Oral Immune Tolerance Research Articles

Microbiota-derived butyrate negatively regulates RORγt+ ILC3 in Peyer’s patch of the terminal ileum

Abstract In the intestinal immune system, anatomical and physiological distinctions in the gut contribute to its regional specialization by microbiota and immunomodulatory agents from the diet. Given that the ileal Peyer’s patch (PP) belongs anatomically to the small intestine, while it is physiologically exposed to an environment similar to the large intestine with respect to microbes and microbial metabolites, its characteristics may differ from those in PPs in the jejunum. As the terminal ileal PP is a key mucosal organ where host defense and oral immune tolerance in the gut develop, identification of the specific factors regionally specialized in the tissue is essential to understand mucosal homeostasis. Among the various cell types present in PPs, group 3 innate lymphoid cells (ILC3s) are closely associated with the regulation of commensal bacteria through the suppression of commensal bacteria-specific CD4+ T cells, although the regulation of ILCs in ileal PPs is poorly defined. In this study, we found that butyrate plays a role as a regional specific factor involved in the repression of ILC3s in PPs of the terminal ileum. This butyrate-mediated negative regulation of ILC3s alleviates the tolerogenic mucosal microenvironment by suppressing regulatory T cells in PPs. Collectively, we conclude that the inhibition of ILC3s by microbiota-derived butyrate can confer the functional ability to induce antigen-specific immunity, and that this network contributes to homeostatic regulation of the mucosal immune inductive site.

Induction of Oral Immune Tolerance in Infant Mice via Breastfeeding from Allergic and Non-allergic Mothers

Induction of Oral Immune Tolerance in Infant Mice via Breastfeeding from Allergic and Non-allergic Mothers

Food allergy and the related diseases of digestive system

Food allergy refers to the body's abnormal immune response to food, which induces disorder and/or injuries of human body, and thus triggers a series of manifestations.Ninety percent of food allergy is due to the following allergen: milk, eggs, peanuts, nuts, seafood, soy, wheat, etc.The symptoms of digestive system are closely associated with the nature of allergen (including protein's family nature, three dimensional structure, biological activity and stability) and the body's own status (genetic factors, integrity of intestinal mucosal barrier and oral immune tolerance). The symptoms of digestive systems are not typical, and differ by various factors like body conditions, ages, regions and allergens, so the diagnosis is difficult.Food allergy is categorized by its mechanism: IgE, non IgE, and mix.The symptoms of digestive systems include vomiting, diarrhea, reflux, constipation and abdominal pain, etc, and relative diseases include food protein-induced enteropathy, food protein-induced enterocolitis syndrome, food protein-induced enterocolitis syndrome, food protein-induced proctocolitis, celiac disease, eosinophilic gastroenteritis, eosinophilic esophagitis and infantile colic, etc.Delayed diagnosis will result in failure to thrive, anemia and hypoproteinemia.Therefore, early diagnosis and treatment can effectively prevent malnutrition and improve the children's animation. Key words: Food allergy; Digestive systems; Child

Oral mucosal immunity

Oral keratinocytes and dendritic cells of the oral mucosa, through molecular pattern recognition receptors, distinguish between commensal and pathogenic microorganisms and mediate the generation of protective immunoinflammatory responses to potentially invading pathogens or mediate immune tolerance toward commensal microorganisms. Oral immune tolerance is the result either of lack of activation of T cells in response to immunogenic presentation of antigens or of suppression of activity of effector T cells by regulatory T cells. Secretory immunoglobulin A (sIgA) antibodies at oral mucosal sites contribute to oral immunity by limiting colonization of microorganisms and their invasion of the epithelium. Ig isotype class switching to IgA is either dependent on or independent of T helper cells and is facilitated by cytokines secreted by dendritic cells and monocytes.

L’enfant allergique à l’arachide : une approche thérapeutique personnalisée

The induction of immune tolerance in peanut allergic patient by of an oral immunotherapy (OIT) protocol is a new therapeutic approach in the management of this allergy. Patients and methodsEnrolled children underwent a personalized oral immune tolerance protocol (OIT) calculated upon peanut threshold dose. Tests of oral provocation (TOP) were performed at the beginning (TOP1) and at the end of the OIT (TOP2). A median period of 8months was observed between both TOP. The threshold doses and the score of clinical severity were compared before (TOP1) and after the administration of the protocol (TOP2). ResultsSixty-seven children with peanut food allergy enrolled in the study (median age: 7years) followed a protocol of specific oral immune tolerance using biscuits blown with peanut. The evolution of the median threshold dose between TOP1 (516mg of peanut) and TOP2 (1066mg of peanut) was significant (P<0.001). Clinical severity score between TOP1 and TOP2 decreased significantly (P=0.0015). ConclusionOur protocol of specific OIT in peanut allergic children, performed at home, with progressive doses of a food containing the peanut allergen allowed to observe a significant increase of the threshold dose and a decrease of the clinical severity score of the patients.

