If ordinary police are confronted with a dangerous situation that they are unable to resolve, they call in the SWAT (special weapons and tactics) team, specialists such as snipers and hostage negotiators, who are trained to target and deal with a specific crisis. When the innate immune system is faced with a prolonged bacterial attack that it cannot handle, the human body uses a similar strategy. It brings in the specialists: antibody-producing B cells and cytotoxic T cells, also called cytotoxic T lymphocytes (CTLs), that are designed to target and kill a specific invading microbe.

The Specialists: Adapting to a Particular Pathogen Challenge

The innate defenses of the body are normally an effective protective force that eliminates invading microbes. Unfortunately, some microbes have developed strategies for evading these innate defenses and surviving in the host. For example, some microbes are able to resist phagocytosis by neutrophils, while others are even able to survive and multiply within phagocytic cells once ingested. To cope with these microbes, the body has evolved a second defense system, the adaptive or acquired immune response, which enables the host to recognize specific pathogens and mount a targeted response against them. In this adaptive immune response, dendritic cells (DCs) internalize the foreign substances that are then processed into smaller pieces and presented on their cell surface to T helper (Th) cells. The Th cells in turn stimulate B cells to produce antibodies that specifically recognize the foreign substance, targeting it for opsonization by phagocytes. The Th cells also stimulate cytotoxic T lymphocytes (CTLs) that recognize and kill infected host cells.

Unlike the innate immune system, which mounts a generalized response against foreign invaders, the adaptive immune system involves specialized cells of the body that change or adapt to target individual invaders in a highly specific way. As such, the adaptive response takes time to adjust and fully develop its specific response, taking up to a week to appear on the scene in full force. However, upon subsequent encounters with the same invader, the specific defenses appear much more rapidly, needing only a day or two to respond. In terms of disease manifestation, this usually means that during the first encounter with a new pathogen the body may have to struggle to overcome the infection and may exhibit symptoms of disease. But, upon future exposure to the same pathogen, the body is prepared to rapidly eliminate the threat and can combat the infection much faster than it did the first time around. Vaccination, which will be covered in greater detail in chapter 17, is a strategy for priming the body with an adaptive immune response against a particular pathogen so as to launch this more rapid response without actually having to endure the first, more unpleasant, encounter.

Understanding how the components of the adaptive immune system are induced and how they protect the body from infection is important for understanding how vaccines work, how they are designed, and why different vaccines are administered in different ways. Although vaccines have been important in preventing disease, the yield of successful vaccines has been disappointingly small. This is due to the fact that scientists are only now beginning to understand certain important nuances of the adaptive immune response, such as autoimmunity, a condition in which the adaptive immune system begins producing an unwanted immune response directed against the host’s own body. In this way, just as the innate immune system has its dark side (septic shock) that we discussed in the previous chapter, the adaptive immune system also has its own dark side (autoimmune reactions). Obviously, it is important that vaccines do not evoke this dark side. New insights into how the adaptive immune response develops, together with new insights into how to better deliver vaccines, are beginning to break down some of the barriers that have prevented development of vaccines and other treatment strategies against diseases such as AIDS, gonorrhea, chlamydial disease, tuberculosis, and malaria, which are serious causes of morbidity and mortality throughout the world.

In this chapter, we start with a description of two of the main products of an adaptive immune response, antibodies and cytotoxic T cells. We will then delve into the complex series of steps that produce these defenses. Finally, we will discuss how the body is able to “remember” past encounters with particular pathogens so as to more rapidly mount an effective immune response upon subsequent attacks by those pathogens.

B Cells: Producers of Antibodies

The Humoral (Antibody) Immune Response

Antibodies are immunoglobulin (Ig) protein complexes produced by mature B lymphocytes (B cells). Humoral immunity refers to the production of antibodies by mature B cells, as well as complement, antimicrobial peptides, and other immune components found in body fluids (archaically called the “humors”), but here we will confine the term to mean primarily the antibody response. Humoral immunity involves activation of naïve B cells through one of two pathways. The first pathway is a T-cell-dependent process, which is followed by clonal expansion and proliferation of the activated mature B cells and their terminal differentiation into plasma cells (mature, nonproliferating B cells that act as professional antibody-producing factories). Alternatively, T-cell-independent activation of B cells can occur through direct binding of highly repetitive structures on their surfaces, which leads to proliferation, maturation, and production of antibodies. Antibodies generated through this T-cell-independent pathway tend to have lower affinity for their targets than those generated through the T-cell-dependent activation of B cells.

Antibodies are found in blood (serum) and tissue fluids, as well as many bodily secretions (e.g., saliva, mucosal fluids, vaginal fluids, and breast milk). Antibodies carry out a number of the adaptive immune system’s critical tasks, including: binding and neutralizing the activities of foreign substances, such as toxins, bacteria, and other pathogens; coating foreign substances for enhanced opsonization and clearance from the body; and targeting infected host cells for the killing action of cytotoxic T cells (CTLs) and natural killer (NK) cells. To understand how different antibodies perform such diverse tasks, it is first necessary to understand how each type of antibody is put together and how they work.

Characteristics of Antibodies and Their Diverse Roles in Preventing Infection

The basic structures of the five types of antibodies (IgG, IgM, IgD, IgE, and IgA/sIgA) are shown in Figure 4-1. The antibody monomer consists of a complex of two heavy chains (H-chains) and two light chains (L-chains). The words “heavy” and “light” refer to the size of the peptide chain, with the H-chain being the larger of the two. The H-chains and L-chains are held together by a combination of disulfide bonds and noncovalent interactions. The H-chains define the type of antibody, with the H-chains for IgE and IgM being longer than those for IgA, IgD, and IgG.

Figure 4-1. Structures of IgG, sIgA, IgM, IgD, and IgE antibodies. Each antibody monomer is a complex comprised of four peptide chains: two identical, larger heavy chains (H-chains) and two identical, smaller light chains (L -chains) that are covalently attached through disulfide bonds. The type of H-chain defines the antibody class, with the H-chains for IgE and IgM being longer than those of IgA, IgD, and IgG. IgG is the major class of circulating antibodies. Each monomer recognizes the target epitope via two antigen-binding sites that are located in the variable regions (Fv) of the Fab portion of the monomer. There are four IgG subtypes (IgG1, IgG2, IgG3, and IgG4). The Fc region of the molecule is responsible for complement activation through binding C1q and for enhanced opsonization through binding to phagocyte Fc receptors. IgA has two subtypes: IgA1, which is found mostly in serum, and IgA2, which forms a dimer of two IgA monomers linked via a polypeptide joining chain (J-chain) and is secreted at mucosal surfaces. The IgA dimer acquires a secretory piece during transport through mucosal epithelial cells and is released as secretory IgA (sIgA) into the lumen, where it binds to mucin. IgD is a monomer that is expressed on the surface of mature B cells or is secreted. IgM is a multimer (mostly pentamer) of IgM molecules linked via disulfide bonds and a J-chain. IgM is highly agglutinating (binds and clumps antigens) and strongly activates complement (thousandfold better than IgG). IgE binds to IgE receptors on mast cells and basophils that, upon binding of antigen, trigger release of histamine and inflammatory cytokines.

Antibodies have two important regions: an N-terminal antigen-binding region (Fab) that harbors the end of the antibody that binds to a substance considered foreign by the body and a C-terminal constant region (Fc) that confers host specificity and interacts with host cells. In the most common antibody, IgG, the Fc region interacts with complement component C1q via a glycosylated region or with phagocytic cells via an Fc receptor-binding region. The Fab region contains a constant region and a variable region (Fv) that binds to a specific antigen. An antigen is defined as any material the body recognizes as foreign (nonself) that binds to an antibody. An antigen that binds to an antibody molecule can be an infectious microbe or some protein, nucleic acid, or carbohydrate component of the microbe. Additional examples of types of antigens are proteins, macromolecules, or organs from noncompatible human donors or other animals, as well as molecules from pets, plants, or insects, including dander, pollens, and toxins.

The humoral adaptive immune system’s ability to recognize a wide range of antigens and subsequently adapt to new invading pathogens relies on the ability of the B cell population to produce a vast array of antibodies with enormous diversity. This vast diversity of antigen specificity is possible because the immunoglobulin (Ig) genes undergo many gene recombination, rearrangement, insertion, deletion, and splicing events from separate, different gene segments encoding different regions of the antibody molecule during B cell development. A detailed description of the mechanism that generates the large, highly diverse population of B cells, each producing a different specific antibody that can bind to the different kinds of antigens, is beyond the scope of this book. But briefly, an amazing DNA recombination process, called V(D)J recombination, randomly shuffles a wide repertoire of variable (V), diversity (D), and joining (J) gene segments that form the N-terminal Fab regions of H-chain proteins (or just V and J gene segments in the case of L-chain proteins) and fuses them to gene segments corresponding to the constant regions in the Ig genes during the development of B cells. The exact pattern of rearrangements occurs independently in each B cell. RNA transcripts of the resulting mosaic Ig genes are further processed to give expression of the specific antibody produced by each B cell in the population. Normally during this developmental process, any B cells that produce Ig molecules that recognize self-antigens are eliminated. All of the antibodies produced by any given B cell have identical antigen-binding sites. But, during clonal expansion, when an antigen and Th cells stimulate a particular subset of B cells, a fairly high rate of spontaneous mutations, termed somatic hypermutation, is introduced, which serves to increase the diversity of the antibody pool even more and leads to proliferation of B cells that produce antibodies with high affinity to their cognate antigens.

