Fundamental Immunology Module 1: Innate and Adaptive Immune Responses Danielle PEREZ-BERCOFF [email protected] BioTech 3 2019-2020 Introduction 3 Modules: • Innate immune response Initiation of adaptive immune response CD4 T cells Danielle Perez Bercoff • T and B ontogeny Humoral response (B cells) (adaptive) Danielle Perez Bercoff • Cytotoxic responses - cellular response : CD8 T cells (adaptive) - NK cells (innate) ?? Module 1: Course outline • Introduction to Immunology • Innate Immunity • The Complement System • Innate response: Cellular effectors - Cellular effectors - Immune sensors - Induction of co-stimulatory molecules • HLA and antigen presentation - Antigen presentation by HLA - Antigen processing • Activation of T lymphocytes Danielle Perez-Bercoff Biotech3:2019-2020 Module 1: Course outline • Introduction to Immunology • Innate Immunity • The Complement System • Innate response: Cellular effectors - Cellular effectors - Immune sensors - Induction of co-stimulatory molecules • HLA and antigen presentation - Antigen presentation by HLA - Antigen processing • Activation of T lymphocytes Danielle Perez-Bercoff Biotech3:2019-2020 Introduction Immunology and vaccinology history Edward Jenner Late of 18th century: Edward Jenner beginning of immunology Vaccinia (mild disease of cowpox) seemed to confer protection against smallpox (often fatal disease) 1796: inoculation with cowpox protected the recipient against smallpox concept of vaccination 1979: WHO announces the eradication of smallpox Immunology and vaccinology history Introduction Robert Koch Paul Ehrlich Nobel Prize 1905 Nobel Prize1908 Robert Koch : Founder of bacteriology. “infectious diseases are caused by specific microorganisms" Identified infectious nature of Tuberculosis, Malaria & Anthrax Tuberculosis is caused by a bacterium (Koch’s bacillus): first to prove it by staining the pathogen, using Ehrlich’s staining methods Louis Pasteur In the 1880s, Louis Pasteur developed a vaccine against cholera in chicken (attenuation) Then developed a rabies vaccine spectacular success upon its first trial in a boy bitten by an infected dog Introduction Basic principles of Immunology Bacteria Viruses Fungi Induction of defense mechanisms 1 Breach in natural barrier Innate Response = Immediate and non specific Local => Inflammatory reaction • primary effectors • cellular effectors 2 Adaptive Response = Specific, acquired Need for time = latency Introduction 2 axes of immune response: overview of immune effectors Immediate elimination of pathogens Non specific response Activation of adaptive IR Not adaptable • Monocytes/M • Dendritic cells • Neutrophils, basophils, eosinophils… • Platelets • NK, NKT, • T-cells Specific response Immune memory • • • • Complement Collectins Cytokines Chemokines • T Lymphocytes • B Lymphocytes • Antibodies • Cytokines • Chemokines Humoral = soluble Innate Immune Response 3 phases in mammalian immune response The response to an initial contamination occurs in three phases: 1 2 • Anatomi barrier • antimicrobial enzymes & peptides • Complement system Cells (innate IR): PAMP PRR 3 Adaptive IR • B + T lymphocytes • Expansion of Ag-specific lymphocytes • memory Introduction Basic principles of Immunology: effector cells Introduction Basic Principles in Immunology: Immune System Network Innate Immunity Adaptive Immunity monocytes macrophages CD4 T lymphocytes basophils eosinophils neutrophils CD8 T lymphocytes Dendritic cells Induction of co-stimulatory molecules platelets B lymphocytes NK , iNKT cells Soluble Factors Cellular interactions Introduction Lymphopoïesis Basic Principles in Immunology: Immune cell precursors Adaptive IR Innate IR Introduction Basic Principles in Immunology: Ilymphoid organs Compartmentalized immune system Primary lymphoid organs (central): • Bone marrow: hematopoiesis & B cell ontogeny • Thymus: T-cell ontogeny Secondary lymphoid organs (or peripheral): Initiation of Adaptive Immune Response lymph nodes (axillary, inguinal, mesenteric) spleen MALT (Peyer‘s patches) + Mucous membrane: skin, intestine, rectum, respiratory system … Trigger of innate response + effector adaptive response Introduction innate Immunity Innate Immunity Becomes an Antigen Presenting Cell (APC) & initiates adaptive Immune Response Epithelial cells Neutrophils Natural Killer Macrophages Pro-inflammatory cytokines & chemokines Dendritic cell Introduction Basic Principles of