Factors That Influence Immunogenicity To protect against infectious disease, the immune system must be able to recognize bacteria, bacterial products, fungi, parasites, and viruses as immunogens. In fact, the immune system actually recognizes particular macromolecules of an infectious agent, generally either proteins or polysaccharides. Proteins are the most potent immunogens, with polysaccha- rides ranking second. In contrast, lipids and nucleic acids of an infectious agent generally do not serve as immunogens unless they are complexed with proteins or polysaccharides. Immunologists tend to use proteins or polysaccharides as immunogens in most experimental studies of humoral im- munity (Table 3-1). For cell-mediated immunity, only pro- teins and some lipids and glycolipids serve as immunogens. These molecules are not recognized directly. Proteins must first be processed into small peptides and then presented to- gether with MHC molecules on the membrane of a cell be- fore they can be recognized as immunogens. Recent work shows that those lipids and glycolipids that can elicit cell- mediated immunity must also be combined with MHC-like membrane molecules called CD1 (see Chapter 8). chapter 3 a73 Immunogenicity Versus Antigenicity a73 Factors That Influence Immunogenicity a73 Epitopes a73 Haptens and the Study of Antigenicity a73 Pattern-Recognition Receptors Antigens S ????????? ???? ??? ?? ?????????? ?? ??? immunoglobulin receptor of B cells, or by the T- cell receptor when complexed with MHC, are called antigens. The molecular properties of antigens and the way in which these properties ultimately contribute to immune activation are central to our understanding of the immune system. This chapter describes some of the molecu- lar features of antigens recognized by B or T cells. The chap- ter also explores the contribution made to immunogenicity by the biological system of the host; ultimately the biological system determines whether a molecule that combines with a B or T cell’s antigen-binding receptor can then induce an im- mune response. Fundamental differences in the way B and T lymphocytes recognize antigen determine which molecular features of an antigen are recognized by each branch of the immune system. These differences are also examined in this chapter. Immunogenicity Versus Antigenicity Immunogenicity and antigenicity are related but distinct immunologic properties that sometimes are confused. Im- munogenicity is the ability to induce a humoral and/or cell- mediated immune response: B cells H11001 antigen n effector B cells + memory B cells g (plasma cells) T cells H11001 antigen n effector T cells + memory T cells g (e.g., CTLs, T H s) Although a substance that induces a specific immune re- sponse is usually called an antigen, it is more appropriately called an immunogen. Antigenicity is the ability to combine specifically with the final products of the above responses (i.e., antibodies and/or cell-surface receptors). Although all molecules that have the property of immunogenicity also have the property of antigenicity, the reverse is not true. Some small molecules, called haptens, are antigenic but incapable, by themselves, of inducing a specific immune response. In other words, they lack immunogenicity. Complementarity of Interacting Surfaces of Antibody (left) and Antigen (right) 8536d_ch03_057-075 8/7/02 9:18 AM Page 57 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: Immunogenicity is not an intrinsic property of an antigen but rather depends on a number of properties of the particu- lar biological system that the antigen encounters. The next two sections describe the properties that most immunogens share and the contribution that the biological system makes to the expression of immunogenicity. The Nature of the Immunogen Contributes to Immunogenicity Immunogenicity is determined, in part, by four properties of the immunogen: its foreignness, molecular size, chemical composition and complexity, and ability to be processed and presented with an MHC molecule on the surface of an anti- gen-presenting cell or altered self-cell. FOREIGNNESS In order to elicit an immune response, a molecule must be recognized as nonself by the biological system. The capacity to recognize nonself is accompanied by tolerance of self, a specific unresponsiveness to self antigens. Much of the ability to tolerate self antigens arises during lymphocyte develop- ment, during which immature lymphocytes are exposed to self-components. Antigens that have not been exposed to im- mature lymphocytes during this critical period may be later recognized as nonself, or foreign, by the immune system. When an antigen is introduced into an organism, the degree of its immunogenicity depends on the degree of its foreign- ness. Generally, the greater the phylogenetic distance be- tween two species, the greater the structural (and therefore the antigenic) disparity between them. For example, the common experimental antigen bovine serum albumin (BSA) is not immunogenic when injected into a cow but is strongly immunogenic when injected into a rabbit. Moreover, BSA would be expected to exhibit greater immunogenicity in a chicken than in a goat, which is more closely related to bovines. There are some exceptions to this rule. Some macromolecules (e.g., collagen and cytochrome c) have been highly conserved throughout evolution and therefore display very little immunogenicity across diverse species lines. Conversely, some self-components (e.g., corneal tissue and sperm) are effectively sequestered from the immune system, so that if these tissues are injected even into the animal from which they originated, they will func- tion as immunogens. MOLECULAR SIZE There is a correlation between the size of a macromolecule and its immunogenicity. The most active immunogens tend to have a molecular mass of 100,000 daltons (Da). Generally, substances with a molecular mass less than 5000–10,000 Da are poor immunogens, although a few substances with a molecular mass less than 1000 Da have proven to be im- munogenic. CHEMICAL COMPOSITION AND HETEROGENEITY Size and foreignness are not, by themselves, sufficient to make a molecule immunogenic; other properties are needed as well. For example, synthetic homopolymers (polymers composed of a single amino acid or sugar) tend to lack im- munogenicity regardless of their size. Studies have shown that copolymers composed of different amino acids or sugars are usually more immunogenic than homopolymers of their constituents. These studies show that chemical complexity contributes to immunogenicity. In this regard it is notable that all four levels of protein organization—primary, sec- ondary, tertiary, and quaternary—contribute to the struc- tural complexity of a protein and hence affect its immuno- genicity (Figure 3-1). LIPIDS AS ANTIGENS Appropriately presented lipoidal antigens can induce B- and T-cell responses. For the stimulation of B-cell responses, lipids are used as haptens and attached to suitable carrier molecules such as the proteins keyhole limpet hemocyanin (KLH) or bovine serum albumin (BSA). By immunizing with these lipid-protein conjugates it is possible to obtain anti- bodies that are highly specific for the target lipids. Using this approach, antibodies have been raised against a wide variety of lipid molecules including steroids, complex fatty-acid de- rivatives, and fat-soluble vitamins such as vitamin E. Such antibodies are of considerable practical importance since many clinical assays for the presence and amounts of med- ically important lipids are antibody-based. For example, a determination of the levels of a complex group of lipids known as leukotrienes can be useful in evaluating asthma pa- tients. Prednisone, an immunosuppressive steroid, is often given as part of the effort to prevent the rejection of a trans- 58 PART II Generation of B-Cell and T-Cell Responses TABLE 3-1 Molecular weight of some common experimental antigens used in immunology Antigen Approximate molecular mass (Da) Bovine gamma globulin 150,000 (BGG) Bovine serum albumin 69,000 (BSA) Flagellin (monomer) 40,000 Hen egg-white lysozyme 15,000 (HEL) Keyhole limpet hemocyanin H110222,000,000 (KLH) Ovalbumin (OVA) 44,000 Sperm whale myoglobin 17,000 (SWM) Tetanus toxoid (TT) 150,000 8536d_ch03_057-075 8/6/02 10:28 AM Page 58 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: planted organ. The achievement and maintenance of ade- quate blood levels of this and other immunosuppressive drugs is important to a successful outcome of transplanta- tion, and antibody-based immunoassays are routinely used to make these evaluations. The extraordinary sensitivity and specificity of assays based on the use of anti-lipid antibodies is illustrated by Table 3-2, which shows the specificity of an antibody raised against leukotriene C 4 . This antibody allows the detection of as little as 16–32 picograms per ml of leukotriene C 4 . Because it has little or no reactivity with sim- ilar compounds, such as leukotriene D 4 or leukotriene E 4 ,it can be used to assay leukotriene C 4 in samples that contain this compound and a variety of other structurally related lipids. T cells