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by: Frankie Kilback

Microbiology MICB 702

Frankie Kilback
GPA 3.83


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This 61 page Class Notes was uploaded by Frankie Kilback on Saturday September 12, 2015. The Class Notes belongs to MICB 702 at West Virginia University taught by Staff in Fall. Since its upload, it has received 14 views. For similar materials see /class/202685/micb-702-west-virginia-university in Microbiology at West Virginia University.

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Date Created: 09/12/15
OBJECTIVES Innate Immunity Students should understand innate immunity with regards to anatomic barriers physiologic barriers endocytic and phagocytic barriers complement amp the complement cascade molecular basis of inflammation pathogen associated molecular patterns PAMPs pattern recognition receptors PRRs Cells and Tissues of the Immune Response 1 Students should be able to identify and distinguish primary and secondary lymphoid organs understand hematopoiesis and describe the features of a pluripotent hematopoietic stem cell Students should be able to describe the organization of the spleen thymus and peripheral lymph nodes Students should be able to distinguish Bcells Tcells and antigenpresenting cells APC on the basis of function and expression of differentiation markers Innate Immunity Cell and Tissues of the Immune System Christopher F Cuff PhD Immunology Derived from Greek immunis39 to be protected from Systems to maintain the integrity of the hosts39 internal environment against 1 pathogens and toxins in the external environment and 2 tumors arising within the internal environment Immunity is mediated by innate nonspecific factors and adaptive speci c factors I The Innate Immune Response Anatomic Physical Barriers Intact epithelium consisting of keratinized epithelium skin and mucous membranes consisting of nonkeratinized or wet epithelium provide an excellent barrier against invasion Mucous on wet epithelium prevents binding of pathogens ortoxins to host cells Cilia on epithelial cells act to block contact between host and parasite Smooth muscle contraction pushes pathogens out ofthe host Physiologic Chemical Barriers temperature salivary mucins lactoferrins peroxidase lysozyme histidinerichproteins prolinerich peptides interferon xB complement acute phase reactants inhibits bacterial growth high mw glycoproteins that form a barrier to penetration concentrate lgA and aggregate bacteria have antibacterial activity through iron binding direct interaction with bacteria generation ofOH radicals mediates the production of thiocyanite which is toxic to bacteria mediates the cleavage of peptidoglycan inhibit bacterial and fungal growth promote the adhesion of symbiotic bacteria inhibits viral replication opsonization chemotactic factor membrane attack mediators of in ammation The In ammatory Respnnse Bland Vessel such occurs in a anavy Lysusume hum 6mm V Recepm Mediated Enaoeymsls and mocymsls Endocytic and Phagocytic Barriers Endocytosis extracellular macromolecules such as bacterial toxins are internalized into cells by nonspecific membrane invagination pinocytosis or by binding to receptors on the surface ofthe cell receptormediated endocytosis to form endocytic vesicles In the cell cytoplasm endocytic vesicles fuse with primam lysosomes which contain reactive oxygen dependent and oxygenindependent antibacterial effector molecules The products ofthis fusion are referred to as secondam lysosomes Antigens are degraded in secondary lysosomes Oxygendependent killing results in production of reactive oxygen intermediates eg superoxide anions 0239 and hydrogen peroxide H202 reactive nitrogen intermediates eg nitric oxide NO Oxygenindependent killing results in production of antibacterial proteins lysozyme and defensins proteolytic enzymes elastase and cathepsin L B and G Inflammation Inflammation is a nonspeci c reaction that occurs as a result of local tissue damage and the presence of foreign substances There are 5 cardinal signs or characteristics ofthe inflammatory response These are 1 pain 2 redness 3 heat 4 swelling and 5 loss of function Local tissue damage induces the release ofa variety of small molecular weight substances that cause increases in capillary permeability and an in ux of effector cells to the site of damage The in ux of cells involves several steps rst cells adhere to the endothelial wall of the capillary margination next the cells move between the endothelial cells into the tissue jdiapedesis nally the cells migrate through the tissue to the damaged area chemotaxisl Phagocytic Cells in Innate Immunity Macrophages Phagocytic cells in innate immunity include macrophages neutrophils and dendritic cells Macrophages are the premier phagocytic cell in the body They are controlled by a variety of methods that will be discussed later in the course For now know that macrophages can be found in the blood or in the tissues referred to as resident macrophages They are generally found in a lower metabolic state called resting but under the in uence of signals provided by bacteria and proteins produced by the body in response to infection these cells can become primed and then activated The differences in the metabolic state are reflected in the functional activity of the cells For now we can say that the more activated macrophages are the greater their capacity to kill In addition macrophages produce molecules called monokines that are important in the in ammatory response They will be discussed in more detail later in the course but we ll identify them now as interleukin1 lL1 tumor necrosis factoral ha NFoc lL6 and interleukin8 IL8 Neutrophils Because of their numerical superiority the neutrophils are the most important type of phagocytic cell in the body Neutrophils are shortlived cells with a lifespan of about 5 days Therefore the body is constantly producing a large number of neutrophils every day Neutrophils typically circulate in the blood stream until they get signals to marginate and diapadese towards the site of infection As described earlier neutrophils roll along endothelial cells This rolling is a result of weak interactions between a molecule expressed on the endothelial cells called E selectin and sialyl Lewisx SLex expressed on the neutrophil As a result of infection tissue macrophages produce lL1 and TNF which causes an additional molecule to be expressed on endothelial cells This new molecule called intercellular adhesion moleculeone lCAM1 binds to LFA1 on the neutrophil This strong binding stops the neutrophil from rolling margination and signals the neutrophil to squeeze out ofthe blood vessel diapadesis The neutrophils then migrate along along a concentration gradient of a variety of chemicals including M produced by macrophages chemokines produced by tissue cells 05a produced as a result ofthe complement cascade and formyl methionine f metl containing peptides produced from phagocytosed bacteria Dendritic Cells Dendritic cells are found in epithelium tissues and blood and are responsible for molding the type of adaptive immune responses that develop following infection The role of these cells in immunity will be discussed in more detail later in the course For now think of dendritic cells as cells that initially encounter antigen and initiate primary immune responses The Complement System Overview Complement is an important mediator of immunity and participates in the in ammatory response It provides important defense against many types of bacteria and some viruses Complement also interacts with the blood clotting system but this aspect is beyond the scope ofthis discussion How important is complement OthenNise normal individuals with inherited deficiencies in the complements system are often Membrane Attack Cnmnlex highly susceptible to infection by a wide range of microorganisms Thus complement is an important part ofthe immune system Complement is not one substance but is a term that describes about 20 proteins that are sequentially activated to mediate the activities of complement These activities include the killing of bacterial cells and enhancement ofthe activity of antimicrobial white blood cells The complement components are produced in an inactive form by the liver and are found in blood serum or plasma Complement components are also found in gingivocrevicular uid the fluid that percolates through the capillary beds in the gingiva and bathes the teeth and gums How does complement destroy bacterial cells Bacterial cells are killed when the activated complement components polymerize to form a pore or channel through the surface ofthe bacterial cell These polymerized complement components are referred to as the membrane attack complex or MAC How does complement enhance the activity of antimicrobial white blood cells Following activation ofthe complement cascade one component of complement binds to the surface of bacterial cells and serves as an opsonin for phagocytic white blood cells to attach to bacteria and then ingest them A second component acts as a chemotactic factor for neutrophils Neutrophils will migrate along the concentration gradient formed by diffusion of this activated complement component towards the site of complement activation There are several other functions of complement components that will be discussed throughout the course Complement components are sequentially activated in a process often referred to as the complement cascade This complement cascade can be activated through 3 distinct pathways The 3 pathways are 1 the classical pathway 2 the alternative pathway 3 lectin activation pathway The classical pathway ofthe complement cascade is initiated by the specific or adaptive immune response so because we are now considering innate immunity we will focus on the alternative and lectin activation pathways The Alternative Pathway of the Complement Cascade The difficult part of understanding the complement cascade is that as mentioned earlier complement is made up of a family of separate proteins that interact sequentially to form the MAC and the phagocytosispromoting molecules Getting the names ofthese proteins and the role of each protein in the cascade is an intellectual challenge Many but not all ofthe complement components are simply called C followed by a number that identi es when it was discovered and characterized So the first complement component that was discovered is called C1 However the alternative pathway begins with component C3 C3 is the most abundant complement component in the blood It is also one ofthe most unstable components C3 slowly but spontaneously breaks down to two fragments called C3a and C3b C3b is very reactive meaning that it will readily bind to free amino or hydroxyl groups Free amino and hydroxyl groups are found on the surface of bacteria so if C3 is cleaved into C3a and C3b near a bacterial cell the C3b binds to the bacterial surface If C3b does not bind to a bacterial surface very quickly lt000006O seconds it will bind to a water molecule and become inert Ultimately this C3b will drive the production ofthe membrane attack complex C3b also acts as the opsonins that are recognized by the neutrophils C3b that is bound to a bacterial cell is stable A second complement component will then bind to C3b This component is termed Factor B This complex is next modi ed by another complement component called Factor D An additional factor called Factor P or Properdin helps stabilize the complex The modi ed complex of C3b and Factor B is referred to as C3bBb What is special about C3bBb that is bound to the bacterial cell This complex can now be called C3 convertase because it will very efficiently and rapidly split more C3 into C3b and C3a The result ofthe C3 convertase is that the bacterial cell can rapidly be coated with C3b Some ofthis newly liberated C3b will associate with the C3bBb to form that s right C3bBb3bl Are we having fun yet So now we have a complex called C3bBb3b on the bacterial cell The C3bBb3b has a new function It can cleave the complement component C5 into C5a and C5b C5b then combines with C6 C7 and C8 to form a stalk that binds to the bacterium The nal component is C9 C9 associates with other C9 molecules around the stalk and the membrane attack complex is formed Again the MAC forms pores in the bacterial cell which causes the cellular cytoplasm to leak out ofthe cell and the cell dies Whatever happened to C3a and C5a Those two components act as chemotactic factors that attract neutrophils to the scene ofthe battle They are sometimes referred to as anaphylatoxins because they are involved in anaphylactic shock to be discussed later See more information in the section on phagocytic cells In older books this pathway of complement activation is sometimes called the Properdin Pathway The Lectin Activation Pathway In addition to the alternative pathway the lectin activation pathway is an important mechanism of complement activation A serum protein called mannosebinding lectin MBL binds to a second protein called mannanbinding lectinassociated serum protease MASP This complex binds to mannose on bacterial cells and then cleaves C4 into C4a and C4b C4b cleaves C2 into C2a and C2b C2a associates with C4b to form a complex called C4b2a C4b2a acts as a C3 convertase C4b2a cleaves C3 to C3a and C3b The C3b associates with C4b2a to form C4b2a3b which is a CS convertase This CS convertase cleaves CS into CSa and CSb From here the MAC is formed just as described above Control of Complement Activation Why aren t the body s own cells damaged by complement There are a number of facors that regulate and control complement activation The two most important factors are decay accelerating factor or DAF which is found on the surface of human cells and accelerates the breakdown of C3 convertase ofthe alternative pathway C3bBb Protectin is another molecule also called CD59 on the surface of human cells that inactivates C3b Summary Both the alternative and mannosebinding lectin system result in the formation of C3 convertase and CS convertase The alternative pathway is initiated as a spontaneous cleavage of C3 into C3a and C3b This is a rather inefficient process but it works The second approach is to use mannosebinding proteins This is more efficient and focuses the activity of complement onto mannosecontaining bacteria Either way produces a MAC on the surface ofthe cell deposition of opsonizing C3b and the production of C5a