Antigen Processing and Presentation Dr. M.S.A. Muthukumar Nadar, Assistant Professor, Dept of Biotechnology, Karunya University, Coimbatore

What does the immune system do? The immune system protects us from infections: ~ 208 viruses ~ 538 bacteria ~ 317 Fungi ~ 287 Worms ~ 57 Parasitic Protozoa Promote normal functioning of the body such as Tissue cleanup, wound healing Removes abnormal cells –including cancer cells But the immune system can also harm the body when not doing the right thing (Allergy, Auto immunity, Transplanted organ rejection, Immuno-deficiency

Distinguishing SELF AND NON-SELF

IMMUNE SYSTEM DESIGN IDENTIFICATION Non-infectious Self and infectious Non-self RECRUITMENT Mobilize Effectors ELIMINATION Clears Pathogen PREVENT RECCURENCE Prevention of Future infection-(Memory) DEACTIVATION Stop fighting when the war is over-Prevention of unwanted effects on host (Reducing Collateral damage)

Components of the immune system Innate immune system Adaptive immune system Response is non-specific Pathogen and antigen specific response Exposure leads to immediate maximal response Lag time between exposure and maximal response Cell-mediated and humoral components No immunological memory Exposure leads to immunological memory Found in nearly all forms of life Found only in jawed vertebrates

Jawless vertebrates (Agnatha) Lamprey

Hagfish: Notice the mouth- No Jaws

Surface Barriers Skin Skin is a barrier that protects and seals the organisms from infection that could be caused by mechanical injury, chemical injury, and biological injury.

Composition of Blood PELLET 19-12

Pathogen-associated molecular patterns- PAMPs PAMPs are molecules associated with groups of pathogens, that are recognized by cells of the innate immune system. These molecules can be referred to as small molecular motifs conserved within a class of microbes. They activate innate immune responses, protecting the host from infection, by identifying some conserved non-self molecules.

PAMPs & Pattern Recognition Receptors Bacterial Lipopolysaccharide (LPS) TLR 4 Flagellin TLR 5 Peptidoglycan Lipoteichoic acid (Gram positive bacteria) Viral dsRNA TLR 3 Unmethylated CpG motifs TLR 9

Leukocytes Have nucleus, mitochondria, & amoeboid ability Can squeeze through capillary walls (diapedesis or extravasation) Granular leukocytes help detoxify foreign substances & release heparin Include eosinophils, basophils, & neutrophils 13-10

Leukocytes Agranular leukocytes are phagocytic & produce antibodies Include lymphocytes & monocytes

Types of T Lymphocytes Helper T cells (CD4)-Activates B-cells (B-cell proliferation and antibody production) Cytotoxic T cells (CD8)– attack and destroy invading agent or antigen Suppressor T Cells (T-reg)– Inhibit or terminate activities of killer cells, plasma cells or T helper cells when their activities are no more needed

T Cell Recognition of Antigen T-cells do not recognize PAMPs or whole pathogens. They recognize antigen peptide fragments bound to specialized cell surface molecules on antigen-presenting cells (APC). Peptides are displayed to T cells as peptide:MHC complexes T cell antigen receptors recognize peptide:MHC complexes Each MHC molecule can bind numerous different peptides

MHC restriction 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 encoded by the most polymorphic gene cluster on the human genome (many alleles)

Before being presented, antigens have to be processed such that they fit in the MHC molecule in a suitable way to be recognized by T-cells

Antigen presentation: 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

Ag is processed through 2 separate pathways: Ag is processed through 2 separate pathways: *MHC I interacts w/ peptides from cytosolic degradation *MHC II interacts w/ peptides from endocytic degradation

Processing Endogenous Antigens: the Cytosolic pathway Cellular proteins are constantly regulated; most have a brief half-life and are “turned over”… the same holds true for endogenous Antigens! Processing of endogenous Ag involves 3 processes: Peptide generation from proteolysis Transport to ER Loading peptide on to MHC I

MHC-I See figure 8-3, Kuby, 6th Edition, p. 194, left

Class I MHC Protein Binding Cleft

10 A

~ 25 A – Can hold Peptides 8-10 amino acids long

~12 A

MHC I binding to peptide Peptide backbone

Ag Site Class I See figure 8-4, Kuby, 6th Edition, p. 195, right

Ag-Bind See figure 8-9(a,b), Kuby, 6th Edition, p. 199

See figure 8-9(c), Kuby, 6th Edition, p. 199 Ag in Site Class I

Class I MHC Space-Filling

H2K and Peptides See figure 7-12, Kuby 4th edition, p. 184

MHC ClassI and Flu

MHC-Class I Polymorphisms Kuby, 6th Edition Figure 8-10(b) p. 201 MHCIPoly Positions of Polymorphic Amino Acid Residues in a Human Class I MHC Protein

Endogenous Ag processing… peptide generation Proteins targeted for lysis combine w/ a small protein  ubiquitin Ubiquitin-protein complex is degraded by a proteosome Specific proteosomes generate peptides which can bind to MHC I

Proteosome 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 synthesized in excess (maintain protein homeostasis) regulatory proteins Only about 1% of the peptide pool is available to immune system

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

26S protease complex 20S proteasome twin 19S caps

TAP, transporter associated with antigen processing structure: TAP-1 and TAP-2 function: transports small peptides (8-13 aa) to the ER

Endogenous Ag processing… transport to ER Peptides from proteolysis bind to a “Transporter protein associated with Antigen Processing” (TAP) TAP is a heterodimer which uses ATP to help transport peptides (8-13 amino acids) to lumen of ER Usually basic amino acids @ COOH end of peptide chain

Trim-peptidases Cytosolic 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 amino acid 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 - mainly involved in epitope destruction (down-regulation enhances presentation).

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 amino acids - endopeptidase activity: trypsin-like specificity There are no carboxypeptidases in the cytosol The proteasomes have to generate C-terminal anchor for MHCI binding

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.

Endogenous Ag processing… peptide binding to MHC I MHC I assembly occurs w/ the aid of chaperone proteins to promote folding (calnexin + MHC I α chain) Tapasin + calreticulin brings TAP/ peptide close to MHC assembly Allows MHC I to bind to peptides MHC I-Ag exits ER to Golgi to plasma membrane

calnexin Structure Function 88kD integral ER membrane chaperone protein Function Binds to a nascent MHC class I a chain after release from a ribosome into the ER lumen so that the a chain will not leave the ER until it binds both a short peptide sequence and b2 microgobulin

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

Molecular chaperones: calnexin, calreticulin,tapasin

Assembly and stabilization of MHC I – Ag complex

Major Histocompatibility Complex Class I Antigen is usually endogenous (e.g. viral proteins). CD8+ cytotoxic T lymphocytes (CTLs) recognize antigen in association with class I MHC on APC. MHC class I is expressed in all nucleated cells.

“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

MHC CLASS II Expressed in Professional Antigen Presenting Cells-APCs Dendritic cells, B-Cells, Macrophages

MHC-II See figure 8-3, Kuby, 6th Edition, p. 194, right

Class II Molecules Antigen is usually extracellular. CD4+ Helper T Lymphocytes recognize antigen in association with class II MHC gene product on APC. Found only on “professional antigen presenting cells”, such as dendritic cells, macrophages, B cells

Peptide binding Cleft

Endocytotic PROCESSING: INVARIANT PROTEIN (Ii) is synthesized with the alpha and beta chains of MHC-II. The invariant chain exists as a trimer and has attached to it three sets of alpha/beta chains.

ANTIGEN PRESENTATION

Antigen Presentation by Macrophage T Cell

Numerous T Cells Interacting with a Single Macrophage

T-cell Receptor and MHC Class I Complex http://www.path.cam.ac.uk/~mrc7/functions/mhc_tcr1.html

SIGNAL TRANSDUCTION BY THE TCR Stimulation of the TCR by antigen-MHC triggers the phosphorylation of tyrosine residues in the cytoplasmic domains of the CD3 chains of the receptor complex. According to a widely accepted model of TCR signaling, Lck and Fyn are responsible for these initial phosphorylation events. 2/9/04

Immune-receptor-Tyrosine-based-Activation-Motif; ITAM