Immunological mechanisms of tolerance: Central, peripheral and the role of T and B cells

Abstract

T and B cells are key components of the adaptive immune system. Through their immune properties and their interactions with other immune cells and cytokines around them, they build a complex network to achieve immune tolerance and maintain homeostasis of the body. This is achieved through mechanisms of central and peripheral tolerance, both of which are associated with advantages and disadvantages. For this reason, the immune system is tightly regulated and their dysregulation can result in the subsequent initiation of various diseases. In this review, we will summarize the roles played by T cells and B cells within immune tolerance with specific examples in the context of different diseases that include allergic disease. In addition, we will also provide an overview on their suitability as biomarkers of allergen-specific immunotherapy.

1. Introduction

Immune tolerance is crucial for the maintenance of homeostasis and health. When immune tolerance is dysfunctional and unable to maintain a balanced homeostatic environment, disease can occur. Immune tolerance is defined differently depending on the disease type. For example, in the case of transplantation, tolerance is defined as graft acceptance without the need for continued immunosuppressive therapy. Whereas in allergy, tolerance manifests as prolonged unresponsiveness to allergen challenge and exposure after desensitization therapy. In the context of autoimmune disease, tolerance is achieved when long-term improvement of disease symptoms has been achieved, accompanied by a decrease in the need for disease-modifying therapy. In other diseases such as cancer, tolerance typically refers to the ability of tumor cells to evade the body’s immune system, resulting in uncontrolled cell proliferation and metastasis.

Immune tolerance represents the long-term or permanent constraint of potentially harmful immune responses towards innocuous stimuli. This regulation is achieved through both central and peripheral immune mechanisms targeting T and B lymphocytes. Central tolerance is a process in which autoreactive lymphocytes are selectively prevented from entering the periphery and occurs in either the bone marrow or the thymus. B cells mature and undergo central tolerance via receptor editing in the bone marrow, while central tolerization of T cells occurs in the thymus via negative selection. In either case, autoreactive cells are efficiently eliminated during this process. Despite the rigor of central tolerance, the system is imperfect, and therefore autoreactive T and B cells may still escape into the periphery. Unwanted peripheral immune activation is inhibited by peripheral tolerance mechanisms, including deletion and anergy [1]. If peripheral tolerogenic balance is lost, various diseases can occur. Therefore, many studies have focused on understanding the balance between effector and regulatory compartments in health and disease in hopes of manipulating the underlying mechanisms for therapeutic benefit. This review will discuss T cell and B cell tolerance separately, focusing on recent advances and research findings in the maintenance of tolerance and homeostatic balance.

2. T cell tolerance

Central tolerance of T cells occurs in the thymus, where hematopoietic lymphoid progenitors migrate to develop into CD4⁺CD8⁺ double-positive (DP) thymocytes expressing the T cell antigen receptor (TCR). DP cells undergo positive and negative selection in the cortex and medulla of the thymus respectively, based on the affinity of their TCR for self-peptide-associated major histocompatibility complex class I (MHC-I) or MHC class II (MHC-II) molecules. In the cortex, all DP thymocytes capable of binding pMHC molecules presented by cortical thymic epithelial cells are positively selected for and continue into the thymic medulla. Those that fail to interact with pMHC molecules undergo apoptosis [2]. In the medulla, the level of TCR affinity for pMHC molecules presented on the surface of thymic dendritic cells (DCs) and medullary thymic epithelial cells determines their fate [3]. T cells carrying TCRs with high affinity are deleted, those with intermediate affinity are diverted into regulatory T cells (Tregs), and those with low affinity become conventional T cells and exit to the periphery (Fig. 1). However, central tolerance is not infallible, and some self-reactive T cells may still escape to the periphery. Peripheral tolerance mechanisms, such as Treg regulation and the exhaustion, anergy, or deletion of self-reactive T cells, are mainly relied upon to suppress the activity of self-reactive T cells (Fig. 1).

Figure 1.

The mechanism of T cell tolerance. Central tolerance of T cells occurs in the thymus. Hematopoietic lymphoid progenitors migrate to cortex, where they are screened by thymic epithelia cells and develop into CD4⁺CD8⁺ double-positive (DP) thymocytes expressing the T-cell antigen receptor (TCR). DP cells migrate to the medulla. After screening for the ability to bind to self-peptide-associated major histocompatibility complex class I (MHC-I) or MHC class II (MHC-II) molecules on the antigen-presenting cell (APC) surface, DP cells develop into CD4⁺CD8⁺ DP thymocytes expressing the TCR. Screening for binding capacity of molecules culminating in Treg CD8⁺ T cells, and CD4⁺ T cells migrate to lymph node undergo peripheral tolerance. Peripheral tolerance mechanisms, such as exhaustion, deletion, anergy, and regulation are mainly relied upon to suppress the activity of self-reactive T cells. Exhaustion and deletion are realized through inhibiting cell function or proliferative capacity of effector T cell and apoptosis. Anergy describes a state of hyporesponsiveness of naive T cells to TCR stimuli after exposure to antigen. Treg cells regulate by inhibiting effector T cell activity and B cell proliferation via interleukin-(IL)-10, Transforming growth factor-β (TGF-beta), and IL-35.