as a chemotactic factor Other complement products including C4a and C3a also affect the inflammatory response to pathogens Tight control ofthe complement system is mediated in part by DAF and CD59 which inactivate complement components that are deposited on human cells inadvertently Complement is an important antimicrobial system in the oral cavity and therefore is important in understanding infection and immunity in the oral cavity The innate arm of the immune system uses receptors pattern recognition receptors to recognize components of microbial pathogens pathogen associated molecular patterns PRRs are Soluble or Found in onin cells Soluble PRRs Mannan Binding Lectin MBL Creactive Protein CRP Serum Amyloid Protein SAP LPS Binding Protein LBP Cell Bound PRRs TollLike Receptors TLRs TLR2 TLR3 TLR4 TLR5 TLR9 found on cell membranes surface or inside Retinoic acid inducible geneIRIG like receptors RLRs RIGl and MDA5 found in cell cytoplasm Nucleotide oligomerization domain containinglike receptors NLRs NOD1 and NOD2 cell cytoplasm PAMPs Bacterial lipopolysaccharide LPS the outer coating of certain bacteria Gram negative Bacterial cell wall peptidoglycan the rigid outer layer of bacteria Bacterial agellin the protein that makes flagella which are used for motility Mannan from yeast the outer layer of yeas Unmethylated CpG DNA nucleic acid bases Double stranded RNA dsRNA from viruses Ill Cells of the Immune System Innate Immunity Granulocytes neutrophils eosinophils and basophils MonocyteMacrophages and Natural Killer NK cells Speci c Immunity Tcells thymusderived Bcells bursaderived Antigen Presenting Cells APC Bcells mediate humoral immunity and Tcells mediate cellular immunity Blymphocytes antibody secreting cells Fully differentiated Bcells that secrete high levels ofantibody are called plasma cells Mature Bcells that come into contact but do not develop into plasma cells are often referred to as memom B These cells may differentiate into plasma cells following subsequent exposure to antigen Bcells recognize soluble or particulate antigens Each individual Bcell expresses a unique antigen receptor and can thus recognize only 1 antigen Tlymphocytes Thelper cells provide signals for the growth and differentiation of Bcells Tcytotoxic cells Tsuppressor cells Each Tcell expresses a unique antigen receptor and can thus recognize only one antigen Tcells recognize antigens found on the surface of antigen presenting cells APCs 8 M CD4 TCR 4lt if Figure legend The membrane receptors on the surfaces of B and Tcells B cells left express surface immunoglobulin slg as an antigen receptor Tcells that express CD4 middle typically act as helper cells but can also be regulatorysuppressor cells and Tcells that express CD8 right typically act as cytotoxic cells CD4 and CD8 Tcells recognize antigen through TCR Each lymphocyte expresses about 105 molecules of identical receptor and each lymphocyte recognizes a single antigen CD8 TCR a variety of cells including dendritic cells macrophages and Bcells They typically have a common function of endocytosing or phagocytosing antigens degrading or processing them into simple molecular fragments and reexpressing fragments of the antigen on the cell surface of the APC This is what Tcells recognize as antigens IV Tissues of the Immune System Primary Lymphoid Organs are organs in which lymphocyte precursors differentiate into immunocompetent lymphocytes Primary lymphoid organs include the bone marrow thymus fetal liver the Bursa of Fabricius in chickens and maybe the gut in mammals Secondary Lymphoid Organs are organs in which lymphocytes proliferate and further differentiate following contact with antigens Secondary lymphoid organs include the spleen lymph nodes and lymphatic vessels and diffuse lymphoid tissue associated with mucosal surfaces gastrointestinal tract respiratory tract and reproductive tract Primary Lymphoid Organs 1 Bone marrow The bone marrow is the site of blood cell formation after birth This process is called hematopoiesis Hematopoiesis is a very complex highly regulated but poorly understood process All blood cells are derived from common progenitor cells referred to as pluripotent hematopoietic stem cells or stem cells39 for short These stem cells have the capacity for selfrenewal and differentiation into every type of cell found in the blood In mammals mature Blymphocytes develop from stem cells in the bone marrow However cells destined to become mature Tcells referred to as preTcells39 leave the bone marrow and migrate to the thymus where they differentiate to mature Tcells ln birds Tcell differentiation is similar to that of mammals but Bcell precursors do not differentiate to mature Bcells in the bone marrow Instead Bcell precursors migrate to the Bursa of Fabricius where they differentiate into mature Bcells Bcells got their name from early experiments in chickens because it was determined that they were bursaderived39l 2 Thymus The thymus is divided into 2 anatomically distinct regions These regions are the cortex and the medulla Histologic examination of the cortex reveals that it is densely packed with lymphoid cells Beneath the cortex is the medulla The medulla contains mainly epithelial like cells and fewer lymphocytes PreTcells migrate from the bone marrow to the thymus and enter the cortex About 90 of the cells die in the thymic cortex The remaining 10 undergo a process of differentiation The 10 surviving cells represent Tcells that can recognize antigens on APC but do not recognize self components The process of selecting these reactive cells actually involves 2 independent processes These are referred to as positive selection39 and negative selection39 The thymus in young animals is much larger than in adult animals relative to total body size As an animal matures the thymus undergoes a process of atrophy referred to as involution In humans involution begins at around the onset of puberty 3 Fetal liver In the fetus the liver is the primary site of hematopoiesis At around the time of birth the bone marrow is seeded with stem cells that take over the task of hematopoiesis 4 Bursa of Fabricius birds Bcell precursors leave the avian bone marrow and migrate into the Bursa of Fabricius where they develop into mature Bcells Early experiments in immunology indicated that antibodies were produced by Bcells by removing the bursa of young chickens These animals had very low levels of antibodies in their blood Secondary Lymphoid Organs 1 Spleen the spleen is a major secondary lymphoid organ Foreign antigens in the circulation are ltered through the spleen and come in contact with lymphocytes and APCs that initiate a speci c immune response 2 Lymph and Lymphatics Lymph is a pale watery proteinaceous tissue uid which ows from intercellular spaces into capillaries and then onto progressively larger lymphatics into the thoracic duct Ultimately lymph enters the blood stream at the left subclavian vein near the heart ll Lymph contains antigens from a site of antigen exposure to a nearby lymph node where the immune system reacts to the antigen The nearby lymph node is referred to as the draining lymph node39 3 Lymph nodes Lymphocytes and APC in lymph nodes are found in 3 anatomically distinct regions These are the cortex the paracortex and the medulla Lymphocytes tend to segregate into these regions Bcells are found mainly in the cortex and Tcells are found mainly in the paracortex There are few lymphocytes in the medulla Lymph Node Afferent lymphatics Cortex LE 3 a 7 Efferent Lymphatic Lymphatic I Blood Supply 4 Splenic Artery 12 The Nature of Antigens Structure and Function of Antibodies Genetics of Antibody Diversity Objectives I Adaptive Immunity The student should be able to distinguish innate and adaptive immune responses II The Nature of Antigens The student should be able to identify the properties of an antigen and be able to predict whether a given substance is antigenic The student should be able to distinguish antigenicity from immunogenicity The student should be able to define the following terms hapten epitope valence mitogen and superantigen The student should understand and identify Bcell and T cell mitogens III The Structure of Immunoglobulins The student should be able to draw a model of an IQ molecule identify the significant features of the molecule and identify the resulting products of enzymatic cleavage The student should be able to list the Ig isotypes know their functions and identify the multimeric forms of immunoglobulin The student should understand genetic mechanisms that result in production of antibodies with diverse specificities I Properties of Specific Adaptive Immune Response 1 Specificig The immune system can react to an almost limitless number of foreign substances antigens by mounting a response that interacts with only that antigen The immune system precisely and constantly distinguishes between self and nonself and does not mount a response against self39 2 Memom Following exposure to an antigen the rst time the immune system mounts a response referred to as a primam immune response This generally takes 714 days The immune response that occurs following subsequent exposure to the same antigen is referred to as a seconda immune response The secondary response is stronger it develops faster less than 7 days and differs qualitatively from a primary immune response Qualitative differences between the primary and secondary responses will be discussed in the next lecture Immune Response Antigen A 1 xA Response Antigen A Antigen B i Time Days Figure ags70 Differences between a primary and secondary immune response When an animal is exposed to a foreign antigen for the first time a primary immune response against the antigen is observed as measured by an increase in antibody titer Following a second exposure to the same antigen a more potent antibody response occurs within a week after exposure This secondary immune response is antigen specific because a concomitant exposure to a different antigen antigen B elicits only a primary immune response This figure illustrates immunologic specificity and immunologic memory II The Nature of Antigens Antigens are molecules that are recognized by and react with the immune system In today39s discussion we will focus primarily on antigens that are recognized by antibodies but we will examine the features of antigens that are recognized by the humoral arm of the immune system Bcells and antibodies and the cellular arm of the immune system F cells Most antigens will also m an immune response These antigens can be additionally referred to as immunogens and are said to be immunogenc There is however a class of antigens that are not immunogenic These antigens will be bound by antibodies but they will not elicit an immune response by themselves Such antigens are referred to as haptens The properties of an immunogen are described below Molecular Size The best immunogens have a molecular size of gt 100000 Some potent immunogens have a molecular size of 10000 100000 Very few molecules of lt10000 mw are potent immunogens The class of antigens that are haptens have very low molecular weights Remember that haptens can react with antibodies but not elicit an immune response Chemical Composition and Heterogeneity Proteins are the most potent immunogens followed by complex polysaccharides Nucleic acids and lipids are typically poorly immunogenic Proteins and complex carbohydrates found in nature are typically more chemically complex than nucleic acids and lipids Degradabilig The rst step in a typical primary immune response is uptake and partial digestion of the immunogen by antigen presenting cells Thus a potent immunogen must be susceptible to partial digestion by antigen presenting cells Foreignness The immune system has the capacity to distinguish between self39 and nonself39 Thus an immunogen must be foreign to the host This is evident by the fact that in a normal individual the immune system does not mount a response against large complex degradable molecules that are constituents of the host Three other factors that play a role in immunogenicity are J the genotype ofthe host 2 the route of immunization and 3 the dose of the immunogen In other words some individuals will react strongly against an immunogen and others individuals will not For example some people are allergic to penicillin while others are not In addition most immunogens are poorly immunogenic if given by the oral route and very small doses of an immunogen may not elicit a potent immune response One can enhance an immune response by coadministering the immunogen with an adjuvant An adjuvant is an agent that enhances the immune response Some adjuvants include components from bacteria such as Mycobacterium tuberculosis and Bordetella pertussis Insoluble aluminum salts have also been found to act as adjuvants Adjuvant research is important for vaccine development A molecule must be large complex and foreign to elicit an immune response However an individual typically mounts an immune response against many different parts of an immunogen These parts of the immunogen are referred to as epitopes or antigenC determnants Consider a protein immunogen Proteins in nature have primary secondary tertiary and in some cases quaternary structure Epitopes recognized by Bcells can be formed by any of these 1 2 3 or 4 structures Epitopes recognized by Tcells are formed only by the 1 structure of a protein Antigenic determinants that are formed by the 1 amino acid sequence of a protein are referred to as linear determnants Determinants formed by 2 3 or 4 structures form conformational determnants Haptens are small molecular weight molecules that react with antibodies but do not elicit an immune response by themselves If this is so then how is an immune response generated against a hapten The answer is that haptens bind to larger molecules to form a new epitope that is not normally on the larger molecule The term valence refers to the number of antigen binding sites on an antigen Thus if the valence of a large antigen is 10 the valence of the same antigen with one molecule of hapten attached to it would be 11 Valence may also refer to the number of antigen binding sites on an immunoglobulin molecule as discussed below Each Bcell and Tcell recognizes only 1 antigenic determinant by expressing a molecule that binds to a unique antigen For Bcells this antigen receptor is called surface immunoglobulin or Bcell receptor BCR The antigenspecific receptor