cancer immunotherapy || mucosal immunity

CHAPTER 6

Mucosal Immunity
Cathryn Nagler, Taylor FeehleyCommittee on Immunology, Department of Pathology, The University of Chicago,Chicago, IL USA
I. OVERVIEWUnique structural and functional adaptations are required at the mucosal surfaces that formthe interface between the external environment and the rest of the body. The anatomy of these

barriers, their specialized mechanisms of protection, and their contribution to immunehomeostasis will be discussed in this chapter.

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II. MUCOSAL SURFACES ARE THE MAJOR PORTALS OF ENTRYFOR ANTIGENThemucus-covered epithelia of the body, namely the respiratory, digestive, and urogenital tracts,

cover an enormous surface area and form the boundary between the external environment

and the underlying tissue. Mucosal surfaces are therefore major routes of antigen entry andare protected by a wide variety of mechanisms, both mechanical and immunological [1].

The antigens that enter through mucosal sites are numerous and varied, including airborne

antigens such as fungal spores, pollen, and dust and ingested antigens such as food and bacteria.The responses mounted at mucosal surfaces depend on the antigens themselves as well as the

environment in which these antigens are sensed. These different responses can be broadly

divided into tolerogenic or inflammatory responses; maintaining a balance between toleranceand immunity is vital for the health of the host.

Although some antigens are processed and presented within the mucosal immune system,others cross the epithelial barrier and traffic throughout the body. In the gut, many antigens

gain access through organized lymphoid structures, known as Peyer’s patches, which are located

in the small intestine (Figure 6.1). Mucosal surfaces are connected by the lymph and/or blood tolymphoid organs such as lymph nodes. The mesenteric and mediastinal lymph nodes drain

the gut and airways respectively via afferent lymphatics (Figure 6.1) [1]. The ability of antigen

that enters at the mucosa to reach distal sites allows for cross talk between the mucosal andsystemic immune systems and the establishment of systemic responses.

Throughout this chapter, emphasis will be placed on the mucosal surfaces of the intestine and

on the responses mounted in the gut associated lymphoid tissue (GALT). However, many ofthe principles to be discussed can be applied to other mucosal sites as well.

III. EPITHELIAL BARRIERThe epithelial barrier at mucosal surfaces typically consists of a single-cell layered columnar

epithelium (with a few notable exceptions, e.g., the squamous epithelium of the esophagus) [2].This seemingly simple barrier is reinforced by a number of specialized protective adaptations,

each of which will be discussed below. These include (1) intercellular tight junctions that restrict

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FIGURE 6.1Antigens enter through mucosal surfaces. Most antigens are either inhaled or ingested and enter the body through the

mucosal surfaces of the respiratory and gastrointestinal tracts. In the gut, many antigens gain access through Peyer’spatches, aggregations of lymphoid follicles found primarily in the distal ileum of the small intestine. Afferent lymphatics

transport antigens from the intestinal mucosa to the draining mesenteric lymph nodes for presentation to naı̈ve T cells

(inhaled antigens are presented in the mediastinal lymph nodes that drain the airways). Antigen sensitized T cells migrateout of the draining node through efferent lymphatics and ultimately enter the systemic circulation through the thoracic duct.(From ref. 1, with permission).

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SECTION 1Principles of Basic Immunology

the passage of even small molecules between the cells of the epithelium, (2) secretory IgA,a mucosa-associated, structurally stabilized form of immunoglobulin which coats all mucosal

surfaces, (3) defensins, antimicrobial peptides secreted by epithelial Paneth cells which act as

natural antibiotics, and (4) the mucus layer itself.