recognize peptides derived from protein antigens when they are presented as peptide-MHC complexes. How- ever, some lipids can also be recognized by T cells. Lipoidal compounds such as glycolipids and some phospholipids can be recognized by T-cell receptors when presented as com- plexes with molecules that are very much like MHC mole- cules. These lipid-presenting molecules are members of the CD1 family (see Chapter 8) and are close structural relatives of class I MHC molecules. The lipid molecules recognized by the CD1–T-cell receptor system all appear to share the com- mon feature of a hydrophobic portion and a hydrophilic head group. The hydrophobic portion is a long-chain fatty acid or alcohol and the hydrophilic head group is composed of highly polar groups that often contain carbohydrates. Recognition of lipids is a part of the immune response to some pathogens, and T cells that recognize lipids arising from Mycobacterium tuberculosis and Mycobacterium leprae, which respectively cause tuberculosis and leprosy, have been isolated from hu- mans infected by these mycobacteria. More about the presen- tation of lipoidal antigens can be found in Chapter 8. Antigens CHAPTER 3 59 SECONDARY STRUCTURE TERTIARY STRUCTURE PRIMARY STRUCTURE α helix β pleated sheet Amino acid sequence of polypeptide chain Domain Monomeric polypeptide molecule QUATERNARY STRUCTURE Dimeric protein molecule —Lys—Ala—His—Gly—Lys—Lys—Val—Leu FIGURE 3-1 The four levels of protein organizational structure. The linear arrangement of amino acids constitutes the primary struc- ture. Folding of parts of a polypeptide chain into regular structures (e.g., H9251 helices and H9252 pleated sheets) generates the secondary struc- ture. Tertiary structure refers to the folding of regions between sec- ondary features to give the overall shape of the molecule or parts of it (domains) with specific functional properties. Quaternary struc- ture results from the association of two or more polypeptide chains into a single polymeric protein molecule. 8536d_ch03_057-075 8/7/02 9:18 AM Page 59 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: SUSCEPTIBILITY TO ANTIGEN PROCESSING AND PRESENTATION The development of both humoral and cell-mediated im- mune responses requires interaction of T cells with antigen that has been processed and presented together with MHC molecules. Large, insoluble macromolecules generally are more immunogenic than small, soluble ones because the larger molecules are more readily phagocytosed and processed. Macromolecules that cannot be degraded and presented with MHC molecules are poor immunogens. This can be illustrated with polymers of D-amino acids, which are stereoisomers of the naturally occurring L-amino acids. Be- cause the degradative enzymes within antigen-presenting cells can degrade only proteins containing L-amino acids, polymers of D-amino acids cannot be processed and thus are poor immunogens. The Biological System Contributes to Immunogenicity Even if a macromolecule has the properties that contribute to immunogenicity, its ability to induce an immune response will depend on certain properties of the biological system that the antigen encounters. These properties include the genotype of the recipient, the dose and route of antigen ad- ministration, and the administration of substances, called adjuvants, that increase immune responses. GENOTYPE OF THE RECIPIENT ANIMAL The genetic constitution (genotype) of an immunized ani- mal influences the type of immune response the animal manifests, as well as the degree of the response. For example, Hugh McDevitt showed that two different inbred strains of 60 PART II Generation of B-Cell and T-Cell Responses TABLE 3-2 Specificity of an antibody against a complex lipid Antibody reactivity* Lipid Structure (on scale of 1 to 100) Leukotriene C 4 100.0 Leukotriene D 4 5.0 Leukotriene E 4 0.5 Prostaglandin D 2 0.001 * The reactivity of the antibody with the immunizing antigen leukotriene C 4 is assigned a value of 100 in arbitrary units. OH OH CH 3 O S O OHNH 2 OH OH O CH 3 HO O ? OH OH CH 3 NH 2 O S O HO O O O N H OH NH ? OH OH CH 3 O S O O N H OH H 2 N 8536d_ch03_057-075 8/6/02 10:28 AM Page 60 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: mice responded very differently to a synthetic polypeptide immunogen. After exposure to the immunogen, one strain produced high levels of serum antibody, whereas the other strain produced low levels. When the two strains were crossed, the F 1 generation showed an intermediate response to the immunogen. By backcross analysis, the gene control- ling immune responsiveness was mapped to a subregion of the major histocompatibility complex (MHC). Numerous experiments with simple defined immunogens have demon- strated genetic control of immune responsiveness, largely confined to genes within the MHC. These data indicate that MHC gene products, which function to present processed antigen to T cells, play a central role in determining the de- gree to which an animal responds to an immunogen. The response of an animal to an antigen is also influenced by the genes that encode B-cell and T-cell receptors and by genes that encode various proteins involved in immune reg- ulatory mechanisms. Genetic variability in all of these genes affects the immunogenicity of a given macromolecule in dif- ferent animals. These genetic contributions to immuno- genicity will be described more fully in later chapters. IMMUNOGEN DOSAGE AND ROUTE OF ADMINISTRATION Each experimental immunogen exhibits a particular dose-re- sponse curve, which is determined by measuring the im- mune response to different doses and different adminis- tration routes. An antibody response is measured by deter- mining the level of antibody present in the serum of immu- nized animals. Evaluating T-cell responses is less simple but may be determined by evaluating the increase in the number of T cells bearing TCRs that recognize the immunogen. Some combination of optimal dosage and route of administration will induce a peak immune response in a given animal. An insufficient dose will not stimulate an immune re- sponse either because it fails to activate enough lymphocytes or because, in some cases, certain ranges of low doses can in- duce a state of immunologic unresponsiveness, or tolerance. The phenomenon of tolerance is discussed in chapters 10 and 21. Conversely, an excessively high dose can also induce tolerance. The immune response of mice to the purified pneumococcal capsular polysaccharide illustrates the impor- tance of dose. A 0.5 mg dose of antigen fails to induce an im- mune response in mice, whereas a thousand-fold lower dose of the same antigen (5 H11003 10 H110024 mg) induces a humoral anti- body response. A single dose of most experimental immuno- gens will not induce a strong response; rather, repeated administration over a period of weeks is usually required. Such repeated administrations, or boosters, increase the clonal proliferation of antigen-specific T cells or B cells and thus increase the lymphocyte populations specific for the im- munogen. Experimental immunogens are generally administered parenterally (para, around; enteric, gut)—that is, by routes other than the digestive tract. The following administration routes are common: a73 Intravenous (iv): into a vein a73 Intradermal (id): into the skin a73 Subcutaneous (sc): beneath the skin a73 Intramuscular (im): into a muscle a73 Intraperitoneal (ip): into the peritoneal cavity The administration route strongly influences which immune organs and cell populations will be involved in the response. Antigen administered intravenously is carried first to the spleen, whereas antigen administered subcutaneously moves first to local lymph nodes. Differences in the lymphoid cells that populate these organs may be reflected in the subsequent immune response. ADJUVANTS Adjuvants (from Latin adjuvare, to help) are substances that, when mixed with an antigen and injected with it, enhance the immunogenicity of that antigen. Adjuvants are often used to boost the immune response when an antigen has low im- munogenicity or when only small amounts of an antigen are available. For example, the antibody response of mice to im- munization with BSA can be increased fivefold or more if the BSA is administered with an adjuvant. Precisely how adju- vants augment the immune response is not entirely known, but they appear to exert one or more of the following effects (Table 3-3): a73 Antigen persistence is prolonged. a73 Co-stimulatory signals are enhanced. a73 Local inflammation is increased. a73 The nonspecific proliferation of lymphocytes is stimulated. Aluminum potassium sulfate (alum) prolongs the persis- tence of antigen. When an antigen is mixed with alum, the salt precipitates the antigen. Injection of this alum precipitate results in a slower release of antigen from the injection site, so that the effective time of exposure to the antigen increases from a few days without adjuvant to several weeks with the adjuvant. The alum precipitate also increases the size of the antigen, thus increasing the likelihood of phagocytosis. Water-in-oil adjuvants also prolong the persistence of antigen. A