on T cells is called Tcell receptor or TCR As part of the immune response to an antigen Tcells and Bcells that recognize antigenic determinants on an antigen will undergo DNA synthesis and divide There is a group an agents that will cause any Tcell or any Bcell to divide These agents are referred to as mitogens A Bcell mitogen is bacterial lipopolysaccharide Two Tcell mitogens are concanavalin A and phytohemagglutinin Pokeweed mitogen will induce both Tcells and B cells to divide Mitogens have been used experimentally to characterize lymphocyte function 7 vtro A superantigen is a class of molecules that will activate a large percentage of lymphocytes irrespective of the antigen specificity of the lymphocyte They are distinguished from mitogens in that mitogens will activate whole populations of lymphocytes whereas superantigens will only activate a percentage like 1020 of that population Several bacterial toxins are superantigens III Structure and Function of Antibodies Following exposure to an immunogenic antigen antibodies are produced by Blymphocytes and appear in serum and in some cases in secretions such as saliva intestinal secretions and milk The antibodies have distinct biological properties including their ability to bind specifically to the antigen and the ability to mediate several other functions It is important to understand the structure of antibodies and how the molecular structure of antibodies confers upon them their biological function Antibodies were first characterized from the serum of experimentally immunized animals Using electrophoresis it was found that antibodies were in the gamma globulin fraction of serum Thus antibodies are generically referred to as gamma gobuins or immunoglobuIhs Immunoglobulins are abbreviated Ig39 There is much molecular heterogeneity among immunoglobulins Initially we will consider the basic structure of the form of antibody which is in the highest concentration in serum This is immunoglobulin G or IgG Each IgG molecule consists of 4 polypeptide chains These are 2 identical polypeptide chains of approximately 50000 mw and 2 identical polypeptide chains of approximately 25000 mw Thus each IgG molecule consists of 4 polypeptide chains with a total molecular weight of approximately 150000 The two larger chains are referred to as heavy chains or H chains and the smaller chains are referred to as light chains or L chains The four polypeptide chains are bound together by disulfide bonds One light chain is associated with 1 heavy chain and the two heavy chains are associated with each other as shown in the figure below Antibodies bind specifically to antigens The portion of the molecule that binds to antigen is formed by the amino terminal ends of the light chains and heavy chains This portion of the molecule is referred to as the antigen binding site As can be seen in the structure the IgG molecule has 2 antigen binding sites and therfore has a valence of 2 Since these sites are formed by identical chains the two antigen binding sites will react with the same antigenic determinant This is said to be the specificity of the IgG molecule The IgG molecule binds to antigen as a result of the physical forces between the amino acids of the antibody molecule and the epitope L I H Constant Variable COOH The structure of the IgG molecule was determined experimentally by treating purified IgG with proteolytic enzymes It was found that treatment with papain resulted in the generation of 2 antigen binding fragments referred to as Fab fragments and 1 fragment which was easily crystallized and thus referred to as an Fc fragment Treatment with the proteolytic enzyme pepsin resulted in the formation of 1 large fragment that contained 2 antigen binding sites referred to as an Fab392 fragment and several smaller fragments It was primarily these experiments that led to the development of the model of IgG structure The model was later confirmed to be accurate with Xray crystallography Fc mu pepsin Fab papal Fab39 2 Immunoglobulin Isotypes and function A closer analysis of the structure of the Ig molecule reveals that the light chains are one of two types designated with the Greek letters kappa K or lambda 7 The heavy chains can be one of 5 different types which are also designated with Greek letters These are gamma g mu p alpha 0c epsilon s and delta 6 One refers to the Ig molecules that bear these heavy chains as IgG IgM IgA IgD and IgE respectively Thus there are 5 different groups or classes of Ig These classes are called isotypes There are also 4 subclasses of IgG and 2 subclasses of IgA based on the structure of the heavy chains These subclasses are designated IgG1 IgG2 IgG3 IgG4 and IgA1 IgA2 Immunoglobulins of the IgM isotype are found as pentamers of the monomeric unit so they are made up of 10 heavy chains and 10 light chains The valence of an IgM molecule is 10 In addition the IgM molecule has a third type of polypeptide chain associated with it called a J chain J stands forjoining Thus IgM is gt5 times larger than an IgG molecule Immunoglobulin of the IgA isotype which is found in high concentrations in secretions such as intestinal fluid tears and saliva is typically found as a dimer consisting of 4 heavy chains 4 light chains and a J chain IgA in secretions has a fourth polypeptide which helps it get translocated into secretions called SC or secretory component IgA will be discussed in more detail in the lecture on mucosal immunity IgD and IgE are found as monomers like IgG Immunoglobulin that is expressed on the surface of Bcels serves as the antigenspeci c receptor forBcels Immunogobulin that is expressed on the surface of Bcels serves as the anh39genspeci c receptor for Bcels Each of these heavy chains confers different biological properties on the Ig molecule Remember that any given Ig monomer has 2 identical light chains and 2 identical heavy chains In summary the association of heavy chains and light chains form an antigenbinding site that confers the specificity of the Ig molecule The heavy chain confers the biological function on the Ig molecule Ig Isotypes Iq the most abundant isotype in the serum 1214 mgml is found as a monomer of 150000 mw and consists of 3 constant region domains IgG antibodies function as opsonins by binding to Fc receptor on phagocytic cells They also activate the complement system The four IgG isotypes are structurally and functionally distinct The structural differences are found mainly within the hinge region the length of the hinge and the number of disulfide bonds between the four heavy chains vary Functional differences include ability to activate complement cross the placenta and bind to Fc receptors on phagocytes IgA accounts for 10 to 15 of serum Ig at about 3 mgml but is the predominant isotype in external secretions and in mucosal associated tissues Serum IgA is primarily a monomer of mw150000 with 3 constant region domains secretory IgA exists as a dimer or a tetramer associated with a J chain and a secretory component IgA does not activate complement However it can bind to Fc receptors for IgA IgA serves an important role in mucosal immunity IgM accounts for 5 to 10 of the serum Ig at 1 to 15 mgml It is expressed as a monomer on the surface of B cells and as a pentamer mw 900000 in the serum comprising 10 antigenbinding sites The pentamer is associated with a J chain which is required for polymerization of the molecule prior to secretion The constant region of IgM has 4 domains IgM is the first isotype generated in a humoral immune response IgM activates complement and binds Fc receptors 139 is present in the serum at 30 mcgml constituting 02 of serum Ig IgD exists mainly as a membrane receptor on mature B cells where it is coexpressed with IgM As a surface protein IgD can bind and internalize antigen and participate in Bcell activation Log occurs in the serum at No3 mcgml MW of the monomeric IgE is 190000 It consists of 4 constant region domains capable of potent biological activity IgE is induced in response to allergens it can bind to Fc receptors on mast cells and basophils and mediate immediate hypersensitivity reactions Properly IgG IgA IgM IgD 19E 7 or u 8 a molecular weight 150000 150000 900000 150000 190000 Daltons 600000 Sedimentation constant Svedberq Units 75 115 19 75 85 CH domains 3 3 4 3 4 Hinge region polymerization serum concentration 1214 3 15 003 00003 mq ml complement activation mast cell degranulation presence in secretions transplacental transfer Property IgG1 IgG2 IgG3 IgG4 IgA1 IgA2 Serum concentration mgml 9 3 1 05 3 05 serum half life days 23 23 8 23 6 6 complement activation transplacental transfer presence in secretions Immunoglobulin molecules are complex proteins Thus puri ed Ig molecules could serve as antigens if they were injected into a host that recognized the Ig as foreign It was found that Ig heavy chains contained antigenic determinants that could be recognized by individual members of the same species These portions of the Ig molecule that differed from one individual to the next within a species are referred to as allotypic determinants Antigenic determinants may also be found in the antigenbinding site of an Ig molecule These determinants are referred to as idiotypic determhants Constant VH Variable ldiotypicdeterminants Allotypic determinants Why care about idiotypic and allotypic determinants Why indeed These determinants become important in understanding immune regulation and autoimmunity later on in the course B cell maturation and differentiation Precursors of B cells are generated within the bone marrow where they go through an antigenindependent phase of differentiation This constitutes a series of stages involving stepwise rearrangements of BCR gene segments and resulting in the expression of BCR at the cellsurface stem cell immature B Exit bone marrow mature B 3990 B cells expressing antigen receptors Ig of the IgM and IgD isotype on their cell surface are released from the bone marrow and circulate through the body Such B cells can encounter antigen within regional lymphoid organs near the site of antigen entry B cells undergo antigen dependent maturation and differentiation at defined sites within these lymphoid organs following contact with the antigen This phase contributes to the humoral immune response III The Genetic Basis for Antibody Diversity The immune system has the capacity to react to a wide range of antigens In fact it is estimated that a mammal can generate antibodies with as many as 108 different speci cities Since antibodies are proteins and proteins are encoded by genes it stands to reason that the diversity of antibodies must arise from diversity in the genome The problem is that the genome does not contain enough genes to encode all of those different Ig speci cities In addition each Bcell produces Ig of only 1 specificity so some mechanism must exist to limit the production of Ig by Bcells to Ig with only 1 specificity The understanding of the genetic basis for generating 10 antibody diversity was one of the major impetuses for the development of modern molecular immunology The following description is intentionally brief Mechanisms for Generating Diversity 1 Heavy chain and light chain combination Essentially any light chain can associate with any heavy chain If there were 104 different light chains and 104 different heavy chains we could derive 108 different specificities 10 x 10 108 2 Multiple V genes In an undifferentiated cell that is destined to become a Bcell the DNA that encodes heavy and light chains is made up of coding regions called exons and vast stretches of DNA that do not encode proteins called introns This DNA is said to be in the germ line con guration During Bcell development the DNA rearranges to bring together the coding exon sequences referred to as gene segments The variable portion of light chains and heavy chains are encoded by gene segments called variable gene segments or V gene segments that are found in the respective gene complexes of the Ig light chain and Ig heavy chain In different species there are different numbers of V gene segments In mice there are approximately 300 different V genes in the heavy chain complex and 300 different V genes in the kappa light chain gene complex There are only 2 different lambda light chain V genes in the mouse Any of these V genes can associate with the constant segment of the light or heavy chain gene The DNA between the V and C regions is not deleted but the RNA transcript from the gene complex is processed so the nal mRNA contains only the complete light chain transcript without the intervening sequences VL1VL2VLn CL Germline DNA VH1VH2VHn quotCH Germline DNA 3 VJ and VDJ Recombination The variable portion of the light and heavy chains are actually encoded only in part by the V genes In the light chain the rest of the variable portion is encoded by another gene segment called the J segment for Joining This is not to be confused with the J chain found in IgM and IgA molecules This is a gene segment that encodes part of the variable domain of the light chain and the heavy chain Any V gene segment can combine with any J segment within the complex In heavy chains another gene segment called the D gene segment for Diversity is located between the V gene and J gene segments Thus to form a variable portion of a heavy chain 3 gene segments come together the VDJ segments 4 Recombination Inaccuracies During the process of recombination segments that are joined together do not always recombine at exactly the same place This results in the addition or subtraction of one or a few bases at ll each site of recombination If these imprecise recombination products can still be transcribed they will yield different transcripts thereby yielding different protein products 5 Somatic mutations Mutations occur within the gene complexes during the lifetime of the cell resulting in the formation of antibodies that are slightly different that the original Sometimes the resulting antibodies will bind to an antigen stronger than the original antibody The new antibody molecule is said to have a higher af nityfor the antigen When antigen is present these cells will preferentially bind antigen proliferate and secrete antibody of a higher affinity This process is referred to as af nity maturation 12 Major Histocompatibility Antigens Antigen Processing and Presentation TCell Receptor Complex Christopher F Cuff PhD OBJECTIVES I Major Histocompatibility Antigens The student should be able to identify cells