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CD4+CD25+Foxp3+ regulatory T cells (Tregs) are of special interest in immunology because of their potent inhibitory function. Many fundamental aspects of Tregs, including their antigenic profile, development and peripheral homeostasis, remain highly controversial. Here, we propose a Treg-centered antigen-non-specific immunoregulation model focused on the T-cell system, particularly on CD4+ T cells. The T-cell pool consists of naive T cells (Tnais), Tregs and effector T cells (Teffs). Regardless of antigen specificity, the ratio of the activated T-cell subsets (Treg/Teff/Tnai) and their temporal and spatial uniformity dictate the differentiation of Tnais. Activated Tregs inhibit the activation, proliferation, induction and activity of Teffs; in contrast, activated Teffs inhibit the induction of Tregs from Tnais but cooperate with Treg-specific antigens to promote the proliferation and activity of Tregs. In many cases, these interactions are antigen-non-specific, whereas the activation of both Tregs and Teffs is antigen-specific. Memory T-cell subsets are essential for the maintenance of adaptive immune responses, but the antigen-non-specific interactions among T-cell subsets may be more important during the establishment of the adaptive immune system to a newly encountered antigen. This is especially important when new and memory antigens are presented closely—both temporally and spatially—to T cells, because there are always baseline levels of activated Tregs, which are usually higher than levels of memory T cells for new antigens. Based on this hypothesis, we further infer that, under physiological conditions, Tregs in lymph nodes mainly recognize antigens frequently released from draining tissues, and that these self-reactive Tregs are commonly involved in the establishment of adaptive immunity to new antigens and in the feedback control of excessive responses to pathogens.
Naive T cells (Tnais) can differentiate into a number of memory T-cell subsets that are roughly defined as effector T cells (Teffs) and regulatory T cells (Tregs). Th1, Th2, Th9, T follicular helper (Thf) and Th17 are typical Teffs, and CD4+CD25+Foxp3+ T cells are the most important of the Tregs.1, 2 Although mutually inhibitory, both Tregs and Teffs can be stimulated in an autocrine fashion. This mutually exclusive induction and antigen-specific recognition is the basis of the adaptive immune response. Antigen-specific regulation provides a clear explanation of why memory status is resistant to conversion after it has been established. Antigen-specific memory T-cell subsets are activated primarily by subsequent invasion of pathogens and maintain memory status by facilitating the induction of Tnais into the appropriate T-cell subsets. However, antigen-specific regulation may play only a small role in the primary establishment of memory status. In addition, it is difficult to explain how the immune system controls the balance between mounting a response to pathogens and avoiding self-destruction by the antigen-specific model. Tregs are capable of not only maintaining self-tolerance but also inhibiting both adaptive and innate responses to pathogens.3 Although the potent inhibitory activity of Treg has been observed in various disease models, many fundamental characteristics, such as the antigenic profile, peripheral tolerance induction and the homeostasis mechanism, are not well understood. In view of the gaps in the antigen-specific model, we propose here the Treg-centered antigen-non-specific model. The two models are logically coherent, and their combination may provide a better understanding of immunoregulation.
CD4+ T cells play a pivotal role in the establishment and maintenance of the adaptive immune response. In peripheral lymphoid organs, Tnais can differentiate into functionally different subsets. Each subset tends to promote self-development and inhibit the differentiation of the other subsets. The competition results in one subset dominating over the others. Apart from the intracellular status of Tnais, many milieu factors may be involved in the differentiation. There is no doubt that properties of antigen-loaded antigen-presenting cells (APCs), such as the expression patterns of cytokines and costimulation molecules, can directly influence T-cell differentiation. The characteristics of the classical MHC II-restricted antigens and the concomitant non-antigenic components (e.g., endogenous or exogenous adjuvant, innate immunity-associated molecules and antibodies) and the capture mode of antigens by APCs (e.g., antigen dose, soluble or particular antigens, antigens from apoptotic or necrotic cells) may be the elements that initially determine the functional status of APCs. The subsequent but even more important factors may develop from activated memory T cells. Subsets of memory T cells may affect Tnai differentiation through both indirect effects on APCs and direct effects on Tnais via cytokine release or cell contact. The effects are essential for adaptive immune memory because of the lack of evidence for the existence of professional antigen-specific memory APCs. If adaptive immunity has been established, antigen-specific memory T-cell subsets are activated primarily by the invading antigens, and the memory status is maintained by facilitating Tnai induction into the same T-cell subsets.
