Key points

Introduction

Oral tolerance is the active process by which the immune system does not respond to an orally administered antigen. The number of studies addressing oral tolerance in humans is surprisingly limited despite the extensive literature from murine models. In fact, animal models have largely been used to study both the mechanism of sensitization to food as well as the resulting allergic response from consuming a food allergen. Most available animal models of food allergy require an artificial sensitization method and may provide only limited insight into the sensitization phase of human food allergic disease. Thus, food allergy researchers have sought to develop an animal model that more closely mimics the sensitization of humans to food antigens. Until such a model, there may not be specific answers to the precise mechanisms that result in establishing oral tolerance or that lead to a break in tolerance. This review provides an overview of some available animal models, comments on other disease states and relevant models, and comments on possible future directions.

Introduction

Oral tolerance is the active process by which the immune system does not respond to an orally administered antigen. The number of studies addressing oral tolerance in humans is surprisingly limited despite the extensive literature from murine models. In fact, animal models have largely been used to study both the mechanism of sensitization to food as well as the resulting allergic response from consuming a food allergen. Most available animal models of food allergy require an artificial sensitization method and may provide only limited insight into the sensitization phase of human food allergic disease. Thus, food allergy researchers have sought to develop an animal model that more closely mimics the sensitization of humans to food antigens. Until such a model, there may not be specific answers to the precise mechanisms that result in establishing oral tolerance or that lead to a break in tolerance. This review provides an overview of some available animal models, comments on other disease states and relevant models, and comments on possible future directions.

Role of the gut immune system

The gut-associated lymphoid tissue (GALT) is the largest immune system in the body. Approximately 30 kg of food proteins reach the human intestine during a year, and 130 to 190 g of these proteins are absorbed daily in the gut. The microbiota in the intestine is an additional major source of natural antigenic stimulation with a perhaps underappreciated number of bacteria colonizing the human intestinal mucosa (∼10 ¹² microorganisms per gram of stool). The physiologic role of the GALT is the ingestion of dietary antigens in a manner that does not result in untoward immune reactions and protection of the organism from pathogens. This represents a careful balancing act, as the mucosal barriers are thin and vulnerable to pathogenic infection. It should be noted that tolerance to food protein affects local and systemic immune responses, whereas tolerance to gut bacteria in the colon does not attenuate systemic responses. Despite these distinct and active processes, the GALT is primarily a tolerogenic environment.

The features of the gut immune system that are important participants in creating the tolerogenic environment have been studied and discussed. Briefly, the inductive sites for immune responses in the gut are Peyer patches and mesenteric lymph nodes (MLNs). MLNs develop distinct from Peyer patches and peripheral lymph nodes and serve as a crossroads between the peripheral and mucosal recirculation pathways. To induce a mucosal immune response, an antigen must gain access to antigen-presenting cells by penetrating the mucus layer and then the intestinal epithelial cell barrier. Dendritic cells (DCs) themselves sample luminal contents by extending their processes through the epithelium without disruption of tight junctions. Another important component of the GALT is the intraepithelial lymphocytes (IELs), which serve to regulate intestinal homeostasis, maintain epithelial barrier function, respond to infection, and regulate adaptive and innate immune responses. Most IELs are CD8+ T cells, which express αβ or γδ T-cell receptors (TCRs). Of note, it has been reported that depletion of γδ T cells impairs induction of oral tolerance. Thus, the combination of commensals, T cells, and DCs set up a tolerogenic environment in the gut. Major factors that condition the gut to be a tolerogenic environment are interleukin-10, retinoic acid, and transforming growth factor-β (TGF-β), which serves as a switch factor for immunoglobulin (Ig)A, the predominant immunoglobulin of the gut.

Regulatory T cells

It is now recognized that there are multiple mechanisms of oral tolerance, and one of the prime determinants is the dose of antigen fed. Low doses favor the induction of regulatory T cell (Tregs), whereas higher doses favor the induction of anergy or deletion. These mechanisms are not exclusive, especially at higher doses. One of the major mechanisms of oral tolerance is the induction of Treg cells, a process that is related to the gut DCs and linked to both TGF-β and retinoic acid. Specifically, it has been shown that mucosal DCs induce forkhead box P3 (Foxp3) Tregs via the production of TGF-β, but that concomitant retinoic acid signaling boosted this process. In fact, all major classes of Tregs can be induced or activated by oral (mucosal) antigen. Even CD8+ Tregs have been shown to play a role in oral tolerance. Interestingly, CD8+ T cells have been shown to recall a tolerant or hyporesponsive phenotype following immune stimulation, suggesting that epigenetic mechanisms are in place to maintain tolerance. As future work progresses, it remains to be elucidated whether similar mechanisms may account for failure of programmed reactive cells to maintain hyporesponsiveness following oral immunotherapy.

Anergy

T-cell unresponsiveness or anergy is one of the primary mechanisms by which tolerance is maintained in self-reactive lymphocytes and anergy is induced in high-dose oral tolerance. The upregulation of anergy-associated genes is largely dependent on nuclear factor of activated T cells. Orally tolerized T cells can form conjugates with antigen-presenting cells, but they are defective in immunologic synapse formation. Similarly, T cells made anergic in vivo following oral antigen can inhibit the migration of responsive T cells in an antigen-independent fashion, indicating that hyporesponsive T cells have broad tolerogenic signals. Using a murine model to examine the role of the thymus in high-dose oral tolerance, researchers found that thymectomized animals were not protected from autoimmune disease. The thymus was actually found to be an important site for the development of CD4+CD25+ Tregs after oral antigen. In fact, clonal deletion was found in the periphery but not the thymus, suggesting that high-dose oral tolerance not only induces deletion but may lead to CD4+CD25+ Tregs that resemble natural Foxp3+ Tregs. These observations are in keeping with results from high-dose oral immunotherapy studies that have reported increased CD4+CD25+ Foxp3+ Tregs in subjects with clinical hyporesponsiveness.

Lessons learned from oral anti-CD3

The investigation of oral tolerance has classically involved the administration of oral antigen followed by challenge with same/similar antigen (albeit usually in an adjuvant) to demonstrate antigen-specific tolerance. One interesting experimental system that has been used to study T-cell function in oral tolerance is the use of TCR transgenic mice, in which all T cells have a common TCR. Using such mice, Dr Weiner and colleagues investigated how oral administration of an antigen affected specific T-cell subsets. These investigators showed a dose-dependent induction of Tregs to the fed antigen. In similar mice that have ovalbumin-specific TCR, high-dose oral administration of ovalbumin led to deletion of Treg subsets.

To translate these findings to humans, it first had to be known whether it was possible to trigger the TCR in wild-type mice in the gut and induce Tregs without using cognate antigen. Previous work had established that anti-CD3 binds to the ϵ chain of the TCR and, given intravenously, deletes T cells and has been shown to be an effective treatment for type 1 diabetes in the nonobese diabetic mouse. It was hypothesized that oral administration of anti-CD3 monoclonal antibody would replace the use of a cognate antigen to trigger the TCR and lead to induction of Tregs when given orally. Using an autoimmune encephalitis murine model, Ochi and colleagues found that oral anti-CD3 suppressed both clinical and pathologic features of the disease. Notably, there was a dose effect observed with disease suppression by oral anti-CD3 seen at lower, but not higher doses. The scientists suggested these findings were consistent with the classic paradigm of oral tolerance: induction of Tregs is seen at lower but not higher doses. Potentially important for all researchers interested in oral tolerance, it demonstrated that induction of Tregs by oral anti-CD3 was not simply related to administering large amounts of antibody to overwhelm breakdown in the gut. Also of significance was the finding that the Fc portion of anti-CD3 was not required, as anti-CD3 Fab’2 fragment was active orally and induced Tregs. The effects of these and similar experiments raise the question of whether it is more advantageous to induce antigen-specific versus antigen nonspecific Tregs for the treatment of relevant diseases, which is an issue being addressed in ongoing trials in humans.