that express Class and Class II MHC antigens The student should know which MHC loci encode Class and Class II MHC antigens The student should be able to draw representative Class and Class II MHC molecules ll Antigen Processing The student should be able to distinguish between the endogenous and exogenous pathways of antigen processing and be able to predict through which pathway a given antigen would be typically processed The student should know what immune responses would be elicited against an antigen that is processed through the endogenous and exogenous pathways The student should be able to distinguish allografts isografts and xenografts and understand the immunologic consequences of each type of graft Ill Tcell Development The student should be able to list the 2 types of Tcell receptors found on mature Tcells The student should understand the processes that occur in the thymus that lead to the production of Tcells Major Histocompatibility Antigens Antigen Processing and Presentation TCell Receptors Christopher F Cuff PhD I Major Histocompatibility Antigens The Tcell arm ofthe immune response is important for maintaining the integrity of the host Cytotoxic Tcells CTL which express the CD8 differentiation marker recognize transformed cells virus infected cells and in some cases host cells that are infected with intracellular bacteria Helper Tcells which express the CD4 differentiation marker secrete cytokines that act as growth and differentiation factors for many different effector cells such as CTL macrophages and Bcells Like Bcells each Tcell has a unique speci city Each Tcell recognizes only 1 antigen However unlike Bcells Tcells do not bind soluble antigen Tcells only bind antigen that is expressed on the surface of antigenpresenting cells APC These APC express fragments of antigen on their cell surface in association with other surface proteins called major histocompatibility antigens Thus a Tcell recognizes a complex formed by the antigen fragment and a major histocompatibility antigen It is said that T cells recognize antigen in the context of major histocompatibility antigens39 Because major histocompatibility antigens play such a critical role in Tcell responses they have been extensively characterized The structure of these major histocompatibility antigens has been de ned and the genes that encode these cell surface proteins have been characterized To understand Tcell function it is necessary to understand major histocompatibility antigens and the genes that encode them Obviously if major histocompatibility antigens are proteins made by the APC they are encoded in the DNA of the APC The region of DNA that encodes these antigens is called the Major Histocompatibility Complex or MHC All vertebrate species have an MHC In humans the MHC is called the HLA which stands for Human Leukocyte Antigen The HLA is located on chromosome 6 in the human genome In mice the MHC is referred to as the H2 and is located on chromosome 17 Although you may not care to think much about mice the study of the murine H2 has been absolutely essential for an understanding of Tcell biology HLA Genetics The human MHC is divided into several regions called loci The singular is locus so we refer to 1 locus or several loci These loci are shown in the Figure below Murine H2 Koc IA 043 IE ocB Some HLA antigens are expressed on all nucleated cells These molecules are encoded by DNA in the HLA A B and C loci The surface proteins encoded by genes in these loci are referred to as Class MHC molecules Class MHC molecules are recognized by CD8 Tlymphocytes Other HLA molecules are expressed only on APC and in humans on activated Tcells These surface proteins are encoded by genes in the D locus and are referred to as Class II MHC molecules Class II MHC molecules are recognized by CD4 Tcells Finally in the Class II loci there are 3 individual genes that encode individual proteins These are referred to as DP DO and DR Thus cytotoxic T cells recognize Class MHC molecules and can interact with any nucleated cell In contrast Thelper cells only interact with APC that express Class II molecules Class III MHC molecules are not relevant to our discussion of Tcell biology Be familiar with the fact that Class III MHC genes encode proteins found in the serum that are associated with complement All humans have MHC genes However there is tremendous variability in the genes from one individual to the other Thus there is an extremely high probability that 2 individuals selected at random will have nonidentical MHC antigens This phenomenon results in variability in the gene products which is referred to as polymorphism This polymorphism is responsible for graft rejections following organ ortissue transplants The recipients39 immune system recognizes polymorphic MHC molecules as foreign antigens Structure of MHC Molecules Understanding the structure of MHCencoded proteins allows one to understand how a complex is formed between a fragment of antigen and the MHC molecule Class and Class II molecules are actually quite similar in structure Both are membrane bound glycoproteins Both are composed of 2 polypeptide chains The major difference in structure is that the Class molecule is composed of a polymorphic 0c chain that is encoded in the MHC and a nonpolymorphic 7 chain referred to as 52 microglobulin 52 microglobulin is encoded by a gene outside the MHC Class II MHCencoded proteins consist of2 chains that are both encoded in the MHC These chains are referred to as the ct chain and the 5 chain Diagrams of the Class and Class II MHC molecules are shown below Representative molecules from both Class and Class II have been puri ed and analyzed by xray crystallography Notice that a groove is formed at the distal portion of the molecule It is believed that protein antigens t into this groove The complex of MHC molecules and peptide antigen is recognized by antigenspeci c Tcells through the Tcell receptor for antigen Class I MHC Class II MHC Antigen Processing T cells recognize the molecular complex of an MHC molecule and an antigenic peptide The antigenic peptide comes from a larger more complex antigen that is broken down into the small peptides that are typically 610 amino acids in length These peptides associate with MHC molecules inside the antigen presenting cell The associated complex is then expressed on the surface ofthe cell There are 2 pathways by which antigenic peptides can be associated with MHC molecules Antigenic peptides that are originally synthesized extracellularly are taken up by the APC processed and presented in association with Class II MHC molecules This process is referred to as the exogenous pathway In order for a cell to serve as an APC for CD4 Thelper cells that bind to Class II MHC molecules the APC must be able to endocytose or phagocytose exogenously produced antigens and the APC must express Class II MHC antigens Thus only cells that express Class II can activate CD4 Thelper cells Some foreign proteins are synthesized intracellularly For example viruses replicate intracellularly They synthesize their viral proteins using the host cells39 metabolic machinery Nonetheless these viral proteins are foreign39 and the immune system can recognize them Foreign proteins that are synthesized intracellularly are expressed on the surface ofthe cell in association with Class MHC molecules This process is referred to as the endogenous pathway Because CD8 CTL recognize Class MHC molecules plus antigen any nucleated cell can serve as an APC for CD8 CTL The pathways are illustrated below exogenous antigen phagocytosed or endolysosome Jsion Golgi complex C a and 3 chains 5 s a associate with invariant chain endoplasmic reticulum Exogenous Class II Endogenous Class I peptide fragments through transported en doplasmi c reticulum TCRCD3 on Tcytotoxic cell APC proteosome fa 5 6 en dogenous protein ll Transplantation Terminology MHC antigens are polymorphic They differ among individuals Because TceIIs recognize MHC antigens TceIIs recognize transplanted tissue as foreign antigens and respond against them thereby mounting a transplant rejection In the context oftransplantation foreign MHC molecules expressed on a transplanted organ or tissues are referred to as alloantigens A transplanted tissue from a different individual is referred to as an allograft An example ofan allograft would be a heart transplant from a donorto a recipient Sometimes individuals receive grafts from one site oftheir bodies to another These transplants are referred to as isografts An example ofthis type oftransplant would be heart bypass surgery Blood vessels removed from a patient39s leg are used to replace clogged arteries in the patient39s heart No foreign MHC antigens are recognized by the patient39s immune system Sometimes individuals receive grafts from animals Grafts from different species are referred to as xenografts An example of a xenograft was the recent case where an HIV infected individual was given bone marrow from a non human primate These cells were not susceptible to HIV Vo ca b u Ia ry Alloantigens Allograft Isograft X enograft Ill TCell Development Approximately 95 of the peripheral TceIIs express a receptor that is composed of 2 polypeptide chains which are designated the ac chain and the 5 chain ofthe TCR Don39t confuse these with the ac and I3 chains in MHC molecules They are different proteins Approximately 5 of peripheral TceIIs express a different form ofthe TCR that is composed ofa y chain and a 6 chain These 6 TCR TceIIs also found in somewhat higher frequencies in the epithelium The function ofthese cells is not understood They do not typically seem to recognize antigen in the context of HLA A B C and D Mature TceIIs develop in the thymus Following migration to the thymus from the bone marrow pre TceIIs undergo rearrangement of the genes encoding the TCR and expression of the TCR lfthe TCR does not bind to either Class or Class II MHC molecules in the thymus the cells die This process is termed positive selection The positively selected cells are then tested again in the thymus for binding to MHC molecules lfthe Tcells bind too strongly they also die This process is referred to as negative selection What is left is a population of Tcells that bind very poorly to self MHC molecules However these cells will bind with high af nity to a molecular complex of self MHC plus antigen This is one method that the hosts39 immune system distinguishes between self and nonself by selecting Tcells in the thymus that recognize only nonselfantigens in association with self MHC The Autolmmune REgulator AIRE protein is a transcription factor that is expressed in thymic medullary epithelial cells AIRE induces these cells to express tissuespecific antigens such as insulin in the thymus Autoreactive Tcells such as insulinspecific Tcells recognize these antigens in association with MHC molecules and are clonally deleted during development People with defects in AIRE develop severe autoimmune diseases Fortunately it is a very rare defect IV CD3 Associated with the ocB heterodimer of the TCR for antigen there is a complex of transmembrane proteins that are collectively referred to as CD3 The CD3 molecular complex appears to function as a signal transduction complex telling the cell that the TCR has come in contact with antigen The complex consists of seven polypeptide chains When one examines the Ig molecule the MHC molecules and the TCR it becomes evident that the genes that encode the proteins and the proteins themselves have many similarities Thus these molecules are sometimes all placed in the same group called the lmmunoglobulin Supergene Family39 selection I IThymic Selection TCRCD3 Complex Cytokines Christopher F Cuff PhD Objectives I The student should be able to define the term cytokine and describe the common features of cytokines including biochemical and functional characteristics The student should be able to distinguish cytokines lymphokines and monokines II The student should be familiar with the sources and functions of cytokines that regulate hematopoiesis initiate and regulate inflammatory responses and regulate the growth and differentiation of lymphocyte populations III The student should understand how various cytokines influence the development of cell mediated or humoral immune responses and know what populations of Th cells elicit cellular humoral responses and inflammatory cells The student should also know the phenotype and function of T regulatory cells IV The student should understand the relationship between the acute phase response endotoxic shock and cytokines Cytokines Christopher F Cuff PhD I Properties of Cytokines Cytokines are secreted proteins that induce cells to 1 proliferate 2 differentiate to mediate some immune function or 3 proliferate and differentiate It is one mechanism by which cells communicate with one another Immune responses require the interaction of several cell types such as APC Tcells and Bcells These cells interact in part through cognate interactions cell to cell contact but much of the interaction is mediated through cytokines Cytokine is a generic term referring to soluble factors produced by any type of cell To be more speci c cytokines that are produced by lymphocytes are referred to as lymphokhesand cytokines that are produced by monocytes or macrophages are referred to as monokhes Some cytokines are produced by both lymphocytes and monocytes so the term cytokine is more useful in describing these molecules Cytokines vary tremendously in function and biochemical properties However cytokines do have several characteristics in common First cytokines are all glycosylated carbohydrate containing proteins Second cytokines act only on cells that express specific receptors for that cytokine Third cytokines may have several functions and they may act on several different types of cells if these cells express cytokinespeci c receptors Therefore cytokines are said to be pleiotropic Finally cytokines are antigen nonspecific Cytokines have both autocrine and paracrine function When a cell produces a cytokine and then the same cell uses the cytokine as a growth or differentiation signal the process is referred to as an autocnhe function When a cytokineproducing cell acts on a different cell the function is referred to as paracnhe function Sometimes paracrine function is referred to as endocrine function but there is little distinction between endocrine and paracrine function II Cytokine Nomenclature In the