Although antigen-specific memory mediated by T cells is central to the adaptive immune response, antigen-non-specific interaction among T-cell subsets may also play an important role in two situations. The first is when antigens are encountered for the first time, when no antigen-specific memory T cells are yet present. The second is when several antigens are presented closely—both temporally and spatially—to different T-cell subsets. Hence, we propose the antigen non-specific model for the induction of Tregs (Figure 1). The key point of this model is that, regardless of antigen specificity, the component ratio of activated T-cell subsets and their temporal and spatial uniformity largely determine Tnai differentiation. If activated Tregs outnumber activated Teffs, Tnais preferentially differentiate into Tregs,4 and vice versa.5 During an individual's lifespan, the peripheral immune system is continually exposed to new endogenous and exogenous antigens. In this setting, there are very few, if any, memory Tregs and Teffs specific for newly encountered antigens. The number of activated Tnais is proportional to the dose of the priming antigens. The number of activated Tregs is determined mainly by the base level of activated Tregs, as well as by the dose and memory status of the concomitant antigens, which also determine the number of activated Teffs. In most cases, newly encountered antigens represent only a fraction of a complex mix of antigens. The concomitant antigens are regarded as making up the remaining portion of the mixed antigens, but they can be presented to the specific memory T-cell subsets.
With respect to tumor cells, newly mutated neo-antigenic peptides and normal cell components are presented to T cells concurrently. The low ratio of mutated tumor antigens to normal cell components, and the preferential recognition of the latter antigens by Tregs, may contribute to the induction of tolerance to tumor cells during the very early stages of oncogenesis. Chronic infection, especially intracellular infection, is more favorable than acute infection for the induction of tolerance because of the decreased level of foreign antigens and the increased level of antigens resulting from the infected tissues (necrotic or apoptotic tissue cells). In addition to antigens, cytokines produced in the tumor and inflammatory milieu determine the fate of Teffs and Tregs. In acute inflammation, IL-6, tumor-necrosis factor-α and other proinflammatory cytokines produced by the innate immune system stimulate immune activity and downregulate Tregs.6 IL-6 also abolishes the suppressive ability of Tregs in inflammatory conditions.7 Moreover, IL-6 enables natural Tregs to become Th17 cells that may assist the immune response in early inflammation.8, 9 It has been shown that tumor cells produce large amounts of transforming growth factor-β, which has an essential role in converting Tnais to Tregs.10, 11, 12, 13 In the late stages of inflammation, transforming growth factor-β becomes the predominant cytokine and subsequently promotes Treg differentiation and development.
In summary, the ratio of the three kinds of antigen components—antigens presented to Tregs, Teffs and Tnais—influences Tnai differentiation. The antigen non-specific interplay requires that the different antigens be presented closely—both temporally and spatially—to the T-cell subsets. This is seen when various APCs present different antigens in the same lymph node or when one APC captures many types of antigens.
The antigen-non-specific model of Treg induction provides new insights into the formation of the antigenic profile of Tregs in the periphery. The thymus begins to produce naturally occurring Tregs (nTregs) in the earliest stages of life.14 Self-antigens expressed in the thymus are involved in the development of these cells.15 Aire is believed to induce expression of a wide repertoire of peripheral tissue antigens in medullary epithelial cells, and this expression controls the clonal selection of thymocytes.16 However, it is possible that Aire does not control the repertoire of T-cell receptors (TCRs) in Foxp3+ Tregs.16, 17 Because it is unnecessary for the thymus to express all types of self-antigens, nTregs might recognize only a representative profile of self-antigens. The nTregs produced in the first several days after birth migrate to peripheral lymphoid organs to become the earliest members of peripheral Tregs. These early nTregs are continuously activated both by systemic self-antigens coming from the blood and by local self-antigens coming from the draining tissues. Besides the inhibitory effects on Teffs, one of the main functions of activated nTregs is to promote the generation of peripherally induced Tregs (iTregs) in an antigen-specific and -non-specific manner (Figure 2). Thus, the antigenic profile of nTregs partially overlaps with that of iTregs. In the case of antigen-non-specific Treg induction, the slight but frequent antigen priming tends to induce iTregs. Because Treg activation, proliferation and induction require challenge of specific antigens, and most of the antigens received by the lymph nodes are those coming from the draining tissues under physiological conditions, Tregs in the lymph node mainly recognize antigens frequently released from the draining tissues. Therefore, the antigenic profile of Tregs may vary among different lymph nodes.
Although iTregs are not efficiently induced in the absence of nTregs during the early life of an individual, the iTreg itself, once it reaches a steady state, can promote the development of a second generation of iTregs, and the peripheral Treg pool no longer requires nTreg supplementation (Figure 2). iTregs may be even more frequent than nTregs after the start of thymus degeneration.
