8536d_ch03_057-075 8/7/02 9: 18 AM Page 57 mac79 Mac 79: 45_BW: Goasby et al./ Immunology 5e Ant gens chapter 3 UBSTANCES THAT CAN BE RECOGNIZED BY THE or cell receptor when complexed with MHC,are called antigens. The molecular properties of antigens and ribute to to our understanding of th mmune system. This chapter describes some of the molecu Complementarity of Interacting Surfaces of Antibody(left) lar features of antigens recognized by B or T cells. The chap- and Antigen(right) 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 m Immunogenicity Versus Antigenicity B or T cells antigen-binding receptor can then induce an im a Factors That Influence Immunogenicity mune response. Fundamental differences in the way B and T determine which molecular features of an antigen are recognized by each branch of the a Haptens and the Study of Antigenicity immune system. These differences are also examined in this chapter a Pattern-Recognition Receptors Immunogenicity versus Antigenicity Immunogenicity and antigenicity are related but distinct Factors That Influer munologic properties that sometimes are confused mmunogenicifyhce munogenicity is the ability to induce a humoral and/or mediated immune response To protect against infectious disease, the immune system must be able to recognize bacteria, bacterial products, fungi B cells antigen effector B cells memory B cells parasites, and viruses as immunogens. In fact, the immune system actually recognizes particular macromolecules of an (plasma cells) infectious agent, generally either proteins or polysaccharides. T cells antigen effector T cells memory T cells Proteins are the most potent immunogens, with polysaccha- (e. g, CTLS, THS an infectious agent generally do not serve as immunogens unless they are complexed with proteins or polysaccharides Although a substance that induces a specific immune re- Immunologists tend to use proteins or polysaccharides as sponse is usually called an antigen, it is more appropriately immunogens in most experimental studies of humoral im- munity(Table 3-1). For cell-mediated immunity, only pro Antigenicity is the ability to combine specifically with teins and some lipids and glycolipids serve as immunogens the final products of the above responses(i.e, antibodies These molecules are not recognized directly. Proteins must and/or cell-surface receptors). Although all molecules that first be processed into small peptides and then presented to- have the property of immunogenicity also have the property gether with MHC molecules on the membrane of a cell be- of antigenicity, the reverse is not true. Some small molecules, fore they can be recognized as immunogens. Recent work called haptens, are antigenic but incapable, by themselves, of shows that those lipids and glycolipids that can elicit cell- inducing a specific immune response. In other words, they mediated immunity must also be combined with MHC-like membrane molecules called CDI(see Chapter 8)
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 polysaccharides 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 immunity (Table 3-1). For cell-mediated immunity, only proteins 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 together with MHC molecules on the membrane of a cell before they can be recognized as immunogens. Recent work shows that those lipids and glycolipids that can elicit cellmediated immunity must also be combined with MHC-like membrane molecules called CD1 (see Chapter 8). chapter 3 ■ Immunogenicity Versus Antigenicity ■ Factors That Influence Immunogenicity ■ Epitopes ■ Haptens and the Study of Antigenicity ■ Pattern-Recognition Receptors Antigens S immunoglobulin receptor of B cells, or by the Tcell 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 molecular features of antigens recognized by B or T cells. The chapter 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 immune 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. Immunogenicity is the ability to induce a humoral and/or cellmediated immune response: B cells antigen n effector B cells + memory B cells g (plasma cells) T cells antigen n effector T cells + memory T cells g (e.g., CTLs, THs) Although a substance that induces a specific immune response 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:
8536d_ch03_057-075 8/6/02 10:28 AM Page 58 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e 58 PART II Generation of B-Cell and T-Cell Respons TABLE 3-1 experimental antigens used in on Molecular weight of some comm to a cow but is strongly immunogenic when injected into a abbit. Moreover, BSA would be expected to exhibit greater immunology immunogenicity in a chicken than in a goat, which is more closely related to bovines. There are some exceptions to Antigen Approximate molecular mass(Da) rule. Some macromolecules (e.g, collagen and cytochrome c) have been highly conserved throughout evolution and Bovine gamma globul 150,000 therefore display very little immunogenicity across diverse specles nents(e. g Bovine serum albumin 69,000 corneal tissue and sperm) are effectively sequestered from the immune system, so that if these tissues are injected even 40,000 into the animal from which they originated, they will func- 15,000 tion as immunogens. MOLECULAR SIZE Keyhole limpet hemocyanin >2,000,000 (KLH) There is a correlation between the size of a macromolecule Ovalbumin(OVA) 44.000 and its immunogenicity. The most active immunogens tend to have a molecular mass of 100,000 daltons( Da). Generally, 17,000 substances with a molecular mass less than 5000-10000 Da are poor immunogens, although a few substances with a Tetanus toxoid () 150,000 molecular mass less than 1000 Da have proven to be im- CHEMICAL COMPOSITION AND HETEROGENEITY Immunogenicity is not an intrinsic property of an antige Size and foreignness are not, by themselves, sufficient to out rather depends on a number of properties of the partic lar biological system that the antigen encounters. The next make a molecule immunogenic; other properties are needed wo sections describe the properties that most immunogens as well. For example, synthetic homopolymers(polymers are and the contribution that the biological system makes composed of a single amino acid or sugar)tend to lack im- to the expression of immunogenicity. munogenicity regardless of their size. Studies have shown are usually more immunogenic than homopolymers f i hat copolymers composed of different amino acids or The Nature of the Immunogen Contributes to Immunogenicity onstituents. These studies show that chemical complexity contributes to immunogenicity. In this regard it is notable mmunogenicity is determined, in part, by four properties of that all four levels of protein organization--primary, sec the immunogen: its foreignness, molecular size, chemical ondary, tertiary, and quaternary-contribute to the struc- composition and complexity, and ability to be processed and tural complexity of a protein and hence affect its immun presented with an MHC molecule on the surface of an anti- genicity(Figure 3-1). gen-presenting cell or altered self-cell. LIPIDS AS ANTIGENS FOREIGNNESS Appropriately presented lipoidal antigens can induce B-and In order to elicit an immune response, a molecule must be T-cell responses. For the stimulation of B-cell responses, recognized as nonself by the biological system. The capacity lipids are used as haptens and attached to suitable carrier to recognize nonself is accompanied by tolerance of self, a molecules such as the proteins keyhole limpet hemocyanin pecific unresponsiveness to self antigens. Much of the ability (KLH)or bovine serum albumin(BSA). By immunizing with to tolerate self antigens arises during lymphocyte develop- these lipid-protein conjugates it is possible to obtain anti ment,during which immature lymphocytes are exposed to bodies that are highly specific for the target lipids. Using this self-components Antigens that have not been exposed to im- approach, antibodies have been raised against a wide variety mature lymphocytes during this critical period may be later of lipid molecules including steroids, complex fatty-acid de- recognized as nonself, or foreign, by the immune system. rivatives, and fat-soluble vitamins such as vitamin E Such When an antigen is introduced into an organism, the degree antibodies are of considerable practical importance since of its immunogenicity depends on the degree of its foreign- many clinical assays for the presence and amounts of med ess. Generally, the greater the phylogenetic distance be- ically important lipids are antibody-based. For example,a tween two species, the greater the structural (and therefore determination of the levels of a complex group of lipids For example, the common experimental antigen bovine tients. Prednisone, an immunosuppressive Stcr,0"? the antigenic)disparity between them. known as leukotrienes can be useful in evaluating asthma serum albumin(BSA) is not immunogenic when injected given as part of the effort to prevent the rejection of a trans
Immunogenicity is not an intrinsic property of an antigen but rather depends on a number of properties of the particular 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 antigen-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 development, during which immature lymphocytes are exposed to self-components. Antigens that have not been exposed to immature 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 foreignness. Generally, the greater the phylogenetic distance between 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 function 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 immunogenic. 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 immunogenicity 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, secondary, tertiary, and quaternary—contribute to the structural complexity of a protein and hence affect its immunogenicity (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 antibodies 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 derivatives, 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 medically 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 patients. 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 2,000,000 (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:
8536d_ch03_057-075 8/7/02 9: 18 AM Page 59 mac79 Mac 79: 45_BW: Goasby et al./ Immunology 5e Antigens CHAPTER 3 -Lys-Ala-His-Gly-Lys-Lys-Va Amino acid sequence a helix PRIMARY STRUCTURE SECONDARY STRUCTURE Monomeric polypeptide molecule meric protein molecule TERTIARY STRUCTURE QUATERNARY STRUCTURE FIGURE The four levels of protein organizational structure. ondary features to give the overall shape of the molecule or parts of The linear arrangement of amino acids constitutes the primary struc- it(domains) with specific functional properties. Quaternary struc- ture. Folding of parts of a polypeptide chain into regular structures ture results from the association of two or more polypeptide chains (e.g, a helices and B pleated sheets) generates the secondary struc- into a single polymeric protein molecule ture. Tertiary structure refers to the folding of regions between sec planted organ. The achievement and maintenance of ade- compounds such as glycolipids and some phospholipids can quate blood levels of this and other immunosuppressive be recognized by T-cell receptors when presented as com drugs is important to a successful outcome of transplanta- plexes with molecules that are very much like MHC mole- tion, and antibody-based immunoassays are routinely used cules. These lipid-presenting molecules are members of the to make these evaluations. The extraordinary sensitivity and CDI family(see Chapter 8)and are close structural relatives specificity of assays based on the use of anti-lipid antibodies of class I MHC molecules. The lipid molecules recognized by is illustrated by Table 3-2, which shows the specificity of an the CDl-T-cell receptor system all appear to share the com antibody raised against leukotriene CA. This antibody allows mon feature of a hydrophobic portion and a hydrophilic head the detection of as little as 16-32 picograms per ml of group. The hydrophobic portion is a long-chain fatty acid leukotriene CA. Because it has little or no reactivity with sim- alcohol and the hydrophilic head group is composed of highly ilar compounds, such as leukotriene Da or leukotriene E4,it olar groups that often contain carbohydrates. Recognition of can be used to assay leukotriene CA in samples that contain lipids is a part of the immune response to some pathogens, his compound and a variety of other structurally related and T cells that recognize lipids arising from Mycobacterium tuberculosis and Mycobacterium leprae, which respectively T cells recognize peptides derived from protein antigens cause tuberculosis and leprosy, have been isolated from hu when they are presented as peptide-MHC complexes. How- mans infected by these mycobacteria. More about the presen- ever,some lipids can also be recognized by T cells. Lipoidal tation of lipoidal antigens can be found in Chapter 8
planted organ. The achievement and maintenance of adequate blood levels of this and other immunosuppressive drugs is important to a successful outcome of transplantation, 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 C4. This antibody allows the detection of as little as 16–32 picograms per ml of leukotriene C4. Because it has little or no reactivity with similar compounds, such as leukotriene D4 or leukotriene E4, it can be used to assay leukotriene C4 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. However, 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 complexes with molecules that are very much like MHC molecules. 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 common 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 humans infected by these mycobacteria. More about the presentation 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 structure. Folding of parts of a polypeptide chain into regular structures (e.g., helices and pleated sheets) generates the secondary structure. Tertiary structure refers to the folding of regions between secondary features to give the overall shape of the molecule or parts of it (domains) with specific functional properties. Quaternary structure 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:
8536d_ch03_057-075 8/6/02 10:28 AM Page 60 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e 60 PART I Generation of B-Cell and T-Cell Response BlE 3-2 Specificity of an antibody against a complex lipid Structure CHa NH 00.0 HO Leukotriene D4 人 OH The reactivity of the antibody with the immunizing antigen leukotriene C4 is assigned a value of 100 in arbitrary units. SUSCEPTIBILITY TO ANTIGEN PROCESSING The Biological System Contributes AND PRESENTATION to Immunogenic The development of both humoral and cell-mediated im mune responses requires interaction of T cells with antigen Even if a macromolecule has the properties that contribute to that has been processed and presented together with MHC immunogenicity, its ability to induce an immune response molecules. Large, insoluble macromolecules generally are vill depend on certain properties of the biological system more immunogenic than small, soluble ones because the that the antigen encounters. These properties include the larger molecules are more readily phagocytosed and genotype of the recipient, the dose and route of antigen ad- presented with MHC molecules are poor immunogens. This adjuvants, that increase immune response, bstances,called processed Macromolecules that cannot be degraded and an be illustrated with polymers of D-amino acids, which are stereoisomers of the naturally occurring L-amino acids. Be GENOTYPE OF THE RECIPIENT ANIMAL cause the degradative enzymes within antigen-presenting The genetic constitution(genotype)of an immunized an cells can degrade only proteins containing L-amino is, mal influences the type of immune response the animal polymers of D-amino acids cannot be processed and thus are manifests, as well as the degree of the response. For example, Poor Immunogens Hugh McDevitt showed that two different inbred strains of
SUSCEPTIBILITY TO ANTIGEN PROCESSING AND PRESENTATION The development of both humoral and cell-mediated immune 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. Because 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 administration, and the administration of substances, called adjuvants, that increase immune responses. GENOTYPE OF THE RECIPIENT ANIMAL The genetic constitution (genotype) of an immunized animal 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 C4 100.0 Leukotriene D4 5.0 Leukotriene E4 0.5 Prostaglandin D2 0.001 * The reactivity of the antibody with the immunizing antigen leukotriene C4 is assigned a value of 100 in arbitrary units. OH OH CH3 O S O NH2 OH OH OH O CH3 HO O † OH OH CH3 NH2 O S O HO O O O N H OH NH † OH OH CH3 O S O O N H OH H2N 8536d_ch03_057-075 8/6/02 10:28 AM Page 60 mac79 Mac 79:45_BW:Goldsby et al. / Immunology 5e:
8536d_ch03_057-075 8/6/02 10:28 AM Page 61 mac79 Mac 79: 45_Bw Glasby et al. Immunology 5e Antigens CHAPTER 3 mice responded very differently to a synthetic polypeptide Intravenous(iv ): into a vein immunogen. After exposure to the im produced high levels of serum antibody, whereas the other Intradermal(id): into the skin strain produced low levels. When the two strains were Subcutaneous(sc): beneath the skin crossed, the Fi generation showed an intermediate response to the immunogen By backcross analysis, the gene control- a Intramuscular(im): into a muscle ling immune responsiveness was mapped to a subregion of Intraperitoneal (ip): into the peritoneal cavity the major histocompatibility complex(MHC). Numerous The administration route strongly influences which immune xperiments with simple defined immunogens have demon strated genetic control of immune responsiveness, largely organs and cell populations will be involved in the response confined to genes within the MHC. These data indicate that Antigen administered intravenously is carried first to the MHC gene products, which function to present processed spleen, whereas antigen administered subcutaneously moves antigen to T cells, play a central role in determining the de- first to local lymph nodes. Differences in the lymphoid cells gree to which an animal responds to an immunogen. that populate these organs may be reflected in the subsequent Immune respons 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- ADJUVANTS ulatory mechanisms. Genetic variability in all of these genes Adjuvants(from Latin adjuvar, to help)are substances that, ffects the immunogenicity of a given macromolecule in dif- ferent animals. These genetic contributions to immuno when mixed with an antigen and injected with it, enhance the genicity will be described more fully in later chapter 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 MMUNOGEN DOSAGE AND ROUTE OF ADMINISTRATION available. For example, the antibody response of mice to im- munization with bsa can be increased fivefold or more if the Each experimental immunogen exhibits a particular dose-re- bsa is administered with an adjuvant. Precisely how adju- sponse curve, which is determined by measuring the im mining the level of antibody present in the serum of immu-(Table 3 3) he immune response is not entirely known, mune response to different doses and different adminis- tration routes. An antibody response is measured by deter nized animals. Evaluating T-cell responses is less simple but Antigen persistence is prolonged may be determined by evaluating the increase in the number ofT cells bearing TCRs that recognize the immunogen. Some. Co-stimulatory signals are enhanced combination of optimal dosage and route of administration Local inflammation