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LECOM-Pharmacy School BMS/PDA II-Immun 4 Adaptive (acquired) immune response Part 1 Antigen & Antibody Dr. Saber Hussein  Learning Objectives 1. Define antigenicity and immunogenicity 2. Know the four characteristics of immunogenic molecule (...

LECOM-Pharmacy School BMS/PDA II-Immun 4 Adaptive (acquired) immune response Part 1 Antigen & Antibody Dr. Saber Hussein  Learning Objectives 1. Define antigenicity and immunogenicity 2. Know the four characteristics of immunogenic molecule (requirements for immunogenicity) – Foreignness – High molecular weight – Chemical complexity – Degradability 3. Define haptens and their functions 4. Define antigenic determinants 5. Capture of protein antigens by antigen presenting cells 6. Antigen recognized by T lymphocytes 7. Antigens recognized by B lymphocytes  Learning Objectives 8. Basic structure of Abs in relation to specificity & diversity 9. Variable & constant regions of light & heavy chain 10.Biological & chemical characteristics of the 5 classes of Ab 11.Compare polyclonal & monoclonal Ab 12.Three characteristics of primary Ag-Ab reaction 13.The forces that foster the primary Ag-Ab reaction 14.Affinity & avidity of Abs 15.Secondary Ag-Ab reaction; lattice formation  Learning Objectives 16.Know the role of receptors in the recognition of antigen in the adaptive immune system: – Antibodies as receptors of B lymphocytes – T cell receptor (TCR) for antigens 17.Development of immune repertoires 1. Maturation of lymphocytes 2. Production of diverse antigen receptors 3. Maturation and selection of B lymphocytes 4. Maturation and selection of T lymphocytes  Antigenicity & Immunogenicity Antigenicity: – The ability to bind an Ab or an activated T cell – Every immunogen is an antigen, BUT not every antigen is an immunogen Immunogenicity: – Ability to elicit immune response – Only proteins can induce cellular immunity – Humoral immunity can be induced by: Proteins Lipopolysaccharides Nucleic acids Other substances  Features of an Immunogen 1. High molecular weight 2. Chemical complexity 3. Solubility or biodegradability 4. Foreignness or nonself Ab cross-reactions with different Ags Abs react most strongly with homologous Ag Sometimes they cross-react with other Ags Cross-reaction – The reaction between an Ag and an Ab that was generated against a different Ag but with some similarity with the cross-reacting Ag Cross-reactions are related to chemical structure of Ags: – i. Chemical nature of hapten’s groups – ii. Position of substitutions – iii. Size of substituted groups – iv. Charge – v. Stereoisomerism  Haptens, Antigenic Determinants (Epitopes) Ag has 2 functional regions: Hapten 1. Hapten Carrier 2. Carrier Ab Epitopes are immunologically active portions of Ag Epitopes on an Ag are recognized by B cells and T cells Antigenic determinants serve as fingerprint of macromolecules Size of an epitope is determined by the size of the Ab’s Ag-binding site Size of recognizable epitope by an Ab: – 6 sugar residues – 15-20 amino acids (Some linear epitopes are as small as 5 aas)  Haptens &Antigenic Determinants Haptens Antigenic Determinants 1. Usually small molecules 1. Small part of the 2. Not immunogenic by themselves molecule 3. Always antigenic with a specific – Few amino acids Ab – A short carbohydrate 4. Immunogenic when combined with moiety- few sugars a carrier molecule (large) 2. Must be accessible to 5. Simple hapten: only 1 antigenic be functional determinant 6. Complex hapten: > 2 antigenic 3. Charge & polarity determinants 4. Conformation dependent  Ag recognized by T lymphocytes T lymphocytes recognize only protein antigens Proteins must be presented in the form of short peptides They must be presented by an APC with the appropriate MHC molecule: – MHC I presents antigen to the cytotoxic T cell – MHC II presents antigen to the helper T cell Antigens recognized by B lymphocytes T-cell independent route of antigen recognition: – B lymphocytes recognize certain antigens without the help of the TH cell – These include: Lipopolysaccharides Nucleic acids: DNA & RNA – No long term immunity results through this route – Only IgM is produced – No memory cells Antigens recognized by B lymphocytes T-cell dependent route: – Recognizes protein antigens – Long term immunity – IgG is produced by class switching – Memory cells  Antigens recognized by B lymphocytes T-cell dependent T-cell independent 1. Recognizes 1. Recognizable Ags: protein antigens Lipopolysaccharides 2. Long term Nucleic acids: DNA & immunity RNA 3. IgG is produced by 2. No long term class switching immunity results through this route 4. Memory cells 3. Only IgM is produced 4. No memory cells  A model of how a T cell receptor Antigen Capture and (TCR) recognizes a complex of a peptide antigen displayed by a Presentation to major histocompatibility (MHC) Lymphocytes molecule – MHC