10-13. Mucosa-associated lymphoid tissue is located in anatomically defined microcompartments throughout the gut

The mucosa-associated lymphoid tissues lining the gut are known as gut-associated lymphoid tissue or GALT. The tonsils and adenoids form a ring, known as Waldeyer's ring, at the back of the mouth at the entrance of the gut and airways. They represent large aggregates of mucosal lymphoid tissue, which often become extremely enlarged in childhood because of recurrent infections, and which in the past were victims of a vogue for surgical removal. A reduced IgA response to oral polio vaccination has been seen in individuals who have had their tonsils and adenoids removed, which illustrates the importance of this subcompartment of the mucosal immune system.

The other principal sites within the gut mucosal immune system for the induction of immune responses are the Peyer's patches of the small intestine, the appendix (which is another frequent victim of the surgeon's knife), and solitary lymphoid follicles of the large intestine and rectum. Peyer's patches are an extremely important site for the induction of immune responses in the small intestine and have a distinctive structure, forming domelike structures extending into the lumen of the intestine (see Fig. 1.10). The overlying layer of follicle-associated epithelium of the Peyer's patches contains specialized epithelial cells. These have microfolds on their luminal surface, instead of the microvilli present on the absorptive epithelial cells of the intestine, and are known as microfold cells or M cells. They are much less prominent than the absorptive gut epithelial cells, known as enterocytes, and form a membrane overlying the lymphoid tissue within the Peyer's patch. M cells lack a thick surface glycocalyx and do not secrete mucus. Hence they are adapted to interact directly with molecules and particles within the lumen of the gut.

M cells take up molecules and particles from the gut lumen by endocytosis or phagocytosis (Fig. 10.17). This material is then transported through the interior of the cell in vesicles to the basal cell membrane, where it is released into the extracellular space. This process is known as transcytosis. At their basal surface, the cell membrane of M cells is extensively folded around underlying lymphocytes and antigen-presenting cells, which take up the transported material released from the M cells and process it for antigen presentation.

Figure 10.17

M cells take up antigens from the lumen of the gut by endocytosis. The cell membrane at the base of these cells is folded around lymphocytes and dendritic cells within the Peyer's patches. Antigens are transported through M cells by the process of transcytosis (more...)

Because M cells are much more accessible than enterocytes to particles within the gut, a number of pathogens target M cells to gain access to the subepithelial space, even though such pathogens then find themselves in the heart of the adaptive immune system of the intestine, the Peyer's patches. We will consider one of these pathogens in Section 10-19.

10-14. The mucosal immune system contains a distinctive repertoire of lymphocytes

In addition to the organized lymphoid tissue in which induction of immune responses occurs within the mucosal immune system, small foci of lymphocytes and plasma cells are scattered widely throughout the lamina propria of the gut wall. These represent the effector cells of the gut mucosal immune system. The life history of these cells is as follows. As naive lymphocytes, they emerge from the primary lymphoid organs of bone marrow and thymus to enter the inductive lymphoid tissue of the mucosal immune system via the bloodstream. They may encounter foreign antigens presented within the organized lymphoid tissue of the mucosal immune system and become activated to effector status. From these sites, the activated lymphocytes traffic via the lymphatics draining the intestines, pass through mesenteric lymph nodes, and eventually wind up in the thoracic duct, from where they circulate in the blood throughout the entire body (Fig. 10.18). They reenter the mucosal tissues from the small blood vessels lining the gut wall and other sites of MALT, such as the respiratory or reproductive mucosa, and the lactating breast; these vessels express the mucosal adressin MAdCAM-1. In this way, an immune response that may be started by foreign antigens presented in a limited number of Peyer's patches is disseminated throughout the mucosa of the body. This pathway of lymphocyte trafficking is distinct from and parallel to that of lymphocytes in the rest of the peripheral lymphoid system (see Fig. 1.11).