past cytokines have been given descriptive names based on their biological function This caused confusion because it became evident that the same cytokine could have many functions pleiotropy In response to this problem researchers formed a committee and designated the use of the term interleukin39 to describe cytokines Interleukin refers to the fact that cytokines mediate communication between white blood cells inter leukocytes Now newly described cytokines are given numerical designations such as interleukin1 interleukin2 interleukin3 etc In order to be designated an interleukin 2 basic criteria must be met First the molecule must be biochemically puri ed and the gene for the cytokine must be cloned and expressed Second the cytokine must be involved in mediating some immunologic or hematopoietic function Some cytokines have kept their descriptive names III Cytokine Function There has been a tremendous amount of new information about cytokine biology over the last 510 years Consequently there is more information to know It can be confusing but one way to get a grasp on cytokine function is to classify cytokines into one of three groups based on their function These groups are 1 cytokines that regulate the immune response 2 cytokines that regulate hematopoiesis 3 cytokines that affect the in ammatory response Remember that cytokines are pleiotropic and therefore many cytokines fall into more than one category However using this approach will at least give you a working knowledge of cytokine biology and help keep them straight C ytoknes that induce the growth and differentiation of lymphocytes and regulate the speci c immune response Specific immune responses are mediated by T and Blymphocytes Activation ofT and B cells requires the presence of antigen Cytokines serve as second signals39 to drive the growth and differentiation of antigenactivated lymphocytes Some cytokines act predominantly on Tcells some on Bcells and some cytokines act on both T and B cells InterleukinZ is the primary growth and differentiation factor for Tcells IL2 causes antigenprimed Thelper cells to proliferate In addition it causes antigenprimed cytotoxic Tcells to proliferate and become aggressively cytotoxic IL2 is produced by CD4 Thelper cells Type II Interferon or Interferony IFNy is produced by Thelper cells It is an important cytokine for activating macrophages An activated macrophage is more phagocytic it processes and presents antigen more ef ciently It produces more cytokines and becomes more bactericidal than resting macrophages Activated macrophages are an important mediator of 4 cellular immunity which is distinguished from antibody mediated humoral immunity IFNy acts antagonistically against other cytokines such as IL 4 Interleukin4 is produced by CD4 Thelper cells IL4 mainly acts as a B cell growth factor for antigen activated Bcells Murine Bcells that are exposed to IL4 proliferate and tend to secrete Ig of the IgE and IgG1 isotypes The role of this cytokine in class switching in human cells is starting to be understood also InterleukinS is similar to IL4 in that it promotes the proliferation and differentiation of Bcells It may preferentially support the growth of IgA producing Bcells in mice Interleukin6 is produced by CD4 Thelper cells endothelial cells and monocytic cells IL6 increases the secretion of antibodies by plasma cells Like IL 1 IL6 is both a proin ammatory cytokine and a cytokine involved in promoting specific immune responses InterleukinlO is produced by a subset of CD4 Thelper cells It appears to suppress the production of IFNy and therefore tends to suppress cell mediated immunity IL10 tends to enhance humoral immunity Interleukin12 is now considered to be a central cytokine in the development and regulation of adaptive immune responses It has the opposite effect of IL10 IL12 is produced by a number of cells including B cells NKcells and most importantly macrophages IL12 enhances IFNy production This is important for immunity to pathogens that are susceptible to cellular effectors Interleukin13 and Interleukin15 are cytokines that appear to act like IL4 and IL2 respectively The functional activities of these cytokines are critical for adaptive immune responses and redundancy might confer an evolutionary advantage to the host Transforming growth factor B or TGFB is produced by a variety of cells including Tregulatory cells that express CD4 CD25 and the intracellular transcription factor FoxP3 and platelets TGFB tends to inhibit the proliferation of lymphocytes in experimental systems and might control the development of potentially pathologic in ammatory responses It also acts as an IgA switch factor to promote IgA production Cytokine Production by Tcell Subsets Thelper cells play an important role in specific immunity by producing cytokines that work predominantly on Bcells or predominantly on macrophages and other Tcells Thus Thelper cell function appears to play a major role on directing the type of response that is elicited following exposure to antigen It is now thought that there are 2 distinct subpopulations of mature Thelper cells There is no good marker surface marker to distinguish these populations they are all CD4 however they can be distinguished by the cytokines that they produce Thelper cells that produce IL2 and IFNy tend to elicit a stronger cellular immune response These Thelper cells are referred to as Th1 cells Thelper cells that produce IL4 and IL5 tend to enhance a humoral immune response and are referred to as Th2 cells Generally speaking particulate antigens or replicating antigens such as viruses tend to elicit Th1associated cellular immune responses while soluble antigens tend to elicit Th2associated antibody responses Extracellular bacteria appear to induce Th17 cells Many factors such as the presence to PAMPs control whether an antigen induces a Th1 Th2 or Th17dominated response However the genetic makeup of the host may also contribute to the tendency to mount a cellular or humoral immune response This is a very active area of research and has important implications in vaccine development Cytoknes that regulate hematopoiesis Several cytokines play a major role in blood cell formation in the bone marrow The best characterized of these are granulocytemonocytecolony stimulating factor GMCSF granulocytecolony stimulating factor GCSF stem cell factor SCF interleukin3 interleukin7 interleukin9 Some of these cytokines are being used to treat patients that have de ciencies in hematopoiesis such as cancer patients whose bone marrow are damaged by anticancer drug therapy GMCSF and GCSF are produced by Thelper cells and promote the production of granulocytes basophils eosinophils and neutrophilsGCSF as well as monocytes GMCSF Interleukin3 is also produced by Thelper cells and increases the production of basophils and progenitor cells Interleukin7 is produced by bone marrow stromal cells It induces lymphoid stem cells to differentiate into progenitor B cells Under some conditions IL7 stimulates the proliferation of thymocytes Stem Cell Factor is also produced by bone marrow stromal cells It synergizes with a number of hematopoietic growth factors including IL7 GMCSF and GCSF Interleukin9 is produced by Thelper cells In appears to increase erythropoiesis and mast cell division and serve as an autocrine growth factor for Tcells C ytoknes that a ect he In ammatory response Cytokines that affect the inflammatory response generally enhance innate immunity These cytokines also play a role in mediating resistance to viral infections and causing the cardinal signs of in ammation pain redness swelling and heat Included in this groups are the Type I Interferons IFNoc and IFNB tumor necrosis factor TNF interleukin1 interleukin6 interleukin8 and interleukin17 Type I IFN is IFNoc and IFNB Type I IFN has several sources and several effects For simplicity know that Type I IFN inhibits viral replication in virusinfected cells IFNoc is made predominantly by neutrophils and IFNB is made predominantly by broblasts WW TNF is made predominantly by monocytes and macrophages following stimulation with bacterial LPS I39NFoc or by activated CD4 Tcells TNFalpha and TNF TNF has many biologic functions including killing tumors and inducing secretion of other in ammatory cytokines It is probably most important in inducing the production of acute phase proteins by the liver It also induces fever This is one of the first cytokines that appear during an in ammatory response TNFoc is sometimes referred to as cachectin TNF is sometimes referred to as lymphotoxin LT Interleukin1 is also made by cells of the monocytemacrophage cell lineage It has many of the same functions as TNF but it typically appears somewhat later in an inflammatory response Like TNF IL1 is an endogenous pyrogen so it induces fever IL1 is also a costimulator of CD4 Thelper cells Interleukin6 like IL1 has 2 major functions that places it both in the group of cytokines that mediate in ammation and the group that regulate the growth and differentiation of lymphocytes IL6 has a function similar to TNF in that it induces the synthesis of acute phase reactants by the liver In addition IL6 also serves as a growth factor for plasma cells Interleukin8 is produced by monocytes IL8 is a chemoattractant for neutrophils It also induces adherence of neutrophils to vascular endothelial cells and aids their migration into tissue spaces Interleukin17 is produced by CD4 Th17 cells IL17 increases differentiation of precursor cells into neutrophils It also induces adherence of neutrophils to vascular endothelial cells by increasing ICAMl expression This cytokine induces IL6 IL8 and GCSF as well as prostaglandins An important function of IL1 and TNFoc is the stimulation of osteoclasts and the inhibition of osteoblast alkaline phosphate levels IL1 has been referred to as osteoclast activating factor or OAF Both TNFoc and IL1 also stimulate broblast and synovial cell activity What is the signi cance to dental immunology Role of C ytoknes 7 he In ammatory Response and Toxic Shock In response to infection or tissue damage the host initiates a series of events referred to as the acute phase response The acute phase response is characterized by both local and systemic events Both local and systemic events are mediated in part by the cytokines described in this section The local reaction involves the activation of the blood clotting system and activation of many chemotactic factors These processes result in the influx of granulocytes monocytes and lymphocytes that participate in clearing antigen The systemic response results in the induction of fever and the new synthesis of acute phase proteins by the liver Systemic infection with Gram negative bacteria can lead to septic shock and death in an infected individual The pathology associated with Gram negative sepsis is caused primarily by the effects of overproduction of IL1 and TNFoc These effects include high fever widespread blood clotting in some regions which paradoxically can lead to uncontrolled bleeding in other areas of the body diarrhea and loss of blood pressure Bacteria that cause Gram negative sepsis with a relatively high frequency include Pseudomonas aeruginosa members of the Enterobacteriaceae and Neisseria meningitidis Some bacterial toxins can also cause overproduction of IL1 and TNF The most notable of these is are the toxins produced by Staphylococcus aureus and Streptococcus pyogenes Tcell Activation and Function Objectives I The student should understand the function of Th17 and Tregulatory cells II The students should understand the molecular mechanisms involved in CTL function III The student should understand characteristics of NK cells and be able to distinguish ADCC from NK and CTL mediated killing IV The student should understand the different requirements for activation of naive and effector Tcells with respect to costimulatory molecules Tcell Activation Tcell Function continued Th 17 cells In 2005 a population of Thelper cells was described that was distinct from Th1 and Th2 cells These cells were CD4 but lacked the transcription factors that were active in Th1 and Th2 cells In addition these previously undescribed Th cells produced the cytokine IL17 IL17 is a pleiotropic cytokine that upregulates expression of ICAM1 on endothelial cells resulting in increased neutrophil marginiation increases mobilization of bone marrow neutrophils and enhances production of in ammatory mediators such as IL6 IL8 and prostaglandins IL17 is also secreted at high levels in animal models of autoimmune disease Together these functions suggest that IL17 play an important role in amplification of the in ammatory response by antigenspecific Tcells Thus these cells could be thought of as a missing link between innate and adaptive immunity Many researchers are trying to understand the importance of Th17 cells in autoimmune diseases and immunity to infection particularly bacterial infection in an effort to develop ways to manipulate the function of Th17 cells for improved health Tregulatory cells The story behind Tregulatory cells I39regs is filled with acrimony bitterness jealousy confusion and unfortunately perhaps some scientific fraud In the 1980s immunologists discovered that under some circumstances Tcells could suppress immune responses both in vitro and in vivo The cells were designated Tsuppressor or Ts cells The conventional wisdom was that there were 3 distinct populations of Tcells CTL Th and Ts and within the Ts population there might be additional subpopulations called Ts1 Ts2 and Ts3 Later an additional population of Tcells was described as Tcontrasuppressor cells that were different than Th or Ts At the time little was know about how Tcells functioned and the Tcell receptor for antigen TCR was not yet characterized Studies on Ts cells indicated that Ts secreted antigenspecific immunosuppressive factors Like Th and CTL Ts were MHC restricted and the MHC molecule that controlled their response was mapped to the Class II MHC region distinct from IA and IE and was designated H In the mid1980s investigators working on CTLs and Th cells were able to clone and expand antigenspecific Tcells in vitro which gave scientists the opportunity to better understand how these cells functioned and it also provided tools necessary to identify the nature of clonotypic TCRs It was clear that the TCRs on CTL and Th were membrane bound and not secreted and that these TCRs recognized antigen fragments complexed with