preparation known as Freund’s incomplete ad- juvant contains antigen in aqueous solution, mineral oil, and an emulsifying agent such as mannide monooleate, which disperses the oil into small droplets surrounding the antigen; the antigen is then released very slowly from the site of injection. This preparation is based on Freund’s complete adjuvant, the first deliberately formulated highly effective adjuvant, developed by Jules Freund many years ago and containing heat-killed Mycobacteria as an additional ingredient. Muramyl dipeptide, a component of the mycobacterial cell wall, activates macrophages, making Antigens CHAPTER 3 61 8536d_ch03_057-075 8/6/02 10:28 AM Page 61 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: Freund’s complete adjuvant far more potent than the in- complete form. Activated macrophages are more phago- cytic than unactivated macrophages and express higher levels of class II MHC molecules and the membrane mole- cules of the B7 family. The increased expression of class II MHC increases the ability of the antigen-presenting cell to present antigen to T H cells. B7 molecules on the antigen- presenting cell bind to CD28, a cell-surface protein on T H cells, triggering co-stimulation, an enhancement of the T- cell immune response. Thus, antigen presentation and the requisite co-stimulatory signal usually are increased in the presence of adjuvant. Alum and Freund’s adjuvants also stimulate a local, chronic inflammatory response that attracts both phagocytes and lymphocytes. This infiltration of cells at the site of the adjuvant injection often results in formation of a dense, macrophage-rich mass of cells called a granuloma. Because the macrophages in a granuloma are activated, this mecha- nism also enhances the activation of T H cells. Other adjuvants (e.g., synthetic polyribonucleotides and bacterial lipopolysaccharides) stimulate the nonspecific pro- liferation of lymphocytes and thus increase the likelihood of antigen-induced clonal selection of lymphocytes. Epitopes As mentioned in Chapter 1, immune cells do not interact with, or recognize, an entire immunogen molecule; instead, lymphocytes recognize discrete sites on the macromolecule called epitopes, or antigenic determinants. Epitopes are the immunologically active regions of an immunogen that bind to antigen-specific membrane receptors on lymphocytes or to secreted antibodies. Studies with small antigens have re- vealed that B and T cells recognize different epitopes on the same antigenic molecule. For example, when mice were im- munized with glucagon, a small human hormone of 29 amino acids, antibody was elicited to epitopes in the amino- terminal portion, whereas the T cells responded only to epi- topes in the carboxyl-terminal portion. Lymphocytes may interact with a complex antigen on sev- eral levels of antigen structure. An epitope on a protein anti- gen may involve elements of the primary, secondary, tertiary, and even quaternary structure of the protein (see Figure 3-1). In polysaccharides, branched chains are commonly present, and multiple branches may contribute to the conformation of epitopes. The recognition of antigens by T cells and B cells is funda- mentally different (Table 3-4). B cells recognize soluble anti- gen when it binds to their membrane-bound antibody. Because B cells bind antigen that is free in solution, the epi- topes they recognize tend to be highly accessible sites on the exposed surface of the immunogen. As noted previously, most T cells recognize only peptides combined with MHC molecules on the surface of antigen-presenting cells and al- tered self-cells; T-cell epitopes, as a rule, cannot be consid- ered apart from their associated MHC molecules. Properties of B-Cell Epitopes Are Determined by the Nature of the Antigen-Binding Site Several generalizations have emerged from studies in which the molecular features of the epitope recognized by B cells have been established. The ability to function as a B-cell epitope is determined by the nature of the antigen-binding site on the antibody molecules displayed by B cells. Antibody binds to an epitope by weak noncovalent interactions, which operate only over short dis- tances. For a strong bond, the antibody’s binding site and the epitope must have complementary shapes that place the in- teracting groups near each other. This requirement poses some restriction on the properties of the epitope. The size of the epitope recognized by a B cell can be no larger than the size of the antibody’s binding site. For any given antigen-an- tibody reaction, the shape of the epitope that can be recog- nized by the antibody is determined by the shape assumed by 62 PART II Generation of B-Cell and T-Cell Responses TABLE 3-3 Postulated mode of action of some commonly used adjuvants POSTULATED MODE OF ACTION Prolongs Enhances Induces Stimulates antigen co-stimulatory granuloma lymphocytes Adjuvant persistence signal formation nonspecifically Freund’s incomplete adjuvant H11001H11001H11001H11002 Freund’s complete adjuvant H11001H11001H11001H11001H11001H11002 Aluminum potassium sulfate (alum) H11001 ? H11001H11002 Mycobacterium tuberculosis H11002 ? H11001H11002 Bordetella pertussis H11002 ? H11002H11001 Bacterial lipopolysaccharide (LPS) H11002H11001H11002H11001 Synthetic polynucleotides (poly IC/poly AU) H11002 ? H11002H11001 8536d_ch03_057-075 8/6/02 10:28 AM Page 62 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: the sequences of amino acids in the binding site and the chemical environment that they produce. Smaller ligands such as carbohydrates, small oligonu- cleotides, peptides, and haptens often bind within a deep pocket of an antibody. For example, angiotensin II, a small octapeptide hormone, binds within a deep and narrow groove (725 ? 2 ) of a monoclonal antibody specific for the hormone (Figure 3-2). Within this groove, the bound pep- tide hormone folds into a compact structure with two turns, which brings its amino (N-terminal) and carboxyl (C-termi- nal) termini close together. All eight amino acid residues of the octapeptide are in van der Waals contact with 14 residues of the antibody’s groove. A quite different picture of epitope structure emerges from x-ray crystallographic analyses of monoclonal antibod- ies bound to globular protein antigens such as hen egg-white lysozyme (HEL) and neuraminidase (an envelope glycopro- tein of influenza virus). These antibodies make contact with the antigen across a large flat face (Figure 3-3). The interact- ing face between antibody and epitope is a flat or undulating surface in which protrusions on the epitope or antibody are matched by corresponding depressions on the antibody or epitope. These studies have revealed that 15–22 amino acids on the surface of the antigen make contact with a similar number of residues in the antibody’s binding site; the surface area of this large complementary interface is between 650 ? 2 and 900 ? 2 . For these globular protein antigens, then, the shape of the epitope is entirely determined by the tertiary conformation of the native protein. Thus, globular protein antigens and small peptide anti- gens interact with antibody in different ways (Figure 3-4). Typically, larger areas of protein antigens are engaged by the antibody binding site. In contrast, a small peptide such as an- giotensin II can fold into a compact structure that occupies less space and fits into a pocket or cleft of the binding site. This pattern is not unique to small peptides; it extends to the binding of low-molecular-weight antigens of various chemi- cal types. However, these differences between the binding of small and large antigenic determinants do not reflect funda- mental differences in the regions of the antibody molecule that make up the binding site. Despite differences in the binding patterns of small haptens and large antigens, Chap- ter 4 will show that all antibody binding sites are assembled from the same regions of the antibody molecule—namely, parts of the variable regions of its polypeptide chains. Antigens CHAPTER 3 63 TABLE 3-4 Comparison of antigen recognition by T cells and B cells Characteristic B cells T cells Interaction with antigen Involves binary complex of membrane Involves ternary complex of T-cell receptor, Ag, Ig and Ag and MHC molecule Binding of soluble antigen Yes No Involvement of MHC molecules None required Required to display processed antigen Chemical nature of antigens Protein, polysaccharide, lipid Mostly proteins, but some lipids and glycolipids presented on MHC-like molecules Epitope properties Accessible, hydrophilic, mobile peptides Internal linear peptides produced by containing sequential or nonsequential processing of antigen and bound to amino acids MHC molecules FIGURE 3-2 Three-dimensional structure of an octapeptide hor- mone (angiotensin II) complexed with a monoclonal antibody Fab fragment, the antigen-binding unit of the antibody molecule. The an- giotensin II peptide is shown in red, the heavy chain in blue, and the light chain in purple. [From K. C. Garcia et al., 1992, Science 257:502.] 