Topic 1: Introduction to the Immune System

The immune system has evolved to protect us from pathogens. Intracellular pathogens infect individual cells (e.g. viruses), whereas extracellular pathogens divide extracellularly within tissues or the body cavities (e.g. many bacteria). Phagocytes and lymphocytes are key mediators of immunity. Phagocytes internalize pathogens and degrade them. Lymphocytes (B and T cells) have receptors that recognize specific molecular components of pathogens and have specialized functions. B cells make antibodies, cytotoxic T lymphocytes (CTLs) kill virally infected cells, and helper T cells coordinate the immune response by direct cellcell interactions and the release of cytokines. Specificity and memory are two essential features of adaptive immune responses. As a result the immune system mounts a more effective response on second and subsequent encounters with a particular antigen. Nonadaptive (innate) immune responses do not alter on repeated exposure to an infectious agent. Antigens are molecules that are recognized by receptors on lymphocytes. B cells usually recognize intact antigen molecules, whereas T cells recognize antigen fragments on the surface of other cells. An immune response occurs in two phases antigen recognition and antigen eradication. In the first phase clonal selection involves recognition of antigen by particular clones of lymphocytes, leading to clonal expansion of specific clones of T and B cells and differentiation to effector and memory cells. In the effector phase, these lymphocytes coordinate an immune response, which eliminates the source of the antigen. Vaccination depends on the specificity and memory of adaptive immunity. Vaccination is based on the key elements of adaptive immunity, namely specificity and memory. Memory cells allow the immune system to mount a much stronger response on a second encounter with antigen. Inflammation is a response to tissue damage. It allows antibodies, complement system molecules, and leukocytes to enter the tissue at the site of infection, resulting in phagocytosis and destruction of the pathogens. Lymphocytes are also required to recognize and destroy infected cells in the tissues. The immune system may fail (immunopathology). This can lead to immunodeficiency, hypersensitivity, or autoimmune diseases. Normal immune reactions can be inconvenient in modern medicine , for example blood transfusion reactions and graft rejection.

1. Adaptive and innate immunity Any immune response involves, firstly, recognition of the pathogen or other foreign material, and secondly, mounting a reaction against to eliminate it. There are two different types of immune response:
Innate or not-adaptive immune responses Adaptive immune responses

The important difference between these is that an adaptive immune response is highly specific for a particular pathogen and improves with each successive encounter with the same pathogen: in effect the adaptive immune system remembers the infectious agent and can prevent it from causing disease later. So the two key features of the adaptive immune response are thus specifity and memory.

2. Antigens Antigens or immnunogen are molecules that initiate adaptive immune responses (e.g. components of pathogenic organisms), generating antibodies. Antigen molecules each have a set of antigenic determinants, also called epitopes. Epitopes are molecular shapes recognized by antibodies and T-cell receptors of the adaptative immune system. Each antibody receptor recognizes one epitope rather than the whole antigen. Ever simple microorganisms have many different antigens which may be protein, lipid or carbohydrate.
Antigens are the initiators and driving forces of all adaptive immune responses. The immune system has evolved to recognize antigens, destroy them, and eliminate the source of their production. When antigen is eliminated, immune responses switch off. Both T cell receptors and immunoglobulin molecules (antibody) bind to their cognate antigens with a high degree of specificit (T cells use their antigen-specific receptors (TCRs) to recognize the antigenic peptides bound to MHC molecules expressed on the surface of the antigens). T cells recognize antigens that originate within other cells, such us as viral peptides from infected cells. They do this by binding specifically to antigenic peptides presented on the surface of the infected cells by molecules encoded by the major histocampatibility complex (TCRs) to recognize the unique combination of MHC molecule plus antigenic peptide. Unlike B cells, which recognizes is made up of residues from the MHC molecule and the antigen peptide. MHC (major histocompatibility complex) a genetic region found in all mammals, encoding more than 100 genes. Class I and class II molecules are primarily responsible for transporting peptide antigens to the surface of a cell for recognition by T cells. Recognition of MHC class I and II molecules also underlies graft rejection.

