The adaptive immune system is found in vertebrate organisms and is composed mainly of antibody-secreting cells (B cells) and specialized T cells (which have distinct functions in immune response) and can eliminate pathogens and generate tolerance to antigens. Antigens are useful for avoiding attacks against the body's own cells and preventing excessive responses against external antigens.²³

Oral tolerance is defined as the suppression of the immune response to antigens that have been previously administered orally.¹ This is a form of peripheral tolerance in which attempts are made to treat external agents that come into contact with the body through the mouth as if they were internal components, thus making them part of the individual. In other words, it is a method to induce immune tolerance systemically.²⁴ The term oral tolerance was first used in 1911 when Wells fed chicken egg proteins to guinea pigs and saw that they were resistant to anaphylaxis.²⁵ Although many researchers have tried to reproduce these results,²⁶ they have not succeeded because tolerance is an active immune event that involves multiple factors such as the dosage of the antigen, the human microbiota, and the co-stimulation of these components.³

When given orally, an antigen is initially found in gut-associated lymphoid tissue (GALT), which is the largest immune organ in the human body.²⁷ The function of this system is the ingestion and recognition of dietary antigens to avoid unwanted immune responses and protect the body against pathogens, thus allowing for a tolerogenic environment.²⁸ GALT comprises epithelial cells, intraepithelial lymphocytes and lamina propria lymphocytes in the form of lymphoid nodes (known as Peyer's patches) located in in the lowest portion of the small intestine and in the mesenteric lymph nodes (Figure 2).

Figure 2
Gut-associated lymphoid tissue system.
Source: Own elaboration.

Another important part of GALT is intraepithelial lymphocytes, which regulate intestinal homeostasis, maintain barrier function, respond to infection, and modify the adaptive and innate immune response. The most important intraepithelial lymphocytes are CD8+T cells. ³

To induce a mucosal immune response, the antigen must gain access to antigen-presenting cells (APCs) by penetrating the mucus layer and the epithelial cell barrier. These antigens are transported through mechanisms, most commonly M cells associated with Peyer's patches,²⁹ which internalize antigenic proteins through phagocytosis and endocytosis, taking them to the extracellular space where they are processed and presented by lymphocytes and macrophages.³⁰ Other mechanisms used by antigens to access APCs is dendritic cell extension into the intestinal lumen³¹ and through enterocytes (located in Peyer's patches), which capture soluble antigens, processing them and presenting them to the effector cells. ⁴

The induction of the immune response occurs after processing the antigen captured in the intestinal lumen. To this end, APCs express the antigen through molecules of the major histocompatibility complex, allowing T lymphocytes to recognize it. In addition, depending on the microenvironment, T-lymphocytes can be classified as regulators or effectors; the former are responsible for oral tolerance, while the latter are involved in cytolytic activity³² (Figure 3).

Figure 3
Antigen presentation and cell selection.
ACP: Antigen-Presenting Cell; MHC: Major Histompatibility Complex; IL: Interleukins; TGF-β: Transforming Growth Factor Beta; TL: T Limphocyte or T Cell. Source: Own elaboration.

Enterocytes play a key role in immune tolerance because, besides being a mechanical barrier against foreign substances, they react intelligently to the heavy antigenic load of the gastrointestinal mucosa. This type of cell has specialized receptors that recognize pathogens such as Toll-like receptors (TLRs) and NOD-like receptors (NLRs), which are sensors of molecular patterns of bacteria and stimulate the inflammatory mechanisms that activate the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). ³³

With respect to the maintenance of immune tolerance, enterocytes express a limited variety of TLRs (subtype TLR-2) in their apical region,⁵ which activate the 3-phosphokinase pathway when stimulated with peptidoglycans, which in turn stimulates a negative regulation of NF-kB.^34,35

Enterocytes have two main proinflammatory cascades, one mediated by NF-kB, as mentioned above, and the other by p38, a mitogen-activated protein kinase. The inhibition exerted by both pro-inflammatory cascades on the enterocytes is the mechanism that maintains immune tolerance in the intestine. This way, NF-Kb activation produces the downregulation of p38 because the former induces the activation of a protein phosphatase kinase-1 (MKP-1) that dephosphorylates the latter³⁶ and promotes tolerance by inhibiting one of the major pro-inflammatory cascades.