molecules are expressed on antigen-presenting cells and function to display peptides derived from protein antigens – Peptides bind to the MHC molecules by anchor residues, which attach the peptides to pockets in the MHC molecules – The TCR of every T cell recognizes some residues of the peptide and some (polymorphic) residues of the MHC molecule Fig. 3-1 The capture and display of microbial antigens Microbes enter the body – through an epithelium and are captured by antigen-presenting cells resident in the epithelium – enter lymphatic vessels or – blood vessels The microbes and their antigens are transported to peripheral lymphoid organs – the lymph nodes, – the spleen, where protein antigens are displayed for recognition Fig. 3-2 by T lymphocytes  The capture and Immature dendritic cells in the epithelium capture presentation of protein microbial antigens by antigens by dendritic cells surface receptors and leave the epithelium The dendritic cells migrate to draining lymph nodes, being attracted there by chemokines produced in the nodes Langerhans cells During their migration, and in response to the microbe, the dendritic cells mature Mature DC express high level MHC & costimulators In the lymph nodes, the dendritic cells present Fig 3-4 antigens to naive T cells Properties of MHC molecules and genes Ann Dunham X Ba Ob Sr. Some of the important features of MHC molecules are listed, with their significance for immune responses Fig 3-8 Role of MHC in Antigen Presentation to T Cells Ag processing – The event whereby the Ag is prepared to be presented to lymphocytes in a form they can recognize – It includes fragmentation of the protein Ag into small peptides in the macrophage and the presentation to T cells Ag-presenting cells (APCs) bind peptide Ags to their MHC II and present it to the CD4+ helper T cells APCs present peptides to CD8+ cytotoxic T cells with their MHC I Ag-presenting cells (APCs) include macrophages and other cells Peptide binding site m Major Histocompatibility Complex (MHC)  MHC Restriction of T Cells The process by which the MHC controls interactions between immune cells It involves the recognition of foreign antigens in association with MHC I or II molecules The following reactions are MHC-restricted: 1. Antigen presentation 2. T- and B-cell cooperation 3. Cytotoxic T-cell interaction with target cells Malignant cells Viral infected cell  What Regions of HLA Complex Encode MHC I & MHC II? Coding Regions: MHC-I coding region: – HLA-A, HLA-B and HLA-C MHC-II coding region: – HLA-D [DN, DO, DP, DQ & DR]  Antigen-MHC Class II Complex What kind of T cell do we see here?  Fig 4-1: Properties of antibodies and T cell antigen receptors (TCRs) Antibodies may be expressed as membrane receptors or secreted proteins TCRs only function as membrane receptors When Ig or TCR molecules recognize antigens, signals are delivered to the lymphocytes by proteins associated with the antigen receptors The antigen receptors and attached signaling proteins form the B cell receptor (BCR) & TCR complexes Single antigen receptors are shown recognizing antigens, Signaling requires the cross-linking of two or more receptors by binding to adjacent antigen molecules Fig 4-1 Fig 4-1 CD4,8 B cell T cell Fig 4-1 (z :zeta) Fig 4-2: The structure of antibodies Schematic diagrams of a secreted IgG (A) and a membrane form of IgM (B) illustrate the domains of the heavy and light chains and the regions of the proteins that participate in antigen recognition and effector functions N and C refer to the amino-terminal and carboxy- terminal ends of the polypeptide chains, respectively Fig 4-2  Fig 4-3: Features of the major isotypes (classes) of antibodies The table summarizes some important features of the major antibody isotypes of humans. Isotypes are classified on the basis of their heavy chains Each isotype may contain either k or l light chain Each of the 5 classes differ in their locations in our body and how they stimulate the innate system to remove antigen The schematic diagrams illustrate the distinct shapes of the secreted forms of these antibodies IgA consists of two subclasses: IgA1 and IgA2 IgG consists of 4 subclasses: IgG1, IgG2, IgG3, & IgG4 The serum concentrations are average values in normal individuals Fig 4-3: Features of the major isotypes of Abs with mother’s milk Breast-fed neonates get it with the mother’s milk Antiparasitic activity Fig 4-3: Features of the major isotypes of Abs Agglutination Diagnostic for acute infections  Polyclonal & Monoclonal Abs Polyclonal Abs Monoclonal Abs 1. Heterogeneous mix of 1. Produced by a single Abs clone of cells 2. With specificity against 2. Resultant Abs are the same Ag identical in all aspects 3. Produced by variety of 3. Same affinity Ab-producing cells 4. Same binding specificity 4. They are many clones of 5. Recognize the same cells epitope 5. Polyclonal Abs recognize 6. They are