Gut-tropic DC precursors contribute to intestinal immune tolerance (120.4)

Abstract Gut-associated tolerogenic and inflammatory dendritic cells (DC) develop from at least two different bone marrow (BM)-derived precursors. Tolerogenic, but not inflammatory gut-associated DC, express CD103 and synthesize retinoic acid (RA), which contributes to the induction of Foxp3+ regulatory T cells (TREG). How CD103+ DC and/or precursors are recruited to the intestinal mucosa and then acquire RA synthesizing capacity remains unknown. Here, we show that the BM contains a distinct population of DC precursors expressing the gut-specific α4β7 and CCR9 receptor (gut-tropic pre-DC). Interestingly, vitamin A-depleted mice exhibited a marked decrease in gut-tropic pre-DC in the BM, which correlated with the absence of RA-producing DC in gut-associated tissues. Furthermore, gut-tropic pre-DC lacking β7 integrins exhibited decreased capacity to home to Peyer’s patches and the small bowel lamina propria. Importantly, gut-tropic pre-DC were required to give rise to RA-producing DC in the mesenteric lymph node and to efficiently induce antigen-specific TREG. Consistent with a pro-tolerogenic role of gut-tropic pre-DC, selective depletion of DC derived from β7-expressing cells promoted an inflammatory milieu, as shown by exacerbated inflammation in experimental colitis and by impaired oral immune tolerance. Thus, gut-tropic pre-DC give rise to RA-producing tolerogenic DC in the intestinal mucosa and are required to maintain immune homeostasis during inflammation and steady state

Gut-Tropic T Cells That Express Integrin α4β7 and CCR9 Are Required for Induction of Oral Immune Tolerance in Mice

Induction of oral immune tolerance (OT) blocks proinflammatory responses to orally administered antigens and might be used to treat autoimmune conditions. We investigated whether gut-tropic T cells that express the integrin α4β7 and the chemokine receptor CCR9 are required for OT. Skin delayed-type hypersensitivity and experimental autoimmune encephalomyelitis were used to monitor OT in mice. To assess the role of receptors that mediate localization of lymphocytes to the gut (gut-homing receptors) in induction of OT, we studied CCR9(-/-) and β7(-/-) mice and also blocked the α4β7 ligand MAdCAM-1 in wild-type mice. We used DEREG and Scurfy mice to assess the role of Foxp3(+) regulatory T cells (Treg) and IL-10(-/-) and IL-10Rβ(-/-) mice to examine the role of interleukin (IL)-10 in induction of OT. OT could not be induced in CCR9(-/-) or β7(-/-) mice, or when MAdCAM-1 was blocked in wild-type mice, indicating that gut-homing receptors are required for oral tolerization. Consistent with the role of all-trans retinoic acid in inducing gut-homing T cells, OT could not be induced in mice depleted of vitamin A. OT was rescued in CCR9(-/-) mice following adoptive transfer of wild-type T cells, but not CCR9(-/-) or β7(-/-) T cells. Gut-homing T cells are therefore necessary and sufficient to induce OT. Wild-type Treg and IL-10 were required to restore OT to CCR9(-/-) mice, indicating that homing and functional differentiation of IL-10-producing Treg in the gut is required for OT. Conversely, transfer of CCR9(-/-) or β7(-/-) T cells to wild-type mice partially inhibited OT. Expression of CCR9 and α4β7 on T cells and their subsequent localization to the gut is required for induction of OT in mice. Therapies designed to block gut-homing receptors might, under some conditions, interfere with normal tolerogenic mechanisms in the intestinal mucosa.