8536d_ch03_057-075 8/7/02 9:18 AM Page 63 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: The B-cell epitopes on native proteins generally are com- posed of hydrophilic amino acids on the protein surface that are topographically accessible to membrane-bound or free anti- body. A B-cell epitope must be accessible in order to be able to bind to an antibody; in general, protruding regions on the surface of the protein are the most likely to be recognized as epitopes, and these regions are usually composed of predom- inantly hydrophilic amino acids. Amino acid sequences that are hidden within the interior of a protein often consist of predominantly hydrophobic amino acids, and cannot func- tion as B-cell epitopes unless the protein is first denatured. In the crystallized antigen-antibody complexes analyzed to date, the interface between antibody and antigen shows nu- merous complementary protrusions and depressions (Figure 3-5). Between 15 and 22 amino acids on the antigen contact the antibody by 75–120 hydrogen bonds as well as by ionic and hydrophobic interactions. B-cell epitopes can contain sequential or nonsequential amino acids. Epitopes may be composed of sequential con- tiguous residues along the polypeptide chain or nonsequen- tial residues from segments of the chain brought together by the folded conformation of an antigen. Most antibodies elicited by globular proteins bind to the protein only when it is in its native conformation. Because denaturation of such antigens usually changes the structure of their epitopes, anti- bodies to the native protein do not bind to the denatured protein. Five distinct sequential epitopes, each containing six to eight contiguous amino acids, have been found in sperm whale myoglobin. Each of these epitopes is on the surface of the molecule at bends between the H9251-helical regions (Figure 3-6a). Sperm whale myoglobin also contains several nonse- quential epitopes, or conformational determinants. The residues that constitute these epitopes are far apart in the pri- mary amino acid sequence but close together in the tertiary structure of the molecule. Such epitopes depend on the 64 PART II Generation of B-Cell and T-Cell Responses (a) (b) (c) FIGURE 3-3 (a) Model of interaction between hen egg-white lysozyme (HEL) and Fab fragment of anti-HEL antibody based on x- ray diffraction analysis. HEL is shown in green, the Fab heavy chain in blue, and the Fab light chain in yellow. A glutamine residue of lysozyme (red) fits into a pocket in the Fab fragment. (b) Representa- tion of HEL and the Fab fragment when pulled apart showing com- plementary surface features. (c) View of the interacting surfaces of the Fab fragment and HEL obtained by rotating each of the mole- cules. The contacting residues are numbered and shown in red with the protruding glutamine (#14) in HEL now shown in white. [From A. G. Amit et al., 1986, Science 233: 747.] FIGURE 3-4 Models of the variable domains of six Fab fragments with their antigen-binding regions shown in purple. The top three an- tibodies are specific for lysozyme, a large globular protein. The lower three antibodies are specific for smaller molecules or very small seg- ments of macromolecules: McPC603 for phosphocholine; BV04 for a small segment of a single-stranded DNA molecule; and 17/9 for a peptide from hemagglutinin, an envelope protein of influenza virus. In general, the binding sites for small molecules are deep pockets, whereas binding sites for large proteins are flatter, more undulating surfaces. [From I. A. Wilson and R. L. Stanfield, 1993, Curr. Opin. Struc. Biol. 3:113.] HyHel-5 HyHel-10 D1/3 McPC603 BV04 17/9 8536d_ch03_064 9/10/02 10:12 AM Page 64 mac46 mac46:385_REB: Antigens CHAPTER 3 65 VISUALIZING CONCEPTS FIGURE 3-6 Protein antigens usually contain both sequential and nonsequential B-cell epitopes. (a) Diagram of sperm whale myoglobin showing locations of five sequential B-cell epitopes (blue). (b) Ribbon diagram of hen egg-white lysozyme showing residues that compose one nonsequential (conformational) epi- tope. Residues that contact antibody light chains, heavy chains, or both are shown in red, blue, and white, respectively. These residues are widely spaced in the amino acid sequence but are brought into proximity by folding of the protein. [Part (a) adapted from M. Z. Atassi and A. L. Kazim. 1978, Adv. Exp. Med. Biol. 98:9; part (b) from W. G. Laver et al., 1990, Cell 61:554.] Heme (145) 146?151 COOH (a) (b) NH 2 15?21 (22) 56?62 113?119 FIGURE 3-5 Computer simulation of an interaction between anti- body and influenza virus antigen, a globular protein. (a) The antigen (yellow) is shown interacting with the antibody molecule; the variable region of the heavy chain is red, and the variable region of the light Antigen Antibody (a) (b) chain is blue. (b) The complementarity of the two molecules is re- vealed by separating the antigen from the antibody by 8 ?. [Based on x-ray crystallography data collected by P. M. Colman and W. R. Tulip. From G. J. V. H. Nossal, 1993, Sci. Am. 269(3):22.] 8536d_ch03_065 9/10/02 10:12 AM Page 65 mac46 mac46:385_REB: native protein conformation for their topographical struc- ture. One well-characterized nonsequential epitope in hen egg-white lysozyme (HEL) is shown in Figure 3-6b. Although the amino acid residues that compose this epitope of HEL are far apart in the primary amino acid sequence, they are brought together by the tertiary folding of the protein. Sequential and nonsequential epitopes generally behave differently when a protein is denatured, fragmented, or re- duced. For example, appropriate fragmentation of sperm whale myoglobin can yield five fragments, each retaining one sequential epitope, as demonstrated by the observation that antibody can bind to each fragment. On the other hand, frag- mentation of a protein or reduction of its disulfide bonds of- ten destroys nonsequential epitopes. For example, HEL has four intrachain disulfide bonds, which determine the final protein conformation (Figure 3-7a). Many antibodies to HEL recognize several epitopes, and each of eight different epitopes have been recognized by a distinct antibody. Most of these epitopes are conformational determinants dependent on the overall structure of the protein. If the intrachain disul- fide bonds of HEL are reduced with mercaptoethanol, the nonsequential epitopes are lost; for this reason, antibody to native HEL does not bind to reduced HEL. The inhibition experiment shown in Figure 3-7 nicely demonstrates this point. An antibody to a conformational determinant, in this example a peptide loop present in native HEL, was able to bind the epitope only if the disulfide bond that maintains the structure of the loop was intact. Infor- mation about the structural requirements of the antibody combining site was obtained by examining the ability of structural relatives of the natural antigen to bind to that an- tibody. If a structural relative has the critical epitopes present in the natural antigen, it will bind to the antibody combining site, thereby blocking its occupation by the natural antigen. In this inhibition assay, the ability of the closed loop to in- hibit binding showed that the closed loop was sufficiently 66 PART II Generation of B-Cell and T-Cell Responses FIGURE 3-7 Experimental demonstration that binding of antibody to conformational determinants in hen egg-white lysozyme (HEL) depends on maintenance of the tertiary structure of the epitopes by intrachain disulfide bonds. (a) Diagram of HEL primary structure, in which circles represent amino acid residues. The loop (blue circles) formed by the disulfide bond between the cysteine residues at posi- tions 64 and 80 constitutes one of the conformational determinants in HEL. (b) Synthetic open-loop and closed-loop peptides corre- sponding to the HEL loop epitope. (c) Inhibition of binding between HEL loop epitope and anti-loop antiserum. Anti-loop antiserum was first incubated with the natural loop sequence, the synthetic closed- loop peptide, or the synthetic open-loop peptide; the ability of the an- tiserum to bind the natural loop sequence then was measured. The absence of any inhibition by the open-loop peptide indicates that it does not bind to the anti-loop antiserum. [Adapted from D. Benjamin et al., 1984, Annu. Rev. Immunol. 