* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download unit-1-5 consise NOTES immunology - E
Survey
Document related concepts
DNA vaccination wikipedia , lookup
Complement system wikipedia , lookup
Immune system wikipedia , lookup
Lymphopoiesis wikipedia , lookup
Molecular mimicry wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Monoclonal antibody wikipedia , lookup
Adaptive immune system wikipedia , lookup
Innate immune system wikipedia , lookup
Immunosuppressive drug wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Transcript
Gregory NEHRU ARTS AND SCIENCE COLLEGE IMMUNOLOGY The immune system is the body’s natural defence in combating organisms. Immunology has developed rapidly over the past decade owing to the refinements made in the molecular tests employed in this area of research. Therefore, the keen reader is encouraged to peruse the ophthalmic and immunological literature in order to keep abreast of the latest developments in this field. Owing to the complex nature of this subject, it is far beyond the scope of this article to cover all aspects of immunology. Rather, the aims of the article are twofold: first to acquaint the busy practitioner with the basic concepts of the immune system; and second, to introduce the reader to the more specific topic of ocular immunology - the study of the ocular immune system. Finally, since it is envisaged that optometrists will one day prescribe therapeutic agents, the discussion is limited to the anterior segment and anterior uvea. Innate & adaptive immune systems The immune system can be thought of as having two “lines of defence”: the first, representing a non-specific (no memory) response to antigen (substance to which the body regards as foreign or potentially harmful) known as the innate immune system; and the second, the adaptive immune system, which displays a high degree of memory and specificity. The innate system represents the first line of defence to an intruding pathogen. The response evolved is therefore rapid, and is unable to “memorise” the same said pathogen should the body be exposed to it in the future. Although the cells and molecules of the adaptive system possess slower temporal dynamics, they possess a high degree of specificity and evoke a more potent response on secondary exposure to the pathogen. The adaptive immune system frequently incorporates cells and molecules of the innate system in its fight against harmful pathogens. For example, complement (molecules of the innate system - see later) may be activated by antibodies (molecules of the adaptive system) thus providing a useful addition to the adaptive system’s armamentaria. A comparison of the two systems can be seen in Table 1. Figure 1 The principle components of the immune system are listed, indicating which cells produce which soluble mediators. Complement is made primarily by the liver, with some synthesised by mononuclear phagocytes. Note that each cell only produces a particular set of cytokines, mediators etc Cells of the innate immune system Phagocytes Although sub-divided into two main types, namely neutrophils and macrophages, they both share the same function - to engulf microbes (phago - I eat, Latin). Neutrophils Microscopically, these cells possess a characteristic, salient feature - a multilobular nucleus (Figure 2). As such, these cells have been referred to as polymorphonuclear leukocytes (PMNs) and have a pivotal role to play in the development of acute inflammation. In addition to being phagocytic, neutrophils contain granules and can also be classed as one of the granulocytes. The granules contain acidic and alkaline phosphatases, defensins and peroxidase - all of which Table 1: Cells and molecules of the innate and adaptive immune systems Immunity Cells Molecules Innate Natural killer (NK) cells Mast cells Dendritic cells Phagocytes Cytokines Complement Acute phase proteins T and B cells Cytokines Adaptive Antibodies Components of the immune system can be seen in Figure 1. Figure 2 Morphology of the neutrophil. This shows a neutrophil with its characteristic multilobed nucleus and neutrophilic granules in the cytoplasm. Giemsa stain, x 1500 represent the requisite molecules required for successful elimination of the unwanted microbe(s). Macrophages Macrophages (termed monocytes when in the blood stream) have a horseshoe-shaped nucleus and are large cells. Properties of macrophages include phagocytosis and antigen presentation to T cells (see later). Unlike neutrophils (which are short-lived cells), they are seen in chronic inflammation as they are long-lived cells. Mononuclear phagocytic system The cells comprising the monocyte phagocytic system are tissue bound and, as a result, are further sub-divided depending on their location. A list of the cells together with their corresponding location can be found in Table 2. Table 2: Examples of cells of the mononuclear phagocytic system and their respective locations Cells Location Monocytes Blood stream Alveolar macrophages Lungs Sinus macrophages Lymph nodes and spleen Liver Kupffer cells Phagocytosis - the process Phagocytosis is the process by which cells engulf microorganisms and particles (Figure 3). Firstly, the phagocyte must move towards the microbe under the influence of chemotactic signals, e.g. complement (see later). For the process to continue, the phagocyte must attach to the microbe either by recognition of the microbial sugar residues (e.g. mannose) on its surface or complement/antibody, which is bound to the pathogen. Following attachment, the phagocyte’s cell surface invaginates and the microbe becomes internalised into a phagosome. The resultant phagosome fuses with multiple vesicles containing O2 free radicals and other toxic proteins known as lysosomes to form a phagolysosome. The microbe is subsequently destroyed. Opsonisation (“to make tasty” - Greek) Opsonins are molecules, which enhance the efficiency of the phagocytic process by coating the microbe and effectively marking them for their destruction. Important opsonins are the complement component C3b and antibodies. Natural killer (NK) cells NK cells are also known as “large granular lymphocytes” (LGLs) and are mainly found in the circulation. They comprise between 5-11% of the total lymphocyte fraction. In addition to possessing receptors for immunoglobulin type G (IgG), they contain two unique cell surface receptors known as killer activation receptor and killer inhibition receptor. Activation of the former initiates cytokine (“communication”) molecules from the cell whilst activation of the latter inhibits the aforesaid action. NK cells serve an important role in attacking virally-infected cells in addition to certain tumour cells. Destruction of infected cells is achieved through the release of perforins and granyzymes from its granules, which induce apoptosis (programmed cell death). NK cells are also able to secrete interferon-γ (IFN-γ ). This interferon serves two purposes: first, to prevent healthy host cells from becoming infected by a virus; and second, to augment the T cell response to other virally infected cells (see later). Figure 3 Phagocytes arrive at a site of inflammation by chemotaxis. They may then attach to microorganisms via their non-specific cell surface receptors. Alternatively, if the organism is opsonised with a fragment of the third complement component (C3b), attachment will be through the phagocyte’s receptors for C3b. If the phagocyte membrane now becomes activated by the infectious agent, it is taken into a phagosome by pseudopodia extending around it. Once inside, lysosomes fuse with the phagosome to form a phagolysosome and the infectious agent is killed. Undigested microbial products may be released to the outside stain with a basic dye. Unlike mast cells, which are present in close proximity to blood vessels in connective tissue, basophils reside in the circulation. Both cell types are instrumental in initiating the acute inflammatory response. Degranulation is achieved either by binding to components of the complement system or by cross-linking of the IgE antibody which results in the release of pro-inflammatory mediators including histamine and various cytokines. The former induces vasodilation and augments vascular permeability whilst the latter are important in attracting both neutrophils and eosinophils. Dendritic cells Dendritic cells consist of Langerhans’ and interdigitating cells and form an important bridge between innate and adaptive immunity, as the cells present the antigenic peptide to the T helper cell (adaptive immunity). Such cells are therefore known as professional antigen presenting cells (APCs). Table 3 illustrates the various types of dendritic cells together with an example of their location. Eosinophils Eosinophils (so called because their granules stain with eosin - Figure 4) are granulocytes that possess phagocytic properties. Despite the fact that they represent only 2-5 % of the total leukocyte population, they are instrumental in the fight against parasites that are too big to be phagocytosed. Table 3: Dendric cells and location Mast cells and basophils Cells Location Morphologically, mast cells and basophils are very similar in that both contain electron dense granules in the cytoplasm. Basophils are so-called owing to the fact that their granules Langerhans cell Limbus, skin Interdigitating cell T cell areas in lymph nodes Figure 4 Morphology of the eosinophil. The multilobed nucleus is stained blue and the cytoplasmic granules are stained red. Leishman stain, x 1800 Molecules of the innate immune system Figure 5 When host cells become infected by virus, they may produce interferon. Different cell types produce interferon-α (IFN-α ) or interferon-β (IFN-β ); interferon-γ There are many molecules, which work in concert with the cells of the innate immune system and which also foster close functional links with their adaptive counterpart. The three major molecules are: • Complement • Acute phase proteins (APP) • Interferons (IFNs) (IFN-γ ) is produced by some types of lymphocyte (TH) after activation by antigen. Interferons act on other host cells to induce a state of resistance to viral infection. IFNγ has many other effects as well Complement The complement system represents a large group of independent proteins (denoted by the letter C and followed by a number), secreted by both hepatocytes (liver cells) and monocytes. Although these proteins maybe activated by both the adaptive immune system (classical pathway) or innate immune system (alternative pathway), the nomenclature is derived from the fact that the proteins help (“complement”) the antibody response. Activation of complement via the microbe itself is known as the alternative pathway. The classical pathway requires the interaction of antibody with specific antigen. The C3 component is the pivotal serum protein of the complement system. Binding of the antigen to C3 results in two possible sequelae. In either case, C3 component becomes enzymatically converted to C3b. The bacterial cell wall can either remain bound to C3b and become opsonised (since phagocytes have receptors for C3b) or act as a focus for other complement proteins (namely C5, 6, 7, 8 and 9). The latter form the membrane attack complex (MAC), which induces cellular lysis. The functions of the complement system may be summarised as follows: • Opsonisation • Lysis (destruction of cells through damage/ rupture of plasma membrane) • Chemotaxis (directed migration of immune cells) • Initiation of active inflammation via direct activation of mast cells It is important that complement is regulated to protect host cells from damage and/or their total destruction. This is achieved by a series of regulatory proteins, which are expressed on the host cells themselves. Acute phase proteins These serum proteins are synthesised by hepatocytes and are produced in high numbers in response to cytokines released from macrophages. Interferons (IFNs) IFNs are a group of molecules, which limit the spread of viral infections (Figure 5). There are two categories of IFNs, namely type I and type II. Type I IFNs maybe sub-divided further into IFN-α and β . IFN-γ is the sole type II interferon. Type I IFNs are induced by viruses, pro-inflammatory cytokines and endotoxins from gram negative bacterial cell walls. Their presence remains vital Table 4: Primary and secondary lymphoid organs Primary lymphoid organs Secondary lymphoid organs Bone marrow Lymph nodes MALT - mucosa associated lymphoid tissue (includes bronchus, gut, nasal and conjunctival associated mucosal tissues) Spleen for the successful eradication of an invading virus by the innate immune system. Type II IFN, IFN-γ , is produced by T Helper cells and NK cells and is able to augment both the antigen presenting properties together with the phagocytic properties of the APCs (e.g. macrophages and dentritic cells). Adaptive immunity As mentioned previously, there is a great deal of synergy between the adaptive immune system and its innate counterpart. The adaptive immune system comprises two main types of leukocyte known as B and T lymphocytes. Before describing these important cell types, it is necessary to acquaint the reader with both the primary and secondary lymphoid organs and tissues in the body. These are summarised in Table 4. The bone marrow represents the dominant site for haemopoiesis (production of blood cells and platelets). Although most of the haemopoietic cells maturate in this region, T lymphocytes do so in the thymus. In the thymus, premature T cells undergo a process of positive and negative selection whereby the former are allowed to progress to maturity whilst the latter are marked for termination via apoptosis (see central tolerance). Lymphocytes Morphologically, there are three types of lymphocytes: T, B and NK cells. However, only T and B lymphocytes exhibit memory and specificity and, as such, are responsible for the unique quality of the adaptive immune system. Resting B lymphocytes are able to react with free antigen directly when it binds to their cell surface immunoglobins which act as receptors. T lymphocytes do not react with free antigen and instead make use of APCs to phagocytose the antigen and then to express its component proteins on the cell surface adjacent to special host proteins called major histocompatibility complex (MHC) class II molecules. As discussed, antigen presenting cells which express MHC class II molecules include dendritic cells and macrophages. This “afferent” phase must occur in order for the T cell to recognise the antigen. The “efferent” phase occurs when activated lymphocytes enter the tissue and meet antigen again. This results in multiplication and secretion of cytokines or immunoglobins in order to destroy the antigen. T cells T cells can be broadly divided into both T helper (TH) and cytotoxic T cells (Tc). Furthermore, TH cells may be sub-divided into TH1 and TH2. The former are pro-inflammatory T cells and stimulate macrophages whilst the latter orchestrate B cell differentiation and maturation and hence are involved in the production of humoral immunity (antibody mediated). T cells express cell surface proteins, described by cluster determination (CD) numbers. TH cells express CD4 molecules on their cell surface, which enable the lymphocyte to bind to a MHC class II molecule. The T cell receptor is unique in that it is only able to identify antigen when it is associated with a MHC molecule on the surface of the cell. Cytotoxic T cells are primarily involved in the destruction of infected cells, notably viruses. Unlike TH cells, cytotoxic cells possess CD8 cell surface markers, which bind to antigenic peptides expressed on MHC class I molecules. B cells and antibodies (immunoglobulins - Ig) B cells are lymphocytes that produce antibodies (immunoglobulins) and can recognise free antigen directly. They are produced in the bone marrow and migrate to secondary lymphoid organs. B cells are responsible for the Table 5: Antibody isotypes and corresponding functions Antibody IgG IgM Figure 6 When a microorganism lacks the inherent ability to activate complement or bind to phagocytes, the body provides antibodies as flexible adaptor molecules. The body can make several million different antibodies able to recognise a wide variety of infectious agents. Thus the antibody illustrated binds microbe 1, but not microbe 2, by its ‘antigen-binding protein’ (Fab). The ‘Fc portion’ may activate complement or bind to Fc receptors on host cells, particularly phagocytes. Characteristics Crosses placenta thus providing newborn with useful humoral immunity High affinity Predominant antibody in blood and tissue fluid Large pentameric structure in circulation Present in monometric form on B cell surface Secreted form is predominant antibody in early immune response against antigen Reaches 75% of adult levels at 12 months of age IgA Exists in both a monometric and dimeric form Secretory IgA (dimeric form) represents 1st line of defence against microbes invading the mucosal surface, e.g. tears IgE Low levels in circulation Increased levels in worm infections Fc region has high affinity for mast cell thus involved in allergy IgD Antigen receptor on B cells Absent from memory cells Table 6: Various cytokines, their sources and functions Cytokine IL-1 Source Macrophages Function 1. T, B cell activation 2. Mobilisation of PMNs 3. Induction of acute phase proteins IL-2 T cells Proliferation of T and NK cells Il-4 Th2 cells Mast cells B cell activation IgE response IL-8 Macrophages T cells Fibroblasts Keratinocytes Chemotaxis of PMNs IL-10 T cells Inhibits other cytokines Macrophages Inflammation TH2 cells Macrophages B cell activation Suppres s macrophages B cells Stimulate TH1 TH2Inhibit IL-12 TGF β (transforming growth factor) TNF α (tumour necrosis factor) development of antibody mediated immunity known as humoral mediated immunity. When activated by foreign antigen, B cells undergo proliferation and mature into antibody secreting plasma cells. The latter are rich in organelles such as rough endoplasmic reticulum and mitochondria, which confer their ability to secrete soluble proteins (antibodies). Not all proliferating B cells develop into plasma cells. Indeed, a significant proportion remain as memory B cells through a process known as clonal selection. This process is vital in eliminating the antigen should the body become re-exposed to it in the future. T cells are also clonally selected and this confers to the production of T memory cells. Although T and B cells behave differently, both are able to recirculate around the body migrating from blood to tissue and vice versa. The ability to recirculate obviously increases the efficiency with which cells of the immune system can home onto the invading antigen. Antibodies Antibodies have two roles to play - the first is to bind antigen and the second is to interact with host