Clonal selection involves proliferation of cells that recognize a specific antigen Each antibody-producing cell (B cell) is programmed to make just one antibody, which is placed on its surface as an antigen receptor (B cell in this example). In this way these cells are stimulated to proliferate and mature into antibody-producing cells, and the longerlive memory cells, all having the same antigen-binding specifity.

Topic 2: Cells involved in the Immune Response

Many cells of different lineages are adapted to carry out specialized functions in the immune response. B and T lymphocytes express specific antigen receptors and other surface molecules (markers) important for their different functions. Antigen-preenting cells are required by T cells to enable them to respond to antigens. B lymphocytes recognize native antigens not processed and presented by other cells. There are functional subpopulations of T lymphocytes which have heper, suppressor and cytotoxic activities. New surface molecules appear on lymphocytes following activation by specific antigens. Phagocytic cells with specific surface markers are found in the circulation (monocytes and granulocytes) and reside in tissues (e.g. Kupffer cells in the liver).

1. Origin of cells involved in the immune response

All hemopoietic cells are derived from pluripotent stem cells which give rise to two main lineages: One for lymhoids cells: the common lymphoid progenitor has the capacity to differentiate into either T cells or B cells depending on the microenvironment to which it homes. In mammals, T cells develop in the thymus while B cells develop in the fetal liver and bone narrow. The precise origin of some antigen-presenting cells (APCs) is uncertain, although they do develop ultimately from the haemopoietic stem cells. NK cells also derive from the common lymphoid progenitor cells. Lymphocytes recirculate through secondary lymphoid tissues. And the other for myeloid cells: myeloid cells differentiate into committed cells whose collective name is granulocytes, and are formed by cells eosinophils, neutrophils and basophils.

Moreover also find: Platelets produced by megakaryocytes are released into the circulation. Blood platelets are not cells, but cell fragments derived from megakaryocytes in the bone marrow. They contain granules, microtubules, and actin/myosin filaments, which are involved in clot contraction. Platelets are also involved in immune responses, especially in inflammation. Monocytes, cells of the mononuclear phagocytic system are found in virtually all organs of the body where the local microenvironment determines their morphology and functional characteristics (e.g. in the lung as alveolar macrophages and in the liver as Kupffer cells). The main role of the mononuclear phagocytes is to remove particulate matter of foreign origin (e.g.microbes) or self origin (e.g. aged erythrocytes). Myeloid progenitors in the bone marrow differentiate into pro-monocytes and then into circulating monocytes, which migrate through the blood vessel walls into organs to become macrophages. Mast cells (mastocyte) is a resident cell of several types of tissues and contains many granules rich in histamine and heparin. Although best known for their role in allergy and anaphylaxis, mast cells play an important protective role as well, being intimately involved in wound healing and defense against pathogens. Interdigitating cells and dendritic cells act as antigen-presenting cells (APCs) in secondary lymphoid tissues. APCs (antigen-presenting cells): a variety of leucocytes that carry antigen in a form that can stimulate lymphocytes.

Bone marrow-derived APCs are found especially in lymphoid tissues, in the skin, and in mucosa. APCs in the form of Langerhans cells are found in the epidermis and are characterized by special granules (the tennis racquet-shaped Birbeck granules; not shown here). Langerhans cells are rich in MHC class II molecules, and carry processed antigens. They migrate via the afferent lymphatics (where they appear as veiled cells) into the paracortex of the draining lymph nodes. Here they make contact with T cells. These interdigitating dendritic cells (IDCs), localized in the T cell areas of the lymph node, present antigen to T helper cells. Antigen is exposed to B cells on the follicular dendritic cells (FDCs) in the germinal centers of B cell follicles. Some macrophages located in the outer cortex and marginal sinus may also act as APCs. In the thymus, APCs occur as IDCs in the medulla. (HEV, high endothelial venule).

