Introduction

The gastrointestinal (GI) tract is the largest immunologic organ in the body. The small intestine itself has the largest surface area in the GI tract due to structural features including villi and microvilli. The purpose of this extensive surface is to facilitate nutrient absorption from ingested foods. From the stomach to the rectum, a single layer of columnar epithelial cells separates the external environment of the gastrointestinal lumen from the body proper. The lumen contains a myriad of microorganisms and dietary proteins. The challenge from an immune perspective is to guard the extensive surface area of the GI tract from breaches by microorganisms, in particular pathogenic microorganisms. In the small intestine, the main antigenic load is from ingested food. Along the proximal to distal axis, the food antigen load decreases as it is digested and absorbed, but the microbial load increases. In the large intestine, there are 10 ¹⁰ –10 ¹² organisms per gram of dry luminal contents. The intestinal immune system must remain nonreactive or tolerant to antigens from food or commensal flora, yet retain the ability to mount a protective immune response to enteropathogens. This function is accomplished by the gastrointestinal associated lymphoid tissue (GALT) that has adapted to its unique environment.

Structure of the Gastrointestinal Associated Lymphoid Tissue (GALT)

The gastrointestinal tract has several types of organized lymphoid tissue comprising the GALT. Underlying the intestinal epithelial layer is a loose connective tissue stroma called the lamina propria (LP), containing a resident population of CD4 and CD8 T lymphocytes, plasma cells, macrophages, dendritic cells (DCs), eosinophils and innate lymphoid cells (ILCs). The LP of the small and large intestine is drained via lymphatics that empty into the mesenteric lymph nodes (MLN). Migratory DCs capture antigens in the LP and deliver those antigens to the MLN. The MLN is a typical secondary lymph node with organized B cell follicles and paracortical T cell areas. Peyer’s patches (PP) are lymph nodes found within the mucosal wall and have direct access to the intestinal lumen. PP are large and visible by eye as bulges on the serosal surface of the intestine. In addition to PPs, the intestine contains smaller structures called isolated lymphoid follicles (ILF), each containing a single B cell follicle with an overlying follicular epithelium. Mouse intestine contains an abundance of these small organized structures. The precursor of the ILF is the cryptopatch, comprised of clusters of lymphoid tissue inducer cells. Bacterial signals promote the enlargement of the ILFs through the recruitment of B cells. The MLN, PP and ILF comprise the inductive sites in the gastrointestinal tract. In addition, T lymphocytes are normally found between epithelial cells (intraepithelial lymphocytes, or IELs). IELs are predominantly CD8 ⁺ T cells in the small intestine and have an oligoclonal repertoire. The immune cells of the LP and the IELs comprise the effector cells of the GALT and are responsible for both the maintenance of tolerance to harmless antigens and immunity against pathogens. Figure 40-1 shows a schematic of the structure of the GALT.

Structure of the GALT. Organized lymphoid structures in the gastrointestinal tract include the mesenteric lymph node ( MLN ) that drains the mucosa via the lymphatics. Peyer’s patches and isolated lymphoid follicles ( ILF ) are present within the mucosal wall and are covered by specialized antigen sampling cells called M cells. Dendritic cells ( DC ) continuously migrate from the lamina propria into the MLN to present antigen. Organized lymphoid structures contain B cell follicles surrounded by T cell areas. Within the lamina propria are scattered T cells, B cells, macrophages ( MP ) and DCs. A population of intraepithelial lymphocytes ( IEL ) is also found through the gastrointestinal tract. A single layer of columnar epithelium separates the mucosal immune system from the luminal contents.

Mechanisms of Antigen Sampling in the Intestinal Mucosa

Food protein antigens are digested by a combination of gastric acid, pancreatic proteases and brush border peptidases, resulting in a mixture of amino acids and di- and tri-peptides, which are then absorbed by the intestinal epithelial cells. Dietary antigens that escape proteolysis in the lumen can be taken up by the intestine in various ways. Soluble antigens are taken up by enterocytes via fluid phase endocytosis by the microvillous membrane, transported in small vesicles and larger phagosomes, and then digested when lysosomes combine to form phagolysosomes. Intact molecules that remain after digestion are deposited in the extracellular space by exocytosis. As a result, approximately 2% of intact proteins reach the intestinal lymph and portal circulation under physiologic conditions. Goblet cells have also been identified as portals of uptake of soluble antigens, delivering these antigens to subepithelial DCs.

Particulate antigens are poorly sampled by enterocytes, where the glycocalyx provides a barrier to even relatively small particles. PPs are overlaid by specialized epithelial cells, referred to as membranous or microfold (M) cells. M cells that overlay PPs have a reduced glycocalyx layer and shortened microvilli that allow for binding of particles that cannot adhere to enterocytes. In addition, they have a sparse flattened cytoplasm and enhanced endocytic activity, allowing rapid antigen delivery into the subepithelial dome region of the Peyer’s patch. The subepithelial dome is rich in DCs that process and present antigen to T lymphocytes or transfer antigen to B lymphocytes.

The intestinal mucosa is densely populated with a network of DCs that function to acquire antigen, migrate to T cell areas of lymph nodes, and present antigen to naïve T cells. They can acquire this antigen after it has been transcytosed across enterocytes, M cells or goblet cells as outlined above. In addition, mononuclear phagocytes with dendritic morphology have been shown to extend dendrites between enterocytes into the intestinal lumen. These dendrites are functional, as antigen sampling extensions can acquire luminal bacteria. This mononuclear phagocyte subset is more similar to macrophages than DCs by transcriptional profiling and does not migrate to the lymph nodes under steady-state conditions. These antigen-sampling resident macrophages provide antigen to DCs, which are the cells that carry antigen to the draining lymph nodes and can prime naïve T cells. Figure 40-2 outlines these major pathways of antigen uptake.

Antigen uptake in the intestine. In the intestinal villus (left), antigen can reach subepithelial dendritic cells ( DC s) by several routes. Macrophages ( MP ) can send extensions between enterocytes to sample particulate antigens directly from the lumen and transfer these antigens to DCs. Alternatively, soluble antigens can be taken up by fluid phase endocytosis by enterocytes and be deposited in the lamina propria for uptake by macrophages or DCs. Goblet cells can also function as conduits for delivery of antigen to DCs. DCs migrate from the lamina propria to the mesenteric lymph node ( MLN ), where antigen can be presented to naïve T cells. In the Peyer’s patch (right), M cells are specialized for uptake of particulate antigens that are delivered to DCs in the subepithelial space. These DCs can then traffic to the interfollicular areas of the Peyer’s patch for presentation to T cells (green) or interaction with B cells (blue).

Antigen transport across the intestinal barrier has been shown to be altered by immunization or allergic sensitization, either enhancing or inhibiting uptake. As discussed in detail later, IgA-, IgG- and IgE-facilitated antigen sampling have been documented in the intestinal mucosa.

Normal Immune Response to Sampled Antigens in the Intestine

Food contains a diverse mix of antigens that are capable of stimulating immune responses if administered by other routes. Administration of antigens by the oral route is one of the most effective means of inducing tolerance. The process of oral tolerance was first defined experimentally in laboratory rodents that displayed systemic unresponsiveness to immunization with antigens to which they had previously been fed. Tolerance can be transferred to a naïve animal by transferring T lymphocytes, demonstrating that this is an active immune-mediated process. Oral tolerance has also been demonstrated in humans by feeding a neo-antigen prior to immunization with that antigen.

Several different phenotypes of regulatory T cells have been shown to contribute to oral tolerance induction to fed antigens, including CD8 ⁺ T _REGS , and different subsets of CD4 ⁺ T _REGS . Of the CD4 ⁺ T _REGS , T helper 3 (Th3) cells and Foxp3 ⁺ CD25 ⁺ cells have been described as contributing to oral tolerance to fed antigens. Th3 cells produce TGF-β together with IL-4 and IL-10, can be identified by surface expression of latency-associated peptide (LAP), and were first described using myelin basic protein (MBP)-specific CD4 ⁺ T cell clones from the mesenteric lymph nodes of MBP-fed mice. Adoptive transfer of these cells from MBP-fed mice suppressed experimental allergic encephalomyelitis, an experimental model of multiple sclerosis. Inhibition of TGF-β with neutralizing antibodies can abrogate tolerance responses in this model. CD25 ⁺ Foxp3 ⁺ T _REGS include both thymic-derived natural T _REGS (nT _REGS ) and induced T _REGS (iT _REGS ) generated in the periphery. There are data both for and against a role for nT _REGS in oral tolerance. Feeding mice induces a population of antigen-specific T regulatory cells that express similar markers as natural T regulatory cells (CD25 ⁺ , Foxp3 ⁺ and CTLA-4) and mediate regulatory responses via TGF-β but not IL-10. Specific depletion of all Foxp3 ⁺ T cells, followed by a rest period to allow nT _REGS to rebound, provides supporting evidence that iT _REGS are the most critical regulatory population mediating oral tolerance. Tr1 cells are another regulatory subset that secrete IL-10. Although several studies indicate that IL-10 is dispensable for the induction of oral tolerance to foods, IL-10 is critical for the suppression of inflammatory responses initiated by the intestinal microbiota.

Naïve T cells must be instructed to become regulatory in phenotype rather than becoming effector Th1, Th2 or Th17 cells. There is growing evidence that the milieu in which food antigens are presented to the naïve T cells by DCs is a critical factor promoting the development of regulatory T cells in the intestine.

The Role of Intestinal Dendritic Cells in Tolerance and Immunity

An abundant network of DCs surrounds the epithelium and fills the lamina propria. The role of DCs in both tolerance and immunity in the intestinal mucosa was first explored using the growth factor FMS-like tyrosine kinase 3 ligand (Flt3L) to expand the DC population in mice. Flt3L treatment enhanced tolerance responses to an innocuous antigen when it was delivered orally. In addition, when antigen was administered with adjuvant to elicit protective immunity, DC expansion also enhanced that response. These studies show that, like elsewhere in the body, DCs are essential for the initiation of an active CD4 ⁺ T cell response, whether regulatory, or effector Th1, Th2 or Th17 in nature.

In the lamina propria, DCs bearing the marker CD103 are derived from DC progenitors and constitutively express CCR7, a chemokine receptor required for lymph node homing. These CD103 ⁺ DCs are required for the generation of oral tolerance. CD103 ⁺ DCs from the mesenteric lymph node preferentially induce the development of Foxp3 ⁺ regulatory T cells that express chemokine receptors and adhesion molecules that support homing back to the intestine. This resulting phenotype is induced by release of retinoic acid and expression of TGF-β by the DCs. The regulatory activity of these DCs is modified by environmental factors. Local tissue factors, including the cytokine GM-CSF and mucins produced by the epithelium, enhance the regulatory function of CD103 ⁺ DCs. In contrast, administration of the mucosal adjuvant cholera toxin can modify these CD103 ⁺ DCs into immunogenic rather than tolerogenic DCs.

Macrophages Have a Regulatory Phenotype in the Intestine

Macrophages are the most abundant phagocytic cells resident in the small and large intestinal LP. They form a band of cells directly beneath the surface epithelium distinct from the localization of DCs. Like DCs, macrophages can take up antigen and present it to T lymphocytes; however macrophages are not migratory, and do not reach lymph nodes for interaction with naïve T cells. Macrophages from the human intestine are adept at both phagocytosis and killing of microbes after uptake. Therefore, they function as a secondary barrier after the epithelium in preventing the influx of microbes from the gut lumen into the body proper. Macrophages from the intestinal mucosa of mouse and human are nonresponsive to microbial stimuli compared to monocytes or macrophages from other sites. This was shown to be due to TGF-β released from the intestinal stroma in humans or autocrine effects of IL-10 in the mouse. IL-10 is clearly important for immune homeostasis in the intestine because mice lacking IL-10 develop spontaneous colitis, as do mice lacking IL-10 receptor specifically in macrophages. Although resident macrophages do not have access to naïve T cells to initiate tolerance to fed antigens, they play an important role in the expansion of T _REGS during the generation of tolerance. CX ₃ CR1 ⁺ macrophages extend dendrites across the epithelium to interact with luminal contents. In mice genetically deficient in CX ₃ CR1, these dendrites cannot form, and oral tolerance to fed antigens is impaired. The role of gastrointestinal DCs and macrophages in the generation of tolerance and immunity is outlined in Figure 40-3 .

Function of antigen presenting cells in the intestine. A subset of DCs in the lamina propria express the marker CD103. These are migratory DCs that acquire antigen and traffic to the MLN. In normal conditions, CD103 ⁺ DCs prime naïve T cells and B cells to generate a gut-homing phenotype expressing CCR9 and α4β7. CD103 ⁺ DCs promote the generation of regulatory T cells expressing Foxp3, and promote IgA secretion from B cells. The T _REGS migrate to the lamina propria, where they interact with IL-10- ⁺ expressing CX ₃ CR1 ⁺ macrophages and are expanded in response to antigen. Mucosal factors such as GM-CSF and mucins (Muc2) promote the regulatory phenotype of DCs, while the presence of the mucosal adjuvant cholera toxin induces CD103 ⁺ DCs to up-regulate OX40L and induce Th2 skewing from responder T cells.

Homing of Lymphocytes to the Intestine

Lymphocytes that differentiate in the inductive sites of the GALT into effector or regulatory T cells or IgA-producing B cells home preferentially to the intestinal lamina propria to carry out their function. Targeted homing of lymphocytes is determined by expression of specific adhesion molecules and chemokine receptors. Homing to the intestine is mediated by the adhesion molecule α4β7 binding to MAdCAM on high endothelial venules. In addition, the chemokine receptor CCR9 promotes migration to the small intestine where constitutive expression of the ligand CCL25 is found. Migration to the large intestine is promoted by the chemokine receptor CCR10 binding to its ligand CCL28. The intestinal migratory phenotype is imprinted on lymphocytes by stromal cells, as well as the CD103 ⁺ population of DCs in the MLN, via a retinoic-acid dependent mechanism.

Microbial Regulation of Mucosal Immunity

The gastrointestinal mucosa is often described as being in a state of ‘physiologic inflammation’. In mice, the genetic deletion of a wide range of immunoregulatory genes results in colitis, a phenotype that is commonly absent if the mice are raised in germ-free conditions. Although microbial signals can induce inflammation and tissue damage if not appropriately controlled, these signals are also necessary for the health of the organism. Our intestinal microbiota contributes locally to the digestion of nutrients, regulates the epithelial barrier, and is essential for maturation of the mucosal immune system in addition to having systemic effects on metabolism and the neuroendocrine system.

Mice reared under germ-free conditions have poorly developed lymphoid structures in the gastrointestinal tract, and colonization with a single strain of bacteria is sufficient to induce significant changes in gene expression in the intestinal epithelium and maturation of the mucosal immune system. Different organisms have differing effects on the development of effector and regulatory responses in the gastrointestinal tract. Segmented filamentous bacteria that are in intimate contact with mouse intestinal epithelium promote the development of Th17 responses in the gastrointestinal tract. Bacteroides fragilis and several Clostridia strains promote the development of regulatory T cells. Humoral immunity is also significantly regulated by the commensal microbiota. Germ-free mice have reduced levels of all isotypes of antibodies with the exception of IgE, which is uniquely elevated. IgA production is carefully regulated by the commensal microbiota, and the specificity of the IgA response adapts to respond to changes in intestinal microbial populations.

Cellular and humoral immune responses in the gastrointestinal tract are influenced by the commensal microbiota, and it is therefore not surprising that the microbiota is a key factor in the development of tolerance or allergy in the gastrointestinal tract. Germ-free mice have a reduced capacity for the generation of oral tolerance, and conversely show increased susceptibility to allergic sensitization through the oral route. Colonization with normal microbiota, or with Clostridia strains, results in the expansion of regulatory T cells and the suppression of allergic sensitization. Toll-like receptor (TLR)2, TLR4 and TLR9 are host receptors that have been shown to contribute to tolerogenic effects of the intestinal microbiota. Metabolic products of the microbiota, such as short chain fatty acids, also promote a tolerogenic tone in the intestine. Although the microbiota has generally been shown to be tolerogenic, susceptibility to food allergy has been shown to be transmissible in mice, suggesting that there may be pro-allergenic bacterial strains as well as pro-tolerogenic strains, although these have yet to be identified.