Immunology: adaptive immunity Adaptive Immunity Active Natural Infection Induced/artificial Vaccination Passive Natural Tansfer of maternal antibodies Induced/artificial Ig Introduction Basic Principles of Immunology: adaptive immunity & memory Immune memory - Immune memory is mediated by T and B cells Primary response ≈ 1st infection, vaccination Secondary response ≈ 2nd infection, vaccination boost Faster more intense longer more diversified Clonal expansion Clonal contraction Latency 1st injection 2nd injection Time Introduction Definitions Antigen (Ag): Substance (toxin, protein) that stimulates an immune response (IR) in the body It is able to specifically react with an antibody or a cellular receptor and able to stimulate antibody production or a cellular reaction Antigen capacity to induce an immune response = immunogenicity Antigen capacity to specifically combine with Ab or T cell receptors = antigenicity Antigen specificity: antigen property to react specifically with a particular antibody or T receptor recognition of a particular antigen structure, the epitope – the site on the surface of the antigen to which an antibody attaches Antibody (Ab): Substance produced in response to antigen administration and able to specifically bind with this antigen After a second infection or injection, the immune response is faster and more intense. It is the immunological memory CELLS of the immune system: All the cells of the immune system are leucocytes originating from the bone marrow from a common precursor, the pluripotent stem cells. Some cells are mature when they leave the bone marrow (BM). Other cells complete their differentiation outside the bone marrow. Introduction Immune system consequences BENEFICIAL HARMFUL - Elimination of infectious pathogens - Elimination of tumoral cells - Immune surveillance - Hypersensitivity (allergy) - Inflammation (chronic diseases) - Auto-immune diseases Module 1: Course outline • Introduction to Immunology • Innate Immunity • The Complement System • Innate respone: Cellular effectors - Cellular effectors - Immune sensors - Induction of co-stimulatory molecules • HLA and antigen presentation - Antigen presentation by HLA - Antigen processing • Activation of T lymphocytes Danielle Perez-Bercoff Biotech3:2019-2020 Innate Immune Response The agents that cause disease Five groups of pathogen: viruses, bacteria, fungi, protozoa and helminths (worms) Two major compartments: extracellular and intracellular Cell damage or killing Innate Immune Response The agents that cause disease Different ways for pathogens to damage tissues Non-secreted constituents of bacterial structure Lipopolysaccharide (LPS) of the outer cell membrane of gram-neg bacteria many of the symptoms of infection (fever, pain, rash, hemorrage, septic shock) are due largely to LPS 2 Innate Immune Response Natural barriers: epithelial surfaces Epithelia = physical barrier • Skin • mucosal epithelia : respiratory, urogenital and gastrointestinal tracts • Tight junctions: seal against external environment • Cilia, flow and movement (peristalsis in the gut) 2 Innate Immune Response Natural barriers: epithelial surfaces Epithelia = physical barrier + microbiological barrier gut commensal flora (= 1014 bacteria) skin, mouth commensal flora produces antimicrobial substances strengthens the barrier functions of epithelia 2 Innate Immune Response Natural barriers: epithelial surfaces Epithelia = physical barrier + microbiological barrier + chemical barrier: Mucus (prevents microorganism adhesion) pH (skin, stomach) Digestive enzymes and fatty acids Soluble factors (antibacterial enzymes and peptides) Innate Immune Response Epithelial damage Pathogens overcome innate host defenses to establish infection Spread countered by inflammatory response & blood clotting Introduction Basic principles of Immunology Humoral = soluble Innate Immune Response Primary effectors : antibacterial enzymes Antimicrobial proteins: - secreted by epithelial cells and phagocytes - mucosal tissue protection Lysozyme (tears, saliva, phagocytes, paneth cells) Enzyme: glycosidase cleavage of peptidoglycan component of the bacterial cell wall more effective on Gram (+) bacteria than Gram (-) because of LPS layer Secretory phospholipase A2 (paneth cells) Highly basic enzyme Insertion in bacterial cell wall phospholipid degradation Death of bacteria PLA2 Innate Immune Response Primary effectors : antimicrobial peptides Antimicrobial peptides: - ancient, evolutionnary conserved peptides - generated by proteolytic processing from inactive propeptides α and β-Defensins (amphipatic structure) (epithelial cells, phagocytes) Disrupt the cell membrane of bacteria and form a pore within minutes Cathelicidins Reg III “Lecticidins” (C-type lectins) Innate Immune Response Primary effectors Platelets: - coagulation, wound healing - production of several mediators: * vaso-active: histamines – serotonine dilation → œdema constriction → pain * inflammatory: prostaglandin - leucotriene – chemoattractants Collectins : Surfactant protein A & D (SP-A and SP-D), MBL - scavenger receptor - present in fluid that bathes epithelial surface of the lung - Pathogen opsonization neutralization + phagocytosis - Activation of complement cascade Opsonization: process of coating the surface of pathogens, making them more susceptible to phagocytosis by macrophages Module 1: Course outline • Introduction to Immunology • Innate Immunity • The Complement System • Innate respone: Cellular effectors - Cellular effectors - Immune sensors - Induction of co-stimulatory molecules • HLA and antigen presentation - Antigen presentation by HLA - Antigen processing • Activation of T lymphocytes Danielle Perez-Bercoff Biotech3:2019-2020 Innate Immune Response Complement system - Introduction • Complement system : • Enhances (complements) the ability of antibodies and phagocytes to clear microbes and damaged cells • Discovered in the 1890s by Jules Bordet as a heat-labile substance (loss of ability to neutralize cholera when serum is heated) • 30 soluble proteins (liver production, plasma circulation) • Enzymatic cascade the majority of proteins are proteases • highly regulated system Jules Bordet Nobel Prize 1919 • Serum from Guinea pigs surviving immunization with cholera kills the bacterium in vitro. • Heat removes this ability • Protective activity maintained when heated serum injected to new Gp 2 components: Heat-labile + heat-resistant Believed complement was aspecific. Paul Ehrlich Nobel Prize1908 • Introduced the term complement • His theory: immunization induces specific receptors (Ab) on cells, which are shed • Named amboceptors b/c bind Ag + complement (heat-labile component) • Believed each Ag had its own specific complement. Believed complement was specific. Complement can act in combination with Ab or on its own Innate Immune Response Complement system - Nomenclature Nomenclature : • Small molecules circulating in blood, produced mainly by hepatocyes or M • Synthesized as inactive proteins which are activated by proteolytic cleavage and activate one-another = zymogens (pro-enzymes) • Inactive complement molecules : Cn factor for classical pathway or letter for alternative pathway • Active complement molecules with suffix ‘a’ (small fragment) or 'b’ (large fragment) • Ex: C4 is cleaved into C4a (small fragment) & C4b (large fragment with catalytic activity) • Exceptions: historically C2a was the large fragment with catalytic activity, but now called C2b. C1q, C1r and C1s 3 different components making up C1 • Complement receptor: CR Innate Immune Response Complement system – Complement cascade Inactive zymogens infection Activation of the cascade by serial proteolytic cleavage Amplification of the signal Stimulation of phagocytes to clear Ag via opsonzation Inflammation to attract additional phagoytes & neutrophils Cell or pathogen lysis by Membrane Attack Complex (MAC) Large fragment Small fragment Large fragment C3b C3a, C4a, C5a C5b Innate Immune Response Complement system - Introduction • 3 biochemical systems activate the complement cascade: 1. Lectin (MBL & ficolins) 2. Classical (C1q – Ab) 3. Alternative (spontaneous hydrolysis) • C3 convertase: - Cleaves C3 C3a + C3b - Lectin & Classical pathways: C4bC2b - Alternative pathway: C3b(2)Bb • C3b: - main central molecule (coating of bacteria) - Influences adaptive immunity: Opsonization enhances pathogen presentation (Ag) to T cells by APCs B cells express complement Rec (CR) Inflammation Phagocytosis Lysis Chap I. Innate Immune Response Complement system - Classical pathway Classical pathway • Initiated by C1 complex: large subunit C1q (pathogen sensor) + 2 serine proteases (C1r and C1s) = C1qr2s2 • C1q: Collectin N-terminal globular domain C-terminal collagen-like domain is a hexamer of trimers • Main function of C1q is to