tissues and effector systems in order to ensure removal of the antigen (Figure 6). There are five different types (known as isotypes) of antibody in the human immune system - namely IgM, IgG, IgA, IgE and IgD. In addition, there are four sub classes of IgG (IgG1-4). The basic antibody unit consists of a glycosylated protein consisting of two heavy and two light, polypeptide chains. The region which binds to the antigen is known as the Fab region, while the constant region, Fc, not only determines the isotype but is the region responsible for evoking effector systems, e.g. mast cell activation. The term immune complex refers to the combination of antigen and antibody and will be discussed later in the article (see type III hypersensitivity). The antibody isotypes together with their corresponding function are illustrated in Table 5. MHC Major histocompatability complex (MHC) are cell surface proteins classified as class I (also termed human leucocytic antigen [HLA] A, B and C), found on all nucleated cells and class II (termed HLA, DP, DQ and DR), found on all antigen presenting cells (APCs). MHC molecules are the sine qua non of T cell induced immunity. Clinically, there is a strong association between HLA and certain systemic and ocular diseases (see later). Cytokines Cytokines (also termed interleukins [IL] meaning “between white blood cells”) are small molecules that act as a signal between cells and have a variety of roles including chemotaxis, cellular growth and cytotoxicity. Owing to their ability to control immune activity, they have been described as the “hormones” of the immune system. Table 6 summarises some of the cytokines pertinent to ocular immunology, their functions and their progenitors. Since interferons have been discussed earlier in the article, they have been omitted from the table. endothelial cells and the presence of junctional complexes linking retinal pigment epithelial cells4. Lymphatic role Since various cells of the immune system are capable of reacting with self-antigens, it is therefore essential that the human body has mechanisms to suppress/eliminate autoreactive cells. Failure to do so, can, in some cases, lead to the development of autoimmune diseases (see later). The fact that skin allografts were not rejected following lymph node removal5 led investigators to hypothesise that immune privilege was solely due to the absence of the same said system at a particular anatomical site. However, although certain immune privileged sites do indeed lack lymphatic drainage, others such as the testes6 and eye7 do possess such a system. It appears that a proportion of the aqueous humour drains via the uveoscleral pathway into the lymphatic vessels in the head and neck. Central tolerance The eye, APCs & MHCs Central and peripheral tolerance: nature’s way of containing the immune response Central tolerance refers to the process whereby both immature B and T cell lymphocytes, which react against normal, healthy cells (self-antigens), are eliminated via apoptosis. Peripheral tolerance This involves the removal of mature lymphocytes, which are not tolerant to healthy cells. Ocular immune privilege There are numerous sites in the body whereby tissue may be grafted with minimal risk of rejection. Such regions include, inter alia, the testis, thyroid lens, anterior chamber, cornea, iris and ciliary body1,2. It is important that immune privilege is not simple interpreted as the host’s inability to initiate an immune response to a transplanted tissue. Rather, it is an area of the body in which there exists a paucity of various elements of the human immune system in response to an antigen. Factors The factors purported by investigators that contribute to the phenomenon of ocular immune privilege include: • Isolation from a vascular supply • Isolation from a lymphatic supply • Presence of a vascular barrier • Ability to suppress the immune response • Anterior chamber associated immune deviation (ACAID) Vascular supply The healthy cornea is a good example of an ocular site devoid of a vascular network. The evidence to support the role a vascular network has to play in the mechanism of graft rejection is unequivocal since the risk of failure correlates positively with the degree of host vascularisation3. Vascular barrier There is a plethora of evidence in the ophthalmic literature to support the existence of a blood-ocular barrier. Furthermore, the same said barrier encompasses different elements including tight junctions between retinal As mentioned previously, APCs, through their ability to express MHC class II molecules, are potent progenitors of the immune response. Moreover, such cells are capable of activating T cells within the tissue itself. It is therefore not unreasonable to assume that a paucity of APCs may play an important role in immune privilege. In addition, failure to express MHC class I molecule would make a tissue immune against the lytic action of the cytotoxic T cells. Although the aforementioned mechanisms are theoretically plausible, cells expressing both MHC class I and II molecules have been detected in the eye. Table 7 illustrates the relationship between histocompatability class and ocular cell type. It is noteworthy that the epithelial cells of the crystalline lens are devoid of class I expression14 and that the Langerhans’ cells (class II expression) are absent from the central cornea15. It is interesting that not all cells, which express MHC class II act as professional APCs in the eye. Indeed, it has been shown that such cells reside in the iris and ciliary body and not only fail to present alloantigens to T cells, but have the ability to suppress mixed lymphocyte reactions16. The failure to incite the inflammatory response has attracted a great deal of interest amongst ophthalmologists and immunologists alike. It appears such suppression is achieved by various factors present in the aqueous humour (e.g. transforming growth factor - β ). Anterior chamber associated immune deviation (ACAID) As a result of experiments with rats, investigators discovered that antigens placed in the anterior chamber resulted in systemic inhibition of delayed type hypersensitivity (DTH or type IV hypersensitivity) reactions to the same said antigens17. This phenomenon has been coined anterior chamber associated immune deviation (ACAID). The anterior chamber is thus able to suppress delayed type hypersensitivity reactions and inhibit the production of complement fixing antibodies18,19. However, it has no inhibitory effect on cytotoxic T cell activity and has a minimal influence on the production of non-complement fixing antibodies. With respect to the endothelium, two adaptations prevent it from immunological injury: first, avoidance of cytotoxic T lymphocyte (CTL)-mediated lysis; and second, inhibition of DTH responses in the anterior chamber22. It achieves the former through the cells inability to express MHC class I molecules21. The corollary of this, however, is that virally infected cells may persist in this region. The râison d’etre of ACAID is to protect the eye from the DTH response to pernicious antigens. As a result of its location in relation to the anterior chamber, the corneal endothelium seems well placed to reap the benefits of ACAID. ACAID is beneficial in reducing the incidence of stromal keratitis in herpes simplex virus infection. It therefore seems reasonable to assume that such an unwanted corneal sideeffect occurs as a result of a DTH response rather than the toxic effect of the virus per se22. Corneal graft rejection may be due, in part, to failure to invoke ACAID. Streilein at al23 not only discovered that the immunosuppressive effects of the cornea were abolished in corneas Table 7: MHC and ocular cell type8-13 MHC class I Ocular cell type Corneal epithelium • Corneal stroma • Corneal endothelium • Trabecular meshwork • Pigmented and non pigmented cells of ciliary body • Anterior iris • RPE cells • MHC class II Corneal limbus • Iris and ciliary body • Uveoscleral pathway Ora serrata • that had ACAID removed via cauterisation or keratoplasty but also abolished in corneas that had been denervated. Ocular immunology Anterior segment immunology may be sub-divided into the following aspects: • Tear film • Corneal immunology • Conjunctival immunology • Scleral immunology • Uveal immunology Tear film a) Mucous layer This layer is produced both by the conjunctival goblet and epithelial cells. The glycocalyx synthesised by the corneal epithelial cells serves to attach the mucous layer and in doing so binds to immunoglobulin in the aqueous24. It has been suggested that the latter immunological sign may have antiviral effects. The evidence to support this is that certain intestinal mucosa, which have similar binding properties to those seen in the eye, are able to exert an inhibitory action against viral replication25. b) Aqueous layer The aqueous layer contains the following antimicrobial factors: 1. Lactoferrin 2. Lysozyme 3. IgA 4. Miscellaneous proteins 1. Lactoferrin Lactoferrin is produced via the acinar cells of the lacrimal gland. Its main function is to bind iron, which is required for bacterial growth. It therefore possesses both bacteriostatic and bacteriocidal properties. Furthermore, it is able to enhance the effects of certain immunoglobulins. 2. Lyszome Produced by type A cells in the lacrimal gland, lysozome constitutes up to 25% of the total tear protein. It is surprising somewhat that despite being effective at lysing gram positive bacterial cell walls, staphylococcus aureus appears recalcitrant to its action26. However, it is instrumental in enhancing IgA against gram negative bacteria. 3. IgA This is the predominant isotype found in the human tears in the non-inflamed eye. Very little is present in serum with the majority secreted through epithelial cells of structures such as the lacrimal gland and the lactating breast. The IgA present in the tears is produced by plasma cells situated underneath the secretory cells lining the acini in the lacrimal gland. IgA is very effective at binding microbes and, as such, prevents the microbe from adhering to the mucosal surface. The antibody achieves this either by interfering with the binding site directly or by agglutination. Successful binding of the microorganism enables the tears to wash them away. IgA possesses other functions including opsonisation and inactivation of various bacterial enzymes and toxins. In addition, it may be involved in antibody dependent cell-mediated cytotoxicity. It must be emphasised, however, that IgA is unable to bind to complement and, as such, is not involved in the classical pathway27. 4. Miscellaneous proteins β -Lysin, a bacteriocidal cationic protein, is present both in the tears and aqueous humour28. Its bacteriocidal properties are conferred through their ability to disrupt the microorganisms’ walls. Various components of complement are present in varying degrees depending on whether the eye is closed or not. Closed eye vs open eye Open eye In addition to the components of complement mentioned above, the tears are rich in lysozyme, lactoferrin and lipocalin (tear pre albumin) together with low levels of IgA29,30. Closed eye In the closed eye environment, levels of IgA and complement components are increased. In addition, neutrophils are also recruited. The eye can be interpreted as being in a state of subclinical inflammation. However, the eye is protected from damage induced by either complement or neutrophils by DAF (Decayed Accelerating Factor [an inhibitory component of the complement system])31 and α 1 antitrypsin33. Corneal immunology Paradoxically, the cornea, an immune privileged site, is capable of evoking an immunological response evidenced by the presence of subepithelial infiltrates, keratic precipitates, epithelial and stromal keratitis under certain clinical conditions. Cytokine release Numerous cytokines are synthesised in the cornea including IL-1, IL-6, IL-8, TNF-α , IFN-γ , C5a and prostaglandins33. Phagocytosis of certain antigens via corneal epithelial cells initiate the release of IL-1 which, in turn, gives rise to the recruitment of Langerhans’ cells from the limbus to the central cornea. It is worthy of note that the same said APC may be recruited centrally in response to chemical or localised damage34,35. Investigators have discovered that stromal fibroblasts synthesise IL-8 in response to infection by the herpes simplex virus36. Furthermore, it seems plausible that the latter cytokine release may be responsible for the neutrophilic infiltration observed in cases of herpetic keratitis. Immunoglobulins The three isotypes detected in the cornea are IgM, G and A. IgG predominates in the central cornea37. By contrast, IgM predominates in the limbal region. The latter antibody is restricted from the central cornea owing to its large size. Antibodies in the cornea are found in the stroma since they are cationically charged. Due to their positive ionic charge, they bind to proteoglycans and the anionic glycosaminoglycans38. Antigen/antibody complexes may sometimes be observed in the corneal stroma in a variety of pathological conditions (e.g. herpes simplex keratitis), and because of their ringed shaped appearance are known as “Wessley rings”. Complement The elements of complement found in the cornea include C1-739 in addition to the regulatory proteins H and I and C1 inhibitor40. Interestingly, C1 is present in high concentrations in the limbus and is restricted from the central cornea. This finding is significant since the limbal region is susceptible to ulceration following complement activation and immune complex deposition. It has been suggested that C1 is produced by corneal fibroblasts whereas the remaining components detected in the cornea appear to be derived from the plasma via linked vessels. Conjunctival immunology The conjunctival associated lymphoid tissue (CALT) is part of the more general mucosa associated lymphoid tissue (MALT). Langerhans’ cells and lymphocytes exist within the conjunctival epithelial layer. In the substantia propria, neutrophils, lymphocytes, IgA and IgG, dendritic cells and mast cells all reside. It is noteworthy that eosinophils and basophils are not present in the healthy conjunctiva. Langerhans’ cells and dendritic cells present the antigenic peptide to the conjunctival T helper cells. Following antigenic presentation, the T cells secrete the cytokine IFN-γ which serves to promote antigen elimination by macrophages. This is the delayed-type hypersensitivity (DTH) response (see later) and is characteristic of conjunctival pathology such as phlyctenulosis. Scleral immunology There exists only a small number of immune cells in the sclera compared to the conjunctiva since it is relatively avascular. In its resting state, IgG appears to be present in large amounts41. However, a sclera under stress, may become immunologically active as a result of migrating cells from the overlying episcleral and underlying choroidal vasculature42,43. Uveal immunology The uveal tract is an important site from an immunological perspective for two reasons: first, it is highly vascular in nature; and second, the majority of vessels are highly fenestrated which facilitates the recruitment of leukocytes during the inflammatory cascade. The uvea contains numerous cellular components of the immune system including macrophages, mast cells, lymphocytes and plasma cells in addition Table 8: Hypersensitivity reactions Hypersensitivity Appearance type time Mediators Mechanism I. Immediate 2-30 mins IgE Mast cell response II. Cytotoxic 5-8 hours IgM and IgG III. Immune complex 2-8 hours IgM and IgG immune complexes 24-72 hours CD4 and CD8 T cells, APCs T cell mediated Macrophages activated Include granulomatous reactions Autoantibodies Autoantibodies against hormone (enhance acute inflammation) IV. Delayed type V. Stimulatory to an appreciable number of immune factors. Although IgG and IgM have been detected in both the choroid and iris, they exist in greater numbers in the former structure. The reason for such disparate levels is the dearth of anionic antibody binding sites on the iris surface44. By contrast, the ciliary body harbours tremendous amounts of IgG by virtue of its anionic tissue sites. Immunopathology and the eye Immunopathology encompasses both pathology as a result of an over-active immune system (hypersensitivity and autoimmunity) together with that acquired through an individuals inability to fight off infection - namely immunodeficiency. Unfortunately, it is far beyond the scope of this article to describe the latter and its ophthalmic correlates. In this section, the classification system pertaining to hypersensitivity reactions will be described and each subtype with an anterior segment manifestation will be discussed. The association between HLA and systemic and ophthalmic disease will be illustrated together with a brief overview of the concepts pertaining to autoimmunity. Hypersensitivity The term hypersensitivity refers to the process whereby the adaptive immune response overreacts to a variety of infectious and inert antigens resulting in damage to the host tissue. Five types of hypersensitivity reactions exist all of which vary in their timing following contact with the antigen (Table 8). • Type I hypersensitivity: allergy Allergies may affect approximately 17% of the population45. The term atopic is used to describe those individuals who possess a genetic predisposition to allergy. Allergies may occur to otherwise innocuous antigens (known as allergens) and infectious agents, e.g. worms. Type I hypersensitivity exists in two phases, the sensitisation and effector phases. Firstly, a harmless allergen causes production of IgE antibody on first exposure. This IgE Antibody and complement Antibody/antigen complexes diffuses throughout the body until it comes into contact with mast cells and basophils. Both these cell types have receptors for IgE antibody. Although the patient experiences no symptoms after the initial binding, reintroduction of the antigen/allergen induces the production of more IgE and, furthermore, increase the likelihood of cross-linking with existing antibodies on the mast cell surface. Such crosslinking induces the mast cell to degranulate and release a host of inflammatory mediators such as histamine, prostaglandins and bradykinin. Histamine characteristically causes the itchy symptoms experienced by patients and as a result of binding to H1 receptors in the eye, induces vasodilation and enhances mucous secretion by the goblet cells. Bradykinin augments vascular permeability, decreases blood pressure and contracts smooth muscle. Prostaglandins are also powerful inflammatory mediators. Figure 7: Corneal thinning and vascularisation in AKC, excess mucous is present Figure 8: Epithelial macroerosion in VKC Ocular correlates: The following anterior segment conditions are