MHC molecules on the surface of antigen presenting cells rather than binding free antigen like antibodies do In contrast Ts could not be routinely cloned and the few clones that existed did not have functional TCR genes Furthermore when Class II MHC gene loci were fully sequenced it was found that there was no IJ region within the Class II locus By the time the 1980s ended many immunologists had reached the conclusion that Ts cells simply did not exist and were an artifact of awed experimental design execution andor interpretation Many very famous immunologists worked on Ts cells including Nobel Prize winner Baruj Benacerraf from Harvard University and Richard Gershon from Yale The concept of immunosuppressive Tcells reemerged in 1995 with a paper by Sakaguchi et al The investigators showed that a subpopulation of thymusderived cells could suppress the development of experimental autoimmune syndromes in mice These cells were rebranded as Tregulatory l39 regs to avoid the stigma associated with the Tsuppressor cell moniker Tregs were found to express CD4 the alpha chain of the IL2 receptor CD25 and perhaps most importantly uniquely expressed a transcription factor called Foxp3 which was not expressed in Th or CTLs A uni ed theory on how these cells function in the immune system has not yet emerged however the current thinking is that these cells are important in controlling the action of autoreactive Tcells that escape central deletion in the thymus and probably are very important in controlling potentially harmful in ammatory responses in the intestine Tregs appear to function mainly by producing antiinflammatory cytokines including IL10 and TGF The role of Tregulatory cells in immunity has a controversial past but their existence is now widely accepted and efforts are being made in determining ways to harness their function to control deleterious immune responses Summary CD4 Tcell Differentiation and Function IFNyILZTNFor Activates macrophage 439 539 IL17 CD25 Foxp3 c025 639 IL39llo I Increased TGFBIL10 Humoral Immunity Neutroph Tolerance IgE prOdUCt39on Productionrecruitment and tissue repair Inflammation Cytotoxic Tlymphocytes Cytotoxic Tlymphocytes or CT L recognize foreign proteins associated with Class I MHC molecules The main function of these cells is to combat pathogens that live intracellularly These intracellular pathogens include viruses intracellular bacteria and intracellular parasites In addition CTL also can kill tumor cells so they might play a role in keeping the host free of cancerous cells As discussed previously naive CTL need to recognize antigen and B7 on specialized APCs to become activated Following activation they express the high affinity form of the IL2R If IL2 is present the IL2 signals the cells to divide and differentiate into an activated CTL Activated CTL can then go kill cells that express the speci c antigen irrespective of whether they express B7 We will now consider how CT L kill infected targets CTL mediated killing In recent years the nature of CTLmediated killing has become clearer It was known for a long time that CTL needed to physically contact a target cell for a few minutes After this physical contact the CTL would release the target and go on to bind to any additional targets in the area Targets that were contacted by CTL didn t die immediately but rather died within a couple of hours after contact Thus it was evident that through this brief physical association CTL caused lethal damage to the target When the target died so did the intracellular pathogen There are generally two distinct pathways that lead to cell death One is necrosis when a cell is damaged from the outside by physical or chemical damage The second pathway is apoptosis or programmed cell death One characteristic of apoptosis is that the cell s DNA is cleaved into small pieces of about 200 base pairs in length Cytotoxic Tlymphocytes appear to act mainly through induction of apoptosis through two distinct mechanisms In vivo it is likely that CTL try to kill a target by every available means CTL killing through cytotoxins Activated CTL have in their cytoplasm large granules that contain a variety of proteolytic enzymes and other proteins Collectively these molecules are referred to as cytotoxins Two classes of cytotoxins include perforins and granzymes After contact with a target cell the CTL can unidirectionally release these granules onto the surface of a target cell The perforins form pores in the target s cell membranes These holes allow free flowing of salt and water which contribute to cell death like poking a hole in a balloon The granzymes are structurally related to trypsin They probably get into the cell through the pores formed by perforins and cleave and denature proteins Additionally the granzymes activate endogenous apoptosis inducing endonucleases CTL killing through fas Perforins Q Q Granzymes r fas CT L Targe Many cells have on their surface a molecule called fas also referred to as APO1 Fas is a member of the receptor family for tumor necrosis factor or TNF CTL secrete TNF Activation of fas occurs following binding to TNF and this event induces the cell to undergo apoptosis Thus by one or both of these mechanisms an activated CTL can kill host cells that express foreign proteins in association with Class I MHC molecules Remember that all nucleated somatic cells express Class I MHC so CTL function is a potent immune mechanism of immune surveillance Introduction Several interactions between a Tcell and an antigen presenting cell APC must take place in order to activate a Tcell Following activation the Tcell will proliferate and differentiate In this lecture we will consider the extracellular and intracellular signals that are required for Tcell activation We will then consider the function of activated Tcells Activation Signals Consider Tcells that are produced by the thymus but have never come in contact with specific antigen These are referred to as naive or virgin Tcells These Tcells travel through the bloodstream and then migrate into lymph nodes and the spleen Once in the lymph nodes or spleen the Tcells bump into a variety of antigenpresenting cells including dendritic cells macrophages and Bcells If the Tcells don t come in contact with antigen they just circulate on through the lymph nodes However if a Tcell recognizes antigen associated with the proper MHC molecule Class I recognized by CD8 cells Class II recognized by CD4 cells the cell stops and undergoes cell division and differentiation Thus the Tcell changes from a naive Tcell to an activated Tcell In addition to this primary signal a costimulator or second signal is also required to activate Tcells This second signal is mediated by additional molecules expressed on the surfaces of the APC and the Tcells One important surface molecule on the APC is called B7 The molecule on the naive Tcell that binds to B7 is CD28 This interaction does not require antigen One result of these interactions is that Tcells start to express an additional molecule called CTLA4 CTLA4 binds very strongly to B7 which strengthens the binding between T cells and APC Although binding of CD28 to B7 activates the cell binding of CTLA4 to B7 deactivates the cell These interactions are illustrated below CD3 TCR and CD4 CTLA4 MHCAg Both interactions must occur for the Tcell to be activated In the absence of the second signal CD28 CTLA4 and B7 the Tcell will not differentiate and proliferate In fact it will become resistant to future activation This phenomenon is termed anergy and the Tcell is said to then be anergic Other interactions between the Tcell and the APC might also contribute to activation Many of these are poorly understood and the relative contribution of each is not known Therefore focus on learning the interactions described above Signal transduction following Tcell activation The next steps in Tcell activation involve transmitting the information that the Tcell has come in contact with antigen to the Tcell s nucleus Many intracellular events occur following Tcell activation Taken together these events are referred to as signal transduction The exact molecular interactions involved in signal transduction are becoming better understood and it is a topic of extreme interest in cell biology today For the purpose of our discussion understand that a cascade of ATPdependent events occur which ultimately increase mRNA transcription for many proteins These proteins mediate the function of T cells and some are expressed on the surface of these cells These molecules include receptors for growth factors the most important of which is the receptor for Interleukin2 IL2 IL2 receptor IL2R expression Once activated a Tcell will proliferate This cell division generates more antigen specific Tcells that can help clear the antigen from the host In order to proliferate the Tcell needs the growth factor IL2 IL2 will be discussed further in the cytokines lecture but for now understand that resting Tcells express on their surface a low af nity receptor for IL2 This lowaffinity receptor is composed of 2 polypeptide chains designated 3 and y Following contact with MHCantigen and B7 a Tcell will express a third component of the IL2 receptor called the X chain Together these 3 chains form a high affinity receptor for IL2 If IL2 is present in the environment it will be bound by the receptor and a signal will be transduced to the cell nucleus to start to undergo cell division This is illustrated below IL2 Antigen Activation Go forth and multiply Natural Killer Cells Another type of killer cells is a population of cells referred to as Natural Killer or NKcells NK cells are not Tcells They do not necessarily develop in the thymus although some might and do not have Tcell receptors They do recognize some tumor cells and play a role in early immune responses to infection particularly infection with intracellular microbes They have a phenotype that is often referred to as large granular lymphocytes NK cells express a surface antigen designated CD16 which is a receptor for the Fc portion of IgG NK cells seem to preferentially attack cells that express lower levels of Class I MHC molecules and they appear to kill targets using mechanisms similar to CTL namely perforins and TNFfi Although NK cells do not have antigenspecific receptors they do participate in antigenspeci c killing via a mechanism referred to as antibodydependent cellular cytotoxicity or ADCC below NK cells also have receptors that recognize decreased levels of Class I MHC molecules on their surfaces and kill these cells Many viruses attempt to escape from CTLs by down regulating expression of Class I MHC Antibody Dependent Cellular Cytotoxicity In addition to NK cells several different cell types including macrophages neutrophils and eosinophils mediate ADCC ADCC is described diagrammatically as shown below The important feature is that specific antibody focuses the effector function of nonspeci c cells In addition cellular activity is generally increased when cells bind to antibodies through the Fc receptors lt Antibody Target Effector cells with Fc Receptors THE HUMORAL IMMUNE RESPONSE OBJECTIVES Kinetics of the humoral Immune response 1 List general features of the humoral immune response 2 Draw an antibody response curve identify major regions of the curve and distinguish between primary and secondary antibodies 3 Define af nity maturation Bcell activation and function 1 Understand the events in B cell activation and differentiation 2 Understand the role of cytokines in antibody response 3 Distinguish between Tdependent and Tindependent antigens Complement and antigen clearance 1 List the key components of the classical pathway of complement activation 2 Define anaphylatoxins and their role in the humoral immune response Polyclonal and Monoclonal Antibody Responses 1 Understand the nature of polyclonal and monoclonal responses 2 Distinguish advantages and disadvantages of each in therapy and diagnostics THE HUMORAL IMMUNE RESPONSE Introduction The humoral immune response is characterized by the production of antibodies directed against the immunizing antigen The antigenbinding capacity of the Ig molecule is coupled with the biological effector functions of the constant region These effector functions facilitate the elimination of exogenous antigen from the host We will review the characteristics of a typical humoral immune response at the outset then elaborate on the cellular events that constitute a humoral immune response and finally understand what regulates the humoral response Characteristics of the humoral immune response Antibodies secreted by terminally differentiated B cells following contact with an exogenous antigen constitute the humoral immune response The response is characterized by distinct phases following exposure to antigen A lag phase immediately follows antigen exposure During this phase B cells undergo clonal selection and differentiate into plasma cells This is followed by a logarithmic increase in levels of antigenspecific antibodies in the serum which peak plateau and then decline The decline in antibody levels marks removal of the antigen Upon reexposure to the same antigen a second response develops albeit with different kinetics A typical response curve is shown in gure above Primary Response The antibody response following the first contact with the antigen is the primary response The kinetics of the primary immune response vary depending on the nature of the antigen the route of administration and the genotype of the host During a primay immune response IgM is secreted initially often followed by IgG A primary immune response can last for varying periods from one week to several weeks depending on the persistence of the antigen Secondary Response Activation of memory B cells formed during a primary antibody response by second exposure to the same exogenous antigen results in the secondary immune response The secondary response is distinguished from the primary response by the following criteria It has a shorter lag phase greater magnitude and a longer duration The specific antibodies display a higher affinity for the antigen and represent a variety of isotypes in addition to IgM Thus a secondary antibody response is much more effective than a primary response in the elimination of a foreign antigen such as a pathogen Af nig maturation In the course of an immune response the af nity of antibodies produced in response to the antigen increases as a function of