Immunology Unit 2

1- Latent or lag phase:

After initial exposure to an antigen, a period of 1 to 2 weeks follows before antibody is detectable in the serum. The actual length of time depends on the species immunized, the nature of the antigen used to stimulate the response, and other factors . The length of the latent period is also greatly dependent on the sensitivity of the assay used to measure the product of the response. The latent period includes the time taken for T and B cells to make contact with the antigen, to proliferate, and to differentiate. B cells must also secrete antibody in sufficient quantity so that it can be detected in the serum. The less sensitive the assay used for detection of antibody is, the more antibody will be required for detection and the longer the apparent latent period will be.

secondary response

Although production of antibody after a priming contact with antigen may cease entirely within a few weeks, the immunized individual is left with a pool of long-lived memory cells capable of mounting a secondary response as well as any other future responses to the antigen. Experimentally, this memory response (also called anamnestic response) becomes apparent when a response is triggered by a second injection of the same antigen used to stimulate a primary response. After the second injection, the lag phase is considerably shorter and antibody may appear in less than half the time required for the primary response. The magnitude of antibody produced in these responses is much greater than that seen in the primary response with significantly higher concentrations of antibody detectable in the serum. The production of antibody may also continue for a longer period, with persistent levels remaining in serum months, or even years, later. There is a marked change in the type of antibody produced in the secondary response, as reflected in the appearance of different Ig classes with the same antigen specificity. This shift is known as class switching, with IgG antibodies appearing at higher concentrations and with greater persistence than IgM antibodies, which may be greatly reduced or disappear altogether. This may be also accompanied by the appearance of IgA and IgE. In addition, affinity maturation occurs, a phenomenon in which the average affinity (binding constant) of the antibodies for the antigen increases as the secondary response develops. The driving force for this increase in affinity may be a selection process during which B cells compete with free antibody to capture a decreasing amount of antigen. Thus, only those B cell clones with high-affinity Ig receptors on their surfaces will bind enough antigen to ensure that the B cells are triggered to differentiate into plasma cells. These plasma cells, which arise from preferentially selected B cells, synthesize this antibody with high affinity for antigen. The capacity to make a secondary response may persist for a long time (years in humans), and it provides an obvious selective advantage for an individual who survives the first contact with an invading pathogen. Establishment of this memory for generating a specific response is, of course, the purpose of public health immunization programs.

the immunoglobulin superfamily

The shared structural features of immunoglobulin HCs and LCs that include the immunoglobulin-fold domains are also seen in a large number of proteins. Most of these have been found to be membrane-bound glycoproteins. Because of this structural similarity, these proteins are classified as members of the immunoglobulin superfamily.

How T Cells Recognize Antigen: The Role of the Major Histocompatibility Complex Introduction

Antibodies play a critical role in the interaction with antigens outside cells, such as viruses or bacteria encountered in blood or at mucosal surfaces. However, many pathogens—particularly viruses, parasites, and some bacteria—invade host cells and live at least part of their life cycle inside them. Antibodies do not enter cells, so once a pathogen gains entry to a host cell, antibodies are ineffective at defending the host. The immune response to pathogens inside host cells is the domain of T cells and their products. T cells mount responses to "harmless" antigens—foreign agents that are not pathogenic but to which we need to respond and eliminate. Antibodies bind to all antigens (proteins, carbohydrates, nucleic acids, or lipids). T cells respond almost exclusively to proteins, or more precisely, small peptides derived from protein catabolism. Proteins are major constituents of pathogens and are also the products of viral infection. In addition, most other antigens are protein in nature. Thus, T cells play a critical role in the response to nearly all potentially harmful agents and the myriad of other antigens to which an individual is exposed. Because T cells deal with pathogens and antigens that infect or are taken into host cells, they use an antigen recognition system distinct from the one used by B cells. T cells interact with antigens expressed on the surface of host cells. However, like B cells, T cells express an antigen-specific receptor, the T-cell receptor (TCR).

Biologic properties of IgM 1- Complement Fixation

Because of its pentameric form, IgM is an excellent complement-fixing or complement-activating antibody Unlike other classes of immunoglobulins, a single molecule of IgM, upon binding to antigen with at least two of its Fab arms, can initiate activation of the classical pathway of complement, making it the most efficient immunoglobulin as an initiator of the complement-mediated lysis of microorganisms and other cells. IgM is the first isotype generated after immunization or infection. This fact together with the complements fixation role make IgM antibodies very important as providers of an early line of immunologic defense against bacterial infections.

Factors that affect the expression of both MHC class I and II molecules

Cytokines released during the response to infectious agents enhance the expression of MHC molecules: IFN α, β, and γ upregulate MHC class I expression, and IFN-γ upregulates MHC class II expression. As a consequence of upregulation, MHC class II expression is induced on cells such as fibroblasts and endothelial cells that do not normally express it, and increased on APCs. Induction and increased expression of MHC class I and II molecules thus enhance T-cell responses to infectious agents. Conversely, some virus infections and tumor development result in decreased expression of MHC molecules.

Representative protein members of the Ig superfamily

Each molecule contains the characteristic Ig-fold structure (loops) formed as a result of intrachain disulfide bonds and consisting of approximately 110 amino acids. These Ig-fold domains are believed to facilitate interactions between membrane proteins (e.g., CD4 molecules on helper T cells and class II major histocompatibility complex [MHC] molecules on antigen-presenting cells).

Structural features of IgA

IgA is found in the serum as a monomeric molecule. It is also the major Ig in external secretions such as saliva, mucus, sweat, gastric fluid, and tears as a dimeric molecule. It is the major immunoglobulin found in the colostrum of milk in nursing mothers, and it may provide the neonate with a major source of intestinal protection against pathogens during the first few weeks after birth. Consists of either two κ LCs or two λ LCs and two α HCs. The α chain is somewhat larger than the γ chain. The molecular weight of monomeric IgA is approximately 165,000 Da. Dimeric IgA has a molecular weight of 400,000 Da. The IgA class of Igs contains two subclasses: IgA1 (93%) and IgA2 (7%). It is interesting to note that if all production of IgA on mucosal surfaces (respiratory, gastrointestinal, and urinary tracts) is taken into account, IgA would be the major immunoglobulin in terms of quantity. Within mucous secretions, IgA exists as a dimer consisting of two four-chain units linked by the same joining (J) chain found in IgM molecules

Importance of IgE in Parasitic Infections and Hypersensitivity Reactions

IgE, also termed reaginic antibody, has a half-life in serum of 2 days, the shortest half-life of all classes of Igs. It is present in serum in the lowest concentration of all Igs. These low levels are due in part to a low rate of synthesis and to the unique ability of the Fc portion of IgE containing the extra CH domain to bind with very high affinity to receptors (Fcε receptors) found on mast cells and basophils. Once bound to these high-affinity receptors, IgE may be retained by these cells for weeks or months. When antigen reappears, it combines with the Fab portion of the IgE attached to these cells, causing it to be cross-linked. The cells become activated and release the contents of their granules: histamine, heparin, leukotrienes, and other pharmacologically active compounds that trigger the immediate hypersensitivity reactions. These reactions may be mild, as in the case of a mosquito bite, or severe, as in the case of bronchial asthma; they may even result in systemic anaphylaxis, which can cause death within minutes IgE is not an agglutinating or complement-activating antibody; nevertheless, it has a role in protection against certain parasites, such as helminths (worms) . This protection is achieved by activation of the same acute inflammatory response seen in a more pathologic form of immediate hypersensitivity responses. Elevated levels of IgE in serum have been shown to occur during infections with ascaris (a roundworm). Immunization with ascaris antigen induces the formation of IgE.

Neutralization of viruses

IgG antibody is an efficient virus-neutralizing antibody. One mechanism of neutralization is that in which the antibody binds with antigenic determinants present on various portions of the virus coat, among which is the region used by the virus for attachment to the target cell. Inhibition of viral attachment effectively arrests infection. Other antibodies are thought to inhibit viral penetration or shedding of the viral coat required for release of the viral DNA or RNA needed to induce infection.

Opsonization

IgG facilitates phagocytosis by opsonization. Within a week to 10 days of generating an antibody response to a particular pathogen, IgG antibodies bind to specific antigenic epitopes via their Fab portions. Once bound, the IgG Fc confers opsonizing property. Many phagocytic cells (macrophages and PMN phagocyes) bear receptors for the Fc portion of IgG. These cells adhere to the antibody-coated bacteria by their Fc receptors. The net effect is a zipper-like closure of the surface membrane of the phagocytic cell around the organism, as receptors for Fc and the Fc regions on the antibodies continue to combine, leading to the final engulfing and destruction of the microorganism

Activation of complement

IgG is important in classical activation of complement. When C1q is exposed to immune complexes of IgG-antigen, downstream components are activated as a result of proteolytic cleavage. This ultimately results in a MAC that causes lysis of the microbe or cell. Some components activated along the way are also opsonins (C3b). They bind to target antigens and direct phagocytes (which carry receptors specific for these opsonins) to focus their phagocytic activity on the target antigen. Other components from complement activation are chemotactic (they attract phagocytic cells). IgG complement activation has profound biologic effects on the host and on the target antigen, whether it is a live cell, a microorganism, or a tumor cell.