produced in & react against different hybridoma between epitopes on the Ag activated B cells and 6. Avidity malignant plasma cells (fusion) Three dimensional representation of the IgG molecule IgG molecule  IgG IgG digestion with papain produces 3 fragments – 2 identical Fab fragments Fab fragments, are capable of binding Ag because they contain the Ag-binding site – Fc fragment: a fragment composed of H chain only. It crystallizes in the cold  Pentameric structure of IgM The structure of IgM is similar to that of IgG except the IgM heavy chain has an extra domain. A small, cysteine-rich protein called J chain initiates cross linking of C3 and C4 of – five IgM monomers to make the circulating, pentameric form of IgM Dimeric structure of IgA Dimeric IgA held together by – J chain and – secretory component J chain Secretory component Secretory IgA IgA represents 15-20% of serum immunoglobulin It constitutes the majority of Ab found in secretions Humans have 2 types of IgA: – IgA1 and IgA2 – IgA1is the prominent subclass in serum and is found mainly as monomer – IgA2 is the prominent Ig in secretions (saliva, gut, respiratory mucus) and occurs as a dimer with two Fc ends of the Abs bound together by a J chain Secretion across the mucosa is mediated by a specific secretory component which binds to a cell receptor  IgE IgE is similar to IgG except – it has an extra constant region domain on the H- chain Functions: – Type I hypersensitivity – Anti-parasitic – Degranulation of mast cells  IgD IgD is similar to the structure of IgG. Its only known function is as part of the signaling complex of B cells  Fig 4-4: Binding of an Ag by an Ab This model of a protein antigen bound to an antibody molecule shows how the antigen-binding site can accommodate soluble macromolecules in their native (folded) conformation. The heavy chains (H) of the antibody are red L chains: yellow Ag is blue  Chemical forces foster Ab-Ag Four noncovalent interactions hold antigenic determinants w/in Ab-binding site: 1. Coulombic (electrostatic, ionic) interactions 2. Van der Waals forces 3. Hydrogen bonds 4. Hydrophobic interactions  Secondary Ag-Ab reaction & Secondary response Secondary Ag-Ab reaction: The conversion of the invisible primary reactions macroscopically visible ones as in the case of precipitation and agglutination Secondary response: The immune response which follows a second encounter with a particular Ag It is usually stronger (affinity maturity)  Lattice formation Ab Ag Occurs when i. Ag-Ab complexes aggregate in form of precipitation in liquid medium - ii. Agglutination, including particulate components, other than Ag and Ab, such as cells  Affinity & Avidity Affinity is the strength of Ag-Ab bonds between a single epitope and an individual Ab’s binding site Avidity: The binding strength between a multivalent Ab (polyclonal Ab) and a multivalent Ag Ag + Ab → Ag..Ab K = [Ag..Ab]/[Ag][Ab] The higher [Ag..Ab], the larger is K (the associated Ab and Ag), the higher is affinity of the Ab to the Ag. K = Equilibrium constant = Association constant = Ab affinity Numerator/denominator  Affinity maturity Ag + Ab → Ag..Ab KD = [Ag][Ab] / [Ag..Ab] The lower the KD (dissociation constant) the higher the affinity Affinity maturity: after repeated exposure to the Ag the affinity increases  Monoclonal Ab production Immunize animals, rats or mice, with Ag When the animals start to make a good Ab response remove their spleens and prepare a cell suspension Fuse spleen cells with a myeloma cell line by the addition of polyethylene glycol (PEG), which promotes membrane fusion – Only a small proportion of the cells fuse successfully The fusion mixture is then set up in culture with medium containing “HAT” – HAT = Mixture of Hypoxanthine Aminopterin (powerful toxin that blocks a metabolic pathway) Thymidine (H & T intermediate metabolites help the cell bypass the pathway when added) – Spleen cells can grow/survive in HAT – Myeloma cells are sensitive to HAT because of metabolic defect that prevents them from using the bypass – HAT culture contains: Spleen cells: die naturally in 1-2 weeks Myeloma cells: Killed by HAT Fused cells (hybridoma): Survive because of immortality of myeloma and HAT-resistance of the spleen cells – Some produce antibody – Any wells containing growing cells are tested for the production of the desired Ab (often by solid phase immunoassay) – Positive ones are cloned by plating out so that there is only one cell in each well – This produces a clone derived from a single progenitor, which is both: » Immortal » Producer of monoclonal Ab Humoral Immune Response  B cells produce Abs B cells are specialized white blood cells produced in the bone marrow. Each B cell contains multiple copies of one kind of antibody as a surface receptor for antigen. The entire population of B cells has the ability to specifically bind to millions of different antigens When the antibody on the surface of a B cell binds to an antigen, the cell can be stimulated to undergo proliferation and differentiation. This process is called clonal selection.  