An antibody monomer has two antigen-binding sites, each of which recognizes and binds to the same specific segment of an antigen. The antigen-binding sites are grooves in the antibody Fv ends that only bind tightly to a molecule having one particular structure, called an epitope or antigenic determinant. An epitope on a protein antigen can vary in size from 4 to 16 amino acids, although most are 5 to 8 amino acids in length. Complex antigens such as microbes contain many possible epitopes, each binding to a different antibody. Epitopes can be continuous or discontinuous, based on whether the antibody recognizes the primary (linear peptide) or tertiary (topographical) structures of the protein, respectively (Figure 4-2).

Figure 4-2. Continuous versus discontinuous epitopes. Epitopes of protein antigens can be continuous based on the linear amino acid sequence (primary structure) of the protein or discontinuous based on the tertiary conformational structure of the protein.

An immunogen is an antigen that elicits an immune response, but it is important to note that not all antigens are immunogens. In practice, only a subset of the epitopes on an antigen dominates the specific response to that antigen. Why some epitopes are highly immunogenic (i.e., elicit a robust antibody or T cell response) while others are only weakly immunogenic is still not well understood. Immunogenicity is often based on the size and complexity of the antigen molecule and is reflected in the antigenicity of different types of macromolecules. In general, proteins are better immunogens than carbohydrates, which are in turn better immunogens than nucleic acids and lipids.

Serum Antibodies

IgG. IgG, produced as a monomer, is the most prevalent type of antibody in blood and extravascular fluid spaces (approximately 80% of circulating antibodies are IgG). IgG is the only antibody type that can cross the placenta (via transcytosis bound to the neonatal Fc receptor, FcRn) and is responsible for protecting an infant during the first six months of life until the infant’s adaptive defenses are developed.

There are four different subtypes of IgG antibodies in humans (Table 4-1), named in the order of their abundance in serum: IgG1 (66%), IgG2 (23%), IgG3 (7%), and IgG4 (4%). These subtypes differ not only in their function, but also in their amino acid sequences, glycosylation (posttranslational decoration of the IgG protein by sugars), length and flexibility of the hinge region, extent of disulfide bond cross-linking of their H-chains (primarily in the Fc portion), and the specificity of Fc receptor interactions. [Warning: The nomenclature used to describe human IgG is not the same as that used to describe murine IgG. So, IgG1 of mice does not necessarily have the same features as IgG1 of humans. We mention this issue because it explains why different papers on the development of the immune response seem to contradict each other, but actually do not. For a comparative summary of the different types of Ig molecules of humans and mice, see Box 4-1.]

Table 4-1. Protective roles of serum antibodies IgG and IgM

Box 4-1.

Are You a Human or a Mouse?

This archaic challenge, issued to someone showing classic symptoms of soft spine syndrome, in the hopes of getting that person to act decisively and courageously, actually applies to antibodies as well. Although humans and mice are much more closely related than many of us would like to admit, there are subtle but real differences between some antibody classes shared by humans and mice. Accordingly, there is also a somewhat different nomenclature used to designate these antibody classes. These different designations can be confusing, especially in the case of the IgG subtypes. A guide to the different antibody designations in humans and mice is provided below:

Why such differences occur between closely related species remains a fascinating but unanswered question.

IgG1 and IgG3, but not IgG2 and IgG4, strongly activate the classical pathway of the complement cascade. The six head domains of the C1q complex bind to the Fc region of IgG monomers bound to the surface of the antigen (see Figure 3-14). This cross-linking activates the C1 complex and initiates the complement cascade. IgG1 and IgG3 are also called opsonizing antibodies because these two subtypes are the most effective in opsonizing microbes due to the fact that they have the highest affinity to Fc receptors on phagocytes (see Figure 3-12). IgG2 and IgG4 opsonize poorly, if at all. IgG2 appears to be best at recognizing carbohydrate or polysaccharide-containing antigens. IgG4 appears to play a regulatory role by dampening Fc receptor-mediated inflammatory responses and is produced when persistent exposure to a particular antigen occurs.

Normally, antibody binding to an antigen on the surface of an extracellular pathogen facilitates ingestion of the opsonized antigen by phagocytic cells or triggers complement-dependent killing at the cell surface (see chapter 3). However, in the case of intracellular infections of tissues, IgG (mostly IgG1 and IgG3, but to some extent also IgG2, as well as IgA and IgE) can also mediate a different response, called antibody-dependent cell-mediated cytotoxicity (ADCC). In ADCC, antigens presented on the surfaces of infected cells bind to the Fab regions of specific IgG molecules. The exposed Fc portions of the IgG molecules are then free to bind to effector cells (PMNs, macrophages, or NK cells) that contain Fc receptors on their surfaces. In the case of NK cells, this linkage triggers a cytotoxic bombardment of the infected cell, as described in chapter 3. In the case of macrophages and PMNs a similar killing response can also be elicited, instead of phagocytosis. Thus, by linking together players of the innate (NK cells, PMNs, and macrophages) and adaptive (antibodies) immune responses, ADCC serves as an important defense against intracellular pathogens (more on this later).

IgM. IgM consists of a complex of five or more monomers that are connected via disulfide bonds to each other and to a peptide called the J-chain (Figure 4-1). IgM is found mostly in serum, where it accounts for 5 to 10% of the total serum Ig, but it is also secreted at mucosal surfaces and in breast milk. IgM is the most effective activator of complement. Because of its polymeric form, a single molecule of IgM has five Fc regions that can be complexed, which is sufficient to bind C1q and strongly activate complement via the classical pathway or to bind and cluster multiple Fc receptors and mediate enhanced opsonization.

IgM is the first antibody type expressed in the fetus (at 20 weeks’ gestation). During B cell differentiation, IgM is the only antibody expressed by naïve B cells. Once B cells mature and proliferate, they start to produce IgG (or IgA) and IgM antibody expression wanes. IgM predominates in the initial (primary) antibody response against a pathogenic microbe, whereas IgG (or IgA at mucosal sites) predominates in response to sustained or subsequent infections (secondary antibody response) by the same microbe (Figure 4-3). Because IgG can circulate in serum for long durations, detectable IgG levels may indicate the presence of a current infection that is well underway or may simply be the residue of a previous infection, whereas IgM levels are detectable during an initial infection but vanish once production of IgG antibodies begins. This aspect of the immune response is actually exploited by diagnostic labs to determine whether a patient is experiencing a first exposure (acute infection) to a particular microbe.

Figure 4-3. The time course of elicitation of antibodies upon initial exposure and subsequent exposure. After exposure to an antigen such as antigen A, IgM is usually the first antibody detected during an acute infection, but levels decrease again after about two weeks. It takes five to seven days for selection and proliferation of B cells producing IgG against antigen A to appear in the blood. After a period of time, the levels of IgG also decrease. Subsequent exposure to antigen A again results in production of IgM at about the same levels as after the first exposure, but the levels of antigen A-specific IgG are now much higher. In contrast, exposure to antigen B along with the second exposure to antigen A does not enhance the antigen B-specific IgG levels.

IgD. IgD monomer is initially coexpressed with IgM on the surface of mature B cells as they exit the bone marrow and migrate to peripheral lymphoid tissues. IgD appears to function primarily to signal B cell activation.

IgE. A serum antibody with a function different from that of IgG and IgM is IgE. IgE is a monomer that is normally found in extremely low concentrations in serum (only 0.05% of total Ig concentration), usually as an Fc receptor-bound complex on the surface of mast cells and basophils. If two IgE molecules bound to mast cells are complexed by a polyvalent antigen (i.e., an antigen with multiple antibody-binding sites), it causes clustering of the Fc receptors, which stimulates the mast cell to release granules containing potent proinflammatory chemokines, in particular the vasoactive amines histamine and serotonin, as well as leukotrienes and cytokines such as IL-4 and IL-13 (Figure 4-4). These IgE-induced responses also initiate the strong inflammatory reactions associated with allergies and asthma (Box 4-2).

Figure 4-4. Antibody-mediated mast cell degranulation. Antigen-mediated clustering of IgE bound to Fc receptors on mast cells triggers degranulation and release of allergic mediators, such as histamines, serotonin, vasoactive amines, and other inflammatory cytokines.

Box 4-2.

The Dark Side of Adaptive Immunity: Allergy and Autoimmunity

Hypersensitivity is a set of undesirable, uncomfortable, and sometimes fatal reactions resulting from an overstimulation of inflammatory responses that cause host damage. Allergens that cause hypersensitivity include plant pollens, fungal spores, insect venom, animal dander, house dust mites, some foods, and certain chemicals (e.g., poison ivy, latex, jewelry, detergents, cosmetics). There are five types of hypersensitivity with differing times of onset and immune mediators:

Type I hypersensitivity occurs within minutes and is mediated through antigen binding and clustering of IgE-Fc receptors on mast cells and basophils, which causes release of histamine and vasoactive cytokines (as in Figure 4-4).

Type II hypersensitivity occurs within hours and is mediated through IgG- or IgM-receptor clustering on a target (infected) host cell, which the immune system then perceives as nonself and targets for attack by MAC by complement and/or ADCC by NK cells or macrophages.

Type III hypersensitivity occurs within hours and is mediated by IgG antibody binding to soluble, circulating antigens and forming a large immune complex that deposits at certain sites, such as joints, and causes local inflammation at those sites.

Type IV hypersensitivity is a delayed response that takes one to two days to manifest and is caused by CD4+ Th1-memory-cell-mediated inflammatory responses to antigens or allergens.

Type V hypersensitivity (autoimmunity) is an IgG- or IgM-mediated immune response (similar to Type II) that is caused by antibodies binding to host cell antigens, which the immune system then perceives as nonself. This type of response sometimes occurs through molecular mimicry, in which a foreign antigen (such as a bacterium or bacterial product) shares structural features (epitopes) with certain host molecules, such that infection or exposure to the foreign antigen elicits antibodies against the common epitopes and thereby generates antibodies also against the host. Certain infections with superantigen-producing bacteria and viruses elicit strong nonspecific T cell or B cell activation and massive inflammatory responses that can also lead to autoimmunity.