2:67.] (b) Synthetic loop peptides CYS 80 64 CYS Open loop Closed loop CYS CYS 80 64 COOH H 2 N (a) Hen egg–white lysosome Disulfide bond COOH H 2 N 8064 16 Ratio of loop inhibitor to anti–loop antiserum 100 Inhibition, % 08 80 60 40 20 Natural loop Closed synthetic loop Open synthetic loop (c) Inhibition of reaction between HEL loop and anti–loop antiserum 8536d_ch03_057-075 8/7/02 9:18 AM Page 66 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: similar to HEL to be recognized by antibody to native HEL. Even though the open loop had the same sequence of amino acids as the closed loop, it lacked the epitopes recognized by the antibody and therefore was unable to block binding of HEL. B-cell epitopes tend to be located in flexible regions of an im- munogen and display site mobility. John A. Tainer and his col- leagues analyzed the epitopes on a number of protein antigens (myohemerytherin, insulin, cytochrome c, myoglo- bin, and hemoglobin) by comparing the positions of the known B-cell epitopes with the mobility of the same residues. Their analysis revealed that the major antigenic de- terminants in these proteins generally were located in the most mobile regions. These investigators proposed that site mobility of epitopes maximizes complementarity with the antibody’s binding site, permitting an antibody to bind with an epitope that it might bind ineffectively if it were rigid. However, because of the loss of entropy due to binding to a flexible site, the binding of antibody to a flexible epitope is generally of lower affinity than the binding of antibody to a rigid epitope. Complex proteins contain multiple overlapping B-cell epi- topes, some of which are immunodominant. For many years, it was dogma in immunology that each globular protein had a small number of epitopes, each confined to a highly accessi- ble region and determined by the overall conformation of the protein. However, it has been shown more recently that most of the surface of a globular protein is potentially antigenic. This has been demonstrated by comparing the antigen-bind- ing profiles of different monoclonal antibodies to various globular proteins. For example, when 64 different mono- clonal antibodies to BSA were compared for their ability to bind to a panel of 10 different mammalian albumins, 25 dif- ferent overlapping antigen-binding profiles emerged, sug- gesting that these 64 different antibodies recognized a minimum of 25 different epitopes on BSA. Similar findings have emerged for other globular proteins, such as myoglobin and HEL. The surface of a protein, then, presents a large number of potential antigenic sites. The subset of antigenic sites on a given protein that is recognized by the immune system of an animal is much smaller than the potential antigenic reper- toire, and it varies from species to species and even among in- dividual members of a given species. Within an animal, cer- tain epitopes of an antigen are recognized as immunogenic, but others are not. Furthermore, some epitopes, called im- munodominant, induce a more pronounced immune re- sponse than other epitopes in a particular animal. It is highly likely that the intrinsic topographical properties of the epi- tope as well as the animal’s regulatory mechanisms influence the immunodominance of epitopes. Antigen-Derived Peptides Are the Key Elements of T-Cell Epitopes Studies by P. G. H. Gell and Baruj Benacerraf in 1959 sug- gested that there was a qualitative difference between the T- cell and the B-cell response to protein antigens. Gell and Benacerraf compared the humoral (B-cell) and cell-medi- ated (T-cell) responses to a series of native and denatured protein antigens (Table 3-5). They found that when primary immunization was with a native protein, only native protein, not denatured protein, could elicit a secondary antibody (hu- moral) response. In contrast, both native and denatured pro- tein could elicit a secondary cell-mediated response. The finding that a secondary response mediated by T cells was in- duced by denatured protein, even when the primary immu- nization had been with native protein, initially puzzled immunologists. In the 1980s, however, it became clear that T cells do not recognize soluble native antigen but rather rec- ognize antigen that has been processed into antigenic pep- tides, which are presented in combination with MHC molecules. For this reason, destruction of the conformation of a protein by denaturation does not affect its T-cell epi- topes. Because the T-cell receptor does not bind free peptides, experimental systems for studying T-cell epitopes must in- clude antigen-presenting cells or target cells that can display the peptides bound to an MHC molecule. Antigenic peptides recognized by T cells form trimolecular complexes with a T-cell receptor and an MHC molecule (Figure 3-8). The structures of TCR-peptide-MHC trimolecular complexes have been determined by x-ray crystallography and are described in Chapter 9. These structural studies of class I or class II MHC molecules crystallized with known T- cell antigenic peptides has shown that the peptide binds to a Antigens CHAPTER 3 67 TABLE 3-5 Antigen recognition by T and B lymphocytes reveals qualitative differences SECONDARY IMMUNE RESPONSE Primary immunization Secondary immunization Antibody production Cell-mediated T DTH response* Native protein Native protein H11001H11001 Native protein Denatured protein H11002H11001 * T DTH is a subset of CD4 H11001 T H cells that mediate a cell-mediated response called delayed-type hypersensitivity (see Chapter 14). 8536d_ch03_057-075 8/6/02 10:28 AM Page 67 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: cleft in the MHC molecule (see Figure 7-8). Unlike B-cell epitopes, which can be viewed strictly in terms of their ability to interact with antibody, T-cell epitopes must be viewed in terms of their ability to interact with both a T-cell receptor and an MHC molecule. The binding of an MHC molecule to an antigenic peptide does not have the fine specificity of the interaction between an antibody and its epitope. Instead, a given MHC molecule can selectively bind a variety of different peptides. For example, the class II MHC molecule designated IA d can bind peptides from ovalbumin (residues 323–339), hemagglutinin (residues 130– 142), and lambda repressor (residues 12–26). Studies revealing structural features, or motifs, common to different peptides that bind to a single MHC molecule are described in Chapter 7. Antigen processing is required to generate peptides that in- teract specifically with MHC molecules. As mentioned in Chapter 1, endogenous and exogenous antigens are usually processed by different intracellular pathways (see Figure 1-9). Endogenous antigens are processed into peptides within the cytoplasm, while exogenous antigens are processed by the endocytic pathway. The details of antigen processing and presentation are described in Chapter 8. Epitopes recognized by T cells are often internal. T cells tend to recognize internal peptides that are exposed by processing within antigen-presenting cells or altered self-cells. J. Roth- bard analyzed the tertiary conformation of hen egg-white lysozyme and sperm whale myoglobin to determine which amino acids protruded from the natural molecule. He then mapped the major T-cell epitopes for both proteins and found that, in each case, the T-cell epitopes tended to be on the “inside” of the protein molecule (Figure 3-9). Haptens and the Study of Antigenicity The pioneering work of Karl Landsteiner in the 1920s and 1930s created a simple, chemically defined system for study- ing the binding of an individual antibody to a unique epitope 68 PART II Generation of B-Cell and T-Cell Responses FIGURE 3-8 Schematic diagram of the ternary complex formed between a T-cell receptor (TCR) on a T H cell, an antigen, and a class II MHC molecule. Antigens that are recognized by T cells yield pep- tides that interact with MHC molecules to form a peptide-MHC com- plex that is recognized by the T-cell receptor. As described in later chapters, the coreceptor, CD4, on T H cells also interacts with MHC molecules. T C cells form similar ternary complexes with class I MHC molecules on target cells however, these cells bear MHC-interacting CD8 coreceptors. CD4 TCR Class II MHC Peptide T H cell Antigen-presenting cell FIGURE 3-9 Experimental evidence that T H cells tend to recognize internal peptides of antigens. This plot shows the relative protrusion of amino acid residues in the tertiary conformation of hen egg-white lysozyme. The known T-cell epitopes in HEL are indicated by the blue bars at the top. Notice that, in general, the amino acid residues that T–cell epitopes of hen egg–white lysozyme 4534 Residue number 9080 120 1291101005040 70603020110 6151 9378 9 0 3 6 1 4 5 2 8 7 Protrusion index correspond to the T-cell epitopes exhibit less overall protrusion. In contrast, note that the B-cell epitope consisting of residues 64–80, which form a conformational determinant in native HEL that is rec- ognized by antibody (see Figure 3-7), exhibit greater overall protru- sion. [From J. Rothbard et al., 1987, Mod. Trends Hum. Leuk., vol. 7.] 