Topic 3: The Lymphoid System

Lymphoid organs and tissues are either primary (central) or secondary (peripheral). Thymus and bone marrow are the primary lymphoid organs. Lymphocytes differentiate from stem cells in the primary organs but migrate to, and function in, secondary organs and tissues. The systemic lymphoid system includes the spleen and lymph nodes. The mucosal system includes all the lymphoid tissues associated with mucosal surfaces. Peyers patches are organized collections of lymphoid tissue present in the wall of the small intestine which process antigens present in the gut. The peripheral lymphoid tissues are equipped with phagocytic cells and other accessory cells that assist the function of T and B lymphocytes located there. Lymphocytes are not sessile there is continuous lymphocyte traffic from the blood stream into lymphoid tissues and back again into the blood via the thoracic duct.

Primary lymphoid organs: Thymus and bone marrow are the primary (central) lymphoid organs. They are the sites of maturation for T and B cells, respectively. It is in the primary lymphoid organs that lymphocytes acquire their repertoire of specific antigen receptors to cope with antigenic challenges received during thir lifespans. The cells are selected for tolerance to autoantigens and are therefore capable of recognizing only non-self antigens when the cells are in the periphery. Cellular and humoral immune responses occur in the secondary (peripheral) lymphoid organs and tissues.

 Secondary lymphoid organs: can be classified according to the body regions they defend. The spleen responds predominantly to blood-borne antigens. Lymph nodes mount immune responses to antigens circulating in the lymph, entering through the skin (subcutaneous lymph nodes) or through mucosal surfaces (visceral lymph nodes). Tonsils, Peyers patches, and other mucosa-associated lymphoid tissues (MALT) (blue boxes) react to antigens that have entered via the surface mucosal barriers. The bone marrow is both a primary and a secondary lymphoid organ because it gives rise to B and NK cells, but is also the site of B cell terminal differentiation (long-lived plasma cells).

Topic 4: Antibodies and their Receptors

Circulating antibodies recognize antigen in serum and tissue fluids. There are 5 classes of antibody: IgG, IgA, IgM, IgD and IgE. Immunoglobulins have a basic unit of two light chains and two heavy chains. The heavy chains differ between classes: IgA and IgM occur as oligomers of the four chain unit. The chains are folded into discrete regions called domains. There are two domains in the light chain and four or five in the heavy chains, depending on their class. Immunoglobulin fragments may be produced by proteolytic cleavage. These are useful experimentally and therapeutically. Papain generates two antigen binding (Fab) fragments and one Fc fragment from each IgG molecule whereas pepsin produces a large F(ab)2 fragment containing both antigen-binding sites. Hypervariable regions form the antigen-binding sites. There are three such regions in the V domains of each light and heavy chain. The folding of the domains causes them to be clustered at the distal tips of the molecule, producing two antigen-binding sites for each four chain unit.

All antibodies are bifunctional, because they exhibit one or more effector functions in addition to antigen binding. These biological activities are localized to sites that are distant from the antigen binding sites (mostly in the Fc region). Immunoglobulin receptor molecules are expressed by mononuclear cells, neutrophils, NK cells, eosinophils and mast cells. They interact with the Fc regions of different classes of immunoglobulins and promote activities such as phagocytosis, tumor cell killing and mas cell degranulation. Most of these Fc receptors are members of the immunoglobulin superfamily and have two or three extracellular immunoglobulin domains.

Antibody receptors There are three classes of cell surface receptor for IgG (FcR) are defined in humans: FcRI (CD64); FcRII (CD32); and FcRIII (CD16). Each receptor is characterized by a glycoprotein chain that binds to antibody and has extracellular domains homologous with immunoglobulin domains that is; they belong to the immunoglobulin superfamily, as do receptors for IgA (FcR) and IgE (FcRI). FcRs are expressed constitutively on a variety of cell types and may be upregulated or induced by environmental factors (e.g. cytokines).

Biological activation results from cross-linking of the FcR and consequent aggregation of immunoreceptor tyrosine-based activation (ITAM) or immunoreceptor tyrosine-based inhibitory (ITIM) motifs in the cytoplasmic sequences. Phosphorylation of the ITAM motif triggers activities such as: phagocytosis; antibody-dependent cell-mediated cytotoxicity (ADCC); mediator release; and enhancement of antigen presentation.

Receptors for Fc in humans belong to the immunoglobulin superfamily, and have either two or three extracellular immunoglobulin domains. Motifs (ITAM, ITIM) on the intracellular segments or on associated polypeptides are targets for tyrosine kinases involved in initiating intracellular signaling pathways.

Topic 5: T-Cell Receptors and MHC Molecules

The T-cell antigen receptor (TCR) is a disulphide-linked heterodimetric (either or ) glycoprotein that enables T cells to recognize a diverse array of antigens. It is associated at the cell surface with a complex of polypeptides known collectively as CD3. The major histocompatibility complex (MHC) encodes two sets of highly polimorfic cell surface molecules, termed HCM class I and HMC class II. The TCR recognizes processed antigen as peptide fragments bound to MHC class I or class II molecules. Both MHC and peptide residues associate with the TCR. A peptide binding cleft is formed by the folding of an MHC molecule. This accommodates peptides that have been processed by the cell, to be presented to T cells. Peptides of 8 or 9 residues can bind to class I molecules, whereas longer peptides can bind to class II molecules. Binding pockets within the clefts are able to accommodate different peptides depending on the haplotype. Since MHC molecules are highly polymorphic, and since a cell can express several different MHC molecules, this explains how the cell can present many different antigenic peptides to a T cell.

T-Cell receptors Antigen recognition by T lymphocytes is central to the generation and regulation of an effective immune response. It was called TCR (or TCR) because it was a heterodimeric molecules comprising an chain and a chain linked by a disulphide bond. The or heterodimers must associate with a series of polypeptide chains collectively termed the CD3 complex for the antigen-binding domains of the TCR to form a complete, functional receptor that is stably expressed at the cell surface and is capable of transmitting a signal upon binding to antigen. The four members of the CD3 complex (, , , and ) are sometimes termed the invariant chains of the TCR because they do not show variability in their amino acid sequences. Organization of the TCR complex The TCR and (or and ) chains each comprise an external V and C domain, a transmembrane segment containing positively charged amino acids, and a short cytoplasmic tail. The two chains are disulfide linked on the membrane side of their C domains. The CD3 , , and chains comprise an external immunoglobulin-like C domain, a transmembrane segment containing a negatively charged amino acid, and a longer cytoplasmic tail. A dimer of , , or is also associated with the complex. Several lines of evidence support the notion that the TCRCD3 complex exists at the cell surface as a dimer. The transmembrane charges are important for the assembly of the complex. A plausible arrangement that neutralizes opposite charges is shown.

Topic 6: The Generation of Diversity

The immune system is able to recognize and respond to many antigens by generating great diversity in the antibodies produced by the B cells, and in the antigen receptors expressed by T cells. Immnoglobulins are composed of heavy and light chains, the light chains being either or. The number of possible antigen-binding sites is the product of the number of heavy and light chains. Inmmnoglobulin light chains are encoded by V and J gene segments; heavy chains are also encoded by V and J segment genes with additional diversity provided by the D gene segment. Diversity is achieved by the recombination of a limited number of V, D and J gene segments to produce a vast number of variable domains. Immunoglobulin heavy and light chains undergo structural modifications after antigen stimulation, called somatic mutation. This does not occur with T-cell receptor. The TCR is generated by flour different sets of genes: and genes are expressed in the majority of peripheral T cells; and genes are expreseed in a subpopulation of thymic T cells and in a minor population of peripheral T cells. Diversification of TCR receptor also occurs by recombination between V, D and J gene segments, with minor variations in detail for each locus. Recombination of V, D and J gene segments for both immunoglobulin and T-cell receptors is controlled, at least in part, by two recombination activating genes (RAG-1 and RAG-2). In addition to simple conbinations of V,D and J regions , diversity in the TCR and immunoglobulins depends upon N-region diversification, joining-site variation and multiple D regions. Immunoglobulin class switching involves recombination of VDJ genes with various C region genes and differential RNA splicing.