Figure 10.18

Anatomy of mucosal immune responses. The left panel shows the afferent immune response. Antigen from pathogenic micro-organisms is presented beneath mucosal surfaces to naive lymphocytes within organized mucosal lymphoid tissue, for example Peyer's patches. (more...)

The distinctiveness of the mucosal immune system from the rest of the peripheral lymphoid system is further underlined by the different lymphocyte repertoires in the different compartments. The T cells of the gut can be divided into two types. One type bears the conventional α:β T-cell receptors in conjunction with either CD4 or CD8, and participates in conventional T-cell responses to foreign antigens as discussed in earlier chapters. The second class is made up of T cells with unusual surface phenotypes such as TCRγ:δ and CD8α:α TCRα:β. The receptors of these T cells do not bind to the normal MHC:peptide ligands but instead bind to a number of different ligands, including MHC class IB molecules. These highly specialized T cells are abundant in the epithelium of the gut and have a restricted repertoire of T-cell receptor specificities. Unlike conventional T cells, many of these cells do not undergo positive and negative selection in the thymus (see Chapter 7), and express receptors with sequences that have undergone no or minimal divergence from their germline-encoded sequences. These cells may be classified in phylogenetic terms as being at the interface between innate and adaptive immunity.

T cells bearing a γ:δ receptor are especially abundant in the gut mucosa compared with other lymphoid tissues. One subset of these γ:δ T cells in humans, which expresses a T-cell receptor that uses the V_δ1 gene segment, carries an activating C-type lectin NK receptor, NKG2D. This latter receptor binds to two MHC-like molecules—MIC-A and MIC-B—that are expressed on intestinal epithelial cells in response to cellular injury and stress. The injured cells may then be recognized and killed by this subset of γ:δ T cells (Fig. 10.19). This illustrates one of the key roles of T cells, which is to patrol and survey the body, destroying cells that express an abnormal phenotype as a result of stress or infection.

Figure 10.19

T cells of the mucosal immune system bearing γ:δ T-cell receptors and an activating NK receptor recognize and kill injured enterocytes. Infection or other injury to enterocytes, the epithelial cells lining the lumen of the gut, stimulates (more...)

The V_δ1-containing receptor on these T cells may also play a part in allowing them to survey tissues for injured cells. Some human T cells expressing this receptor bind to CD1c, one of the isotypes of the CD1 family of MHC class I-like molecules that we encountered in Section 10-5. This protein, which shows increased expression on activated monocytes and dendritic cells, presents endogenous lipid and glycolipid antigens to some types of T cell. In response to antigen presentation by CD1c, these T cells secrete IFN-γ, which may have an important role in polarizing the response of conventional T cells bearing α:β receptors toward a T_H1 response. This is closely analogous, although opposite in effect, to the polarization toward T_H2 cells induced by secretion of IL-4 by NK 1.1⁺ T cells responding to CD1d discussed in Section 10-5.

A second group of specialized mucosal T cells, so far only characterized in mice, express α:β T-cell receptors together with a CD8 α:α homodimer, instead of the normal CD8 α:β heterodimer that characterizes MHC class I-restricted cytotoxic T cells. These cells can be found in the gut of mice lacking conventional MHC class I molecules, which shows that their development is not dependent on positive selection in the thymus by peptides bound to classical MHC class I molecules. They are, however, absent in mice lacking expression of β₂-microglobulin, which is necessary for the expression of MHC class IB molecules. The ligand recognized by these T cells in mice has been identified as the nonpolymorphic MHC class IB molecule known as Qa-2. These cells are likely to represent a further class of T cells that have a major role in maintaining the integrity of the gut mucosa by recognizing and destroying injured mucosal cells.