Many of the symptoms of infections caused by metazoal parasites (such as helminths, also known as parasitic worms) are traceable to elevated levels of IgE during infection. The release of mast cell granules in the vicinity of the intestinal wall may provoke an allergic response in the host that leads to ejection of the metazoal parasites from the intestinal or pulmonary mucosal sites. An interesting fact to ponder is that the human body evolved over millions of years to respond to worm infestations, with IgE functioning as part of that response. However, in recent times, particularly in developed countries, worms have been almost entirely eliminated from the human intestinal landscape. As such, in these more developed areas where metazoal infections are relatively uncommon, IgE is actually most often associated with noninfectious diseases such as allergies or asthma.

The most serious complication of the massive release of mast cell granules is anaphylaxis, which can rapidly kill a person. Is the rise in allergies and asthma seen in developed countries due to an immunological imbalance caused by elimination of a former enemy, thereby leaving the worm-oriented part of the specific and nonspecific defenses with nothing to do except cause trouble? Rest assured that this is not the start of a “bring back the worms” initiative, but it is interesting to contemplate the potential negative consequences of an abrupt change (in evolutionary terms) in our exposure to invaders that have been with us since we first appeared as a species.

Secretory Antibodies: Antibodies That Protect Mucosal Surfaces

IgA. In its monomeric form, IgA represents about 10 to 15% of the total serum antibody content. The role of IgA in blood and tissue is to aid in the clearance of antigen-antibody immune complexes from the blood. Because IgA monomer is a poor opsonin and activator, IgA in blood binds to the Fc receptor on immune effector cells, stimulating inflammatory responses and causing ADCC. By far the most important form of IgA, however, is the dimeric secretory IgA (sIgA), the dominant antibody in mucosal secretions of the gastrointestinal, urogenital, and respiratory tracts, including tears, salivary glands, sweat, bile, colostrum, and milk.

Dimeric sIgA consists of two IgA antibody monomers joined end-to-end through disulfide bonds to a J-chain peptide and to another tightly bound peptide, called the secretory piece (Figure 4-1), acquired during transport out into the mucin layer (more on this later). The main role of sIgA is to attach to incoming microbes or toxic microbial components and trap them in the mucus layer, thus preventing them from reaching the epithelial surface. Because sIgA is also secreted into mother’s milk, sIgA, like IgG, serves as an important protection against infection for infants who have not yet developed their own set of immune responses.

IgA is heavily glycosylated in the hinge region, which protects it from proteolysis. The secretory piece also helps protect the protease-sensitive sites in the H-chain from cleavage by bacterial and host proteases. Humans have two subtypes of IgA, IgA1 and IgA2. An interesting evolutionary development is the production of an IgA1-specific protease by a number of pathogenic bacteria (e.g., Neisseria gonorrhoeae), which is thought to have provided a selective pressure that resulted in human development of another gene encoding IgA2 that lacks the sites recognized by the IgA1 protease.

Pathogen and Toxin Neutralization by Antibodies

Just as sIgA antibodies are able to bind to microbes and prevent mucosal binding and entry into the host epithelium, both IgG and IgM antibodies bind to the surfaces of invading bacteria and viruses and prevent them from attaching to and entering target host cells (neutralization of the pathogen) (see Table 4-1). Antibodies are able to act as neutralizing agents because they are bulky molecules that can block interaction between microbial surface proteins and the receptors they recognize on host cells. Antibodies can also neutralize toxic proteins produced and secreted by bacteria by binding to the toxins and preventing them from binding to host cell surface receptors, thereby blocking their toxic effects (toxin neutralization). In addition, antibodies can directly neutralize the catalytic activities of secreted bacterial enzymes such as proteases, nucleases, or glycosidases that normally work to degrade host extracellular molecules and allow the pathogen to disseminate throughout the body.

An example of protective antibody neutralization of a bacterial toxin is the antibody response to Corynebacterium diphtheriae, the cause of diphtheria, a serious toxin-mediated disease of children. Corynebacteria often live innocuously in the upper respiratory tract, but when they become infected with a corynebacteriophage encoding the diphtheria toxin gene, they are then able to produce and secrete the diphtheria toxin, which enters cells lining the respiratory tract and bloodstream. Diphtheria toxin is one of the most potent bacterial toxins known and can kill many types of human cells. The action of the toxin in the throat is evident from a grayish or whitish “pseudo-membrane” patch that consists of dead epithelial cells and mucus. In some cases, this membrane can grow to the point at which it causes asphyxiation. If the toxin makes it through the bloodstream to the heart, it can cause death due to heart failure. The most effective protective response to infection by toxin-producing C. diphtheriae, which is also the response elicited by the antidiphtheria vaccine, is the production of antibodies that bind to diphtheria toxin and prevent it from binding to and killing human cells (neutralization of the toxin).

Affinity and Avidity

A characteristic of antibody binding to antigens, which is of critical importance in assessing the effectiveness of the antibody, is the avidity of the antigen-binding site for the epitope it binds. Avidity is a combination of affinity (the strength of the binding interaction between an antigen-binding site and an epitope) and valence (the number of antigen-binding sites available for binding epitopes on an antigen). A single epitope can elicit a mixture of antibodies that vary considerably in affinity. This variation may arise because the body cannot know in advance what epitopes it will encounter, so it produces a variety of antibodies with differing antigen-binding sites, some of which will have a high affinity for a particular antigen. In fact, as the antibody response to an epitope develops, the B cells producing antibodies with the highest affinity will proliferate the most, and eventually those high-affinity antibodies will predominate.

High affinity is important, but it is not sufficient to ensure that an antibody bound to an epitope will retain its hold on the epitope. Because the binding between antigen-binding sites and epitopes is noncovalent, the interaction is reversible. Thus, there is an off-rate, as well as an on-rate, associated with antibody binding to an epitope. The importance of valence is that an antibody with a higher valence will be significantly less likely to detach from the antigen to which it is bound. If two antigen-binding sites of an antibody monomer bind to two adjacent epitopes on an antigen, the probability that both of them will detach at the same time is much lower than the probability that a single antigen-binding site will detach from its epitope. Thus, higher valence can improve the apparent strength of binding of an antibody to an epitope by orders of magnitude.

The differences in affinity and avidity between the first responders IgM/IgA and the adaptive IgG antibodies become important when one considers the roles that these different antibodies play in the immune response. Antibodies, such as IgM and sIgA, that appear early and have lower affinity and less specificity for the invading microbe need to have a higher valence number so that they can increase the avidity for the foreign antigen by binding more epitopes, up to 4 for sIgA or 10 for IgM. High avidity for an antigen is more effective at neutralizing microbes and toxins, blocking their binding and entry into cells. For IgM, high avidity significantly enhances the activation of complement and clustering of receptors to stimulate immune cells. Moreover, high avidity leads to agglutination that enhances opsonization of the microbes by phagocytic cells and clearance of the microbe from the circulation. Rapid clearance is a desirable feature because the longer antibody-antigen complexes remain in circulation, the more likely they are to deposit in the kidneys or other blood-filtering organs, where the complexes can activate complement and cause an inappropriate inflammatory response that damages the organ.

In contrast, IgG is a monomer and can bind only two epitopes, but because it is the product of an adaptive immune response, its low valency and hence low avidity is countered by its much higher affinity and specificity for the foreign antigen. Circulating IgG antibodies can not only bind very tightly to neutralize a specific microbe or toxin, but also mediate opsonization of the specific microbe through binding to Fc receptors on phagocytes. Antigen-bound IgG can also mediate complement activation through binding to a C1 complex.

Cytotoxic T Cells, Also Known as Cytotoxic T Lymphocytes (CTLs)

Cytotoxic T Lymphocytes: Critical Defense against Intracellular Pathogens

Like PMNs and NK cells (in ADCC), cytotoxic T cells, also called CD8+ T cells or cytotoxic T lymphocytes (CTLs), kill infected host cells, but through a different mechanism. By entering cells, intracellular pathogens are protected from antibodies and complement. As such, killing infected host cells is often the only way to attack these invaders. CTLs and NK cells (in ADCC) are important parts of this defense response. The difference between CTLs and NK cells is that CTLs have T cell receptors (TCRs) that are specific to a particular epitope from a microbial antigen. Thus, while NK cells kill host cells infected with a variety of intracellular pathogens (see previous sections and chapter 3), CTLs kill only cells infected with a specific intracellular pathogen (see Figure 3-3).

CTLs have two mechanisms for killing infected cells. In the first, the CTL binds to a microbial antigen displayed on the surface of an infected cell using a TCR on its surface. (How the microbial antigen comes to be displayed on the cell surface will be described later.) This interaction then triggers the CTL to release granules containing two proteins, perforin (a pore-forming cytolysin that pokes holes in the host cell membrane) and proteolytic enzymes (granzymes) that enter the cell through the pore and trigger programmed cell death (apoptosis) in the infected cell (Figure 4-5). This type of attack kills the infected host cell but not the microbes. Instead, the released microbes are taken up by nearby activated macrophages that are better able to kill them. In the second mechanism, the CTL also releases a second pore-forming cytolysin from the granules, called granulysin. Granulysin is somewhat ineffective in lysing host cells, but it is very effective at killing bacteria. Presumably, granulysin kills bacteria the way perforin kills eukaryotic cells: by creating holes in the bacterial membranes and collapsing the proton motive force that bacteria use to gain energy. Additionally, perforin may also help deliver other yet-to-be-discovered antibacterial lysins or toxins into intracellular compartments of host cells.