Topic 7: Antigen Recognition

Antibodies are highly specific for three-dimensional conformation of eptopo. Antibody affinity is a measure of the strength of the bond between an antibodys combining site and a single epitope. The functional affinity or avidity of the interaction also depends on the number of binding sites on the antibody and their ability to react with multiple epitopes on the antigen T cells recognize cell-bound antigen in association with MHC class I or class II molecules. Peptide fragments from processed antigen bind to grooves of MHC antigens. MHC class I and class II molecules present peptides derived, respectively, from endogenous and exogenous antigens. This is reflected by the intracellular site which processed antigen accesses and binds to the MHC molecules. Peptides that bind to MHC class I molecules are produced in the cytoplasm, probably due to the activity of an intracellular organelle called a proteosome. Peptides are transported across the endoplasmtico reticulum by an ABC family transporter. The ternary complex of class I heavy chain/2- microglobulin/peptide moves to the cell surface. Peptides that bind to class II molecules are derives from endocytosed exogenous antigen which has been processed in an endosoma/lysosomal compartment. Class II molecules complexed with invariant (Li) chain are transported through the Golgi complex to an acidic endosomal compartment, where dissociation of Li and peptide loading occurs. Peptide-MHC molecule complexes on the cell surface can be recognized by a specific T-cell receptor. However, a variety of additional interactions involving accessory molecules are required for T-cell activation.

1. Antibodies form multiple non-covalent bonds with antigen The antigenantibody interaction results from the formation of multiple non-covalent bonds. These attractive forces consist of: hydrogen bonds; electrostatic bonds; van der Waals forces; and hydrophobic forces.

Each bond is relatively weak in comparison with covalent bonds, but together they can generate a highaffinity interaction. Thus interacting groups must be in quite intimate contact before these attractive forces come into play. For a paratope to combine with its epitope the interacting sites must be complementary in shape, charge distribution, and hydrophobicity, and in terms of donor and acceptor groups capable of forming hydrogen bonds. The affinity with which antibody binds antigen is the sum of the attractive and repulsive forces between them. A high affinity antibody implies a good fit and conversely, a low affinity antibody implies a poor fit

2. Kinetics of antibody-antigen reactions Measurements of antibody affinity relate to equilibrium conditions. Affinity indicates the tendency of the antibodies to form stable complexes with the antigen. However, for many biological activities of antibodies, it is possible that kinetics of the reaction may also be significant. Kinetics measures the forward rate (on rate) constant K1,2 (mol s ) and the reverse rate (off rate). At equilibrium the ratio of the two constants gives the equilibrium constant or affinity of the antibody.
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3. T Cell-Antigen Recognition The T cell receptor or TCR is a molecule found on the surface of T lymphocytes (or T cells) that is responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. The binding between TCR and antigen is of relatively low affinity and is degenerate: that is, many TCR recognize the same antigen and many antigens are recognized by the same TCR. The TCR is composed of two different protein chains (that is, it is a heterodimer). In 95% of T cells, this consists of an alpha () and beta () chain, whereas in 5% of T cells this consists of gamma and delta (/) chains. When the TCR engages with antigen and MHC, the T lymphocyte is activated through a series of biochemical events mediated by associated enzymes, co-receptors, specialized accessory molecules, and activated or released transcription factors. 4. Antigen processing Antigen processing is a biological process that prepares antigens for presentation to special cells of the immune system called T lymphocytes. This process involves two distinct pathways for processing of antigens from an organism's own (self) proteins or intracellular pathogens (e.g. viruses), or from phagocytosed pathogens (e.g. bacteria); subsequent presentation of these antigens on class I or class II MHC molecules is dependent on which pathway is used. Both MHC class I and II are required to bind antigen before they are stably expressed on a cell surface. While the conventional distinction between the two pathways is useful, there are instances where extracellular-derived peptides are presented in the context of MHC class I and cytosolic peptides are presented in the context of MHC class II (this often happens in dendritic cells).

5. Antigen presentation Antigen presentation is a process in the body's immune system by which macrophages, dendritic cells and other cell types capture antigens and then enable their recognition by T-cells. The basis of adaptive immunity lies in the capacity of immune cells to distinguish between the body's own cells and infectious pathogens. The hosts cells express self antigens that identify them as such. These antigens are different from those in bacteria ("non-self" antigens) or in virally infected host cells (missing -self). The ability of the adaptive immune system to survey for infection requires specialized pathways of enabling recognition of pathogen-derived antigens by T cells.