due, if not only in part, to type I hypersensitivity: • Seasonal allergic conjunctivitis (type I) • Giant papillary conjunctivitis (types I and IV) • Vernal keratoconjunctivitis (VKC) (type I and IV) • Atopic keratoconjunctivitis (AKC) (types I and IV) (Figure 7) Seasonal allergic conjunctivitis can be easily recognised by chemosis and hyperaemia of the conjunctiva, lid swelling and excessive lacrimation. The cornea is not affected. GPC is well known to optometrists as an allergic response to contact lenses, prosthetic lenses, protruding corneal sutures and scleral buckles. Histological examination of eyes suffering from GPC have revealed degranulated mast cells (type I hypersensitivity)46 and CD4+ T cells (type IV)47, thus corroborating the theory that such a condition is mediated by both types of hypersensitivity. Vernal conjunctivitis can present either in a palpebral form characteristically exhibiting Figure 9: Corneal plaque in VKC giant cobblestone papillae, or in a limbal form with gelatinous deposits known as Trantas’-dots, which represent degenerating epithelial cells and eosinophils. The first corneal change is a punctate epithelial keratitis, which if left unchecked develops into a macroerosion (Figure 8) and finally a corneal plaque develops (Figure 9). The conjunctiva itself is characteristically oedematous and contains an array of immunological cells including lymphocytes and mast cells. Evidence of type I involvement includes the histological detection of, inter alia, degranulated mast cells, eosinophils and increased levels of IgE in affected eyes49. The detection of CD4+ T cells and macrophages is indicative of a delayed inflammatory component49. Type II hypersensitivity: cytotoxic This classification of hypersensitivity involves either IgG or IgM antibodies, which may induce cellular lysis due to the involvement of the classical complement pathway (as seen in blood transfusion reactions) or recruit and activate inflammatory cells via complement. The components of complement include the C5a, which serves to attract inflammatory cells to the site of interest. The hypersensitivity reaction is a result of the excessive amount of extracellular mediators released by the inflammatory cells to antigens that are too big to be completely phagocytosed. Antibodies to self-antigens, such as the acetylcholine receptor in myasthenia gravis, is not only another example of type II hypersensitivity but also an example of autoimmune disease. Ocular manifestations • Mooren’s ulcer • Cicatrical pemphigoid Mooren’s ulcer is a rare peripheral ulcerative keratitis that exists either as a unilateral, non-progressive form which has a predilection for elderly patients or as a more severe, progressive form affecting both eyes of relatively young individuals. The signs range from a small patch of grey infiltrate near the margin to frank ulceration involving the entire corneal circumference and, in some cases, the central region as well. The healing process results in a thin, vascularised, opaque cornea. Investigators have identified a significant number of lymphocytes, neutrophils and plasma cells50 in the cornea, thus providing unequivocal evidence to support the theory that this condition has an immunopathological aetiology. Cicatrical pemphigoid is a chronic, blistering disease, which has a predilection for both the ocular and oral mucous membranes. Unlike its self-limiting counterpart, pemphigus vulgaris, cicatrical pemphigoid rarely affects the skin. Ocular cicatrical pemphigoid is a serious, bilateral condition that represents effective shrinkage of the conjunctiva. Although the initial presentation may be subacute and non-specific, it frequently progresses to symblepharon, entropion with secondary trichiasis, dry eye, ankyloblepharon and conjunctival fornix shortening. There is evidence to support the presence of IgG antibodies directed against self-antigen in the basement membrane of both the skin and eye51. Binding of the aforementioned antibodies may activate complement with subsequent recruitment of inflammatory cells into the area. The process of cicatrisation is achieved through the secretion of collagen via fibroblasts as a result of stimulation via cytokines released from the invading inflammatory cells. Type III hypersensitivity: immune complex Large pathogens with multiple antigenic sites have several antibodies bound to them forming immune complexes. Normally, these complexes are removed by the mononuclear phagocytes in the liver and spleen with no adverse sequelae. However, persistence of immune complexes does occur in certain individuals leading to their deposition in tissue. As a consequence of the latter action, complement may be activated thus paving the way for inflammatory cells to enter the deposition site. Since blood vessels (which filter plasma at high pressure and exhibit a great deal of tortuosity) are more susceptible for immune complex deposition, the ciliary body is particularly vulnerable to this type of hypersensitivity reaction. Ocular manifestations • Uveitis (Crohn’s disease) • Peripheral corneal lesions associated with rheumatoid arthritis • Stevens Johnson syndrome • Sjögren’s syndrome The signs and symptoms of the above will be described in future articles in the series. Type IV hypersensitivity: delayed-type hypersensitivity (DTH) The term DTH has been used to describe such a reaction owing to its prolonged time-scale relative to the other hypersensitivity types. Although DTH can be transferred by T cells that have been previously sensitised by an antigen, it cannot be transferred in serum. The sequence of events leading to DTH begins with initial presentation of the antigen peptide to T cells by APCs (e.g. Langerhans’ cells). The primed T cell migrates to the site of antigenic entry whereby it releases pro-inflammatory mediators such as TNF. The release of these cytokines facilitates blood flow and extravasation of plasma contents to the area. The activation of CD4 T helper and CD8 cells results in the release of IFN-γ and, as a consequence, enhances macrophage activity in that area. Resolution of DTH is dependent on the efficacy with which such phagocytes can remove the offending antigen. Recalcitrant infectious agents result in a chronic DTH that causes the chronically activated macrophages to fuse together and form multinucleated giant cells. In an attempt to contain the infectious agent, macrophages may undergo further inter connections to resemble an epithelial layer. Owing to the similarity to this layer they are referred to as epitheloid cells. Both epitheloid and multinucleated giant cells secrete factors that induce fibrosis resulting in granuloma formation. Thus granulomata are the hallmark of chronic inflammation. Damage and loss of function of the neighbouring tissues frequently ensues until the agent is removed either chemically or surgically. Ocular manifestations • Ocular allergies (VKC, AKC GPC) • Idiopathic uveitis • Sympathetic ophthalmia • Phlyctenulosis Type V hypersensitivity This relatively new category encompasses the concept of autoantibodies binding to hormone receptors that mimic the hormone itself. This results in stimulation of the target cells. Examples include thyrotoxicosis. Autoimmunity The ability to react against self-antigens is known as autoimmunity. However, a significant number of people exist who harbour autoantibodies and yet remain asymptomatic. The corollary of this is that the presence of autoreactive cells per se is not sufficient to trigger autoimmune disease. In fact, autoimmune disease is a result of breakdown of one of the immunoregulatory mechanisms. Furthermore, autoimmune disease may be classified as either being organ specific (e.g. insulin dependent diabetes mellitus, Grave’s disease) or non-organ specific (e.g. Sjögren’s syndrome, ankylosing spondylitis). It is important to realise that the causes of autoimmune diseases are multifactorial. The main predisposing factors are age, gender, infection and genetics. However, one of the most important factors of interest to immunologists and clinicians alike is the association between HLA and autoimmune disease. When determining the likelihood of contracting a disease both epidemiologists and clinicians refer to the relative risk. In the case of HLA antigen, the relative risk compares the chance of a person who has a particular HLA antigen acquiring a disease to those individuals who do not have such an antigen. The association is exemplified by the relative risk of suffering from ankylosing spondylitis (AS) in individuals who possess HLA-B27. In patients suffering from AS, the prevalence of HLA-B27 is 90% and this figure rises to 95% in patients who suffer both with the disease and acute iritis. Indeed, in the UK approximately 45% of patients who present with acute iritis will harbour HLA-B2752. It is therefore important that patients who present with anterior uveitis are screened for HLA-B27 because although a positive result may not necessarily be diagnostic, its presence will certainly improve the sensitivity of further radiological tests. Table 9 compares the HLA associations with both ophthalmic disorders together with those systemic disorders relevant to the ophthalmic practitioner. Conclusion This article has only broached the fascinating subject of ocular immunology. A basic understanding of immunology is required if practitioners are to therapeutically manage their patients. Further articles in the series will help to reinforce the concepts of this challenging subject. . Disease Table 9: HLA and ophthalmic disease HLA association Relative risk 54 Ocular manifestation Ankylosing spondylitis B27 90 Anterior uveitis Reiter’s disease B27 33 Mucopurulent conjunctivitis Anterior uveitis Keratitis Rheumatoid DR4 7 Keratoconjunctivitis sicca Keratitis Scleritis DR3, DR5 10 Keratoconjunctivitis sicca DR3 Not known Anterior uveitis (acute and chronic) Dacryoadentis Retinal vasculitis, neovascularisation, Optic nerve granulomata DQw7 Not known Shrinkage of conjunctiva B5 3 Sympathetic ophthalmia DR4, A11, B40 Not known Systemic lupus erythematosus DR2, DR3 3 Primary Sjögrens’ syndrome Sarcoidosis Cicatrical pemphigoid Behcet’s disease Multiple choice questions - Basic immunology 1. Which one of the following is not part of the innate immune system? a. Mast cells b. Complement c. Phagocytes d. T cells 2. Which one of the following statements is correct regarding the innate immune system? a. It is specific b. It evokes a more potent response on secondary exposure c. It represents the first line of defence d. It is able to memorise pathogens on subsequent exposures 3. Which one of the following statements is correct regarding the cells of the innate system? a. Basophils are important phagocytes b. During phagocytosis the pathogen becomes initially internalised as a phago-lysosome c. Eosinophils play an important role in combating virally-infected cells d. Langerhans’ cells form a bridge between innate and adaptive immunity 4. Which one of the following statements is correct regarding the adaptive immune system? a. It consists of all types of lymphocytes b. T cells produce antibodies c. T cells maturate in the thymus d. B cells are produced in the spleen 5. Which one of the following statements is correct regarding T cells? a. T cells can be subdivided into TH1 and TH2 subtypes only b. T cells alone can identify any type of antigen c. T cells express cell surface proteins denoted by cluster determinant (CD) numbers d. All T cells are involved in initiating the inflammatory response 6. Which one of the following statements is incorrect? a. B cells have antibodies as their cell surface receptor b. There are five types of antibody c. IgE is an important antibody in allergies d. All B cells differentiate into plasma cells 7. Which one of the following statements is correct regarding ocular immune privilege? a. There is an absence of Langerhans’ cells in the central cornea b. Aqueous humour has no role c. Abundant vascular supply is vital d. All ocular cells express MHC class II 8. Which one of the following statements concerning ocular immunology is incorrect? a. Levels of IgA and complement increase when the eyes are closed b. IgA is the predominant antibody in blood and tissue fluid c. IgM, IgG and IgA antibody isotypes have been identified in the cornea d. The sclera contains a smaller number of immune cells than the conjunctiva Anterior uveitis Retinitis, periphlebitis, retinal oedema Panuveitis Punctate epithelial keratopathy Keratopathy Necrotising scleritis Retinal lesions (cotton wool spots) Autoimmune optic neuropathy Please note there is only one correct an 9. In a patient suffering from vernal conjunctivitis, which one of the following statements is correct? a. Trantas’-dots represent infiltrating T cells b. Affected eyes have increased levels of IgD c. Histologically, mast cells, lymphocytes and macrophages have been identified d. Is due to type III Hypersensitivity 10. Which one of the following statements is incorrect? a. HLA-B27 is a risk factor for both anterior uveitis and ankylosing spondylitis b. Granulomas are present in type IV hypersensitivity reactions c. Histamine is an important vasoconstrictor d. IgE mediated hypersensitivity is of rapid onset 11. What proportion of patients with acute iritis will harbour HLA-B27? a. 15% b. 25% c. 45% d. 65% 12. Which one of the following statements is correct regarding a type I hypersensitivity reaction? a. It always occurs in isolation b. It is characterised by the presence of macrophages c. It is associated with myasthenia gravis d. It involves the degranulation of mast cells following the cross-linking of IgE bound to its cell surface POLYCLONAL SERUM Most antigens offer multiple epitopes and therefore induce proliferation and differentiation of a variety of B-cell clones, each derived from a B cell that recognizesa particular epitope. The resulting serum antibodies are heterogeneous, comprising a mixture of antibodies, each specific for one epitope. Such a polyclonal antibody response facilitates the localization, phagocytosis, and complement-mediated lysis of antigen; it thus has clear advantages for the organism in vivo. Unfortunately, the antibody heterogeneity that increases immune protection in vivo often reduces the efficacy of an antiserum for various in vitro uses. Polyclonal antibodies are a minor component in a complex mixture of serum proteins and are a heterogeneous mixture of molecules with a wide range of binding affinities. Therefore such antisera lack the degree of definition required for many of the current molecular techniques, where an increase in assay sensitivity is often counteracted by a decrease in serological specificity. Nonetheless, the fact that polyclonal antibodies can bind a particular antigen from so many different perspectives is of great technical advantage and they are therefore excellent for: (a) routine affinity purification of the native antigen; and (b) relating an expressed partial gene sequence to the mature gene product. However, they have the disadvantage that non-specific or crossreactive binding reactions can be a serious problem when antisera are used to identify or quantify antigens, e.g. the study of differentiation or tumour antigens, or in the clinical laboratory for immunodiagnosis of disease. This is because of other components in an antiserum. MONOCLONAL ANTIBODIES Most antigens offer multiple epitopes and therefore induce proliferation and differentiation of a variety of B-cell clones, each derived from a B cell that recognizesa particular epitope. The resulting serum antibodies are heterogeneous, comprising a mixture of antibodies, each specific for one epitope. Such a polyclonal antibody response facilitates the localization, phagocytosis, and complement-mediated lysis of antigen; it thus has clear advantages for the organism in vivo. Unfortunately, the antibody heterogeneity that increases immune protection in vivo often reduces the efficacy of an antiserum for various in vitro uses. For most research, diagnostic, and therapeutic purposes, monoclonal antibodies, derived from a single clone and thus specific for a single epitope, are preferable. Direct biochemical purification of a monoclonal antibody from a polyclonal antibody preparation is not feasible. In 1975, Georges Köhler and Cesar Milstein devised a method for preparing monoclonal antibody, which quickly became one of immunology’s key technologies. By fusing a normal activated, antibody-producing B cell with a myeloma cell (a cancerous plasma cell), they were able to generate a hybrid cell, called a hybridoma, that possessed the immortal growth properties of the myeloma cell and secreted the antibody produced by the B cell . The resulting clones of hybridoma cells, which secrete large quantities of monoclonal antibody, can be cultured indefinitely. B-cell hybridomas that secrete antibody with a single antigenic specificity, called monoclonal antibody, in reference to its derivation from a single clone, have revolutionized not only immunology but biomedical research as well as the clinical laboratory. Basis of fusion and selection: Spleen cells, prepared from immunized mice or rats, are induced to fuse with myeloma cells using polyethylene glycol. Many cells show cytoplasmic fusion; a lower proportion complete the nuclear fusion. This procedure results in a heterogeneous mixture of fused and unfused cells, although there is a preferential association of ontogenetically similar cells: myeloma cells tend to ‘rescue’ large, recently activated B lymphocytes. After dispensing into culture wells, the cell mixture is cultured in a selective medium that positively selects for fusion hybrids. Culture supernatants are tested for antibody activity after 1–3 weeks. Positive cultures are cloned by conventional cell cloning techniques. This is done by the use of a myeloma cell line deficient in the enzyme responsible for incorporation of hypoxanthine into DNA. Briefly, cells can synthesize DNA in two ways, either by de novo synthesis or via the so-called ‘salvage’ pathway using exogenous or endogenous sources of preformed bases, as summarized in. If myeloma cells are grown in the presence of a purine analogue, for example 8-azaguanine or 6-thioguanine, the hypoxanthine guanine phosphoribosyltransferase (HGPRT) enzyme catalyses the incorporation of the purine analogue into DNA where it interferes with normal protein synthesis and so the cells die. Gene coding for the HGPRT enzyme is on the X chromosome, so only a single copy per cell is expressed. Cells will arise that are deficient in the HGPRT gene and therefore do not incorporate the purine analogue, i.e. HGPRT-deficient cells which are unable to utilize hypoxanthine and therefore can only synthesize ribonucleotides by de novo synthesis. A selective medium containing aminopterin (or amethopterin, methotrexate), hypoxanthine and