecules ER: antigen peptide—class Ⅰ MHC complexes Maturation and loading of class I MHC Peptide Peptide Peptide Endoplasmic reticulum B2-M Calnexin binds binds and to nascent stabilises class I chain floppy until b2-M binds 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 4. Presenting to CD8+T cells Antigen peptide-class Ⅰ MHC molecuels presented on cell membrane by exocytosis Fate of class I MHC Exported to the cell surface Sent to lysosomes for degradation Ag(cytosolic protein) Proteasome proteolytic degradation Ag peptide TAP complex transporting into ER antigen peptide-class Ⅰ MHC complexes Golgi complex exocyotsis Presenting to CD8+T cells CD8+T cells CD8+T cells CD4+T cells SectionⅢ Cross-presentation of antigens Cross-priming: • Class Ⅰ MHC molecules also present exogenous antigens to CD8+T cells • Class Ⅱ MHC molecules also present endogenous antigens to CD4+T cells CD8+T cell(Tc) CD4+T cell(Th) T cell Receptor T cell Receptor Peptide CD4 MHC Class II Peptide CD8 MHC Class I Antigen Presenting Cell Target cell