Clinical Aspects of Pediatric Food Allergy and Failed Oral Immune Tolerance

Food allergy seems to represent a new spectrum of disease that has elicited significant community concern and extended waiting lists for allergists and gastroenterologists alike. The apparent rise in prevalence of IgE-mediated food allergy (and associated risk of anaphylaxis) has been postulated to result from effects of a "modern lifestyle" but as yet clear environmental risk factors have not yet emerged. Family history seems to contribute to risk suggesting that gene-environment interactions will be important for identifying a subpopulation with increased susceptibility to any identified lifestyle effects. Non-IgE-mediated food allergy (including food-induced enteropathies and colitides, eosinophilic esophagitis, and Crohn's disease) with potentially similar environmental triggers resulting in diverse immune dysregulatory mechanisms. The evidence underpinning the putative rise in food allergy is discussed and potential mechanisms of disease explored. Clinical aspects of various food allergic conditions including non-IgE-mediated food allergy are outlined.

Induction of oral immune tolerance in systemic lupus erythematosus-like murine model by recombinant nucleosomal universal Th cell epitope

To explore the feasibility of oral immune tolerance of systemic lupus erythematosus (SLE)-like model induced by nucleosomal Th cell epitope via the attenuated Salmonella typhimurium. SLE-like murine model was established by immunization with apoptotic syngeneic lymphocytes. The recombinant strains were orally administrated to induce immune tolerance. The levels of serum autoantibodies, such as anti-ANA, ds-DNA, and antinucleosome antibody, leukopenia, proteinuria and kidney injuries were evaluated. SLE-like murine model was successfully established. Compared with controls, it was shown that CTLA4-Ig-H2B group could dramatically reduce the levels of serum autoantibodies, such as anti-ANA, ds-DNA and antinucleosome antibody and ameliorate leukopenia and proteinuria (all P < 0.05). Immune complex deposits of IgG in glomeruli were lower in CTLA4-Ig-H2B (1.35 +/- 0.16) than in CTLA4-Ig (1.66 +/- 0.23) and H2B (1.69 +/- 0.24) (both P < 0.05). The score of glomeruli lesion of CTLA4-Ig-H2B (1.26 +/- 0.14) was significantly lower than those of CTLA4-Ig (1.73 +/- 0.25) and H2B (1.71 +/- 0.20) (both P < 0.05). Combined with CTLA4-Ig, it is feasible to induce oral immune tolerance of SLE models with nucleosomal Th cell epitope via the attenuated Salmonella typhimurium. This may provide a novel way to prevent and treat SLE by oral immune tolerance.

β-Glycoglycosphingolipid-Induced Alterations of the STAT Signaling Pathways Are Dependent on CD1d and the Lipid Raft Protein Flotillin-2

Beta-glucosylceramide has been shown to affect natural killer T cell function in models of inflammation. We, therefore, investigated the effects of different beta-glycosphingolipids, including beta-glucosylceramide, on STAT (signal transducers and activators of transcription) signaling pathways and determined whether these effects were mediated by lipid raft microdomains and/or CD1d molecules. The effects of alpha- and beta-structured ligands on the lipid raft protein flotillin-2 were studied in both natural killer T hybridoma cells and leptin-deficient mice. To determine whether CD1d was involved in the effects of the beta-glycosphingolipids, an anti-CD1d blocking antibody was used in a cell proliferation assay system. The downstream effects on the protein phosphorylation levels of STAT1, STAT3, and STAT6 were examined in both immune-mediated hepatitis and hepatoma models. The effects of beta-glycosphingolipids on the STAT signaling pathways were found to be dependent on CD1d. Lipid rafts were affected by both the dose and ratio of the beta-glycosphingolipids and the acyl chain length, and these effects were followed by downstream effects on STAT proteins. Our results show that beta-glycosphingolipids have beneficial effects in natural killer T cell-dependent immune-mediated metabolic and malignant animal models in vivo.

The Intestinal Intraepithelial Lymphocytes with T Cell Receptor αβ Express Toll-Like Receptor 4 and Are Responsive to Lipopolysaccharide

Background: Intestinal intraepithelial lymphocytes (iIELs) play an important role in intestinal innate immunity and oral immune tolerance. To compare the differences in gene expression between murine iIELs and splenic T lymphocytes, we established the cDNA subtractive library of iIELs and analyzed the iIELs special genes. Our study focused on the relationship between Toll-like receptor 4 (TLR4), TLR5 and iIELs. Methods: Ninety percent purified iIELs and splenic T lymphocytes were isolated by density-gradient centrifugation in a Percoll and nylon column, respectively. We then established the cDNA subtractive library of iIELs via improved subtractive hybridization. The special expressed sequence tags of iIELs were screened by reverse Northern blot. The expressions of TLR4 and TLR5 were analyzed by RT-PCR and fluorescence staining. The proliferation of T cells was determined by ³H-TdR incorporation. Results: TLR4, but not TLR5, was detected in iIELs by RT-PCR and fluorescence staining. However, TLR4 was only found in αβ iIELs. Furthermore, iIELs were observed to proliferate in response to lipopolysaccharide in vitro, with upregulation of IRAK-1 mRNA expression. Conclusion: αβ iIELs can recognize lipopolysaccharide via TLR4, which may play an important role in the intestinal innate immunity.

Oral immune tolerance mediated by suppressor T cells may be responsible for the poorer prognosis of foregut cancers

The poor prognosis of foregut cancers might, in part, be due to the immune tolerising effect of tumour antigens which are shed into the gastrointestinal tract and processed by the gut immune system. This would create a tumour specific tolerance without compromise of global immune functions. Experimental data shows that orally fed cancer tissue induces a non cross reactive attenuation of the cellular anti tumour host responses and confers a growth advantage specific to individual cancers. Although the cellular basis of such pro-tumourogenic responses has yet to be established, it is likely, based on studies of oral tolerance mechanisms, that recruitment of immune suppressive T cells (T(regs)) may be responsible. Abrogation of oral immune tolerance to the tumour by immune based therapy could represent a significant advance in the management of upper gastrointestinal cancers.

Oral administration of a cholera toxin B subunit–insulin fusion protein produced in silkworm protects against autoimmune diabetes

The oral administration of disease-specific autoantigens can induce oral immune tolerance and prevent or delay the onset of autoimmune disease symptoms. Here, we describe the construction of an edible vaccine consisting of a fusion protein composed of cholera toxin B subunit (CTB) and insulin that is produced in silkworm larvae at levels of up to 0.3 mg/ml of hemolymph. The silkworm bioreactor produced this fusion protein vaccine as the pentameric CTB–insulin form, which retained the GM1-ganglioside binding affinity and the native antigenicity of CTB and insulin. Non-obese diabetic mice fed hemolymph containing microgram quantities of the CTB–insulin fusion protein showed a prominent reduction in pancreatic islet inflammation and a delay in the development of symptoms of clinical diabetes. These results demonstrate that the silkworm bioreactor is a feasible production and delivery system for an oral protein vaccine designed to develop immunological tolerance against T-cell-mediated autoimmune diabetes by regulatory T-cell induction.

Prolongation of skin allograft survival by combined feeding of donor spleen cells and cyclosporine in mice

Background Oral immune tolerance is a method for inducing donor-specific immunotolerance and prolonging graft survival. Objectives We studied the effect of feeding donor spleen cells in combination with cyclosporine (CsA) on skin allograft survival in mice. Methods Tail skins from BALB/c ( H-2 d ) female mice were transplanted onto C57BL/6 ( H-2 b ) female mice. The animals were divided into four groups, each with eight mice: group I, untreated controls; group II, treated with spleen cells; group III, treated with CsA; and group IV, treated with spleen cells and CsA. All grafts were inspected daily. Rejection was diagnosed when the graft loss was >80% to 90%. The immune responses of C57BL/6 toward donor mice were examined by delayed-type hypersensitivity (DTH). Results Survival times of allogeneic skin grafts in groups I, II, III, and IV were 9.9 ± 0.6, 13.1 ± 0.6, 14.7 ± 0.9, and 20.0 ± 0.7 days, respectively. When compared with group I, the survival times of groups II, III, and IV were prolonged significantly ( P < .01). The survival time for group IV was prolonged significantly compared with groups II and III ( P < .01). The DTH responses of group IV were decreased significantly in contrast to groups II and III ( P < .01). Conclusions Feeding donor spleen cells prolonged the survival of skin allografts in mice; combination with CsA led to further prolongation of skin allograft survival.

Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) and IL-4.

Induction of specific immunological unresponsiveness by feeding protein antigens is termed oral tolerance and may be a potential therapy for autoimmune diseases. Whereas oral tolerance therapy may be both simple and effective, the requirement for large amounts of protein will limit clinical testing of autoantigens, which are difficult to produce. We have previously demonstrated transgenic plant production and direct oral delivery of a beta cell autoantigen murine GAD67 to prevent autoimmune diabetes in nonobese diabetic mice. Mucosal adjuvants such as cholera toxin B subunit may lower the level of autoantigen required, but the development of neutralizing mucosal antibody responses may limit usefulness in enhancing long-term oral tolerance. IL-4, being an endogenous protein, would avoid this result and possibly enhance oral tolerance but has not been tested as a mucosal adjuvant. In this study, human GAD65 (hGAD65), as well as murine IL-4, was expressed in transgenic plants for feeding trials. Both IL-4 and hGAD65 plant tissue were required to protect nonobese diabetic mice from diabetes, and no benefit was found if either was used alone. Combined therapy enhanced levels of IgG1 anti-GAD antibodies, increased splenocyte IL-4/IFN-gamma cytokine responses, and produced protective regulatory T cells. These results demonstrate that orally administered plant IL-4 remains biologically active and is synergistic when given with hGAD65 in inducing robust oral immune tolerance. Using transgenic plants expressing IL-4 and GAD65 may be a novel clinical approach to the prevention of human type 1 diabetes by oral tolerance.

Oral immune tolerance to tumor specific antigens may confer growth advantage to esophageal and gastric cancers*

Oral administration of antigens induces antigen-specific systemic immune tolerance (Oral Tolerance). We postulate that the poorer prognosis of foregut cancers might, in part be explained by the systemic immune tolerizing effect of tumor specific antigens shed into and processed by the gut immune system thus conferring a growth advantage specific to individual cancers. Immunocompetent Balb/c mice were fed by gavage, either tumor tissue (JBS/CarB) in phosphate buffered saline (PBS) or PBS alone, daily for 14 days. On day 15 either subcutaneous tumors were induced or animals were immunized with cells in adjuvant. JBS tumors appeared earlier and grew faster in the JBS tumor fed mice than in either the PBS (P = 0.025) or CarB (P = 0.168) fed animals. The delayed type hypersensitivity response in tolerized mice was significantly abrogated (P < 0.01) compared to controls. These experiments demonstrate antigen specific oral immune tolerance for tumors, which is reflected in a faster growth rate and impaired delayed type hypersensitivity response. Similar mechanisms may be operational in human esophagogastric malignancy and may in part explain their poorer outcome.

Breastfeeding does not influence the development of inhibitors in haemophilia.

Our aim was to test the hypothesis that breastfeeding may reduce development of inhibitors in male infants with haemophilia by inducing an oral immune tolerance to factor VIII. To achieve that goal, we performed a structured epidemiological survey comprising all males born with severe haemophilia A (in all 100 patients, 19 with inhibitors) or haemophilia B (in all 16 patients, six with inhibitors) in Sweden in 1980-99. Our results show no protective effect of breastfeeding.

A transforming growth factor-beta1-mediated bystander immune suppression could be associated with remission of chronic idiopathic thrombocytopenic purpura.

Bystander immune suppression has been demonstrated in experimental models of oral immune tolerance induction. This phenomenon is associated with expression of transforming growth factor (TGF)-beta1 and T-helper cell (Th) 2 cytokines. We have studied serum levels of Th cytokines and B- and T-lymphocyte subsets in chronic idiopathic thrombocytopenic purpura (ITP), a disorder in which the production of platelet autoantibodies might be caused by a cytokine network dysregulation. Forty-six patients with ITP were separated into three groups depending on the platelet count (pltc): (1) < 50 x 10(9)/l, (2) 50-150 x 10(9)/l and (3) > 150 x 10(9)/l. We found significantly elevated plasma levels of the Th3 cytokine TGF-beta1 in patients with pltc >150x10(9)/l (23.5+/-2.8ng/ml), compared with patients with pltc <50x10(9)/l (2.3+/-0.6 ng/ml; P<0.0001), patients with pltc 50-150x 10(9)/l (7.2+/-1.7 ng/ml; P<0.0001) and healthy volunteers (9.8+/-1.3 ng/ml; P<0.01). The serum levels of the Thl cytokines interleukin (IL)-2 and interferon (IFN)-y were below the detection limits of the assays. Likewise, the Th2 cytokine IL-4 was not detectable or was very low both in patients and controls. The serum levels of IL-10, a Th2 cytokine, were within the assay range and patients with pltc <50 x 10(9)/l had significantly lower levels (0.6+/-0.1 pg/ml) than both patients with pltc 50-150 x 10(9)/l (1.8 +/- 0.1 pg/ml; P<0.005) and healthy volunteers (1.4+/-0.1 pg/ml; P<0.005). Furthermore, patients with pltc <50 x 10(9)/l and splenectomised patients had significantly higher levels of CD4 + CD25 + activated T cells [26.2 +/- 14.8% (P<0.05) and 26.7+/-11.9% (P<0.005), respectively] than healthy controls (16.5+/-4.0%). Also, the number of natural killer (NK) cells among patients with pltc >150 x 10(9)/l were significantly elevated (26.6+/-16.0%; P<0.05) compared with controls (17.4+/-7.6%). In conclusion, our data corroborate previous findings of elevated numbers of activated T cells in chronic ITP patients with active disease, but neither a clear-cut Th1 nor a Th2 serum cytokine profile could be established. However, ITP in remission was associated with elevated TGF-beta1, which might be a part of a bystander immune suppression. We propose that the effect of possible expression of TGF-beta1 by oral immune tolerance induction deserves to be explored in ITP patients with an active disease.

Liver-associated lymphocytes expressing NK1.1 are essential for oral immune tolerance induction in a murine model.

Oral tolerance is the induction of immunological hyporesponsiveness towards orally administered antigens. Tolerance initiation involves induction of anti-inflammatory (Th2) lymphocytes, with downregulation of pro-inflammatory (Th1) lymphocytes. The liver was previously shown to play a critical role in oral tolerance induction. The aim of the present study was to test whether liver-associated-lymphocytes expressing the NK1.1 marker (NK1.1+ LAL) are substantial for induction of oral tolerance in an experimental colitis model. Colitis was induced in C57 mice by intracolonic instillation of trinitrobenzensulfonic acid (TNBS). Mice received five oral doses of colonic proteins extracted from TNBS-colitis colonic wall. Anti-NK1.1 monoclonal antibodies were injected before tolerance induction. Colitis was assessed by standard clinical, macroscopic, and microscopic scores. Serum IFN-gamma, TGF-beta1, and IL4 levels were measured by enzyme-linked immunosorbent assay. To evaluate the role of NK1.1+ LAL in keeping the balance between immunogenic and tolerogenic subsets of cells, we tested whether peripheral lymphocytes harvested from tolerized and NK1.1-depleted nontolerized mice can adoptively transfer the tolerance into naive irradiated rats. Depletion of NK1.1+ LAL prevented immune tolerance induction in the experimental colitis model. NK1.1+ LAL-depleted nontolerized mice, disclosed severe clinical, macroscopic, and microscopic parameters of colitis. These mice had significantly lower TGF-beta1, IL4, and higher IFN-gamma serum levels, and their lymphocytes failed to transfer the tolerance into naive animals. In contrast, the feeding of colitis-extracted proteins, without NK1.1+ LAL depletion, markedly alleviated the disease. Tolerized mice had higher IL4 and TGF-beta1 and lower IFN-gamma serum levels, and adoptive transfer of their suppressor splenocytes markedly alleviated colitis in naive recipients. NK1.1+ LAL plays a critical role in oral tolerance induction. Depletion of this subset of LAL prevents a shift from Th1 to a Th2 type of immune response, hindering the ability to induce immune tolerance.

The effect of portacaval shunt on delayed-hypersensitivity responses following antigen feeding

The antibody responses to ingested antigens are normally enhanced following a portacaval shunt (PCS), but the cell-mediated immune response has not been examined. It is also known that prior feeding induces tolerance to soluble protein antigens. This study examines the effect of PCS on oral cell-mediated tolerance. Normal Lewis rats ( n = 16) and those ( n = 10) with an end-to-side portacaval shunt surgically created 7 days earlier were fed either 200 mg soluble ovalbumin (OVA) or water by daily gastric gavage for 7 days. One week later, all animals were challenged subcutaneously with 250 μg OVA admixed in complete Freund's adjuvant. Twenty-one days later, delayed-hypersensitivity (DTH) responses were measured 24 hr after injection of 100 μg OVA (in media), or an irrelevant antigen (SRBCs), into the pinna of the ear. The intensity of the response was reflected by the increment in ear thickness, as well as the histological intensity of the mononuclear cell infiltrate. Oral administration of OVA significantly and specifically abrogated the DTH response to subsequent challenge with OVA ( P < 0.001). This DTH hyporesponsiveness was significantly reversed by the creation of a PCS ( P < 0.001). We conclude that the initial processing of ingested antigen within the liver is essential for the development of oral cell-mediated immune tolerance.