8536d_ch03_057-075 8/7/02 9:18 AM Page 68 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: on a complex protein antigen. Landsteiner employed various haptens, small organic molecules that are antigenic but not immunogenic. Chemical coupling of a hapten to a large pro- tein, called a carrier, yields an immunogenic hapten-carrier conjugate. Animals immunized with such a conjugate pro- duce antibodies specific for (1) the hapten determinant, (2) unaltered epitopes on the carrier protein, and (3) new epi- topes formed by combined parts of both the hapten and car- rier (Figure 3-10). By itself, a hapten cannot function as an immunogenic epitope. But when multiple molecules of a sin- gle hapten are coupled to a carrier protein (or nonimmuno- genic homopolymer), the hapten becomes accessible to the immune system and can function as an immunogen. The beauty of the hapten-carrier system is that it provides immunologists with a chemically defined determinant that can be subtly modified by chemical means to determine the effect of various chemical structures on immune specificity. In his studies, Landsteiner immunized rabbits with a hapten- carrier conjugate and then tested the reactivity of the rabbit’s immune sera with that hapten and with closely related hap- tens coupled to a different carrier protein. He was thus able to measure, specifically, the reaction of the antihapten antibod- ies in the immune serum and not that of antibodies to the original carrier epitopes. Landsteiner tested whether an anti- hapten antibody could bind to other haptens having a slightly different chemical structure. If a reaction occurred, it was called a cross-reaction. By observing which hapten modifications prevented or permitted cross-reactions, Land- steiner was able to gain insight into the specificity of antigen- antibody interactions. Using various derivatives of aminobenzene as haptens, Landsteiner found that the overall configuration of a hapten plays a major role in determining whether it can react with a given antibody. For example, antiserum from rabbits im- munized with aminobenzene or one of its carboxyl deriva- tives (o-aminobenzoic acid, m-aminobenzoic acid, or p- aminobenzoic acid) coupled to a carrier protein reacted only with the original immunizing hapten and did not cross-react with any of the other haptens (Table 3-6). In contrast, if the overall configuration of the hapten was kept the same and the hapten was modified in the para position with various nonionic derivatives, then the antisera showed various de- grees of cross-reactivity. Landsteiner’s work not only demon- strated the specificity of the immune system, but also demon- strated the enormous diversity of epitopes that the immune system is capable of recognizing. Many biologically important substances, including drugs, peptide hormones, and steroid hormones, can function as haptens. Conjugates of these haptens with large protein car- riers can be used to produce hapten-specific antibodies. These antibodies are useful for measuring the presence of various substances in the body. For instance, the original home pregnancy test kit employed antihapten antibodies to determine whether a woman’s urine contained human chori- onic gonadotropin (HCG), which is a sign of pregnancy. However, as shown in the Clinical Focus, the formation of drug-protein conjugates in the body can produce drug aller- gies that may be life-threatening. Pattern-Recognition Receptors The receptors of adaptive and innate immunity differ. Anti- bodies and T-cell receptors, the receptors of adaptive immu- nity, recognize details of molecular structure and can discriminate with exquisite specificity between antigens fea- turing only slight structural differences. The receptors of in- nate immunity recognize broad structural motifs that are highly conserved within microbial species but are generally absent from the host. Because they recognize particular over- all molecular patterns, such receptors are called pattern- recognition receptors (PRRs). Patterns recognized by this type of receptor include combinations of sugars, certain pro- teins, particular lipid-bearing molecules, and some nucleic acid motifs. Typically, the ability of pattern-recognition receptors to distinguish between self and nonself is perfect because the molecular pattern targeted by the receptor is produced only by the pathogen and never by the host. This contrasts sharply with the occasional recognition of self Antigens CHAPTER 3 69 FIGURE 3-10 A hapten-carrier conjugate contains multiple copies of the hapten—a small nonimmunogenic organic compound such as dinitrophenol (DNP)—chemically linked to a large protein carrier such as bovine serum albumin (BSA). Immunization with DNP alone elicits no anti-DNP antibodies, but immunization with DNP- BSA elicits three types of antibodies. Of these, anti-DNP antibody is predominant, indicating that in this case the hapten is the immuno- dominant epitope in a hapten-carrier conjugate, as it often is in such conjugates. Hapten–carrier conjugate HaptenCarrier Antibodies to hapten Antibodies to carrier Antibodies to conjugate of hapten and carrier Immunize rabbit Injection with: Antibodies formed: Hapten (DNP) None Protein carrier (BSA) Anti–BSA Hapten–carrier conjugate (DNP-BSA) Anti–DNP (major) Anti–BSA (minor) Anti–DNP/BSA (minor) 8536d_ch03_057-075 8/7/02 9:18 AM Page 69 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: antigens by receptors of adaptive immunity, which can lead to autoimmune disorders. Like antibodies and T-cell recep- tors, pattern-recognition receptors are proteins. However, the genes that encode PRRs are present in the germline of the organism. In contrast, the genes that encode the enormous diversity of antibodies and TCRs are not present in the germline. They are generated by an extraordinary process of genetic recombination that is discussed in Chapter 5. Many different pattern-recognition receptors have been identified and several examples appear in Table 3-7. Some are present in the bloodstream and tissue fluids as soluble circu- lating proteins and others are on the membrane of cells such as macrophages, neutrophils, and dendritic cells. Mannose- binding lectin (MBL) and C-reactive protein (CRP) are solu- ble pattern receptors that bind to microbial surfaces and promote their opsonization. Both of these receptors also have the ability to activate the complement system when they are bound to the surface of microbes, thereby making the invader a likely target of complement-mediated lysis. Yet another soluble receptor of the innate immune system, lipopolysaccharide-binding protein, is an important part of the system that recognizes and signals a response to lipopolysaccharide, a component of the outer cell wall of gram-negative bacteria. Pattern-recognition receptors found on the cell mem- brane include scavenger receptors and the toll-like receptors. Scavenger receptors (SRs) are present on macrophages and many types of dendritic cells, and are involved in the binding and internalization of gram-positive and gram-negative bac- teria, as well as the phagocytosis of apoptotic host cells. The exact roles and mechanisms of action of the many types of scavenger receptors known to date are under active investiga- tion. The toll-like receptors (TLRs) are important in recog- nizing many microbial patterns. This family of proteins is ancient—toll-like receptors mediate the recognition and generation of defensive responses to pathogens in organisms as widely separated in evolutionary history as humans and flies. Typically, signals transduced through the TLRs cause transcriptional activation and the synthesis and secretion of cytokines, which promote inflammatory responses that bring macrophages and neutrophils to sites of inflammation. 70 PART II Generation of B-Cell and T-Cell Responses TABLE 3-6 Reactivity of antisera with various haptens REACTIVITY WITH Antiserum against Aminobenzene (aniline) o-Aminobenzoic acid m-Aminobenzoic acid p-Aminobenzoic acid Aminobenzene H11001 000 o-Aminobenzoic acid 0 H11001 00 m-Aminobenzoic acid 00H11001 0 p-Aminobenzoic acid 0+ KEY: 0 H11005 no reactivity; H11001H11005strong reactivity SOURCE: Based on K. Landsteiner, 1962, The Specificity of Serologic Reactions, Dover Press. Modified by J. Klein, 1982, Immunology: The Science of Self-Nonself Discrimination, John Wiley. NH 2 NH 2 COOH NH 2 COOH NH 2 COOH Lipoproteins Lipoarabinomannan LPS (Leptospira) LPS (P. gingivalis) PGN (Gram-positive) Zymosan (Yeast) GPI anchor (T. cruzi) LPS (Gram-negative) Taxol (Plant) F protein (RS virus) hsp60 (Host) Fibronectin (Host) Flagellin CpG DNA TLR2 TLR4 TLR5 TLR9TLR6 MD-2 FIGURE 3-11 Location and targets of some pattern-recognition re- ceptors. Many pattern-recognition receptors are extracellular and tar- get microbes or microbial components in the bloodstream and tissue fluids, causing their lysis or marking them for removal by phagocytes. Other pattern-recognition receptors are present on the cell membrane and bind to a broad variety of microbes or microbial products. Engagement of these receptors triggers signaling path- ways that promote inflammation or, in the case of the scavenger re- ceptors, phagocytosis or endocytosis. dsRNA H11005 double stranded RNA; LPS H11005 lipopolysaccharide. [S. Akira et al., 2001, Nature Im- munology 2:675.] 