Figure 4-5. Cytotoxic T cell (CTL) action on infected host cells. Cytotoxic T cells (CTLs) mediate their action on infected cells through the release of perforin, granzyme, and granulysin from granules. (A) The cytotoxic T cell recognizes an infected target cell (e.g., macrophage) containing bacteria through T-cell receptor binding to a bacterial antigen displayed via MHC I. (B) The cytotoxic T cell releases granules containing perforin, granzymes, and granulysin. Perforin creates pores in the infected target cell membrane. Granzyme and granulysin enter the target cell through the pores created by perforin. (C) Granzyme triggers signaling pathways that lead to apoptosis of the infected cell. (D) Granulysin binds to and kills intracellular bacteria by creating pores in the bacterial membranes. The infected host cell then processes the degraded bacteria into antigens.

Antigen Presentation to the Immune System

Processing of Protein Antigens by Dendritic Cells

When microbes or their products first enter the body, professional phagocytes called antigen-presenting cells (APCs) engulf, process, and present antigens on their surfaces, which then direct other cells in the adaptive immune system to develop into cells with specific antibacterial activity. There are three types of APCs: dendritic cells (DCs), macrophages, and B cells. As described in chapter 3, macrophages and DCs are important players in the innate immune system, but they also serve as links to the adaptive immune system. Macrophages, as part of the innate immune system, are produced in an immature form (monocytes) and migrate through the bloodstream before moving into tissue where an infection is taking place. They help clear debris from dead human cells that may be circulating in blood or deposited in tissue. Macrophages also play a critical role in initiating and organizing the adaptive immune response.

DCs, like macrophages, initiate and organize the adaptive immune response, but because they are found mainly in localized areas of the dermis, the mucosal lining of the intestinal tract, and lymphoid tissue, they are the first APCs on the scene to process microbial antigens and stimulate the adaptive immune response. B cells can also function as APCs, albeit not as efficiently as macrophages or DCs. In addition to producing and secreting soluble antibodies, B cells also produce membrane immunoglobulins (mIg) that they display on their surfaces. When the mIg captures an antigen, the mIg-antigen complex is internalized and the antigen is processed and presented to the T helper cells.

Peptide Antigens. Figure 4-6 shows an overview of how APCs process protein antigens and display the resulting peptide epitopes on their surfaces bound to a protein complex called the major histocompatibility complex (MHC). Two main classes of MHC molecules, MHC I and MHC II, form complexes with peptide epitopes (Figure 4-7). MHC I molecules are produced by all nucleated cells in the body, while only professional phagocytic APCs, such as DCs, macrophages, B cells, and certain activated epithelial cells, produce MHC II molecules in addition to MHC I molecules. In the case of peptide antigens, the type of MHC used to display the epitope determines the type of immune response that will be elicited (more on this later).

Figure 4-6. MHC I, MHC II, and CD1 pathways of antigen processing by APCs and presentation to T cells leads to activation and increased proliferation of T cells. (A) In the MHC I pathway, protein antigens present in the cytosol are processed by the proteasome, and the resulting peptides are transported to the endoplasmic reticulum (ER) via the transporter associated with antigen processing (TAP). In the endosome, the peptide antigens are further processed by an endosomal protease into smaller peptide epitopes, which then bind to MHC I, and the complex traffics to the cell surface, where it binds to the T cell receptor (TCR) and CD8 on the surface of CD8+ T cells. (B) In the class II MHC pathway, extracellular protein antigens are endocytosed into late endosomal and lysosomal vesicles, where they are processed into peptides that displace the invariant chain of the MHC II molecule. An accessory protein, called H-2M in mice (HLA-DM in humans), facilitates the displacement of the invariant chain with the peptide epitope. The peptide epitope-MHC II complex is then transported to the cell surface, where the complex binds to the T cell receptor (TCR) and CD4 on the surface of CD4+ T cells. (C) The CD1 antigen presentation pathway. Uptake of foreign glycolipid or lipid antigens occurs through multiple pathways. Glycolipid antigens bind to APCs via pattern recognition molecules such as CD14 and the mannose receptor. The low-density lipoprotein receptor (LDLR) can bind to the lipoprotein transporter ApoE. The mannose receptor can also mediate the uptake and trafficking of such lipid or glycolipid antigens through the endosomal pathway where, at acidic pH, the lipid portions of the glycolipid antigens are released and displace the self-lipids at the binding cleft of the CD1 molecule. Accessory proteins, called saposins, and possibly CD1e help process the lipid antigen and displace the self-lipid on the CD1 with the lipid antigen. An ER protein, called microsomal triglyceride transfer protein (MTP), facilitates the loading of the self-lipids onto CD1. The antigen-CD1 complex then traffics to the cell surface, where it is recognized by the CD1-specific T cell receptor. CD1-mediated antigen presentation occurs through TCRs in both αβ T cells and γδ T cells.

Figure 4-7. Major histocompatibility complexes I and II. Shown are the structures of major histocompatibility complex I (MHC I) and II (MHC II) proteins, which bind to peptide antigens and present them to T cell receptors (TCRs) on the surface of Th1 and Th2 cells, respectively. NOTE: In humans, the corresponding MHC molecules are called human leukocyte antigens (HLA).

How does an APC decide whether to display an epitope on MHC I or MHC II? This question has received a great deal of attention because understanding what leads to each type of presentation is critical for developing effective vaccines. From cumulative data so far, some basic rules have emerged. Intracellular pathogens such as viruses and some bacteria, particularly those that can enter the cytoplasm or nucleus of an APC, are most likely to elicit an MHC I display of the epitopes (Figure 4-6A). Additionally, large particulate or aggregated antigens that do not escape the phagocytic or pinocytic vesicle seem to elicit primarily an MHC I-linked display. By contrast, epitopes processed from soluble antigens, such as peptides or proteins secreted from bacteria or those exposed on the surface of extracellular pathogens, are displayed almost exclusively by MHC II (Figure 4-6B). Thus, if one wishes to use peptides to elicit a CTL response, it is necessary to present these peptides in particulate form (e.g., bound up in a large immune complex, or to deliver them directly into the cytosol to ensure their processing will occur via the MHC I pathway). Alternatively, these peptides could be expressed intracellularly using mammalian expression vectors (more on this in chapter 17).

Differences in immunogenicity have important practical consequences. For example, it is now possible to produce epitope-sized peptides synthetically. Peptides are not only much cheaper to produce than full-length proteins, which must be purified through time-consuming biochemical procedures, but they also make it possible to target a specific antibody or CTL response toward one or more defined regions of an antigen. Epitopes differ in the way they are recognized by the immune system, and only a small subset of the potential epitopes present in any given antigen will elicit an immune response. Epitopes that are processed and presented on the surface of APCs bound as MHC complexes, which are then recognized by T cell receptors on Th cells, are referred to as T cell epitopes. T cell epitopes that bind to MHC I complexes are typically 8 to 11 amino acid residues in length, whereas T cell epitopes that bind to MHC II complexes are 13 to 17 amino acid residues in length. Epitopes that are recognized by antibodies expressed on the surface of the B cells are called B cell epitopes. B cell epitopes vary from 5 to 10 amino acids in length and are found on the exposed surface of the native conformation of the antigen (i.e., they are not processed by APCs). B cell epitopes can elicit strong antibody responses, while T cell epitopes can also elicit strong cell-mediated CTL responses.

Directing the adaptive immune response toward particular epitopes is important because not all immunogenic epitopes elicit protective responses. Some immunogenic epitopes, for example, are buried within a folded protein and are therefore inaccessible to the antibody or are expressed inside a microbe and are thus not exposed to circulating antibodies. Eliciting an antibody response to such an epitope is useless because the antibody will not be able to bind and neutralize the antigen or enhance its opsonization. Furthermore, some microbial proteins have regions that vary considerably from one strain of microbe to another. Antibodies or cytotoxic T cells that recognize highly variable regions of microbial proteins will only be useful against a limited number of strains. A better strategy is to target regions of microbial proteins that are exposed and highly conserved (i.e., found in all strains of the microbe). Using peptide epitopes as vaccines makes it possible to program a specific immune response directed toward conserved epitopes that are exposed on the surface of the antigen. We will return to this concept in chapter 17.

Other Kinds of Macromolecular Antigens. Unfortunately, many of the bacterial surface antigens recognized by the immune system are lipid, carbohydrate, or lipid-carbohydrate combinations. Gram-negative LPS and Gram-positive LTA and PG are excellent examples. In the past, immunologists and vaccinologists have focused almost exclusively on peptide antigens because they are easier to characterize than carbohydrate or lipid-containing molecules and because peptides elicit a strong immune response. Peptide antigens consist of different amino acids linked by a single type of bond, the peptide bond, which can be readily processed into defined epitopes. Carbohydrate oligomers, by contrast, can be linked by any of 12 types of glycosidic linkages. Lipids also contain more than a single type of linkage. Because of the great diversity of carbohydrate, lipid, and lipid-carbohydrate molecules, it is not surprising that the mechanism for how these carbohydrate and lipid antigens are processed has been largely neglected until recently.

A major breakthrough in this area came with the discovery of CD1 molecules. Mycobacterium tuberculosis, the cause of tuberculosis, provides a cor nucopia of lipid-saccharide and lipid-peptide antigens not found in most other bacteria, which enabled researchers to identify the first CD1 complexes that were bound to lipid or glycolipid antigens. Since then, five forms of human CD1 have been found: CD1a through CD1e. The steps in processing and displaying lipid antigens appears to be similar to those for peptide antigens (Figure 4-6C), with CD1 taking the place of the MHC I molecule for presentation of antigens to CD1-specific CTLs (for CD1a, CD1b, and CD1c) or NK cells (for CD1d). CD1e is the only isoform that does not appear to be expressed on the surface of DCs, and it is suspected that it might play a role in antigen processing, rather than in display of the antigen. CD1 is related, at the amino acid sequence and overall domain structure level, to MHC I, but has obviously diverged during evolution. CD1 proteins have deeper and larger binding cavities to accommodate the hydrophobic alkyl chains of lipid antigens. The more hydrophilic head group of the lipid antigen faces outward and interacts with the TCR on the CTLs and NK cells.