The selectively permeable epithelial barrier is regulated by both tight junctions and adherens

junctions located at the apical intercellular space (Figure 6.2) [2]. Tight junctions are formedthrough cellecell interactions of proteins, such as occludin and members of the claudin

family. Adherens junctions confer stability to the epithelial barrier and are found just basal to

the tight junctions. The molecules that make up the tight and adherens junctions interact withactin and myosin rings adjacent to the junctions, allowing them to regulate contraction,

tightening or relaxing the contact between neighboring cells and serve to provide a selectively

permeable barrier that can regulate the passage of solutes based on both size and charge [2,4].These junctions ensure that cellecell contact, and thus barrier integrity, are maintained under

E-cadherin

ActinMyosinOccludin

Desmoglein

Keratin

Desmoplakin

Desmocollin

-catenin

-catenin

ClaudinsZ01

MLCK

Tightjunction

Desmosome

Adherensjunction

Microvilli

Mucus

Goblet cell

Unstirred layer

Epithelial cells

Intraepitheliallymphocyte

Basementmembrane

Plasma cell

Apicaljunctional complex

Lamina proprialymphocyte

(a) (b)

FIGURE 6.2The epithelial barrier. a. The intestinal mucosa is lined by single cell layered columnar epithelium. Goblet cells, whichsynthesize and release mucin, as well as other specialized cell types, are contained within the epithelial layer. The tightjunction, a component of the apical junctional complex, seals the paracellular space between epithelial cells. Intraepitheliallymphocytes reside above the basement membrane, subjacent to the tight junction. Lymphocytes are readily detectable

in the lamina propria. b. An electron micrograph, and the corresponding line drawing, of the junctional complex of an

intestinal epithelial cell. Just below the base of the microvilli the plasma membranes of adjacent cells seem to fuse at the tight

junction, where claudins, zonula occludens 1(ZO1), occludin and F-actin interact. E-cadherin, a-catenin, b-catenin,catenind1 and F-actin interact to form the adherens junction (Adapted from ref. 2, with permission).

CHAPTER 6Mucosal Immunity

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homeostatic conditions. Two kinds of transport across the epithelial barrier have been
described: paracellular and transcellular [2,5]. Paracellular transport is passive and involves thepassage of ions through the space between epithelial cells while transcellular transport is active

and harnesses the energy of electrochemical gradients across the epithelium into the lamina

propria for transport of other nutrients across the barrier. Passive, paracellular transport allowsproteins or polysaccharides to pass through the barrier between cells down their concentration

gradients. Due to the size and charge selectivity of the epithelial barrier, however, nothing as

large as a bacterium or whole cell can pass freely into the lamina propria. Certain stimuli,including infectious pathogens and environmental toxins may, however, disrupt the barrier

allowing foreign antigens to enter the lamina propria. For example, proinflammatory cyto-

kines such as TNFa [6] and IFNg [7] can alter the conformation of the tight junctions to inducebarrier dysfunction and increase permeability [2].

Another protective mechanism at mucosal surfaces is the production of secretory IgA [8e10].IgA exists in both a monomeric and dimeric form; the monomeric form is found predomi-

nantly in serum while dimeric IgA is found at epithelial barriers, particularly in the gut.

Dimeric IgA is held together by J (joining) chain, a small protein that links the Fc portions ofthe two immunoglobulinmolecules to form a dimer. The production of the J chain seems to be

limited to mucosal surfaces, making the dimeric form of IgA a unique feature of these sites.

Secretory IgA is produced at the phenomenal rate of 40e60 mg/kg body weight/day [1]. Itssecretion into the lumen is regulated by the polymeric Ig receptor (pIgR). This receptor allows

dimeric IgA to traffic from the basolateral to the luminal surface, against what might be

considered the normal flow of transport across the epithelium. The pIgR is a transmembraneprotein that binds to the J chain. Upon binding to the pIgR, IgA traffics in endosomes from the

basolateral surface to the apical surface where it is released into the lumen. Release requires

the cleavage of pIgR from the membrane, leaving only a small peptide bound to the J chaincalled the secretory component (SC). In the lumen SC prevents proteolytic degradation of IgA.


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IgA’s utility at mucosal surfaces is largely related to its ability to neutralize and sequesterantigen. Some of the IgA produced under homeostatic conditions is generated by T-cell-

independent responses from B1 B cells; this IgA is low affinity because it does not undergo

somatic hypermutation in a germinal center [9,10]. Much of this T-independent IgA responseis thought to be bacteria specific and broadly reactive against the microbiome because its

production is induced upon colonization of germ free mice with intestinal commensalbacteria [10,11,12]. High affinity, T-dependent IgA responses to the commensal microbiota

have also been documented [10]. In addition to guarding the epithelium from antigen entry,

IgA can bind bacteria or bacterial antigens that do gain access to the lamina propria andtransport them back into the lumen. In this role IgA functions as an antigen “sump pump,”

constantly removing potentially damaging stimuli and helping to control inflammation. IgA

can also bind to bacteria in the lumen; this may be important to limit overgrowth of themicrobiota as well as block invasion by pathogenic microbes. Indeed, it has recently been

appreciated that, by regulating the composition of gut bacterial communities, IgA plays

a critical role in the maintenance of immune homeostasis [12].

A third specialized adaptation for the protection of the epithelial barrier is the production of

antimicrobial peptides [13,14]. Specialized epithelial cells called Paneth cells, which are

located in the crypts of the intestinal villi, secrete these peptides. Antimicrobial peptideproduction is constitutive, and its mode of action is antigen nonspecific, allowing for broad-

spectrum antibacterial protection. Paneth cell-secreted peptides provide an extra layer of

protection that prevents bacteria from invading the crypts, thereby guarding the site ofepithelial cell regeneration. There are several types of antimicrobial peptides, including

defensins, trefoil peptides, C-type lectins, and phospholipases [15]. Given their varied modes

of action, these peptides serve as an important first line of defense against certain intestinalpathogens and can limit aberrant expansion of the microbiota.

The goblet cell is another specialized epithelial cell type; these very large cells are distributedthroughout the epithelium and are responsible for secreting mucus (see Figure 6.2). Mucus

covers the apical face of epithelial cells and is a complex polysaccharide matrix that forms

a protective barrier between the epithelial surface and the gut lumen [2]. The mucus layer isparticularly important in separating the microbiota from the epithelium, effectively parti-

tioning microbial communities [16]. Although certain bacteria can be associated with enter-

ocytes, most remain embedded in mucus or free in the lumen, limiting their ability to interactdirectly with the epithelium and trigger inflammatory responses.

Secretory IgA, antimicrobial peptides and mucus are specialized adaptations of the mucosathat provide reinforcement to the physical barrier of the epithelium itself. The combined effect

of these defense mechanisms is a well-maintained divide between the lumen and the lamina

propria that is not often breached under conditions of health and homeostasis.

IV. INDUCTIVE AND EFFECTOR SITES IN THEMUCOSA-ASSOCIATED LYMPHOID TISSUEUnique structural features of the intestine contribute to its role as an immunological “organ.”

The intestinal epithelium has a distinct organization and forms folds called villi; these villi actto increase the surface area of the intestine to aid in the absorption of water and nutrients.

Intestinal villi contain primarily columnar epithelial cells, also called enterocytes, which have

finger-like protrusions on their apical/luminal side called microvilli. The microvilli furtherincrease the surface area (and corresponding absorptive capacity) of each enterocyte. At the

ends of these microvilli are glycoproteins that form a brush border which, along with the

mucus layer, creates a thick glycocalyx. Within the small intestine, however, there are cells thathave a different morphology and are interspersed between enterocytes, typically as part of

organized mucosal lymphoid structures. These cells, called M cells, lack the brush border


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glycocalyx and are specialized to acquire and transcytose antigen from the gut lumen to thelamina propria of the intestine [17,18]. M cells facilitate transport of antigen or material that is

larger than that which passes by paracellular or transcellular transport, including whole

microbes [17]. Thus, these specialized cells regulate the amount of antigen that gains access tothe mucosa and provide a controlled route for antigen trafficking.

Antigen-presenting cells (APCs) of the lamina propria often reside below the M cells wherethey have access to newly imported antigens [19]. The population of cells in the lamina

propria that can phagocytose and present antigen is highly heterogeneous. Some of these APC

are migratory dendritic cells (DC) and can carry antigen to the mesenteric lymph node forpresentation to naı̈ve T cells at that site. The migratory DC subset is often identified by its

expression of the surface markers CD11c and CD103 [20]. Other DCs are tissue-resident and

sample antigen directly from the intestinal lumen by extending dendritic processes betweenepithelial cells [21,22]. These DCs are identified by their expression of CD11c and CX3CR1,

a chemokine receptor, on their cell surface. A third class of APC that is present in the intestinal

lamina propria is a population of nonmigratory, CX3CR1þ macrophages. These macrophages,

although partially defined by the same surface receptor as the DCs, are also CD11bþ and

F4/80þ, do not extend processes and are hypothesized to be potent producers of IL-10, an

immunosuppressive cytokine important for limiting inflammation and maintaining regula-tory T-cell populations [23]. They do not seem to present antigen effectively to naı̈ve T cells but

process antigen efficiently, suggesting a predominant role in the clonal expansion of effector

cells that home back to the lamina propria [19].

In addition to epithelial cells (which are derived from the stromal compartment) and APCs,

mucosal surfaces are associated with a wide range of other hematopoietic cells. There aremore antibody-producing B cells in mucosal tissues than in the spleen and lymph nodes

combined [2]! One abundant and unique population of cells is the intestinal intraepithelial

lymphocytes (IEL). These atypical and nonmigratory T cells reside between enterocytes,above the basement membrane. Expression of a CD8aa homodimer (rather than the CD8ab

heterodimer expressed by CD8þ T cells at other sites) correlates with the activated/memory

phenotype of IEL. Indeed many IEL are constitutively cytolytic directly ex vivo [24,25]. TheIEL population consists largely of gd T cells (referring to the structure of their T-cell receptor,

TCR), but there are also ab TCRþ IELs. IELs act as sentinels to detect and repair damaged

epithelium [26]. They have also been implicated in protection against colitis as well asintestinal infections [26].

The intestine has gut-associated lymphoid tissue (GALT), the lungs have bronchus-associatedlymphoid tissue (BALT), and the nasal passages have nasal-associated lymphoid tissue (NALT).

Within these dedicated lymphoid tissues, there are both inductive sites and effector sites.

Antigen-specific responses are initiated at inductive sites. Effector sites are populated withmemory effector T and B cells which man the barriers, poised to respond quickly and effec-

tively to subsequent antigen challenge [1]. Trafficking between inductive and effector sites is

regulated by highly specific homing receptors. Each mucosal tissue has a zip code-like addressof chemokine and adhesion/addressin molecules that direct lymphocytes back to these

sites upon infection or stimulation. For the gut, homing is directed by CCR9/CCL25 and

a4b7/MADCAM1 engagement, while in the lung homing is dependent on CCR10/CCL28 anda4b1/VCAM1 receptor/ligand interactions [27,28]. Given these distinct molecular signatures

imparted on lymphocytes as part of their education, cells can migrate from inductive to

effector sites and from systemic circulation back to a mucosal surface. Another factor thatdirects lymphocyte trafficking and education at mucosal surfaces is the presence of vitamins

and their metabolites. This is of particular importance in the gut. Dietary vitamin A can be

converted to retinoic acid (RA) by DCs that possess the retinaldehyde dehydrogenase(RALDH) enzyme; when these DCs interact with cognate T cells in the presence of RA, the

T cells are induced to upregulate gut homing receptors [29]. A similar pathway operates in


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the skin; DCs convert sunlight-induced vitamin D3 to its active form 1,25(OH)2D3, whichinduces the expression of CCR10 on activated T cells, allowing their migration into the

epidermis [30].

Using the gut as a model mucosal surface again, inductive sites include the mesenteric

lymph node, which drains the lymph and trafficking lymphocytes from the lamina propria,

and two kinds of tertiary lymphoid organs, the Peyer’s patches (Figure 6.3) and the isolatedlymphoid follicles (ILFs) [1]. Peyer’s patches and ILFs are clusters of lymphocytes in the lamina

propria that can support antigen presentation to T cells and the formation of germinal

centers for generation of high affinity antibodies [9,10]. All three of these sites contain antigen-loaded APCs that encounter naı̈ve T cells [10]. When these T cells interact with APC loaded

with their cognate antigen, their TCR is engaged and they differentiate into an effector T cell

(Teff) or a regulatory T cell (Treg). This fate decision is dictated by signals from the APCand the cytokine environment of the inductive site. Cytokines that are of particular

relevance for this fate decision are TGFb, RA, and IL-6. The presence of TGF-b and RA

supports the differentiation of Tregs by upregulating expression of the transcription factor

Tight junctionsM cell

DC

SED

B-cell follicle

T (interfolIicular region)

(a)

LPBT

IELDC

Lumen(b)

FIGURE 6.3The gut associated lymphoid tissue. The gut associated lymphoid tissue contains inductive (Peyer’s patch) and effector(lamina propria) sites. a. The Peyer’s patch contains B-cell follicles similar to those found in lymph nodes. A follicle-

associated epithelium (FAE) lines the dome of the Peyer’s patch. Antigen is transported across the epithelial barrier by

specialized epithelial cells called M cells and by dendritic cells (DCs) that reach into the intestinal lumen. Tight junctions atthe apical surface of epithelial cells maintain barrier integrity. The abundance of DCs and macrophages present in the

subepithelial dome (SED) of the Peyer’s patch facilitates the uptake, processing and presentation of antigen transported

across the epithelium. b. The intestinal villus epithelium contains an unusual population of intraepithelial lymphocytes (IEL),

which reside above the epithelial basement membrane. The lamina propria of the intestinal villi is richly populated with both

lymphoid effector cells (e.g., memory T cells and IgA secreting B cells) and antigen-presenting cells (DC and macrophages).

(Adapted from ref. 1, with permission).


Foxp3 [29,31,32]. By contrast when TGF-b and IL-6 dominate the cytokine milieu, T cellsupregulate another transcription factor, RORgt, which drives the proinflammatory Th17

effector program [33e35]. The balance between these lineages, as well as the other effector

T-cell subsets (Th1, Th2), is important for the maintenance of homeostasis at mucosal surfaces[36] and TGF-b plays a particularly critical role. In addition to regulating the differentiation

of Tregs, TGF-b governs class switching to the IgA isotype [8e10]. Naı̈ve B cells in inductivesites can acquire and present antigen and interact with antigen-experienced T cells in B-cell

follicles. When B and T cells that recognize the same antigen interact, the B cell undergoes

T-dependent class-switch recombination and somatic hypermutation to produce high affinityantibodies. The choice of antibody isotype produced by these B cells is controlled by cytokines.

In the TGF-b rich microenvironment of the GALT, IgA is preferentially produced. T-dependent

IgA is antigen specific, meaning it undergoes somatic hypermutation that results ultimately inhigh affinity antibodies; this is distinctly different from the barrier-protective, low affinity

secretory IgA discussed earlier. Other cytokines drive switching to different isotypes (e.g., IL-4

for IgG1 switching), creating a varied repertoire of high affinity antibodies with differentfunctions. Once antigen-specific T and B lymphocytes have clonally expanded, they can traffic

back to the lamina propria and villus epithelium, which are the effector sites for the gut. When

these cells are reintroduced to their cognate antigen at this effector site, they are engaged again,and can rapidly mount an appropriate immune response.

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V. THE MICROBIOME AND MUCOSAL SURFACESOne major challenge to maintaining homeostasis at mucosal surfaces is the presence of the

microbiome. The microbiome is defined as the community of bacteria, the commensalmicrobiota, that colonize all mucosal sites in the body; there are trillions of individual bacteria

representing up to 1000 unique bacterial species [37]. Although at least 28 bacterial phyla have

been described, in a healthy individual the commensal microbiota is dominated by bacteriafrom only two of these phyla: Bacteroidetes and Firmicutes [38]. On a species level, the

composition of the microbiota varies from site to site and individual to individual. It can alsobe affected by the environment, age of the host and genetics. The commensal microbiota is

essential for the health of the host, providing energy, nutrients, and metabolites that the host is

unable to produce itself, including short chain fatty acids such as acetate and butyrate [39].

The microbiome, however, also presents a major conundrum for the immune system. How

does the host tolerate and promote homeostasis with the microbiota while mounting the

necessary response in the presence of invading intestinal pathogens? Innate immune sensorsincluding Toll-like receptors (TLRs), Nod-like receptors (NLRs), and C-type lectin receptors

(CLRs) detect bacterial surface molecules or nucleic acids [40]. Many bacterial pathogen

associated molecular patterns (PAMPS), including lipopolysaccharide (LPS), peptidoglycan,and flagellin, are common to both pathogens and members of the microbiota. Responses to

the same PAMPS, which trigger inflammation and an adaptive immune response, must be

downregulated to avoid a response to the commensals that inhabit mucosal surfaces. Yet,clearance of pathogens often necessitates an inflammatory response. Different classes of

pathogens elicit different effector responses at mucosal sites; intracellular bacteria primarily

drive Th1 responses, helminth infection drives Th2 responses, and acute bacterial infectionsdrive Th17 responses.

VI. TOLERANCE TO DIETARY ANTIGEN AND THE MICROBIOMEAlthough pathogens typically evoke host protective effector responses, the commensalmicrobiome does not. The exact mechanism for maintaining nonresponsiveness to the

microbiome has not been fully elucidated. Several mechanisms have been proposed, however,

including reduced mucosal responsiveness to major PAMPs such as LPS and alterations in thestructure of PAMPs on commensals that change the signaling pathways they induce [41].


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There is also compelling new evidence for induction of bacteria-specific Foxp3þTregs [42].These regulatory cells are potent producers of the immunoregulatory cytokines IL-10 and TGF-

b, which can both prevent the induction of effector responses or limit their duration.

A final hypothesis is that it is not the mere presence of the microbiota or individual isolated

species, but the composition of the community, the microbiome, that allows the host to

tolerate their presence. The host adaptive immune system, having co-evolved with an entericmicrobiome, requires stimuli from the microbiota in order to promote the generation of

a balanced lymphocyte compartment with both Teff and Tregs [37]. Recent work has

demonstrated that individual species or defined microbial cocktails can have potent immu-nomodulatory effects such as the induction of Tregs or the induction of pro-inflammatory Th1

and Th17 cells. Single species have even been shown to do one or more of these things under

different conditions, and have thus been deemed “pathobionts,” referring both to theirpresence as normal members of the microbiome and their potential to become pathogenic

under conditions of dysbiosis or excessive external stress. Dysbiosis refers to the state in which

the homeostatic balance of the microbiome is lost; it can be caused by a variety of environ-mental factors such as diet, antibiotic use, and pathogen exposure and is also influenced by

host genetics. Prolonged dysbiosis often leads to increased inflammatory responses and may

drive the development of various diseases. This suggests that the homeostasis of the mucosalimmune system is regulated by a balance of activating and inhibitory signals provided by both

the bacteria themselves and the lymphocytes present to regulate them. Given this model of

microbial-immune cross talk, it follows that changes in the microbiota can influence generalhealth and homeostasis.

Along with the microbiome, there is another major class of antigens to which the immunesystemmust become tolerant: food antigens. Mucosal and systemic nonresponsiveness to food

is called oral tolerance. The mechanisms regulating tolerance to dietary antigen are likely to

have both similarities and differences to those regulating tolerance to the microbiome. Theinduction of oral tolerance has been the focus of extensive research and has recently been

found to be more complicated than originally appreciated. Briefly, food antigen is taken up by

APCs in the intestine and presented to naı̈ve T cells, which then are educated not to respond tothis antigen on reexposure [43]. Since these food antigens are acquired in an environment

that favors anti-inflammatory responses, T cells may undergo anergy or deletion if they are

reactive to food antigens, in a mechanism similar to central tolerance. A second mechanism oftolerance is also employed in the intestine, namely the induction of Tregs. The fate of these

food antigen-specific T cells was initially hypothesized to depend on the dose of food antigen

present; high doses of antigen-induced anergy or deletion while frequent, low-dose exposurefavored the induction of Tregs [43]. This theory about antigen dose has recently fallen out of

favor among many investigators in light of more compelling models [19]. The actual subsets

of APCs that are required for this tolerance, the location of APC/T cell interaction, and thecytokine environment required to promote oral tolerance are still incompletely understood.

Recent work promotes a two-step model of oral tolerance where induction of Tregs is the

primary mechanism for preventing food-specific effector responses [44,45]. In the first step,antigen can be taken up through a variety of methods, including transcytosis through M cells

or enterocytes and diffusion between epithelial cells. Once antigen reaches the lamina propria,

it is phagocytosed by the CD103þ subset of migratory DC. This DC population then processesthis antigen and traffics to the MLN, where antigen-loaded DCs present their antigen to

naı̈ve T cells. Because these DCs can metabolize retinoic acid and TGF-b, they have been

shown to be particularly good at inducing Foxp3þ Treg differentiation in food antigen-specificT cells [29]. It has been shown that after feeding/intragastric gavage with soluble antigen,

the frequency of antigen-specific CD4þFoxp3þ Tregs increases markedly [44]. It is thought that

the environment of the MLN also promotes/supports the induction of Tregs. These inducedTregs, instead of being deleted or becoming nonresponsive to antigenic stimulation, become

highly effective producers of IL-10 and can help to suppress effector responses by other


79

cells that may escape tolerization [46,47]. When naı̈ve T cells are stimulated by antigenpresented by these DCs, they upregulate key gut homing receptors such as a4b7 and CCR9,

allowing them to traffic back to the lamina propria, the second step required for the induction

of tolerance in this model [44,45]. Once in the lamina propria, Tregs expand markedly andform the predominant population of lymphocytes at this site [46]. This expanded Treg

population contributes to the anti-inflammatory environment in which APCs see antigen,further limiting the chance of generating an effector response to food antigens. While it is not

completely known what drives this expansion, there is evidence that it is dependent on the

CX3CR1þ macrophage population that is resident in the LP [44]. Oral tolerance is impaired in

mice deficient in this macrophage population [47]. Significantly fewer Tregs are detectable in

the lamina propria of mice lacking CX3CR1þ macrophages; bacterial translocation and

proinflammatory cytokines are increased [47].

Other cells have also been implicated in oral tolerance, albeit with less clearly defined roles or

strict necessity. Antigen-presenting cells in the liver have been proposed to be important for

promoting the systemic nonresponsiveness that is characteristic of, and necessary for,successful oral tolerance [19,48]. Plasmacytoid DCs, in particular, have been implicated in

inducing anergy in antigen-specific T cells after oral antigen administration [49,50]. Of note,

the CX3CR1þ DC subset is related to plasmacytoid DCs based on gene-expression profiles [51].

Overall, oral tolerance is a classical process of education of the immune system that takes place

uniquely at mucosal sites. When oral tolerance breaks down, multiple pathologies can occur

that have both local (mucosal) and systemic effects.

The mucosal surfaces of the body are important sites for the regulation of immune responses.

The epithelium and its various protective mechanisms form a physical barrier between theexternal and internal environments and contain and control responses to the commensal

microbiota. Mucosal immune responses regulate the choice between tolerogenic or inflam-

matory responses to luminal antigen. When all of these specialized mechanisms work inconcert, the health and homeostasis of the host is maintained.

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