chapter 4 and 5 essay questions

4-22 A. What is the basic structural difference between the immunoglobulins produced by B cells and their descendants before antigen encounter and after antigen encounter? B. Say which cell type(s) produce each form. C. In which way do these different molecular forms resemble each other?

4-22 A. Before antigen encounter, antibodies are produced in a membrane-bound form. After antigen encounter, antibodies are secreted in a soluble form. B. Immature, mature, and memory B cells produce membrane-bound antibodies. Plasma cells secrete soluble antibodies. C. The membrane-bound and soluble forms of antibody produced by a particular B cell possess the same antigen specificity.

4-47 Explain how mature, naive B cells co-express IgM and IgD

4-47 Naive B cells express IgM and IgD simultaneously through a mechanism involving alternative ways of processing the RNA transcript before translation. A primary transcript containing leader (L), V, D, J, Cμ, and Cδ is produced first. This transcript contains two distinct polyadenylation signal sequences, one following the Cμ exons (pA1) and the other following the Cδ exons (pA2). Processing results in the removal of either Cμ or Cδ exons (plus introns) through alternative splicing. The resulting mRNAs, which encode either Cμ or Cδ, are polyadenylated at the pA1 or pA2 site, respectively.

5-67 T-cell receptors do not undergo isotype switching. Suggest a possible reason for this.

5-67 T-cell receptors are not made in a secreted form, and their constant regions do not contribute to T-cell effector function. Other molecules secreted by T cells are used for effector functions. There is therefore no need for isotype switching in T cells, and the T-cell receptor loci do not contain numerous alternative C genes.

5-76 A. What is the difference between MHC variation due to multigene families and that due to allelic polymorphism? B. How does MHC variation due to multigene families and allelic polymorphism influence the antigens that a person's T cells can recognize?

5-76 A. Multigene family refers to the presence of multiple genes for MHC class I and MHC class II molecules in the genome, encoding a set of structurally similar proteins with similar functions. MHC polymorphism is the presence of multiple alleles (in some cases several hundreds) for most of the MHC class I and class II genes in the human population. B. T cells recognize peptide antigens in the form of peptide:MHC complexes, which they bind using their T-cell receptors. To bind specifically, the T-cell receptor must fit both the peptide and the part of the MHC molecule surrounding it in the peptide-binding groove. (i) Because each individual expresses a number of different MHC molecules from the MHC class I and class II multigene families, the T-cell receptor repertoire is not restricted to recognizing peptides that bind to just one MHC molecule (and thus all must have the same peptide-binding motif). Instead, the T-cell receptor repertoire can recognize peptides with different peptide-binding motifs during an immune response, increasing the likelihood of antigen recognition and, hence, T-cell activation. (ii) The polymorphism in MHC molecules is localized to the regions affecting T-cell receptor and peptide binding. Thus, a T-cell receptor that recognizes a given peptide bound to variant 'a' of a particular MHC molecule is likely not to recognize the same peptide bound to variant 'b' of the same MHC molecule. Polymorphism also means that the MHC molecules of one person will bind a different set of peptides from those in another person. Taken together, these outcomes mean that because of MHC polymorphism, each individual recognizes a somewhat different range of peptide antigens using a different repertoire of T-cell receptors.

5-77 What evidence supports the proposal that MHC diversity evolved by natural selection caused by infectious pathogens rather than exclusively by random DNA mutations?

5-77 MHC polymorphisms are non-randomly localized, predominantly to the region of the molecule that makes contact with peptide and T-cell receptors. Random DNA mutations, in contrast, would be scattered through the gene, giving rise to amino acid changes throughout MHC molecules and not just in those areas important for peptide binding and presentation.

4-21 A. What is an epitope? B. Define the term multivalent antigen. C. How does a linear epitope differ from a conformational epitope? D. Do antibodies bind their antigens via noncovalent bonding or via covalent bonding?

4-21 A. An epitope is the specific part of the antigen that is recognized by an antibody and binds to the complementarity-determining regions in the antibody variable domains and are sometimes referred to as antigenic determinants. Epitopes can be part of a protein or can be carbohydrate or lipid structures present in the glycoproteins, polysaccharides, glycolipids, and proteoglycans of pathogens. B. Multivalent antigens are complex macromolecules that contain more than one epitope. C. Linear epitopes are epitopes in proteins that comprise a contiguous amino acid sequence. They are also called continuous epitopes. In contrast, a conformational epitope is formed by amino acids that are brought together as a result of protein folding and are not adjacent in the protein sequence. Conformational epitopes are also known as discontinuous epitopes. D. Antibodies bind antigens via noncovalent bonding such as hydrogen bonds, hydrophobic interactions, van der Waals forces, and electrostatic attraction.

4-24 A. Explain why catalytic antibodies are attracting attention in the medical field. B. Provide two potential examples.

4-24 A. Catalytic antibodies bind with a high degree of specificity to the target antigen and facilitate a chemical conversion of that antigen in a similar manner to the action of enzymes. If this reaction results in the alteration of that antigen so that it is no longer able to carry out its undesirable effect in the body, the catalytic antibody has therapeutic value. B. Examples would include: (1) converting toxic substances to less harmful molecules, and (2) converting addictive drugs to derivatives that no longer possess psychostimulating effects.