Foreign protein or antigen (1) is taken up by an antigen-presenting cell (2). The antigen is processed and displayed on a MHC II molecule (3), which interacts with a T helper cell (4). In the lower pathway; whole foreign proteins are bound by membrane antibodies (5) and presented to B lymphocytes (6), which process (7) and present antigen on MHC II (8) to a previously activated T helper cell (10), spurring the production of antigen-specific antibodies (9).

Topic 8: Cell Cooperation in the Antibody Response

Immune activation to stimulate antibody production involves cell interaction between T cells and APC, and later between these primed T cells and B cells. T-cell activation involves antigen-specific interaction, cell adhesion molecules, and cytokines. The most potent co-stimulatory molecule is B7 (CD80 or CD86). In the absence of appropriate co-stimulation, antigen recognition by nave T cells may result in clonal anergy. Lymphocyte proliferation is indirect, occurring via the induction of receptors for lymphocyte growth factors following cell activation. Lymphocyte growth factors (e.g. IL-2) are chiefly produced by T cells. Two types of antigens induce antibody responses T-dependent and T-independent. T-dependent antigens induce secondary immune responses characterized by IgG production and affinity maturation. Cytokines are intercellular signaling proteins intimately involved in most biological processes cell growth, activation, inflammation, immunity, repair, fibrosis, chemotaxis. Cytokines include proteins also known as interferons, interleukins, colony-stimulating factors and other growth factors. Cytokines act at low molarity because they act on very high affinity receptors.

Cytokines
Cytokines are small cell-signaling protein molecules that are secreted by numerous cells and are a category of signaling molecules used extensively in intercellular communication. Cytokines can be classified as proteins, peptides, or glycoproteins; the term "cytokine" encompasses a large and diverse family of regulators produced throughout the body by cells of diverse embryological origin. The term "cytokine" has been used to refer to the immunomodulating agents, such as interleukins and interferons. Virtually all nucleated cells, but especially endo/epithelial cells and resident macrophages (many near the interface with the external environment) are potent producers of IL-1, IL-6, and TNF-. Each cytokine has a matching cell-surface receptor. Subsequent cascades of intracellular signalling then alter cell functions. This may include the upregulation and/or downregulation of several genes and their transcription factors, resulting in the production of other cytokines, an increase in the number of surface receptors for other molecules, or the suppression of their own effect by feedback inhibition. A classification useful in clinical and experimental practice divides immunological cytokines into those that enhance cellular immune responses, type 1 (IFN-, TGF-, etc.), and type 2 (IL-4, IL-10, IL-13, etc.), which favor antibody responses. A key focus of interest has been that cytokines in one of these two sub-sets tend to inhibit the effects of those in the other. Dysregulation of this tendency is under intensive study for its possible role in the pathogenesis of autoimmune disorders.

Topic 9: Cell Cooperation in the Antibody Response

Non-adaptative immune defences allow leucocytes to detect and respond to the presence of pathogens, without involving the more recently evolved antigen-specific receptors of B cells and T cells. Microbial structures are recognized early in a reaction, while specific immune responses are developing. Release of cytokines is a major determinant of the type of response that is subsequently activated. The specific immune response is directed by helper T cells (Th), which respond to and release different cytokine profiles, and so drive distinct patterns of effector function. Cell-mediated immune responses comprise numerous distinct effector functions. Activation of an inappropriate effector function can lead to failure to eliminate the pathogen and to chronic immunopathology. There are two major types of cell-mediated effector mechanism. The first aims to destroy infected cells (with or without the help antibody). The second involves pathways that activate phagocytes to kill organisms and tissue cells to resist infection. Cytokine mediators play a central role in positive and negative regulation of immune reactions, and in integrating them with other physiological compartments such as the endocrine and haematopoietic systems. Granuloma formation or chronic tissue-destructive inflammation occur when cell-mediated reactions are nor resolved, due either to failure to eliminate an infection or an inability to clear an antigen which has become persistent. Immnunopathology can result from microvascular damage secondary to excessive cytokine release, or from direct destruction of essential cells.