Clonal selection The cells produced make the same Ab, but become memory cells and plasma cells – Memory cells insure that subsequent infections by the pathogen receive a more rapid response. – Plasma cells secrete large amounts of the Ag-specific Ab T helper cells are required for the clonal selection of B cells Ab secreted by plasma cells forms complexes with free pathogens and their toxic products The complexes can: – inactivate pathogens & – stimulate other innate systems including phagocytes and complement to eliminate the danger from our extracellular fluids  Abs and their diversity An Ab immunoglobulin is a "Y" shaped molecule made up of two identical "light" and "heavy" chains of amino acids. The variable region includes the N-terminal 110- 130 amino acids of the light and heavy chains, and is responsible for binding to antigen. The constant region is the C-terminal end and contains similar amino acids for each class of Ab.  Abs Diversity (con) When a stem cell changes to become a B cell, DNA segments for both heavy (VDJ) and light (VJ) chains are randomly combined. Each B cell ends up with functional genes for making one light and one heavy chain coding for an Ab as a membrane-bound receptor. Ab specificity depends on the gene fragments used. Abs are produced that can react with almost any chemical structure in nature.  Abs Diversity The immune system creates billions of different Abs with a limited number of genes by rearranging DNA segments during B cell development, prior to Ag exposure. Mutation can also increase genetic variation in Abs  Ab Class switching At first, B cells contain IgM molecules only. Class switching occurs after Ag binding, when plasma cells are produced. Class switching refers to a DNA rearrangement changing the heavy chain constant gene in memory cells. Loss of coding regions for the constant part of the heavy chain causes IgG, IgA, and IgE to be produced. Ab Class switching to produce IgA  Part II Effector Function of Antibodies (Humoral Immunity)  Learning Objectives Know the phases of B cell development/humoral immune response Know the primary and secondary antibody response Know the role of complement (C3d) in B cell activation Understand affinity maturation of antibodies Understand the mechanism of feedback inhibition of B cell by IgG-antigen complex Know the effector functions of antibodies: – Neutralization, opsonization, ADCC, …. Know the mechanisms by which microbes evade antibodies  Phases of humoral immune responses Naive B lymphocytes recognize antigens Under the influence of helper T cells and Fig.7-1 other stimuli the B cells are activated to proliferate This gives rise to: – clonal expansion, and – differentiation into antibody-secreting effector cells Some of the activated B cells undergo: – heavy chain class switching and – affinity maturation Some become long- lived memory cells  Primary and secondary Features of primary and antibody responses differ in several respects. secondary Ab responses In a primary response – naive B cells in peripheral Fig.7-2 lymphoid tissues are activated to proliferate and differentiate into antibody- secreting cells and memory cells – Some antibody-secreting plasma cells may migrate to and survive in the bone marrow for long periods In a secondary response, – memory B cells are activated to produce larger amounts of antibodies, often with more heavy chain class switching and affinity maturation  Features of primary and secondary Ab responses Fig.7-2 Features of secondary responses such as: heavy chain class switching and affinity maturation are seen mainly in responses to protein antigens, because these changes in B cells are stimulated by helper T cells and only proteins activate T cells. The kinetics of the responses may vary with different antigens and types of immunization.  The role of the complement protein C3d in B cell activation Activation of complement by microbes leads to the binding of a complement Fig7-4 breakdown product, C3d, to the microbes. The B cell Ig receptor recognizes a microbial Complement antigen and the CR2 receptor receptor recognizes bound AgR C3d CR2 is attached to a complex of proteins (CD19, CD81) that are involved in delivering activating signals to the B cell  Affinity maturation in antibody responses Analysis of several individual antibodies produced by different clones of B cells against one antigen at different stages of primary, secondary, and tertiary immune responses shows that: – with time and repeated immunization the antibodies that are produced contain increasing numbers of mutations in their antigen-binding regions (the complementarity-determining regions [CDRs]) The antibodies also show increasing affinities for the