Describe the structure of an antibody molecule and how this structure enables it to bind to a specific antigen. Include the following terms in your description: heavy chain (H chain), light chain (L chain), variable region, constant region, Fab, Fc, antigen-binding site, hypervariable region, and framework region.

4-3 An antibody molecule is made of four polypeptide chains—two identical heavy chains and two identical and smaller light chains, with a total molecular weight of approximately 150 kDa. Each chain is made up of a series of structurally similar domains known as immunoglobulin domains. The amino-terminal portion of each H chain combines with one L chain, and the two carboxy-terminal portions of the H chains combine with each other, forming a Y-shaped quaternary structure. Disulfide bonds hold the H and L chains together, hold the two H chains together (interchain disulfide bonds), and stabilize the domain structure of the chains (intrachain disulfide bonds). The arms of the antibody molecule are called Fab (fragment antigen binding) and interact with antigen. The stalk is called Fc (fragment crystallizable) and is made up of H chains only. The amino-terminal domains of an H and an L chain together make up a site that binds directly to antigen and varies greatly between different antibodies. These domains are referred to as the variable region, and each antibody has two identical antigen-binding sites. The remaining domains of both H and L chains are the same in all antibodies of a given class (isotype). These domains are referred to as the constant region. The variable region of each chain includes hypervariable regions of amino acid sequences that differ the most between different antibodies. These are nested within less variable sequences known as the framework regions. The hypervariable regions make loops at one end of the domain structure and are also known as complementarity-determining regions because they confer specificity on the antigen-binding site.

4-37 A. Explain briefly how a vast number of immunoglobulins of different antigen specificities can be produced from the relatively small number of immunoglobulin genes present in the genome. Include the following terms in your explanation: somatic recombination; germline configuration; V, D, and J segments. B. What is the final arrangement of gene segments in the rearranged immunoglobulin heavy-chain gene V region, and in what order do these gene segment rearrangements occur? C. In what order do the various immunoglobulin loci rearrange?

4-37 A. In developing B cells, gene rearrangements within the genetic loci for immunoglobulin light and heavy chains can produce an almost unlimited variety of different variable regions, and thus produce the huge repertoire of antibodies with different specificities for many types of antigen. This gene rearrangement mechanism is called somatic recombination. In the germline configuration, before gene rearrangement, the immunoglobulin loci in progenitor B cells are composed of sequences encoding the constant regions and families of gene segments encoding different portions of the variable region. Heavy-chain loci contain a series of gene segments called variable (V), diversity (D), and joining (J). Light-chain loci contain only V and J gene segments. In somatic recombination in developing B cells, one of each family of gene segments is randomly selected and joined together to give a complete variable-region sequence, which is subsequently expressed as an immunoglobulin heavy or light chain. Immunoglobulin gene rearrangement is irreversible, leading to permanent alteration of the chromosome; it occurs exclusively in B cells. B. A D gene segment first joins to a J to form DJ, followed by a V becoming joined to DJ to form VDJ, which encodes a complete variable region. C. The heavy-chain locus rearranges before the light-chain loci. For light chains in humans, the κ locus rearranges first and is followed by the λ locus only if both κ loci fail to produce a successful rearrangement.

4-39 What would be the effect of a genetic defect that resulted in a lack of somatic recombination between V, D, and J segments?

4-39 An individual with this genetic defect would be unable to rearrange either immunoglobulin or T-cell receptor genes somatically. There would be a severe combined immunodeficiency (SCID) owing to the absence of mature B cells and T cells.

4-42 How do recombination signal sequences ensure that gene segment rearrangement occurs in the right order?

4-42 Gene rearrangement by somatic recombination involves recombination signal sequences (RSSs) that flank V, D, and J segments and are recognized by the enzymes involved in cutting and rejoining the gene segments. An RSS is composed of a conserved nonamer sequence and heptamer sequence separated by a spacer region. There are two types of RSS, one with a spacer of 12 bp and one with a spacer of 23 bp. To ensure that segments are brought together in the right order, an RSS with a 12-bp spacer is always brought together with one with a 23-bp spacer. This is called the 12/23 rule. This ensures that in the heavy-chain locus, V rearranges to DJ and not directly to J or another V, and in the light-chain locus, V rearranges to J and not to another V.

4-43 How is additional diversity introduced into the variable region by the molecular mechanism of somatic recombination? Include the following terms in your answer: junctional diversity, P nucleotides, N nucleotides, terminal deoxynucleotidyl transferase (TdT).

4-43 The rejoining and repair of DNA during the recombination process leads to additional variation in sequence at the junctions between the rearranged gene segments. This is called junctional diversity and contributes considerably to the final diversity of immunoglobulin specificities. Two sources of junctional diversity are introduced: P (palindromic) and N (nontemplated) nucleotides. P nucleotides are generated through endonuclease activity and repair around a hairpin loop at the ends of the gene segments to be joined. N nucleotides are nucleotides added at random at the junctions by terminal deoxynucleotidyl transferase (TdT) activity.

4-44 The third hypervariable region (CDR3) is the most variable site in an immunoglobulin V region. It differs in its composition between the light-chain and heavy-chain V regions. Explain what this difference is and how the diversity in CDR3 is generated.

4-44 CDR3 of the light chain is composed mainly of the coding joint between the V and J segments, which is formed during somatic recombination, with junctional diversity being generated by the addition of P and N nucleotides. CDR3 of the heavy chain is composed mainly of the D gene segment plus its coding joints with a V gene segment on one side and a J gene segment on the other. P and N nucleotides are also added to these joints during recombination. In addition, the D gene segment sequences differ between immunoglobulins.