Interaction between APCs and T Cells: The T-Cell-Dependent Response

When an APC displays a particular MHC I-epitope or MHC II-epitope complex on its surface, only a small number of the vast pool of available immature precursor T cells will have a TCR capable of recognizing that particular MHC-epitope combination and stimulating the signals needed to induce proliferation and production of cytokines (Figure 4-8). All T cells express CD3 complexes (comprised of γ, δ, and ε chains) and TCR complexes (comprised of α, β, and two ζ chains). The CD3 and ζ chains are invariant, but the large repertoire of TCRs with surface receptors that recognize a wide variety of different MHC-bound epitopes is generated through variation of the αβ chains, which undergo V(D)J recombination during T cell development in the thymus (analogous to the process mentioned earlier that leads to antibody diversity).

Figure 4-8. The T cell receptor (TCR) complex. Shown is a schematic illustration of the complex formed between the epitope-MHC II complex on the APC and the CD3-TCR complex on the CD4+ Th cell. Note: Similar complex formation occurs for MHC I recognition by CD3-CD8+ T cells. CD4 and CD8 are expressed on two different types of αβ T cells, where CD4 recognizes MHC II complexes and CD8 recognizes MHC I complexes on APCs. Activation of the T cells requires stimulation by two signals: signal 1 occurs through specific interaction of the epitope-MHC II with the CD4-TCR and CD3 complexes and signal 2 occurs upon costimulation through the CD28 binding to CD80 or CD86 on the APC.

Antigen presentation and binding to the TCR stimulates the T cell to become either a CTL or a T helper (Th) cell (Figure 4-9). Display of intracellularly derived peptide epitopes on MHC I allows the APC to activate and stimulate the proliferation of CTLs. CTLs are distinguished by the presence of CD8 molecules on their surfaces (hence the name CD8+ cytotoxic T cells). The CD8 molecules, along with the T cell receptor (TCR), are responsible for recognizing only antigen-bound MHC I complexes on the APC. CD8 binds to MHC I and stabilizes the interaction between epitope-bound MHC I and the TCR. CTLs are particularly well equipped to recognize infected cells because virtually all cells of the body produce MHC I. If a cell is infected, it displays epitopes from the invading microbe on its surface. Binding of a CTL to the surface of an infected cell causes the CTL to release cytolytic or apoptotic proteins that can kill the infected cell, as described earlier (see Figure 4-5). In addition, as described in chapter 3, NK cells also use the amount of MHC I on cell surfaces as an indicator of cell health, because infected cells generally express less MHC I than normal cells.

Figure 4-9. T-cell-mediated immunity and memory. Characteristics of the antigen (Ag) determine whether the antigen is presented via APCs through MHC I complexes or through MHC II complexes. Ag-MHCI complexes bind to TCRs on CD8+ T cells that trigger the CTL response. Ag-MHC II complexes that bind to TCRs on CD4+ Th0 cells trigger IL-2 cytokine production and maturation into CD4+ Th1 cells, leading to cellular immunity or, in the subsequent presence of IL-4 into CD4+ Th2 cells, leading to stimulation of the antibody-producing B cell response. Cytokines and the presence of Th17 cells help direct the type of cell-mediated immunity and memory that occurs. Treg cells help control the immune response, especially once the pathogen has been cleared from the system.

In contrast, if an epitope is presented by the APC in complex with MHC II rather than MHC I, a different class of T cells, T helper (Th) cells, is stimulated. Complex formation between TCRs and coreceptor CD4 molecules on the surface of precursor Th cells (called CD4+ Th0 cells) with epitope-bound MHC II molecules on the surface of APCs leads to production of IL-2 and then IL-4 by the Th0 cell and proliferation and maturation. Proliferating Th cells come in two types: Th1 and Th2 cells. These Th cells influence a variety of immune responses through release of cytokines, and how they do this is a very active area of research in the field of immunology.

Other proteins on the surface of the APC and the Th cell, called costimulatory molecules (e.g., CD54, CD11a/CD18, CD58, CD2), must also interact to make the binding between the APC and the Th cell tight enough to stimulate the APC to release cytokines that stimulate the Th cell to proliferate and become activated. Th1 cells are stimulated to proliferate by IL-12 and IL-2 released by APCs, which causes the Th1 cells to release more IL-2 (positive feedback), IFN-γ, and TGF-β. Recognition of MHC II-epitope by CD4+ Th1 cells also stimulates the production and release of IL-2 and IFN-γ, which in turn activate macrophages. Th1-cell-mediated responses lead to cellular immunity, which is most effective against intracellular pathogens. The necessity for contacts between different surface proteins of the APC and the Th cell helps ensure that only specific binding of an MHC-epitope complex to its cognate TCR will result in Th cell activation.

Th2 cells are stimulated to proliferate by IL-4, which causes the cells to release more IL-4 (positive feedback), as well as other cytokines (IL-5, IL-9, IL-10, and IL-13), which modulate other immune cells. MHC II-epitope stimulation of CD4+ Th2 cells stimulates naïve B cells to proliferate into B cells and mature into antibody-producing plasma cells (i.e., humoral immunity) and memory B cells. IL-4 also stimulates B cells to produce IgE antibodies, which in turn stimulate mast cells to release their cytokines (histamine, serotonin, and leukotrienes). The Th2 response is most effective against extracellular pathogens.

Once activated, T cells begin to proliferate, with most of the resulting effector Th cells becoming involved in combating the invading microbes. A few of the T cells, however, become quiescent memory T cells. Memory T cells persist for long periods in the body. They are generally present in higher numbers than naïve T cells with the same TCR, and they are more easily stimulated to proliferate and produce stronger cytokine responses when they encounter their specific epitopes on APCs during subsequent infections. Memory T cells thus allow the body to respond to a second encounter with a particular microbial invader much faster and more strongly than it did after the initial encounter.

Some bacteria and viruses produce proteins called superantigens that interfere with this highly specific progression of events in an interesting way that will be described in greater detail in chapter 12. Superantigens force a close association between APCs and T cells through abnormal association of MHC and the TCRs without a matched antigen. Normally, only a small fraction of T cells will interact with APCs presenting antigens on their surface. Superantigens, on the other hand, can cause up to 20% of T cells to participate in such interactions. As the cytokine signaling begins, cytokines are produced at higher levels than normal and this overreaction can trigger shock. The term “septic shock” is sometimes used to describe this phenomenon; however, as described in chapter 3, LPS or other bacterial surface components usually initiate what is considered septic shock. The term “T-cell-mediated shock” or “toxic shock” might be more appropriate to describe the shock initiated by superantigens, to distinguish it from septic shock.

Th-(Th1/Th2/Th17)-Cell-Mediated Immunity

A paradigm for the process by which T helper (Th) cells influence the development of different types of immunity (such as cytotoxic T cells, activated macrophages, antibodies, and the IgE response) has emerged in recent years. The simplest form of this paradigm is the Th1/Th2 cell model, in which there are two subtypes, Th1 and Th2, that control the development of acquired immunity (Figure 4-9). Both Th1 and Th2 cells are descended from the same cell type, Th0. The decision to produce Th1 cells is triggered in part by PMN production of IL-12, which stimulates NK cells to produce IFN-γ, which in turn stimulates Th0 cells to differentiate into the Th1 form. Extracellular antigens stimulate differentiation of Th0 cells to develop into Th2 cells. IL-4 is required for Th2 differentiation and is later produced by mature Th2 cells. Cytokines produced by Th1 cells, such as IFN-γ, or by Th2 cells, such as IL-4, induce a positive feedback loop that leads to further differentiation of Th0 cells into Th1 or Th2 cells, respectively, and inhibits production of the other cell type. Later in the course of combating infection, the balance between Th1 and Th2 differentiation is eventually restored.

A third, more recently discovered pathway is the Th17 pathway (Figure 4-9), so called because Th17 cells are CD4+ Th cells and CD8+ cytotoxic T cells that produce IL-17. The main protective role of the Th17 pathway is thought to stimulate proinflammatory reactions that lead to recruitment of PMNs and neutrophils in tissues. Th17 cells also produce other cytokines, including IL-22, which induces antimicrobial peptide production by epithelial cells that protect against extracellular bacteria and fungi.

Th2 cells activate eosinophils and stimulate B cells to produce antibodies of the IgG1 class (the most effective opsonizing antibodies) as well as antibodies of the IgE class (the class associated with the allergic or antimetazoan response). Th2 cells also produce IL-10 and IL-13, cytokines that downregulate some cells of the immune system. Thus, the Th1-type response is the most desirable type of response to viral and intracellular bacterial infections. The Th2-type response produces the more effective opsonizing antibodies, which are important for clearing extracellular bacterial pathogens. An example of the type of Th1 or Th2 response that is elicited and its importance in disease outcome is illustrated in Box 4-3.

Box 4-3.

The Th1/Th2 Response and the Outcome of Leprosy and Tuberculosis

Some people who contract tuberculosis, caused by Mycobacterium tuberculosis, or leprosy, caused by Mycobacterium leprae, develop a systemic form of the disease. In the case of tuberculosis this is called miliary tuberculosis, while in the case of leprosy this is called lepromatous leprosy. Other people develop a more localized form of the disease and, although there is damage in the area of infection, bacteria do not spread so readily to internal organs. What causes some people to develop the localized type of infection and others to develop the more dangerous systemic form of infection? What both M. tuberculosis and M. leprae have in common is that they enter and replicate in macrophages. An explanation based on the Th1/Th2 response has been proposed and seems to be supported by the evidence so far.