10-15. Secretory IgA is the antibody isotype associated with the mucosal immune system

The dominant antibody isotype of the mucosal immune system is IgA. This class of antibody is found in humans in two isotypic forms, IgA1 and IgA2. The expression of IgA differs between the two main compartments in which it is found—blood and mucosal secretions. In the blood, IgA is mainly found as a monomer and the ratio of IgA1 to IgA2 is approximately 4:1. In mucosal secretions, IgA is almost exclusively produced as a dimer and the ratio of IgA1 to IgA2 is approximately 3:2. A number of common intestinal pathogens possess proteolytic enzymes that can digest IgA1, whereas IgA2 is much more resistant to digestion. The higher proportion of plasma cells secreting IgA2 in the gut lamina propria may therefore be the consequence of selective pressure by pathogens against individuals with low IgA2 levels in the gut. The mechanism of isotype switching to IgA is discussed in Section 9-14.

There are special mechanisms for the secretion of polymeric IgA and IgM antibody into the gut lumen (see Section 9-13). Polymeric IgA and IgM are synthesized throughout the gut by plasma cells located in the lamina propria and are transported into the gut by immature epithelial cells located at the base of the intestinal crypts. These express the polymeric immunoglobulin receptor on their basolateral surfaces. This receptor binds polymeric IgA or IgM and transports the antibody by transcytosis to the luminal surface of the gut. Upon reaching the luminal surface of the enterocyte, the antibody is released into the secretions by proteolytic cleavage of the extracellular domain of the polymeric IgA receptor. Secreted IgA and IgM bind to the mucus layer overlying the gut epithelium where they can bind to and neutralize gut pathogens and their toxic products (Fig. 10.20).

Figure 10.20

The major antibody isotype present in the lumen of the gut is secretory polymeric IgA. This is synthesized by plasma cells in the lamina propria and transported into the lumen of the gut through epithelial cells at the base of the crypts. Polymeric IgA (more...)

10-16. Most antigens presented to the mucosal immune system induce tolerance

We are continuously exposed to a huge array of foreign antigens in the form of foods, but these do not normally induce an adaptive immune response. For example, IgA antibodies with high affinity to food antigens do not normally develop. This lack of response occurs despite the fact that the repertoire of lymphocyte antigen receptors has not been negatively selected to remove those specific for food antigens. This is because, like any other foreign antigen, food antigens do not play a part in the central mechanisms of lymphocyte tolerance to self, which are established in the thymus and bone marrow (see Chapter 7).

The feeding of foreign antigens leads typically to a state of specific and active unresponsiveness, a phenomenon known as oral tolerance. Thus, no antibody response follows the feeding of a foreign protein such as ovalbumin, although a strong antibody response to this protein can be induced by injecting it subcutaneously, especially if an adjuvant is given as well. However, the feeding of ovalbumin is followed by a prolonged period during which the administration of ovalbumin by injection, even in the presence of adjuvant, elicits no antibody response. This suppression is antigen-specific, because antibody responses to other injected antigens are not affected. These experiments show that there are antigen-specific mechanisms for suppressing peripheral immune responses to antigens delivered by mouth. The mechanisms of oral tolerance are partly understood but, before considering them, we will first discuss the contrasting immune responses that are seen in response to bacterial infections of the gut.

10-17. The mucosal immune system can mount an immune response to the normal bacterial flora of the gut

We each harbor more than 400 species of commensal bacteria, which are present in the largest numbers in the colon and ileum. Despite the fact that these bacteria collectively weigh approximately 1 kg and outnumber us by approximately 10¹⁴ to 1, for most of the time we cohabit with our intestinal bacterial flora in a happy symbiotic relationship.

One protective activity of our normal gut flora is that of competition against pathogenic bacteria for space and nutrients, preventing their colonization of the gut (see Fig. 2.4). This activity is dramatically illustrated by one of the adverse effects of antibiotics. Taking an antibiotic kills large numbers of commensal gut bacteria and thereby offers an ecological niche to bacteria that would not otherwise be able to compete successfully with the normal flora and grow in the gut. One example of a bacterium that grows in the antibiotic-treated gut and can cause a severe infection is Clostridium difficile; this produces two toxins, which can cause severe bloody diarrhea associated with mucosal injury (Fig. 10.21).