Structural Features of IgG

IgG is the predominant immunoglobulin in blood, lymph fluid, cerebrospinal fluid, and peritoneal fluid. The IgG molecule consists of two γ HCs of molecular weight approximately 50,000 Da each and two LCs (either κ or λ) of molecular weight approximately 25,000 Da each, held together by disulfide bonds. Thus, the IgG molecule has a molecular weight of approximately 150,000 Da. Electrophoretically, the IgG molecule is the least anodic of all serum proteins, and it migrates to the γ range of serum globulins, hence its earlier designation as γ-globulin or 7S immunoglobulin. Contains 4 subclasses in humans (IgG1, IgG2, IgG3, and IgG4) named in order of their abundance in serum (IgG1 being the most abundant). Except for their variable regions, all Igs within a class have ~ 90% homology in their amino acid sequences but only 60% homology between classes (e.g., IgG and IgA). Antiserum made in mice against human IgG may include antibodies against all IgG class members. Other antisera may be specific for determinants in only one subclass such as IgG2. This variation was first detected antigenically using antibodies against various γ chains. The IgG subclasses differ in their chemical properties and, more importantly, in their biologic properties. Represents about 15% of the total protein in the serum of human adults. IgG is distributed approximately equally between the intravascular and extravascular spaces. Except for IgG3, which has a rapid turnover (half-life is 7 days), the half-life of IgG is ~ 23 days (the longest of all Ig isotypes). Persistence in the serum makes IgG the most suitable for passive immunization by transfer of antibodies. As the serum concentration of IgG increases (multiple myeloma or after transfer of very high concentrations of IgG), the rate of catabolism of IgG increases, and its half-life decreases to 15-20 days or even less.Biologic Properties of IgG

Immobilization of bacteria

IgG molecules are efficient in immobilizing various motile bacteria. The reaction of antibodies specific for the flagella and cilia of certain microorganisms causes them to clump. Clumping arrests bacterial movement and prevents their ability to spread or invade tissue.

Agglutination and Formation of Precipitate

IgG molecules can cause the agglutination or clumping of particulate (insoluble) antigens such as microorganisms. The reaction of IgG with soluble, multivalent antigens can generate precipitates. Insoluble antigen-antibody complexes are easily phagocytized and destroyed by phagocytic cells. IgG molecules may be made to aggregate by: 1- Precipitation with alcohol (a method employed in the purification of IgG). 2- Heating at 56° C for 10 minutes (a method used to inactivate complement). Aggregated IgG can still combine with antigen. Many of the properties that are attributed to antigen-antibody complexes are exhibited by aggregated IgG (without antigen) such as attachment to phagocytic cells, as well as complement activation and other biologically active substances that may be harmful to the body. Such activation is due to the juxtaposition of Fc domains by the aggregation process in a way analogous to that produced by antigen-induced immune complex formation. It is therefore imperative that no aggregated IgG be present in passively administered IgG (e.g., gamma globulin preparations used to treat patients exposed to venomous snake bites, certain hepatitis viruses, etc.).

Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

IgG molecules play an important role in antibody-dependent, cell-mediated cytotoxicity (ADCC). In this form of cytotoxicity, the Fab portion of IgG binds with the target cell, whether it is a microorganism or a tumor cell, and the Fc portion binds with specific Fc receptors that are found on certain large granular lymphocytic cells called natural killer (NK) cells. By this mechanism, the IgG molecule focuses the killer cells on their target, and the killer cells destroy the target, not by phagocytosis but with the various toxic substances contained in cytoplasmic granules that they release.

Isohemagglutinins

IgM antibodies include the isohemagglutinins—the naturally occurring antibodies against the red blood cell antigens of the ABO blood groups. These antibodies are presumed to arise as a result of immunization by bacteria in the gastrointestinal and respiratory tracts, which bear determinants similar to the oligosaccharides of the ABO blood groups. Without prior immunization, people with the type O blood group have isohemagglutinins to the A and B antigens; those with the type A group have antibodies to the B antigens; and those with the B antigen have antibodies to the A antigen. Those of the AB group has neither anti-A nor anti-B Fortunately, the IgM isohemagglutinins do not pass through the placenta, so incompatibility of the ABO groups between mother and fetus poses no danger to the fetus. However, transfusion reactions, which arise as a result of ABO incompatibility, and in which the recipient's isohemagglutinins react with the donor's red blood cells, may have disastrous consequences.

Biologic properties of IgM

IgM is found predominantly in the intravascular spaces. The half-life of the IgM molecule is ~ 5 days. In contrast to IgG, the IgM antibodies are not very versatile; they are poor toxin-neutralizing antibodies, and they are not efficient in the neutralization of viruses. IgM in monomeric form is also found on the surface of mature B cells together with IgD, where it serves as an antigen-specific BCR. Once the B cell is activated by antigen following ligation of the BCR, it may undergo class switching and begin to secrete and express other membrane Ig isotypes (e.g., IgG)

MHC Class I and CD8+ T cells

MHC class I molecules interact with CD8, whose expression defines the T cell subset called CD8+ T cells. CD8+ T cell responses are restricted by MHC class I molecules. MHC class I molecules are expressed on all nucleated cells (thus, not on red blood cells), any of which may be infected by a pathogen (a virus, bacterium, or parasite). The main function of CD8+ T cells is to kill pathogen-infected host cells, as well as tumors and transplanted tissue. Thus, MHC class I molecules and CD8+ T cells play critical roles in the responses to pathogens that infect host cells. In addition to their interaction with CD8 expressed on CD8+ T cells, MHC class I molecules also interact with molecules expressed on natural killer (NK) cells. This interaction prevents NK cells from killing normal host cells.

MHC Class II and CD4+ T cells

MHC class II molecules interact with CD4, whose expression defines the T cell subset called CD4+ T cells. CD4+ T cell responses are restricted by MHC class II molecules. MHC class II molecules have a more limited distribution than MHC class I molecules: They are expressed constitutively (that is, under baseline conditions) only on antigen-presenting cells (APCs) but can be induced on other cell types. In humans, the principal APCs that express MHC class II are dendritic cells, macrophages, and B lymphocytes; thymic epithelial cells also express MHC class II molecules.In the absence of inducing factors, most cells (for example, liver and kidney tissue cells) express MHC class I but not MHC class II molecules; by contrast, APCs constitutively express both MHC class I and class II molecules.In response to activation, CD4+ T cells synthesize a vast array of cytokines, and hence cooperate with multiple types of cells, including helping B cells synthesize antibody. Thus, MHC class II molecules and CD4+ T cells play critical roles in the responses to agents—pathogens and antigens—that are taken into APCs.

MHC Role in Antigen Presentation to T cells Key functions of MHC molecules

MHC molecules have two key functions: (1) To selectively bind to peptides produced when proteins are processed inside cells of the host(2) To present peptides on the surface of a host cell to a T cell with the appropriate TCR The critical role played by MHC molecules in binding processed antigen and presenting it in T-cell responses is referred to as the MHC restriction of T-cell responses.MHC molecules also play a key role during the differentiation of T cells in the thymus Thus, MHC molecules play important interrelated roles in both the differentiation of immature T cells and in the responses of mature T cells. Multiple copies of each MHC molecule are expressed on the surface of a host cell, and each MHC molecule can bind many peptides (one peptide at a time). By binding to peptides inside the cell, MHC molecules "sample" the internal environment of host cells and present information on the cell surface that allows T cells to identify whether a particular host cell has been infected, or contains some foreign component. The combination of MHC molecule plus foreign peptide expressed on the surface of a host cell is a key signal to host T cells that they need to respond. T cells do not respond to host cells in the absence of foreign peptide or infectious agents inside the cells.

T cells and transplants

Other early studies of transplantation in mice indicated that T cells played an important role in the rejection response Taken together, these transplantation studies demonstrated an important but not well-understood connection between the MHC and T-cell responses. Because individuals do not normally undergo transplants, the function of the MHC in "everyday" T-cell responses became the focus of intense investigation.

MHC Role in Antigen Presentation to T cells Events that occur in a host cell after the entry of a protein antigen

Protein antigens are broken down (catabolized or processed) to peptides (linear fragments) of varying length. Some peptides bind to an MHC molecule inside the cell. This binding is selective (not all peptides bind to MHC). The peptide-MHC combination moves to the cell surface and is recognized at the cell surface by a T cell that expresses the "appropriate" or "correct" TCR—one of the billions of different TCRs the host can generate. The three critical components of T-cell recognition of antigen: peptide, an MHC molecule expressed on the surface of a host cell, and the TCR expressed on a T cell.