4-48 Describe the process responsible for altering the expression of membrane-bound immunoglobulin to secreted antibody.

4-48 Whether immunoglobulin is expressed as a transmembrane-anchored protein or a secreted protein is determined by alternative processing of the heavy-chain RNA transcript. All the heavy-chain C genes contain MC (membrane-coding) exons, which encode the transmembrane region and cytoplasmic tail, and an SC (secretion-coding) exon, which encodes the carboxy terminus of the secreted antibody. The primary RNA transcript contains the MC and SC exons. In naive resting B cells or memory B cells, cleavage and polyadenylation of the transcript at a site (pAm) following the MC exons and deletion of the SC exon by RNA splicing produces the membrane-bound immunoglobulin. On B-cell activation and differentiation into plasma cells, the SC exon is retained in the transcript, and a polyadenylation signal sequence, pAs, immediately following it is used to produce an mRNA encoding the secreted form of the heavy chain.

4-52 A. What are the functions of the Igα and Igβ proteins? B. Explain why it is desirable that they do not vary in sequence from cell to cell in the same way that immunoglobulins do.

4-52 A. Igα and Igβ are essential for escorting immunoglobulins from the endoplasmic reticulum membrane to the cell membrane, where they remain associated with the immunoglobulin to form the functional B-cell antigen receptor. The long cytoplasmic tails of Igα and Igβ contain amino acid motifs that interact with intracellular signaling proteins after the receptor has been activated by the binding of antigen to the immunoglobulin. B. Igα and Igβ proteins have no need to be variable, because they do not interact directly with antigen. Igα and Igβ perform specific signaling functions, which require particular amino acid sequences and also have evolved a sequence and structure that enable them to interact with all the different immunoglobulin isotypes. Extensive variation in Igα and/or Igβ could therefore compromise their interaction with immunoglobulins and their signal transduction capabilities.

4-66 A. What is affinity maturation and what molecular process enables it to occur? B. Describe this process and its consequences.

4-66 A. Affinity maturation is the phenomenon observed during a B-cell response in which antibodies with increasing affinity for the antigen are produced as the response proceeds. This occurs as a result of the process known as somatic hypermutation. B. In somatic hypermutation, which occurs only in activated B cells, random point mutations are introduced into the rearranged V regions of H-chain and L-chain genes at a rate six orders of magnitude higher than spontaneous mutation. Some of these mutations give rise to immunoglobulin with higher affinity for the antigen than the original immunoglobulin. Those B cells producing higher-affinity surface immunoglobulin will be preferentially selected for activation by the antigen and will come to dominate the response, differentiating into plasma cells producing high-affinity antibodies.

4-67 A. What is isotype switching? B. Explain the molecular mechanism of isotype switching. C. Why is isotype switching important?

4-67 A. Isotype switching is the process by which antibodies change their heavy-chain constant regions so as to acquire different effector functions, while preserving the variable region and antigen specificity. The light chain is unaffected. B. The molecular mechanism involves a recombination between sequences, called switch regions, which lie upstream (on the 5′ side) of heavy-chain C genes. All heavy-chain C genes except Cδ have a switch region. Recombination between two switch regions results in the excision of DNA (as a circular DNA molecule) between the two and the movement of the new heavy-chain C gene next to the preserved V region. Transcription will produce an mRNA encoding the same V-region sequence and the new C region. Switching can occur between the first switch region and any other switch region that lies downstream (on the 3′ side). Isotype switching is not random but is influenced by T-cell cytokines. C. Isotype switching is important because the different antibody isotypes have different effector functions, and efficient immune responses rely upon the production of the most appropriate effector function to combat the particular pathogen.

4-68 Which immunoglobulin isotypes (out of IgM, IgG1, IgG2, IgG3, IgG4, IgA, IgE, and IgD) participate in (a) neutralization; (b) opsonization; (c) sensitization for killing by NK cells; (d) sensitization of mast cells; (e) activation of complement? Which isotypes (f) are transported across epithelium; (g) are transported across the placenta; (h) diffuse into extravascular sites?

4-68 A. Neutralization: IgM, IgG1, IgG2, IgG3, IgG4, IgA B. Opsonization: IgG1, IgG2, IgG3, IgG4, IgA C. Sensitization for killing by NK cells: IgG1, IgG3 D. Sensitization of mast cells: IgG1, IgG3, IgE E. Activation of complement: IgM, IgG1, IgG2, IgG3, IgA F. Transport across epithelium: IgM, IgA (dimer) G. Transport across placenta: IgG1, IgG2, IgG3, IgG4 H. Diffusion into extravascular sites: IgM, IgG1, IgG2, IgG3, IgG4, IgA (monomer), IgE

4-69 Isotype switching and immunoglobulin gene rearrangement by somatic recombination are both recombinational processes but have very different outcomes. Give four ways in which they differ from each other.

4-69 (1) Gene rearrangements affect the variable region of immunoglobulins, whereas isotype switching affects the constant region. (2) Different recombination-signal sequences and enzymes are used for the two processes. (3) Isotype switching occurs only after antigen stimulation, whereas gene rearrangement occurs only during B-cell maturation in the bone marrow. (4) All isotype switch recombinations are productive, but not all gene rearrangements are. (5) Only heavy chains are involved in isotype switching, whereas both heavy-chain and light-chain genes are involved in somatic recombination.