Figure 10.21

Treatment with antibiotics causes massive death of the commensal bacteria that normally colonize the colon. This allows pathogenic bacteria to proliferate and occupy an ecological niche that is normally occupied by harmless commensal bacteria. Clostridium (more...)

There are some circumstances in which the normal bacterial inhabitants of the gut cause disease, for example, following breakdown of the integrity of the mucosa lining the gut. This may occur following poor blood flow in the gut, or following endotoxemia (see Chapter 2). In these circumstances, normally innocuous gut bacteria, such as nonpathogenic Escherichia coli, can cross the mucosa, invade the bloodstream, and cause fatal systemic infection. This illustrates the vital importance of the barrier to infection provided by the mucosal surfaces of the body. The normal gut flora also becomes an important cause of systemic infection in patients with immunodeficiency. This illustrates the role of the adaptive immune system in host defense against the flora of the gut, but also shows that this response does not result in the elimination of bacteria from the lumen of the gut, but rather a state resembling symbiosis.

The scale of the normal immune response to gut bacteria is illustrated by the study of animals delivered by Caesarian section into a sterile environment in which there is no colonization of the gut by microorganisms. These are known as germ-free or gnotobiotic animals. These animals have marked reductions in the size of all secondary lymphoid organs and reduced levels of antibodies of all isotypes.

10-18. Enteric pathogens cause a local inflammatory response and the development of protective immunity

In spite of the array of innate immune mechanisms in the gut and stiff competition from the indigenous flora, the gut is a frequent site of infection by pathogenic microorganisms. These include many species of viruses, enteric pathogenic bacteria including Salmonella, Yersinia, Shigella, and Listeria, and protozoa such as Entamoeba histolytica and Cryptosporidium. These organisms cause disease in many different ways, but there are certain common features of infection that are crucial to understanding how these pathogens stimulate an immune response by the host, in contrast to the immunological tolerance shown to the foreign antigens ingested in food.

The most important consequence of infection in the gut, as elsewhere in the body, is the development of an inflammatory response. The release of cytokines and chemokines in this response is key to the induction of an adaptive immune response. The inflammatory mediators stimulate the maturation of dendritic cells and other antigen-presenting cells, so that they express the co-stimulatory molecules that provide the additional signals for activation and expansion of naive lymphocytes.

Some intestinal pathogens infect enterocytes, the absorptive cells that line much of the intestine. Enterocytes do not act as passive victims of infection but signal infection by releasing cytokines and chemokines. These include the chemokine IL-8, which is a potent neutrophil chemoattractant, and CC chemokines such as MCP-1, MIP-1α and β, and RANTES, which are chemoattractants for monocytes, eosinophils, and T lymphocytes (see Fig. 2.33). In this way, the onset of infection triggers an influx of inflammatory cells and lymphocytes, leading to the induction of an immune response to the antigens of the infectious agent. Injury and stress to the enterocytes lining the gut may also stimulate the expression of nonclassical MHC molecules, such as MIC-A and MIC-B (see Section 10-14). These act as ligands to the receptors on γ:δ T cells at the base of the crypts, which kill the infected mucosal cell, thereby promoting repair and recovery of the injured mucosa.

A number of pathogens directly exploit the M cell as a means of invasion. Some viruses are transported through the M cell by transcytosis and from the subepithelial space are able to establish systemic infection. For example, from this site polio and retroviruses enter intestinal neuronal cells and spread to the central nervous system. HIV, which we discuss in detail in Chapter 11, may use a similar route into the lymphoid tissue of the rectal mucosa, where it first encounters and infects macrophages.

Many of the most important enteric bacteria that cause infections in humans gain entry to the body through M cells. Invasion by this route delivers bacteria straight to the lymphoid system of the host. Depending on the pathogenicity of the organism and the strength of the host adaptive immune response, infections that breach the gut mucosa may be cleared with little tissue injury, cause a local inflammatory response, or invade the bloodstream or lymphatics and result in a systemic infection. Bacteria that specifically target M cells include Salmonella typhi, the causative agent of typhoid, and S. typhimurium, a major cause of bacterial food-poisoning. These bacteria cause a brisk local and systemic inflammatory response associated with the induction of T_H1-type T-cell responses and antibody responses of the IgG and IgA classes.

10-19. Infection by Helicobacter pylori causes a chronic inflammatory response, which may cause peptic ulcers, carcinoma of the stomach, and unusual lymphoid tumors

There is one exceptional bacterial infection of the stomach, in which the inflammatory and immune response to the organism causes the disease instead of clearing the infection. Helicobacter pylori, which infects many millions of people around the world, adheres to the mucosa of the stomach and causes a local inflammatory response, with the release of IL-8 and the influx of leukocytes. In the majority of those infected there is no overt disease, but up to 5% of infected people make either one of two very different responses to the infection. In some, there is an excessive release of the hormone gastrin, which stimulates acid production and the development of peptic ulcers. In others, chronic inflammation has the opposite effect, leading to atrophy of the stomach, which is associated with reduced acid production and an increased risk of carcinoma of the stomach (Fig. 10.22). Rarely, lymphomas known as MALT lymphomas, because of their origin from the mucosa-associated lymphoid tissue, arise from the B lymphocytes that accumulate in the chronic inflammatory lymphoid infiltrates of the stomach. These are very extraordinary tumors, because some of them, despite being monoclonal proliferations with a transformed phenotype, are still dependent on antigenic or other inflammatory stimulation by H. pylori. These tumors may regress if the H. pylori infection is effectively eliminated by antibiotics alone.

Figure 10.22

Helicobacter pylori infects the stomachs of many millions of people. In some, it stimulates the G cells of the stomach to secrete gastrin, which stimulates excess acid production by the stomach, causing peptic ulceration. In others, it causes chronic (more...)

10-20. In the absence of inflammatory stimuli, the normal response of the mucosal immune system to foreign antigens is tolerance

We have seen that there are two possible and opposite outcomes of exposure to foreign antigens entering through the mucosa of the gut. These are tolerance in the case of food antigens, which is contrasted with a vigorous antibody and T-cell response after exposure to pathogens. The essential difference between antigenic challenge by food compared with that by pathogens is that the pathogens cause inflammation, whereas food does not. Both the antigens within food and the antigens within pathogens are presented by antigenpresenting cells to T lymphocytes, but the contexts in which these two sources of antigen are presented are quite different.

Three different responses of T cells to the presentation of peptides derived from foods and other antigens delivered via the mucosa may account for the phenomenon of oral tolerance. The first is the deletion of antigen-specific T cells by the induction of apoptosis, which has been found to occur in experimental animals in response to oral intake of very large, and probably nonphysiological, doses of antigen. This is probably not the most important mechanism of oral tolerance, although it may contribute.

The second response is anergy, in which T cells presented with peptide in the absence of co-stimulatory signals become refractory to further stimulation with antigen (see Section 8-11). The development of anergy in response to a food antigen was demonstrated by feeding ovalbumin to mice that had large numbers of T cells carrying a transgenically expressed T-cell receptor for an ovalbumin peptide epitope (see Appendix I, Section A-46). Following feeding of ovalbumin, T cells bearing the transgenic T-cell receptor could still be detected, but these were totally refractory to further stimulation by ovalbumin in vitro and in vivo, even when ovalbumin was injected systemically together with an adjuvant (Fig. 10.23).

Figure 10.23

T-cell anergy following feeding of antigen. Mice were injected with CD4 T cells bearing a transgenic receptor specific for an ovalbumin peptide. Two days later they were fed ovalbumin or a control protein. Four days later mice were injected with the relevant (more...)

The third response involves the development of regulatory T cells, which can actively suppress antigen-specific responses following rechallenge with antigen. One subset of T cells has been described that produces IL-4, IL-10, and TGF-β on stimulation with antigen. These cells have been called T_H3 cells. A similar population secretes TGF-β in an IL-10-dependent manner and has been named T_R1 (T regulatory 1 cells). This pattern of cytokine secretion in response to antigen-specific stimulation inhibits the development of T_H1 responses and is associated with low levels of antibody and virtually absent inflammatory T-cell responses. The γ:δ T cells that are abundant throughout the mucosal immune system may also have a role in oral tolerance, because tolerance appears to be reduced in mice lacking this subset of T lymphocytes.

Antigen-specific suppression is a form of oral tolerance that can be transferred experimentally to recipient animals by lymphocytes derived from animals that have been fed antigen. When an animal that has been injected with such lymphocytes is exposed to the same antigen for the first time, these regulatory cells respond to the antigen and inhibit the responses of naive T cells in the recipient animal. This contrasts with anergic T cells, which cannot transfer oral tolerance following transfer to a naive animal.

In order to understand the phenomenon of oral tolerance, it is essential to understand how orally delivered antigens are presented to T cells. Two routes of antigen presentation of soluble food antigens have been characterized that may induce T-cell responses favoring tolerance rather than immune activation. The first is presentation of soluble food antigens by the antigen-presenting cells of the gut and other peripheral lymphoid organs. In the absence of inflammatory stimuli, antigen presentation by dendritic cells favors the induction of tolerance rather than T-cell activation. Dendritic cells in Peyer's patches have been shown to express IL-10 and IL-4, in contrast to similar cells in peripheral lymph nodes which express IFN-γ and IL-12. However, this heterogeneity of cytokine responses does not fully explain tolerance to food antigens. These may be detected in the bloodstream after feeding and there is evidence that the induction of tolerance to food antigens takes place in lymph nodes and spleen as well as in the mucosal lymphoid system. The second possible route of presentation of food antigens is by the enterocytes of the gut, which express MHC class I and MHC class II molecules in the absence of co-stimulatory molecules and thus may induce anergy on presenting antigens to intraepithelial lymphocytes.

We will discuss each of these mechanisms of tolerance further in Chapter 13, where we consider how the loss of tolerance to self tissues may contribute to the development of autoimmune disease. As we will see in Chapter 14, one of the strategies for treating allergy and autoimmune disease is to attempt to manipulate the nature of the antigen-specific response to stimulate T cells with the properties of such regulatory T cells.

Summary

The immune system can be divided into a series of functional anatomical compartments, of which the two most important are the peripheral lymphoid system made up of the conventionally studied spleen and lymph nodes, and the mucosal lymphoid system. Specific homing mechanisms for lymphocytes to each of these compartments serve to maintain a separate population of lymphocytes in each. The mucosal surfaces of the body are highly vulnerable to infection and possess a complex array of innate and adaptive mechanisms of immunity. The adaptive immune system of the mucosa-associated lymphoid tissues differs from that of the rest of the peripheral lymphoid system in several respects. The types and distribution of T cells differ, with significantly greater numbers of γ:δ T cells in the gut mucosa compared with peripheral lymph nodes and blood. The major antibody type secreted across the epithelial cells lining mucosal surfaces is secretory polymeric IgA. The mucosal lymphoid system is exposed to a vast array of foreign antigens from foods, from the commensal bacteria of the gut, and from pathogenic microorganisms and parasites. No immune response can normally be detected to food antigens. Indeed, soluble antigens taken by mouth may induce antigen-specific tolerance or antigen-specific suppression. In contrast, pathogenic microorganisms induce strong protective T_H1 responses. It is an important challenge to understand these contrasting specific immune responses. The key distinction between tolerance and the development of powerful protective adaptive immune responses is the context in which peptide antigen is presented to T lymphocytes in the mucosal immune system. In the absence of inflammation, presentation of peptide to T cells by MHC molecules on antigen-presenting cells occurs in the absence of co-stimulation. By contrast, pathogenic microorganisms induce inflammatory responses in the tissues, which stimulate the maturation and expression of co-stimulatory molecules on antigen- presenting cells. This form of antigen presentation to T cells favors development of a protective T_H1 response.