Biologic Properties of IgG

Recent studies have explained the prolonged survival of IgG relative to other serum proteins and why its half-life decreases at high concentrations. A saturable IgG protection receptor (FcRp; the Brambell receptor) binds to the Fc region of this isotype. FcRp in cellular endosomes selectively recycles endocytosed IgG back to the circulation following endocytosis of antigen-antibody immune complexes. This helps cleanse IgG antibody of antigen and harvest antigen for presentation without antibody destruction. High IgG levels saturate FcRp rendering catabolism of excess IgG indistinguishable from albumin or other Ig isotypes.

Antiviral activity of IgA

Secretory IgA is an efficient antiviral antibody. It prevents the viruses from entering host cells. In addition, secretory IgA is an efficient agglutinating antibody.

Biologic properties of IgA

Serum IgA, which has no known biologic function, has a half-life of 5.5 days. The IgA present in serum is predominantly monomeric (one four-chain unit) and has presumably been released before dimerization so that it fails to bind to the secretory component. Secretory IgA is very important biologically, but little is known of any function for serum IgA . Most IgA is present not in the serum, but in secretions such as tears, saliva, sweat, and mucus, where it serves an important biologic function as part of the mucosa-associated lymphoid tissue (MALT) IgA-secreting plasma cells synthesize the IgA molecules and the J chains, which form the dimers. Such plasma cells are located in the connective tissue called lamina propria that lies immediately below the basement membrane of many surface epithelia in the parotid (salivary) gland, along the gastrointestinal tract in the intestinal villi (in the gut), in tear glands, in the lactating breast (lactating mammary cells), or beneath bronchial mucosa (respiratory epithelia). When these dimeric molecules are released from plasma cells, they bind to the poly-Ig receptor and the complex undergoes transcytosis within vesicles across the cell. The receptor (expressed on the basal membranes of adjacent epithelial cells) transports the molecules via the epithelial cells and releases them into extracellular fluids (gut or bronchi). Release of IgA from the cell is facilitated by enzymatic cleavage of the poly-Ig receptor from the complex at the apical surface, leaving a large 70,000 Da pentameric fragment of the poly-Ig receptor (the secretory component) still attached to the Fc piece of the dimeric IgA molecule The secretory component protects the dimeric IgA (in the lumen of organs in contact with the external environment) from proteolytic cleavage. It also binds & transports pentameric IgM to mucosal surfaces in small amounts.

The Major Histocompatibility Complex and transplants

The term derives from transplantation research that started in the mid-twentieth century. Experiments provided insight into the rules governing acceptance or rejection of tissue transplants (histocompatibility) between different mice. Rapid transplant rejection was determined by a single gene, which was called the major histocompatibility gene. Later studies indicated that this gene was a complex (a set of closely linked genes inherited as a unit) & it became known as the major histocompatibility complex. We know now that every vertebrate species has an MHC containing multiple genes. The human MHC is known as HLA (human leukocyte antigen).

Immunoglobulin Heavy Chain Isotypes

The Ig HCs, whose constant regions are derived from Ig HC genes are designated with Greek letters. Therefore, the genes encoding these constant (C) regions responsible for the μ, δ, γ, α, and εHCs are called Cμ, Cδ, Cγ, Cα, and Cε, respectively. Any individual of a species makes all five Ig isotypes, in proportions characteristic of the species, but, just as the case with LCs, in any one antibody molecule both HCs are always identical (e.g., 2γ or 2ε, etc.). An antibody molecule of the IgG class could have the structure κ2γ2 with two identical kappa light chains and two identical gamma heavy chains. Alternatively, it could have the structure λ2γ2 with two identical lambda light chains and two identical γ heavy chains. An antibody of the IgE class could have the structure κ2ε2 or λ2ε2. Further characterization of these isotypes by specific antisera has led to the designation of several subclasses that have more subtle differences among themselves. Thus, the major class of human IgG can be subdivided into the subclasses IgG1, IgG2, IgG3, and IgG4. IgA has been divided similarly into two subclasses, IgA1 and IgA2. The subclasses differ from one another in numbers and arrangement of interchain disulfide bonds, as well as by alterations in other structural features. These alterations, in turn, produce some changes in functional properties.

Bactericidal activity of IgA

The IgA molecule does not contain receptors for complement, and thus IgA is not a complement-activating or complement-fixing immunoglobulin. Consequently, it does not induce complement-mediated bacterial lysis. However, IgA has been shown to possess bactericidal activity against Gram-negative organisms, but only in the presence of lysozyme, which is also present in the same secretions that contain secretory IgA.

Structural and Biologic properties of IgD

The IgD molecule consists of either two κ or two λ light chains and two δ heavy chains. IgD is present as a monomer with a molecular weight of 180,000 Da. No heavy chain allotypes or subclasses have been reported for the IgD molecule. IgD is present in serum in very low and variable amounts. Why? 1- IgD-secreting plasma cells are rare 2- IgD is highly susceptible to proteolytic degradation due to its long hinge region. 3- Following B-cell activation, transcription of the δ heavy chain protein is rapidly downregulated. IgD is co-expressed with IgM on the surface of mature B cells and, like IgM, functions as an antigen-specific BCR. During ontogeny of B cells, IgD expression lags behind that of IgM. Thus, IgD serves as a marker of the differentiation of B cells to a more mature form. Expression of membrane IgD correlates with the elimination of B cells that generate self-reactive antibodies. During development, the major biologic significance of IgD may be in silencing autoreactive B cells. In mature B cells, IgD serves as an antigen-binding surface Ig together with co-expressed IgM.

Passage through the Placenta and Absorption in Neonates

The IgG isotype (except for subclass IgG2) is the only class of Ig that can pass through the placenta, enabling the mother to transfer her immunity to the fetus. Placental transfer is facilitated by expression of an IgG protection receptor (FcRn) expressed on placental cells. FcRn was shown to be identical to the IgG protection receptor (FcRp) found in the cellular endosomes. At the third or fourth month of pregnancy there is a rapid increase in the concentration of IgG. This IgG must be of maternal origin, since the fetus is unable to synthesize Igs at this age. Then, during the fifth month of pregnancy, the fetus begins to synthesize IgM and trace amounts of IgA. The infant begins to synthesize its own IgG 3 or 4 months after birth, when the level of inherited maternal IgG drops as a result of catabolism (half-life ~ 23 days). The infection resistance of the fetus and the neonate is conferred by the mother's IgG. Passage across the placenta is mediated by the Fc portion. IgG Fab fragments do not cross the placenta. The IgG protection receptor (FcRn) on placental cells is transiently super expressed in the neonatal intestinal tissue. Absorption of maternal IgG contained in the colostrum of nursing mothers is achieved by binding to these high density receptors in intestinal tissue. FcRn are down regulated in intestinal tissue at 2 weeks of age. It is not until 3 or 4 months after birth, when the level of inherited maternal IgG drops as a result of catabolism (the half-life of IgG is 23 days), that the infant begins to synthesize its own IgG antibodies. While passage of IgG molecules across the placenta confers immunity to infection on the fetus, it may also be responsible for hemolytic disease of the newborn (erythroblastosis fetalis). This is caused by maternal antibodies to fetal red blood cells. The maternal IgG antibodies, produced by a previously sensitized (immunized) Rh− mother, to Rh antigen, pass across the placenta and attack the fetal red blood cells that express Rh antigens (Rh+).

Neutralization of toxins

The IgG molecule is an excellent antibody for the neutralization of toxins such as tetanus and botulinus, or for the inactivation of, for example, snake and scorpion venoms. Because of its ability to neutralize such poisons (mostly by blocking their active sites) and because of its long half-life, compared to that of other isotypes, the IgG molecule is the isotype of choice for passive immunization (i.e., gamma globulin injections) against toxins and venoms.