In people who are able to elicit a robust Th1-type immune response that leads to generation of activated macrophages, the numbers of invading bacteria are greatly reduced and the infection tends to be localized. In people who do not mount a strong Th1-type immune response, but instead mount a Th2-type response, which does not contribute to activation of macrophages but rather to production of antibodies, the bacteria reach high numbers in macrophages and subsequently disseminate throughout the body. Thus, balance between the Th1- and Th2-initiated paths of immune response to infection has critical practical implications for patients. This is one of many reasons why immunologists are trying to understand the complex interplay of cytokines that controls this crucial decision. Obviously, understanding this decision is crucial to the development of effective vaccines and therapies. Indeed, in cases for which a vaccine might not be available, development of pharmaceutical drugs capable of directing the Th1-Th2 decision branching could potentially manipulate the immune response to favor the more effective one against a particular infection. Recent evidence suggests that Treg and Th17 cells play important roles in directing the Th1-Th2 decision branching.

Source:

Modlin RL. 1994. Th1-Th2 paradigm: insights from leprosy. J Invest Dermatol 102:828–832.[PubMed][CrossRef]

Sadhu S, Khaitan BK, Joshi B, Sengupta U, Nautiyal AK, Mitra DK. 2016. Reciprocity between regulatory T cells and Th17 cells: relevance to polarized immunity in leprosy. PLoS Negl Trop Dis 10:e0004338.

A fourth pathway that contributes to T cell regulation, the so-called Treg pathway (or Th3 pathway), has now been identified. The Treg cells produce additional regulatory compounds that control the differentiation of Th0 cells. Treg cells maintain tolerance to self-antigens and modulate autoimmune responses and serve to suppress other immune cells, particularly once invading pathogens have been cleared from the body. Cytokines produced by Treg cells can direct the outcome of an immune response, tipping the balance between one dominated by a Th1 or a Th2 pathway (as in Box 4-3).

Production of Antibodies by B Cells

Two signals are required to activate B cells to differentiate into plasma cells and produce antibodies (Figure 4-10). This failsafe costimulatory mechanism makes sense, considering the dire consequences of not strictly controlling antibody production. One mechanism that activates B cells is the associated Th cells that mature in the thymus gland (thymus-dependent antigens). Resting B cells are not dividing, but they do express a single type of membrane immunoglobulin (mIg) on their surfaces. There are an enormous number (>1010) of resting B cells displaying mIg molecules with different antigen specificities on their surfaces! The mIg molecules interact with a membrane-bound transducer protein to form the B cell receptor (BCR). When an antigen binds to the Fab regions of two mIg molecules, linking them together, a signal is sent to the B cell that can lead to cell proliferation, if a second signal is detected. The second signal is generated by endocytosis and presentation of the antigen on the MHC II-antigen complexes on the surface of the same B cell. The use of MHC II helps B cells find the activated Th cells that are displaying TCRs specific to the displayed antigen, which ensures specific responses to a particular antigen. Binding of a Th cell to the B cell involves a set of multiprotein contacts similar to those that occur between other APCs and T cells (Figure 4-10). This binding causes the Th cell to produce additional surface receptors that lead to even tighter binding between the antigen-stimulated B cell and the Th cell. This tight binding stimulates the B cell to produce and display cytokine receptors and the Th cell to produce cytokines, which in turn stimulate the B cell to proliferate and differentiate into the mature form of B cells, called plasma cells, that secrete antibodies.

Figure 4-10. The Th2 cell binds and activates B cells presenting the same epitope on their MHC II. The B cell receptor complex is comprised of membrane-bound immunoglobulin (mIg), which can be of isotype IgM, IgD, IgA, IgG2, or IgE, the signaling mediator proteins Igα and Igβ, and the CD21 complement receptor that recognizes iC3b and promotes B cell activation. For simplicity, many of the other proteins involved in binding and activating the B cell (e.g., CD54, CD11a/CD18, CD2, CD58, CD80/70, CD28) are omitted. Stimulation of B cells through interactions of CD40 on the B cells with CD40 ligand expressed by Th cells and through cytokines produced by Th cells results in proliferation of the B cells and conversion into antibody producing cells, called plasma cells. A fraction of the activated B cells become memory B cells.

A fraction of activated B cells become quiescent memory B cells. In subsequent encounters with the same antigen and again with memory Th cells against that same antigen, the memory B cells respond with a much shorter lag period and produce more antibodies for a longer period of time than in the primary response of the naïve Th and B cells. In addition, this secondary response produces antibodies with higher affinity, primarily of the IgG isotype.

Links between the Innate and Adaptive Defense Systems

Treating the innate and adaptive defense systems as separate subjects in separate chapters makes pedagogical sense because it is generally easier to understand complex systems by first separating them into their constituent components. However, it is important to note that for the human body to have the innate defenses acting independently of the adaptive defenses would be counterproductive, because the two types of defense need to act synergistically. Some examples of links are cytokines and chemokines, which are produced by and act on both types of immune responses; PMNs that serve as first responders and contribute through cytokine production to the Th1/Th2 decision-making process; DCs that opsonize pathogens and present the processed antigens to the adaptive immune cells; and antibodies that are made as an adaptive response but, when bound to bacterial cells, activate one arm of the complement cascade (i.e., the classical pathway).

We now add yet another link: the interaction between natural killer cells and B cells. It may be simply a coincidence that natural killer (NK) cells have a number of similarities, morphologically and cytokinologically, to T cells. Because T cells interact with B cells on other planes of existence, it is somewhat satisfying to learn that NK cells interact with B cells, too. Briefly, NK cells can stimulate B cells to proliferate (as T helper cells do) but by some factor that is not one of the known cytokines. B cells in turn induce NK cells to secrete more IFN-γ, which, as we have already seen, is an important factor in macrophage activation.

T-Cell-Independent Antibody Responses

In addition to the CD1-dependent T cell pathway discussed earlier (Figure 4-6C), nonpeptide antigens such as polysaccharides (e.g., bacterial capsule components), nucleic acids (e.g., bacterial DNA or RNA), and to a lesser extent lipids and glycolipids (e.g., bacterial membrane components) can also stimulate T-cell-independent antibody responses. T-cell-independent antigens provoke an antibody response by directly interacting with B cells (Figure 4-11), without the involvement of T cells. Polysaccharides are a well-studied class of T-cell-independent antigens. Those B cells displaying mIg with affinity for the sugar repeat units will bind to the polysaccharide chain. Because these epitopes are repeated many times, numerous mIgs and BCRs are linked together, which causes them to cluster and activate cellular signaling pathways. This acts as one signal to stimulate the B cells to proliferate and differentiate into mature antibody-producing plasma cells. A second signal, such as binding of cytokines produced at the infection site, is also required to initiate the T-cell-independent response.

Figure 4-11. T-cell-independent production of antibodies. Macromolecules with highly repetitive structures, such as carbohydrates (polysaccharides, capsules) and nucleic acids, are able to directly stimulate naïve B cells to proliferate, mature, and produce antibodies through antigen-mediated clustering of IgG2 and IgM antibodies on the surface of the B cells.

The T-cell-independent response is particularly important for protection against bacterial pathogens that can avoid phagocytosis by covering themselves with an exopolysaccharide layer (capsule), which is not effectively opsonized through coating with C3b. Such bacterial pathogens are only ingested and killed by phagocytes if antibodies that bind to capsular antigens are elicited and act as opsonins.

Although the T-cell-independent response provides protection against capsule-producing bacteria, it has some important drawbacks. First, the antibody response elicited by T-cell-independent antigens is not as strong as the T-cell-dependent response. It is also not long-lasting, because no memory B cells are developed. Second, the main antibodies elicited by T-cell-independent antigens are IgM and IgG2. IgG2 does not opsonize and IgM does so less effectively than IgG1 and IgG3 (Table 4-1). Third, infants do not mount a T-cell-independent response. Polysaccharides and lipids can elicit an immune response in children and adults, but not in infants under the age of two years. Thus, the ability to respond to T-cell-independent antigens, such as capsule exopolysaccharide, is acquired after birth. This is an important consideration because it means that vaccines consisting of T-cell-independent antigens are not effective until an infant has become old enough to respond to these antigens. Unfortunately, infants are one of the highest risk groups for contracting serious infections due to capsule-producing bacteria (e.g., pneumonia and meningitis).

A strategy for improving the immune response to T-cell-independent antigens and extending this response to infants is to covalently link epitopes of the polysaccharide capsule to a protein. This type of vaccine is called a conjugate vaccine (more about this in chapter 17). APCs process conjugate vaccines as if they were proteins and elicit a Th2-cell-dependent response that culminates in production of antibodies that also recognize the polysaccharide antigens. This immune response is long-lived because it involves T cell activation and memory cell generation and produces opsonizing IgG1 antibodies, IgG3, and IgG4.

Mucosal Immunity: IgA/sIgA Antibodies

An important immune defense against infectious diseases, but one that is much less well understood than the humoral or cell-mediated responses, is the immune system that produces sIgA. The first step in many microbial infections is colonization and invasion of a mucosal surface. sIgA can prevent such infections by blocking colonization. Thus, while the cell-mediated or humoral antibody responses may cause collateral damage to tissues in the area where infection is occurring, the sIgA-mediated defense is usually innocuous to the host because it occurs in the mucus layer.