thymidine (HAT medium) is used. Aminopterin (analogue of folic acid) binds folic acid reductase and blocks the coenzymes required for de novo synthesis of DNA. To grow in this medium a cell must make DNA via the ‘salvage’ pathway. If HGPRT- deficient plasmacytoma cells are fused with normal lymphoid cells and then placed in HAT medium, only the hybrids between plasmacytoma and normal cells will grow (plasmacytoma cell provides immortality; plasma cell provides the HGPRT enzyme. SCREENING: The initial screen for antibody activity should be carried out as soon as growth of hybrid cells is seen under the microscope or when the pH indicator dye has become yellow. Although cells are diluted to limit the number of independent hybrid cells per well, several hybrids may grow, perhaps at different rates, each producing their own clone of cells. This might affect the screening assay in two ways: (i) A positive clone (secreting the desired antibody) may be detected soon after fusion, but then might be lost by overgrowth of a negative or other positive clones. (ii) No activity may be detected during the first assay due to the cells of a positive clone being in a minority. Once antibody activity has been detected in any particular well, it is essential to clone and retest the cells as soon as possible. The type of assay used to detect antibody is determined by the nature of the antigen and the type of antibody desired. Antibody-secreting hybrid cells from positive culture wells must be cloned to ensure that the antibody is homogeneous and monospecific. CLONING: Cloning is necessary to ensure that non-producers, arising either in the original fusion wells or as spontaneous variants, do not outgrow the antibody-secreting hybrids. If continuous growth of a hybrid line is required, it will be necessary to repeat the cloning and positive selection procedure at regular intervals. USES: ORGANS OF THE IMMUNE SYSTEM A number of morphologically and functionally diverse organsand tissues have various functions in the development of immune responses. These can be distinguished by function as the primary and secondary lymphoid organs (Figure 2-13). The thymus and bone marrow are the primary (or central) lymphoid organs, where maturation of lymphocytes takes place. The lymph nodes, spleen, and various mucosalassociated lymphoid tissues (MALT) such as gut-associated lymphoid tissue (GALT) are the secondary (or peripheral) lymphoid organs, which trap antigen and provide sites for mature lymphocytes to interact with that antigen. Once mature lymphocytes have been generated in the primary lymphoid organs, they circulate in the blood and lymphatic system, a network of vessels that collect fluid that has escaped into the tissues from capillaries of the circulatory system and ultimately return it to the blood. PRIMARY LYMPHOID ORGANS Immature lymphocytes generated in hematopoiesis mature and become committed to a particular antigenic specificity within the primary lymphoid organs. Only after a lymphocyte has matured within a primary lymphoid organ is the cell immunocompetent (capable of mounting an immune response). T cells arise in the thymus, and in many mammals—humans and mice for example—B cells originate in bone marrow. THYMUS: The thymus is the site of T-cell development and maturation. It is a flat, bilobed organ situated above the heart. Each lobe is surrounded by a capsule and is divided into lobules, which are separated from each other by strands of connective tissue called trabeculae. Each lobule is organized into two compartments: the outer compartment, or cortex, is densely packed with immature T cells, called thymocytes, whereas the inner compartment, or medulla, is sparsely populated with thymocytes. Both the cortex and medulla of the thymus are crisscrossed by a three-dimensional stromal-cell network composed of epithelial cells, dendritic cells, and macrophages, which make up the framework of the organ and contribute to the growth and maturation of thymocytes. Many of these stromal cells interact physically with the developing thymocytes (Figure 2-14). Some thymic epithelial cells in the outer cortex, called nurse cells, have long membrane extensions that surround as many as 50 thymocytes, forming large multicellular complexes. THE THYMUS AND IMMUNE FUNCTION: The role of the thymus in immune function can be studied in mice by examining the effects of neonatal thymectomy, a procedure in which the thymus is surgically removed from newborn mice. These thymectomized mice show a dramatic decrease in circulating lymphocytes of the T cell lineage and an absence of cell-mediated immunity. Other evidence of the importance of the thymus comes from studies of a congenital birth defect in humans (DiGeorge’s syndrome) and in certain mice (nude mice) in which the thymus fails to develop. In both cases, there is an absence of circulating T cells and of cell-mediated immunity and an increase in infectious disease. Aging is accompanied by a decline in thymic function. This decline may play some role in the decline in immune function during aging in humans and mice. The thymus reaches its maximal size at puberty and then atrophies, with a significant decrease in both cortical and medullary cells. BONE MARROW In humans and mice, bone marrow is the site of B-cell origin and development. Its having two regions1) Vascular region2) Hematopoietic region. The bone marrow weighs about 3 Kg in adult. The vascular region supplies nutrients and removes the waste materials during hematopoiesis. The hematopoietic region is also called as yellow marrow. Arising from lymphoid progenitors, immature B cells proliferate and differentiate within the bone marrow, and stromal cells within the bone marrow interact directly with the B cells and secrete various cytokines that are required for development. Bone marrow is not the site of B-cell development in all species. In birds, a lymphoid organ called the bursa of Fabricius, a lymphoidtissue associated with the gut, is the primary site of B-cell maturation. In mammals such as primates and rodents, there is no bursa and no single counterpart to it as a primary lymphoid organ. In cattle and sheep, the primary lymphoid tissue hosting the maturation, proliferation, and diversification of B cells early in gestation is the fetal spleen. SECONDARY LYMPHOID ORGAN Lymph nodes and the spleen are the most highly organized of the secondary lymphoid organs; they comprise not only lymphoid follicles, but additional distinct regions of Tcell and B-cell activity, and they are surrounded by a fibrous capsule. Less-organized lymphoid tissue, collectively called mucosal-associated lymphoid tissue (MALT), is found in various body sites. MALT includes Peyer’s patches (in the small intestine), the tonsils, and the appendix, as well as numerous lymphoid follicles within the lamina propria of the intestines and in the mucous membranes lining the upper airways, bronchi, and genital tract. As blood circulates under pressure, its fluid component (plasma) seeps through the thin wall of the capillaries into the surrounding tissue.Much of this fluid, called interstitial fluid, returns to the blood through the capillary membranes. The remainder of the interstitial fluid, now called lymph, flows from the spaces in connective tissue into a network of tiny open lymphatic capillaries and then into a series of progressively larger collecting vessels called lymphatic vesselsWhen a foreign antigen gains entrance to the tissues, it is picked up by the lymphatic system (which drains all the tissues of the body) and is carried to various organized lymphoid tissues such as lymph nodes, which trap the foreign antigen. As lymph passes from the tissues to lymphatic vessels, it becomes progressively enriched in lymphocytes. Thus, the lymphatic system also serves as a means of transporting lymphocytes and antigen from the connective tissues to organized lymphoid tissues where the lymphocytes may interact with the trapped antigen and undergo activation. LYMPH NODES Lymph nodes are the sites where immune responses are mounted to antigens in lymph. They are encapsulated beanshaped structures containing a reticular network packed with lymphocytes, macrophages, and dendritic cells. Clustered at junctions of the lymphatic vessels, lymph nodes are the first organized lymphoid structure to encounter antigens that enter the tissue spaces. As lymph percolates through a node, any particulate antigen that is brought in with the lymph will be trapped by the cellular network of phagocytic cells and dendritic cells (follicular and interdigitating). The overall architecture of a lymph node supports an ideal microenvironment for lymphocytes to effectively encounter and respond to trapped antigens. Morphologically, a lymph node can be divided into three roughly concentric regions: the cortex, the paracortex, and the medulla, each of which supports a distinct microenvironment (Figure 2-18). The outermost layer, the cortex, contains lymphocytes (mostly B cells), macro-phages, and follicular dendritic cells arranged in primary follicles. After antigenic challenge, the primary follicles enlarge into secondary follicles, each containing a germinal center. In children with B-cell deficiencies, the cortex lacks primary follicles and germinal centers. Beneath the cortex is the paracortex, which is populated largely by T lymphocytes and also contains interdigitating dendritic cells thought to have migrated from tissues to the node. These interdigitating dendritic cells express high levels of class II MHC molecules,which are necessary for presenting antigen to TH cells. The innermost layer of a lymph node, the medulla, is more sparsely populated with lymphoidlineage cells; of those present, many are plasma cells actively secreting antibody molecules. As antigen is carried into a regional node by the lymph, it is trapped, processed, and presented together with class II MHC molecules by interdigitating dendritic cells in the paracortex, resulting in the activation of TH cells. The initial activation of B cells is also thought to take place within the T-cell-rich paracortex. Once activated, TH and B cells form small foci consisting largely of proliferating B cells at the edges of the paracortex. Some B cells within the foci differentiate into plasma cells secreting IgM and IgG. These foci reach maximum size within 4–6 days of antigen challenge. Within 4–7 days of antigen challenge, a few B cells and TH cells migrate to the primary follicles of the cortex. It is not known what causes this migration.Within a primary follicle, cellular interactions between follicular dendritic cells, B cells, and TH cells take place, leading to development of a secondary follicle with a central germinal center. Some of the plasma cells generated in the germinal center move to the medullary areas of the lymph node, and many migrate to bone marrow. Lymph coming from the tissues percolates slowly inward through the cortex, paracortex, and medulla, allowing phagocytic cells and dendritic cells to trap any bacteria or particulate material (e.g., antigen-antibody complexes) carried by the lymph. SPLEEN The spleen plays a major role in mounting immune responses to antigens in the blood stream. It is a large, ovoid secondary lymphoid organ situated high in the left abdominal cavity. While lymph nodes are specialized for trapping antigen from local tissues, the spleen specializes in filtering blood and trapping blood-borne antigens; thus, it can respond to systemic infections. Unlike the lymph nodes, the spleen is not supplied by lymphatic vessels. Instead, bloodborne antigens and lymphocytes are carried into the spleen through the splenic artery. Experiments with radioactively labeled lymphocytes show that more recirculating lymphocytes pass daily through the spleen than through all the lymph nodes combined. The spleen is surrounded by a capsule that extends a number of projections (trabeculae) into the interior to form a compartmentalized structure. The compartments are of two types, the red pulp and white pulp, which are separated by a diffuse marginal zone (Figure 2-19). The splenic red pulp consists of a network of sinusoids populated by macrophages and numerous red blood cells (erythrocytes) and few lymphocytes; it is the site where old and defective red blood cells are destroyed and removed.Many of the macrophages within the red pulp contain engulfed red blood cells or iron pigments from degraded hemoglobin. The splenic white pulp surrounds the branches of the splenic artery, forming a periarteriolar lymphoid sheath (PALS) populated mainly by T lymphocytes. Primary lymphoid follicles are attached to thePALS. These follicles are rich in B cells and some of them contain germinal centers. The marginal zone, located peripheral to the PALS, is populated by lymphocytes and macrophages. Blood-borne antigens and lymphocytes enter the spleen through the splenic artery, which empties into the marginal zone. In the marginal zone, antigen is trapped by interdigitating dendritic cells, which carry it to the PALS. Lymphocytes in the blood also enter sinuses in the marginal zone and migrate to the PALS. The initial activation of B and T cells takes place in the Tcell- rich PALS. Here interdigitating dendritic cells capture antigen and present it combined with class II MHC molecules to TH cells. Once activated, these TH cells can then activate B cells. The activated B cells, together with some TH cells, then migrate to primary follicles in the marginal zone. Upon antigenic challenge, these primary follicles develop into characteristic secondary follicles containing germinal centers (like those in the lymph nodes), where rapidly dividing B cells (centroblasts) and plasma cells are surrounded by dense clusters of concentrically arranged lymphocytes. The effects of splenectomy on the immune response depends on the age at which the spleen is removed. In children, splenectomy often leads to an increased incidence of bacterial sepsis caused primarily by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Splenectomy in adults has less adverse effects, although it leads to some increase in bloodborne bacterial infections (bacteremia). LYMPHOCYTE TRAFFIC AND REGULATION Lymphocytes are capable of a remarkable level of recirculation, continually moving through the blood and lymph to the various lymphoid organs (Figure 15-1). After a brief transit time of approximately 30 min in the bloodstream, nearly 45% of all lymphocytes are carried from the blood directly to the spleen, where they reside for approximately 5 h. Almost equal numbers (42%) of lymphocytes exit from the blood into various peripheral lymph nodes, where they reside for about 12 h. A smaller number of lymphocytes (10%) migrate to tertiary extralymphoid tissues by crossing between endothelial cells that line the capillaries. An individual lymphocyte may make a complete circuit from the blood to the tissues and lymph and back again as often as 1–2 times per day. Cell-Adhesion Molecules: The vascular endothelium serves as an important “gatekeeper,” regulating the movement of blood-borne molecules.A number of endothelial and leukocyte CAMs have been cloned and characterized, providing new details about the extravasation process. Most of these CAMs belong to four families of proteins: the selectin family, the mucin-like family, the integrin family, and the immunoglobulin (Ig) superfamily. Lymphocyte Extravasation: Various subsets of lymphocytes exhibit directed extravasation at inflammatory sites and secondary lymphoid organs. The recirculation of lymphocytes thus is carefully controlled to ensure that appropriate populations of B and T cells are recruited into different tissues. Extravasation of lymphocytes involves interactions among a number of cell-adhesion molecules (Table 15-1). The overall process is similar to what happens during neutrophil extravasation and comprises the same four stages of contact and rolling, activation, arrest and adhesion, and, finally, transendothelial migration. High-Endothelial Venules Are Sites of Lymphocyte Extravasation: Some regions of vascular endothelium in postcapillary venules of various lymphoid organs are composed of specialized cells with a plump, cuboidal (“high”) shape; such regions are called high-endothelial venules, or HEVs (Figure 15-4a, b). Their cells contrast sharply in appearance with the flattened endothelial cells that line the rest of the capillary. Each of the secondary lymphoid organs, with the exception of the spleen, contains HEVs The general process of lymphocyte extravasation is similar to neutrophil extravasation. An important feature distinguishing the two processes is that different subsets of lymphocytes migrate differentially into different tissues. This process is called trafficking, or homing. The different trafficking patterns of lymphocyte subsets are mediated by unique combi- nations of adhesion molecules and chemokines; receptors that direct the circulation of various populations of lymphocytes to particular lymphoid and inflammatory tissues are called homing receptors.Researchers have identified a number of lymphocyte and endothelial celladhesion molecules that participate in the interaction of lymphocytes with HEVs and with endothelium at tertiary sites or sites of inflammation. chemokines, play a major role in determining the heterogeneity of lymphocyte circulation patterns. Adhesion-Molecule Interactions Play Critical Roles in Extravasation: The extravasation of lymphocytes into secondary lymphoid tissue or regions of inflammation is a multistep process involving a cascade of adhesion-molecule interactions similar to those involved in neutrophil emigration from the bloodstream. Figure 15-7 depicts the typical interactions in extravasation of naive T cells across HEVs into lymph nodes. The first step is usually a selectin-carbohydrate interaction similar to that seen with neutrophil adhesion. Naive lymphocytes initially bind to HEVs by L-selectin, which serves as a homing receptor that directs the lymphocytes to particular tissues expressing a corresponding mucinlike vascular addressin such as CD34 or GlyCAM-1. Lymphocyte rolling is less pronounced than that of neutrophils. In the second step, an integrin-activating stimulus is mediated by chemokines that are either localized on the endothelial surface or secreted locally. The thick glycocalyx covering of the HEVs may function to retain these soluble chemoattractant factors on the HEVs. If, as some have proposed, HEVs secrete lymphocyte-specific chemoattractants, it would explain why neutrophils do not extravasate into lymph nodes at the HEVs even though they express L-selectin. Chemokine binding to G-protein–coupled receptors on the lymphocyte leads to activation of integrin molecules on the membrane, as occurs in neutrophil extravasation. Once activated, the integrin molecules interact with Igsuperfamily adhesion