Chapter 11 Antigen Processing and Presentation

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

Cell surface peptides of Ag Antigens must be processed in order to be recognised by T cells Y T T cell response No T cell response No T cell response No T cell response No T cell response Soluble native Ag Cell surface native Ag Soluble peptides of Ag Cell surface peptides of Ag presented by cells that express MHC molecules ANTIGENPROCESSING APC

Contents  Part Ⅰ Introduction--concepts  Part Ⅱ Characteristics of APCs  Part Ⅲ Ag Processing and presentation Chapter 11 Antigen Processing and Presentation

Part Ⅰ 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

Y The site of pathogen replication or mechanism of antigen uptake determines the antigen processing pathway used Y Cytosolic compartment Endogenous processing (Viral, tumor antigens ) Vesicular Compartment Contiguous with extracellular fluid Exogenous processing (Streptococcal, tumor antigens) INTRACELLULAR REPLICATION EXTRACELLULAR OR ENDOSOMAL REPLICATION

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

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 class Ⅱ MHC molecules Dendritic cell Macrophage B lymphocyte Facultative APC: endothelial cells, epithelial cells, fibroblast, etc

APC Express class Ⅰ, 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

Part Ⅱ 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)Identification of DC: Typical morphology — spinelike projection MLR — stimulate na ï ve T cells activation Surface markers : CD1a, CD11c, CD83(human) high expression of class Ⅱ MHC co-stimulatory molecules-- CD80,CD86 others — CKs, CAMs, R (2) Source of DC: pluripotent hematopoietic stem cells myeloid DC myeloid progenitor lymphoid DC lymphoid progenitor 1. Dendritic cell (DC) GM-CSF, IL-4

(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 1. Dendritic cell (DC)

Interdigitating DC( IDC ） Express high level of class Ⅰ, Ⅱ MHC molecules and B7 ， lack of FcR and CR, can stimulate T cells.

FDC B cell Folicular DC （ FDC ） Lie in follicle of LN, no expression of class Ⅱ MHC, high level of FcR and C3bR.

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.

(4) Development and Maturation of DC Pre-DC phase Immature DC （ iDC ） phase Migration phase Mature DC （ mDC ） phase 1. Dendritic cell (DC)

Pre-DC Blood Non-lymphoid tissue Differentiation ImmatureDC Distribute 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

Difference between iDC and mDC Ability of uptaking and processing antigens decreases. Ability of antigen presentation increases. Express high level of MHC, co- stimulatory molecules(CD80,CD86), CAMs(ICAM-1). Ability to stimulate na ï ve T cell activation increases.

(5) Antigens capturing: Phagocytosis—cell, bacteria Pinocytosis—soluble antigen Receptor-mediated endocytosis FcγR Ⅱ, 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 1. Dendritic cell (DC)

2. Macrophage （ MФ ） Stem from monocytes in blood Have strong phagocytosis (big phagocyte) Can not stimulate na ï ve T cells Capture antigens by phagocytosis, pinocytosis, receptor-mediated endocytosis

Function : Phagocytosis Presentation of antigens Unactivated macrophage Activated macrophage: Class Ⅱ MHC molecules and co-stimulatory molecules 2. Macrophage （ MФ ）

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

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

Part Ⅲ Ag Processing and Presentation  Class Ⅱ MHC pathway exogenous antigens  Class Ⅰ MHC pathway endogenous antigens  Cross – presentation of antigen

Section Ⅰ 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 class Ⅱ 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

Y Y Pinocytosis Phagocytosis Membrane Ig receptor mediated uptake Y Uptake of exogenous antigens Complement receptor mediated phagocytosis Y Fc receptor mediated phagocytosis

2. Processing of Ag endosome + lysosome Ag antigen peptides(10-30aa) Cathepsin

Endosomes Exogenous pathway Cell surface To lysosomes Uptake Protein antigens In endosome 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 ) Ii chain ① Promote formation of class Ⅱ MHC dimer ② Preventing endogenous peptide from combining with class Ⅱ MHC molecules within ER ③ Leading class Ⅱ MHC molecules into endosome from ER Endosome (MIIC) *Ii chain: Ia-associated invariant chain

A peptide of the invariant chain blocks the MHC molecule binding site. This peptide is called the CLass Ⅱ associated Invariant chain Peptide (CLIP) Invariant chain CLIP peptide  and  chains of MHC class II molecules CLIP

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 Ii chain CLIP

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

Endosomes Cell surface Uptake 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

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 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 peptide exchange Sequence in cytoplasmic tail retains HLA-DM in endosomes HLA-DM HLA-DR

5.Presenting to CD4 + T cells Antigen peptide-class Ⅱ MHC molecuels presented on cell membrane by exocytosis

MIIC compartment sorts peptide-MHC complexes for surface expression or lysosomal degradation Surface expression of class II MHC - peptide complexes Exported to the cell surface (t1/2 = 50hr) Sent to lysosomes for degradation

CD4 + T cells

section Ⅱ 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 The components of the proteasome include LMP2, LMP7, MECL-1 （ LMP10 ） *MECL-1 ： Multicatalytic endopeptidase complex subunit Cytoplasmic cellular proteins, including non-self proteins are degraded continuously by a multicatalytic protease Binding ubiquitin

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)

ENDOPLASMIC RETICULUM CYTOSOL Peptide antigens produced in the cytoplasm are physically separated from newly formed class I MHC Newly synthesised class I MHC molecules Peptides need access to the ER in order to be loaded onto class I MHC molecules

ER membrane Lumen of ER Cytosol Transporters associated with antigen processing (TAP1 & 2) Transporter has preference for >8 amino acid peptides with hydrophobic C termini. 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

3. Peptide loading of class Ⅰ MHC molecules ER: antigen peptide—class Ⅰ MHC complexes

Endoplasmic reticulum Calnexin binds to nascent class I  chain until  2-M binds 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 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 class I MHC

4. Presenting to CD8 + T cells Antigen peptide-class Ⅰ MHC molecuels presented on cell membrane by exocytosis

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

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

CD4 + T cells

Section Ⅲ 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

CD4 + T cell(Th) CD8 + T cell(Tc) T cell Receptor Peptide MHC Class II T cell Receptor Peptide MHC Class I Antigen Presenting Cell Target cell CD4CD8