As we mentioned in chapter 2, skin and mucosal surfaces all have attendant mucosa-associated lymphoid tissues (MALT) located just below the epithelial layer in the lamina propria. The mucosal surface of the small intestine is underlain with the GALT, while the lungs have BALT, the upper respiratory tract has NALT, and the vaginal tract has vaginal-associated lymphoid tissue (VALT). Skin also has a similar system (called SALT).

The intestinal cells that comprise the GALT are visible as a collection of follicles, called Peyer’s patches, which are most highly concentrated in the ileum and rectum of the intestine. Similar mucosal lymphoid tissues are found in the respiratory and vaginal tracts, although they are not as pronounced. The cells that form the Peyer’s patches are illustrated in Figure 4-12. M (microfold) cells take up antigens from the lumen of the intestinal tract and pass them to closely associated GALT macrophages, which act as the APCs of the GALT. M cells have never been successfully cultivated in vitro, so little is known about the activities of M cells. The mechanism by which GALT macrophages process antigens and elicit production of cytotoxic T cells or antibodies is the same as that described in earlier sections, except that the macrophages, B cells, and T cells of the GALT reside specifically in the lamina propria of mucosal surfaces. Two additional types of T cells, Th17 and Tregs, appear to be important for IgA production in the intestine, both of which appear to help modulate the B cells.

Figure 4-12. Cells of the GALT that confer mucosal immunity. M cells and their associated macrophages and lymphoid cells (T and B cells) are sometimes called follicles. Collections of such follicles in the gut are called Peyer’s patches. M cells sample the contents of the gut lumen and transfer the antigens to closely associated resident macrophages, which in turn ingest the bacteria and present antigens to the underlying T cells that then stimulate nearby B cells to produce IgA. The IgA binds to receptors on the basal surface of the mucosal epithelial cell and is transcytosed across the cell and secreted into the lumen of the gut as sIgA.

When the GALT is stimulated, one outcome is production of IgA (see Figure 4-12). Dimeric IgA is produced by plasma cells in the lamina propria at mucosal sites. The secretory piece is acquired when IgA dimer is transported through the mucosal epithelial cell into mucosal secretions covering various mucosal surfaces. IgA dimer binds to the poly-Ig receptor on the basal surface of mucosal cells and is then taken up by endocytosis and transported in vesicles, through a process called transcytosis, to the apical surface, where it is released into the lumen of the gut. Release involves proteolytic cleavage of the poly-Ig receptor, where the secretory piece comes from the portion of the receptor that remains attached to the IgA, making it sIgA. sIgA can trap microbes in mucus because the Fc portion of sIgA binds to glycoprotein constituents of the mucin and the Fab portion binds to antigens on the microbes in the gut. By trapping the microbes in the mucus layer, the sIgA-antigen-mucin complex essentially forms a protective barrier that blocks the microbes and their toxic products from gaining access to the epithelial cell layer. Mucus laden with sIgA-coated microbes is then sloughed off and excreted from the body.

The mucosal and skin tissues that have contact with the external environment are also constantly patrolled by DCs (APCs), which engulf and kill the invading microbes and process the antigens for presentation to the adaptive immune system. Once activated, DCs migrate to the lymph nodes, where they present the antigens to Th cells and stimulate adaptive immunity. The Langerhans cells of the epidermis are the DCs of SALT.

As part of the mucosal immune system, some T cells and B cells stimulated by antigen processing at the GALT can migrate to other mucosal sites and vice versa. Thus, stimulation at one of the MALT sites can transfer to other sites, resulting in general mucosal immunity. The first evidence for this feature of the mucosal immune system came from elegant experiments performed by Husband and Gowans in the late 1970s. These researchers excised a segment of the small intestine from a rat, preserving its vascular and lymphatic supplies, and reconnected the ends of the intestinal segment to the skin surface of the animal, forming a so-called Thiry-Vella loop (Figure 4-13). They then introduced an antigen, in this case cholera toxin (CT), into the loop and found that sIgA was secreted in not only the immunized loop segment, but also in a second such loop (when made) and in the main intestine. Their results demonstrated that introduction of an immunogen at one site could confer mucosal immunity at a remote site. This characteristic of the MALT system is what makes oral vaccines feasible. Initially, oral vaccines stimulate the GALT, but sIgA against vaccine antigens is later detectable in other MALT sites. Thus, an oral vaccine can also be used to elicit immunity to respiratory and, presumably, urogenital pathogens.

Figure 4-13. Classic experiment by Husband and Gowans demonstrating mucosal immunity at remote sites. The experimental setup involved isolating Thiry-Vella loops of the small intestine of rats and connecting them to the skin, preserving the associated vascular and lymphatic systems attached to the loops. The IgA immune response to administering immunogen (cholera toxin) to the main intestine through the oral route or through the skin opening of the loop could then be observed. Results showed that local immunization through the isolated loops that contained Peyer’s patches generated Th cells and B cells that circulated through the lymph and blood to populate other mucosal sites. Based on Husband AJ, Gowans JL. 1978. J Exp Med 148:1146–1160.

Currently, efforts are being made to develop vaccines administered by inhalation, so that stimulation of the nasal MALT (NALT) would produce an sIgA response at other MALT sites. These vaccines would have the advantage of not having to pass through the stomach. Developing vaccines that target the GALT means developing vaccines capable of surviving the low-pH/protease-rich stomach environment, a barrier that has proven problematic in many cases. Administering vaccines by rectal or vaginal suppositories is theoretically possible, but this strategy has not been actively pursued to date. On the other hand, the SALT is gaining in attraction as a target for vaccine development (more on vaccines in chapter 17).

Activation of the GALT can also lead to production of cytotoxic T cells. These cells probably remain on the basal side of the mucosa, although it is possible that during an infection some of them migrate to the apical surface, especially in areas where damage to the mucosa has occurred. GALT cytotoxic T cells are important for protection against viral infections of the GI tract and some bacterial infections in which the bacteria multiply inside mucosal cells.

One of the many mysteries swirling around the intestinal immune system is the role of a particular type of mucosal cell called γδ T cells. The majority of γδ T cells are CD8+ T cells and would thus be grouped with CTLs. However, whereas CTLs have TCRs composed of αβ chains (Figure 4-8), the intestinal epithelial lymphocytes (IELs) have T-cell receptors composed of related but somewhat different γδ chains. These γδ T cells account for less than 4% of circulating CD8+ T cells, but they account for as much as 10 to 15% of the mucosal T cells found in the GI tract. In some regions, such as the colon, the levels may be as high as 40%.

Unlike αβ T cells, γδ T cells seem to recognize only a limited number of cell-surface antigens. Also, γδ T cells seem to bypass antigen presentation by MHCI and MHCII on the surfaces of macrophages and DCs and directly recognize nonpeptide antigens that have not been processed. Indeed, they are believed to play a predominant role in lipid-antigen recognition via CD1 complexes (Figure 4-6C). γδ T cells also respond to two human protein complexes related to MHC I, MICA and MICB. These proteins are displayed on the surface of cells that are stressed (e.g., by being infected) and are recognized by NKG2D receptors present on γδ T cells, as well as NK cells, which results in activation of cytotoxic responses that kill the stressed cells. γδ T cells also produce cytokines that stimulate αβ CD8+ cytotoxic T cells to migrate to the area and eliminate infected cells.

As with other immune responses, the GALT has a downside. Normally, bacteria and other viable antigens that pass through M cells are killed by the resident GALT macrophages. Some bacterial pathogens, however, have acquired the ability to avoid this fate and exploit the GALT as an entryway into the body. Because the M cell is an antigen-sampling cell, it usually does not take up substantial amounts of an antigen because only a few bacteria or other antigens are sufficient to stimulate a mucosal immune response. The bacteria that use the GALT as an entryway into the body, such as Salmonella enterica or Yersinia pseudotuberculosis, invade M cells or use them to move across the epithelial layer (see Figure 4-14).

Figure 4-14. Scanning electron micrographs of mouse Peyer’s patches (a) before and (b) after incubation with Yersinia pseudotuberculosis. Both images depict a central M cell surrounded by enterocytes (and part of a brush cell in the lower left of panel b). The M cell in panel a lacks adherent bacteria but, in contrast, the association of bacteria with the M cell in panel b is accompanied by disruption of the normal surface morphology of the M cell. Bars, 2 µm. Reprinted with permission from Clark MA, Hirst BH, Jepson MA. 1998. Infect Immun 66:1237–1243.

Development of the Adaptive Immune System from Infancy to Adulthood

Human infants do not develop fully effective immune defenses until they are one to two years of age. Infants have very low levels of NK cells and are deficient in mannose-binding proteins, one of the acute-phase proteins produced by the liver that helps opsonize some types of microbes. For the period up to one year of age, maternal antibodies that were transferred through the placenta during gestation or ingested in milk during infancy protect the infants at least partially. The infant’s antibody production begins to kick in at about two to three months of age, but the rate of production is still relatively slow with antibodies being short-lived and of low avidity. T-cell-independent responses do not start until about 18 months of age. The transition between the protection conferred by maternal antibodies and the development of the infant’s own immune system provides pathogenic microbes with a window of opportunity and is one of the reasons why children under the age of two are particularly vulnerable to infectious diseases.

Interestingly, circulating maternal antibodies can actually interfere with an immune response to some antigens, because high levels of antibody to a particular epitope discourage the development of an antibody response to that epitope. This is a consequence of immune regulation that normally protects the body from overreacting to a particular epitope. The dampening effect of circulating maternal IgG is the reason why some vaccines are not given to infants less than one year of age.