molecules (e.g., ICAM-1), so the lymphocyte adheres firmly to the endothelium. The molecular mechanisms involved in the final step, transendothelial migration, are poorly understood. Cluster of differentiation The cluster of differentiation (cluster of designation) (often abbreviated as CD) is a protocol used for the identification and investigation of cell surface molecules present on White blood cells. CD molecules can act in numerous ways, often acting as receptors or ligands (the molecule that activates a receptor) important to the cell. A signal cascade is usually initiated, altering the behavior of the cell (see cell signaling). Some CD proteins do not play a role in cell signaling, but have other functions, such as cell adhesion. CD for humans is numbered up to 350 most recently (as of 2009). Nomenclature The CD nomenclature was proposed and established in the 1st International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), which was held in Paris in 1982.This system was intended for the classification of the many monoclonal antibodies (mAbs) generated by different laboratories around the world against epitopes on the surface molecules of leukocytes (white blood cells). Since then, its use has expanded too many other cell types, and more than 320 CD unique clusters and subclusters have been identified. The proposed surface molecule is assigned a CD number once two specific monoclonal antibodies (mAb) are shown to bind to the molecule. If the molecule has not been well-characterized, or has only one mAb, it is usually given the provisional indicator "w" (as in "CDw186"). Cell markers The CD system is commonly used as cell markers, allowing cells to be defined based on what molecules are present on their surface. These markers are often used to associate cells with certain immune functions. While using one CD molecule to define populations is uncommon (though a few examples exist), combining markers has allowed for cell types with very specific definitions within the immune system. CD molecules are utilized in cell sorting using various methods including flow cytometry. Cell populations are usually defined using a '+' or a '–' symbol to indicate whether a certain cell fraction expresses or lacks a CD molecule. For example, a "CD34+, CD31–" cell is one that expresses CD34, but not CD31. This CD combination typically corresponds to a stem cell, opposed to a fully-differentiated endothelial cell. Type of cell CD markers stem cells CD34+,CD31- all leukocyte groups CD45+ Granulocyte CD45+,CD15+ Monocyte CD45+,CD14+ T lymphocyte CD45+,CD3+ T helper cell CD45+,CD3+,CD4+ Cytotoxic T cell CD45+,CD3+,CD8+ B lymphocyte CD45+,CD19+ or CD45+,CD20+ Thrombocyte CD45+,CD61+ Natural killer cell CD16+,CD56+,CD3- Two commonly-used CD molecules are CD4 and CD8, which are, in general, used as markers for helper and cytotoxic T cells, respectively. These molecules are defined in combination with CD3+, as some other leukocytes also express these CD molecules (some macrophages express low levels of CD4; dendritic cells express high levels of CD8). Human immunodeficiency virus (HIV) binds CD4 and a chemokine receptor on the surface of a T helper cell to gain entry. The number of CD4 and CD8 T cells in blood is often used to monitor the progression of HIV infection. Other uses It is important to note that, while CD molecules are very useful in defining leukocytes, they are not merely markers on the cell surface. While only a fraction of known CD molecules have been thoroughly characterised, most of them have an important function. In the example of CD4 & CD8, these molecules are critical in antigen recognition. ANTIBODIES Antibodies are the antigen binding proteins present on the B-cell membrane and secreted by plasma cells. Membrane-bound antibody confers antigenic specificity on B cells; antigen specific proliferation of B-cell clones is elicted by the interaction of membrane antibody with antigen. Secreted antibodies circulate in the blood, where they serve as the effectors of humoral immunity by searching out and neutralizing antigens or marking them for elimination.All antibodies share structural features, bind to antigen, and participate in a limited number of effector functions. BASIC STRUCTURE OF ANTIBODIES: Blood can be separated in a centrifuge into a fluid and a cellular fraction. The fluid fraction is the plasma and the cellular fraction contains red blood cells, leukocytes, and platelets. Plasma contains all of the soluble small molecules and macromolecules of blood, including fibrin and other proteins required for the formation of blood clots. If the blood or plasma is allowed to clot, the fluid phase that remains is called serum. It has been known since the turn of the century that antibodies reside in the serum. The first evidence that antibodies were contained in particular serum protein fractions came from a classic experiment by A. Tiselius and E. A.Kabat, in 1939. They immunized rabbits with the protein ovalbumin (the albumin of egg whites) and then divided the immunized rabbits’ serum into two aliquots. Electrophoresis of one serum aliquot revealed four peaks corresponding to albumin and the alpha (_), beta (_), and gamma (_) globulins. The other serum aliquot was reacted with ovalbumin, and the precipitate that formed was removed; the remaining serum proteins, which did not react with the antigen, were then electrophoresed. A comparison of the electrophoretic profiles of these two serum aliquots revealed that there was a significant drop in the _-globulin peak in the aliquot that had been reacted with antigen (Figure 4-1). Thus, the _-globulin fraction was identified as containing serum antibodies, which were called immunoglobulins, to distinguish them from any other proteins that might be contained in the _-globulin fraction. The early experiments of Kabat and Tiselius resolved serum proteins into three major nonalbumin peaks—_, _ and _.We now know that although immunoglobulin G (IgG), the main class of antibody molecules, is indeed mostly found in the _-globulin fraction, significant amounts of it and other important classes of antibody molecules are found in the _ and the _ fractions of serum. ANTIBODIES ARE HETERODIMERS: Antibody molecules have a common structure of four peptide chains (Figure 4-2). This structure consists of two identical light (L) chains, polypeptides of about 25,000 molecular weight, and two identical heavy (H) chains, largerpolypeptides of molecular weight 50,000 or more. Like the antibody molecules they constitute, H and L chains are also called immunoglobulins. Each light chain is bound to a heavy chain by a disulfide bond, and by such noncovalent interactions as salt linkages, hydrogen bonds, and hydrophobic bonds, to form a heterodimer (H-L). Similar noncovalent interactions and disulfide bridges link the two identical heavy and light (H-L) chain combinations to each other to form the basic four-chain (H-L)2 antibody structure, The first 110 or so amino acids of the amino-terminal region of a light or heavy chain varies greatly among antibodies of different specificity. These segments of highly variable sequence are called V regions:VL in light chains and VH in heavy. All of the differences in specificity displayed by different antibodies can be traced to differences in the amino acid sequences of V regions. In fact, most of the differences among antibodies fall within areas of the V regions called complementarity- determining regions (CDRs), and it is these CDRs, on both light and heavy chains, that constitute the antigenbinding site of the antibody molecule. The regions of relatively constant sequence beyond the variable regions have been dubbed C regions, CL on the light chain and CH on the heavy chain.Antibodies are glycoproteins; with few exceptions, the sites of attachment for carbohydrates are restricted to the constant region.We do not completely understand the role played by glycosylation of antibodies, but it probably increases the solubility of the molecules. Inappropriate glycosylation, or its absence, affects the rate at which antibodies are cleared from the serum, and decreases the efficiency of interaction between antibody and the complement system and between antibodies and Fc receptors. Our knowledge of basic antibody structure was derived from a variety of experimental observations. In a key experiment, brief digestion of IgG with the enzyme papain produced three fragments, two of which were identical fragments and a third that was quite different (Figure 4-3). The two identical fragments (each with a MW of 45,000), had antigen-binding activity and were called Fab fragments (“fragment, antigen binding”). The other fragment (MW of 50,000) had no antigenbinding activity at all. Because it was found to crystallize during cold storage, it was called the Fc fragment (“fragment, crystallizable”).Digestion with pepsin, a different proteolytic enzyme, also demonstrated that the antigen-binding properties of an antibody can be separated from the rest of the molecule. Pepsin digestion generated a single 100,000- MW fragment composed of two Fab-like fragments designated the F(ab_)2 fragment, which binds antigen. The Fc fragment was not recovered from pepsin digestion because it had been digested into multiple fragments. A key observation in deducing the multichain structure of IgG was made when the molecule was subjected to mercaptoethanol reduction and alkylation, a chemical treatment that irreversibly cleaves disulfide bonds. If the sample is chromate graphed on a column that separates molecules by size following cleavage of disulfide bonds, it is clear that the intact 150,000MW IgG molecule is, in fact, composed of subunits. Each IgG molecule contains two 50,000-MW polypeptide chains, designated as heavy (H) chains, and two 25,000-MW chains, designated as light (L) chains (see Figure 4-3). Immunoglobulin Fine Structure The structure of the immunoglobulin molecule is determined by the primary, secondary, tertiary, and quaternary organization of the protein. The primary structure, the amino acid sequence, accounts for the variable and constant regions of the heavy and light chains. The secondary structure is formed by folding of the extended polypeptide chain, back and forth upon itself into an antiparallel _ pleated sheet (Figure 4-4). The chains are then folded into a tertiary structure of compact globular domains, which are connected to neighboring domains by continuations of the polypeptide chain that lie outside the _ pleated sheets. Finally, the globular domains of adjacent heavy and light polypeptide chains interact in the quaternary structure (Figure 4-5), forming functional domains that enable the molecule to specifically bind antigen and, at the same time, perform a number of biological effector functions. Antibody Classes and Biological Activities: The various immunoglobulin isotypes and classes have been mentioned briefly already. Each class is distinguished by unique amino acid sequences in the heavy-chain constant region that confer class-specific structural and functional properties. In this section, the structure and effector functions of each class are described in more detail. The molecular properties and biological activities of the immunoglobulin classes are summarized in Table 4-2 (page 90). The structures of the five major classes are diagramed in Figure 4-13 (page 91). Immunoglobulin G (IgG): IgG, the most abundant class in serum, constitutes about 80% of the total serum immunoglobulin. The IgG molecule consists of two _ heavy chains and two light chains (see Figure 4-13a). There are four human IgG subclasses, distinguished by differences in _-chain sequence and numbered according to their decreasing average serum concentrations: IgG1, IgG2, IgG3, and IgG4 (see Table 4-2). The amino acid sequences that distinguish the four IgG subclasses are encoded by different germ-line CH genes, whose DNA sequences are 90%–95% homologous. The structural characteristics that distinguish these subclasses from one another are the size of the hinge region and the number and position of the interchain disulfide bonds between the heavy chains (Figure 4-14, page 92). The subtle amino acid differences between subclasses of IgG affect the biological activity of the molecule: _ IgG1, IgG3, and IgG4 readily cross the placenta and play an important role in protecting the developing fetus. _ IgG3 is the most effective complement activator, followed by IgG1; IgG2 is less efficient, and IgG4 is not able to activate complement at all. _ IgG1 and IgG3 bind with high affinity to Fc receptors on phagocytic cells and thus mediate opsonization. IgG4 has an intermediate affinity for Fc receptors, and IgG2 has an extremely low affinity. Immunoglobulin M (IgM): IgM accounts for 5%–10% of the total serum immunoglobulin, with an average serum concentration of 1.5 mg/ml. Monomeric IgM, with a molecular weight of 180,000, is expressed as membrane-bound antibody on B cells. IgM is secreted by plasma cells as a pentamer in which five monomer units are held together by disulfide bonds that link their carboxyl- terminal heavy chain domains (C_4/C_4) and their C_3/C_3 domains (see Figure 4-13e). The five monomer subunits are arranged with their Fc regions in the center of the pentamer and the ten antigen-binding sites on the periphery of the molecule. Each pentamer contains an additional Fc-linked polypeptide called the J (joining) chain, which is disulfidebonded to the carboxyl-terminal cysteine residue of two of the ten _ chains. The J chain appears to be required for polymerization of the monomers to form pentameric IgM; it is added just before secretion of the pentamer. IgM is the first immunoglobulin class produced in a primary response to an antigen, and it is also the first immunoglobulin to be synthesized by the neonate. Because of its pentameric structure with 10 antigen-binding sites, serum IgM has a higher valency than the other isotypes. An IgM molecule can bind 10 small hapten molecules; however, because of steric hindrance, only 5 or fewer molecules of larger antigens can be bound simultaneously.Because of its high valency, pentameric IgM is more efficient than other isotypes in binding antigens with many repeating epitopes such as viral particles and red blood cells (RBCs). For example, when RBCs are incubated with specific antibody, they clump together into large aggregates in a process called agglutination. It takes 100 to 1000 times more molecules of IgG than of IgM to achieve the same level of agglutination. A similar phenomenon occurs with viral particles: less IgM than IgG is required to neutralize viral infectivity. IgM is also more efficient than IgG at activating complement. Complement activation requires two Fc regions in close proximity, and the pentameric structure of a single molecule of IgM fulfills this requirement. Because of its large size, IgM does not diffuse well and therefore is found in very low concentrations in the intercellular tissue fluids. The presence of the J chain allows IgM to bind to receptors on secretory cells,which transport it across epithelial linings to enter the external secretions that bathe mucosal surfaces. Although IgA is the major isotype found in these secretions, IgM plays an important accessory role as a secretory immunoglobulin. Immunoglobulin A (IgA): Although IgA constitutes only 10%–15% of the total immunoglobulin in serum, it is the predominant immunoglobulin class in external secretions such as breast milk, saliva, tears, and mucus of the bronchial, genitourinary, and digestive tracts. In serum, IgA exists primarily as a monomer, but polymeric forms (dimers, trimers, and some tetramers) are sometimes seen, all containing a J-chain polypeptide (see Figure 4-13d). The IgA of external secretions, called secretory IgA, consists of a dimer or tetramer, a J-chain polypeptide, and a polypeptide chain called secretory component (Figure 4-15a, page 93). As is explained below, secretory component is derived from the receptor that is responsible for transporting polymeric IgA across cell membranes. The J-chain polypeptide in IgA is identical to that found in pentameric IgM and serves a similar function in facilitating the polymerization of both serum IgA and secretory IgA. The secretory component is a 70,000-MW polypeptide produced by epithelial cells of mucous membranes. It consists of five immunoglobulin-like domains that bind to the Fc region domains of the IgA dimer. This interaction is stabilized by a disulfide bond between the fifth domain of the secretory component and one of the chains of the dimeric IgA. The daily production of secretory IgA is greater than that of any other immunoglobulin class. IgA-secreting plasma cells are concentrated along mucous membrane surfaces. Binding of secretory IgA to bacterial and viral surface antigens prevents attachment of the pathogens to the mucosal cells, thus inhibiting viral infection and bacterial colonization. Complexes of secretory IgA and antigen are easily entrapped in mucus and then eliminated by the ciliated epithelial cells of the respiratory tract or by peristalsis of the gut. Secretory IgA has been shown to provide an important line of defense against bacteria such as Salmonella, Vibrio cholerae, and Neisseria gonorrhoeae and viruses such as polio, influenza, and reovirus. Breast milk contains secretory IgA and many other molecules that help protect the newborn against infection during the first month of life (Table 4-3). Because the immune system of infants is not fully functional, breast-feeding plays an important role in maintaining the health of newborns. Immunoglobulin E (IgE): The potent biological activity of IgE allowed it to be identified in serum despite its extremely low average serum concentration (0.3g/ml). IgE antibodies mediate the immediate hypersensitivity reactions that are responsible for the symptoms of hay fever, asthma, hives, and anaphylactic shock. The presence of a serum component responsible for allergic reactions was first demonstrated in 1921 by K. Prausnitz and H. Kustner, who injected serum from an allergic person intra-dermally into a nonallergic individual. When the appropriate antigen was later injected at the same site, a wheal and flare reaction (analogous to hives) developed there. This reaction, called the P-K reaction (named for its originators, Prausnitz and Kustner), was the basis for the first biological assay for IgE activity. IgE binds to Fc receptors on the membranes of blood basophils and tissue mast cells. Cross-linkage of receptorbound IgE molecules by antigen (allergen) induces basophils and mast cells to translocate their granules to the plasma membrane and release their contents to the extracellular environment, a process known as degranulation. As a result, a variety of pharmacologically active mediators are released and give rise to allergic manifestations (Figure 