bind to the constant region of antibodies (Fc portion) attached to pathogens via their Agbinding-site (Fab) links complement to recognition provided by adaptive immunity (natural Ab = Ab produced without infection) • Mainly binds pentameric IgM and IgG (ideally 6 IgG complexed to Ag) • C1q can bind to the surface of pathogens • Acts as link between innate & adaptive immunity Chap I. Innate Immune Response Complement system - Classical pathway Classical pathway • Binding of C1q globular heads to pathogen conformational change in (C1r:C1s)2 Activation of the C1r (serine protease) autocleavage C1r cleaves the 2 C1s (serine protease) C1s cleaves: C4 C4a + C4b C2 C2a + C2b Binding of C4b + C2b = C4b2b = classical C3 convertase C3 C3b C3a N.B: C2b is the active component of C3 & C5 convertases Innate Immune Response • Similar to Classical pathway Instead of C1q • Complement system - Lectin pathway Lectin pathway Initiated by: - Mannan-Binding Lectin (MBL) a collectin synthesized in the liver N-terminal collagen-like domain C-terminal C-type lectin domain Assembled in trimers High Avidity for repetitive carbohydrate structures present on microbial surface - Ficolins (L-, M- and H- ficolin) Synthesized in liver, lung and blood cells N-terminal collagen-like domain C-terminal fibrinogen-like domain Specificity for acetylated sugars • Associated with serine protease: MBL-associated serine protease (MASP-1 & -2) instead of C1r & C1s • Binding of MBL to mannose activates MASP-1 (~C1r) Activation of MASP-2 (~C1s) Chap I. Innate Immune Response Complement system - Classical pathway Lectin pathway • Binding of MBL or Ficolin to pathogen surface conformational change in MBL Activation of MASP-1 (serine protease) MASP-1 cleaves MASP-2 (serine protease) MASP-2 cleaves: C4 C4a + C4b C2 C2a + C2b Binding of C4b + C2b = C4b2b = C3 convertase C3 C3b C4b2b C4b2b C4b2b C2b C4b2b C4b2b C3a Innate Immune Response Complement system - Alternative pathway Alternative pathway : amplification of C3b • Alternative pathway C3 convertase : C3bBb • Special place because it can generate more of itself once some C3b has been formed by whichever pathway, the alternative pathway acts as an amplification loop C3b • Activation by two different ways: * classical or lectin pathway : C3b linked to surface of pathogen alters the conformation of factor B factor D (a protease) cleaves Factor B into Ba + Bb = C3 convertase (C3bBb) * spontaneous hydrolysis Innate Immune Response Complement system - Alternative pathway Alternative pathway: Spontaneous hydrolysis Spontaneous hydrolysis (C3H2O) due to breakdown of internal thioester bond If no pathogen C3b rapidly inactivated by Factor H & Factor I If Pathogen ++++ Hydrolyzed C3b reacts with –OH or -NH3+ group on surface of pathogen No lysis by factor H Binding of factor B Factor B is cleaved by Factor D (protease) Alternative C3 convertase C3bBb or C3(H2O)Bb C3bBb is stabilized by properdin (factor P) Innate Immune Response Complement system - Alternative pathway Amplification of C3b lysis • Stabilized C3 convertase C3bBbP cleaves more C3 C3b binds B, D & P (+) feedback loop & signal amplification Alternative pathway acts as amplifcation system Role of C3 convertase : decorate Bacteria with C3b • C3bBb accumulates on non-self surface & binds a 2nd C3b C3bBbC3bP = C5 convertase • C3 signal on cell surface is limited by endogenous regulatory proteins : CD35, CD46, CD55 (DAF), CD59 Compete with Bb for binding C3b (Factor H, CR1, DAF) or cleave it (Factor I) no self-destruction • Pathogens lack complement inhibitors Distinction between self and non-self • N.B: some pathogens evade complement response by expressing complement regulatory proteins Innate Immune Response Complement system - C3b & C5 convertase • C3 & C5 convertase activity: C4b2b • Different C3 convertases cleave C3 to form C3b + C3a C5 convertase = - Classical pathway = C4b-C2b-C3b - Alternative pathway = C3b2-Bb C3a intiates local inflammation • opsonizes pathogen to facilitate phagocytosis C4b2b3b C4b2b3b C3b • C4b2b3b & = C5 convertase C3bBb3b • C5 convertases bind C5 cleave C5 • N B: C3 is the most abundant complement protein in plasma (1.2 mg/mL) up to 1000 molecules of C3b can bind in the vicinity of a single active C3 convertase C4b2b3b C2b C4b2b3b Innate Immune Response Complement system C3 convertase: - Classical pathway = C4bC2b Alternative pathway = C3bBb C5 Convertase: - Classical