3. Exhaustion

Although not typically associated with tolerance, T cell exhaustion represents an important mechanism of tolerance in specific immunological situations. Phenotypically, exhausted T Cells (Tex) have evidence of reduced functional and proliferative capacities and display elevated levels of inhibitory receptor expression, including that of Programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte-activation gene 3 (LAG3), Cluster of differentiation 244 (CD244), Cluster of differentiation 160 (CD160), T Cell immunoreceptor with Ig and ITIM domains (TIGIT), Cluster of differentiation 38 (CD38), Cluster of differentiation 39 (CD39), and T cell immunoglobulin (Ig) domain and mucin domain-containing protein 3 (TIM3) [4]. These exhausted T cells are classically associated with chronic viral infection and cancer; both instances in which T cells experience prolonged antigen stimulation. In fact, T-cell failure or “exhaustion” was first described in chronic viral infections in mice [5]. However, in recent times it has been argued that Tex cells’ reduced functional capacity may not be an unavoidable consequence of prolonged antigen stimulation, but a designated differentiation program [6]. There has yet to be definitive evidence that Tex cells in chronic infection derive from those active T cells seen in early infection, in fact some T cells seen in early infection already display some of the characteristics associated with exhausted T cells such as a monofunctional cytokine signature [7]. It is therefore possible that T cells with ‘exhausted’ phenotypes are generated in parallel with Tconv cells in early infection, but their population dominance occurs later in infection, during the chronic phase. In cases of T cell exhaustion in cancer, this non-functional phenotype can be reversed through use of checkpoint blockade of PD-1/PD-L1, indicating the cells’ functional capacity is not itself diminished. The exhausted phenotype may therefore describe cells experiencing increased resistance to activation rather than being intrinsically incapable of such.

Although counterintuitive at first, this less aggressive, exhausted T cell state has been proposed to protect the host whilst still managing the disease to optimise disease outcome. In other words, it is a form of tolerance designed to protect the host [8]. In a murine study by Cornberg et al [9], they describe how mice infected with lymphocytic choriomeningitis virus (LCMV) at high concentrations experienced T cell exhaustion and very little immunopathology, however mice given an intermediate dose of virus experienced only partial T cell exhaustion but suffered significant immunopathology in the liver and lungs, leading to severe disease and death. This outcome strongly supports the idea that exhaustion is a protective mechanism designed to protect the host during periods of prolonged activation. The benefits of this are clear in terms of autoimmunity to self-antigen, graft vs. host disease [10] and in response to chronic viral infections. Through prevention of ongoing inflammation, tissue damage, and chronic auto-reactivity which are damaging to the host, Tex cells may be seen as essential for long-term host survival.

Although T cell exhaustion can increase the severity of chronic disease by leading to tumour growth and persistent viral infections, there is also evidence of their ability to continue to enact disease control. For example, one hallmark feature of multiple chronic infections is mutated T cell epitopes, the presence of which evidences T-cell driven selective pressure, despite their ‘exhausted’ state in cases of established chronic infection. In addition, it has been shown that removal of CD8 T cells in Macaques infected with chronic SIV results in rapid increases in viral titre [11], supporting the idea that T cells still maintain some functionality and in this case display continued anti-viral activity despite prolonged antigen stimulation. Overall, although T cell exhaustion is not typically considered a peripheral tolerance mechanism, it has been shown to be essential for long-term host survival and is in essence an immune mechanism which prevents immune cell activity that is damaging to the host, which is the core of what immunological tolerance is.

4. Anergy

T cell anergy describes a state of hyporesponsiveness of naive T cells to TCR stimuli after exposure to antigen. Anergy is established by the activation of intracellular pathways triggered by the interaction of the TCR with pMHC in the absence of costimulation [12]. When T cells encounter antigens, they require 2 signals to be fully activated. The first signal is the binding of the TCR to the antigen presented on the antigen-presenting cell (APC), and the second signal is the binding of costimulatory molecules on the APC. In the absence of costimulation, T cells become unresponsive.

This differs from quiescence and ignorance. Quiescence is independent of antigen-TCR interaction and refers to a state in which T cells are at rest or inactive but still alive and metabolically active. Ignorance occurs when T cells are not able to recognize a particular antigen, either because the antigen is not properly presented to them or because the T cells lack the specific receptor required to recognize the antigen.