antigen, as revealed by the lower dissociation constants (Kd) These results imply that the mutations are responsible for the increased affinities of the antibodies for the immunizing antigen Secondary and tertiary responses refer to responses to the second and third immunizations with the same antigen  Affinity maturation in antibody responses Fig7-11 Selection of high-affinity B cells in germinal centers Some of the B cells that are activated by antigen with help from T cells – Migrate into follicles to form germinal centers – There, they undergo rapid proliferation and – Accumulate mutations in their Ig V genes The mutations generate B cells with different affinities for the antigen Follicular dendritic cells (FDCs) display the antigen Only B cells that recognize the antigen are selected to survive  Selection of high-affinity B Fig 7-12 cells in germinal centers FDCs display antigens: – by binding immune complexes to Fc receptors or – by binding immune complexes with attached C3b and C3d complement proteins to C3 receptors As more Ab is produced, the amount of available Ag decreases, so the B cells that are selected must express receptors with higher affinities to bind the Ag FDCs express CD40L, and germinal centers contain a few T cells that also express CD40L CD40L delivers survival signals to the B cells that recognize Ag on the FDCs Fig 7-14 Fig 7-14  Secreted IgG Abs form immune Mechanism of Ab complexes (Ag-Ab complexes) feedback inhibition with residual antigen The complexes interact with B Fig 7-15 cells specific for the Ag in 2 ways: 1. BCR-Ig recognizes & binds the Ag 2. Fc receptor (Fc RII) recognizes the Ab of the Ag-Ab complexes The Fc receptors block activating signals from the Ag receptor → thus terminate B cell activation The cytoplasmic domain of B cell Fc RII contains an immunoreceptor tyrosine-based inhibition motif (ITIM) that binds enzymes that ITIM inhibit antigen receptor-mediated B cell activation.  Fig 8-1 A Effector functions of Antibodies  The effector functions of antibodies Abs are produced by the activation of B Lymphocytes by Ags and other signals Abs of different heavy chain classes(isotypes) perform different effector functions Fig 8-1B  Neutralization of microbes and toxins by antibodies Fig8-2A A. Antibodies prevent the binding of microbes to cells and thus block the ability of the microbes to infect host cells  Neutralization of microbes and toxins by antibodies Fig8-2B B. Antibodies inhibit the spread of microbes from an infected cell to an adjacent uninfected cell.  Neutralization of microbes and toxins by antibodies Fig8-2C C. Antibodies block the binding of toxins to cells and thus inhibit the pathologic effects of the toxins  Antibody-mediated opsonization and phagocytosis of microbes A. Antibodies of certain IgG subclasses bind to microbes and are then recognized by Fc receptors on phagocytes Signals from the Fc receptors promote the phagocytosis of the opsonized microbes and activate the phagocytes to destroy these microbes Fig 8-3A Antibody-mediated opsonization and phagocytosis of microbes B. The different types of human Fc receptors, and their cellular distribution and functions, are listed Fig 8-3B  Splenectomy, phagocytosis & infections The spleen contains large number of phagocytes It is important for phagocytic clearance of opsonized bacteria ➔ Splenectomy makes patient susceptible to disseminated infections by encapsulated bacteria Encapsulated bacteria are cleared by antibody- mediated phagocytosis Without opsonization bacteria with capsule are able to escape phagocytosis Antibody-dependent cellular cytotoxicity (ADCC) Antibodies of certain IgG subclasses bind to cells (e.g., infected cells), and the Fc regions of the bound antibodies are recognized by an Fcg receptor on NK cells The NK cells are activated and kill the antibody-coated cells Fig 8-4A Antibody-dependent cellular cytotoxicity (ADCC) IgE antibodies bind to helminthic parasites, and the Fc regions of the bound antibodies are recognized by Fce receptors on eosinophils. The eosinophils are activated to release their granule contents, which kill the parasites Fig 8-4B  Transport of IgA through epithelium In the mucosa of the gastrointestinal and respiratory tracts, – IgA is produced by plasma cells in the lamina propria and is actively transported through epithelial cells by an IgA-specific Fc receptor (called the poly-Ig receptor because it recognizes IgM as well). – On the luminal surface, the IgA with a portion of the bound receptor is released Fig8-9 Here the Ab recognizes ingested or inhaled PIgR microbes and blocks their entry through the epithelium  Evasion of humoral immunity by microbes The principal mechanisms by which microbes evade humoral immunity are listed, with illustrative examples (Streptococcus pneumoniae)  Vaccination strategies Different types of vaccines induce different protective immune responses