4-16). Immunoglobulin D (IgD): IgD was first discovered when a patient developed a multiple myeloma whose myeloma protein failed to react with antiisotype antisera against the then-known isotypes: IgA, IgM, and IgG.When rabbits were immunized with this myeloma protein, the resulting antisera were used to identify the same class of antibody at low levels in normal human serum. The new class, called IgD, has a serum concentration of 30g/ml and constitutes about 0.2% of the total immunoglobulin in serum. IgD, together with IgM, is the major membranebound immunoglobulin expressed by mature B cells, and its role in the physiology of B cells is under investigation. No biological effector function has been identified for IgD. HEMATOPOIESIS All blood cells arise from a type of cell called the hematopoietic stem cell (HSC). Stem cells are cells that can differentiate into other cell types; they are selfrenewing—they maintain their population level by cell division. In humans, hematopoiesis, the formation and development of red and white blood cells, begins in the embryonic yolk sac during the first weeks of development.Here, yolk-sac stem cells differentiate into primitive erythroid cells that contain embryonic hemoglobin. In the third month of gestation, hematopoietic stem cells migrate from the yolk sac to the fetal liver and then to the spleen; these two organs have major roles in hematopoiesis from the third to the seventh months of gestation. After that, the differentiation of HSCs in the bone marrow becomes the major factor in hematopoiesis, and by birth there is little or no hematopoiesis in the liver and spleen. It is remarkable that every functionally specialized, mature blood cell is derived from the same type of stem cell. Incontrast to a unipotent cell, which differentiates into a single cell type, a hematopoietic stem cell is multipotent, or pluripotent, able to differentiate in various ways and thereby generate erythrocytes, granulocytes, monocytes, mast cells, lymphocytes, and megakaryocytes. hematopoietic stem cells are maintained at stable levels throughout adult life; however, when there is an increased demand for hematopoiesis, HSCs display an enormous proliferative capacityEarly in hematopoiesis, a multipotent stem cell differentiates along one of two pathways, giving rise to either a common lymphoid progenitor cell or a common myeloid progenitor cell (Figure 2-1). The types and amounts of growth factors in the microenvironment of a particular stem cell or progenitor cell control its differentiation. During the development of the lymphoid and myeloid lineages, stem cells differentiate into progenitor cells, which have lost the capacity for self-renewal and are committed to a particular cell lineage. Common lymphoid progenitor cells give rise to B, T, and NK (natural killer) cells and some dendritic cells. Myeloid stem cells generate progenitors of red blood cells (erythrocytes), many of the various white blood cells (neutrophils, eosinophils, basophils, monocytes, mast cells, dendritic cells), and platelets. Progenitor commitment depends on the acquisition of responsiveness to particular growth factors and cytokines. When the appropriate factors and cytokines are present, progenitor cells proliferate and differentiate into the corresponding cell type, either a mature erythrocyte, a particular type of leukocyte, or a platelet-generating cell (the megakaryocyte). Red and white blood cells pass into bone marrow channels, from which they enter the circulation. Development of B and T lymphocytes Lymphocytes constitute 20%–40% of the body’s white blood cells and 99% of the cells in the lymphThe lymphocytes can be broadly subdivided into three populations—B cells, T cells, and natural killer cells—on the basis of function and cell-membrane components. Natural killer cells (NK cells) are large, granular lymphocytes that do not express the set of surface markers typical of B or T cells. Resting B and T lymphocytes are small, motile, nonphagocytic cells,which cannot be distinguished morphologically. B and T lymphocytes that have not interacted with antigen— referred to as naive, or unprimed—are resting cells in the G0 phase of the cell cycle. Known as small lymphocytes, these cells are only about 6 _m in diameter; their cytoplasm forms a barely discernible rim around the nucleus. Small lymphocytes have densely packed chromatin, few mitochondria, and a poorly developed endoplasmic reticulum and Golgi apparatus. The naive lymphocyte is generally thought to have a short life span. Interaction of small lymphocytes with antigen, in the presence of certain cytokines discussed later, induces these cells to enter the cell cycle by progressing from G0 into G1 and subsequently into S, G2, and M (Figure 2-7a). As they progress through the cell cycle, lymphocytes enlarge into 15 _m-diameter blast cells, called lymphoblasts; these cells have a higher cytoplasm:nucleus ratio and more organellar complexity than small lymphocytes (Figure 2-7b). Lymphoblasts proliferate and eventually differentiate into effector cells or into memory cells. Effector cells function in various ways to eliminate antigen. These cells have short lifespans, generally ranging from a few days to a few weeks. Plasma cells—the antibody-secreting effector cells of the Bcell lineage—have a characteristic cytoplasm that contains abundant endoplasmic reticulum (to support their high rate of protein synthesis) arranged in concentric layers and also many Golgi vesicles (see Figure 2-7). The effector cells of the T-cell lineage include the cytokine-secreting T helper cell (TH cell) and the T cytotoxic lymphocyte (TC cell). Some of the progeny of B and T lymphoblasts differentiate into memory cells. The persistence of this population of cells is responsible for life-long immunity to many pathogens. Memory cells look like small lymphocytes but can be distinguished from naive cells by the presence or absence of certain cellmembrane molecules. EXPRESSION OF IG GENES As in the expression of other genes, post-transcriptional processing of immunoglobulin primary transcripts is required to produce functional mRNAs (see Figures 54 and 5-5). The primary transcripts produced from rearranged heavy-chain and light-chain genes contain intervening DNA sequences that include noncoding introns and J gene segments not lost during V-(D)-J rearrangement. In addition, as noted earlier, the heavy-chain C-gene segments are organized as a series of coding exons and noncoding introns. Each exon of a CH gene segment corresponds to a constant-region domain or a hinge region of the heavy-chain polypeptide. The primary transcript must be processed to remove the intervening DNA sequences, and the remaining exons must be connected by a process called RNA splicing. Processing of the primary transcript in the nucleus removes each of these intervening sequences to yield the final mRNA product. The mRNA is then exported from the nucleus to be translated by ribosomes into complete H or L chains. DIFFERENTIAL RNA PROCESSING Processing of an immunoglobulin heavy-chain primary transcript can yield several different mRNAs,which explains how a single B cell can produce secreted or membranebound forms of a particular immunoglobulin and simultaneously express IgM and IgD. EXPRESSION OF MEMBRANE OR SECRETED IMMUNOGLOBULIN A particular immunoglobulin can exist in either membrane-bound or secreted form. The two forms differ in the amino acid sequence of the heavy-chain carboxyl-terminal domains (CH3/CH3 in IgA, IgD, and IgG and CH4/CH4 in IgE and IgM). The secreted form has a hydrophilic sequence of about 20 amino acids in the carboxylterminal domain; this is replaced in the membrane-bound form with a sequence of about 40 amino acids containing a hydrophilic segment that extends outside the cell, a hydrophobic transmembrane segment, and a short hydrophilic segment at the carboxyl terminusThe C_4 exon contains a nucleotide sequence (called S) at its 3_ end that encodes the hydrophilic sequence in the CH4 domain of secreted IgM. Two additional exons called M1 and M2 are located just 1.8 kb downstream from the 3_ end of the C_4 exon. The M1 exon encodes the transmembrane segment, and M2 encodes the cytoplasmic segment of the CH4 domain in membrane-bound IgM. Later DNA sequencing revealed that all the CH gene segments have two additional downstream M1 and M2 exons that encode the transmembrane and cytoplasmic segments. The primary transcript produced by transcription of a rearranged _ heavy-chain gene contains two polyadenylation signal sequences, or poly-A sites, in the C_ segment. Site 1 is located at the 3_ end of the C_4 exon, and site 2 is at the 3_ end of the M2 exon (Figure 5-16b). If cleavage of the primary transcript and addition of the poly-A tail occurs at site 1, the M1 and M2 exons are lost. Excision of the introns and splicing of the remaining exons then produces mRNA encoding the secreted form of the heavy chain. If cleavage and polyadenylation of the primary transcript occurs instead at site 2, then a different pattern of splicing results. In this case, splicing removes the S sequence at the 3_ end of the C_4 exon, which encodes the hydrophilic carboxyl-terminal end of the secreted form, and joins the remainder of the C_4 exon with the M1 and M2 exons, producing mRNA for the membrane form of the heavy chain. Thus, differential processing of a common primary transcript determines whether the secreted or membrane form of an immunoglobulin will be produced. SIMULTANEOUS EXPRESSION OF IgM and IgD Differential RNA processing also underlies the simultaneous expression of membrane-bound IgM and IgD by mature B cells. If the heavy-chain transcript is cleaved and polyadenylated at site 2 after the C_ exons, then the mRNA will encode the membrane form of the _ heavy chain (Figure 5-17b); if polyadenylation is instead further downstream at site 4, after the C exons, then RNA splicing will remove the intervening C_ exons and produce mRNA encoding the membrane form of the heavy chain (Figure 5-17c). Since the mature B cell expresses both IgM and IgD on its membrane, both processing pathways must occur simultaneously. Likewise, cleavage and polyadenylation of the primary heavy-chain transcript at poly-A site 1 or 3 in plasma cells and subsequent splicing will yield the secreted form of the _ or _ heavy chains, respectively. SYNTHESIS, ASSEMBLY, AND SECRETION OF IMMUNOGLOBULINS Immunoglobulin heavy- and light-chain mRNAs are translated on separate polyribosomes of the rough endoplasmic reticulum (RER). Newly synthesized chains contain an amino-terminal leader sequence, which serves to guide the chains into the lumen of the RER, where the signal sequence is then cleaved. The assembly of light (L) and heavy (H) chains into the disulfide-linked and glycosylated immunoglobulin molecule occurs as the chains pass through the cisternae of the RER. The complete molecules are transported to the Golgi apparatus and then into secretory vesicles, which fuse with the plasma membrane (Figure 5-18). The order of chain assembly varies among the immunoglobulin classes. In the case of IgM, the H and L chains assemble within the RER to form half-molecules, and then two half-molecules assemble to form the complete molecule. In the case of IgG, two H chains assemble, then an H2L intermediate is assembled, and finally the complete H2L2 molecule is formed. Interchain disulfide bonds are formed, and the polypeptides are glycosylated as they move through the Golgi apparatus. If the molecule contains the transmembrane sequence of the membrane form, it becomes anchored in the membrane of a secretory vesicle and is inserted into the plasma membrane as the vesicle fuses with the plasma membrane (see Figure 5-18, insert). If the molecule contains the hydrophilic sequence of secreted immunoglobulins, it is transported as a free molecule in a secretory vesicle and is released from the cell when the vesicle fuses with the plasma membrane. PLASMA CELLS AND MEMORY CELLS Lymphocytes are the central cells of the immune system, responsible for adaptive immunity and the immunologic attributes of diversity, specificity, memory, and self/nonself recognition. The other types of white blood cells play important roles, engulfing and destroying microorganisms, presenting antigens, and secreting cytokines. The lymphocytes can be broadly subdivided into three populations—B cells, T cells, and natural killer cells— on the basis of function and cell-membrane components. B and T lymphocytes that have not interacted with antigen— referred to as naive, or unprimed—are resting cells. When a naive B cell (one that has not previously encountered antigen) first encounters the antigen that matches its membrane bound antibody it enlarge into 15 _m-diameter blast cells, called lymphoblasts; these cells have a higher cytoplasm: nucleus ratio and more organellar complexity than small lymphocytes the binding of the antigen to the antibody causes the cell to divide rapidly; its progeny differentiate into memory B cells and effector B cells called plasma cells. Memory B cells have a longer life span than naive cells, and they express the same membrane-bound antibody as their parent B cell. Plasma cells produce the antibody in a form that can be secreted and have little or no membrane-bound antibody. Although plasma cells live for only a few days, they secrete enormous amounts of antibody during this time. It has been estimated that a single plasma cell can secrete more than 2000 molecules of antibody per second. Secreted antibodies are the major effector molecules of humoral immunity. Plasma cells—the antibody-secreting effector cells of the Bcell lineage—have a characteristic cytoplasm that contains abundant endoplasmic reticulum (to support their high rate of protein synthesis) arranged in concentric layers and also many Golgi vesicles (see Figure 2-7). Some of the progeny of B and T lymphoblasts differentiate into memory cells. The persistence of this population of cells is responsible for life-long immunity to many pathogens. Memory cells look like small lymphocytes but can be distinguished from naive cells by the presence or absence of certain cellmembrane molecules. THE LECTIN PATHWAY Lectins are proteins that recognize and bind to specific carbohydrate targets. (Because the lectin that activates complement binds to mannose residues, some authors designate this the MBLectin pathway or mannan-binding lectin pathway.) The lectin pathway, like the alternative pathway, does not depend on antibody for its activation.However, the mechanism is more like that of the classical pathway, because after initiation, it proceeds, through the action of C4 and C2, to produce a C5 convertase (see Figure 13-2). The lectin pathway is activated by the binding of mannose- binding lectin (MBL) to mannose residues on glycoproteins or carbohydrates on the surface of microorganisms including certain Salmonella, Listeria, and Neisseria strains, as well as Cryptococcus neoformans and Candida albicans. MBL is an acute phase protein produced in inflammatory responses. Its function in the complement pathway is similar to that of C1q, which it resembles in structure. After MBL binds to the surface of a cell or pathogen, MBL-associated serine proteases,MASP-1 and MASP-2, bind to MBL. The active complex formed by this association causes cleavage and activation of C4 and C2. The MASP-1 and -2 proteins have structural similarity to C1r and C1s and mimic their activities. This means of activating the C2–C4 components to form a C5 convertase without need for specific antibody binding represents an important innate defense mechanism comparable to the alternative pathway, but utilizing the elements of the classical pathway except for the C1 proteins. REGULATION OF THE COMPLEMENT SYSTEM The complement system are capable of attacking host cells as well as foreign cells and microorganisms, elaborate regulatory mechanisms have evolved to restrict complement activity to designated targets. A general mechanism of regulation in all complement pathways is the inclusion of highly labile components that undergo spontaneous inactivation if they are not stabilized by reaction with other components. In addition, a series of regulatory proteins can inactivate various complement components . For example, the glycoprotein C1 inhibitor (C1Inh) can form a complex with C1r2s2, causing it to dissociate from C1q and preventing further activation of C4 or C2. The reaction catalyzed by the C3 convertase enzymes of the classical, lectin, and alternative pathways is the major amplification step in complement activation, generating hundreds of molecules of C3b. The C3b generated by these enzymes has the potential to bind to nearby cells, mediating damage to the healthy cells by causing their opsonization by phagocytic cells bearing C3b receptors or by induction of the membraneattack complex. Damage to normal host cells is prevented because C3b undergoes spontaneous hydrolysis by the time it has diffused 40 nm away from the C4_b2_a or C3_bB_bconvertase enzymes, so that it can no longer bind to its target site. The potential destruction of healthy host cells by C3b is further limited by a family of related proteins that regulate C3 convertase activity in the classical and alternative pathways. These regulatory proteins all contain repeating amino acid sequences (or motifs) of about 60 residues, termed short consensus repeats (SCRs). All these proteins are encoded at a single location on chromosome 1 in humans, known as the regulators of complementactivation (RCA) gene cluster. In the classical and lectin pathways, three structurally distinct RCA proteins act similarly to prevent assembly of C3 convertase). These regulatory proteins include soluble C4b-binding protein (C4bBP) and two membranes- bound proteins, complement receptor type 1 (CR1) and membrane cofactor protein (MCP). Each of these regulatory proteins binds to C4b and prevents its association with C2a. Once C4bBP, CR1, or MCP is bound to C4b, another regulatory protein, factor I, cleaves the C4b in to bound C4d and soluble C4c). A similar regulatory sequence operates to prevent assembly of the C3 convertase C3_bB_b in the alternative pathway. In this case CR1,MCP, or a regulatory component called factor H binds to C3b and prevents its association with factor B (Figure 13-9a(4)). A serum protein called S protein can bind to C5b67, inducing a hydrophilic transition and thereby preventing insertion of C5b67 into the membrane of nearby cells CYTOKINES Properties of Cytokines: Cytokines are low-molecularweight regulatory proteins or glycoproteins secreted by white blood cells and various other cells in the body in response to a number of stimuli.Cytokines bind to specific receptors on the membrane of target cells, triggering signal-transduction pathways that ultimately alter gene expression in the target cells (Figure 12-1a).The activity of cytokines was first recognized in the mid- 1960s. The susceptibility of the target cell to a particular cytokine is determined by the presence of specific membrane receptors. In general, the cytokines and their receptors exhibit very high affinity for each other. A particular cytokine may bind to receptors on the membrane of the same cell that secreted it, exerting autocrine action; it may bind to receptors on a target cell in close proximity to the producer cell, exerting paracrine action; in a few cases, it may bind to target cells in distant parts of the body, exerting endocrine action. For example, cytokines produced by activated TH cells can influence the activity of B cells, T C cells, natural killer cells, macrophages, granulocytes, and hematopoietic stem cells, thereby activating an entire network of interacting cells. Cytokines exhibit the attributes of pleiotropy, redundancy, synergy, antagonism, and cascade induction, which permit them to regulate cellular activity in a coordinated, interactive way. Many cytokines are referred to as interleukins, a name indicating that they are secreted by some leukocytes and act upon other leukocytes. Interleukins 1–25 have been identified. Cytokines Belong to Four Structural Families: Once the genes encoding various cytokines had been cloned, sufficient quantities of purified preparations became available for detailed studies on their structure and function. Cytokines generally have a molecular mass of less than 30 kDa. Structural studies have shown that the cytokines share a similar polypeptide fold, with four _-helical regions (A–D) in which the first and second helices and the third and fourth helices run roughly parallel to one another and are connected by loops. Cytokines Have Numerous Biological Functions: Although a variety of cells can secrete cytokines, the two principal producers are the TH cell and the macrophage. Cytokines released from these two cell types activate an entire network of interacting cells (Figure 12-5). Among the numerous physiologic responses that require cytokine involvement are development of cellular and humoral immune responses, induction of the inflammatory response, regulation of hematopoiesis, control of cellular proliferation and differentiation, and the healing of wounds. Although the immune response to a specific antigen may include the production of cytokines, it is important to remember that cytokines act in an antigen-nonspecific manner. TRANSPLANTATION Transplantation as the term used in immunology, refers to the act of transferring cells, tissues, or organs from one site to another. Many diseases can be cured by implantation of a healthy organ, tissue, or cells (a graft) from one individual (the donor) to another in need of the transplant. The seriousness of the donor organ shortage is reflected in the fact that, as of November 2000, an estimated 73,000 patients in the United States were on the waiting list for an organ transplantation. The immune system has evolved elaborate and effective mechanisms to protect the organism from attack by foreign agents, and these same mechanisms cause rejection of grafts from anyone who is not genetically identical to the recipient. Alexis Carrel reported the first systematic study of transplantation in 1908; he interchanged both kidneys in a series of nine cats. Some of those receiving kidneys from other cats maintained urinary output for up to 25 days. Although all the cats eventually died, the experiment established that a transplanted organ could carry out its normal function in the recipient. The first human kidney transplant, attempted in 1935 by a Russian surgeon, failed because there was a mismatch of blood types between donor and recipient. This incompatibility caused almost immediate rejection of the kidney, and the patient died without establishing renal function. The rapid immune response experienced here, termed hyperacute rejection, is mediated by antibodies. The first successful human kidney transplant, which was between identical twins, was accomplished in Boston in 1954. Today, kidney, pancreas, heart, lung, liver, bone-marrow, and cornea transplantations are performed among non identical individuals with ever increasing frequency and success. A variety of immunosuppressive agents can aid in the survival of the transplants, including drugs and specific antibodies developed to reduce the immunologic attack on grafts, but the majority of these agents have an overall immunosuppressive effect. New methods of inducing specific tolerance to the graft without suppressing other immune responses are being developed and promise longer survival of transplants without compromise of host immunity. IMMUNOLOGIC BASIS OF GRAFT REJECTION The degree of immune response to a graft varies with the type of graft. The following terms are used to denote different types of transplants: Autograft is self-tissue transferred from one body site to another in the same individual. Transferring healthy skin to a burned area in burn patients and use of healthy blood vessels to replace blocked coronary arteries are examples of frequently used autografts. Isograft is tissue transferred between genetically identical individuals. In inbred strains of mice, an isograft can be performed from one mouse to another syngeneic mouse. In humans, an isograft can be performed between genetically identical (monozygotic) twins. Allograft is tissue transferred between genetically different members of the same species. In mice, an allograft is performed by transferring tissue or an organ from one strain to another. In humans, organ grafts from one individual to another are allografts unless the donor and recipient are identical twins. Xenograft is tissue transferred between different species (e.g., the graft of a baboon heart into a human). Because of significant shortages in donated organs, raising animals for the specific purpose of serving as organ donors for humans is under serious consideration. Autografts and isografts are usually accepted, owing to the genetic identity between graft and host. Obviously, xenografts exhibit the greatest genetic dissimilarity and therefore show a vigorous graft rejection. TRANSPLANTATION ANTIGEN: Tissues that are antigenically similar are said to be histocompatible; such tissues do not induce an immunologic response that leads to tissue rejection. Tissues that display significant antigenic differences are histoincompatible and induce an immune response that leads to tissue rejection. The MHC in mice is called as the H-2 complex and in humans it is called as HLA complex. HLA Typing: Since differences in blood group and major histocompatibility antigens are responsible for the most intense graft-rejection reactions, various tissue-typing procedures to identify these antigens have been developed to screen potential donor and recipient cells. Initially, donor and recipient are screened for ABO blood-group compatibility. The bloodgroup antigens are expressed on RBCs, epithelial cells, and endothelial cells. Antibodies produced in the recipient to any of these antigens that are present on transplanted tissue will induce antibodymediated complement lysis of the incompatible donor cells. HLA typing of potential donors and a recipient can be accomplished with a microcytotoxicity test (Figure 21-4a, b). In this test, white blood cells from the potential donors and recipient are distributed into a series of wells on a microtiter plate, and then antibodies specific for various class I and class II MHC alleles are added to different wells. After incubation, complement is added to the wells, and cytotoxicity is assessed by the uptake or exclusion of various dyes (e.g., trypan blue or eosin Y) by the cells. If the white blood cells express the MHC allele for which a particular monoclonal antibody is specific, then the cells will be lysed upon addition of complement, and these dead cells will take up a dye such as trypan blue. HLA typing based on antibody-mediated microcytotoxicity can thus indicate the presence or absence of various MHC alleles. Even when a fully HLA-compatible donor is not available, transplantation may be successful. In this situation, a one-way mixed-lymphocyte reaction (MLR) can be used to quantify the degree of class II MHC compatibility between potential donors and a recipient (Figure 21-4c). Lymphocytes from a potential donor that have been x-irradiated or treated with mitomycin C serve as the stimulator cells, and lymphocytes from the recipient serve as responder cells. Proliferation of the recipient T cells, which indicates T-cell activation, is measured by the uptake of [3H]thymidine into cell DNA. The greater the class II MHC differences between the donor and recipient cells, the more [3H]thymidine uptake will be observed in an MLR assay. Intense proliferation of the recipient lymphocytes indicates a poor prognosis for graft survival. The advantage of the MLR over microcytotoxicity typing is that it gives a better indication of the degree of TH-cell activation generated in response to the class II MHC antigens of the potential graft. The disadvantage of the MLR is that it takes several days to run the assay. If the potential donor is a cadaver, for example, it is not possible to wait for the results of the MLR, because the organ must be used soon after removalfrom the cadaver. In that case, the microcytotoxicity test, which can be performed within a few hours, must be relied on. HLA matching is most important for kidney and bonemarrow transplants; liver and heart transplants may survive with greater mismatching. MHC identity of donor and host is not the sole factor determining tissue acceptance. When tissue is transplanted between genetically different individuals, even if their MHC antigens are identical, the transplanted tissue can be rejected because of differences at various minor histocompatibility loci. The tissue rejection induced by minor histocompatibility differences is usually less vigorous than that induced by major histocompatibility differences. Still, reaction to these minor tissue differences often results in graft rejection. For this reason, successful transplantation even between HLA-identical individuals requires some degree of immune suppression. MECHANISM OF GRAFT REJECTION: The process of graft rejection can be divided into two stages: (1) A sensitization phase, in which antigen-reactive lymphocytes of the recipient proliferate in response to allo- antigens on the graft. (2) An effector stage, in which immune destruction of the graft takes place. SENSITIZATION STAGE: During the sensitization phase, CD4+ and CD8+ T cells recognize alloantigens expressed on cells of the foreign graft and proliferate in response. Both major and minor histocompatibility alloantigens can be recognized. The response to major histocompatibility antigens involves recognition of both the donor MHC molecule and an associated peptide ligand in the cleft of the MHC molecule. The peptides present in the groove of allogeneic class I MHC molecules are derived from proteins synthesized within the allogeneic cell. The peptides present in the groove of allogeneic class II MHC molecules are generally proteins taken up and processed through the endocytic pathway of the allogeneic antigen-presenting cell. Depending on the tissue, different populations of cells within a graft may function as APCs. Because dendritic cells are found in most tissues and because they constitutively express high levels of class II MHC molecules, dendritic cells generally serve as the major APC in grafts. APCs of host origin can also migrate into a graft and endocytose the foreign alloantigens (both major and minor histocompatibility molecules) and present them as processed peptides together with self-MHC molecules. In some organ and tissue grafts (e.g., grafts of kidney, thymus, and pancreatic islets), a population of donor APCs called passenger leukocytes has been shown to migrate from the graft to the regional lymph nodes. These passenger leukocytes are dendritic cells, which express high levels of class II MHC molecules (together with normal levels of class I MHC molecules) and are widespread in mammalian tissues, with the chief exception of the brain. Because passenger leukocytes express the allogeneic MHC antigens of the donor graft, they are recognized as foreign and therefore can stimulate immune activation of T lymphocytes in the lymph node. Recognition of the alloantigens expressed on the cells of a graft induces vigorous T-cell proliferation in the host. Both dendritic cells and vascular endothelial cells from an allogeneic graft induce host T-cell proliferation. The major proliferating cell is the CD4+ T cell, which recognizes class II alloantigens directly or alloantigen peptides presented by host antigen-presenting cells. This amplified population of activated TH cells is thought to play a central role in inducing the various effector mechanisms of allograft rejection. EFFECTOR STAGE A variety of effector mechanisms participate in allograft rejection (Figure 21-6). The most common are cell-mediated reactions involving delayed-type hypersensitivity and CTLmediated cytotoxicity; less common mechanisms are antibodyplus- complement lysis and destruction by antibody-dependent cell-mediated cytotoxicity (ADCC). Graft rejection shows Sensitization and Memory: The rate of allograft rejection varies according to the tissue involved. In general, skin grafts are rejected faster than other tissues such as kidney or heart. If an inbred mouse of strain A is grafted with skin from strain B, primary graft rejection, known as first-set rejection, occurs (Figure 21-1b). The skin first becomes revascularized between days 3 and 7; as the reaction develops, the vascularized transplant becomes infiltrated with lymphocytes, monocytes, neutrophils, and other inflammatory cells. There is decreased vascularization of the transplanted tissue by 7–10 days, visible necrosis by 10 days, and complete rejection by 12– 14 days. Immunologic memory is demonstrated when a second strain-B graft is transferred to a previously grafted strain-A mouse. In this case, a graft-rejection reaction develops more quickly, with complete rejection occurring within 5–6 days; this secondary response is designated second-set rejection. TUMOR IMMUNOLOGY From an immunologic perspective, cancer cells can be viewed as altered self-cells that have escaped normal growth-regulating mechanisms. The immune responses that develop to cancer cells, as well as the methods by which cancers manage to evade those responses, are then described. In most organs and tissues of a mature animal, a balance is usually maintained between cell renewal and cell death. The various types of mature cells in the body have a given life span; as these cells die a new cells are generated by the proliferation and differentiation of various types of stem cells. Under normal circumstances, the production of new cells is regulated so that the number of any particular type of cell remains constant. Occasionally, though, cells arise that no longer respond to normal growthcontrol mechanisms. These cells give rise to clones of cells that can expand to a considerable size, producing a tumor, or neoplasm. A tumor that is not capable of indefinite growth and does not invade the healthy surrounding tissue extensively is benign. A tumor that continues to grow and becomes progressively invasive is malignant; the term cancer refers specifically to a malignant tumor. In addition to uncontrolled growth, malignant tumors exhibit metastasis; in this process, small clusters of cancerous cells dislodge from a tumor, invade the blood or lymphatic vessels, and are carried to other tissues, where they continue to proliferate. In this way a primary tumor at one site can give rise to a secondary tumor at another site (Figure 22-1). Types of Tumor (or) Cancer: Malignant tumors or cancers are classified according to the embryonic origin of the tissue from which the tumor is derived. Most (>80%) are carcinomas, tumors that arise from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. The majority of cancers of the colon, breast, prostate, and lung are carcinomas. The leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow and account for about 9% of cancer incidence in the United States. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Sarcomas, which arise less frequently are derived from mesodermal connective tissues such as bone, fat, and cartilage. Causes of Tumor: Transformation-Treatment of normal cultured cells with chemical carcinogens, irradiation, and certain viruses can alter their morphology and growth properties. In some cases this process, referred to as transformation, makes the cells able to produce tumors when they are injected into animals. Such cells are said to have undergone malignant transformation. Chemical Agents- Various chemical agents (e.g.,DNA-alkylating reagents) and physical agents (e.g., ultraviolet light and ionizing radiation) that cause mutations have been shown to induce transformation. Methylcholanthrene and ultraviolet light are two carcinogens that have been used extensively to generate lines of tumor cells Viruses- A number of DNA and RNA viruses have been shown to induce malignant transformation. Two of the best-studied are SV40 and polyoma. Most RNA viruses replicate in the cytosol and do not induce malignant transformation. The exceptions are retroviruses, which transcribe their RNA into DNA by means of a reverse-transcriptase enzyme and then integrate the transcript into the host’s DNA. This process is similar in the cytopathic retroviruses such as HIV-1 and HIV-2 and in the transforming retroviruses, which induce changes in the host cell that lead to malignant transformation. In some cases, retrovirusinduced transformation is related to the presence of oncogenes, or “cancer genes,” carried by the retrovirus. One of the best-studied transforming retroviruses is the Rous sarcoma virus. Oncogenes-They are tumor causing genes. Oncogenes and tumor suppressor genes have been shown to play an important role in this process, by regulating either cellular proliferation or cell death. TUMOR ANTIGENS: The subdiscipline of tumor immunology involves the study of antigens on tumor cells and the immune response to these antigens. Two types of tumor antigens have been identified on tumor cells: tumor-specific transplantation antigens (TSTAs) and tumor-associated transplantation antigens. Tumor-specific antigens are unique to tumor cells and do not occur on normal cells in the body. Tumor-associated antigens, which are not unique to tumor cells, may be proteins that are expressed on normal cells during fetal development when the immune system is immature and unable to respond but that normally are not expressed in the adult. Tumor specific antigen (e.g) Chemically or Physically induced Tumor antigens, viral antigens. Tumor associated antigen (e.g) 1)Oncofetal tumor antigen: Oncofetal tumor antigens, as the name implies, are found not only on cancerous cells but also on normal fetal cells. These antigens appear early in embryonic development, before the immune system acquires immunocompetence; if these antigens appear later on cancer cells, they are recognized as nonself and induce an immunologic response. 