T cell anergy is considered a peripheral tolerance mechanism. T cell anergy can be broadly categorized as either clonal anergy or adaptive tolerance. Clonal anergy is a state in which T cells do not proliferate but still produce a limited amount of effector cytokines. Its function is maintained without continuous antigen exposure, and this form of T cell anergy can be reversed by interleukin (IL)-2 [13]. Conversely, adaptive tolerance maintains anergy in response to prolonged antigen stimulation, inhibiting both cell proliferation and cytokine production. Strong TCR signaling may induce anergy or deletion in CD8⁺ T cells, which generally express both inhibitory receptors CTLA-4 and C-C chemokine receptor type 7 (CCR7). It has not been fully determined whether differences in TCR signaling affect the induction of CD4⁺ T cell anergy. However, it has been suggested that low-affinity TCR interaction with pMHC can induce anergy, resulting in CD4⁺ T cells that lack the capacity to proliferate or produce IL-2 [14]. Alteration of the timing of the TCR-pMHC interaction can also lead to CD4⁺ T cell anergy, for example, a short-duration TCR-pMHC interaction or low TCR signaling [14, 15]. Overall, the identification of anergic CD4⁺ T cells in humans has been challenging, especially considering the lack of distinct markers. Evidence currently exists in murine models in which a high expression of CD73 and FR4 has been observed in anergic cells. Whether they are involved in the induction or maintenance of anergy function, and whether they can be used exclusively as markers of anergic CD4⁺ T cells in humans, is currently subject to further study [16].

Although stimulation of the TCR is the main driving force behind the hyporesponsiveness and resulting dysfunction of both Tex and anergic T cells, these states are unique. The main difference between anergy and exhaustion is that the signals that induce these 2 cell types are different. Anergic T cells are a product of co-stimulation-deficient T-cell activation, while Tex cells are T cells which have experienced prolonged TCR and costimulatory molecule stimulation [17].

Anergy helps to prevent the immune system from attacking healthy tissues [18]. However, T cell unresponsiveness has also been implicated in disease, such as cancer or chronic infection, where T cells become unresponsive and fail to generate an effective immune response. Again highlighting the careful balancing act that is immune tolerance, and its potential to be exploited in novel theraputic therapies [19].

5. Deletion

T cell deletion is a tolerance mechanism most associated with central tolerance; however, there is evidence that this also occurs in the periphery. Evidence for this form of tolerance was shown by Chen et al [18]. They demonstrated that oral administration of antigen led to apoptotic deletion of antigen-specific T cells peripherally in the Peyer’s patches of mice [20]. Following this initial discovery, the cellular mechanism of this form of peripheral tolerance was investigated by Davey et al [19]. Using a transgenic OVA-expressing murine model, it was shown that tolerance against OVA-specific CD8⁺ T cells was maintained by cross-presentation of OVA by DCs and subsequent deletion of OVA-specific T cells. Investigation of the molecular mechanism found that this deletion failed upon overexpression of BCL-2, indicating BCL-2 suppression is essential for this form of peripheral tolerance [21]. The location of peripheral deletion investigated by Carlow et al [20]. They found that when female mice, that were made to express T cells specific for a male antigen, were injected with male lymphoid cells, apoptotic phenotypes of their male antigen-specific CD8⁺ T cells were quickly observed in secondary lymphoid organs. This suggested that secondary lymphoid organs are the site of peripheral T cell deletion [22, 23]. Collectively, there is mounting evidence that cell deletion is a peripheral tolerance mechanism for T cells. However, these studies have been conducted in mice and therefore may not perfectly describe the tolerance mechanisms in humans. This form of peripheral tolerance is important for termination of immune responses. Towards the ends of an immune reaction, T cells begin expressing death ligands such as Fas which facilitate their deletion and terminate the immune response. The importance of this has been demonstrated in mice. It has been shown that mice lacking Fas ligand experience extensive self-reactivity and autoimmune reactions [24]. Similarly, in the human disease, ALPS in which there are mutations in Fas or FasL leading to autoimmune reactions and lymphadenopathy [25].

6. Regulation: Tregs

Regulation is an important mechanism of T cell tolerance, mediated mainly by Tregs. In the periphery, Tregs with a self-skewed TCR repertoire are very sensitive to self-antigens and microbial antigens; much lower concentrations (from 10 to 100 fold) of peptide/MHC will activate Tregs [22]. Tregs suppress the activity of both autoreactive T cells that escaped central deletion and T cells that cross-react with self-antigen as a result of molecular similarity with microbial antigens [23]. In the early 2000s, human CD25⁺CD4⁺ Tregs were discovered and were thought to have the same phenotype and function as mouse Tregs, playing an important role in antitumor, antimicrobial, and graft immunity. Following the discovery of CD25⁺CD4⁺ natural Tregs (nTregs), it was discovered that these cells specifically expressed transcription factor Foxp3, and that deletion or mutation of FoxP3 led to the absence or hypofunctioning of nTregs. Loss of nTreg function results in a range of systemic immune diseases or infections, such as type I diabetes, allergies, and inflammatory bowel disease [26]. There are more than 70 Foxp3 mutant genes reported so far, 40% of which can cause severe autoimmune disease [27], and the rest can also cause varying degrees of symptoms. Immune dysregulation polyendocrinopathy enteropathy, X-linked (IPEX) syndrome is one example of a disorder that is characterized by dysfunction of Tregs, which can result in immune dysregulation. The first report of Treg transplantation for the treatment of immunodeficiency diseases was carried out in FoxP3 mutant mice, which provided the basis for the subsequent treatment of human IPEX disease and other autoimmune diseases [26].