8536d_ch03_057-075 8/7/02 9:18 AM Page 70 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: Antigens CHAPTER 3 71 TABLE 3-7 Receptors of innate and adaptive immunity Characteristic Innate immunity Adaptive immunity Specificity Specific for conserved Specific for details of antigen molecular patterns or types structure Self/nonself Perfect: evolutionarily selected Excellent: but imperfect. discrimination to distinguish phylogenetic Occasional reaction with differences. Never recognizes self antigens self. RECEPTORS OF THE ADAPTIVE IMMUNE SYSTEM Receptor Target (location) (source) Effect of recognition Antibody Specific components of Labeling of pathogen for (B-cell membrane, pathogen destruction and removal blood, tissue fluids) T-cell receptor Proteins or certain lipids of Induction of pathogen- (T-cell membrane) pathogen specific humoral and cell- mediated immunity RECEPTORS OF THE INNATE IMMUNE SYSTEM Complement Microbial cell-wall Complement activation, (bloodstream, components opsonization tissue fluids) Mannose-binding lectin (MBL) Mannose-containing Complement activation, (bloodstream, tissue fluids) microbial carbohydrates opsonization (cell walls) C-reactive protein (CRP) Phosphatidylcholine Complement activation, (bloodstream, tissue fluids) (microbial membranes) opsonization LPS-binding protein (LBP) Bacterial lipopolysaccharide Delivery to cell-membrane LPS receptor (bloodstream, tissue fluids) (LPS) (TLR-CD14-MD-2 complex*) TLR2 Cell-wall components of gram-positive Attracts phagocytes, activates macrophages, (cell membrane) bacteria, LPS*. Yeast cell-wall component dendritic cells. Induces secretion of (zymosan) several cytokines TLR3 Double-stranded RNA (dsRNA) Induces production of interferon, (cell membrane) (replication of many RNA viruses) an antiviral cytokine TLR4 LPS* Attracts phagocytes, activates macrophages, (cell membrane) dendritic cells. Induces secretion of several cytokines TLR5 Flagellin Attracts phagocytes, activates macrophages, (cell membrane) (flagella of gram-positive dendritic cells. Induces secretion of and gram-negative bacteria) several cytokines TLR9 CpG Attracts phagocytes, macrophages, (cell membrane) dendritic cells. Induces secretion of several cytokines Scavenger receptors (many) Many targets; gram-positive and gram- Induces phagocytosis or endocytosis (cell membrane) negative bacteria, apoptotic host cells * LPS is bound at the cell membrane by a complex of proteins that includes CD14, MD-2, and a TLR (usually TLR4). 8536d_ch03_057-075 8/6/02 10:28 AM Page 71 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: TLR signaling can also result in the recruitment and activa- tion of macrophages, NK cells, and dendritic cells, key agents in the presentation of antigen to T cells. The links to T cells and cytokine release shows the intimate relationship between innate and adaptive responses. A search of the human genome has uncovered 10 TLRs, and the functions of six members of this PRR family have been determined. TLR2, often with the collaboration of TLR6, binds a wide variety of molecular classes found in mi- crobes, including peptidoglycans, zymosans, and bacterial lipopeptides. TLR4 is the key receptor for most bacterial lipopolysaccharides, although TLR2 also binds some vari- eties of LPS. The binding of LPS by either of these TLRs is complex and involves the participation of three additional proteins, one of which is the lipopolysaccharide-binding protein mentioned above, abbreviated LBP. The first step in the process is the binding of LPS by circulating LBP, which then delivers it to a complex of TLR4 (or TLR2) with two ad- ditional proteins, CD14 and MD2. The engagement of LPS by this complex causes its TLR component to initiate a sig- nal-transduction process that can produce a cellular re- sponse. Another family member, TLR5, recognizes flagellin, the major structural component of bacterial flagella. TLR3 recognizes the double-stranded RNA (dsRNA) that appears after infection by RNA viruses. As shown in Table 3-7, dsRNA is also recognized by dsRNA-activated kinase. Finally, TLR9 recognizes and initiates a response to CpG (unmethy- lated cytosine linked to guanine) sequences. These sequences are represented in abundance in microbial sequences but are much less common in mammalian sequences. Table 3-7 summarizes the receptors of adaptive immunity and lists many pattern-recognition receptors of innate immunity. The microbial targets and physiological sites of many PRRs are shown in Figure 3-11. 72 PART II Generation of B-Cell and T-Cell Responses Since WorldWar II, penicillin has been used to successfully treat a wide variety of bacterial infec- tions. However, the penicillin family of antibiotics is not without drawbacks. One is the role of penicillins and other antibiotics in the evolution of antibiotic- resistant bacterial strains. Another is their capacity to induce allergic reactions in some patients. Penicillin and its rela- tives are responsible for most of the recorded allergic reactions to drugs and 97% of the deaths caused each year by drug allergies. Allergies to penicillin and other drugs can be induced by small doses and are not consequences of the pharmacologi- cal or physiological effects of the drugs. An allergic response usually occurs about a week or so after the patient’s first exposure to the agent, with typically mild symptoms often including hives, fever, swelling of lymph nodes, and occasion- CLINICAL FOCUS Drug Allergies—When Medicines Become Immunogens ally an arthritis-like discomfort. Subse- quent treatments with the drug usually cause much more rapid and often more severe reactions. Within minutes the throat and eyelids may swell. Grave dan- ger arises if these symptoms progress to anaphylaxis, a physiological collapse that often involves the respiratory, circulatory, and digestive systems. Hives, vomiting, abdominal pain, and diarrhea may be a preamble to respiratory and circulatory problems that are life threatening. Wheezing and shortness of breath may be accompanied by swelling of the larynx and epiglottis that can block airflow, and a profound drop in blood pressure causes shock, frequently accompanied by weakened heart contractions. The treatment of choice for anaphy- laxis is injection of the drug epinephrine (adrenaline), which can reverse the body’s slide into deep anaphylaxis by raising blood pressure, easing constric- tion of the air passages, and inhibiting the release from mast cells and ba- sophils of the agents that induce ana- phylaxis. Other drugs may be used to raise the low blood pressure, strengthen heart contractions, and expand the blocked airways. After a case of drug-in- duced anaphylaxis, affected individuals are advised to carry a notice warning future healthcare providers of the drug allergy. Most drugs, including penicillin, are low-molecular-weight compounds that cannot induce immune responses un- less they are conjugated with a larger molecule. Intensive investigation of al- lergy to penicillin has provided critical in- sight into the basis of allergic reactions to this and other drugs. As shown in the accompanying figure, penicillin can react with proteins to form a penicilloyl-pro- tein derivative. The penicilloyl-protein behaves as a hapten-carrier conjugate, with the penicilloyl group acting as a haptenic epitope. This epitope is readily recognized by the immune system, and antibodies are produced against it. Some individuals respond to penicillin by pro- ducing significant amounts of a type of antibody known as immunoglobulin E (IgE). Once generated, these IgE anti- bodies are dispersed throughout the body and are bound by IgE receptors on the surfaces of mast cells and basophils, 8536d_ch03_057-075 8/6/02 10:28 AM Page 72 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: Antigens CHAPTER 3 73 Penicillin R CO C HN HC C(CH 3 ) 2 CH COOHO N S O R C C C H C Protein HN HC H C H C Penicillenic acid R C C O C N C(CH 3 ) 2 SH 2 CH COOHO N H C C(CH 3 ) 2 (CH 2 ) 4 CH COOHN NHO S H N Protein Penicilloyl-protein Same or different protein Reaction of isomeric structures with body proteins produces a variety of major and minor determinants Isomerization When nucleophiles such as amino groups or hydroxyl groups are present on soluble proteins or on the membrane of cells, they can react with penicillin and its relatives to form covalent linkages between host macromolecular structures and the drug. This is illustrated by the reaction of the free amino group of a lysine residue with penicillin (or with its sponta- neously forming isomeric compounds, such as penicillenic acid) to produce protein-drug or cell-surface–drug derivatives. Such adducts are the major immunogenic species that elicit immune responses to this antibiotic. However, as indicated, other hapten-carrier conjugates of somewhat different struc- ture are also formed and, because of their structural similarity, can also induce immune responses to penicillin. [Adapted from N. F. Adkinson, 1995, in Manual of Clinical Laboratory Immunology, N. Rose et al., eds., American Society for Microbiology, Washington, D.C.] where they can remain for a long time. If a person with penicillin-specific IgE anti- body bound to mast cells is subse- quently treated with penicillin, there may be an allergic reaction. In fact, between 1 and 5 percent of people treated with penicillin develop some degree of allergy to it. Penicillin is not the only drug against which patients can develop allergies. Others include streptomycin, aspirin, the so-called “sulfa-drugs” such as the sul- fonamides, some anesthetics (e.g., suc- cinyl choline), and some opiates. All of these small molecules first react with proteins to form drug-protein deriva- tives. When this happens, there is a pos- sibility that the immune system will pro- duce an anti-hapten response to the drug, just as with penicillin. Drugs (and their metabolites) that are incapable of forming drug-protein conjugates rarely elicit allergic reactions. SUMMARY