The primary aim of the immune system is to clear invading pathogens; however, excessive responses result in self-damage. Activation-induced cell death might be an intracellular mechanism to control excessive immune responses, but this negative feedback may be unable to detect an excessive response that threatens to cause self-destruction. Here, we propose that antigen-non-specific interplay between Tregs and Teffs is one of the three most important mechanisms for preventing self-inflicted damage. First, activated Tregs inhibit the activation, proliferation and activity of Teffs, but activated Teffs synergize with Treg-specific antigens to promote the activation, proliferation and activity of Tregs. Second, without the help of activated Teffs, Tregs remain at low levels of activation and proliferation and are susceptible to apoptosis. Third, the interplay is antigen-non-specific in most cases, whereas the activation of both Tregs and Teffs is antigen-specific. Although the suppression mediated by antigen-specific Tregs is more effective than that of polyclonal Tregs, it is unlikely that antigen-specific Tregs are often involved in negative feedback. If tolerance has been maintained by antigen-specific Tregs, subsequent antigen challenge may not lead to excessive responses. If excessive responses have been triggered by the dominance of Teff over Treg levels, there may be little chance to generate enough Treg numbers in an antigen-specific manner because activated Teffs inhibit the induction of Tregs from Tnais.5
In light of the abovementioned Treg antigenic profile, the non-specific interplay between Tregs and Teffs may occur most often between the draining tissue-derived self-antigens and invading foreign antigens. The antigen non-specific model might advance our understanding of how the immune system regulates the balance between mounting a response to pathogens and avoiding self-destruction. Under physiologic conditions, T cells in the peripheral lymphoid organs are frequently, but slightly, challenged by self-antigens, especially the draining organ-specific antigens. These self-antigens are preferentially presented to Tregs by APCs. As a result, the potentially pathogenic response to self-tissues is controlled. On the other hand, when Tregs are challenged by specific antigens in the absence of help from activated Teffs, Tregs frequency cannot increase much. Once newly encountered antigens are of a sufficient dose, Teffs would be readily induced and activated, resulting in amplification of an immune defense cascade. When a strong immune response brings about self-destruction, organ-specific self-antigens from damaged tissues are released in proportion to the extent of destruction, and these antigens are presented to Tregs in the draining lymph nodes. The proliferation and activity of self-reactive Tregs are increased via the synergy between self- and foreign antigens, establishing a negative feedback mechanism to counteract excessive response in an antigen-non-specific manner. After clearance of pathogens, self-reactive Tregs decrease to homeostatic levels. If an infection becomes chronic, the elevated activity and numbers of self-reactive Tregs help the low dose of foreign antigens to induce additional specific Tregs. Self-reactive Tregs may also suppress B-cell responses to foreign antigens in an antigen-non-specific manner.
The novel antigen-non-specific model is based on the findings of a number of recent studies. In vitro studies have shown that Tregs require TCR engagement for their activation. However, once activated, Tregs can suppress both CD4+ and CD8+ T cells in a non-specific manner.18 An in vivo model has also shown that antigen-specific Tregs displayed broad suppression of the induction of experimental autoimmune encephalomyelitis.19 Tregs preferentially recognize self-antigens.20, 21 There are always baseline levels of activated Tregs in the steady state.21, 22, 23 Continuous low-dose antigen stimulation has led to Treg-mediated tolerance.24 In vitro studies have shown that Tregs can be induced from CD4+CD25−Foxp3− Tnais and that Treg itself facilitates this induction.4, 10, 11 Foreign antigen-specific Tregs can be induced in vivo under certain circumstances during antigen exposure.25, 26 IL-2 is produced mainly by activated Teffs but not Tregs and can promote Treg proliferation.27 Tregs play a central role in infectious tolerance.28 The self-injury caused by burns can enhance the suppressive activity of Tregs and inhibit both the innate and adaptive immune responses.29
The interplay between self- and foreign antigens has also been observed recently. We found that simultaneous administration of bowel tissue antigens attenuated the immune responses to enteric bacterial antigens and raised the frequency of Tregs in mesenteric lymph nodes (Hu GZ, Zheng SG, unpubl. data, 2010).
In developing this hypothesis, we highlighted Treg-centered antigen-non-specific regulation with a focus on the interactions between self-reactive Tregs, which recognize antigens from the draining tissues, and Tnais challenged by new antigens. The interactions may be commonly involved in tolerance or immune establishment of adaptive immunity to new antigens and in feedback control of excessive responses to pathogens.
This hypothesis may provide new insights into therapeutic strategies for immunity-related diseases. Concurrent administration of certain types of self-antigens and objective antigens may favor the induction of antigen-specific tolerance. This may be particularly feasible for the induction of transplantation tolerance.