2)Oncogene proteins as tumor antigen: A number of tumors have been shown to express tumorassociated antigens encoded by cellular oncogenes. These antigens are also present in normal cells encoded by the corresponding proto-oncogene. In many cases, there is no qualitative difference between the oncogene and proto-oncogene products; instead, the increased levels of the oncogene product can be recognized by the immune system. IMMUNE RESPONSE TO TUMOR: Humoral And Cell-Mediated Immune Response: In experimental animals, tumor antigens can be shown to induce both humoral and cell-mediated immune responses that result in the destruction of the tumor cells. In general, the cell-mediated response appears to play the major role. CTLS Tumor Specific: A number of tumors have been shown to induce tumor-specific CTLs that recognize tumor antigens presented by class I MHC on the tumor cells. However as discussed below, expression of class I MHC molecules are decreased in a number of tumors, thereby limiting the role of specific CTLs in their destruction. Role of NK cells: The recognition of tumor cells by NK cells is not MHC restricted. Thus, the activity of these cells is not compromised by the decreased MHC expression exhibited by some tumor cells. In some cases, Fc receptors on NK cells can bind to antibody-coated tumor cells, leading to ADCC. Macrophages: Numerous observations indicate that activated macrophages also play a significant role in the immune response to tumors. For example, macrophages are often observed to cluster around tumors, and their presence is often correlated with tumor regression. IMMUNE SURVEILLANCE MECHANISM: The immune surveillance theory was first conceptualized in the early 1900s by Paul Ehrlich.He suggested that cancer cells frequently arise in the body but are recognized as foreign and eliminated by the immune system. Some 50 years later, Lewis Thomas suggested that the cell-mediated branch of the immune system had evolved to patrol the body and eliminate cancer cells.According to these concepts; tumors arise only if cancer cells are able to escape immune surveillance, either by reducing their expression of tumor antigens or by impairment in the immune response to these cells. Among the early observations that seemed to support the immune surveillance theory was the increased incidence of cancer in transplantation patients on immunosuppressive drugs. Other findings, however, were difficult to reconcile with this theory. Nude mice, for example, lack a thymus and consequently lack functional T cells. According to the immune surveillance theory, these mice should show an increase in cancer, instead, nude mice are no more susceptible to cancer than other mice. Furthermore, although individuals on immunosuppressive drugs do show an increased incidence of cancers of the immune system, other common cancers (e.g., lung, breast, and colon cancer) are not increased in these individuals, contrary to what the theory predicts. One possible explanation for the selective increase in immune system cancers is that the immunosuppressive agents themselves may exert a direct carcinogenic effect on immune cells. Experimental data concerning the effect of tumor-cell dosage on the ability of the immune system to respond also are incompatible with the immune surveillance theory. For example, animals injected with very low or very high doses of tumor cells develop tumors, whereas those injected with intermediate doses do not. The mechanism by which a low dose of tumor cells “sneaks through” is difficult to reconcile with the immune surveillance theory. Finally, this theory assumes that cancer cells and normal cells exhibit qualitative antigen differences. In fact, as stated earlier, many types of tumors do not express tumor-specific antigens, and any immune response that develops must be induced by quantitative differences in antigen expression by normal cells and tumor cells. However, tumors induced by viruses would be expected to express some antigens encoded by the viral genome. These antigens are qualitatively different from those expressed by normal tissues and would be expected to attract the attention of the immune system. In fact, there are many examples of specific immune responses to virally induced tumors. Nevertheless, apart from tumors caused by viruses, the basic concept of the immune surveillance theory—that malignant tumors arise only if the immune system is somehow impaired or if the tumor cells lose their immunogenicity, enabling them to escape immune surveillance—at this time remains unproved. In spite of this, it is clear that an immune response can be generated to tumor cells, and therapeutic approaches aimed at increasing that response may serve as a defense against malignant cells. IMMUNOLOGIC ENHANCEMENT OF TUMOR GROWTH: Although the immune system clearly can respond to tumor cells, the fact that so many individuals die each year from cancer suggests that the immune response to tumor cells is often ineffective. Following the discovery that antibodies could be produced to tumorspecific antigens, attempts were made to protect animals against tumor growth by active immunization with tumor antigens or by passive immunization with antitumor antibodies. Much to the surprise of the researchers, these immunizations did not protect against tumor growth; in many cases, they actually enhanced growth of the tumor. Modulation of Tumor antigens: Certain tumor-specific antigens have been observed to disappear from the surface of tumor cells in the presence of serum antibody and then to reappear after the antibody is no longer present. This phenomenon, called antigenic modulation, is readily observed when leukemic T cells are injected into mice previously immunized with a leukemic T-cell antigen Reduction in Class I MHC molecule: Since CD8+ CTLs recognize only antigen associated with class I MHC molecules, any alteration in the expression of class I MHC molecules on tumor cells may exert a profound effect on the CTL-mediated immune response. Malignant transformation of cells is often associated with a reduction (or even a complete loss) of class I MHC molecules, and a number of tumors have been shown to express decreased levels of class I MHC molecules. ENZYME-LINKED IMMUNOSORBENT ASSAY Enzyme-linked immunosorbent assay, commonly known as ELISA (or EIA), is similar in principle to RIA but depends on an enzyme rather than a radioactive label. An enzyme conjugated with an antibody reacts with a colorless substrate to generate a colored reaction product. Such a substrate is called a chromogenic substrate. A number of enzymes have been employed for ELISA, including alkaline phosphatase, horseradish peroxidase, and galactosidase. These assays approach the sensitivity of RIAs and have the advantage of being safer and less costly. There Are Numerous Variants of ELISA: A number of variations of ELISA have been developed, allowing qualitative detection or quantitative measurement of either antigen or antibody. Each type of ELISA can be used qualitatively to detect the presence of antibody or antigen. Alternatively, a standard curve based on known concentrations of antibody or antigen is prepared, from which the unknown concentration of a sample can be determined. INDIRECT ELISA: Antibody can be detected or quantitatively determined with an indirect ELISA (Figure 6-10a). Serum or some other sample containing primary antibody (Ab1) is added to an antigen- coated microtiter well and allowed to react with the antigen attached to the well. After any free Ab1 is washed away, the presence of antibody bound to the antigen is detected by adding an enzyme-conjugated secondary anti-isotype antibody (Ab2), which binds to the primary antibody. Any free Ab2 then is washed away, and a substrate for the enzyme is added. The amount of colored reaction product that forms is measured by specialized spectrophotometric plate readers, which can measure the absorbance of all of the wells of a 96-well plate in seconds. Indirect ELISA is the method of choice to detect the presence of serum antibodies against human immunodeficiency virus (HIV), the causative agent of AIDS. In this assay, recombinant envelope and core proteins of HIV are adsorbed as solid-phase antigens to microtiter wells. Individuals infected with HIV will produce serum antibodies to epitopes on these viral proteins. Generally, serum antibodies to HIV can be detected by indirect ELISA within 6 weeks of infection. SANDWICH ELISA: Antigen can be detected or measured by a sandwich ELISA (Figure 6-10b). In this technique, the antibody (rather than the antigen) is immobilized on a microtiter well. A sample containing antigen is added and allowed to react with the immobilized antibody. After the well is washed, a second enzyme- linked antibody specific for a different epitope on the antigen is added and allowed to react with the bound antigen. After any free second antibody is removed by washing, substrate is added, and the colored reaction product is measured. COMPETITIVE ELISA Another variation for measuring amounts of antigen is competitive ELISA (Figure 610c). In this technique, antibody is first incubated in solution with a sample containing antigen. The antigen-antibody mixture is then added to an antigencoated microtiter well. The more antigen present in the sample, the less free antibody will be available to bind to the antigen-coated well. Addition of an enzyme-conjugated secondary antibody (Ab2) specific for the isotype of the primary antibody can be used to determine the amount of primary antibody bound to the well as in an indirect ELISA. In the competitive assay, however, the higher the concentration of antigen in the original sample, the lower the absorbance. IMMUNOFLUORESCENCE In 1944, Albert Coons showed that antibodies could be labeled with molecules that have the property of fluorescence. Fluorescent molecules absorb light of one wavelength (excitation) and emit light of another wavelength (emission). If antibody molecules are tagged with a fluorescent dye, or fluorochrome, immune complexes containing these fluorescently labeled antibodies (FA) can be detected by colored light emission when excited by light of the appropriate wavelength. Antibody molecules bound to antigens in cells or tissue sections can similarly be visualized. The emitted light can be viewed with a fluorescence microscope,which is equipped with a UV light source. In this technique, known as Immunofluorescence or Fluorescent antibody technique, fluorescent compounds such as fluorescein and rhodamine are in common use, but other highly fluorescent substances are also routinely used, such as phycoerythrin, an intensely colored and highly fluorescent pigment obtained from algae. These molecules can be conjugated to the Fc region of an antibody molecule without affecting the specificity of the antibody. Each of the fluorochromes below absorbs light at one wavelength and emits light at a longer wavelength: Fluorescein- an organic dye that is the most widely used label for immunofluorescence procedures, absorbs blue light (490 nm) and emits an intense yellow-green fluorescence (517 nm). Rhodamine- another organic dye, absorbs in the yellow-green range (515 nm) and emits a deep red fluorescence (546 nm). Because it emits fluorescence at a longer wavelength than fluorescein, it can be used in two-color immunofluorescence assays. An antibody specific to one determinant is labeled with fluorescein, and an antibody recognizing a different antigen is labeled with rhodamine. Phycoerythrin-is an efficient absorber of light (~30-fold greater than fluorescein) and a brilliant emitter of red fluorescence, stimulating its wide use as a label for immunofluorescence. Fluorescent-antibody staining of cell membrane molecules or tissue sections can be direct or indirect (Figure 6-14). In direct staining, the specific antibody (the primary antibody) is directly conjugated with fluorescein; In indirect staining, the primary antibody is unlabeled and is detected with an additional fluorochrome-labeled reagent. A number of reagents have been developed for indirect staining. APPLICATION: 1. Immunofluorescence has been applied to identify a number of subpopulations of lymphocytes, notably the CD4_ and CD8_ T-cell subpopulations. 2. The technique is also suitable for identifying bacterial species. 3. For detecting Ag-Ab complexes in autoimmune disease. 4. For detecting complement components in tissues, and localizing hormones and other cellular products stained in situ. 5. Major application of the fluorescent-antibody technique is the localization of antigens in tissue sections or in subcellular compartments. RADIO IMMUNO ASSAY One of the most sensitive techniques for detecting antigen or antibody is radioimmunoassay (RIA). The technique was first developed in 1960 by two endocrinologists, S. A. Berson and Rosalyn Yalow, to determine levels of insulin–anti-insulin complexes in diabetics. It’s used for measuring hormones, serum proteins, drugs, and vitamins at concentrations of 0.001 micrograms per milliliter or less. In 1977, some years after Berson’s death, the significance of the technique was acknowledged by the award of a Nobel Prize to Yalow. The principle of RIA involves competitive binding of radiolabeled antigen and unlabeled antigen to a high-affinity antibody. The labeled antigen is mixed with antibody at a concentration that saturates the antigenbinding sites of the antibody. Then test samples of unlabeled antigen of unknown concentration are added in larger amounts. The antibody does not distinguish labeled from unlabeled antigen, so the two kinds of antigen compete for available binding sites on the antibody. As the concentration of unlabeled antigen increases, more labeled antigen will be displaced from the binding sites. The decrease in the amount of radiolabeled antigen bound to specific antibody in the presence of the test sample is measured in order to determine the amount of antigen present in the test sample. The antigen is generally labeled with a gamma- emitting isotope such as 125I, but beta-emitting isotopes such as tritium (3H) are also routinely used as labels. The radiolabeled antigen is part of the assay mixture; the test sample may be a complex mixture, such as serum or other body fluids, that contains the unlabeled antigen. The first step in setting up an RIA is to determine the amount of antibody needed to bind 50%– 70% of a fixed quantity of radioactive antigen (Ag) in the assay mixture. This ratio of antibody to Ag is chosen to ensure that the number of epitopes presented by the labeled antigen always exceeds the total number of antibody binding sites. Consequently, unlabeled antigen added to the sample mixture will compete with radiolabeled antigen for the limited supply of antibody. Even a small amount of unlabeled antigen added to the assay mixture of labeled antigen and antibody will cause a decrease in the amount of radioactive antigen bound, and this decrease will be proportional to the amount of unlabeled antigen added. To determine the amount of labeled antigen bound, the Ag-Ab complex is precipitated to separate it from free antigen (antigen not bound to Ab), and the radioactivity in the precipitate is measured. A standard curve can be generated using unlabeled antigen samples of known concentration (in place of the test sample), and from this plot the amount of antigen in the test mixture may be precisely determined. Several methods have been developed for separating the bound antigen from the free antigen in RIA. One method involves precipitating the Ag-Ab complex with a secondary anti-isotype antiserum. After removal of the complex by either of these methods, the amount of free labeled antigen remaining in the supernatant can be measured in a radiation counter; subtracting this value from the total amount of labeled antigen added yields the amount of labeled antigen bound.Various solid-phase RIAs have been developed that make it easier to separate the Ag-Ab complex from the unbound antigen. In some cases, the antibody is covalently crosslinked to Sepharose beads. The amount of radiolabeled antigen bound to the beads can be measured after the beads have been centrifuged and washed.Alternatively, the antibody can be immobilized on polystyrene or polyvinylchloride wells and the amount of free labeled antigen in the supernatant can be determined in a radiation counter. In another approach, the antibody is immobilized on the walls of microtiter wells and the amount of bound antigen determined. For example, a microtiter RIA has been widely used to screen for the presence of the hepatitisB virus (Figure 6-9). RIA screening of donor blood has sharply reduced the incidence of hepatitis B infections in recipients of blood transfusion. IMMUNOLOGY-Important questions 1. Inflammatory response 2. Complement fixation test 3. Phagocytosis 4. Immunoflurescense 5. Primary and secondary immune response 6. Cascade regulation 7. Interferon & Interleukins 8. Precipitation test 9. Agglutination test 10. Write about Humoral and cell mediated immune response. 11. Write in detail about the different pathways of complement activation 12. Give the principle and procedures of immunoelectrophoresis 13. Describe the various antigen-antibody reactions and their characteristics 14. How B cells & T cells are get activated 15. Write about the structure and function of lymphoid organs with neat Diagram 17. Describe hypersensitive reactions and its types 18. Write in detail about the cells involved in the immune response 19. Explain the innate and Acquired immune response of our body 20. Define antibody? Explain the different types of immunoglobulins with neat labeled sketch 21. Write about monoclonal antibody and its application 22. Define Hematopoiesis? Development of B and t lymphocytes 23. Give detailed notes on immunoglobulin Gene expression 24. Write about primary and secondary Immunodeficiency disorderes. 25. Write about auto immune diseases 26. Write about transplantation and mechanism of graft rejection 27. Give detailed notes on Tumor immunology and different types of ELISA 28. Write about the principle and application of RIA 29. Give detailed notes on HLA typing 30. Write about Cytokine structure and its function 31. Explain Tolerance and Suppression in Immunoregulation 32. Describe Plasma and Memory cells 33. Explain antigen its types and application 34. Write in detail about cell mediated immune response 35. Write about the historical developments in Immunology All the Best