time The average affinity of antibody for a given antigen at any time during the immune response depends on the dose of the immunizing antigen As antigen levels decline over time following immunization competition for available antigen increases As a result only those B cells with high affinity for the antigen can bind sufficient antigen to be activated and expanded This process is complemented with the ability of proliferating B cells to mutate their Ig genes While somatic mutation has the potential to generate antibodies with lower or higher binding af nity for the antigen the antigen selects only those B cells producing higher affinity antibodies as it becomes limiting A combination of these two mechanisms leads to affinity maturation a hallmark of the secondary response Hallmarks of the adaptive immune response Specificity for antigen Induction of memory a In 5 2 antiA 939 Mainly 19 U 2 1 antiA g g Mainly IgM A t39 A E E 1 antIB II IgM 1 o 14 o 6 14 Time Days Sequence of events within the lymphoid organ When an antigen enters the body it is carried to and concentrated in the draining lymph nodes and to the spleen by antigentransporting cells eg Langerhans cells or dendritic cells Here they deliver the antigen to B cells in the cortex and to T cells in the paracortex The initial activation of B and T cells is thought to take place in the paracortex populated mainly by T cells macrophages and dendritic cells The antigen is endocytosed and processed by macrophages and dendritic cells and presented to CD4 T cells within the paracortex T cells proliferate within the paracortex and secrete cytokines B cells specific for the antigen migrate from the cortex to the Tcell rich paracortex Minute amounts of antigen can be bound endocytosed and processed very efficiently by antigenspecific activated B cells through interaction of the antigen with antigenspecific surface immunoglobulin One antigen is taken up by Bcells in the presence of an additional stimulus such as a TLRligand or adjuvant the B cells increase in size increase expression of surface Ig and importantly increase expression of MHC Class II and B7 molecules Endocytosed antigen is processed through the endogenous antigen processing pathway and fragments of the endocytosed antigen are expressed on the surface of the Bcell associated with newly expressed Class II MHC molecules In this fashion the Bcells become excellent antigenpresenting cells The interaction between CD40 on B cells and its ligand on T cells is required for and permits the formation of TB conjugates gure below leading to increased secretion of cytokines by T cells B cells proliferate in response to the cytokines secreted by T cells in the conjugates and form small foci at the edges of Tcell rich zone B cells in these foci differentiate into plasma cells to secrete antibodies that constitute the primary antibody response Th2 cell APC directional secretion of cytokines A few days later some of the activated and proliferating B and T cells migrate to the cortex to form primary follicles Here follicular dendritic cells deliver antigen to the B cells via iccosomes or immune complexes formed during the primary immune response and retained by the dendritic cells on their surface The B cells process and present the antigen to activated T cells forming TB conjugates and driving the secretion of cytokines This leads to the proliferation and differentiation of B cells giving rise to a secondary follicle and thence to a germinal center Activated Bcells leave the germinal centers and migrate into the medulla where they become plasma cells and secrete antibody or further differentiate into memory B cells Memory B cells either remain in the follicular mantle surrounding the germinal center or leave via efferent lymphatics to circulate to other parts of the body particularly the bone marrow Three other phenomena are associated with B cells that are stimulated within the germinal center First the cells are actively proliferating and the resulting daughter cells can differentiate into plasma cells that secrete high levels of antibody Seconds a process called somatic hypermutation introduces base changes within the Ig variable regions leading to changes in the amino acid composition of the antigenbinding site This leads to a loss or gain in the affinity strength of binding for the antigen Increase in af nity of the overall antibody response is termed af nig maturation B cells that lose af nity for the antigen die and are rapidly phagocytosed by tingible body macrophages Third actively proliferating B cells also switch Ig isotype the class to which they switch is determined by the nature of the antigen and the cytokines produced by T cells Cytokines in the humoral immune response The initial activation of CD4 helper T cells in the paracortex stimulates them to produce IL2 IL2 acts as an autocrine factor and induces proliferation of CD4 T cells that in turn produce more IL2 Depending on the antigen and additional signals delivered by cytokines and TLRs the activated and proliferating T cells may develop into the Th1 Th2 or Th17 subsets Th2 cells a produce IL4 IL5 and IL10 IL4 acts as a Bcell growth factor initially inducing proliferation and later differentiation of activated B cells while IL5 primarily supports Bcell differentiation IL10 inhibits the production of IFNy by the Th1 subset ofT cells IL4 and IL5 also stimulate differentiation of activated B cells into plasmablasts following TB conjugate formation IL6 is produced primarily by macrophages and boosts antibody secretion by terminally differentiated plasma cells Immunoglobulin isotype switching also requires cytokines In the inbred mouse and in humans IL4 induces a switch to the IgG1and IgE isotypes In the mouse IFNy is required for a switch to IgG3 and IgG2a TGFB produced by macrophages and B cells can induce a switch to IgG2b and IgA Clearance of AntigenAntibody complexes Following the discovery of immunoglobulins it was demonstrated that antibacterial antibodies were able to agglutinate bacteria by themselves but could cause lysis of bacterial cells only in the presence of a heatlabile substance present in the serum The substance that assists or complements the lytic function of antibodies was termed complement We now know that complement is not a single protein but a series of proteins that form highly regulated functional cascades to provide many of the effector functions of the humoral immunity and inflammation The complement system is comprised of at least 20 different proteins and glycoproteins that are synthesized largely by hepatocytes These components constitute 15 by weight of the serum globulin fraction and are found in the circulation in functionally inactive forms The complement components are designated by numerals C1 through C9 or by letter symbols or by names The components become activated upon cleavage the ragments are then denoted by letters a or b a being the smaller fragment and b being the larger Activation of complement involves cascades of proteolysis which allow for tremendous amplification since each fragment functioning as an enzyme can generate multiple activated enzyme molecules at the next step Multiple complement fragments can also interact to form functional complexes The main biologic functions of the complement system are the following Complement components opsonize antibodybound particles opsonization enhances receptor mediated phagocytosis of these particles Opsonization is largely due to a fragment of C3 called C3b specific receptors for which are expressed on a large number of leukocytes Certain proteolytic fragments of complement components C3a C4a and C5a act as anaphylatoxins and can induce degranulation of mast cells C5a fragment also acts as a chemoattractant for neutrophils This leads to localized in ammation and hypersensitivity reactions at sites of complement activation Certain activated complement components polymerize on the cell surface of pathogens to form the membraneattack complex These complexes constitute pores in the cell membranes and leading to the eventual lysis of the pathogen Certain complement components bind to circulating immune complexes and induce their clearance from circulation Complement activation and function in the humoral immune response The classical pathway of complement function is activated by antigenantibody complexes Activation of C1 occurs when it binds the CH3 domains of IgM or CH2 domains of IgG The binding brings about a conformational change in C1 leading to its enzymatic activation Fragments C1r and 015 are sequentially generated activating each of them Activated C1s cleaves C4 and CZ generating 04b and 02a which deposit on the pathogen and form the C4b2a complex while C4a and 02b are released C4b2a a 03 convertase cleaves 03 to yield 03b and 03a 03a is released while 03b joins C4b2a to yield C4b2a3b C4b2a3b is a C5 convertase which cleaves C5 to 05b and 05a 05b is the rst component of the membraneattack complex Activationof the classical pathway of complement is therefore dependent on the productionof antigenspecific antibodies The C5b generated as a result of complement activation by the classical pathway can combine with CS C7 CB in that order to form a C5b678 complex which attaches stably to the surface of the target cell Several 09 molecules polymerize at the site of C5b678 attachment to generate the membraneattack complex MAC which can form pores in the target cellmembrane and cause celldeath by osmotic lysis Circulating leukocytes express one or more of receptors for the various complement components or their cleavage products These receptors mediate many of the biologic activities of the complement components and also play an important role in the regulation of the complement system Tdependent v Tindependent antigens Certain bacterial components can induce antibody responses independently of Tcell help These are referred to as Tindependent antigens Tindependent antigens are either repeating large molecular weight polysaccharides or they contain bacterial LPS In either case strong enough signals are sent to the Bcell to produce IgM Tindependent antigens do not induce IgG class switching except in rare circumstances beyond the scope of the course do not induce memory Bcells and usually induce only low af nity antibodies Antibodies in Diagnosis and Treatment of Disease The high speci city and biologic stability of antibodies make them excellent reagents for therapy diagnostics and as research tools As therapeutic reagents antibodies are used as a treatment for some infections and intoxications as well as for treatment against cancer and other chronic diseases In dentistry efforts have been made to use antibodies in the treatment of periodontal disease by applying antibodies specific for periodontal pathogens to the gums of patients Antibodies are used for diagnosing a wide range of conditions including infectious diseases such as HIV infection and strep throat as well as other conditions such as pregnancy Monoclonal and Polyclonal Antibodies So how does one obtain antibodies that could be used in diagnosis and treatment One method is to immunize an animal or human with an antigen and then collect blood serum from the immunized subject The serum will contain antibodies against the antigen and these antibodies can be further purified by additional biochemical methods Immunization and collection of antibody containing serum has been performed since the late 1800s and still goes on today Remember that an antigen often has many epitopes and each epitope is recognized by an individual Bcell clone Thus the antibody response is really a collection of antibodies produced by each antigen activated clone This type of response is referred to as a polyclonal response It is important to remember that during the response to a complex antigen each antibody is the product of a single Bcell clone In the mid1970 s a technique was developed to obtain large amounts of antigenspecific antibody from single Bcell clones that are cultivated in vitro This technique eliminated the need to repeatedly immunize and bleed animals or human volunteers This technique involves first immunizing a single animal and then harvesting Bcells from the immune animal These Bcells by themselves have a nite lifespan of only a few days once taken out of the animal However the Bcells can be biochemically fused to a tumor cell that can grow in vitro indefinitely This resulting type of cell is said to be immortalized and is referred to as a hybridoma meaning that it is a hydrid between the tumor cell and the Bcell Not only will this cell now live and divide in vitro it will also produce the antibody that was produced by the Bcell used to make the hybridoma The antibody is secreted into the culture fluid at high levels from where it can be purified biochemically and used as described above This antibody the product of a single Bcell clone is referred to as a monoclonal antibody There are advantages and disadvantages to using either monoclonal or polyclonal antibodies Polyclonal antibodies are initially easier to produce but do not yield a limitless supply The specificity of polyclonal antibodies is less well defined but in some cases the combination of specificities against multiple epitopes makes polyclonal antibodies sometimes more effective Monoclonal antibodies generally have a limitless supply and once produced are can be made forever Most antibodybased therapeutic and diagnositic reagents being developed and produced today are monoclonals HYPE RSE NSITIVITY Immune responses against exogenous antigens and pathogens are routinely accompanied by cell death and local and systemic injury to body tissues However such pathologic effects are minor and they abate as the foreign antigen is eliminated Some antigens have the property to induce abnormal reactions in the host which constitute significant pathological damage to host tissue Such reactions are termed Hypersensitivig H ypersensitivig can be defined as the pathologic consequence of an altered reaction to an antigen Hypersensitivity can develop in the course of humoral or cell mediated immune responses following second exposure to certain antigens The antigen that is responsible for induction of hypersensitivity is recognized as allergen and the first exposure to the antigen constitutes sensitization The effectors of hypersensitivity include pharmacological agents released by various in ammatory cells immunoglobulins and components of the