Idiotypes

The combining site of a specific antibody molecule is made up of a unique combination of amino acids in the variable regions of the LCs and HCs. Since this combination is not present in other antibody molecules, it should be immunogenic and capable of stimulating an immunologic response against itself in an animal of the same species. This prediction was actually found to be accurate. If one immunizes mice to generate an antibody response to the immunogen and then isolates the antigen-specific antibodies from the immune sera, these antibodies are capable of stimulating anti-antibody responses in mice of the same strain. These anti-antibody responses are polyclonal in nature as they have been shown to be specific for several epitopes present on the antibodies used in the inoculation. Given the fact that the donors and recipients of the antiserum used in the immunization protocol were members of the same strain and therefore genetically identical, shouldn't the antibodies fail to stimulate a response due to the fact that they should be considered "self" antigens to which we are tolerant? The anti-antibody responses were, in fact, stimulated by the collective variable regions on the H and L chains that constitute the antigen-specific regions of the antibody molecules contained in the inoculum. These portions of antibody molecules are called idiotypes. Thus, a more accurate designation of the antibodies produced in the antibody-immunized mice described above is anti-idiotype antibodies. Evidence has suggested that anti-idotype responses occur normally within individuals. One proposed explanation for these findings is that anti-idiotypic antibodies play a physiologic role in regulating or turning off the antibody response to the antigen that stimulated the initial antibody response. Indeed, in some cases, anti-idiotypic sera prevent binding of the antibody with its antigen, in which event the idiotypic determinant is considered to be in or very near the combining site itself. Anti-idiotypic sera, which do not block binding of antibody with antigen, are probably directed against variable determinants of the framework area, outside the combining site There are examples of immunization of experimental animals using anti-idiotypic internal images as immunogens. Such immunogens induce antibodies capable of reacting with the antigen that carries the epitope to which the original idiotype is directed. Thus, these antibodies are induced without the immunized animal ever having seen the original antigen. An anti-idiotypic antibody with a combining site complementary to that of the idiotype resembles the epitope, which is also complementary to the idiotypes' combining site. Thus, the anti-idiotype may represent a facsimile or an internal image of the epitope. Different types of variation between immuoglobulins: Isotypes: Differences between constant regions due to usage of different HC and LC constant region genes. Allotypes: Differences due to different alleles of the same constant region gene Idiotypes: Within a given isotype (e.g., IgG), differences due to particular rearranged VH and VL genes

Kinetics of the Antibody Response Following Immunization Primary Response

The first exposure of an individual to a particular immunogen is referred to as the priming immunization and the measurable response that ensues is called the primary response. The primary antibody response may be divided into several phases: 1- Latent or lag 2- Exponential: The antibody concentration in the serum increases exponentially. 3- Steady state: Antibody production & degradation are balanced. 4- Declining phase: he immune response begins to shut down & the antibody concentration in serum declines rapidly. The first antibody class detected in primary responses is generally IgM, which, in some instances, may be the only class of Ig that is made. If IgG production ensues, its appearance is generally accompanied by a rapid cessation of production of IgM.

Biologic properties of IgM 2- Neonatal Immunity & First Line of Humoral Defense

Unlike IgG, IgM antibodies do not pass through the placenta; however, since this is the only class of Igs that is synthesized by the fetus beginning at approximately 5 months of gestation, elevated levels of IgM in the fetus are indicative of congenital or perinatal infection . IgM is the isotype synthesized by children and adults in appreciable amounts after immunization or exposure to T-independent antigens, and it is the first isotype that is synthesized after immunization. Thus, elevated levels of IgM usually indicate either recent infection or recent exposure to antigen.

summary

1- Igs of all classes have a fundamental four-chain structure, consisting of two identical LCs and two identical HCs. Through disulfide bonds, each LC is linked to a HC, and the two HCs are linked to each other. 2- In the native state, L and H chains are coiled into domains stabilized by an intrachain disulfide bond. A group of other proteins (e.g., TCR, CD4, class I and class II MHC molecules) also contain these Ig-fold domains, making them all members of the immunoglobulin superfamily. 3- Igs are expressed in two forms: a membrane-bound antibody present on the surface of B cells and a secreted antibody produced by plasma cells. Membrane-bound antibodies associate with a heterodimer called Igα/Igβ to form the BCR. 4- The N-terminal domains of both heavy and light chains are the variable (V) regions and contain the hypervariable regions, also called complementarity-determining regions (CDRs), which make up the combining site of the antibody and vary according to the specificity of the antibody. 5- The constant (C) region domains of L and H chains are similar within each of the L and H chain isotypes, respectively. 6- The Fc regions of the heavy chains are responsible for the different biologic functions carried out by each class of antibody.Summary 7- Ig heavy and light chain isotypes are distinguished by the structure of their constant regions. Differences in regions of the HC constant regions are due to different genetic alleles causing even a one or two amino acid change. These are called allotypes, and they distinguish individuals within a species. 8- By contrast, idiotypic markers are represented by the unique combinations of amino acids that make up the antigen-combining site of an antibody molecule; thus, they are unique for that particular antibody. 9- IgG is a versatile class of antibody, capable of carrying out numerous biologic functions that range from neutralization of toxins to activation of complement and opsonization. IgG is the only class of immunoglobulin that passes through the placenta and confers maternal immunity on the fetus. The half-life of IgG (23 days) is the longest of all immunoglobulin classes. 10. IgM is expressed on the surface of mature B cells (as a monomer) and is secreted as a pentameric antibody held together by a J chain; of all classes of immunoglobulin it functions as the best agglutinating and complement-activating antibody. 11. Monomeric IgA is found in serum, whereas dimeric IgA is found in secretions and is referred to as secretory IgA. Secretory IgA is an important antiviral immunoglobulin. 12. IgD is present on the surface of mature B cells and is co-expressed and shares antigen-specificity with IgM. The functional properties of IgD have not been fully elucidated. 13. IgE is of paramount importance in allergic reactions. It also appears to be of importance in protection against parasitic infections. The Fc portion of IgE binds with high affinity to receptors on certain cells including mast cells. On contact with antigen, IgE triggers the degranulation of such cells, resulting in the release of pharmacologically active substances that mediate the hypersensitivity (allergic) reactions. 14. Following first exposure to an antigen, a primary antibody response occurs that consists mainly of the production of IgM antibodies. The second exposure to the same antigen results in a secondary or anamnestic (memory) response, which is more rapid than the primary response and in which the response shifts from IgM production to the synthesis of IgG and other isotypes. The secondary response lasts much longer than the primary response.

Multiple Antigens Activate γδ T Cells

A subset of T cells uses a two-chain molecule known as γδ, rather than αβ as its TCR. γδ T cells respond to many different types of antigens derived from both pathogens and damaged host cells. γδ T cells respond to phospholipid antigens and other small nonprotein molecules, known as phosphoantigens, as well as heat-shock proteins. How these different antigens are presented to γδ T cells is not completely understood, but it does not appear to involve polymorphic MHC molecules. Lipid molecules are likely presented by CD1 molecules and heat-shock proteins by MHC class I-like molecules, such as MHC class I polypeptide-related sequence (MIC) A and MICB.

Structure of MHC Class I and Class II MoleculesMHC class I molecule

An MHC class I molecule is a transmembrane glycoprotein (molecular weight approximately 43 kDa), expressed on the cell surface in noncovalent association with a small invariant (identical on all cells) polypeptide called β2-microglobulin (β2m; molecular weight 12 kDa). β2m is encoded by a gene on a chromosome that is separate from the MHC. The MHC class I molecule is referred to as the α or heavy chain and comprises three extracellular Ig-like domains—α1, α2, and α3. β2m has a structure homologous to a single Ig domain; indeed, β2m and MHC class I are members of the Ig superfamily. At the cell surface, MHC class I plus β2m has the appearance of a four-domain molecule—α1 paired with α2 on the exterior of the MHC class I molecule and α3 and β2m paired closer to the membrane. The peptide-binding groove is a deep groove or cleft in the part of the molecule farthest from the membrane that is composed of parts of the α1 and α2 domains. This groove can hold one peptide 8-9 amino acids in length in a linear array. MHC class II molecule is a transmembrane glycoprotein comprising two chains: α and β (molecular weight of approximately 35,000 and 28,000 Da, respectively). Like MHC class I, every MHC class II molecule is expressed at the cell surface as a four-domain structure: the α1 domain is paired with β1, and α2 with β2. The chains α and β have cytoplasmic tails and extracellular Ig-like domains; they are also members of the Ig superfamily. Like MHC class I, the MHC class II molecule contains a peptide-binding groove at the top of the molecule, which holds one peptide. However, in MHC class II, the groove is formed by interactions between α1 & β1 domains. Panel C indicates that the floor and walls of the MHC class II cleft have the same β-pleated sheet and α-helical structures found in the MHC class I molecule. In contrast to the 8- to 9-amino-acid peptides that bind to the cleft in the MHC class I molecule, the MHC class II groove binds peptides varying in length from 12 to approximately 17 linearly arranged amino acids. Panels C and D show that the ends of the peptide are outside the peptide-binding groove. As with MHC class I molecules, each MHC class II molecule binds to peptides with specific anchor residues. A peptide that binds to a typical MHC class II molecule has 3 (sometimes 4) anchor residues in its central region that bind to the allele-specific pockets of MHC class II. Because the other amino acids in the peptide outside the anchor residues may vary, MHC class II molecules are also capable of binding a wide selection of peptides. 4-6 of the peptide's amino acids contact the TCRs (only two are shown). Like class I, MHC class II molecules are composed of variable (polymorphic) & invariant (nonpolymorphic) regions. CD4 binds to the invariant region of all MHC class II molecules, specifically in the β2 domain

Allotypes

Another form of variation in the structure of Igs is allotypy. It is based on genetic differences between individuals. Different allelic forms (allotypes) of the HC or LC constant region genes give rise to different forms of the same gene at a given locus. As a result of allotypy, a HC or LC constituent of any Ig can be present in some members of a species and absent in others. Despite these allotypic differences among Ig classes within a species, the vast majority of the protein sequences of the constant regions (H or L) for a given class is highly conserved. Allotypic differences at known HC- and LC- gene loci usually result in changes in only one or two amino acids in the constant region of a chain. With a few exceptions, the presence of allotypic differences in two identical Ig molecules does not generally affect binding with antigen, but it serves as an important marker for analysis of Mendelian inheritance. Some known allotype markers constitute a group on the γ chain of human IgG (called Gm for IgG markers), a group on the κ chain (called Km), and a group on the α chain (called Am).