is increased. will induce a peak immune response in a given animal. An insufficient dose will not stimulate an immune re- he nonspecific proliferation of lymphocytes is sponse either because it fails to activate enough lymphocytes or because, in some cases, certain ranges of low doses can in- Aluminum potassium sulfate(alum) prolongs the persis duce a state of immunologic unresponsiveness, or tolerance. tence of antigen. When an antigen is mixed with alum, the The phenomenon of tolerance is discussed in chapters 10 salt precipitates the antigen Injection of this alum precipitate and 21. Conversely, an excessively high dose can also induce results in a slower release of antigen from the injection site, so tolerance. The immune response of mice to the purified that the effective time of exposure to the antigen increases pneumococcal capsular polysaccharide illustrates the impor- from a few days without adjuvant to several weeks with the tance of dose. A 0.5 mg dose of antigen fails to induce an im- adjuvant. The alum precipitate also increases the size of the mune response in mice, whereas a thousand-fold lower dose antigen, thus increasing the likelihood of phagocytosis of the same antigen(5 X 10 mg)induces a humoral anti Water-in-oil adjuvants also prolong the persistence of body response. A single dose of most experimental immuno- antigen. A preparation known as Freund s incomplete ad gens will not induce a strong response; rather, repeated juvant contains antigen in aqueous solution, mineral oil, administration over a period of weeks is usually required. and an emulsifying agent such as mannide monooleate, ch repeated administrations, or boosters, increase the which disperses the oil into small droplets surrounding the clonal proliferation of antigen-specific T cells or B cells and antigen; the antigen is then released very slowly from the thus increase the lymphocyte populations specific for the im- site of injection. This preparation is based on Freund's munogen. complete adjuvant, the first deliberately formulated Experimental immunogens are generally administered highly effective adjuvant, developed by Jules Freund many parenterally(para, around; enteric, gut)that is, by routes years ago and containing heat-killed Mycobacteria as an other than the digestive tract. The following administration additional ingredient Muramyl dipeptide, a component of routes are commor the mycobacterial cell wall, activates macrophages, making
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 F1 generation showed an intermediate response to the immunogen. By backcross analysis, the gene controlling immune responsiveness was mapped to a subregion of the major histocompatibility complex (MHC). Numerous experiments with simple defined immunogens have demonstrated 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 degree 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 regulatory mechanisms. Genetic variability in all of these genes affects the immunogenicity of a given macromolecule in different animals. These genetic contributions to immunogenicity will be described more fully in later chapters. IMMUNOGEN DOSAGE AND ROUTE OF ADMINISTRATION Each experimental immunogen exhibits a particular dose-response curve, which is determined by measuring the immune response to different doses and different administration routes. An antibody response is measured by determining the level of antibody present in the serum of immunized 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 response either because it fails to activate enough lymphocytes or because, in some cases, certain ranges of low doses can induce 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 importance of dose. A 0.5 mg dose of antigen fails to induce an immune response in mice, whereas a thousand-fold lower dose of the same antigen (5 104 mg) induces a humoral antibody response. A single dose of most experimental immunogens 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 immunogen. 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: ■ Intravenous (iv): into a vein ■ Intradermal (id): into the skin ■ Subcutaneous (sc): beneath the skin ■ Intramuscular (im): into a muscle ■ 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 immunogenicity or when only small amounts of an antigen are available. For example, the antibody response of mice to immunization with BSA can be increased fivefold or more if the BSA is administered with an adjuvant. Precisely how adjuvants augment the immune response is not entirely known, but they appear to exert one or more of the following effects (Table 3-3): ■ Antigen persistence is prolonged. ■ Co-stimulatory signals are enhanced. ■ Local inflammation is increased. ■ The nonspecific proliferation of lymphocytes is stimulated. Aluminum potassium sulfate (alum) prolongs the persistence 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 adjuvant 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: