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T cell immunity to oral allergens Thomas T MacDonald

Considerable light has been thrown on the mechanisms of oral tolerance (or, more correctly, orally-induced systemic tolerance) in the past 1218 months. While it is very clear that T cell anergy and apoptosis can occur after being fed antigen, a major pathway that has been described in different models is the induction of regulatory T cells which secrete transforming growth factor . These cells have been designated Th3 cells but their relation to the in-vitro-generated Tr cells, which inhibit tissue-damaging T cell responses in the gut mucosa, is not known. An important discovery is that food antigens have major systemic effects on T cells, similar in many ways to those seen following intravenous injection of soluble antigens. This conceptually moves us away from the notion that there is something special about mucosal (compared to systemic) lymphoid tissue to the notion that it is the type of antigens seen in the gut (i.e. digested, soluble polypeptides) which dictates the types of response seen there. After initial excitement, clinical trials using oral tolerance to treat autoimmune disease have been somewhat disappointing.
Addresses Department of Paediatric Gastroenterology, St Bartholomews and the Royal London School of Medicine and Dentistry, St Bartholomews Hospital, London EC1A 7BE, UK; e-mail: t.t.macdonald@mds.qmw.ac.uk Current Opinion in Immunology 1998, 10:620627 http://biomednet.com/elecref/0952791501000620 Current Biology Ltd ISSN 0952-7915 Abbreviations CT cholera toxin EAE experimental autoimmune encephalomyelitis MBP myelin basic protein OVA ovalbumin PLP proteolipid peptide PP Peyers patch RA rheumatoid arthritis TGF-b transforming growth factor

mesenteric nodes and few (TCR+) T cells in their gut, although (TCR+) T cells are abundant in the epithelium [3]. The responses to the flora and foods are, however, linked; if gut bacteria elicit a large response in PPs, the size of the follicle-associated epithelium will increase and pari passu, uptake of dietary antigens will increase. This does not mean, however, that food antigens are ignored. Instead, in the past few years, it has become very clear that food antigens (and autoantigens added to food) are recognised by the immune system but that the net effect of recognition is downregulation of responsiveness oral tolerance. There has been a clear implication since the beginning of this research that the study of oral tolerance should shed insight into the mechanisms by which some individuals become hyper-reactive to foods, as in celiac disease (hypersensitivity to wheat) and cows-milk intolerance; in both cases there is an intestinal lesion caused by an excessive Th1-type immune response [4]. In fact this question has virtually never been addressed because it is so hard to elicit T cell lesions to nominal antigens in the gut in rodents and there is an understandable reluctance amongst clinicians to attempt oral tolerance in the treatment of food-sensitivityrelated diseases. Instead the standard protocol has been to orally immunise and then systemically challenge, so in this case it is more correct to use the term orally-induced systemic tolerance. The title of this review also alludes to the disease-inducing properties of macromolecules given orally, in that the term allergen is used rather than antigen. Although these words nowadays have slightly different connotations, decades ago they were interchangeable. There is a small literature on the consequences of feeding defined allergens but the interests of completeness demand that the response to all oral antigens must be considered. Such differences are, anyway, artificial since many of the most interesting recent advances in this area have arisen through the use of transgenic mice, in particular mice transgenic for TCRs which recognise ovalbumin (OVA) peptides in the context of MHC class II; and allergy to eggs is rather common in children. Three mechanisms of tolerance are well documented deletion, anergy and active suppression. In normal mice or rats only the last can be studied with any precision since anergy and deletion can only really be studied at the clonal level. In normal animals there is no doubt that being fed nominal antigens or self antigens (especially at low doses) induces regulatory cells (that are either CD4+ or CD8+ depending on the system) in PPs [5]. These cells then migrate to the periphery; on re-encounter with the antigen that was present in food, they secrete immunosuppressive cytokines (transforming growth factor [TGF-], IL-4 and IL-10), which dampen potentially tissue-damaging Th1type responses (Figure 1). It is more likely that this happens

Introduction
The intestine contains more T cells than the rest of the body combined. In an individual, there are dozens of mesenteric lymph nodes, hundreds of Peyers patches (PPs), tens of thousands of isolated lymphoid follicles and innumerable T cells in the epithelium and lamina propria [1]. The notion that the epithelium is an impermeable barrier to dietary macromolecules is no longer tenable: the function of the M-cells in the follicle-associated epithelium is to transport macromolecules and particles from the gut into the organised lymphoid tissue; and after being fed proteins, intact protein molecules can be detected in the blood. It should be stated from the outset, however, that the stimulus for the abundance of gut T cells is not food antigens it is the antigens of the normal microbial flora [2]. Germ-free mice, eating an autoclaved diet, have very small PPs and

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Figure 1

Food antigens Gut lumen Self antigens

Peyer's patch Th3 TGF-?

Th0 Tr Th2 Emigration to periphery IL-10?

Site of inflammation (e.g. joint, CNS) Th1 Th2

(a)

IL-4 TGF- IL-10 (b) Efferent lymphatic from site of inflammation

Th2 cytokines switch off Th1 cells and their pathogenic effects

Th2

Lymph node (draining either site of immunisation or site (c) of inflammation) Efferent lymphatic from site of immunization Th1

IL-4 TGF- IL-10

(d)

Th2 cytokines prevent Th1 cells responding to antigens ab initio

Current Opinion in Immunology

Regulation of inflammation by orally induced regulatory cells. Gutderived food antigens and self antigens can induce the development of Th2 cells (and perhaps Tr or Th3 cells) in Peyers patches. Following cell emigration it is likely that Th2 cells could mediate immune suppression at different sites. (a) If Th2 cells have been induced by self antigens they may be able to switch off pathogenic Th1 cells at sites of inflammation via the cytokines IL-4, TGF- and

IL-10. (b) Self antigens from inflammatory sites, as well as (c) food antigens deliberately given parenterally in complete Freunds adjuvant, will also be transported via efferent lymphatics to draining lymph nodes where (d) Th2 cells derived from the gut will also be able to prevent continuing activation of pathogenic Th1 cells. It is this latter case that occurs when animals are parenterally immunised after being fed food antigens.

in the lymph nodes connected to lymphatics that drain the site of systemic immunisation (antigen is almost always given with Freunds adjuvant to elicit a Th1-type response) than in the target organ. This is why it is easier to give antigen in food and then suppress de novo responses than it is to suppress ongoing, polarised, Th1-type, tissue-damaging responses although there are some examples of the latter [6,7]. An important feature of active suppression is the bystander effect, in which the response to any antigen is downregulated by the immunosuppressive cytokines. This was best shown by Weiner and co-workers [8], who showed

that feeding OVA to rats allowed protection against experimental autoimmune encephalomyelitis (EAE) when OVA was incorporated into the normally encephalitogenic vaccination with myelin basic protein (MBP) in adjuvant. There are publications that clearly show that antigens in food inhibit the IL-4 response to subsequent in vitro challenge [9,10], which would seem at odds with the general notion that antigens in food promote Th2-type responses. This is largely a matter of semantics, however, dependent on which cytokines are measured. Thus,

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Figure 2 Changes in T cell populations in Peyers patches and peripheral lymphoid tissues induced by high or low doses of fed antigens. The use both of mice transgenic for T cell receptors and of MHCpeptide tetramers means that clonotypic T cell populations can be followed in vivo. In this Figure, indicates an increase in specific T cells and indicates a decrease. Analysis was done a few hours after feeding (early) or after a period of days or weeks (late). Three main conclusions can be drawn from these studies: first, being fed antigens can affect sites distant from the gut (e.g. the spleen); second, being fed low doses of antigens increases the number of specific T cells in the Peyers patches at later stages; and third, being fed high doses of antigen leads to deletion of T cells in Peyers patches at later stages.

Low dose Antigen

High dose

Peyers patch

Early Late

(activation)

Early Late

(activation) (deletion)

Mesenteric nodes Early Late ? Early ? Late ?

Spleen

Early Late

Early Late

(after an intermediate recovery)

Current Opinion in Immunology

although IL-4 may be suppressed, without measuring IL-10, IL-13 or TGF- one cannot really say whether Th2-type responses are suppressed or not. There is also no reason why an IL-4-secreting cell cannot be anergised or deleted in such experiments. In this review I will also concentrate on studies where antigens have been given in food that has no adjuvants. There is a great deal of interest by vaccinologists towards agents such as cholera toxin (CT), nontoxic mutants of CT, Escherichia coli labile toxin (LT), LTB or CTB subunits and liposomes which can modulate oral tolerance, but lack of space precludes their inclusion in this review.

Is TGF-b the key cytokine that mediates oral tolerance?

There has been particular focus on the potent immunosuppressive cytokine TGF- and the way in which oral immunisation elicits a subset of T cells that are capable of secreting this cytokine the so-called Th3 cells. There is compelling in vivo evidence that neutralisation of endogenous TGF- is effective at breaking oral tolerance: it was first shown in rats that the antiencephalitogenic

effects on EAE seen if MBP was fed to animals beforehand was prevented by injecting the tolerised animals with anti-TGF- antibodies [11]; if haptenised self-proteins are fed to mice, the animals are resistant to trinitrobenzene sulphonic acid (TNBS)-induced colitis and this is also abolished by anti-TGF- [12]; Balb/c mice transgenic for a TCR which recognises the OVA peptide OVA323339 in the context of the MHC class II molecule IAd develop tracheal eosinophilia following intratracheal infusion of OVA this is prevented if mice are fed OVA beforehand and is re-established if the fed mice are injected with anti-TGF- [13]. Thus in three separate systems by three separate groups of investigators, in vivo administration of anti-TGF- has profound effects on oral tolerance. Since TGF- is made by a whole variety of different cell types and oral tolerance can be transferred with T cells, an obvious question is whether food that contains antigens elicits T cells which preferentially make TGF-. A number of studies have been done to address this. Most studies have utilised mice, for which clonotypic markers are available to follow individual T cells, as described in [13]; however,

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there is a problem in using these mice to study events in the gut. While the splenic T cells of these mice express a naive phenotype (CD45RBhi CD69) as would be expected PP T cells and lamina propria T cells display an activated phenotype (i.e. CD45RBlo CD69+) [14]. This is because in the gut the transgenic T cells are responding to the antigens of the flora through endogenous nontransgenic TCRs. When crossed onto recombination-activating gene 1 (RAG1)knockout mice to prevent endogenous TCR expression, gut T cells show a naive phenotype [14]. There are now data from a number of different groups to show that being fed antigens results in the production of T cells which secrete TGF-. Spleen cells from DO11.10 mice that are fed OVA over a wide range of doses secrete TGF- when re-stimulated in vitro [15]. In mice fed OVA for a short time there is priming for interferon- production and an increase in cell numbers in PPs; however, with prolonged feeding this disappears and the T cells secrete TGF-, IL-4 and IL-10 while OVA-specific T cells are lost [16]. Systemic administration of anti-IL-12 modestly enhances oral tolerance and increases TGF- production by T cells isolated from mice that are fed high doses of antigen [17,18]. Similar types of results have been obtained by other workers who have shown that, following oral tolerance induction, the addition of IL-12 to a mixture of OVA and the less toxic adjuvant dimethyldioctadyl ammonium bromide reverses oral tolerance when given by subcutaneous injection [19]. Feeding low doses of mouse MBP to mice transgenic for a TCR that recognises Ac1-11 (the major encephalitogenic peptide of MBP) in the context of I-Au also results in T cells which secrete TGF-, IL-4 and IL-10 [20]. Some of the most compelling data to show that this occurs in man followed the observation that short-term T cell lines made from the blood of patients with multiple sclerosis who had been fed MBP or proteolipid peptide (PLP) had a high frequency of cells that secreted TGF-1, compared to untreated patients [21]. Very recently attempts have been made to determine the location in vivo of cells that secrete TGF- and other cytokines [22]. It was demonstrated that, within hours of feeding OVA to OVA-TCR-transgenic mice, there is an increase in cells that are immunoreactive for IL-4 and IL 10 in the domes of the PPs. Increased numbers of TGF--containing cells were not seen in the domes, but were seen in the interfollicular zone. Double staining revealed that the TGF--containing cells were CD4+ T cells and macrophages. Increased TGF--containing cells were also seen in the lamina propria a few hours after feeding. Many of the T cells activated within animals being fed antigens secrete IL-10 as well as TGF- and it is the in vivo experiments cited above which seem to suggest that the latter cytokine is more important than the former; however, T cells from OVA-TCR-transgenic mice cultured in vitro with IL-10 and OVA develop into a cell type with a distinct cytokine profile high levels of IL-10 and IL-5, low levels of IL-2 and IL-4 and variable amounts of TGF- [23].

When these in-vitro-cultured cells are adoptively transferred into mice with severe combined immunodeficiency that are reconstituted with CD45RBhi cells, the animals develop colitis (as they do if given CD45RBhi cells alone); however, if the mice are fed OVA during the reconstitution phase, colitis is prevented. These cells have been termed Tr cells and are clearly able to suppress colitis by bystander tolerance.

The effects of being fed antigens on T cell numbers in the gut and peripheral lymphoid tissues
Transgenic mice are also ideal as the tool to investigate whether antigens in food induce cells to undergo apoptosis, anergy or proliferation. It was demonstrated several years ago that feeding extremely high doses of OVA to DO11.10 mice induces apoptosis in PP T cells [15]. At low doses of OVA, there was an increase in T cell numbers and the appearance of Th2-type cells [15]. It is difficult to extrapolate these findings to man, since the high dose of OVA used, 500 mg given 35 times, is equivalent to 1.75 kg OVA 35 times in a 70 kg adult human. These findings have been essentially repeated in a transfer model in which cells from OVA-TCR-transgenic mice are transferred into syngeneic BALB/c recipients [16]. Using the same transfer system, slightly different results have been obtained in mice that were similarly fed high doses of OVA (100 mg). Only a slight deletion of cells was observed, although the mice were unresponsive in terms of antigen-specific proliferation [24]. In a rather complex analysis, evidence was found that the unresponsiveness was due to anergy [24] confirming earlier reports that being fed autoantigens or OVA could also induce anergy [25,26]. An additional finding of this study was that being fed antigen did not result in migration of transgene-bearing T cells into the gut mucosa [24]. This is in contrast to another series of experiments using mice that were transgenic for a TCR that sees the SIINFEKL peptide (single-letter code is used for amino acids) of OVA in the context of the MHC class I molecule H-2Kb [27]. In these mice, intraperitoneal immunisation with OVA resulted in the migration of TCR+ CD8+ cells into the mucosa and gut epithelium. Finally, OVA-TCR-transgenic mice have been immunised with OVA in alum and pertussis toxin (to generate a Th2type response and IgE) [28]. Being fed low doses of OVA tolerised the Th2-type response and decreased IL-4 production. The number of clonotype+ cells decreased in peripheral lymphoid tissue but the number of CD4+ T cells did not, suggesting that tolerance was not due to clonal deletion but was due to TCR downregulation. In a paper which is difficult to interpret at present, Garside et al. [29] showed that in normal animals made orally tolerant and then systemically immunised with OVA in adjuvant all the lymph node T cells underwent apoptosis in vitro. The mechanisms responsible for this bystander apoptosis are not understood. The best way to study antigen-specific responses in vivo is to use antigenic ligands as the probe. It is now possible to construct biotinylated tetrameric peptideMHC reagents that bind to specific T cells and follow T cell activation and

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expansion in vivo. This technique has been used to determine the effects of feeding pigeon cytochrome C to mice transgenic for a TCR chain which sees pigeon cytochrome C in the context of the MHC class II molecule I-Ek [30] . A single feeding resulted in the expansion and activation of T cells in the spleen and mesenteric nodes. Multiple feeding resulted in a loss of specific peripheral T cells and functional unresponsiveness that was not due to immune deviation. An important observation was that, six hours after feeding, splenic antigen-presenting cells could stimulate transgenic T cells in vitro. The authors conclude that oral tolerance is similar to tolerance induced by intravenous soluble proteins and, importantly, that dietary antigens have access to, and can influence, peripheral T cell function. The way in which the dose of oral antigen can alter T cell numbers in the PPs and periphery is shown in Figure 2.

Why do oral antigens induce Th2-type responses?

The key question relates to the cellular and molecular basis for the production of immunoregulatory T cells in PPs. Is their anything unique about PPs or are all the responses, described above, the inevitable consequence of proteolytic digestion and would they be seen at any site exposed to soluble antigen fragments? It should also be remembered that the cytokines that downregulate systemic Th1-type responses are also the same ones that promote IgA responses [31]. IgA responses are presumably a good thing which is why all healthy people make 45 g of IgA every day and it could be that orally-induced systemic unresponsiveness is simply derived from the curiosity of immunologists and the discovery of complete Freunds adjuvant. It has been proposed that effective secretory immunity and oral tolerance are mutually exclusive [32] but mechanistically and teleologically this makes little sense and probably reflects our lack of understanding. It has been claimed that there are fundamental differences in PP or splenic dendritic cells, in that the former induce T cells to make IL-4 whereas the latter induce T cells to make interferon- [33,34]. Whether this is due to differences in co-stimulatory molecules or dendriticcell-derived cytokines is not known. In a very interesting recent study, Viney and co-workers [35] injected mice with Flt3 ligand to expand dendritic cells in vivo in all tissues. Somewhat surprisingly, Flt3-treated mice were more susceptible to oral tolerance, becoming tolerant at lower doses than control mice.

well with earlier reports that T cells could break oral tolerance [38]. It has also been suggested that interferon--knockout mice lack oral tolerance [39 ]. Feeding mice with PLP increases the amount of the chemokine monocyte chemotactic protein 1 (MCP-1) in the gut and prevents the EAE that is elicited with PLP in adjuvant [40]. Antibody neutralisation of MCP-1 during feeding renders mice susceptible to EAE. There have been very few studies where the effect of being fed antigens on local immunity has been studied. Grdic et al. [41] have, however, provided evidence that CD8+ cells downregulate IgA responses in the gut. Finally, oral tolerance has been studied in mice infected with the nematode Helimosomoides polygyrus, which induces a strong antiparasite Th2-type response in the gut. Feeding OVA to normal mice suppressed both Th1- and Th2-type cytokine secretion by peripheral lymph node T cells [10]. In infected mice, however, Th2-type responses were not suppressed.

Clinical studies
Undoubtedly the major impetus for enthusiasm in oral tolerance was the demonstration, in two small clinical trials, that feeding chicken collagen to patients was of some benefit in treating rheumatoid arthritis (RA) and that feeding bovine myelin to patients with multiple sclerosis also helped a subset of them [42,43]; however in a double-blind, placebo controlled, multicenter, phase III trial with 515 patients, a single dose of bovine myelin was ineffective [44]. More encouraging results were obtained with a phase II, doubleblind, dose-ranging trial using long-term feeding of chicken type II collagen in 280 patients with RA [45], in which an effect was seen at the lowest dose (20 ug). Interestingly a large analysis of five studies involving 1200 RA patients also seems to support the notion that low doses are more effective than high doses [44] and a phase III trial, in which patients with RA are fed 60 ug collagen, is underway. Preliminary clinical studies involving the feeding of insulin to newly diagnosed type I diabetic patients, the feeding of a cross-reacting MHC peptide or bovine S-antigen to patients with uveitis and the feeding of nickel to patients with nickel allergy are showing positive trends but larger studies need to be done [44]. Oral Der f 2 antigen, from house dust mites, inhibits airway inflammation in a mouse model [46]. It has been reported that mite-induced rhinoconjunctivitis in patients is ameliorated by tablets containing chemically modified Dermataphagoides pteronyssinus and D. farinae allergens [47]. The tablets were dissolved in the mouth for 12 minutes and then swallowed. This confirms and extends many previous studies, cited in [47], showing that sublingual immunotherapy has benefit on rhinitis induced by pollen and house dust mites. The immunological basis for this treatment is not known but it must be different from that seen following standard subcutaneous desensitisation regimes, which result in decreased IL-4 and increased IL-12 [48,49]. Perhaps sublingual therapy activates Tr or Th3 cells.

Other regulatory pathways in oral tolerance

It is not yet possible to integrate all the available data into a unified hypothesis of oral tolerance and there are several intriguing papers which offer to give new insights into this phenomenon. It has been reported that feeding OVA to -knockout mice does not induce tolerance to subsequent parenteral immunisation [36]. This fits in well with the observation from Holts group that tolerance to inhaled antigens is mediated by T cells [37] but does not fit in

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The type of T cell responses to oral allergens in man

Peanuts are the most dangerous oral allergens every year several people in the UK die after inadvertently coming into contact with foods containing peanut extracts. As would be expected of an allergen which elicits strong IgE responses, antigen-specific T cell clones from patients with food allergy show a marked Th2-type skewing [50,51] and there is some evidence that in whole-blood cultures from patients with peanut allergy and atopic dermatitis there is an increased IL-4 response by lymphocytes to antigen challenge in vitro [52]. The most common food allergen, however, is cows milk and it is estimated that up to 5% of all bottle-fed infants develop adverse effects. Cows-milk allergy is very heterogeneous clinically. The majority of infants show classical food allergy with IgE antibodies, immediate symptoms, a skin rash and anaphylaxis [53]. A second group develops symptoms after a few hours, sometimes has skin lesions or bronchitic symptoms and does not have specific IgE. The third group develops symptoms over a period of hours to a few days and has gut symptoms with or without eczema or bronchitic symptoms. There is a very large literature on proliferative responses of blood T cells to cows-milk proteins in patients with cows-milk allergy and there is no doubt that there is increased reactivity in these patients; however, studies on cytokines are fewer and are so far inconclusive. Mitogen-stimulated blood T cells from patients with immediate-onset symptoms to cows milk produce less interferon- than cells from control patients [54,55]. At the clonal level things are less clear. Nakajima et al. [56] produced T cell lines reactive to casein from patients with cows-milk allergy. There was a high frequency of CD8+ lines produced from the patients, which made both interferon- and IL-4. A high frequency of CD8+ T cell clones that secreted interferon- was also produced by Reekers et al. [57] from patients with food allergy and atopic dermatitis. In a similar type of study of patients with milk-responsive dermatitis, Werfel et al. [58] also produced CD4+ T cell clones that were reactive to casein and that secreted interferon-. Enzyme-linked immunosorbent spot assays (ELISPOTs) have been used to analyse the cytokine profile of CD4+ T cells that were freshly isolated from the blood of patients with late-onset symptoms to cows milk [59]. In these patients there is an exaggerated interferon-, IL-4, IL-5 and IL-10 response in the blood and lamina propria, although interferon- dominates. Children with immediate-onset cows-milk allergy had a very high frequency of IL-4-secreting cells in their blood. The best-investigated food sensitivity is celiac disease. CD4+ T cells reactive to gluten can be isolated from the blood and intestinal lamina propria of celiacs and the cells show a Th0 or Th1 phenotype [60]. In the tissue, glutenreactive T cells show a marked Th1-type response [61].

Celiac disease is unusual in that it is exquisitely linked to HLA-DQ2 and there is intriguing evidence that native gliadin can be enzymatically modified by tissue transglutaminase to make it more immunogenic to gut T cells [62]. There are, however, no data on oral tolerance in hypersensitivity to foods in man. The only data available follow observations of children with atopic dermatitis and exanthematous reactions in a double-blind, placebo-controlled challenge using cows milk; this reduces the amount of interferon- secreted by blood T cells stimulated with milk antigens in vitro [63]. Extensive preclinical work needs to be carried out before oral tolerance could be considered as a therapy for food hypersensitivity.

Conclusions
All three types of immunological tolerance (deletion, anergy and suppression) can be demonstrated to occur if a subject is fed antigen. The dose of antigen seems to be quite important in determining which of these pathways is activated. Low doses elicit suppressor cells and higher doses result in anergy or deletion. The suppressor cells elicited when subjects are fed antigen produce IL-4, IL-10 and TGF-; these cells have been designated Th3 cells. The relationship of these cells to in-vitro-generated Tr cells, which make predominantly IL-10, remains to be elucidated. In patients there will be difficulties in giving very large doses of antigens orally. In this regard it is encouraging that the most positive clinical data have been achieved with feeding low doses of chicken collagen to patients with RA. Although the equivalent effect has not been shown in RA, feeding low doses of bovine myelin induces Th3-like cells in patients with multiple sclerosis. Given that the technology is now available to express mammalian self-antigens in plants, it is possible that future therapy may be dietary rather than medicinal [64].

Acknowledgements
Work in the authors laboratory is supported by the Wellcome Trust, Crohns in Childhood Research Appeal, the Biotechnology and Biological Sciences Research Council and the Special Trustees of St Bartholomews Hospital.

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

of special interest of outstanding interest

1. Brandtzaeg P, Farstad IN, Helgeland L: Phenotypes of T cells in the gut. Chem Immunol 1998, 71:1-26. This provides an extremely comprehensive review of the phenotype of gut T cells in man. 2. Crabbe PA, Nash DR, Bazin H, Eyssen H, Heremans JF: Immunohistochemical observations on lymphoid tissues from conventional and germ-free mice. Lab Invest 1971, 22:448-457. Guy-Grand D, Cerf-Bensussan N, Malissen B, Malassis-Seris M, Briottet C, Vassalli P: Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J Exp Med 1991, 173:471-481. MacDonald TT: Effector and regulatory lymphoid cells and cytokines in mucosal sites. Curr Top Microbiol Immunol 1998, in press.

3.

4.

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5. Weiner HL: Oral tolerance: immune mechanisms and treatment of autoimmune diseases. Immunol Today 1997, 18:335-343. This is an extremely good review of oral tolerance as it applies to the treatment of autoimmune diseases. 6. Higgins PJ, Weiner HL: Suppression of experimental autoimmune encephalomyelitis by oral administration of myelin basic protein and its fragments. J Immunol 1988, 140:440-445. Gaupp S, Hartung HP, Toyka K, Jung S: Modulation of experimental autoimmune neuritis in Lewis rats by oral application of myelin antigens. J Neuroimmunol 1997, 79:129-137. Miller A, Lider O, Weiner HL: Antigen-driven bystander suppression after oral administration of antigens. J Exp Med 1991, 174:791-798. Garside P, Steel M, Worthey EA, Staskar A, Alexander J, Bluethmann H, Liew FY, Mowat AM: T helper 2 cells are subject to high dose oral tolerance and are not essential for its induction. J Immunol 1995, 154:5649-5655.

22. Gonnella PA, Chen Y, Inobe J-I, Komagata Y, Quartulli M, Weiner HL: In situ immune response in gut-associated lymphoid tissue (GALT) following oral antigen in TCR-transgenic mice. J Immunol 1998, 160:4708-4718. This gives a detailed immunohistological and immunofluorescent study of the expression of cytokines in the gut following OVA challenge. 23. Groux H, OGarra A, Bigler M, Rouleau M, Antonenlo S, de Vries JE, Roncarlo MG: A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 1997, 389:737-742. This is conceptually a very important paper. In-vitro-generated Tr cells from OVATCR-transgenic mice which make IL-10 can suppress colitis following transfer of CD45RBhi cells into mice with severe combined immunodeficiency as long as the animals are fed OVA; a good example of bystander tolerance. 24. Van Houten N, Blake SF: Direct measurement of anergy of antigenspecific T cells following oral tolerance induction. J Immunol 1996, 157:1337-1341. 25. Whitacre CC, Gienapp IE, Orosz CG, Bitar DM: Oral tolerance in experimental encephalomyelitis. III. Evidence for clonal anergy. J Immunol 1991, 147:2155-2163. 26. Melamed D, Friedman A: Direct evidence for anergy in T lymphocytes tolerized by oral administration of ovalbumin. Eur J Immunol 1993, 23:935-942. 27. Kim S-K, Reed DS, Heath WR, Carbone F, Lefrancois L: Activation and migration of CD8 T cells in the intestinal mucosa. J Immunol 1997, 159:4295-4306. Using mice transgenic for an OVA peptide which is seen in the context of MHC class I, this paper suggests that systemic immunisation leads to the rapid recruitment of cells to the gut epithelium. This observation stands out from a wealth of data which show that intraepithelial lymphocytes (IELs) are renewed extremely slowly and that systemic immunisation has little effect on IELs. 28. Xu X-M, Nakashima M, Watanabe T: Selective suppression of antigen-specific Th2 cells by continuous micro-dose oral tolerance. Eur J Immunol 1998, 28:134-142. This is one of the few papers where an attempt has been made to tolerise mice after sensitisation, akin to the clinical situation. 29. Garside P, Steel M, Worthey EA, Kewin PJ, Howie SE, Harrison DJ, Bishop D, Mowat AM: Lymphocytes from orally tolerized mice display enhanced susceptibility to death by apoptosis when cultured in the absence of antigen in vitro. Am J Pathol 1996, 149:1971-1979. 30. Gtgemann I, Fahrer AM, Altman JD, Davis MM, Chein Y-H: Induction of rapid T cell activation and tolerance by systemic presentation of an orally adminstered antigen. Immunity 1998, 8:667-673. This is a very important paper which uses MHCpeptide tetramers to track T cell numbers and activation status after feeding antigen. The authors show that there is a loss of cells in the periphery and, importantly, that feeding antigens has systemic effects. They suggest that oral tolerance is similar to the systemic tolerance that is induced by intravenous soluble antigen. 31. Lebman DA, Coffman RL: Cytokines in the mucosal immune system. In Handbook of Mucosal Immunology. Edited by Ogra PL, Mestecky J, Lamm ME, Strober W, McGhee JR, Bienenstock J. San Diego: Academic Press; 1994:243-250. 32. Mowat AM: Oral tolerance and regulation of immunity to dietary antigens. In Handbook of Mucosal Immunology. Edited by Ogra PL, Mestecky J, Lamm ME, Strober W, McGhee JR, Bienenstock J. San Diego: Academic Press; 1994:185-202. 33. Harper HM, Cochrane L, Williams NA: The role of small intestine antigen-presenting cells in the induction of T cell reactivity to soluble protein antigens: association between aberrant presentation in the lamina propria and oral tolerance. Immunology 1996, 89:449-456. 34. Everson MP, Lemak DG, McDuffie S, Koopman WJ, McGhee JR, Beagley KW: Dendritic cells from Peyers patch and spleen induce different T helper cell responses. J Interferon Cytokine Res 1998, 18:103-115. Evidence is presented here to show that PP dendritic cells are fundamentally different from spleen dendritic cells in the type of T helper subset they activate. 35. Viney JL, Mowat AM, OMalley JM, Williamson E, Fanger NA: Expanding dendritic cells in vivo enhance the induction of oral tolerance. J Immunol 1998, 160:5815-5825. In this report Flt3 ligand was used to expand dendritic cells in the gut, a procedure which should be of practical use in the future. In these mice small doses of antigen, which were not tolerogenic in normal mice, did induce tolerance.

7.

8. 9.

10. Shi HN, Ingui CJ, Dodge I, Nagler-Anderson C: A helminth-induced mucosal Th2 response alters nonresponsiveness to oral administration of a soluble antigen. J Immunol 1998, 160:2449-2455. This reports an interesting experiment in which nematode infection was used to produce a polarised Th2 response in the gut and then oral tolerance to OVA was studied. In this situation, Th2 responses to OVA were not tolerised. This may be of relevance to the third world, where intestinal helminthiases are common. 11. Miller A, Lider O, Roberts AB, Sporn MB, Weiner HL: Suppressor T cells generated by oral tolerization to myelin basic protein suppress both in vitro and in vivo immune responses by the release of TGFb following antigen specific triggering. Proc Natl Acad Sci USA 1992, 89:421-425. 12. Neurath MF, Fuss I, Kelsall BL, Presky DH, Waegall W, Strober W: Experimental granulomatous colitis in mice is abrogated by induction of TGF-b-mediated oral tolerance. J Exp Med 1996, 183:2605-2616. 13. Haneda K, Sano K, Tamura G, Sato T, Habu S, Shirato K: TGF-b induced by oral tolerance ameliorates experimental tracheal eosinphilia. J Immunol 1997, 159:4484-4490. Lung eosinophilia is produced by intratracheal injection of OVA in OVATCRtransgenic mice. This paper shows that prior feeding of OVA reduces the response and that treating tolerised mice with anti-TGF- breaks tolerance. 14. Hurst SD, Sitterding SM, Ji S, Barrett TA: Functional differentiation of T cells in the intestine of T cell receptor transgenic mice. Proc Natl Acad Sci USA 1997, 94:3920-3925. This demonstrates that in DO11.10 mice, gut T cells respond to the normal flora via endogenously expressed TCRs. Thus when OVA is fed to these mice, the antigen-specific T cells in the gut do not have a naive phenotype. 15. Chen Y, Inobe J-I, Marks R, Gonnella PA, Kuchroo VK, Weiner HL: Peripheral deletion of antigen-reactive T cells in oral tolerance. Nature 1995, 376:177-180. 16. Chen Y, Inobe J-I, Weiner HL: Inductive events in oral tolerance in the TCR transgenic adoptive transfer model. Cell Immunol 1997, 178:62-68. 17. Marth T, Strober W, Kelsall BL: High dose oral tolerance in ovalbumin TCR-transgenic mice: systemic neutralisation of IL-12 augments TGF-b secretion and T cell apoptosis. J Immunol 1996, 157:2348-2357.

18. Marth T, Strober W, Seder RA, Kelsall BL: Regulation of transforming growth factor beta production by interleukin-12. Eur J Immunol 1997, 27:1213-1220. 19. Claessen AME, Von Blomberg BME, De Groot J, Wolvers DAE, Kraal G, Scheper RJ: Reversal of mucosal tolerance by subcutaneous administration of interleukin-12 at the site of attempted sensitization. Immunology 1996, 88:363-373. 20. Chen Y, Inobe J-I, Kuchroo VK, Baron JL, Janeway CA Jr, Weiner HL: Oral tolerance in myelin basic protein T cell receptor transgenic mice: suppression of autoimmune encephalomyelitis and dose dependent induction of regulatory cells. Proc Natl Acad Sci USA 1996, 93:388-391. 21. Fukura H, Kent SC, Pietrusewicz MJ, Khoury SJ, Weiner HL, Hafler DA: Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor b-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J Clin Invest 1996, 98:70-77.

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36. Ke Y, Pearce K, Lake JP, Ziegler HK, Kapp JA: gd T lymphocytes regulate the induction and maintenance of oral tolerance. J Immunol 1997, 158:3610-3618. This paper shows that TCR-knockout mice fail to develop oral tolerance after being fed antigen. The dose used was quite low (three doses of 2.5 mg OVA) and it would be interesting to see if the mice were made tolerant by high doses. Since this should involve deletion, the absence of TCR T cells should have no effect. 37. McMenamin C, Pimm C, McKersey M, Holt PG: Regulation of IgE responses to inhaled antigens in mice by antigen-specific gd T cells. Science 1994, 265:1869-1871.

grass pollen immunotherapy. J Allerg Clin Immunol 1997, 99:254-260. 50. Higgins JA, Lamb JR, Lake RA, OHehir RE: Polyclonal and clonal analysis of human CD4+ T-lymphocyte responses to nut extracts. Immunology 1995, 84:91-97. 51. de Jong EC, Spanhaak S, Martens BP, Kapsenberg ML, Penninks AH, Wierenga EA: Food allergen (peanut)-specific Th2 clones generated from the peripheral blood of a patients with peanut allergy. J Allerg Clin Immunol 1996, 98:73-81. 52. Laan MP, Tibbe GJM, Oranje AP, Bosmans EPE, Neijens HJ, Savelkoul HFJ: CD4+ cells proliferate after peanut-extract-specific and CD8+ cells proliferate after polyclonal stimulation of PBMC of children with atopic dermatitis. Clin Experiment Allergy 1998, 28:35-44. 53. Hill DJ, Hosking CS: The cow milk allergy complex: overlapping disease profiles in infancy. Eur J Clin Nutr 1995, 49(suppl 1):S1-S12. 54. Andre F, Pene J, Andre C: Interleukin-4 and interferon-gamma production by peripheral blood mononuclear cells from foodallergic patients. Allergy 1996, 51:350-355. 55. Hill DJ, Ball G, Hoskings CS, Wood PR: g-interferon production in cows milk allergy. Allergy 1993, 48:75-80. 56. Nakajima H, Hachimura S, Nishiwaki S, Katsuki T, Shimojo N, Ametani A, Kohno Y, Kaminogawa S: Establishment and characterization of alpha s1-casein specific T-cell lines from patients allergic to cows milk: unexpected higher frequency of CD8+ T-cell lines. J Allerg Clin Immunol 1996, 97:1342-1349. 57. Reekers R, Beyer K, Niggemann B, Wahn U, Freihorst J, Kapp A, Werfel T: The role of circulating food antigen-specific lymphocytes in food allergic children with atopic dermatitis. Br J Dermatol 1996, 135:935-941.

38. Fujihashi K, Taguchi T, Aicher WK, McGhee JR, Bluestone JA, Eldridge JH, Kiyono H: Immunoregulatory functions for murine intraepithelial lymphocytes: g/d T cell receptor-positive (TCR+) T cells abrogate oral tolerance, while a/b TCR+ T cells provide B cell help. J Exp Med 1992, 175:695-707. 39. Kweon M-N, Fujihashi K, VanCott JL, Higuchi K, Yamamoto M, McGhee JR, Kiyono H: Lack of orally induced systemic unresponsiveness in IFN-g knockout mice. J Immunol 1998, 160:1687-1693. It is claimed that interferon (IFN)--knockout mice have defective oral tolerance; however the data presented do not appear to show this. For antibody responses, an absence of tolerance appears because the IFN--knockout controls that are not fed OVA only make a weak response to OVA compared to wild-type mice. The inhibition of cytokine responses by oral feeding seems identical in the knockout and wild-type mice. 40. Karpus WJ, Kennedy KJ, Kunkel SL, Lukacs NW: Monocyte chemotactic protein 1 regulates oral tolerance induction by inhibition of T helper cell 1-related cytokines. J Exp Med 1998, 187:733-741. This is an interesting paper which shows that feeding 2 mg of PLP 139151 peptide, which binds with high affinity to the MHC class II molecule I-As, leads to increased MCP-1 in the gut mucosa, as does a control peptide which also binds to I-As. If these mice are then injected with PLP 139-151 in adjuvant to induce EAE, there is an increase in IL-4 and a decrease in IL-12 in the gut mucosa and the mice are protected from EAE. In vivo inhibition of MCP-1 restores EAE. 41. Grdic D, Hrnquist E, Kjerrulf M, Lycke NY: Lack of local suppression in orally tolerant CD8-deficient mice reveals a critical regulatory role of CD8+ T cells in the normal gut mucosa. J Immunol 1998, 160:754-762. In this report CD8-knockout mice were used to show that after feeding antigen, PPs contain a population of CD8+ cells which downregulates mucosal antibody responses. 42. Weiner HL, Mackin GA, Matsui M, Orav EJ, Khoury SJ, Dawson DM, Hafler DA: Double-blind pilot trial of oral tolerization with myelin antigens in multiple sclerosis. Science 1993, 259:1321-1324. 43. Trentham DE, Dynesius-Trentham RA, Orav EJ, Combitchi D, Lorenzo C, Sewell KL, Hafler DA, Weiner HL: Effects of oral administration of type II collagen on rheumatoid arthritis. Science 1993, 261:1727-1730. 44. Liu LM, Weiner HL: T cell response to orally administered antigens and its role in the treatment of autoimmune diseases. Chem Immunol 1998, 71:139-160. 45. Barnet ML, Kremer JM, St Clair EW, Clegg DO, Furst D, Weisman M, Fletcher MJ, Chasan-Taber S, Finger E, Morales A et al.: Treatment of rheumatoid arthritis with oral type II collagen. Arthritis Rheum 1998, 41:290-297. This describes a double-blind, placebo-controlled trial using oral chicken collagen for patients with RA. Positive findings were seen in those patients who were fed low doses. 46. Yasue M, Yokota T, Kajiwara Y, Suko M, Okudaira H: Inhibition of airway inflammation in rDer f 2-sensitized mice by oral administration of recombinant Der f 2. Cell Immunol 1997, 181:30-37. 47. Passalacqua G, Albano M, Fregonese L, Riccio A, Pronzato C, Mela GS, Canonica GW: Randomised controlled trial of local allergoid immunotherapy on allergic inflammation in mite-induced rhinoconjuctivitis. Lancet 1998, 351:629-632. Sublingual therapy, to desensitise patients with pollen rhinitis, has been used for several years. This reports that it is likely that a lot of the antigen is swallowed. 48. Secrist H, Chelen CJ, Wen Y, Marshall JD, Umetsu DT: Allergen immunotherapy decreases interleukin 4 production in CD4+ T cells from allergic individuals. J Exp Med 1993, 178:2123-2130. 49. Hamid QA, Schotman E, Jacobson RM, Walker SM, Durham SR: Increases in interleukin 12 messenger RNA+ cells accompany inhibition of allergen-induced late skin responses after successful

58. Werfel T, Ahlers G, Schmidt P, Boeker M, Kapp A, Neumann C: Milk-responsive atopic dermatitis is asociated with caseinspecific lymphocyte response in adolescent and adult patients. J Allerg Clin Immunol 1997, 99:124-133. 59. Hauer AC, Breese EJ, Walker-Smith JA, MacDonald TT: The frequency of cells secreting interferon-g, IL-4, IL-5 and IL-10 in the blood and duodenal mucosa of children with cows milk hypersensitivity. Pediat Res 1997, 42:629-638. This is the only paper in the literature where ongoing T cell responses have been studied in children with cows-milk intolerance. These children have a very high number of interferon--secreting cells in their duodenal lamina propria but cells secreting IL-4, IL-5 and IL-10 are also increased in number. 60. Nilsen EM, Lundin KEA, Krajci P, Scott H, Sollid LM, Brandtzaeg P: Gluten-specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon-g. Gut 1995, 37:766-776. 61. Nilsen EM, Jahnsen FL, Lundin KEA, Johansen F-E, Fausa O, Sollid LM, Jahnsen J, Scott H, Brandtzaeg P: Gluten induces an intestinal cytokine response strongly dominated by interferon gamma in patients with celiac disease. Gastroenterology 1998, 115:564-572. 62. Molberg O, McAdam SN, Korner R, Quartsen H, Kristiansen C, Madsen L, Fugger L, Scott H, Noren O, Roepstorff P et al.: Tissue transglutaminase selectively modifies gliadin peptides that are recognised by gut-derived T cells in celiac disease. Nat Med 1998, 4:713-717. This paper is of fundamental importance to understanding autoimmune disease. Tissue transglutaminase is an enzyme produced to repair tissue injury. This study found that a peptide of gliadin could be deamidated by transglutaminase; following this process it bound with greater affinity to the celiac restriction element, HLA-DQ2, and was then recognised by gut-derived T cells from celiac patients. It raises the possibility that gut infections and injury may be important in determining whether someone develops celiac disease or not and, as tissue transglutaminase is widely expressed, that enzymatic modification of self-peptides could create altered peptides which could bind to self-MHC and be recognised by T cells. 63. Stas Y, Hurme M, Isolauri E: Oral cow milk challenge abolishes antigen-specific interferon-g production in the peripheral blood of children with atopic dermatitis and cow milk allergy. Clin Experiment Allergy 1997, 27:277-283. 64. Ma SW, Zhao DL, Yin ZQ, Mukherjee R, Singh B, Qin HY, Stiller CR, Jevnikar AM: Transgenic plants expressing autoantigens fed to mice to induce oral tolerance. Nat Med 1997, 3:793-796.

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