Antibody structure and function

Antibodies are soluble proteins that circulate freely and exhibit properties that contribute specifically to immunity and protection against foreign material. Antibodies belong to a class of proteins called globulins because of their globular structure. Antibodies are collectively known as immunoglobulins (Igs) .Immunoglobulins can be membrane-bound or secreted.

Structural features Variable region

Constitutes the part of the molecule that binds to the antigen for which the antibody is specific A major problem for immunologists was to determine how so many individual specificities, which are required to meet the enormous variety of antigenic challenges, are generated from the variable region gene This issue has been largely resolved and is explained by the phenomenon of gene rearrangement associated with B cells (and T cells for the TCR) The concept of hypervariability regions of Igs relates to the concept of antibody specificity. When the amino acid sequences of immunoglobulin molecules derived from sera or urine of individuals suffering with multiple myeloma were examined, significant insights into the antigen-binding region of antibodies were obtained. Why were these sera and urine samples chosen for examination? The sera in multiple myeloma patients often contains copious amounts of Ig molecules, all identical in structure and specificity by virtue of their production by the neoplastic plasma cells causing the disease. Urine from such patients contains large amounts of light chain molecules associated with these myeloma proteins (i.e., Bence Jones proteins). Using these sera and urine samples, it was found that the greatest variability in sequence existed in the N-terminal 110 amino acids of both the light and heavy chains. Kabat and Wu compared the amino acid sequences of many different VL and VH regions. They plotted the variability in the amino acids at each position in the chain and showed that the greatest amount of variability occurred in three regions of the light and heavy chains. These regions are called hypervariable regions. The less variable stretches, which occur between these hypervariable regions, are called framework regions It is now clear that the hypervariable regions participate in the binding with antigen and form the region complementary in structure to the antigen. Consequently, hypervariability regions are termed complementarity-determining regions (CDRs) of the light and heavy chains: CDR1, CDR2, and CDR3. Variability of amino acids representing the N-terminal residues of VH in a representative immunoglobulin molecule The hypervariable regions, although separated in the linear, two-dimensional model of the peptide chains, are actually brought together in the folded form of the intact antibody molecule, and together they constitute the combining site, which is complementary to the epitope CDRs variability provides the diversity in the shape of the combining site that is required for the function of antibodies of different specificities. Antigen-antibody interactions are via weak, noncovalent forces such as ionic, hydrogen-bonding, van der Walls forces, and hydrophobic interactions. It is therefore necessary that there be a close fit between antigen and antibody over a sufficiently large region to allow a total binding force that is adequate for stable interaction. Contributions to this binding interaction by both heavy and light chains are involved in the overall association between epitope and antibody. Two antibody molecules with different antigenic specificities must have different amino acid sequences in their hypervariable regions Antibody molecules with similar sequences will generally have similar specificities. However, it is possible for two antibodies with different amino acid sequences to have specificity to the same epitope. In this case, the binding affinities of the antibodies with the epitope will probably be different because there will be differences in the number and types of binding forces available to bind identical antigens to the different binding sites of the two antibodies. Another variability source is the size of the combining site on the antibody, which is usually (but not always) considered to take the form of a depression or cleft. In some instances, especially when small hydrophobic haptens are involved, the epitopes do not occupy the entire combining site, yet they achieve sufficient affinity of binding. Antibodies specific for such a hapten may, in fact, react with other antigens that have no obvious similarity to the hapten (e.g., dinitrophenol and sheep red cells). These dissimilar antigens bind either to a larger area or to a different area of the combining site on the antibody. Thus, a particular antibody-combining site may have the ability to combine with two (or more) apparently diverse epitopes, a property called redundancy. The ability of a single antibody molecule to cross-react with an unknown number of epitopes may reduce the number of different antibodies needed to defend an individual against the range of antigenic challenges.

Structural features Domains

Each domain is designated by a letter that indicates whether it is on a LC or a HC and a number that indicates its position. The first domain on LCs and HCs is highly variable, in terms of amino acid sequence, from one antibody to the next, and it is designated VL or VH accordingly. The second and subsequent domains on both HCs are much more constant in amino acid sequence and are designated CH1, CH2, and CH3. In addition to their interchain disulfide bonding, the globular domains bind to each other in homologous pairs, largely by hydrophobic interactions, as follows: VHVL, CH1CL, CH2CH2, and CH3CH3.

Structure of light & heavy chains The second discovery

Edelman discovered that when γ-globulin was reduced by treatment with mercaptoethanol, the molecule fell apart into four polypeptide chains: Two identical light chains (LCs)with MW =22,000Da Two identical heavy chains (HCs) with MW=53,000Da Porter and Edelman shared the Nobel prize for the elucidation of antibody structure. Summary: All Ig molecules consist of a basic unit of four polypeptide chains held together by several disulfide bonds.

Endogenous Antigens: Generation of MHC Class I-Peptide Complexes

Endogenous antigens are proteins synthesized inside a cell and are generally derived from pathogens (such as viruses, bacteria, and parasites) that have infected a host cell. The next figure illustrates the processing and presentation of a typical endogenous antigen, a viral protein synthesized after a cell has been infected by a virus. Processing occurs in the cytoplasm. The major mechanism for generating peptide fragments is via a giant cytoplasmic protein complex known as the proteasome. The proteasome is also involved in normal turnover (the routine degradation) of cellular proteins and breaks them down into peptides about 15 amino acids in length. Cytosolic enzymes (aminopeptidases) remove even more amino acids from the peptides. Some peptides are destroyed, but some, 8-15 amino acids in length (such as the three shown in the figure), are selectively transported into the ER by a two-chain peptide transporter (TAP). Peptides transported from the cytoplasm into the ER bind to newly synthesized MHC class I molecules. MHC class I and β2m chains are synthesized separately in the rough ER and associate in this cellular compartment. As with the synthesis of MHC class II molecules, chaperones stabilize the structure of the assembled MHC class I with their β2m chains in the ER and direct transport of the complex through the cell. MHC class I molecules preferentially bind peptides 8 to 9 amino acids in length. he normal fate of peptides that reach the ER is degradation by an aminopeptidase, which removes amino acids one at a time until the peptides are completely degraded. Some peptides with the appropriate binding characteristics (8-9 amino acids in length and with sufficient affinity to bind to the MHC class I binding groove) are "rescued" from this fate by binding to a newly synthesized MHC class I. A peptide that binds to an MHC class I molecule in the ER moves via the Golgi apparatus to the cell surface, where it is displayed and presented to a CD8+ T cell expressing the appropriate antigen receptor. As described for the interaction of peptides with MHC class II molecules, only those peptides that bind to MHC class I molecules trigger CD8+ T-cell responses. These are the immunodominant epitopes for the CD8+ T-cell response specific for that antigen. One such immunodominant epitope is the peptide derived from the catabolism of the virus protein shown in green in the previous figure. Because pathogens can infect almost any cell in the body, CD8+ T cells "scan" MHC class I and peptide combinations expressed on any nucleated host cell to identify whether it has been infected.

Selective binding of processed peptides by different MHC molecules

Every person will make a T-cell response to this viral protein because of the selectivity of peptide-MHC binding, individuals who express different MHC molecules respond to different parts of the same protein.

Antigen Processing and Presentation: How MHC Molecules Bind Peptides and Create Ligands That Interact with T Cells

Exogenous Antigens and Generation of MHC Class II-Peptide Complexes Endogenous Antigens: Generation of MHC Class I-Peptide Complexes

Exogenous Antigens and Generation of MHC Class II-Peptide Complexes

Exogenous antigens are antigens that come from outside a host cell and are taken inside, normally by endocytosis or phagocytosis. Exogenous antigens can be derived from pathogens (such as bacteria or viruses) or from foreign proteins (such as vaccines) that do not injure the host but activate an immune response. The specialized cells that take up exogenous antigens (and present it to T cells) are known as APCs. The main APCs are dendritic cells, macrophages, and B cells, all of which express MHC class II molecules constitutively. The protein is internalized, contained in an intracellular vesicle that fuses with endosomal or lysosomal vesicles that are highly acidic (pH approximately 4.0). These vesicles contain an array of degradative enzymes, including proteases and peptidases. Proteases, known as cathepsins, which function at low pH, cut proteins into peptides in these vesicles. Catabolism of a typical protein antigen yields several peptides. The acid vesicles containing peptides intersect inside the cell with vesicles containing MHC class II that have been synthesized on ribosomes of the rough ER (endoplasmic reticulum).

Decreased MHC Class I Expression in Virus-Infected Cells

Factors such as cytokines synthesized in response to infectious agents induce or increase expression of MHC class I and class II molecules. This leads to enhanced immune responses to the inducing pathogen. Conversely, some viruses (the herpes simplex virus, adenovirus & cytomegalovirus) synthesize proteins that interfere with steps in the MHC class I pathway. They inhibit the synthesis of MHC class I or interrupt transport of peptide-MHC class I complexes to the cell surface. Thus, the virus decreases MHC class I expression and so subverts the potential host CD8+ T-cell response to the virus.

Structure of Immunoglobulins

Features include specificity and biologic activity. Specificity is attributed to a defined region of the antibody molecule containing the hypervariable or complementarity-determining region (CDR). This restricts the antibody to combine only with substances that have a particular antigenic structure. The existence of a vast array of potential epitopes promoted the evolution of a system for producing an enormous repertoire of antibody molecules, each of which is capable of combining with a particular antigenic structure. Thus, antibodies collectivelyexhibit great diversity. Diversity is in terms of the types of molecular structures with which they are capable of reacting. Individually, they exhibit a high degree of specificity, since each is able to react with only one particular antigenic structure. Despite the large numbers of antigen-specific antibodies, the biologic effects of antigen-antibody reactions are rather few in number. The biologic activity depends on the nature of the antigen to which the antibody is specific. Differences in activities are attributed to structural properties conferred by the germline-encoded portions of the Ig molecule. Thus, not all antibody molecules are equal in the performance of their biologic activities.

Structural features of IgM

IgM is the first immunoglobulin produced following immunization. Its name derives from its initial description as a macroglobulin (M) of high molecular weight (900,000 Da). It has an extra CH domain. In comparison to the IgG molecule, which consists of one four-chain structure, IgM is a pentameric molecule composed of five such units. Each unit consists of two LCs and two HCs, all joined together by additional disulfide bonds between their Fc portions and by a polypeptide chain termed the J chain The J chain is synthesized in the B cell or plasma cell (like light and heavy chains) The J chain has a molecular weight of 15,000 Da. This pentameric ensemble of IgM, which is held together by disulfide bonds, comes apart after mild treatment with reducing agents such as mercaptoethanol. Surprisingly, each pentameric IgM molecule appears to have a valence of 5 (i.e., five antigen-combining sites), instead of the expected valence of 10 predicted by the 10 Fab segments contained in the pentamer. This apparent reduction in valence is probably the result of conformational constraints imposed by the polymerization. It is known that pentameric IgM has a planar configuration, such that each of its 10 Fab portions cannot open fully with respect to the adjacent Fab, when it combines with antigen, as is possible in the case of IgG. Thus, any large antigen bound to one Fab may block a neighboring site from binding with antigen, making the molecule appear pentavalent (or of even lesser valence).

Biologic properties of IgM 3- Agglutination

IgM molecules are efficient agglutinating antibodies. Because of their pentameric form, IgM antibodies can form macromolecular bridges between epitopes on molecules that may be too distant from each other to be bridged by the smaller IgG antibodies. Because of their pentameric form and multiple valence, the IgM antibodies are particularly well suited to combine with antigens that contain repeated patterns of the same antigenic determinant, as in the case of polysaccharide antigens or cellular antigens, which are multiply expressed on cell surfaces.

summary

MHC molecules play a crucial role in the response of T cells to antigens that are taken into or live inside cells of the body: MHC molecules selectively bind peptides derived from protein antigens and present them to T cells with the appropriate receptor. Thus, T-cell responses are said to be MHC restricted. The MHC is a complex set of genes inherited as a unit. The MHC codes for two major categories of cell surface transmembrane molecules, MHC class I & MHC class II. MHC class I is expressed on all nucleated cells in association with a small invariant peptide, β2-microglobulin. Antigenic peptide bound to MHC class I interacts with the TCR of a CD8+ T cell, so responses of CD8+ T cells are MHC class I restricted. MHC class II is expressed constitutively only on cells that present antigen to T cells. Peptide bound to MHC class II interacts at the cell surface with the TCR of a CD4+ T cell. Thus, the response of CD4+ T cells is MHC class II restricted. The expression of MHC molecules is inducible on many cell types, particularly in response to cytokines generated in the response to infectious agents. This induction of expression enhances T-cell responses directed against the pathogen. Viruses inhibit MHC class I expression and so subvert the T-cell response directed against them. Within one individual, the same MHC class I and II molecules are expressed on all cells of the body. Different individuals express different MHC class I and II molecules. This diversity arises because individuals within a species have a huge range of inheritable forms of MHC class I and II genes (genetic polymorphism)—the MHC is the most genetically diverse system in the population. Because of the extensive polymorphism of MHC genes, every individual has an almost unique array of inherited MHC genes. The outer region of every MHC class I and II molecule contains a deep cleft called the peptide-binding groove that binds a peptide derived from the catabolism (processing) of protein antigens. The binding of peptides to MHC molecules is selective; that is, each MHC molecule binds a range of peptides but favors the binding of peptides with particular motifs MHC class I & II molecules bind peptides derived from proteins processed in different ceullular compartments. MHC class I molecules bind peptides 8-9 amino acids long derived from proteins catabolized in the cytoplasm ("endogenous antigens"); peptides are transported into the endoplasmic reticulum where they interact with newly synthesized MHC class I and β2m chains. MHC class II binds peptides 12-17 amino acids long derived from proteins taken into cells ("exogenous" antigens) and catabolized in acid vesicles in APCs. DCs have a unique pathway(cross-presentation) in which antigen taken into the cell associates with MHC class I and peptides are presented to CD8+ T cells. Because proteins are generally structurally complex, they usually generate at least one peptide able to bind to an MHC molecule, ensuring that a T-cell response is made to at least some part of a foreign antigen. MHC class I molecules also interact with receptors on NK cells. This interaction prevents NK cells from killing normal cells of the host. Decreased expression of MHC class I after infection by certain viruses and in some tumors triggers NK cell killing of the altered host cell MHC molecules bind peptides derived from self-components as well as from foreign antigens, but self-components do not normally activate a T-cell response. This is mainly because under normal conditions self-molecules do not generate the co-stimulator (second) signals needed to activate naïve T cells. The T-cell response is focused on the response to foreign molecules, particularly components of pathogens, which induce these co-stimulator signals. Susceptibility and resistance to many autoimmune and inflammatory conditions are associated with the expression of a particular MHC molecule.

Exogenous Antigens and Generation of MHC Class II-Peptide Complexes Stages for removal of CD74 from the complex

Initially, the invariant chain is degraded proteolytically, leaving a fragment known as CLIP (class II-associated invariant polypeptide) bound to the MHC class II groove. Vesicles containing MHC class II with bound CLIP fuse with acid vesicles (endosomes or lysosomes) containing peptides derived from the catabolism of exog. antigens. In this compartment, HLA-DM catalyzes the peptide exchange between the MHC class II-CLIP complex and peptides derived from the exogenous antigen. A peptide-MHC class II complex is generated, which moves to the cell surface where it is displayed and available to interact with (be presented) to a CD4+ T cell expressing the appropriate antigen receptor. Although catabolism of a typical protein yields several peptides (three were shown on the previous figure), not all the peptides formed bind to MHC molecules because MHC binding to peptides is selective. The next figure shows the situation with three of the many peptides that are derived from the catabolism of a larger protein.

Structural features Disulfide bonds

Interchain disulfide bonds hold the chains together. They hold together light and heavy chains, as well as the two heavy chains. Intrachain disulfide bonds (in each of the heavy and light chains) form loops within the chain Intrachain disulfide bonds create immunoglobulin-fold domains to create antiparallel β-pleated sheet structures characteristic of antibody molecules. Other Ig superfamily molecules share this structural feature The presence of intrachain disulfide bonds at regular, approx. equal intervals (100-110 amino acids) led to the prediction that each loop in the peptide chains should form a compactly folded globular domain

Structural features LCs

LCs have two domains each Two major classes of LCs: κ and λ Any one individual of a species produces both types of LC, but the ratio of κ chains to λ chains varies with the species (mouse: 95% κ; human: 60% κ) In any one immunoglobulin molecule, the LCs are always either both κ or both λ, never one of each.

Inability to Respond to an Antigen

Limited numbers of different MHC class I and II molecules are expressed on the cells of any one person. For an antigen to generate a T-cell response, at least one peptide derived during processing must bind to one of these MHC molecules. A peptide that does not bind to an MHC molecule does not activate a T-cell response; thus, if an entire antigen fails to generate a single peptide able to bind to an MHC molecule, the individual will not mount a T-cell response to that particular antigen. Naturally occurring pathogens are generally large and complex and contain multiple epitopes that stimulate responses by both T and B cells; thus, some sort of response to a pathogen is more or less certain. However, unresponsiveness to a large antigen can occur in the case of synthetic polymers of amino acids that contain a very limited number of epitopes. Another important situation is in the response to a small peptide, such as a vaccine comprising a single, small peptide. Since the population expresses many different types of MHC molecules, the MHC molecules expressed in some people may not bind this particular peptide. How to resolve this?In vaccination, this problem has been circumvented by coupling the peptide to a large protein, a carrier, which enhances the response to the peptide.

Structure of MHC Class I and Class II Molecules Selectivity of Peptide Binding to MHC Class I Molecules

Looking at differences in sequence among MHC class I molecules, most of the differences in amino acids are confined to a limited region in the extracellular α1 and α2 domains, and particularly in the floor and walls of the peptide-binding groove (see panel C in the previous figure). These differences in amino acid sequence and hence structure of the binding groove play a critical role in determining which peptides bind to a particular MHC molecule. The pockets forming the floor of the groove also help to align peptides so they can be recognized by specific TCRs (Figure on the next slide). Peptide binding to an MHC class I molecule is selective: One MHC molecule will bind with high affinity to only certain peptides. A single MHC class I molecule preferentially binds peptides with specific anchor residues: invariant or closely related amino acids at certain positions in the 8- or 9-amino-acid sequence. A peptide that binds to an MHC class I molecule typically has two anchor residues, which interact with the allele-specific pockets in the MHC molecule. The other positions in the peptide may vary. As a result of the previous, one MHC molecule can bind a large number of peptides with different sequences. This helps explain how only a maximum of six MHC class I molecules in an individual can display many different peptide antigens. It also helps explain why with very few exceptions T-cell responses are made to at least one epitope from almost all proteins and why failure to respond to a protein antigen is so rare. Peptide bound in the cleft and parts of the MHC class I molecule interact with the TCR. 1-4 peptide amino acids make contact with the TCR (only a small number of contacts with the peptide are critical for TCR recognition)

Variability of MHC Class I and MHC Class II Molecules

MHC class I and class II molecules differ from individual to individual within the population, and these differences are genetically determined; that is, MHC-distinct individuals express MHC molecules with somewhat different sequences. These differences arise from two sources: polygenicity and polymorphism. Polygenicity means that MHC class I and II molecules are coded for by multiple independent genes. The human HLA complex (found on chromosome 6) contains three independent genes (HLA-A, HLA-B, and HLA-C) that code for MHC class I molecules Each HLA class I molecule is expressed at the surface in association with a small molecule, β2-microglobulin (β2m), coded for outside the HLA complex. Because every cell has two sets of chromosomes (one paternally derived and one maternally derived), every nucleated cell may express up to six different HLA class I molecules, each capable of binding peptides. Similarly, the HLA complex codes for three different two-chain MHC class II molecules: HLA-DP, HLA-DQ, and HLA-DR. Thus, human APCs may express up to six different HLA class II molecules, each capable of binding to peptides. The mechanisms used to generate the diversity of MHC structures differ from the mechanisms used to generate the diversity of the antigen-specific receptors of B and T cells (Ig and TCR, respectively) that arises from rearrangement of DNA and which produces one type of receptor per cell. In contrast, although MHC molecules are diverse within the population, each cell in a particular individual (liver, kidney, lymphocytes, etc.) expresses the same set of HLA class I and class II molecules.

Biologic activities of antibodies

Neutralization of toxins Immobilization of microorganisms Neutralization of viral activity Agglutination of microorganisms & antigenic particles Ability to cross the placenta (from mother to fetus) Some activities constitute examples of how the adaptive collaborates with the innate immune system: 1- Binding with soluble antigen leads to the formation of precipitates. Precipitated antigens are readily phagocytized and destroyed by phagocytic cells 2- Binding with antigen activates complement to facilitate microbial lysis (complement-mediated opsonization also results in phagocytosis & destruction of microbes)

Structure of MHC Class I and Class II Molecules CD8 Binding to Invariant Region of MHC Class I Molecules

Outside the peptide-binding cleft the sequences of different MHC class I molecules are very similar. Thus an individual MHC class I molecule can be divided into a polymorphic or variable region (sequence unique to that molecule) in the area in and around the peptide-binding groove, and a nonpolymorphic or invariant region that is similar in all MHC class I molecules. CD8, the molecule that characterizes the CD8+ T-cell subset, binds to the invariant region of all MHC class I molecules, specifically in the α3 domain

Papain and Pepsin digestion

Papain digestion of the Ig molecule results in cleavage N-terminally to the disulfide bridge between the HCs at the hinge region, yielding two monovalent Fab fragments and an Fc fragment. Pepsin digestion results in cleavage C-terminally to the disulfide bridge, resulting in a divalent fragment F(ab')2, consisting of two Fab fragments joined by the disulfide bond and several Fc subfragments. The globular structure of Igs, and the ability of enzymes to cleave these molecules at very restricted positions into large entities instead of degrading them to oligopeptides and amino acids, is indicative of a very compact structure.

Variability of MHC Class I & MHC Class II Molecules Polymorphism

Polymorphism means that multiple stable forms of each MHC gene exist in the population. The MHC is the most highly polymorphic gene system in the body and hence in the population: In humans over a thousand slightly different versions (alleles) of the gene that codes for the MHC class I molecule HLA-B and MHC class II molecule HLA-DRB have been identified. Other important examples of genetic polymorphism in humans are the different forms of the red blood cell antigens (A, B, and O) and of hemoglobin molecules. Recent studies comparing gene sequences from different individuals (by identifying single nucleotide polymorphisms) have found that many genes show allelic variation, for example, genes coding for cytokine receptors and liver detoxification enzymes. However, none of these genes is as variable within the population as HLA. The extensive polymorphism of human MHC genes makes it very unlikely that two random individuals will express identical sets of HLA class I & class II molecules. The enormous diversity of MHC molecules and the genes that code for them is a major barrier to successful transplantation of organs and tissues

Structure of light & heavy chains The first discovery

Porter found that proteolytic treatment with Papain split the Ig molecule (MW 150,000Da) into 3 fragments of about equal size. Two fragments retained the antibody's ability to bind antigen specifically, but they cannot precipitate antigen from solution. These two fragments are referred to as Fab (fragment antigen-binding) and are considered to be univalent, possessing one binding site each and being in every way identical to each other. The third fragment could be crystallized out of solution, a property indicative of its apparent homogeneity. This fragment is called the Fc fragment (fragment crystallizable). It cannot bind antigen but, as was subsequently shown, is responsible for the biologic functions of the antibody molecule after antigen has been bound to the Fab part of the intact molecule.

Membrane-bound antibody

Present on the surface of B cells where it serves as the antigen-specific receptor. Associated with a heterodimer called Igα/Igβ to form the BCR. The Igα/Igβ heterodimer mediates the intracellular signaling mechanisms associated with B-cell activation. Secreted antibodies are produced by plasma cells. These are terminally differentiated B cells that serve as antibody factories that reside largely within the bone marrow.

Which Antigens Trigger Which T-Cell Responses?

Proteins from bacteria, most viruses, allergens, and completely harmless antigens all trigger CD4+ T-cell responses. In contrast, only infectious pathogens, particularly viruses, create epitopes via the endogenous or cross-presentation pathways and are presented by MHC class I molecules; thus, they are the only types of antigen that activate CD8+ T cells. Generally, but not always, infectious agents activate both CD4+ T cells and CD8+ T cells because these agents are taken up by APCs However, some viruses do not evoke CD4+ T cell responses and activate CD8+ T cells almost exclusively. Transplantation responses—in which host T cells respond to nonself MHC molecules expressed on cells of the graft—generally activate both CD4+ and CD8+ T-cell responses. Immune responses to tumors are generally mediated by CD8+ cells.