Tregs can be divided into various subsets based on their developmental pathway; thymus-derived Tregs (tTregs), peripherally-derived pTregs, and in vitro-induced Tregs (iTregs). tTregs, differentiate from CD4⁺ thymocytes [27]. They mainly recognize self-antigens and constitute a significant proportion of FoxP3⁺ Tregs within the periphery. While some CD4⁺CD25-conventional T cells (Tconvs) can express Foxp3 stably, they eventually become peripherally-derived Tregs (pTregs). Tconvs that are stimulated under special conditions in vitro, can differentiate into iTregs with the expression of Foxp3. The specific TCR of pTreg can recognize infectious antigens and harmless microbial antigens, an important process for mucosal tolerance. In mice, this distinction between pTregs and tTregs can be made using the surface marker Neuropilin 1 (NRP1), which is found exclusively on pTregs [28]. However, there is no way to distinguish pTregs from tTregs in humans yet. Despite the lack of distinction, in general, Tregs are defined as Foxp3⁺CD25⁺CD127^low/−. The immunosuppressive function of Tregs in vivo or in vitro relies on multiple mechanisms. CD39 and CD73, which are highly expressed on the surface of Tregs, catabolize ATP into adenosine. Adenosine is then able to bind the adenosine A2a Receptor (ADORA2A). Binding of ADORA2A on Teff cells can promote their differentiation into Tregs, skewing immune activity towards tolerance. In addition, adenosine can also promote tolerogenic APC activity, reducing their ability to present antigen to and activate Teff cells. Adenosine thus both directly and indirectly inhibits Teff cell activity [29]. Contact between FoxP3⁺ Treg-expressing CTLA-4 and DCs induces DCs to express indoleamine-2, 3-dioxygenase (IDO), which activates the kynurenine metabolic pathway. In this pathway, tryptophan is metabolised into kynurenine. This significantly affects Teff cells which are highly dependent on tryptophan for many of their effector functions [30]. In addition to the above mechanisms, immunosuppression by Tregs can also be achieved by humoral factors such as anti-inflammatory cytokines (IL-2, IL-10, Transforming growth factor-β [TGF-β], and IL-35) or secreted/intracellular molecules (granzyme, cyclic AMP, and IDO) [31].

7. Inhibitory molecules

7.1. Interleukin-10

IL-10 is an important anti-inflammatory cytokine that regulates immune responses and maintains immune homeostasis. It is produced by various cells of the immune system, including T cells, B cells, macrophages, and DCs. IL-10 contributes to the suppression of immune responses that may lead to inflammation and tissue damage. For example, it inhibits Tconv cell proliferation and activation, accelerates depletion, suppresses the antigen-presenting function of APC cells, and also induces iTreg cells, which in turn can produce high levels of IL-10 to help maintain their suppressive function and promote immune tolerance [32]. IL-10 can activate the STAT3 signaling pathway to promote Treg development and expansion [33] and can inhibit the production of pro-inflammatory cytokines such as IL-6 and IL-12 by DCs, which in turn promotes Treg expansion and suppressive functions [34, 35]. IL-10 also enhances Treg expansion and suppressive functions by upregulating CTLA-4 and PD-1 [36]. IL-10 also plays a role in regulating the differentiation of naive T cells into Teff cells. In the absence of IL-10 signaling, naive T cells are more likely to differentiate into pro-inflammatory Type-1 T helper cells (Th1) or Type-17 T helper cells (Th17) cells, thereby promoting self-immune responses. However, IL-10 signaling can redirect the differentiation of naïve T cells to a regulatory phenotype, leading to the development of Treg and the promotion of immune tolerance [37]. Overall, IL-10 plays a key role in maintaining T cell tolerance by regulating Treg function and promoting the differentiation of naïve T cells into regulatory phenotypes.

In cancer and autoimmune disease, IL-10 can act as both positive and negative regulator of the immune response. As a negative regulator of the antitumor immune response, IL-10 can inhibit the activation and function of immune cells (including T cells and natural killer cells) involved in the antitumor immune response. On the other hand, it has been shown that IL-10 enhances CD8⁺ T cell-mediated antitumor immunity by regulating DCs [38]. Similarly, in autoimmune diseases, the presence of increased expression of IL-10 [39] can help suppress inflammatory responses and limit tissue damage, while they also play a role in mediating pro-inflammatory effects in certain autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis [40, 41].

In inflammatory diseases, the effect of IL-10 is more favorable, wherein it plays a role in inhibiting the production of pro-inflammatory cytokines, such as TNF-α and IL-6. In allergic inflammatory reactions, IL-10 inhibits the production of pro-inflammatory cytokines and chemokines associated with allergic reactions. It also promotes the differentiation and function of Treg cells, contributing to the suppression of allergic responses and maintenance of immune tolerance. Studies have shown that IL-10 levels are reduced in allergic individuals, suggesting that IL-10 deficiency may contribute to the development and severity of allergic reactions [42].

Overall, IL-10 has a complex and multifaceted role in regulating immune responses in autoimmune diseases, and further studies are needed to fully understand its function and potential therapeutic applications.

7.2. Transforming growth factor-β

TGF-β is a multifunctional cytokine that plays a key role in the regulation of immune responses, including T cell tolerance. It is produced by a variety of immune cells, such as Tregs, DCs, and macrophages. It acts on T cells and APCs to inhibit their activation and differentiation and plays a crucial role in the induction and maintenance of T cell tolerance. Similar to IL-10, TGF-β has a role in regulating the differentiation of naive T cells toward a regulatory phenotype during their development, leading to the development of Treg cells and the promotion of immune tolerance; conversely, in the absence of TGF-β signaling, naive T cells are more likely to differentiate into pro-inflammatory Th1 or Th17 cells, thus promoting autoimmune responses [43]. TGF-β also promotes the differentiation of Th17 cells to Tregs [44]. In addition, TGF-β inhibits the function and survival of APCs [43, 45]. TGF-β also promotes the differentiation of Th17 cells to Tregs [44]. In addition, TGF-β inhibits the function and survival of APCs [45], subsequently preventing the activation of self-reactive T cells and promoting their deletion or anergy in the thymus and peripheral tissues [45].

The role of TGF-β in disease development and immunotherapy is complex. Depending on the stage of the disease and the microenvironmental context, it can both promote and suppress the immune response. In cancer immunotherapy, for example, TGF-β can have both tumor-promoting and tumor-suppressive effects. In the early stages of cancer, TGF-β promotes the activation and proliferation of effector T cells and natural killer cells to enhance the immune response against cancer cells, thereby attacking and killing them. In the late stages of cancer, however, TGF-β can induce the differentiation of Tregs, thereby inhibiting the activation of effector T cells and promoting tumor growth and metastasis. Suppression of TGF-β signaling has been proposed as a potential strategy for cancer therapy [46]. However, the potential side effects of TGF-β inhibition on normal tissue homeostasis and wound healing should be carefully considered [47]. In allergic diseases such as asthma, TGF-β has been shown to promote disease progression by stimulating collagen production, inhibiting collagen breakdown, and activating fibroblast, which can induce airway remodeling [48, 49]. On the other hand, TGF-β has also been shown to enhance immune tolerance and attenuate excessive immune responses by promoting the differentiation of Tregs, thereby relieving asthma [50]. In addition, TGF-β can promote the production of anti-inflammatory cytokines (eg, IL-10), further reducing allergic inflammation.

In other diseases, such as autoimmune diseases or transplant rejection, TGF-β may be used to induce the differentiation of Treg cells or tolerogenic DCs, thereby suppressing autoimmunity or preventing transplant rejection. However, TGF-β may also suppress normal immune responses, leading to infections and other complications resulting in graft failure. Overall, TGF-β signaling can have both beneficial and detrimental effects on the immune response. A better understanding of the role of TGF-β in various diseases and the development of more selective and targeted TGF-β modulators are needed to achieve optimal immunotherapy.

7.3. Interleukin-35

IL-35 is a member of the IL-12 family, a potent anti-inflammatory cytokine, and a heterodimer consisting of Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. It is produced mainly by Treg, Breg [51], endothelial cells, smooth muscle cells, and monocytes.

IL-35 has a protective role in autoimmune diseases, transplant rejection, and allergic diseases. It has been shown to inhibit the activity of autoreactive T cells, prevent them from attacking auto-antigens and transplanted tissues, and promote the activity of Treg cells, thereby suppressing the activity of pro-inflammatory immune cells, promoting the production of anti-inflammatory cytokines, and ultimately enhancing immune tolerance. IL-35 expression has been found to be upregulated in patients with autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus (SLE) [52, 53]. Shamji et al [54] reported that IL-35 and IL-35-induced Tregs (iTR35) cells are potential novel immune regulators induced by grass pollen sublingual immunotherapy (SLIT). Moreover, Zeng et al [55] show that IL-35 inhibits apoptosis, adhesion, migration, and activation of eosinophils in AR patients.

The role of IL-35 in tumors is more complex, and its action may depend on the type of tumor and the tumor microenvironment. Many studies have shown that IL-35 can induce an immunosuppressive environment by interacting with other immune cells, such as Treg, Th17, or tumor-infiltrating lymphocytes [56]. At the same time, they are known to promote tumors by secreting cytokines such as IL-6 and G-CSF [57], and inhibiting anti-inflammatory cytokine IFN-γ [58].

8. B cell tolerance

B cells are classically renowned for their capacity to differentiate into antibody-secreting plasma cells, providing protection from invading pathogens. B cells also have important antigen presenting capacity, an understudied field which is often overshadowed by the other professional APCs such as DCs and monocytes. An example of this is the dominant APC role B cells play in the maintenance and differentiation of primed T cells into germinal centre Tfh cells, providing co-stimulatory signals through CD80, CD86 and ICOSL [59].

B cells have also demonstrated involvement in chronic inflammatory responses, for example in allergic and autoimmune diseases. However, B cells also have a regulatory capacity through the secretion of immunosuppressive cytokines (Fig. 2). In this section the central and peripheral tolerance relating to B cells will be discussed.

Figure 2.