a73 All immunogens are antigens but not all antigens are im- munogens. a73 Immunogenicity is determined by many factors including foreignness, molecular size, chemical composition, com- plexity, dose, susceptibility to antigen processing and pre- sentation, the genotype of the recipient animal (in particular, its MHC genes), route of administration, and adjuvants. a73 The sizes of B-cell epitopes range widely. Some are quite small (e.g., small peptides or small organic molecules), and are often bound in narrow grooves or deep pockets of the antibody. Protein B-cell epitopes are much larger and interact with a larger, flatter complementary surface on the antibody molecule. a73 T-cell epitopes are generated by antigen processing, which fragments protein into small peptides that combine with class I or class II MHC molecules to form peptide-MHC complexes that are displayed on the surface of cells. T-cell activation requires the formation of a ternary complex between a T cell’s TCR and peptide-MHC on antigen- presenting or altered self cells. a73 Haptens are small molecules that can bind to antibodies but cannot by themselves induce an immune response. However, the conjugate formed by coupling a hapten to a large carrier protein is immunogenic and elicits produc- tion of anti-hapten antibodies when injected into an ani- mal. Such injections also produce anti-carrier and anti- hapten/carrier antibodies as well. a73 In the body, the formation of hapten-carrier conjugates is the basis of allergic responses to drugs such as penicillin. Go to www.whfreeman.com/immunology Self-Test Review and quiz of key terms 8536d_ch03_057-075 9/6/02 9:01 PM Page 73 mac85 Mac 85:365_smm:Goldsby et al. / Immunology 5e: a73 The innate immune system uses pattern-recognition re- ceptors to recognize and respond to broad structural mo- tifs that are highly conserved within microbial species but are generally absent from the host. References Berzofsky, J. A., and J. J. Berkower. 1999. Immunogenicity and antigen structure. In Fundamental Immunology, 4th ed., W. E. Paul, ed., Lippincott-Raven, Philadelphia. Dale, D., and D. Federman, eds. 1997. Drug allergy. In Scientific American Medicine. Chapter VIII, Hypersensitivity and allergy, p. 27. Demotz, S., H. M. Grey, E. Appella, and A. Sette. 1989. Charac- terization of a naturally processed MHC class II-restricted T- cell determinant of hen egg lysozyme. Nature 342:682. Grey, H. M., A. Sette, and S. Buus. 1989. How T cells see antigen. Sci. Am. 261(5):56. Landsteiner, K. 1945. The Specificity of Serological Reactions. Harvard University Press, Cambridge, Massachusetts. Laver, W. G., G. M. Air, R. G. Webster, and S. J. Smith-Gill. 1990. Epitopes on protein antigens: misconceptions and realities. Cell 61:553. Peiser, L., S. Mukhopadhyay, and S. Gordon. 2002. Scavenger re- ceptors in innate immunity. Curr. Opin. Immunol. 14:123. Stanfield, R. L., and I. A. Wilson. 1995. Protein-peptide interac- tions. Curr. Opin. Struc. Biol. 5:103. Tainer, J. A., et al. 1985. The atomic mobility component of pro- tein antigenicity. Annu. Rev. Immunol. 3:501. Underhill, D. M., and A. Ozinsky. 2002. Toll-like receptors: key mediators of microbe detection. Curr. Opin. Immunol. 14:103. USEFUL WEB SITES http://www.umass.edu/microbio/rasmol/ RASMOL is free software for visualizing molecular structures that can be run on Windows-based, Macintosh, or Unix PCs. With it one can view three-dimensional structures of many types of molecules, including proteins and nucleic acids. http://www.expasy.ch/ This is the excellent and comprehensive Swiss Institute of Bioinformatics (SIB) Web site, which contains extensive in- formation on protein structure. From it one can obtain pro- tein sequences and three-dimensional structures of proteins, as well as the versatile Swiss-PdbViewer software, which has several advanced capabilities not found in RASMOL. Study Questions CLINICAL FOCUS QUESTION Consider the following situations and provide a likely diagnosis or appropriate response. a. Six hours after receiving a dose of penicillin, a young child who has never been treated with penicillin develops a case of hives and diarrhea. The parents report the illness and ask if it might be an allergic reaction to penicillin. b. A patient who has never taken sulfonamides but is known to be highly allergic to penicillin develops a bladder infec- tion that is best treated with a “sulfa” drug. The patient wonders if “sulfa” drugs should be avoided. c. A student who is unaware that he had developed a signifi- cant allergy to penicillin received an injection of the an- tibiotic and within minutes experienced severe respiratory distress and a drop in blood pressure. An alert intern administered epinephrine and the patient’s condition improved quickly. Frightened but impressed by the effectiveness of the treatment, he asked the intern why the shot of adrenaline made him feel better. d. A pet owner asks whether the same mechanism that causes his allergy to penicillin could also be responsible for his dog’s development of a similar allergy to the drug. (Please go beyond yes or no.) 1. Indicate whether each of the following statements is true or false. If you think a statement is false, explain why. a. Most antigens induce a response from more than one clone. b. A large protein antigen generally can combine with many different antibody molecules. c. A hapten can stimulate antibody formation but cannot combine with antibody molecules. d. MHC genes play a major role in determining the degree of immune responsiveness to an antigen. e. T-cell epitopes tend to be accessible amino acid residues that can combine with the T-cell receptor. f. Many B-cell epitopes are nonsequential amino acids brought together by the tertiary conformation of a protein antigen. g. Both T H and T C cells recognize antigen that has been processed and presented with an MHC molecule. h. Each MHC molecule binds a unique peptide. i. All antigens are also immunogens. j. Antibodies can bind hydrophilic or hydrophobic com- pounds, but T-cell receptors can only bind peptide-MHC complexes. 2. What would be the likely outcome of each of the develop- ments indicated below. Please be as specific as you can. a. An individual is born with a mutation in C-reactive pro- tein that enables it to recognize phospholipids in both bac- terial and mammalian cell membranes. b. A group of mice in which the CD1 family has been “knocked out” are immunized with Mycobacterium tuber- culosis. Spleen cells from these mice are isolated and di- vided into two batches. One batch is treated with a lipid extract of the bacteria and a second batch is treated with a protein derived from the bacteria known as purified pro- tein derivative (PPD). 3. Two vaccines are described below. Would you expect either or both of them to activate T C cells? Explain your answer. a. A UV-inactivated (“killed”) viral preparation that has re- tained its antigenic properties but cannot replicate. 74 PART II Generation of B-Cell and T-Cell Responses 8536d_ch03_057-075 8/6/02 10:28 AM Page 74 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: b. An attenuated viral preparation that has low virulence but can still replicate within host cells. 4. For each pair of antigens listed below, indicate which is likely to be more immunogenic. Explain your answer. a. Native bovine serum albumin (BSA) Heat-denatured BSA b. Hen egg-white lysozyme (HEL) Hen collagen c. A protein with a molecular weight of 30,000 A protein with a molecular weight of 150,000 d. BSA in Freund’s complete adjuvant BSA in Freund’s incomplete adjuvant 5. Indicate which of the following statements regarding haptens and carriers are true. a. Haptens are large protein molecules such as BSA. b. When a hapten-carrier complex containing multiple hap- ten molecules is injected into an animal, most of the in- duced antibodies are specific for the hapten. c. Carriers are needed only if one wants to elicit a cell-medi- ated response. d. It is necessary to immunize with a hapten-carrier complex in order to obtain antibodies directed against the hapten. e. Carriers include small molecules such as dinitrophenol and penicillenic acid (derived from penicillin). 6. For each of the following statements, indicate whether it is true only of B-cell epitopes (B), only of T-cell epitopes (T), or both types of epitopes (BT) within a large antigen. a. They almost always consist of a linear sequence of amino acid residues. b. They generally are located in the interior of a protein anti- gen. c. They generally are located on the surface of a protein anti- gen. d. They lose their immunogenicity when a protein antigen is denatured by heat. e. Immunodominant epitopes are determined in part by the MHC molecules expressed by an individual. f. They generally arise from proteins. g. Multiple different epitopes may occur in the same antigen. h. Their immunogenicity may depend on the three-dimen- sional structure of the antigen. i. The immune response to them may be enhanced by co- administration of Freund’s complete adjuvant. Antigens CHAPTER 3 75 8536d_ch03_057-075 8/6/02 10:28 AM Page 75 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e: