Topic 5 - Biology immunology adaptive immune system

29. What are the main mechanisms of antibody diversification in mice and humans?

(1) Combinatorial joining of gene segments (2) Junctional diversification during gene segment joining (3) Combinatorial joining of L and H chains (4) Somatic hypermutation + class-switch recombination

Fx of mast cell?

(1) mast cell w/ histamine containing secreotry vesciel (2) IgE binds to Fc receptors (3) multivalent antigen cross-link adj. IgE mol (4) histamine relase by exocytosis

29. POSITIVE and NEGATIVE SELECTION OF T CELLS?

- A combination of positive and negative selection processes operates during T cell development in the thymus to shape the TCR repertoire. These processes help to ensure that only T cells with potentially useful receptors survive and mature, while all of the others die by apoptosis. (1) First, T cells that can respond to peptides complexed with self MHC proteins are positively selected (2) subsequently, the T cells in this group that can react strongly with self peptides complexed with self MHC proteins are eliminated. Helper and cytotoxic T cells that leave the thymus with receptors that could react with self antigens are eliminated, functionally inactivated, or actively suppressed when they recognize self antigens on nonactivated dendritic cells.

29. Molecules with multiple antigenic determinants (A)?

- A globular protein is shown with a number of different antigenic determinants. - Different regions of a polypeptide chain usually come together in the folded structure to form each antigenic determinant on the surface of the protein, as shown for three of the four determinants. - (B) A polymeric structure is shown with many identical antigenic determinants

28. How to antibody and antigen interact? (antibody-antigen interactions)

- ANTIBODY: Because of their two antigen-binding sites, they are described as bivalent - As long as an antigen has three or more epitopes, bivalent antibody molecules can cross-link it into a large lattice that macrophages can readily phagocytose and degrade. The efficiency of antigene-binding and cross-linking is greatly increased by the flexible hinge region in most antibodies, which allows the distance between the two antigen-binding sites to vary - The protective effect of antibodies is not due simply to their ability to bind and cross-link antigen. The tail of the Y-shaped molecule mediates many other activities of antibodies. - Antibodies with the same antigene-binding sites can have any one of several different tail regions. Each type of tail region gives the antibody different functional properties, such as the ability to activate the complement system, to bind to phagocytic cells, or to cross the placenta from mother to fetus. - Antigen binding to antibody In this highly schematized diagram, an antigenic determinant on a macromolecule is shown interacting with one of the antigen-binding sites of two different antibody molecules, one of high affinity and one of low affinity. - Various weak noncovalent forces hold the antigenic determinant in the binding site, and the site with the better fit to the antigen has a greater affinity. Note that both the light and heavy chains of the antibody molecule usually contribute to the antigen-binding site.

29. How can B cells switch the class of antibody they make?

- All B cells begin their antibody- synthesizing lives by making IgM molecules and inserting them into the plasma membrane as receptors for antigen. - After the B cells leave the bone marrow, but before they interact with antigen, they begin making both IgM and IgD molecules as membrane-bound antigen receptors, both with the same antigen- binding sites - Stimulation by antigen and helper T cells activates many of these cells to become IgM- secreting effector cells, so that IgM antibodies dominate the primary antibody response - Later in the immune response, however, when activated B cells are undergoing somatic hypermutation, the combination of antigen and helper-T-cell-derived cytokines stimulates many of the B cells to switch from making membrane-bound IgM and IgD to making IgG, IgA, or IgE antibodies - a process called class switching. - Some of these cells become memory cells that express the corresponding class of antibody molecules on their surface, while others become effector cells that secrete the antibodies. SECONDARY CLASSES OF ANTIBODIES - The IgG, IgA, and IgE molecules are collectively referred to as secondary classes of antibodies, because they are produced only after antigen stimulation, dominate secondary antibody responses, and make up the secondary antibody repertoire. CLASS SWITCH RECOMBINATION - As we saw earlier, each different class of antibody is specialized to attack pathogens in different ways and in different sites - The constant region of an antibody heavy chain determines the class of the antibody - Thus, the ability of B cells to switch the class of antibody they make without changing the antigen-binding site implies that the same assembled VH region coding sequence (which specifies the antigen-binding part of the heavy chain) can sequentially associate with different CH-coding sequences. This has important functional implications. It means that, in an individual animal, a particular antigen-binding site that has been selected by environmental antigens can be distributed among the various classes of antibodies, thereby acquiring the different biological properties of each class - When a B cell switches from making IgM and IgD to one of the secondary classes of antibody, an irreversible change at the DNA level occurs—a process called class-switch recombination - It entails the deletion of all the CH-coding sequences between the assembled VDJ-coding sequence and the particular CHcoding sequence that the cell is destined to express. CLASS-SWITCH RECOMBINATION vs. V(D)J RECOMBINATION - Class-switch recombination differs from V(D)J recombination in several ways. (1) It happens after antigen stimulation, mainly in germinal centers, and depends on helper T cells (2) It uses different recombination signal sequences, called switch sequences, which are composed of short motifs tandemly repeated over several kilobases (3) (3) It involves cutting and joining the switch sequences, which are non-coding sequences, and so the coding sequence is unaffected (4) (4) Most importantly, the molecular mechanism is different. It depends on AID, which is also involved in somatic hypermutation, rather than on RAG, which is responsible for V(D)J recombination. - The cytokines that activate class switching induce the production of transcription factors that activate transcription from the relevant switch sequences, allowing AID to bind to these sequences. Once bound, AID initiates switch recombination by deaminating some cytosines to uracil in the vicinity of these switch sequences. Excision of these uracils by uracil-DNA glycosylase is thought to lead somehow to double-strand breaks in the participating switch regions, which are then joined by a form of nonhomologous endjoining. - Thus, whereas the primary antibody repertoire in mice and humans is generated by V(D)J joining mediated by RAG, the secondary antibody repertoire is generated by somatic hypermutation and class-switch recombination, both of which are mediated by AID. a. Constant region is involved in Ig class switching - Generally a B cell make only a class at a time - Class switching: can occur in which BNB cell changes Ig class it syntehsises Ex. B cell making IgM can switch class - Early in its life, B cell produces IgM molecuels, which are receptors resp for recognition of specific antigen - At this time, constant region of heavy chain is encoded by in this case; 1st constant region gene, the "mu (symbol for micro)" gene - IF B cell later becomes plasma cell during humoral immune response => another deletion occurs in cell´s DNA of part of of constatnt region gene cluster => get different constant region of heavy chain => modification leads to class switching - However.. Ig has same variable region - therefore same antigen specificity - as IgM produced by parent B cell - New Ig proteins fall into 1 of 4 other classes (IgA, IgD, IgE, or IgG) => depending on which constant region genes is placed adj. to variable region genes i. What trigger class switching? - Th cells direct course of immune response and determine nature of attack on antigen - These Th cells induce class switching by sending cytokine signals - Cytokines binds to receptor on target B cell => generate signal transduction cascades => recombination and altered expression of Ig genes

29. TCR and MHC proteins?

- All T cells express cell-surface, antibodylike receptors (TCRs), which are encoded by genes that are assembled from multiple gene segments during T cell development in the thymus. - TCRs recognize fragments of foreign proteins that are displayed on the surface of host cells in association with MHC proteins - T cells are activated in peripheral lymphoid organs by antigen-presenting cells, which express peptide-MHC complexes, co-stimulatory proteins, and various cell -cell adhesion molecules on their cell surface - The most potent of these antigen-presenting cells are dendritic cells, which are specialized for antigen presentation and are required for the activation of naïve T cells. MHC CLASS I + II Class I and class II MHC proteins have crucial roles in presenting foreign protein antigens to T cells: (1) class I MHC proteins - present antigens to cytotoxic T cells (2) class II MHC proteins - present antigens to helper and regulatory T cells -- • Whereas class I proteins are expressed on almost all vertebrate cells, class II proteins are normally restricted to those cell types that interact with helper T cells, such as dendritic cells, macrophages, and B lymphocytes • Both classes of MHC proteins have a single peptide-binding groove, which binds small peptide fragments derived from proteins. Each MHC protein can bind a large set of peptides, which are constantly being produced intracellularly by normal protein degradation processes. However, class I MHC proteins mainly bind fragments produced in the cytosol, while class II MHC proteins mainly bind fragments produced in endocytic compartments • After they have formed inside the target cell, the peptide-MHC complexes are transported to the cell surface. Complexes that contain a peptide derived from a foreign protein are recognized by TCRs, which interact with both the peptide and the walls of the peptide-binding groove of the MHC protein. • T cells also express CD4 or CD8 co-receptors, which simultaneously recognize nonpolymorphic regions of MHC proteins on the antigen-presenting cell or target cell: helper cells and regulatory cells express CD4, which recognizes class II MHC proteins, while cytotoxic T cells express CD8, which recognizes class I MHC proteins.

1. B cells and antibodies?

- Antibodies => Synthesized exclusively by B cells - antibodies are produced in billions of forms, each with a different AA sequence. - Collectively called immunoglobulins (abbreviated as Ig), they are among the most abundant protein components in the blood, constituting about 20% of the total protein in plasma by weight - Mammals make five classes of antibodies, each of which mediates a characteristic biological response following antigene-binding. B cells make antibodies as both cell-surface antigen receptors and secreted proteins (1) The first antibodies made by a newly formed B cell are not secreted but are instead inserted into the plasma membrane, where they serve as receptors for antigen. - Each B cell has approximately 105 such receptors in its plasma membrane - Each of these receptors is stably associated with a complex of transmembrane proteins that activate intracellular signaling pathways when antigen on the outside of the cell binds to the receptor - Each B cell clone produces a single species of antibody, with a unique antigen-binding site - When an antigen (with the aid of a helper T cell) activates a naïve or a memory B cell, that B cell proliferates and differentiates into an antibody-secreting effector cell. Such effector cells make and secrete large amounts of soluble (rather than membrane-bound) antibody, which has the same unique antigen-binding site as the cell-surface antibody that served earlier as the antigen receptor

29. The constant and variable regions of Ig-chains?

- Antibody light and heavy chains consist of constant and variable regions - Comparison of the AAsequences of different antibody molecules reveals a striking feature with important genetic implications. Both light and heavy chains have a variable sequence at their N-terminal ends but a constant sequence at their C-terminal ends - Consequently, when we compare the amino acid sequences of many different chains, the C-terminal halves are the same or show only minor differences, whereas the N-terminal halves all differ - Light chains have a constant region about 110 amino acids long and a variable region of the same size - The variable region of the heavy chains is also about 110 amino acids long, but the constant region is about three or four times longer (330 or 440 amino acids), depending on the class.