Isolation and characterization of Immunoglobulins

Serum is the antibody-containing blood component. It is the liquid portion left when blood has been withdrawn and allowed to clot. When serum is subjected to electrophoresis at slightly alkaline pH (8.2), five major components can be visualized. The slowest, in terms of migration toward the anode, is called γ-globulin and contains the immunoglobulins (Igs). It was shown later that antibody activity is present not only in the γ-globulin fraction but also in a slightly more anodic area. All globular proteins with antibody activity are generically referred to as Igs, as exemplified by the γ peak. Analysis of the structural characteristics of antibody molecules began in 1959 with two discoveries:

Other Types of Antigen That Activate T-Cell ResponsesLipids and Glycolipids

Some T cells can recognize lipids and glycolipids found in the cell walls of pathogens such as Mycobacterium tuberculosis that live inside macrophages; these T cells also respond to many host glycolipids. Lipids and glycolipids are presented by a family of molecules known as CD1 (CD1a through CD1d), which is expressed by APCs (macrophages & dendritic cells). CD1 molecules are cell surface glycoproteins coded for outside the MHC region; like MHC class I molecules, they are expressed on the surface of APCs in association with β2m. The structure of a CD1 molecule is similar to that of an MHC class I molecule, but CD1 contains a larger binding groove with a deep cavity. The cavity binds the hydrophobic backbone of a lipid antigen, exposing the polar region of the lipid or glycolipid for binding to the T-cell receptor. Binding of lipid antigens to CD1 takes place in acidic cellular compartments, similar to the loading of exogenous peptides onto MHC class II molecules. Like the interaction of some viruses with MHC class I, recent evidence indicates that herpes simplex virus 1 down regulates expression of CD1d, inhibiting glycolipid antigen presentation.

Other Types of Antigen That Activate T-Cell Responses Super antigens

Super antigens in humans are predominantly exotoxins produced by pathogenic bacteria. Examples:1- The enterotoxin released by staphylococcal organisms (the cause of food poisoning) 2-The toxin responsible for toxic shock syndrome. Supe rantigens are not processed. The intact molecule binds to MHC class II molecules outside the peptide-binding groove and cross-links MHC II with the variable region of the TCR β chain expressed by a CD4+ T cell. Each superantigen binds to T cells expressing a particular Vβ TCR gene product. There are only about 50 different Vβ TCR genes in humans & a superantigen may bind to more than one Vβ. Thus, superantigens are able to activate more than 10% of the total T-cell population. This is an enormous response compared to the triggering of a few T-cell clones by a peptide-MHC class II complex derived from a conventional antigen. the key biologic & clinical feature of superantigens is that they activate huge numbers of CD4+ T cells, which proliferate and secrete high levels of cytokines, and can pass into the circulation. The systemic effects of the toxin activating so many T cells can have clinical consequences, including fever and cardiovascular shock, and can be fatal.

Exogenous Antigens and Generation of MHC Class II-Peptide ComplexesThe invariant chain (Ii, CD74)

The MHC class II α and β chains are synthesized individually in the endoplasmic reticulum and are assembled there with invariant chain (Ii, CD74). The invariant chain binds to the groove of the newly formed MHC class II molecule, preventing the binding of peptides that may be present in the ER, such as peptides derived from the processing of endogenous antigens. The invariant chain also acts as a chaperone for the newly synthesized MHC class II chains. In other words, interaction with the invariant chain allows the MHC class II α and β chains to leave the ER and enter the Golgi complex, and from there they proceed into the acid vesicle endocytic pathway.

Structural features Hinge region

The hinge region is composed of a short segment of amino acids (relatively long in the case of IgD and IgE) and is found between the CH1 and CH2 regions of the HCs This segment is made up predominantly of cysteine and proline residues. The cysteines are involved in formation of interchain disulfide bonds, and the proline residues prevent folding in a globular structure. Is a region of the HC that provides an important structural characteristic of immunoglobulins: 1- It permits flexibility between the two Fab arms, allows them to open and close to accommodate binding to two identical antigenic epitopes, separated by a fixed distance, as might be found on the surface of a bacterium. 2- Additionally, since this stretch of amino acids is open and as accessible as any other nonfolded peptide, it can be cleaved by proteases, such as papain, to generate the Fab and Fc fragments

MHC Molecules Bind Peptides Derived from Self-Molecules

The process of antigen processing and presentation includes the catabolism of proteins and movement of products from compartment to compartment inside a cell. These are all aspects of normal cell physiologic pathways. The proteins normally found inside cells, self-proteins, "turn over" and are catabolized using the same pathways described for the processing of protein antigens. Ribosomal and mitochondrial proteins are broken down inside cells and peptides derived from these molecules can associate with MHC molecules. Indeed, MHC molecules extracted from cells nearly always contain peptides derived from such self-proteins. From our description of protein processing, proteins from inside host cells would be expected to associate with MHC class I molecules, and indeed this is observed. However, self-peptides bound to MHC molecules do not normally activate T cells. One reason is that T cells reactive to many self-molecules are removed or inactivated during differentiation in the thymus. However, we know that mature T cells with the potential to react with self-molecules are detectable outside the thymus. Why are these T cells not activated? Equally important, an individual's cells are bathed in a sea of self-proteins that they are continually processing and binding to their MHC molecules—how can a person respond to a tiny amount of foreign protein? A T cell must be able to distinguish between a normal host cell, to which no response is required, and a cell that has been infected by a pathogen. The answer appears to be that the major effect induced by pathogens is co-stimulator function (also referred to as second signals) in the specialized APCs that present antigen to T cells. These co-stimulator signals are required to activate naïve T cells. n contrast to APCs, normal tissue cells (such as those of the liver or pancreas) displaying peptides derived from self-molecules do not express co-stimulator function; thus, T cells are not activated. Even if peptides derived from a self-molecule are presented by an APC in the tissue, T cells are not activated because, in the absence of foreign antigen or an inflammatory response, APCs in tissue do not express co-stimulator signals. This signaling requirement ensures that T cells do not normally respond to peptides derived from self-components but do respond to peptides derived from nonself, potentially harmful, antigens. The previous also helps to explain why the induction of T-cell responses to even some foreign antigens, such as harmless antigens or some vaccines, can be difficult. To develop strong T-cell (and antibody) responses, such antigens are frequently administered with an adjuvant (literally, adding to the response). Adjuvants are generally products that activate APCs such as dendritic cells, macrophages, and B cells

Decreased MHC Class I Expression in Tumor Cells: Role of NK Cells.

Tumor cells frequently show decreased expression of MHC class I molecules compared to normal cells, so reducing a potential antitumor response by CD8+ T cells. The decrease in MHC class I expression also triggers the response of NK cells to a virus-infected or tumor cell.MHC class I molecules are negative regulators of NK cell function. That is, an MHC class I expressed on a normal host cell interacts with a killer-cell inhibitory receptor (KIR) expressed on an NK cell and so prevents the NK cell from killing the host cell. If a host cell does not express MHC class I (tumors or virus-infected cells) and the KIR-MHC class I interaction does not occur, NK cells are activated and kills

Structural features HCs

Two glycosylated HCs HCs have 4 or 5 domains, separated by a short unfolded stretch HCs can be divided into five different classes or isotypes: IgM, IgD, IgG, IgA, and IgE. These are distinguished from one another based upon so called constant regionsof the HCs, which differ from one another with regard to their protein sequences, carbohydrate content, and size. The nature of the HC confers on a molecule its unique biologic properties, such as its half-life in circulation, its ability to bind to certain receptors, and its ability to activate complement on combination with antigen.

Immunoglobulin variants Isotypes

We have already introduced the term isotype. Why has the immune system evolved to provide this level of immunoglobulin diversity? 1- To optimize humoral immune defenses against infectious pathogens and other foreign substances, a variety of mechanisms, each dependent on a somewhat different property or function of an immunoglobulin molecule, has developed. 2- Thus, when a specific antibody molecule combines with a specific antigen such as a pathogen, several different effector mechanisms come into play. 3- These different mechanisms derive from the different classes of Igs (isotypes), each of which may combine with the same epitope but each of which triggers a different biologic response. 4- These differences result from structural variations in the constant regions of the HCs, which have generated domains that mediate a variety of functions. A summary of the properties of the immunoglobulin classes is given in the following two tables.

Cross-Presentation: Exogenous Antigens Presented in the MHC Class I Pathway

in addition to their ability to process exogenous antigens in the MHC class II pathway, APCs (and particularly dendritic cells) have a unique pathway, called cross-presentation, for generating peptides derived from exogenous protein antigens and presenting them to CD8+ T cells. In the cross-presentation pathway, exogenous antigens associate with MHC class I molecules. The dendritic cell takes up exogenous antigens (such as those derived from a virus-infected or dying cell) by either phagocytosis or pinocytosis. How exogenous peptides intersect with MHC class I molecules inside the cell is not completely understood. One pathway is thought to involve transferring antigen from acid compartments into the cytosol for processing by proteasomes. Peptides are then transported into the ER where they bind to newly synthesized MHC class I molecules. A second pathway involves the loading of peptides directly onto MHC class I in acid vesicles, with peptide-MHC complexes then trafficking to the cell surface. The Cross-presentation is believed to play an important role in activating CD8+ T cells to respond to tissue cells infected by some viruses that are not taken up by APCs. It is also thought to play a role in the response to dying cells. In some circumstances endogenous antigens may also associate with MHC class II molecules. This may occur during autophagy, an intracellular pathway in which proteins in the cytoplasm are transported into lysosomes for degradation. Self-peptides can bind to MHC class II in these acid vesicles.