pathway = C4bC2bC3b Alternative pathway =C3b2Bb Innate Immune Response Complement system - opsonization and phagocytose First function of complement system: opsonization • Facilitate the uptake and destruction of pathogens by phagocytic cells • Occurs through the specific recognition of bound complement by complement receptors (CRs) * CR1 (CD35) = C3b Rec with the help of C5a (Rec for C5a is G-prot) * CR2 (CD21), CR3 (CD11b), CR4 (CD11c) and CRIg bind C3b or its inactive forms • Phagocytosis by M: pathogen destruction Phagocytosis by DCs: pathogen destruction + Ag presentation • deficiency in C3 susceptibility to infection by a wide range of extracellular bacteria (Streptococcus pneumonia) Cellular distribution of CR CR1 (CD35) : mono, PMN, B cells, fDC CR2 (CD21) : B cells, fDC CR3 (CD11b/CD18) : mono, fMN, DC CR4 (CD11c/CD18) : mono, PMN, DC Innate Immune Response Complement system - Local inflammatory response Second function of complement system: local inflammatory response • The small complement fragments C3a & C5a act on Chemokine (CC) Receptors on endothelial cells and Mast cells to produce inflammatory response • N.B: If produced in a large amounts, C5a & C3a could induce anaphylactic shock referred to as anaphylatoxins (C5a > C3a > C4a) • Increase vascular permeability • Induce contraction of smooth muscles • Induce synthesis of adhesion molecules by endothelial cells • Induce release of inflammatory molecules (TNF-) by Mast cells • Recruit Ab, complement and phagocytic cells out of blood to the site of infection • Increased fluid hastens the movement of APC to the LN initiation of adaptive IR Chap I. Innate Immune Response Complement system - Membrane-Attack Complex (MAC) Third function of complement system: MAC • First step: C5 convertase cleaves C5 release of C5b • C5b triggers assembly of a complex of one molecule of each C6, C7 & C8 • Conformational change of C7 hydrophobic site inserted in the membrane • C8 binds to C5b hydrophobic domain of C8- inserts in mb & hydrophilic domain of C8- induces polymerization • Up to 16 molecules are added to the assembly generate a channel disrupt the bacterial cell membrane and kill the bacteria • N.B: these components seem to be quite limited in host defenses opsonizing and inflammatory actions of the complement system are more important for host defense against infection Chap I. Innate Immune Response Complement system - Regulation Regulation of complement activation • Activation events must remain confined to site of infection /bacterium where it is initiated • Cleavage of C4b exposes a highly reactive thioester bond (thioester bond: S-C=O) C4b can bind covalently carbohydrates on pathogen or adjacent proteins If no bond, thioester bond is cleaved by H2O inactivation • C2 susceptible to cleavage by C1s only when bound to C4b C2b active only when bound to C4b i.e in form of C3 convertase C4b2b Innate Immune Response Complement system - Regulatory proteins 27 Regulatory proteins of complement system (on cell surface: avoid cleavage of cells) • C1 inhibitor (C1 INH, serpin) = a serine protease inhibitor C4b2b b C2b C4b2b • DAF(CD55), C4BP and CR1 displace C2b from C4b + cleave C4b to an inactive form C4b2b • Factor I (protease) cleaves C3b to iC3b (with help of co-factors) • CR1 (CD35) and Factor H displace C3b and act as cofacteurs in the cleavage of C3b by Factor I • CD59 prevents final assembly of MAC at the C8 to C9 stage C4b2b3b C4b2b Innate Immune Response Complement system - Regulatory proteins Summary of Regulatory proteins b b Regulation of all three pathways of complement activation to protect the host from destructive effects Innate Immune Response Pathogens produce several types of proteins that can inhibit complement activation: • Express proteins that bind to soluble regulatory proteins (C4BP or Factor H) Ex: Factor H binding protein (fHbp) & PorA Neisseria meningitidis inactivation of C3b • Secrete proteins that directly inhibit components of complement Ex: Staphylococcus aureus produces: - Staphylococcal protein A (Spa) binds to Ab and interferes with C1 recruitment - Staphylokinase (SAK) cleaves Ig Inhibition of complement activation + avoid phagocytosis - Staphylococcal component inhibitor (SCIN) binds C3 convertase Inhibition from classical & alternative complement pathways Inhibition of complement by pathogen Innate Immune Response Complement system – summary Complement cascade