29. The control of V(D)J recombination ensures that B cells are monospecific - How?

- B cells are monospecific. That is, all the antibodies that any one B cell produces have identical antigen-binding sites. This property enables antibodies to crosslink antigens into large aggregates, thereby promoting antigen elimination. It also means that an activated B cell secretes antibodies with the same specificity as that of its membrane-bound antibody receptor, guaranteeing the specificity of antibody responses. - To achieve monospecificity, each B cell must make only one type of VL region and one type of VH region. Since B cells, like other somatic cells, are diploid, each cell has six loci encoding antibody chains: two heavy-chain loci (one from each parent) and four light-chain loci (one k and one l from each parent). - If DNA rearrangements occurred independently in each heavy-chain locus and each light-chain locus, a single B cell could make up to eight different antibodies, each with a different antigen-binding site. In fact, however, each B cell uses only two of the six antibody loci: one of the two heavy-chain loci and one of the four light-chain loci. - Thus, each B cell must choose not only between its kappa and λ light-chain loci, but also between its maternal and paternal light-chain and heavy-chain loci. This second choice is called allelic exclusion. - Allelic exclusion also occurs in the expression of some genes that encode T cell receptors and genes that encode olfactory receptors in the nose. However, for most proteins that are encoded by autosomal genes, both the maternal and paternal gene copies in a cell are expressed about equally. - Allelic exclusion and kappa versus λ light-chain choice during B cell development depend on negative feedback regulation of the V(D)J recombination process. A functional rearrangement in one antibody locus suppresses rearrangements in all remaining loci that encode the same type of antibody chain. In B cell clones isolated from transgenic mice expressing a rearranged m-chain gene, for example, the rearrangement of all of the endogenous heavy-chain genes is usually suppressed. - Comparable results have been obtained for light chains. The suppression does not occur if the product of the rearranged gene fails to assemble into a receptor that inserts into the plasma membrane. It has therefore been proposed that either the receptor assembly process itself or extracellular signals that act on the receptor suppress further gene rearrangements. - Although no biological differences between the constant regions of k and l light chains have been discovered, there is an advantage in having two separate loci encoding light-chain variable regions. Having two separate loci increases the chance that a pre-B cell that has successfully assembled a VH-region coding sequence will then successfully assemble a VL-region coding sequence to become a B cell. This chance is further increased because, before a developing pre-B cell produces ordinary light chains, it makes surrogate light chains, which assemble with m heavy chains. - The resulting receptors are displayed on the cell surface and allow the cell to proliferate, producing large numbers of progeny cells, some of which are likely to succeed in producing light chains. - The production of a functional B cell is a complex and highly selective process: in the end, all B cells that fail to produce intact antibody molecules die by apoptosis.

CLASS-SWITCH RECOMBINATION vs. V(D)J RECOMBINATION

- Class-switch recombination differs from V(D)J recombination in several ways. (1) It happens after antigen stimulation, mainly in germinal centers, and depends on helper T cells (2) It uses different recombination signal sequences, called switch sequences, which are composed of short motifs tandemly repeated over several kilobases (3) (3) It involves cutting and joining the switch sequences, which are non-coding sequences, and so the coding sequence is unaffected (4) (4) Most importantly, the molecular mechanism is different. It depends on AID, which is also involved in somatic hypermutation, rather than on RAG, which is responsible for V(D)J recombination. - The cytokines that activate class switching induce the production of transcription factors that activate transcription from the relevant switch sequences, allowing AID to bind to these sequences. Once bound, AID initiates switch recombination by deaminating some cytosines to uracil in the vicinity of these switch sequences. Excision of these uracils by uracil-DNA glycosylase is thought to lead somehow to double-strand breaks in the participating switch regions, which are then joined by a form of nonhomologous endjoining. - Thus, whereas the primary antibody repertoire in mice and humans is generated by V(D)J joining mediated by RAG, the secondary antibody repertoire is generated by somatic hypermutation and class-switch recombination, both of which are mediated by AID.

1. Cellular immune response?

- Direcvted against antigens that have become establiushed within cell on host animal - Detects and destroys virus-infected or mutateted cells, such as cancer cells expressing unique proteins caused by mutaitons - T cells in lymph nodes, bloodstream, intercellular spaces carry out cellular immune responses - T cell have integral membrane proteins - T cell receptors - recognize and bind to antigens - T cell receptor similar to antibodies in structure and fx, each including specific molecular configuration that bind to speicifc antigens - Once T cell bound to antigen => initiates immune repsosne => result in total destruction of antigen-containing cell a. 2 types of effector T cells (1) T-helper cells (Th) (2) Cytotoxic T ells - Work along with proteins of major histocompatibility complex (MHC proteins) -. Present antigen on surfaces of cells an d contribute to immune systems tolerance for body´s own cells b. T cell receptors bind to antigens on cell surfaces - Like B cells, T cell possess specific membrane receptors - T cell receptor is not an Ig, but a glycoprotein with molecular weight of about ½ that of IgG i. Structure - Made up of 2 polypeptide chains - each encoded by separate gene - 2 chains have distinct regions with constant + variable AA seq. - Variable region: as in Ig, variable regions provide site for specific binding to antigens; but one major difference: whereas antibody can bind to any antigen, whether present on surface of cell or not; T cell receptor binds ONLY to antigen displayed by MHC protein on surface of antigen-presenting or target genes!!! ii. Activation of T cell - Activated by contact with specific antigen => proliferates and forms a clone - It s descendants form clones of 2 types of effector T cells (1) Cytotoxic T cells (Tc cells) - recognize virus-infected OR mutated cells and kill them by inducing lysis (2) T helper cells (Th cells) - assist both cellular + humoral immune responses c. MHC PROTEINS PRESENT ANTIGEN TO T CELLS - T cell receptor do not bind directly to antigen; theey bidn to antigens bound to cell surface glycoprotein- MHC protein - MHC protein is a cell surface marker of genetic individuality - Diversity of MHC protein => means many possibilities for presenting antigen to T cell receptor i. 2 classes of MHC proteins; both fx to present antigens to diff T lymphocytes - Tenk at du må oppnå 8; - MHC I * CD8 => 8 - MHC II* CD4 => 8 (1) Class I MHC - Proteins are present on surface of every nucleated cell in body - Enable Tc cells to recognize virus-infected cells and kill them (or cancer cell?) - Viral protein fragment that are antigenic are complexed with MHC I inside the cell and then the complex is carried to plasma membrane - A T c cell with appropriate T cell receptor bind to MHC-antigen copmpelx - TO ensure binding, Tc cell also has cell surface protein called CD8 => recognize MHC I (2) Class II MHC - Proteins found mostly on surfaces of B cells, macrophages, and other antigen present cells incl. dendritic cells - When one of these cells ingest pathogen such as bacterium, vbacterial antigens broken down ion pahosoem - MHC II moplecule may bind to one of fragment and carry it to cell surface => present to Th cells - Th cell have surface protein called CD4 - that recognizes and bind to MHC II i. Mechanism a) Antigen-rpeeesenting cell takes up antigen by phagocytosis b) Cell breaks down antigen into fragment in phagosome c) Class II MHC protein bind to antigen fragment d) MHC present antigen to Th cell

a. Ig Diversity result from DNA rearrangement and other mutations?

- Each mature B cell makes one- and only one - specific antibofdy targeted to signle epitope - 1 antibody gene: 2,100 bP DNA - 10 million different antibofies => 21 billion bp DNA!!! => 7x size of entire human genoime! Must be another way to genereate antibody diversity!!! i. Diversity - So.. instead of single gene encoding each Ig, genome of differentiating B cell has limited number of alleles for each of several regions (domain s) of protein => combination of these alleles generate diversisty - Shuffling this genetic deck => generate enormous immunological diversity - Each gene encoding Ig chjains is in reality a "supergene" assembled by means of genetic recombination from several clusters of smaller genes scattered along part of chromosome - Every cell in body has hundreds of Ig genes located in separate clusters that are potentially capable of participating in synthesis of both variable and constant regions of Ig chains; most body cells and tissues - these genes remain intact and separated from one another. But.. during B cell development - genes are cut out, arranged, and joined together in DNA recombination event => one gene form each cluster is chosen randomly for joining, and others are deleted - => in this manner, unique Ig supergene is assembled from randomly selected "parts" - each B-cell precursor assembles 2 supergene - (1) for for specific heavy chain and (2) assembled independently, for specific light chain EXAMPLE: Mice variable region of heavy chain assembled from 100 V, 30 D, and 6 J genes; each B cell randomly select one gene from each of these clusters to make final coding seq. (VDJ) of heavy chain variable region; so number of different heavy chains can be made through this random recombination is quite large 100 V*30D * 6J* => 18,000 possible combinations If we assume similar amount of diversity in light chain variable region, number of possible combinations of light-and heavy-chaisn varable region is: 18,000 different light chains x 18,000 diffgerent heavy chains => 324 million possibilities! - Evevn more diversity generated by various kinds of mutation that occur during recombination events - Mutation can occur through imprecise recombination and high spontaneous mutation rates in Ig genes - Genetic events irreversible - once final coding swq. Assembled for variable regions of B cells light and heavy chains => B cells epitope specificity cannot change! - => generate enormous diversity of IG from same starting genome - Once pre-transcriptiopnal processing compelteted, each supergene transcribed and then translated to produce Ig light chain or heavy chains => combine to form active Ig protein Variable region for heavy chain of specific antibofdy: - Encoded by one V gene, one D gene, one J gene - Each of these genes taken form ppool of like genes - V: variable gens - D: diversity genes - J: joining genes - join to constant region Constant region: selected from another pool of genes - Number of combination to make Ig heavy chain from tehse ppools of genes is (100V)(30D)(6J)(8C) => 144,000 - C: constant genes V,D,J: => makes up light chain C:= > make heavy chain a. Constant region is involved in Ig class switching - Generally a B cell make only a class at a time - Class switching: can occur in which BNB cell changes Ig class it syntehsises Ex. B cell making IgM can switch class - Early in its life, B cell produces IgM molecuels, which are receptors resp for recognition of specific antigen - At this time, constant region of heavy chain is encoded by in this case; 1st constant region gene, the "mu (symbol for micro)" gene - IF B cell later becomes plasma cell during humoral immune response => another deletion occurs in cell´s DNA of part of of constatnt region gene cluster => get different constant region of heavy chain => modification leads to class switching - However.. Ig has same variable region - therefore same antigen specificity - as IgM produced by parent B cell - New Ig proteins fall into 1 of 4 other classes (IgA, IgD, IgE, or IgG) => depending on which constant region genes is placed adj. to variable region genes i. What trigger class switching? - Th cells direct course of immune response and determine nature of attack on antigen - These Th cells induce class switching by sending cytokine signals - Cytokines binds to receptor on target B cell => generate signal transduction cascades => recombination and altered expression of Ig genes

29. IgD

- IgD molecules function mainly as an antigen receptor on B cells that have not been exposed to antigens - It has been shown to activate basophils and mast cells to produce antimicrobial factors.

29. Heavy and light chains?

- In addition to the five classes of heavy chains found in antibody molecules, higher vertebrates have two (2) types of light chains, kappa (k) and lambda (l) - which seem to be functionally indistinguishable - Either type of light chain may be associated with any of the heavy chains. An individual antibody molecule, however, always contains identical light chains and identical heavy chains: an IgG molecule, for instance, may have either k or l light chains, but not one of each. As a result, an antibody's antigen-binding sites are always identical - Such symmetry is crucial for the cross- linking function of secreted antibodies - All classes of antibody can be made in a membrane-bound form, as well as in a soluble, secreted form => The two forms differ only in the C-terminus of their heavy chain. The heavy chains of membrane-bound antibody molecules have a transmembrane hydrophobic C- terminus, which anchors them in the lipid bilayer of the B cell's plasma membrane. The heavy chains of secreted antibody molecules, by contrast, have instead a hydrophilic C-terminus, which allows them to escape from the cell. - The switch in the character of the antibody molecules made occurs because the activation of B cells by antigen (and helper T cells) induces a change in the way in which the H-chain RNA transcripts are made and processed in the nucleus.

29. How does imprecise joining of gene segments greatly increases the diversity of V regions?

- In the process of V(D)J recombination, site-specific recombination joins separate antibody gene segments together to form a functional VL- or VH-region coding sequence - Conserved recombination signal sequences flank each gene segment and serve as recognition sites for the joining process, ensuring that only appropriate gene segments recombine. Thus, for example, a light-chain V segment will always join to a J segment but not to another V segment. - An enzyme complex called the V(D)J recombinase mediates joining. This complex contains two proteins that are specific to developing lymphocytes, as well as enzymes that help repair damaged DNA in all our cells. - Two closely linked genes called Rag1 and Rag2 (Rag = recombination activating genes) encode the lymphocyte-specific proteins of the V(D)J recombinase, RAG1 and RAG2. - To mediate V(D)J joining, the two proteins come together to form a complex (called RAG), which functions as an endonuclease, introducing double-strand breaks precisely between the gene segments to be joined and their flanking recombination signal sequences. - RAG then initiates the rejoining process by recruiting enzymes involved in DNA double- strand repair in all cells. Mice or humans deficient in either of the two Rag genes or in non-homologous end joining are highly susceptible to infection because they are unable to carry out V(D)J recombination and consequently do not have functional B or T cells, a condition called severe combined immunodeficiency (SCID). - RAG-1 element evolved from an ancient transposable element related to the Transib superfamily - During the joining of antibody (and T cell receptor) gene segments, as in nonhomologous end-joining, a variable number of nucleotides are often lost from the ends of the recombining gene segments, and one or more randomly chosen nucleotides may also be inserted. This random loss and gain of nucleotides at joining sites is called junctional diversification, and it enormously increases the diversity of V-region coding sequences created by V(D)J recombination, specifically in the third hypervariable region. This increased diversification comes at a price, however. In many cases, it will shift the reading frame to produce a nonfunctional gene. Because roughly two in every three rearrangements are "nonproductive" in this way, many developing B cells never make a functional antibody molecule and consequently die in the bone marrow. - B cells making functional antibody molecules that bind strongly to self antigens in the bone marrow would be dangerous. Such B cells maintain expression of the RAG proteins and can undergo a second round of V(D)J recombination in a light-chain locus (usually a k locus), thereby changing the specificity of the cell-surface antibody they make—a process referred to as receptor editing. - To provide a further layer of protection, clonal deletion eliminates those self-reactive B cells that fail to change their specificity.

What is the antibody hypervariable region?

- It is the N-terminal ends of the light and heavy chains that come together to form the antigen-binding site, and the variability of their AA sequences provides the structural basis for the diversity of antigen-binding sites - The greatest diversity occurs in three (3) small hypervariable regions in the variable regions of both light and heavy chains; the remaining parts of the variable region, known as framework regions, are relatively constant - Only about 5-10 amino acids in each hypervariable region form the actual antigen-binding site. As a result, the size of the epitopes that an antibody recognizes is generally comparably small. It can consist of fewer than 10 amino acids on the surface of a globular protein, for example.

29. How are antibody genes assembled from separate gene segments during B cell development?

- Mice and humans produce their primary antibody repertoire by joining separate antibody gene segments together during B cell development. - Each type of antibody chain kappa (k) light chains, λ (l) light chains, and heavy chains - is encoded by a separate locus on a separate chromosome - Each locus contains a large number of gene segments encoding the V region of an antibody chain, and one or more gene segments encoding the C region - During the development of a B cell in the bone marrow (or fetal liver), a complete coding sequence for each of the two antibody chains to be synthesized is assembled by site-specific genetic recombination. In addition to bringing together the separate gene segments of the antibody gene, these rearrangements also activate transcription from the gene promoter through changes in the relative positions of the enhancers and silencers acting on the gene. Thus, a complete antibody chain can be synthesized only after the DNA has been rearranged - Each light-chain V region is encoded by a DNA sequence assembled from two gene segments - a long V gene segment and a short joining, or J gene segment, which is encoded elsewhere in the genome). - The SLIDE illustrates the sequence of events involved in the production of a human k light-chain polypeptide from its separate gene segments - Each heavy-chain V region is similarly constructed by combining gene segments, but here an additional diversity segment, or D gene segment, is also required - The large number of inherited V, J, and D gene segments available for encoding antibody chains contributes substantially to antibody diversity, and the combinatorial joining of these segments (called combinatorial diversification) greatly increases this contribution - Any of the 40 V segments in the human k light-chain locus, for example, can be joined to any of the 5 J segments, so that this locus can encode at least 200 (40 x 5) different kappa-chain V regions - Similarly, any of the 40 V segments in the human heavy-chain locus can be joined to any of the 25 D segments and to any of the 6 J segments to encode at least 6000 (40 x 25 x 6) different heavy- chain V regions - The combinatorial diversification resulting from the assembly of different combinations of inherited V, J, and D gene segments is an important mechanism for diversifying the antigen-binding sites of antibodies. By this mechanism alone, called V(D)J recombination, a human can produce 320 different VL regions (200 k and 120 l) and 6000 different VH regions. - In principle, these could then be combined to make about 1.9 x 106 (320 x 6000) different antigen-binding sites - The joining mechanism itself greatly increases this number of possibilities (probably more than 108-fold), making the primary antibody repertoire much larger than the total number of B cells (about 1012) in a human - In the "germ-line" DNA (where the antibody genes are not rearranged and are therefore not being expressed), the cluster of five J gene segments is separated from the C-region coding sequence by a short intron and from the 40 V gene segments by thousands of nucleotide pairs - During the development of a B cell, a randomly chosen V gene segment (V3 in this case) is moved to lie precisely next to one of the J gene segments (J3 in this case). The "extra" J gene segments (J4 and J5) and the intron sequence are transcribed (along with the joined V3 and J3 gene segments and the C-region coding sequence) and then removed by RNA splicing to generate mRNA molecules with contiguous V3, J3, and C sequences, as shown. - These mRNAs are then translated into k light chains. A J gene segment encodes the C-terminal 15 or so amino acids of the V region, and a short sequence containing the V-J segment junction encodes the third hypervariable region of the light chain, which is the most variable part of the V region.

29. Ig domains?

- The light and heavy chains are composed of repeating Ig domains - Both light and heavy chains are made up of repeating segments - each about 110 AA long and each containing one (1) intra-chain disulfide bond - Each repeating segment folds independently to form a compact functional unit called an immunoglobulin (Ig) domain. - As shown in, a light chain consists of one variable (VL) and one constant (CL) domain. - VL pairs with the variable (VH) domain of the heavy chain to form the antigen-binding region - CL pairs with the first constant domain of the heavy chain (CH1), and the remaining constant domains of the heavy chains form the Fc region, which determines the other biological properties of the antibody - Most heavy chains have three constant domains (CH1, CH2, and CH3), but those of IgM and IgE antibodies have four.

29. IgG

- The major class of immunoglobulin in the blood is IgG, which is a four-chain monomer produced in large quantities during secondary antibody responses. - Besides activating complement, the tail region of an IgG molecule binds to specific receptors on macrophages and neutrophils. Largely by means of such Fc receptors (so-named because antibody tails are called Fc regions), these phagocytic cells bind, ingest, and destroy infecting microorganisms that have become coated with the IgG antibodies produced in response to the infection - Some IgG subclasses are the only antibodies that can pass from mother to fetus via the placenta.

1. Structures of antibodies?

- The simplest antibodies are Y-shaped molecules with two identical antigene- binding sites, one at the tip of each arm of the Y. - The basic structural unit of an antibody molecule consists of four (4) polypeptide chains, two (2) identical light (L) chains (each containing about 220 amino acids) and two (2) identical heavy (H) chains (each usually containing about 440 aminoacids) - A combination of noncovalent and covalent (disulfide) bonds holds the four chains together. The molecule is composed of two identical halves, each with the same antigen-binding site - Both light and heavy chains usually cooperate to form the antigen-binding surface. a. Structure of Ig - Several types of Ig, but all contain tetramer - consisting of 4 polypeptiode chains - Each Ig molecule - 2 polypeptides are identical light chains, and 2 are identical heavy chains - Disulfide bond: hold chains together i. Polypeptide chain - Each polypeptide chain has a constant region + variable region 1) Constant region: AA seq. - similar among Ig; determine the destination and fx - the class of each Ig 2) Variable region: AA seq. are different for each specific Ig; 3D antigen-binding sites are determined by their 2nd strucutres and are responsible for antibody specisifity - 2 antigen-binding sites on each IG are identical, making antibody bivalent "("two-binding") => ability to bind 2 antigen molecules at one ; along with presence of multiple epitopes on surface of many antigens (incl. large proteins, viruses, bacteria) =>mpermit antibodies to form large complexces with antigen => complexes easy target for ingestion and breakdown by phagocytes

29. IgE

- The tail region of IgE molecules, which are four-chain monomers, binds with unusually high affinity to yet another class of Fc receptors. - These receptors are located on the surface of mast cells in tissues and of basophils in the blood. - Antigene-binding triggers the mast cell or basophil to secrete a variety of cytokines and biologically active amines, especially histamine. - The histamine causes blood vessels to dilate and become leaky, which in turn helps white blood cells, antibodies, and complement components to enter sites where mast cells have been activated - The release of amines from mast cells and basophils is largely responsible for the symptoms of such allergic reactions as hay fever, asthma, and hives - In addition, mast cells secrete factors that attract and activate WBC called eosinophils - Eosinophils also have Fc receptors that bind IgE molecules, and they can kill extracellular parasitic worms, especially if the worms are coated with IgE antibodies.

29. Antigen-driven Somatic hypermutation => fine-tunes antibody responses => HOW?

- With the passage of time after immunization, there is usually a progressive increase in the affinity of the antibodies produced against the immunizing antigen. This phenomenon, known as affinity maturation, is due to the accumulation of point mutations in both heavy-chain and light-chain V region coding sequences. - The mutations occur long after the coding regions have been assembled. After B cells have been stimulated by antigen and helper T cells in a peripheral lymphoid organ, some of the activated B cells proliferate rapidly in the lymphoid follicles and form structures called germinal centers. Here, the B cells mutate at the rate of about one mutation per V-region coding sequence per cell generation. - Because this is about a million times greater than the spontaneous mutation rate in other genes and occurs in somatic cells rather than germ cells, the process is called somatic hypermutation. Very few of the altered antibodies generated by hypermutation will have an increased affinity for the antigen. Because the same antibody genes produce the antigen receptors on the B cell surface, the antigen will stimulate preferentially those few B cells that do make such antibodies with increased affinity for the antigen - Clones of these altered B cells will preferentially survive and proliferate, especially as the amount of antigen decreases to very low levels late in the response. Most other B cells in the germinal center will die by apoptosis. Thus, as a result of repeated cycles of somatic hypermutation, followed by antigen- driven proliferation of selected clones of effector and memory B cells, antibodies of increasingly higher affinity become abundant during an immune response, providing progressively better protection against the pathogen. (In some mammals, including sheep and cows, a similar somatic hypermutation also plays a major part in diversifying the primary antibody repertoire before B cells encounter their antigen.) - Somatic hypermutation is carried out with an enzyme called activation-induced deaminase (AID). It is expressed specifically in activated B cells and deaminates cytosine (C) to uracil (U) in transcribed V- region coding DNA. The deamination produces U:G mismatches in the DNA double helix, and the repair of these mismatches produces various types of mutations, depending on the repair pathway used. - Somatic hypermutation affects only actively transcribed V-region coding sequences, possibly because the AID enzyme is specifically loaded onto RNA transcripts. AID is also required when activated B cells switch from IgM production to the production of other classes of antibody.

29. What are the main stages in B cell development?

1) Pro-B cells (make only "mu"-chains) => pre-B cells 2) Pre-B cells (mu chains associate with surrogate light chains => insert into plasma membrane) 3) Light chains combine with mu chains - replace surrogate light chains => form 4-chain IgM molecules (each with 2 "mu" chains + 2 light chains) 4) Insert into plasma membrane=> fx as receptors for antigen => immature naïve B cell 5) Leave bone marrow 6) Cell produce cell-surface IgD molecules w/ same antigen-binding site as IgM molecules => now called mature naïve B cell 7) Mature naïve b cell => respond to foreign antigen in peripheral lymphoid organs 8) Foreign antigen + helper T cells in peripheral lymphoid organs => mature naïve B cell proliferate + differentiate into either antibody-secreting cells or memory cells - All of the stages occur independently of antigen. The first cells in the B cell lineage that make Ig are pro-B cells, which make only μ chains, but they remain in the endoplasmic reticulum until surrogate light chains are made - They give rise to pre-B cells, in which the μ chains associate with so-called surrogate light chains and insert into the plasma membrane. - Signaling from this pre-B cell receptor is required for the cell to progress to the next stage of development, where it makes normal light chains. Although not shown, all of the cell-surface Ig molecules are associated with transmembrane proteins that help convey signals to the cell interior - The light chains combine with the μ chains, replacing the surrogate light chains, to form four-chain IgM molecules (each with two μ chains and two light chains). These molecules then insert into the plasma membrane, where they function as receptors for antigen - At this point, the cell is called an immature naïve B cell. After leaving the bone marrow, the cell starts to produce cell-surface IgD molecules as well, with the same antigen-binding site as the IgM molecules. It is now called a mature naïve B cell. It is this cell that can respond to foreign antigen in peripheral lymphoid organs. When the immunoglobulins are activated by their specific foreign antigen and helper T cells in peripheral lymphoid organs, mature naive B cells proliferate and differentiate into either antibody-secreting cells or memory cells (not shown)

29. How is the organization of the DNA sequences that encode the constant region of an antibody?

Constant region of antibody: - The similarity in their domains suggests that antibody chains arose during evolution by a series of gene duplications, beginning with a primordial gene coding for a single 110 amino acid domain of unknown function - Each domain of the constant region of a heavy chain is encoded by a separate coding sequence (exon), which supports this hypothesis - The coding sequences (exons) for each domain and for the hinge region are separated by noncoding sequences (introns). - The intron sequences are removed by splicing the primary RNA transcripts to form mRNA. The presence of introns in the DNA is thought to have facilitated accidental duplications of DNA segments that gave rise to the antibody genes during evolution

29. IgA?

DIMERIC IgA is the principal class of antibody in secretions, including saliva, tears, milk, and respiratory and intestinal secretions - IgA is a four-chain monomer in the blood which is assembled into a dimer by the addition of two other polypeptide chains before it is released into secretions - It is transported through secretory epithelial cells from the extracellular fluid into the secreted fluid by transcytosis mediated by another type of Fc receptor that is unique to secretory epithelia.

29. How is generation of antibody diversity? - MECHANISM (2)

MECHANISM Antibody diversity is generated in two steps (1) First, before antigen stimulation, developing B cells join together separate gene segments in DNA in order to create the genes that encode the primary repertoire of low-affinity IgM and IgD antibodies (2) Second, after antigen stimulation, the assembled antibody-coding genes can undergo two further changes, MUTATIONS that can increase the affinity of the antigen-binding site and DNA rearrangements that switch the class of antibody made Together, these changes produce the secondary repertoire of high-affinity IgG, IgA, and IgE antibodies. --- Antibodies are proteins, and proteins are encoded by genes. Antibody diversity therefore poses a special genetic problem: how can an animal make more antibodies than there are genes in its genome? This problem is not quite as formidable as it might first appear. - Recall that the variable regions of the light and heavy chains of antibodies usually combine to form the antigen-binding site. Thus, an animal with 1000 genes encoding light chains and 1000 genes encoding heavy chains could, in principle, combine their products in 1000 x 1000 different ways to make 106 different antigen-binding sites (although, in reality, not every light chain can combine with every heavy chain to make an antigen- binding site). - Nonetheless, unique genetic mechanisms have evolved to enable adaptive immune systems to generate an almost unlimited number of different light and heavy chains in a remarkably economical way.

29. IgM?

PENTAMERIC ACTIVATE COMPLEMENT SYSTEM - IgM is not only the first class of antibody to appear on the surface of a developing B cell; It is also the major class secreted into the blood in the early stages of a primary antibody response, on first exposure to an antigen - Unlike IgM, IgD molecules are secreted in only small amounts and seem to function mainly as cell-surface receptors for antigen. - In its secreted form, IgM is a pentamer composed of five (5) four-chain units, giving it a total of 10 antigen-binding sites (5x2 = 10) - Each pentamer contains one copy of another polypeptide chain, called a J (joining) chain. The J chain is produced by IgM-secreting cells and is covalently inserted between two adjacent tail regions - When an antigen with multiple identical epitopes binds to a single secreted pentameric IgM molecule, it alters the structure of the pentamer, allowing it to activate the complement system.

29. Primary and secondary antibody repertoire?

PRIMARY ANTIBODY REPERTOIRE (Preimmune) - Even in the absence of antigen stimulation, a human can probably make more than 1012 different antibody molecules—its preimmune, primary antibody repertoire. - The primary repertoire consists of IgM and IgD antibodies and is apparently large enough to ensure that there will be an antigen-binding site to fit almost any potential epitope, albeit with low affinity. SECONDARY ANTIBODY REPERTOIRE - After stimulation by antigen (and helper T cells), B cells can switch from making IgM and IgD to making other classes of antibodies - a process called class switching - In addition, the affinity of these antibodies for their antigen progressively increases over time - a process called affinity maturation. - Thus, antigen stimulation => generates a secondary antibody repertoire, with a greatly increased diversity of both Ig classes and antigen- binding sites

29. What are the 5 classes of antibodies?

The five (5) classes of antibodies there are five classes of antibodies: "GAMED" - IgG (gamma) => major class secreted during secondary immune response => activate complement + opsonize (+ only type that cross placenta) - IgA (alfa) => form dimer, principal antibody in secretions - IgM (μ) => major class secreted during primary immune response => activate complement system - IgE (ε) => mast cells in tissues, basophils in blood => secretete active amines, especially histamine! Mast cell secrete also factor that attract WBC called eosinophils => kill parasitic worm exctracellularly if worm coated with IgE - IgD (delta) => function mainly as an antigen receptor on B cells that have not been exposed to antigens. It has been shown to activate basophils and mast cells to produce antimicrobial factors. Each with its own class of heavy chain (gamma, alfa, mu, e, delta) - IgA molecules have alfa chains - IgG molecules have gamma chains, and so on. - In addition, there are a number of subclasses of IgG and IgA immunoglobulins; for example, there are four (4) human IgG subclasses (IgG1, IgG2, IgG3, and IgG4), having gamma1, gamma2, gamma3, and gamma 4 heavy chains, respectively. - The various heavy chains give a distinctive conformation to the hinge and tail regions of antibodies, so that each class (and subclass) has characteristic properties of its own - IgM, which has μ heavy chains, is ALWAYS the first class of antibody that a developing B cell makes, although many B cells eventually switch to making other classes of antibody when an antigen stimulates them -- a. The 5 classes of Ig - Constant region of heavy chain detemrioen class of Ig - ex. Wheteher it will be an integral membrane receptor (e.g. on surface of B cell)= or soluble antibody that is secreted into bloodstream (1) IgG - General structure: monomer - Location: free in blood plasma; about 80% of circulating antibodies - Function: most abundant antibody in primary + secondary immjune resposnes; crosses placenta and provides passive immunization to fetus - Most abundant class is IgG => soluble antibody protein make up 80% of total Ig content of bloodstrum; made in greatest quantity during 2nd immune response - IgG defend body in several way; ex. Binds to antigens => become attached by heavy chains to macrophages => attachment permits macrophages to destroy antigen by phagocytosis (2) IgM - General structure: pentamer - Location: surface of B cell; free in blood plasma - Function: antigen receptor on B cell membrane; 1st class of antibodies released by B cell during primary response (3) IgD - General structure: monomer - Location: Surface of B cell - Function: cell surface receptor of mature B cell; important in B cell activation (4) IgA - General structure: dimer - Location: saliva, tears, milk, ando thjer body secretions - Function: protects mucosal surfaces; prevents attachment of pathogens to epithelial cells (5) IgE - General structure: monomer - Location: secreted by plasma cells in skin and tissues lining GI and respiratory tract - Function: binds to mast cells and basophils to sensitize them to subsequent bidning of antgen => trigger release of histamine => contribuees to inflammation + some allergic responses

29. WHAT ARE THE 3 MAIN FX DISTINCT CLASS OF T CELLS?

There are three (3) main functionally distinct classes of T cells (1) Cytotoxic T cells - kill infected cells directly by inducing them to undergo apoptosis (2) Helper T cells - help activate B cells to make antibody responses - help activate cytotoxic T cells to kill their target cells - help activate dendritic cells to stimulate T cell responses - help activate macrophages to destroy microorganisms that either invaded the macrophage or were ingested by it. (3) Regulatory T cells - suppress the activity of effector T cells and dendritic cells and are crucial for self tolerance