4-70 Monoclonal antibodies are used for a wide range of applications including serological assays and diagnostics probes in the laboratory, and as therapeutic reagents in the clinic. Discuss why 'humanizing' monoclonal antibodies is necessary for use as therapeutic reagents but is not necessary when monoclonal antibodies are used as serological or diagnostic reagents.

4-70 Mice are used routinely to generate monoclonal antibodies. The constant regions of mouse antibodies are sufficiently different from the constant regions of human antibodies in amino acid composition that, if mouse antibodies are infused into a patient, an immune response will be stimulated and directed against the mouse constant-region epitopes. This immune response neutralizes the monoclonal antibody and in practice limits its intended use to one effective dose. When monoclonal antibodies are used for serological or diagnostic purposes in the laboratory, the monoclonal antibodies do not need to be humanized because laboratory assays are performed in vitro.

4-71 What would be the effect of a genetic defect that resulted in a lack of recombination between the switch regions in the immunoglobulin C-region genes?

4-71 The B cells in a person carrying such a defect would be unable to switch antibody isotype and would be unable to produce any antibody other than IgM. Because IgM antibodies can implement fewer effector functions than IgG antibodies, which constitute the main class of antibody produced in an adaptive immune response, one would expect that immunity would be impaired. In addition, no IgA antibodies could be produced, leaving the person highly vulnerable to infection through mucosal surfaces. There are, in fact, rare inherited genetic deficiencies that result in an inability to switch isotype. They are called hyper IgM immunodeficiencies because the patient is unable to produce any antibody other than IgM. The most frequent one affects the expression of a cell-surface molecule called CD40 ligand in T cells, which is required for the interaction between T cells and B cells that stimulates isotype switching, as we shall learn later in this book.

4-72 Influenza virus contains two proteins, called hemagglutinin and neuraminidase, exposed on the surface of the virion. Two additional proteins, located on the interior of the virion, are called matrix protein and nucleoprotein. Which of these four proteins will generate a better antibody response and why?

4-72 Hemagglutinin and neuraminidase epitopes, because epitopes exposed on the surface of pathogens stimulate antibodies.

4-73 A. Identify the four types of antibody used for therapeutic purposes. B. How is each produced? C. (i) Which is most desirable for the treatment of chronic conditions? (ii) Why? (iii) Provide an example.

4-73 A. The four types of therapeutic antibody include (i) mouse monoclonal antibodies, (ii) chimeric monoclonal antibodies, (iii) humanized monoclonal antibodies, and (iv) fully human monoclonal antibodies. B. (i) Mouse monoclonal antibodies are produced from hybridoma cell lines obtained by immortalizing mouse B cells by fusing them with a tumor cell. Hybridomas secreting antibody with the appropriate specificity for antigen are cloned and propagated. (ii) Chimeric monoclonal antibodies are produced by fusing the coding regions of the variable regions of mouse monoclonal antibodies, known to have specificity for a particular antigen, with the coding regions of human constant regions. (iii) Humanized monoclonal antibodies retain only the complementarity-determining regions of mouse monoclonal antibodies, and all remaining regions are replaced with human-derived regions. (iv) Fully human monoclonal antibodies are made either by using human hybridoma cell lines or by using transgenic mice whose immunoglobulin genes have been replaced by human immunoglobulin genes. C. (i) Fully human monoclonal antibodies are the most desirable, because (ii) they will not stimulate an anti-constant region antibody response in the recipient and can therefore be used for repeated treatment in chronic diseases without complications and without reducing therapeutic efficacy. (iii) Adalimumab is an example of a fully human monoclonal antibody used to treat rheumatoid arthritis. It neutralizes the inflammatory cytokine TNF-α to decrease inflammation of the joints.

4-74 A. What is the difference between polyclonal antibodies and monoclonal antibodies? B. How is each produced?

4-74 A. Polyclonal antibodies are a mixture of antibodies of different specificities and affinities for a particular antigen. They are the product of numerous different B cells. Monoclonal antibodies have a single specificity and affinity for a given antigen. They derive from a single B cell. B. Polyclonal antibodies are produced in vivo by immunizing an animal with antigen, allowing sufficient time for an immune response to occur and then preparing antiserum containing the antibodies from the blood. Monoclonal antibodies are made in vitro from individual cell lines derived from single B cells. This is achieved by producing a hybrid immortalized cell line through the fusion of an antibody-producing B cell with a myeloma tumor cell to produce an antibody-producing 'hybridoma.' A hybridoma producing the desired antibody can then be cloned and grown on to produce unlimited amounts of monoclonal antibody.

5-15 Explain the importance of promiscuous binding specificity exhibited by MHC class I and class II molecules.

5-15 Each MHC molecule can bind to a very large number of peptides made up of different sequences of amino acids. The consequence of this promiscuity is that humans need only encode a relatively small number of MHC molecules in their genome if they are to bind to the huge number of pathogen-derived peptides encountered during a lifetime of infections. Because MHC molecules are coexpressed on the cell surface, this also ensures that an appropriate density of MHC molecules populates the cell surface to ensure efficient T-cell engagement and subsequent activation.

5-23 Explain how mycobacteria avoid immune recognition by T cells during infection.