The mechanism of B cell tolerance. The B cell precursor completes central tolerance in the bone marrow. Immature B cell receptor binds to self-antigen and may cause high acitivity, resulting in clonal deletion or anergy, whereas immature B cells that only undergo moderate activity may move to the lymph nodes to participate in peripheral tolerance. Self-antigen interacts with B cell receptor (BCR), and B cells with strong interaction will undergo apoptosis and deletion, while B cells with weak interaction will develop into anergic B cells. Immature B cells without interaction, will develop into mature B cells. Breg cells play an important role in peripheral tolerance. It can induce and promote Treg proliferation, inhibit monocytes activity, inhibit mast cell degranulation, and also inhibit T cell activity.

9. Central tolerance

In 1890, the concept of B cell tolerance was proposed by Ehrlich et al [60] when they studied the specificity of serum. B cell tolerance was understood to be an acquired property of the immune system that controls the specificity of antibodies in a way that avoids autotoxicity. But this definition does not comprehensively explain the auto-specific reaction in healthy individuals, which may be meaningful to health. The tolerogenic process that undifferentiated B cells undergo in the bone marrow is known as central tolerance. Central tolerance is mainly achieved through the deletion, anergy, and B cell receptor (BCR) editing of B cells. When a self-antigen bind to the BCR with high affinity the B cell undergoes clonal deletion whereas when the self-antigen binds with lower affinity this can cause inactivation of the B cell without deletion, also known as anergy [61, 62]. Fulcher et al [63] and Cyster et al [64] extend the concept of anergy by suggesting that peripheral anergic B cells are excluded from lymphoid structures and are gradually deleted as they compete with nonautoreactive cells.

During the development of BCR B cells, a considerable part of the B cells will edit the BCR receptor because of self-reactivity, though the exact proportion is currently unknown. Studies have shown that 20% of immature B cells downregulate the expression of BCR and actively carry out tolerance-induced editing. Ongoing Ig light (L) chain gene recombination alters B cell antigen specificity and in so doing, rescues auto‐reactive B cells from deletion [59, 65–67]. If auto-reactive B cells not edited, or their heavy (H) chain features are not effectively corrected to L chain features, these cells may face apoptosis [68]. The control of apoptosis on B cell central tolerance is similar to that of editing, which is incomplete. Through central tolerance, autoreactive B cells will be regulated, resulting in the decrease of their frequency in the B cell repertoire, their affinity for self-tissue, or their functionality. Following this process, B cells will then enter the spleen, lymph nodes, or other tissues for further peripheral tolerance. It is accepted that B cell immune tolerance is achieved through the loss of autoreactive B cells, receptor editing, or reduced cell viability/cell function.

10. Peripheral tolerance

Regulatory B (Breg) cells play a key role in peripheral tolerance and can help maintain tolerance, limit ongoing immune responses, and restore immune homeostasis. The role of Bregs in suppressing the pathology associated with aggravating inflammatory responses in autoimmunity and transplant rejection has been consistently demonstrated, and recent studies have shown that Bregs also play an important role in other diseases such as infection, allergy, cancer, and chronic metabolic diseases [69, 70]. Unlike Treg cells that can be identified by markers such as Foxp3, Breg cells are not as easily identified, though their ability to secrete IL-10 remains one of the key factors that allow their identification [71, 72]. In addition to IL-10, expression of CD9, TIM-1, CD80, and CD86 have also been loosely described. For example, Xiao et al [73] demonstrated in murine models that TIM-1 not only regulates the production of IL-10 by Breg cells but also is one of the main regulators of other inhibitory cytokines and co-repressive molecules, such as Ebi3, GITRL, Fgl2, CLTLA-4, Lag3, and TIGIT. This also explains why TIM-1-deficient B cells cause a more severe inflammatory response than those lacking TIGIT or IL-10 [73]. Despite this promising evidence, the role of TIM-1 is still unclear as a marker of human Breg cells. Reportedly, there are about 40% of all Transitional B (TrB) cells express both TIM-1 and IL-10, and 90% of all TIM-1⁺ TrB cells express IL-10. In SLE patients, the expression of TIM-1 increased and correlated with IL-10 expression [74].

11. Inhibitory cytokines

Breg cells can induce tolerance through their secretion of inhibitory cytokines. These cytokines traditionally encompass IL-10, and more recently IL-35 and TGF-β.

11.1. Interleukin-10

IL-10⁺ Breg cells can convert naïve T cells into Treg cells and IL-10-secreting type-1 regulatory CD4⁺ T cells (Tr1). In addition, they can also suppress immunogenic CD4⁺ T cells. These functions are intact in healthy people but are dysregulated in those with immune diseases due to a reduced proportion of IL-10⁺ Breg cells and impaired function. However, these functions are enhanced in patients with chronic viral disease. In the mouse model of listeria infection, Horikawa et al [75] found that IL-10⁺ Breg cells can inhibit bacterial clearance by suppressing macrophage function and CD4⁺ T cell proliferation. Furthermore, in a study of Mycobacterium tuberculosis infections, active tuberculosis is associated with high levels of active Breg cells and their inhibition of Th17 [76] thereby indicating that an effective tuberculosis treatment would be associated with a reduction in Breg cells [77]. In the case of patients with an immunocompromised immune system (ie, SLE, RA, and type 1 diabetes), however, lower proportion of Breg cells results in an inefficient inhibition of bacterial infection and viral clearance [78, 79].

Although the immunosuppressive nature of Breg cells has been vilified in cancer and viral and bacterial infections, Breg cells play an important positive role in allergic disease, autoimmunity, and improved transplantation tolerability [80, 81]. Desensitization immunotherapy in allergic patients causes increased IL-10⁺ Breg cells [82, 83]. Kim et al [84] demonstrated that increased IL-10 produced by Breg cells helps to inhibit the degranulation of murine mast cells, a crucial mechanism of allergic disease. In studies of allergic airway disease and food allergy, again it has been demonstrated that IL-10-producing Bregs not only suppress Th2 and Th17 inflammatory responses but also promote Treg generation [85]. In transplantation, several studies have indicated that lower numbers of transitional B cells or impaired suppressor function are associated with a higher risk of renal transplant rejection [86]. Whereas, increased proportions of IL-10⁺ Breg cells in patients have been associated with improved lung transplantation tolerance compared with patients who develop chronic graft dysfunction [87]. Besides, IL-10 Breg cells also exert mechanisms of tolerance to the fetus during pregnancy in mice and humans, including the generation of tolerogenic DCs and Tregs and the reduction of Th17 cells [88, 89] shown to be important for maintaining the homeostasis and successful pregnancy outcomes.

11.2. Transforming growth factor-β

TGF-β is a multifunctional cytokine that plays a vital role in B cell regulation. Although TGF-β is mainly produced by Treg cells, there is evidence that some Breg cells can also produce TGF-β. Yang et al [90] categorized Breg cells into different subtypes based on the gene expression profile. They found that the expression of genes that promote TGF-β production was enriched in one subset, and they could activate the TGF-β pathway and IL-35-mediated pathway, but TGF-β⁺ Breg could not produce IL-10 [90]. Further evidence shows that TGF-β produced by TGF-β⁺ Breg can induce naïve CD4⁺ T cells to differentiate into Treg cells, thereby limiting T cell-related immune responses. TGF-β in Treg-mediated gastrointestinal tract immunosuppression plays a key role [91] with TGF-β⁺ Breg cells being key players in the inhibition of Th2 inflammation through the influence of Tregs, thereby inhibiting colitis caused by food allergy in mice. Similarly, mice infected with parasites can also cooperate with anti-inflammatory macrophages to suppress Th1 and Th2 inflammatory responses, thereby controlling colitis. In respiratory diseases, TGF-β⁺ Breg cells have been found to be reduced in the alveoli of patients with interstitial lung disease [92]. In contrast, allergic airway inflammation in mice can be controlled by transplanting TGF-β⁺ Bregs from hilar lymph nodes. In addition, it has been reported that TGF-β⁺ Breg-mediated hyporesponsiveness leads to anergy of CD4⁺ and CD8⁺ T cells [93]. TGF-β⁺ Bregs can also induce immature DCs to differentiate into tolerogenic DCs, leading to reduced antigen-presentation capacity and thus limiting the Th1/Th17 inflammatory response. A study involving B cell deficiency in TGF-β transgenic mice demonstrated acceleration of experimental autoimmune encephalomyelitis development, which is related to the activation of myeloid DCs and increased Th1/Th17 responses in the central nervous system [94].

11.3. Interleukin-35

IL-35 can induce the conversion of B cells into the Breg subsets, though most experimental evidence is based on murine models [54, 95]. Murine studies illustrated that EBI3 or IL-12p35 knock-out mice were unable to recover from experimental autoimmune encephalomyelitis and experimental autoimmune uveoretinitis (EAU), highlighting the importance of IL-35 cytokine. Progression of EAU was shown to be controlled by IL-35 injection which resulted in the expansion of Breg cells in mice, an increase in IL-10 and IL-35 production, and inhibition of Th1/Th17 inflammatory responses. Despite the benefit of IL-35 injection for EAU, this resulted in the heightened risk of Salmonella typhimurium infection [96]. Studies in human have demonstrated that IL-35⁺ Bregs play a key role in promoting tumor growth with their levels being upregulated in late-stage gastric cancer [97] and pancreatic cancer [98]. In the context of autoimmune disease, IL-35⁺ Bregs and IL-35 in the peripheral blood of patients with SLE displayed a more positive role in which they negatively correlated with the disease activity index [99]. Furthermore, decreased levels of IL-35 have also been demonstrated in patients with ankylosing spondylitis [100], allergic asthma [100], and allergic rhinitis [54]. These highlight the importance of IL-35 and their potential use as a therapeutic target for multiple diseases.

12. Immune tolerance as a therapeutic strategy

There are several immunotherapeutic strategies to treat diseases arising from tolerance imbalance. A prime example of this is allergen-specific immunotherapy (AIT), which consists of a repeated administration of allergen extracts over several years, restoring tolerance and potentially eliminating the need for symptom-alleviating medication [101–103]. Adoptive cell therapies using Tregs have shown promising preclinical and clinical results for the treatment of autoimmune diseases, graft-versus-host disease, and transplantation [104–107]. Immunotherapy aims to restore or enhance the body’s natural tolerance mechanisms to achieve long-term control or even cure of disease. However, there are still challenges associated with the treatment, such as developing effective strategies to induce and maintain immune tolerance, identifying appropriate biomarkers to predict and monitor treatment responses, and managing potential side effects of therapy.

13. Biomarkers of allergen-specific immunotherapy

AIT remains the only disease-modifying treatment for allergic rhinitis, with tolerance induction observed in patients who receive treatment for a duration of 3 years or longer. While AIT is effective in many patients, it is ineffective in a small proportion of patients, and it remains a challenge to stratify these nonresponders from responders. Currently, there is no validated biomarker that can be used to measure desensitization, efficacy, and response to AIT, though several candidate cellular biomarkers have been proposed with promising evidence. These biomarkers can be broadly categorized into in vitro biomarkers (ie, humoral, cellular, and metabolic) and in vivo biomarkers.

14. T and B cells as in vitro cellular biomarkers of allergen-specific immunotherapy

Mechanisms of tolerance induction following AIT has been explored thoroughly and has been implicated with various modulation of the immune system that includes dampening of pro-allergic T cell responses (ie, Th2 and Tfh cells), immune deviation toward a Th1 response, induction of T and B regulatory cells and induction of neutralizing antibodies. Elevated levels of natural and inducible Treg cells have been demonstrated following a successful AIT [54, 108, 109]. In particular, induction of natural Treg was observed in patients with Japanese cedar pollinosis who received SLIT treatment [110] while induction of iTreg was observed in grass pollen allergic patients who received SLIT treatment [54]. Similarly, Breg cells have also been shown to be induced following AIT. In a recent study, induction of IL-10⁺ Breg cells was demonstrated following a 3-week short course of Lolium Perenne immunotherapy [111, 112]. Furthermore, allergen-specific B memory cells have been reported to have an altered phenotype following AIT, with upregulated CD29 surface expression [113]. Though validation via clinical trial is required to further confirm these observations, Treg and Breg cells possess tolerogenic properties that may indicate their suitability as potential biomarkers of AIT.

15. Humoral biomarkers

The success of AIT may also be assessed by monitoring the levels of different Ig classes. These humoral biomarkers include total and specific IgE, IgG, and IgA subclasses. Although widely used to diagnose allergic patients, changes in specific IgE levels do not correlate well with clinical response and therefore are not a validated for assessment of efficacious AIT [114–116]. A better, but still imperfect, tool of assessment is IgG1, IgG2, and IgG4 levels. Mechanistically, IgG2 and IgG4 likely help regulate allergic responses through neutralizing allergens rather than immune activation due to their reduced capacity to activate effector cells. Indeed, the observed success of IgG blocking activity in vitro, investigated using IgE-facilitated allergen binding assays, has made IgG one of the more promising humoral biomarkers of AIT success [117]. However, although IgG subclasses have shown increases in successful SCIT-treated patients [118], it is uncertain whether their induction correlates with clinical response [119–122]. IgA is another potential humoral biomarker for measuring AIT success. Induction of IgA was shown following SLIT in GRASS trial patients and like IgG, IgA can compete with IgE for allergen [118]. Although each Ig subclass discussed has the potential to act as a biomarker for AIT success, their use in-clinic is still controversial. To date, there is no universally accepted biomarker of AIT efficacy; acceptance of any promising biomarker will require larger clinical trial-based efforts.

16. In vivo biomarkers

Determination of efficacious AIT treatment relies on observation of validated in vivo biomarkers. Among those used in clinical practice are intradermal dilution testing, allergen provocation testing, and the allergen exposure chamber. The allergen exposure chamber is particularly useful for assessing the responses of patients to airborne allergens. By controlling the concentration of allergen released, symptomatic changes of patients across different allergen concentrations may be assessed [123]. However, the most widely used method is skin prick testing (SPT). Proof of principle for this method was demonstrated by Moreno et al [124] and Sun et al [125], who observed statistically significant reductions in the size of SPT wheals in AR patients who underwent SCIT with DPT for 3 years. Clinically, the utility of this method was demonstrated by Hajdu et al [126], who showed patients who underwent AIT treatment for atopic dermatitis and AR showed both the absence of symptoms and concomitant negative SPT results [126].

17. Conclusion

Immune tolerance is an extremely complex immune process, which is achieved through a complex network of mechanisms that prevent the immune system from attacking the body’s own cells and tissues. These mechanisms include central and peripheral tolerance mechanisms which both have associated advantages and disadvantages. These advantages include the prevention of autoimmune disease, allergy, and transplant rejection. While major drawbacks include the exploitation of tolerogenic mechanisms in cancer, creating an immunosuppressive microenvironment, and aiding disease progression. Deepening our knowledge of immune tolerance mechanisms not only provides us with a greater understanding of underlying disease mechanism and progression, but also unveils a plethora of immune pathways that may be exploited for therapeutic advancement.

Conflicts of interest

The authors have no financial conflicts of interest.

Author contributions

Xun Meng prepared the initial draft of the review. Mohamed H. Shamji provided the outline of the review. Janice A. Layhadi, Sean T. Keane and Natanya J.K. Cartwright reviewed, amended and provided feedback to the manuscript.