Immune tolerance is defined as the absence of a specific response against specific actively acquired antigens. Tolerance mechanisms occur via T cells and B cells and can be established centrally (central tolerance) during cell genesis and differentiation, and peripherally (peripheral tolerance) on already differentiated adult cells:^6,7

Central tolerance. During the embryonic stage and neonatal period, immature cells migrate from the bone marrow to the thymus to express receptors that recognize peptides of the major histocompatibility complex. T cells that recognize these complexes with high avidity survive; this process is known as positive selection.³⁷ In contrast, T cells that weakly recognize such complexes die, and this is known as negative selection,³⁸ which is the main mechanism for regulating self-tolerance;³⁹ the surviving T-cells migrate to secondary lymphoid organs.⁶ An additional mechanism of immune tolerance is the modification of the T cell receptor to allow it to bind to interleukine-7 (IL-7)-like cytokines, which induces the formation of regulatory T cells (Treg) or lymphocytes.⁸Peripheral tolerance. Mechanisms of peripheral tolerance include: anergy, immunological ignorance, clonal deletion, active suppression and Treg cells.

1. 1. Anergy: It occurs when, despite the existence of the first signal (CD3) to trigger T-lymphocyte response - in other words, the recognition of the MHC+ peptide junction-, there is no second signal (co-stimulation), which causes no response from the T-lymphocytes.⁸
2. 2. Immunological ignorance: It occurs in the absence of T-cell activation due to low concentrations of the antigen, which does not induce the first signal.⁴⁰
3. 3. Clonal deletion: It refers to lymphocyte apoptosis by caspase activation.⁴¹
4. 4. Activa suppression: Cell activity is suppressed through the secretion of inhibitory cytokines such as transforming growth factor B (TGF-β) and interleukin 10 (IL-10) by Treg cells. ⁸
5. 5. Treg cells: They are dominantly responsible for controlling the immune response. ⁹

Central tolerance. B cells are formed, expressed, and matured in the bone marrow; however, they can be highly avid (leading to clonal deletion) or moderately avid (leading to receptor editing) due to autoantigens. ⁴²

Peripheral tolerance. Surviving B cells migrate to the periphery and those that are highly avid for autoantigens are eliminated through the intrinsic apoptosis pathway. Low-affinity B cells enter into partial anergy because if they are exposed to high doses of the antigen, they can be re-recruited. ⁴³ It should be noted that the dose of antigen taken orally is a fundamental determinant of the immune response: low doses induce tolerance via Treg lymphocytes, while high doses induce anergy or clonal deletion.^25,44,45

One of the best ways to understand oral tolerance is the identification of the role of dendritic cells, retinoic acid and CD103+ lymphocyte differentiation cluster in their induction. ³ The expansion of dendritic cells in vivo improves oral tolerance, since they induce the expression of Treg FOXP3+ cells when found in the mucosa by means of TGF-β and retinoic acid signaling pathways.³ Dendritic cells in the mucosa can be divided into two types: CD103+ (tolerogenic) and CD103- (non-tolerogenic). Tolerogenic cells produce retinoic acid and induce FOXP3+ Treg cells if given TGF-β. ^12,46

There are other innate immune cells that are relevant for oral tolerance. On the one hand, macrophages are found in the lamina propia of the intestine and produce IL-10. On the other hand, dendritic cells are found in the gut; they produce p-catenin, which stimulates the production of retinoic acid, IL-10 and TGF-Β; trigger the proliferation of Treg cells; and inhibit the response of effector T cells.³

The importance of the tolerance mechanisms in the intestinal mucosa described above lies in understanding that regulatory lymphocytes do not travel from other lymphatic organs to the site of response, but that oral antigens induce the formation of Treg cells.