5-23 Both the MHC class I and MHC class II pathways are subverted by mycobacteria during intracellular growth and replication. Although mycobacteria are obligate intracellular pathogens their proteins do not enter the cytosol, so proteasomes are unable to generate mycobacteria-derived peptides for the MHC class I pathway. Mycobacteria are also resistant to degradation by lysosomal enzymes because they inhibit phagolysosome formation. This interferes with the MHC class II pathway.

5-24 Identify the three functions of the invariant chain.

5-24 1. Invariant chain protects the peptide-binding groove of MHC class II molecules from binding to endoplasmic reticulum-derived peptides. 2. Binding of invariant chain to MHC class II molecules stabilizes their conformation so that they are eventually able to bind peptides. 3. Invariant chain facilitates the transport of MHC class II molecules from the ER to the MIIC cellular compartment, where they can bind peptides.

5-25 Explain specifically how interferon-γ produced during an infection enhances (A) antigen processing in the MHC class I pathway, and (B) antigen presentation in the MHC class II pathway.

5-25 A. Interferon-γ causes a shift from the production of constitutive proteasomes to that of immunoproteasomes. This is accomplished through increased expression of alternative subunits (LMP2 and LMP7) that are present in the immunoproteasome. These proteasomes exhibit modified protease activities favoring the production of peptides (antigen processing) that can bind to MHC class I molecules. Specifically, cleavage after hydrophobic residues is enhanced and cleavage after acidic residues is decreased. B. Interferon-γ increases the expression of HLA-DM but not HLA-DO. This causes a shift in the balance of these two molecules, resulting in an overall decrease in the antagonist activity of HLA-DO. If HLA-DM is more abundant, it has the ability to catalyze the release of CLIP from MHC class II molecules and facilitate the replacement of CLIP with other peptides for presentation to CD4 T cells (antigen presentation). Another way in which interferon-γ increases antigen presentation in the MHC class II pathway is by increasing the expression levels of MHC class II molecules on both professional and non-professional antigen-presenting cells.

5-26 Discuss how T-cell receptors differ from immunoglobulins in the way that they recognize antigen. Use the following terms in your answer: peptides, antigen-presenting cells, MHC molecules, and antigen-binding sites.

5-26 First, T-cell receptors can bind to only one type of antigen, namely protein fragments called peptides. Immunoglobulins can bind to peptides, intact proteins, carbohydrates, and lipids. Second, unlike immunoglobulins, T-cell receptors cannot bind to a free antigen directly, but instead require accessory antigen-presenting cells that present the peptide antigens in association with cell-surface glycoproteins called MHC class I and class II molecules. Third, T-cell receptors possess a single antigen-binding site; immunoglobulins have at least two binding sites for antigen, and more in the case of secreted dimeric IgA (four sites) and secreted pentameric IgM (ten sites).

5-27 Pathogens that infect the human body replicate either inside cells (such as viruses) or extracellularly, in the blood or in the extracellular spaces in tissues. A. Identify (i) the class of T cells that are stimulated by intracellular pathogens, (ii) their co-receptor, (iii) the MHC molecule used for recognition of antigen and (iv) the T-cell effector function. B. Repeat this for the classes of T cells that are stimulated by extracellular pathogens. For the purposes of this question, count those pathogens (such as mycobacteria) that can survive and live inside intracellular vesicles after being taken up by macrophages as extracellular pathogens.

5-27 A. (i) Pathogens that are propagating freely within cells (for example viruses) are eradicated by the actions of cytotoxic T cells. (ii) Cytotoxic T cells express a glycoprotein called CD8, a T-cell co-receptor that interacts with (iii) MHC class I on antigen-presenting cells. (iv) Once activated, cytotoxic T cells kill cells infected with the pathogen, which are displaying pathogen peptides on MHC class I molecules, and thereby inhibit further replication of the pathogen and infection of neighboring cells. B. (i) Pathogens that reproduce in extracellular spaces, for example encapsulated bacteria such as Streptococcus pneumoniae, are eradicated after the activation of other cell types by helper T cells, namely the classes TH1 and TH2. (ii) TH1 and TH2 cells express a glycoprotein called CD4, a T-cell co-receptor that interacts with (iii) MHC class II molecules on antigen-presenting cells. (iv) TH1 cells activate macrophages that are displaying pathogen peptides (derived from phagocytosed pathogen) on MHC class II molecules on their surface. This stimulates increased phagocytosis by the macrophage and destruction of pathogens inside phagolysosomes. Activated macrophages also secrete inflammatory mediators that have an important part in eradicating the infection by helping to induce inflammation which recruits phagocytic cells and effector lymphocytes to the site of infection. TH1 cells also induce switching of B cells to certain antibody isotypes. TH2 cells activate B cells displaying antigen-derived peptides on MHC class II molecules, resulting in the differentiation of the B cells into plasma cells and the production of antibodies that remove the extracellular pathogen or its toxins as a result of neutralization, opsonization, and complement activation.

5-38 Explain how professional antigen-presenting cells optimize antigen presentation to T cells despite the relatively limited capacity of any particular MHC molecule to bind different pathogen-derived peptides.

5-38 Professional antigen-presenting cells express several different types of MHC molecule on the cell surface, and each type has the potential to bind to different peptides. In addition, MHC molecules are highly polymorphic, so that most individuals are heterozygous and encode different allelic forms at each gene locus. The variety of peptides that can bind to these MHC molecules is therefore increased.