Adaptive Defense Systems in Nonmammals

Scientists have found primitive versions of the mammalian adaptive defense system in many different organisms, ranging from insects to sharks (Table 4-2). Insects such as Drosophila melanogaster (fruit fly) and slime molds such as Dictyostelium also have primitive defense systems that resemble mammalian defense responses, especially the innate immune response. Zebra fish not only have an innate immune response but also have a response that parallels the Th1/Th2 adaptive response. Sharks produce antibodies that can bind to human pathogens.

Table 4-2. Defense systems in higher organisms

Even plants have a primitive defense system in which proteins produced by the plant (R proteins) bind to invading bacteria, a reaction that produces apoptosis in the surrounding plant cells. This area of dead, dry tissue, which appears as black spots on leaves or fruit, traps the bacteria in the area. Bacteria need to move throughout the plant via the xylem and phloem in order to cause serious disease, and the plant’s defense response is designed to prevent this.

Assertions that systems in nonmammals are “immune systems” should be viewed with some skepticism. In the case of sharks, plants, and zebra fish, there is evidence that the systems are actually a response that protects the organism from infection. In the case of Drosophila, however, the Toll receptors that led to the discovery of mammalian Toll-like receptors, which are a central part of the mammalian innate immune response, appear to be involved primarily in development of the insect or in feeding behavior. Only one Toll receptor appears to play a role in defending the insect against fungal infections. Nonetheless, it is intriguing to see that parallels of what we know as the mammalian innate and adaptive defense systems are so widespread in other creatures. Interest in the evolution of the mammalian immune system is a relatively new area of research focus, so there are likely to be many more insights in the future as this research progresses.

The Dark Side of the Adaptive Defenses: Autoimmune Disease

Complex figures such as those shown in this chapter illustrate the manifold interactions that occur between cells of the immune system when they activate each other. All of those proteins labeled CD followed by a number have an important role. They help ascertain that the contacts between the immune cells are occurring in response to true foreign antigen presentation events and not accidental associations between cells of the immune system and other cells in the body. They serve as insurance that the immune system will not go out of control and attack self cells.

Unfortunately, this safeguard is sometimes breached, like when bacterial antigens contain epitopes that mimic human antigens. In such cases, the adaptive defenses can produce antibodies or CTLs that mount an attack against the host, thereby creating a set of conditions that can lead to autoimmune disease. If the targeted tissue is the heart or another vital organ, the misguided attack can be lethal. In later chapters, we will encounter examples of autoimmunity initiated by the adaptive immune defenses against bacterial components that mimic host antigens. Bacteria that elicit an autoimmune response are particularly difficult targets for vaccine development. Such a vaccine could induce an autoimmune response when the vaccinated person is exposed to the bacterium, which could make the disease worse rather than preventing it. Thus, one of the concerns of vaccine producers is to make sure that their vaccine does not elicit an autoimmune response.

SELECTED READINGS

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Bemark M. 2015. Translating transitions—how to decipher peripheral human B cell development. J Biomed Res 29:264–284.[PubMed][CrossRef]

Blum JS, Wearsch PA, Cresswell P. 2013. Pathways of antigen processing. Annu Rev Immunol 31:443–473.[PubMed][CrossRef]

Campbell KS, Hasegawa J. 2013. Natural killer cell biology: an update and future directions. J Allergy Clin Immunol 132:536–544.[PubMed][CrossRef]

Clark MA, Hirst BH, Jepson MA. 1998. M-cell surface β1 integrin expression and invasin-mediated targeting of Yersinia pseudotuberculosis to mouse Peyer’s patch M cells. Infect Immun 66:1237–1243.[PubMed]

Cullen SP, Martin SJ. 2008. Mechanisms of granule-dependent killing. Cell Death Differ 15:251–262.[PubMed][CrossRef]

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Husband AJ, Gowans JL. 1978. The origin and antigen-dependent distribution of IgA-containing cells in the intestine. J Exp Med 148:1146–1160.[PubMed][CrossRef]

Kabelitz D, Peters C, Wesch D, Oberg HH. 2013. Regulatory functions of γδ T cells. Int Immunopharmacol 16:382–387.[PubMed][CrossRef]

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Mabbott NA, Donaldson DS, Ohno H, Williams IR, Mahajan A. 2013. Microfold (M) cells: important immunosurveillance posts in the intestinal epithelium. Mucosal Immunol 6:666–677.[PubMed][CrossRef]

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Soypayrac L. 2016. How the Immune System Works, 5th ed. Wiley Blackwell, West Sussex, United Kingdom.

Questions

1. In the short statement that began the chapter, the innate immune system was likened to ordinary police and the adaptive immune system was likened to specialized backups such as snipers. For those of you who follow true crime or mystery stories, you might find it amusing—and helpful in your studies—to try to come up with other criminal justice analogies. For example, you might make an analogy between “antibody production by B cells” and “smart bombs, surveillance, and tracking devices.” See what you can do with T helper cells, cytokines, APCs, and NK cells.

2. Does the fact that sIgA does not activate complement make it less effective in preventing infection than IgG? Hint: Consider its location and function.

3. Explain how the structure of an antibody is designed to facilitate the function of the particular antibody (e.g., why an antibody has at least two active sites, why it has an Fc portion, and why it might be cross-linked as a multimer).

4. Why is a Th1 or Th2 cell needed to mediate APC-initiated antibody production? Why do the B cells not bind directly to the APCs?

5. Explain why direct antigen activation of cytotoxic T cells and B cells is more important than APC-initiated activation in subsequent encounters with a microbe. Why is the APC-initiated pathway needed at all?

6. There are many interactions between the innate and adaptive defense systems. List as many of them as you can and explain how they work. Why are these interactions important? Are all of them beneficial?

7. What are the two ways that polysaccharides, DNA, and lipid antigens can elicit an immune response? How are these responses different than that elicited by a peptide antigen?

8. Why is the level of specific IgM antibodies of diagnostic significance?

Solving Problems in Bacterial Pathogenesis

1. A group of researchers have isolated a new pathogenic bacterium Q from lungs and lymph nodes of cystic fibrosis patients that produces an unusual polysaccharide capsule, called QPS. They can see QPS on the surface of the bacteria in electron micrographs from fresh isolates (i.e., bacteria just obtained from patients). In addition to the capsule, bacterium Q produces a protease, called QP, which the researchers believe is responsible for degrading host sIgA.

a. The researchers propose that QPS and QP might make good targets for vaccine development against bacterium Q. What led the researchers to propose this?

b. What potential problems could the researchers have in using QPS as a vaccine?

c. How are QP antigens presented to immune cells?

d. The researchers find that the QPS vaccine does not elicit a long-lasting immune response. How does QPS elicit an immune response? What vaccine strategy could be used to generate long-lasting immunity targeting QPS?

e. The researchers believe that they can develop an oral vaccine that will be effective against lung infections with bacterium Q using QP as a vaccine component. What specific experimental evidence supports their rationale for using this proposed approach?

f. If QP were used as a vaccine, what immune response would be primarily responsible for controlling lung infection caused by bacterium Q?

2. Listeria monocytogenes is a foodborne, Gram-positive bacterium that causes infections (listeriosis) in individuals with compromised immune systems. Listeria is predominantly an intracellular pathogen. After phagocytosis by intestinal macrophages, Listeria escapes the phagosome and replicates in the cytosol before spreading to other neighboring cells through a special invasion process that allows it to remain intracellular while spreading from cell to cell.

a. What advantage is there for Listeria to invade and spread intracellularly?

b. What immune response is primarily responsible for controlling Listeria infection in healthy but unimmunized individuals?

c. What immune response is primarily responsible for controlling Listeria infection in healthy but immunized individuals or those having prior exposure?

d. How are Listeria antigens presented to immune cells?

3. Lyme disease is a common zoonotic vector-borne disease in the United States. It is caused by the Gram-negative spirochete Borrelia burgdorferi, which is transmitted through the bite of infected deer ticks. After a period ranging from 3 to 30 days, a unique red rash with a classic bull’s-eye appearance occurs in ∼75% of infected persons at the site of the bite (shown in Figure 1). Though such a rash does not always appear, the presence of this characteristic rash is diagnostic for Lyme disease (there are no other known causes for it), so physicians often prescribe antibiotics without further diagnostic testing. In most cases, antibiotics such as tetracycline and penicillin eliminate the infection and its symptoms. However, delayed or inadequate treatment can lead to more serious symptoms much later, including effects on the joints (chronic arthritis, inflammation), the heart (atrioventricular blockage, abnormal heart rate), and the central nervous system leading to facial palsy (paralysis), meningitis (headaches, neck stiffness), or encephalitis (memory loss, mood changes). Upon injection of the bacteria into the skin by the tick, the bacteria multiply and migrate slowly outward within the dermis. Without antibiotic treatment, the bacteria eventually spread throughout the body, despite generation of high titers of antispirochete antibodies. Microscopic examination of various tissues during infection reveals that these spirochetes are not found inside host cells, but instead reside within the extracellular matrix of skin, joints, and heart, peripheral, and central nervous systems.

Figure 1. The characteristic bull’s-eye rash (erythema migrans) caused by Lyme disease. Image from CDC-PHIL ID#9875, courtesy of CDC/James Gathany.

a. What is the cause of the characteristic bull’s-eye rash that appears around the tick bite without antibiotic treatment? Be sure to provide a plausible explanation for the bull’s-eye appearance.

b. Which cells would normally be the first on the scene to eliminate the spirochetes from the bite site? Be specific.

c. Interpret what it means to have the occurrence of strong antibody responses against B. burgdorferi in chronically infected individuals in the absence of sterilizing immunity.

d. Provide two possible explanations with rationale for why these cells might not respond effectively to clear the spirochete infection. For each of these possibilities, provide at least one definitive experiment that could be performed to confirm the explanation.

e. Provide a possible explanation with rationale for the later symptoms of inflammation and arthritis associated with chronic infections.

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