When dendritic cells recognize antigens in the gastrointestinal tract, the inflammatory response istriggered, resulting in up to 80% anergy and deletion for each antigen. This is known as primary response and its effector organ is the liver; the secondary immune response occurs in the mesenteric nodules with the presentation of antigens by the tolerogenic dendritic cells.¹⁰

Treg cells are the most widely studied cells in relation to oral tolerance induction.^11,47 Currently, operational tolerance after transplantation, which is understood as the long-term survival of a graft in the absence of maintenance immunosuppressive therapy, ^13,48 is mediated by an antigen-specific response caused by FOXP3 expressing CD4+ CD25(high) regulatory T cells. These cells control the immune response against the donor's alloantigens. ⁴⁹

Of all Treg cells, the most relevant for transplantation are CD4+ CD25+ regulatory T cells²² and FOXP3+ transcription factor expressing cells.⁵⁰ The introduction of this transcription factor into CD4+ CD25+ T cells gives them the ability to suppress and amplify the transcription of specific regulation genes. ⁴⁹

Type 3 and 4 immunoglobulin transcriptase enzymes favor the induction of co-stimulatory molecules in CD4+ CD25+ Treg cells. This makes the latter tolerant to donor antigens by stimulating CD4+ T cells and natural killer cells, which produce positive feedback on CD4+ CD25+ Treg cells to express the FOXP3 protein. As a result of this expression, an antigenic microchimerism is formed, and it allows immune tolerance and prevents rejection of the transplanted organ (Figure 4).⁵¹

Figure 4
Antigenic microchimerism.
Ig: immunoglobulin; CD: cluster of differentiation; NK: natural killer cell; FOXP3: forkhead box P3. Source: Own elaboration.

The main advantage of immune tolerance is its specificity since it maintains the immune response against neoplasms and microorganisms. However, there is a decreased response to alloantigens in the transplanted organ.⁴⁹

It is known today that the administration of oral antigens induces the production of Treg cells, particularly, induced CD4+ CD25+ FOXP3+ Treg cells, natural CD25+ FOXP3+Treg cells, Tr1 and CD8+ cells.³ Regarding these subclasses, Tr1 cells act through cell contact, unlike the others, which do so through suppressive cytokines such as IL-10 and TGF-β. Tr1 cells suppress the response of virgin T cells, the expression of co-stimulatory cells, and the secretion of pro-inflammatory cytokines by APCs (Figure 5). ⁽⁴⁹

Figure 5
Mechanisms of immunological tolerance.
CD: cluster of differentiation; TGF-β: transforming growth factor-beta; IL: interleukin; Treg: regulatory T lymphocyte; Th: helper T lymphocyte; MAMPs: microorganism associated molecular pattern; TLR: Toll-like receptor; FOXP3: fork-head box P3; PIP3: phosphatidylinositol 3-phosphate; NF-Kb: nuclear factor kappa-light-chain-enhancer of activated B cells; NLRs: NOD-like receptors. Source: Own elaboration.

Because Treg cells are known to mediate tolerance induction and maintenance of their effect over time, researchers such as Scalea et al.¹¹ state that adoptive transfer of this type of receptor-derived cells may lead to stable graft tolerance.

During solid organ transplantation, the immune system generates a response that is attributed to rejection, which is mediated by antibodies, T cells or vascular disturbances; in this response, antibody rejection is the worst prognosis.^52,53

Immunosuppressive drugs used in transplants produce a large number of side effects,⁵⁴ including nephrotoxicity, malignancy, hypertension, diabetes and infections. Cytomegalovirus infection is one of the most common. ^55-57

In Colombia, the first kidney transplant was performed at the Hospital San Juan de Dios in Bogotá in 1963.⁵⁸ Since then, transplantation surgeries and the amount of places where these procedures are performed have increased, and today there are about 25 hospitals authorized to perform solid organ transplantations in the country.⁵⁹ Nevertheless, several obstacles must be faced to achieve successful transplants, for example, graft rejection due to lack of immunological tolerance.

As described above, one of the methods for achieving immunological tolerance is the oral route since the intake of alloantigens or soluble human leukocyte antigen from the donor allows the immune system to make an initial recognition through the APCs and the major histocompatibility complex in the gastrointestinal tract. This way, when the transplant is done, the recipient's immune system will not trigger a rejection response, as tolerance to the donor's antigens will already have been generated.

One of the most promising results in induction of immune tolerance to a transplanted graft was found in a study by Kelman et al.,¹⁴ which describes what happens to the immune system during pregnancy. These authors state that there is tolerance between the molecules of the human leukocyte antigen system of the fetus and the immune system of the mother and report a correlation between oral sex and swallowing sperm and a lower incidence of preeclampsia. Said results allow us to presume that the oral route is a useful mechanism to induce tolerance.