5-62 Provide an explanation of why it is believed that MHC class I genes are the evolutionary ancestors of MHC class II genes.

5-62 MHC class I molecules not only have the role of presenting antigen to T cells, but they also possess additional functions in the body not associated with MHC class II molecules. For example, they participate in iron homeostasis, IgG uptake in the gastrointestinal tract, and the regulation of NK-cell function in innate immunity. In addition, MHC class I and class I-like genes are not confined to chromosome 6, in contrast with MHC class II genes. Finally, vertebrates exist (such as Atlantic cod) that have only MHC class I genes in their genome, and lack MHC class II genes.

5-65 Describe (A) five ways in which T-cell receptors are similar to immunoglobulins, and (B) five ways in which they are different (other than the way in which they recognize antigen).

5-65 A. Similarities. (1) The T-cell receptor has a similar overall structure to the membrane-bound Fab fragment of immunoglobulin, containing an antigen-binding site, two variable domains, and two constant domains. (2) T-cell receptors and immunoglobulins are both generated through somatic recombination of sets of gene segments. (3) The variable region of the T-cell receptor contains three complementarity-determining regions (CDRs) encoded by the Vα domain and three CDRs encoded by the Vβ domain, analogous to the CDRs encoded by the VH and VL domains. (4) There is huge diversity in the T-cell receptor repertoire and it is generated in the same way as that in the B-cell repertoire (by combination of different gene segments, junctional diversity due to P- and N-nucleotides, and combination of two different chains). (5) T-cell receptors are not expressed at the cell surface by themselves but require association with the CD3 γ, δ, ε, and ζ chains for stabilization and signal transduction, analogous to the Igα and Igβ chains required for immunoglobulin cell-surface expression and signal transduction. B. Differences. (1) A T-cell receptor has one antigen-binding site; an immunoglobulin has at least two. (2) T-cell receptors are never secreted. (3) T-cell receptors are generated in the thymus, not the bone marrow. (4) The constant region of the T-cell receptor has no effector function and it does not switch isotype. (5) T-cell receptors do not undergo somatic hypermutation.

5-66 Compare the organization of T-cell receptor α and β genes (the TCRα and TCRβ loci) with the organization of immunoglobulin heavy-chain and light-chain genes.

5-66 The organization of the TCRα locus resembles that of an immunoglobulin light-chain locus, in that both contain V and J gene segments and no D gene segments. The TCRα locus on chromosome 14 contains about 80 V gene segments, 61 J gene segments, and 1 C gene. The immunoglobulin light-chain loci, λ and κ, are encoded on chromosomes 22 and 2, respectively. The λ locus contains about 30 V gene segments and 4 J gene segments, each paired with a C gene. The κ locus contains about 35 V gene segments, 5 J segments, and 1 C gene segment. The arrangement of the κ locus more closely resembles that of the TCRα locus except that there are more J segments in the T-cell receptor locus. The organization of the TCRβ locus resembles that of the immunoglobulin heavy-chain locus; both contain V, D, and J gene segments. The TCRβ locus contains about 52 V gene segments, 2 D gene segments, 13 J gene segments, and 2 C genes, encoded on chromosome 7. Each C gene is associated with a set of D and J gene segments. The immunoglobulin heavy-chain locus on chromosome 14 contains about 40 V segments, 23 D segments, and 6 J segments, followed by 9 C genes, each specifying a different immunoglobulin isotype. The heavy-chain C genes determine the effector function of the antibody.

5-71 A. (i) Describe the structure of an MHC class I molecule, identifying the different polypeptide chains and domains. (ii) What are the names of the MHC class I molecules produced by humans? Which part of the molecule is encoded within the MHC region of the genome? (iii) Which domains or parts of domains participate in the following: antigen binding; binding the T-cell receptor; and binding the T-cell co-receptor? (iv) Which domains are the most polymorphic? B. Repeat this for an MHC class II molecule.

5-71 A. (i) The complete MHC class I molecule is a heterodimer made up of one α chain and a smaller chain called β-microglobulin. The α chain consists of three extracellular domains α1, α2, and α3—a transmembrane region and a cytoplasmic tail. β2-Microglobulin is a single-domain protein noncovalently associated with the extracellular portion of the α chain, providing support and stability. (ii) The polymorphic class I molecules in humans are called HLA-A, HLA-B, and HLA-C. The α chain is encoded in the MHC region by an MHC class I gene. The gene for β2-microglobulin is elsewhere in the genome. (iii) The antigen-binding site is formed by the α1 and α2 domains, the ones farthest from the membrane, which create a peptide-binding groove. The region of the MHC molecule that binds to the T-cell receptor encompasses the α helices of the α1 and α2 domains that make up the outer surfaces of the peptide-binding groove. The α3 domain binds to the T-cell co-receptor CD8. (iv) The most polymorphic parts of the α chain are the regions of the α1 and α2 domains that bind antigen and the T-cell receptor. β2-Microglobulin is invariant; that is, it is the same in all individuals. B. (i) MHC class II molecules are heterodimers made up of an α chain and a β chain. The α chain consists of α1 and α2 extracellular domains, a transmembrane region, and a cytoplasmic tail. The β chain contains β1 and β2 extracellular domains, a transmembrane region, and a cytoplasmic tail. (ii) In humans there are three polymorphic MHC class II molecules called HLA-DP, HLA-DQ, and HLA-DR. Both chains of an MHC class II molecule are encoded by genes in the MHC region. (iii) Antigen binds in the peptide-binding groove formed by the α1 and β1 domains. The α helices of the α1 and β1 domains interact with the T-cell receptor. The β2 domain binds to the T-cell co-receptor CD4. (iv) With the exception of HLA-DRα, which is dimorphic, both the α and β chains of MHC class II molecules are highly polymorphic. Polymorphism is concentrated around the regions that bind antigen and the T-cell receptor in the α1 and β1 domains.

5-72 What is meant by the terms (A) antigen processing and (B) antigen presentation? (C) Why are these processes required before T cells can be activated?

5-72 A. Antigen processing is the intracellular breakdown of pathogen-derived proteins into peptide fragments that are of the appropriate size and specificity required to bind to MHC molecules. B. Antigen presentation is the assembly of peptides with MHC molecules and the display of these complexes on the surface of antigen-presenting cells. C. Antigen processing and presentation must occur for T cells to be activated because (1) T-cell receptors cannot bind to intact protein, only to peptides, and (2) T-cell receptors do not bind antigen directly, but rather must recognize antigen bound to MHC molecules on the surface of antigen-presenting cells.

5-73 A. Describe in chronological order the steps of the antigen-processing and antigen-presentation pathways for intracellular, cytosolic pathogens. B. (i) What would be the outcome if a mutant MHC class I α chain could not associate with β2-microglobulin, and (ii) what would happen if the TAP transporter were lacking as a result of mutation? Explain your answers.

5-73 A. Proteins derived from pathogens located in the cytosol are broken down into small peptide fragments in proteasomes. The peptides are transported into the lumen of the endoplasmic reticulum (ER) using the transporter associated with antigen processing (TAP), which is a heterodimer of TAP-1 and TAP-2 proteins anchored in the ER membrane. Meanwhile, MHC class I molecules are assembling and folding in the ER with the assistance of other proteins. Initially, the MHC class I α chain binds calnexin through an asparagine-linked oligosaccharide on the α1 domain. After folding and forming its disulfide bonds, the α chain binds to β2-microglobulin, forming the MHC class I heterodimer. At this stage, calnexin is released and the heterodimer joins the peptide-loading complex composed of tapasin, calreticulin, and ERp57, which position the heterodimer near TAP, stabilize the peptide-loading complex, and render the heterodimer in an open conformation until a high-affinity peptide binds to the heterodimer through a process known as peptide editing. The heterodimer consequently changes its conformation, is released from the peptide-loading complex, and leaves the ER as a vesicle. Arrival at the Golgi apparatus induces final glycosylation, and finally the peptide:MHC class I heterodimer complex is transported in vesicles to the plasma membrane, where it presents peptide to CD8 T cells. B. (i) If an MHC class I α chain is unable to bind β2-microglobulin, it will be retained in the ER and will not be transported to the cell surface. It will remain bound to calnexin and will not fold into the conformation needed to bind to peptide. Thus, antigens will not be presented using that particular MHC class I molecule. (ii) If TAP-1 or TAP-2 proteins are mutated and not expressed, peptides will not be transported into the lumen of the ER. Without peptide, an MHC class I molecule cannot complete its assembly and will not leave the ER. A rare immunodeficiency disease called bare lymphocyte syndrome (MHC class I immunodeficiency) is characterized by a defective TAP protein, causing less than 1% of MHC class I molecules to be expressed on the cell surface in comparison with normal. Thus, T-cell responses to all pathogen antigens that would normally be recognized on MHC class I molecules will be impaired.

5-75 A. Describe in chronological order the steps of the antigen-processing and antigen-presentation pathways for extracellular pathogens. B. What would be the outcome (i) if invariant chain were defective or missing, or (ii) if HLA-DM were not expressed?

5-75 A. Extracellular pathogens are taken up by endocytosis or phagocytosis and degraded by enzymes into smaller peptide fragments inside acidified intracellular vesicles called phagolysosomes. MHC class II molecules delivered into the ER and being transported to the cell surface intersect with the phagolysosomes, where these peptides are encountered and loaded into the antigen-binding groove. To prevent MHC class II molecules from binding to peptides prematurely, invariant chain (Ii) binds to the MHC class II antigen-binding site in the ER. Ii is also involved in transporting MHC class II molecules to the phagolysosomes via the Golgi as part of the interconnected vesicle system. Ii is removed from MHC class II molecules once the phagolysosome is reached. Removal is achieved in two steps: (1) proteolysis cleaves Ii into smaller fragments, leaving a small peptide called CLIP (class II-associated invariant chain peptide) in the antigen-binding groove of the MHC class II molecule; and (2) CLIP is then released by HLA-DM catalysis. Once CLIP is removed, HLA-DM remains associated with the MHC class II molecule, enabling the now empty peptide-binding groove to sample other peptides until one binds tightly enough to cause a conformational change that releases HLA-DM. Finally, the peptide:MHC class II complex is transported to the plasma membrane. B. (i) Defects in the invariant chain would impair normal MHC class II function because invariant chain not only protects the peptide-binding groove from binding prematurely to peptides present in the ER but is also required for transport of MHC class II molecules to the phagolysosome. (ii) If HLA-DM were not expressed, most MHC class II molecules on the cell surface would be occupied by CLIP rather than endocytosed material. This would compromise the presentation of extracellular antigens at the threshold levels required for T-cell activation.

5-8 A. Identify which features of the RAG genes have similarity to the transposase gene of transposons. B. Explain how the mechanisms for immunoglobulin and T-cell receptor rearrangement may have evolved in humans.

5-8 A. RAG genes do not contain introns, and they function to facilitate the cleavage of double-stranded DNA. B. It has been proposed that the evolution of rearranging antigen-receptor genes began with the insertion of a transposable element into a gene encoding an innate immune receptor. This gene was not only split into two segments, but also became flanked by repetitive DNA sequences donated by the transposon. A later chromosomal rearrangement event translocated the transposase genes to a different chromosome, where they evolved into the ancestral RAG-1 and RAG-2 genes. The repetitive DNA sequences left behind at the original receptor gene location evolved into the recombination signal sequences (RSSs), and the segments of the receptor gene evolved into V and J sequences. Eventually this led to a family of rearranging genes on five chromosomes encoding the immunoglobulin heavy- and light-chain genes, and the T-cell receptor α, β, γ, and δ genes.

5-86 A. How many HLA-DR α:β combinations can be made by an individual who is heterozygous at all HLA-DRβ loci, inherits the DRβ haplotype DRB1 from their mother, the DRβ haplotype DRB1, DRB4 from their father, and also inherits different allelic forms of DRA from each parent? B. Repeat this exercise given the same information except that the maternal DRβ haplotype is DRB1, DRB3

5-86 m and p denote maternal and paternal allotypes, respectively. A. The answer is 6. The possible combinations are as follows: (1) DRA-m:DRB1-m; (2) DRA-m:DRB1-p; (3) DRA-m:DRB4-p; (4) DRA-p:DRB1-m; (5) DRA-p:DRB1-p; and (6) DRA-p:DRB4-p. B. The answer is 8. The possible combinations are as follows: (1) DRA-m:DRB1-m; (2) DRA-m:DRB3-m; (3) DRA-m:DRB1-p; (4) DRA-m:DRB4-p; (5) DRA-p:DRB1-m; (6) DRA-p:DRB3-m; (7) DRA-p:DRB1-p; (8) DRA-p:DRB4-p.

5-89 A. What is the maximum number of MHC class I and class II molecules that a heterozygous individual could theoretically express? Explain your answer. (Ignore the possibility of MHC class II molecules composed of chains from different isotypes.) B. How does this relatively small number of MHC molecules have the potential to bind the huge number of antigenic peptides encountered in the environment, and what features of a peptide determine whether it will be bound by a given MHC molecule?

5-89 A. There are three MHC class I isotypes in humans (HLA-A, HLA-B, and HLA-C) and they are expressed from both chromosomes. Assuming that each gene is heterozygous, the maximum number of different MHC class I α chains that could be expressed is 6. Because β-microglobulin is invariant, this means that six different MHC class I molecules could be produced. For MHC class II molecules, assuming complete heterozygosity and the presence of two functional DRB genes (DRB1 and DRB3, 4, or 5) on both chromosomes, the maximum number of MHC class II molecules that could be expressed is 16 (Figure A5-89). Therefore, the total number of different MHC class I and MHC class II molecules that can be expressed is 22. <<insert Figure A5-89>> Figure A5-89 The number of HLA molecules that can be expressed in a single individual. m, maternal chromosome; p, paternal chromosome. B. MHC molecules have promiscuous binding specificity, which means that one MHC molecule is able to bind a wide range of peptides with different sequences. For all MHC molecules, only a few of the amino acids in the antigen peptide are critical for binding to amino acids in the peptide-binding groove. The critical amino acids in the peptide are called anchor residues; they are the same or similar in all peptides that bind to a given MHC molecule. The other amino acid residues in the peptides can be different. The pattern of anchor residues that binds to a given MHC molecule is called the peptide-binding motif. Hence, a very large number of discrete peptides can bind to each MHC isoform, the only constraint being the possession of the correct anchor residues at the appropriate positions in the peptide. MHC class I molecules also bind peptides that are typically nine amino acids long, whereas MHC class II molecules bind longer peptides with a range of lengths.

5-90 (A) Explain the difference between interallelic conversion and gene conversion, and (B) provide an example for both.

5-90 A. Interallelic conversion is a recombination between homologous alleles of the same gene. Gene conversion is a recombination between non-homologous alleles of different genes. B. An example of interallelic conversion would involve recombination between HLA B*5101 and HLA B*3501. An example of gene conversion would involve recombination between HLA B*1501 and HLA Cw*0102.

5-91 In the context of MHC isoforms, what is the difference between balancing selection and directional selection?

5-91 Balancing selection maintains a variety of MHC isoforms in a population, whereas directional selection replaces older isoforms with newer variants.

5-92 A. What are alloantibodies? B. How do alloantibodies arise naturally? C. Why are alloantibodies problematic for transplantation?

5-92 A. Alloantibodies are antibodies specific for variant antigens encoded at polymorphic genes within a species. produced following introduction of an alloantigen( present in some, but not all individuals of a species; genetically determined) into the system of an individual lacking that particles antigen. B. They arise naturally during pregnancy when the mother's immune system encounters fetal cells expressing variant antigens derived from the father but not expressed by the mother. C. If present, alloantibodies with specificity for transplanted organs will mediate graft rejection.