complement system Four major types of hypersensitivity reactions can be distinguished based on differences in the nature of the allergen the primary cell types involved and the mechanism of tissue damage These are listed below kinetics mediator type nature of mechanism of allergen tissue damage small protein degranulation of immediate antibody I low mast cells and IgE concentration basophils complement and cell or matrix Fc receptor II bound mediated antibody IgG cytotoxicity IgM ADCC NK cells phagocytosis immune complex deposition III soluble inflammation localized tissue and vascular damage small specialized delayed T cell IV molecules cytokines and lytic can complex enzymes edema with skin proteins Type I hypersensitivig reaction is induced by certain antigens called allergens An allergen induces a humoral immune response by the same mechanisms as any other antigen However this class of antigen induces the production of antigenspecific IgE antibodies that bind to receptors on mast cells and basophils through high affinity Fc receptors for IgE present on the surface of these cells Cells thus coated with IgE are said to be sensitized Reexposure to the allergen crosslinks the IgEbound Fc receptors thereby inducing degranulation of the sensitized mast cells and basophils The release of pharmacologically active substances from degranulated mast cells and basophils mediate effects such as smoothmuscle contraction increased vascular permeability and vasodilation Type I reactions can induce annoying localized conditions such as hay fever or eczema as well as serious lifethreatening reactions such as systemic anaphylaxis his tamine pro s tag landins de granul ate le uko trie nes P AF kinino g e nas e secrete 1L4 IL13 1L3 1L5 GMCSF mast cell Type II hypersensitivig is mediated by IgM andor IgG antibodies Antibodies induced against an exogenous antigen may crossreact with structures on surface of circulating cells or xed body tissues Cells or tissues coated with such autoreactive antibodies activate and become the target of complementmediated destruction Cell bound antibodies can also mediate destruction of target cells by antibodydependent cell mediated c otoxicig ADCC where the effector cells are natural killer cells that express Fc receptors for IgG This type of hypersensitivity is commonly encountered following the administration of certain drugs sensitivity to Penicillin is quite common Type II reactions also occur in individuals given blood transfusions from blood group incompatible donors Hemolytic disease of the newborn called erythroblastosis fetalis which develops due to incompatibility of the Rh antigen is another example of type II hypersensitivity Type III hypersensitivity is induced by immune complexes Immune complexes formed during the course of an immune response are normally cleared by Fc or complement receptormediated uptake and degradation In some cases large amounts of immune complexes develop that cannot be cleared ef ciently This frequently happens in the event of antigen excess This activates the complement system Deposition of the immune complexes induces chemoattraction of in ammatory cells at the site of deposition The majority of damage is caused by the release of lytic enzymes by neutrophils that make unsuccessful attempts to ingest the immune complexes Localized type III reactions include the Arthus reaction which typically develops 28 hours after antigen is injected into tissue The complement components C3a C4a and C5a released at the site of reaction function as anaphylatoxins and cause degranulation of mast cells in the area leading to edema and erythema When large amounts of immune complexes are present in the bloodstream it can lead to generalized vasculitis lymphadenopathy arthritis and sometimes glomerulonephritis including poststreptococcal glomerulonephritis Autoimmune diseases such as Systemic Lupus Erythematosis SLE Rheumatoid arthritis and Goodpasture s syndrome are examples of type III hypersensitivity Serum Sickness though rarely seen anymore is a classic example of type III hypersensitivity reactions Type IV hypersensitivig reactions are mediated by specialized Th1 type helper T cells called TDTH Certain antigens enter the body through skin or mucosa where they bind to cellsurface proteins on epidermal keratinocytes or Langerhans cells The antigen is presented to CD4 T cells Cytokines secreted by the T cells cause macrophages to accumulate at the site The lysosomal enzymes released by macrophages cause damage to local tissue Contact dermatitis as well as skin and lung lesions resulting from infection with Mycobacteria species are examples of chronic delayedtype hypersensitivity reactions Since Mycobacteria induce TDTH cells in humans a skin test with tuberculin or lepromin is widely used as an indicator of possible exposure to the pathogen In addition poison ivy is an example of a type IV hypersensitivity reaction that is caused by the urushiol resin binding to self MHC antigens to make them look like nonself Mediators of hypersensitivity reactions Histamine a bioactive amine released by mast cells and basophils immediately following and as a result of degranulation Histamine binds to two types of receptors on a variety of cells Binding of histamine to H1 receptors induces contraction of smooth muscle cells but induces separation of cell junctions on endothelial cells leading to increased vascular permeability Binding to H2 receptors also increases vascular permeability Leukotrienes produced as a result of breakdown of arachidonic acid via the lipoxygenase pathway They induce vasodilation and bronchoconstriction They can also act as chemoattractants for in ammatory cells Prostaglandins produced by the breakdown of arachidonic acid via the cyclo oxygenase pathway Induce vasodilation and bronchoconstriction and platelet aggregation Platelet aggregating factor released from mast cells and macrophages causes platelets to aggregate leading to release of platelet factor 4 PL4 and thromboxane TxB4 PL4 release leads to increased platelet aggregation particularly in the lung TxB4 causes increased pulmonary arteriole pressure and prolonged systemic hypotension Kiningenerating proteases responsible for production of bradykinin which in turn induces smooth muscle contraction increased vascular permeability and stimulates pain bres Eosinophil and neutrophil chemoattractants Induce migration of eosinophils and neutrophils to the site of in ammation Immunoglobulin and complement IgM and IgG can mediate lysis of host cells they bind to via ADCC Immune complexes are deposited in host tissues where they activate the complement cascade The release of anaphylatoxins C3a C4a and C5a recruits inflammatory cells to the site of complex deposition resulting in release of lytic enzymes and tissue destruction Cytokines sensitized CD4 TDTH cells in the skin secrete macrophage chemotactic factor MCF and migration inhibition factor MIF These cytokines recruit macrophages to the site of antigen entry where they release lysosomal enzymes which cause dermal capillaries to leak plasma This results in formation of edema and brin deposition TOLERANCE The mammalian germline has the capacity to mount an immune response to an infinite number of antigens by virtue of the numerous immunoglobulin and TCR variable region gene segments that can recombine and diversify to give a large number of specificities However the immune system normally does not mount a response against self antigens This is because lymphocytes with receptors recognizing self antigens are held in check by specialized mechanisms Similarly the immune system cannot respond to extremely small amounts or very large amounts of foreign antigen in order to avoid excessive pathological damage to host tissues This lack of immune response is described as tolerance Tolerance can therefore be de ned as antigeninduced block in the development growth or differentiation of specific lymphocytes MECHANISMS OF TOLERANCE TO SELF ANTIGENS Two mechanisms for induction of tolerance in T and B lymphocytes are clonal deletion and clonal anergy While clonal deletion refers to the physical elimination of antigenreactive lymphocytes clonal anergy refers to a functional silencing of antigenspecific lymphocytes Clonal deletion is the major means of eliminating potentially selfreactive lymphocytes within primary lymphoid organs such as the thymus and the bone marrow during development Interaction of immature lymphocytes with self antigen within the thymus or the bone marrow leads to elimination of lymphocytes with receptors recognizing such antigens The AutoImmune Regulator AIRE protein induces transcription of tissue speci c genes in the thymus thereby enabling the thymus to express tissue speci c antigens that one wouldn t normally expect to be expressed in the thymus eg insulin Thus insulin specific Tcells as well as other autoantigenspeci c Tcells can be negatively selected in the thymus Lymphocytes recognizing antigens that are not presented at these primary sites may be eliminated or silenced in the periphery Induction of clonal anergy frequently results from absence of a costimulatory signal upon interaction with antigen for T cells or through as yet unknown mechanism for B cells Anergized cells suffer a block in activation which in some instances can be reversed under experimental conditions B cells can also be tolerized by exogenous antigens that are multivalent Polysaccharides containing repeating homopolymers and polymeric proteins are potent tolerogens Immature B cells are also easily tolerized by high doses of exogenous antigen Hyperimmunization with an antigen frequently leads to high levels of circulating specific antibody titers This can shut down an ongoing immune response and shut down retriggering of an immune response When tolerance occurs in the primary lymphoid organs ie thymus and bone marrow it is referred to as central tolerance When tolerance occurs in the secondary lymphoid tissues it is referred to as peripheral tolerance REGULATION OF IMMUNE RESPONSES Regulation of specific immune responses is a complex phenomenon The nature Intensity and duration of immune responses is in uenced by prior host exposure to antigen the nature and the concentration of antigen circulating antibodies and immune complexes cytokines etc Antigen exerts a major effect on the type of immune response generated Chemically different antigens stimulate different types of immune responses Whereas proteins induce both humoral and cellmediated immunity responses to polysaccharides and lipids are typically T cell independent and consist largely of IgM antibodies Antigen dose frequently dictates the magnitude of the immune response low doses of antigen are poorly immunogenic and elicit few clones of antigenspecific highaf nity antibodies whereas larger doses elicit a broader range of antibodies Very high doses lead to nonspeci c responses or tolerance The port of antigen entry also influences the outcome Antigens that enter through the skin are usually immunogenic over a wide dose range whereas large amounts of antigens administered orally or by intravenous route can induce a state of speci c unresponsiveness This is due in large part because of the potent activity of Langerhan s cells and dermal dendritic cells that reside in layers of the skin and actively take up antigens that breech the protective epidermis It has been observed that individuals vary in their responsiveness to the same foreign antigen Unresponsiveness may be due to the absence of antigen speci c lymphocytes Alternately polymorphisms in the MHC antigens may influence the presentation of speci c antigen epitopes even if specific T cells are present Thus the genotype of the host can regulate the immune response Antibody exerts feedback on its production This is thought to be due to competition of antibody with antigenreactive B cells for binding to antigen If antibody is administered passively following antigen this antibody competes with B cells that bind the antigen with low af nity and drives the response toward high affinity antibodies Antigenantibody complexes have been shown to enhance or to suppress immune responses to distinct antigens The complexes may bind to Fc receptors on the surface of a variety of cells and exert their effect indirectly Cytokines secreted by Th1 and Th2 subsets ofT cells are distinct and the cytokine pattern determines whether a humoral or a cellmediated immune response predominates following antigen exposure Selflimitation is an important property of the immune system because it ensures that immune responses persist only as long as they are needed The products of lymphocytes that are activated by antigen such as immunoglobulin and cytokines are secreted for brief periods and are shortlived Similarly the effector lymphocytes such as plasma cells and CTLs are terminally differentiated nonselfrenewing and shortlived When antigen becomes limiting activation of additional B andor T cells halts These features ensure selflimitation of immune responses The network theory states that B and T cell responses against an antigen are regulated in part by within through the recognition of idiotypic determinants on immunoglobulin and TCR molecules For example the idiotopes on the primary antibody Ab1 against an antigen induce the production of the antiidiotype antibody Ab2 which in turn induces the production of the antiantiidiotype antibody or Ab3 Ab3 often resembles Ab1 and the network begins to limit itself Some of the Ab2 may appear as an internal image of the exogenous antigen Passively administered Ab2 may either stimulate or suppress the production of Ab1 in the absence of antigen Based on experimental systems a population ofT cells called Tregulatory Treg cells has been demonstrated Removal of these cells in experimental animals causes induction of autoimmune disease The mechanism of action of these cells is not well understood but Tregs do produce TGF which is immuno suppressive Finally the central nervous system interacts with the immune system through neuroendocrine mediators Lymphocytes display receptors for a large number of neuroendocrine substances Many of the neuroendocrine and neurotransmitter substances released during a stress response have been shown to either enhance or suppress the immune response A good example of this is reactivation of oral herpes simplex during times of physical or emotional stress


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