Most of the evidence found comes from murine models with heart, kidney, cornea, and bone marrow transplants, as shown in Table 2.

Table 2
Studies of oral tolerance induction and their results.

Probiotics, herbal medicine, COX-2 inhibitors, monoclonal antibodies and CD4 lymphocytes from the donor are some of the antigens that have been used to induce immunological tolerance.

The use of probiotics in transplant recipients was considered an alternative when changes in the microbiota were evident in the subjects (humans and mice) that rejected the graft. In this regard, Finney et al.⁷² found increased Bacteroides and Ruminococcus colonies in specimens that rejected liver transplantation in murine models; in contrast, Taur et al.²⁰ observed decreased Bacteroides colonies in human models that presented acute rejection to kidney transplantation. Therefore, the use of probiotics has been proposed as a way to modify the microbiota and the immune system, not only at a local level but also at a systemic level. ^2,21

Probiotics have demonstrated good survival rates after bone-marrow transplantation in murine models because its administration stimulates both anti-inflammatory and pro-inflammatory signals. ⁶² These microorganisms are responsible for maintaining the integrity of the epithelial barrier of the gastrointestinal tract by stimulating immunoglobulin A and the secretion of mucus and defensins, and by altering the adhesion of bacteria to the epithelium.

In 2004, Gerbitz et al.⁶³ found that the administration of Lactobacillus rhamnosus before transplantation improved long-term survival and reduced rejection associated with decreased CD3 levels.

The COX enzyme is responsible for prostaglandin secretion, which has two isoforms: COX-1, a constitutive enzyme, and COX-2, induced by the action of cytokines and tumor promoters. In 1990, Stonc et al.²² proved that the use of COX-2 inhibitors prolongs the survival time of cardiac grafts in mice, and in 2005, Yokayama et al.¹⁶ noted that its use prolongs transplant survival beyond 100 days versus 11 days in the control group.

Given their immune system modulating characteristics, medicinal plants are another type of antigen used throughout history to sensitize the donor to transplants. ⁶⁴ For example, in Asia, these plants have been used as alternative therapy for different diseases in humans for more than 3 000 years; in the case of transplants, it has been reported that their use in murines with heart transplants increases the survival time of the graft. ¹⁷

Medicinal plants have been studied at the molecular level, finding that they are made up of multiple components. For example, the administration of Tokishakuyaku-san, also known as Tsumura TJ-23, at a minimum dose of 2 g/kg in murine models has yielded good results in terms of cardiac graft survival. ⁶⁴

Oral tolerance induction studies in humans also include the use of anti-CD3 monoclonal antibodies (OKT3). Its immunological effects in peripheral blood were the suppression of Th1 and Th17 lymphocyte response, increased expression of Treg cell markers, increased TGF-β/IL-10 and decreased expression of IL-23/IL-6 in dendritic cells without side effects. ¹⁸ Likewise, studies in mice have shown how the intake of CD4 lymphocytes from the donor decreases the induction of pro-inflammatory cascades and increases IL-10 levels in cases of spleen transplantation. ^19,65-67

Finally, combined therapy of CD4 lymphocytes and anti-CD3 orally demonstrated promising results in kidney and heart transplantation in murine models by prolonging the survival of the grafts by more than 100 days. ^68,69,71 None of the interventions described above have been studied in humans, so despite having a physiological basis, these results cannot be extrapolated or presumed to be safe in a human context. However, the use of oral immunological tolerance induction strategies opens a door to the study of new practices to treat chronic diseases and manage side effects of immunosuppressive drugs, immune transplant rejection, autoimmune diseases, among others, which may generate high-quality evidence to create novel strategies in the field.

^*Corresponding author: Juan Felipe Rivillas-Reyes. Grupo de Trasplante de Órganos y Tejidos Humanos, Departamento de Morfología, Facultad de Medicina, Universidad Nacional de Colombia. Carrera 30 No. 45-03, building: 471, office: 124. Telephone number: + 57 1 3165000, ext.: 15105; mobile number: +57 3134066589. Bogotá D.C. Colombia. Email: jfrivillasr1994@gmail.com.