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Regulatory T cells have been shown to prevent the development of autoimmune disease, and can modulate immune responses during infections or following tissue transplantation. Recently, the processes by which CD4CD25 regulatory T cells are produced during immune repertoire formation have begun to be elucidated. This review focuses on the role of self-peptides in mediating CD4CD25 regulatory T cell selection in the thymus. How self-peptides continue to have an important influence on the accumulation of CD4CD25 regulatory T cells in the periphery is also discussed.

CD25 CD4 T cells (T) are a naturally arising subset of regulatory T cells important for the preservation of self-tolerance and the prevention of autoimmunity. Although there is substantial data that TCR specificity is important for T development and function, relatively little is known about the antigen specificity of naturally arising T. Here, we will review the available evidence regarding naturally arising T TCR specificity in the context of T development, function, and homeostasis.

The seminal work of Le Douarin and colleagues (Ohki et al. 1987; Ohki et al. 1988; Salaun et al. 1990; Coutinho et al. 1993) first demonstrated that peripheral tissue-specific tolerance is centrally established in the thymus, by epithelial stromal cells (TEC). Subsequent experiments have shown that TEC-tolerance is dominant and mediated by CD4 regulatory T cells (Treg) that are generated intrathymically by recognition of antigens expressed on TECs (Modigliani et al. 1995; Modigliani et al. 1996a). From these and other observations, in 1996 Modigliani and colleagues derived a general model for the establishment and maintenance of natural tolerance (MM96) (Modigliani et al. 1996b), with two central propositions: (1) T cell receptor (TCR)-dependent sorting of emergent repertoires generates TEC specific Treg displaying the highest TCR self-affinities below deletion thresholds, thus isolating repertoires undergoing positive and negative selection; (2) Treg are intrathymically committed (and activated) for a unique differentiative pathway with regulatory effect functions. The model explained the embryonic/perinatal time window of natural tolerance acquisition, by developmental programs determining (1) TCR multireactivity, (2) the cellular composition in the thymic stroma (relative abundance of epithelial vs hemopoietic cells), and (3) the dynamics of peripheral lymphocyte pools, built by accumulation of recent thymic emigrants (RTE) that remain recruitable to regulatory functions. We discuss here the MM96 in the light of recent results demonstrating the promiscuous expression of tissue-specific antigens by medullary TECs (Derbinski et al. 2001; Anderson et al. 2002; Gotter et al. 2004) and indicating that Treg represent a unique differentiative pathway (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003), which is adopted by CD4 T cells with high avidity for TEC-antigens (Bensinger et al. 2001; Jordan et al. 2001; Apostolou et al. 2002). In the likelihood that autoimmune diseases (AID) result from Treg deficits, some of which might have a thymic origin, we also speculate on therapeutic strategies aiming at selectively stimulating their de novo production or peripheral function, within recent findings on Treg responses to inflammation (Caramalho et al. 2003; Lopes-Carvalho et al., submitted, Caramalho et al., submitted).

In short, the MM96 argued that natural tolerance is dominant, established and maintained by the activity of Treg, which are selected upon high-affinity recognition of self-ligands on TECs, and committed intrathymically to a unique differentiative pathway geared to anti-inflammatory and antiproliferative effector functions. By postulating the intrathymic deletion of self-reactivities on hemopoietic stromal cells (THC), together with the inability of peripheral resident lymphocytes to engage in the regulatory pathway, the MM96 simultaneously explained the maintenance of responsiveness to non-self in a context of suppression mediating dominant self-tolerance. The major difficulty of the MM96 is related to the apparent tissue specificity of Treg repertoires generated intrathymically. This difficulty has now been principally solved by the work of Hanahan, Kyewski and others (Jolicoeur et al. 1994; Derbinski et al. 2001; Anderson et al. 2002; Gotter et al. 2004), demonstrating the selective expression of a variety of tissue-specific antigens by TECs, in topological patterns that are compatible with the MM96, but difficult to conciliate with recessive tolerance models (Kappler et al. 1987; Kisielow et al. 1988). While the developmentally regulated multireactivity of TCR repertoires (Gavin and Bevan 1995), as well as the peripheral recruitment of Treg among RTE (Modigliani et al. 1996a) might add to this process, it would seem that the establishment of tissue-specific tolerance essentially stems from the “promiscuous expression of tissue antigens” by TEC. The findings of AID resulting from natural mutations (reviewed in Pitkanen and Peterson 2003) or the targeted inactivation (Anderson et al. 2002; Ramsey et al. 2002) of the AIRE transcription factor that regulates promiscuous gene expression on TECs support this conclusion.

The observations on the correlation of natural or forced expression of the Foxp3 transcription factor in CD4 T cells with Treg phenotype and function (Fontenot et al. 2003; Hori et al. 2003; Khattri et al. 2003) provided support for the MM96 contention that Treg represent a unique differentiative pathway that is naturally established inside the thymus. Furthermore, Caton and colleagues (Jordan et al. 2001), as well as several other groups (Bensinger et al. 2001; Apostolou et al. 2002), have provided direct evidence for our postulate that Treg are selected among differentiating CD4 T cells with high affinity for ligands expressed on TECs (Modigliani et al. 1996b).

Finally, the demonstration by Caramalho et al. that Treg express innate immunity receptors (Caramalho et al. 2003) and respond to pro-inflammatory signals and products of inflammation (Caramalhoet al., submitted) brought about a new understanding on the peripheral regulation of Treg function. Together with the observation that Treg also respond to ongoing activities of “naï ve/effector” T cells—possibly through the IL-2 produced in these conditions—these findings explain the participation of Treg in all immune responses (Onizuka et al. 1999; Shimizu et al. 1999; Annacker et al. 2001; Curotto de Lafaille et al. 2001; Almeida et al. 2002; Shevach 2002; Bach and Francois Bach 2003; Wood and Sakaguchi 2003; Mittrucker and Kaufmann 2004; Sakaguchi 2004), beyond their fundamental role in ensuring self-tolerance (e.g., Modigliani et al. 1996a; Shevach 2000; Hori et al. 2003; Sakaguchi 2004; Thompson and Powrie 2004). Thus, anti-inflammatory and anti-proliferative Treg are amplified by signals that promote or mediate inflammation and proliferation, accounting for the quality control of responses (Coutinho et al. 2001). In turn, such natural regulation of Treg by immune responses to non-self may well explain the alarming epidemiology of allergic and AID in wealthy societies (Wills-Karp et al. 2001; Bach 2002; Yazdanbakhsh et al. 2002), where a variety of childhood infections have become rare or absent. Thus, it is plausible that Treg were evolutionarily set by a given density of infectious agents in the environment. With hindsight, it is not too surprising that natural Treg performance falls once hygiene, vaccination, and antibiotics suddenly (i.e., 100 years) plunged infectious density to below some critical physiological threshold. As the immune system is not adapted to modern clean conditions of postnatal development, clinical immunologists must now deal with frequent Treg deficiencies (allergies and AID) for which they have no curative or rational treatments. It is essential, therefore, that basic immunologists concentrate on strategies to selectively stimulate the production, survival, and activity of this set of lymphocytes that is instrumental in preventing immune pathology. We have argued that the culprit of this inability of basic research to solve major clinical problems has been the self-righteousness of recessive tolerance champions, from Ehrlich to some of our contemporaries. It is ironical, however, that none of us—including the heretic opponents of horror autotoxicus—had understood that self-tolerance, or its robustness at least, is in part determined by the frequency and intensity of the responses to non-self.

In the evolution of ideas on immunological tolerance, the time might be ripe for some kinds of synthesis. First, conventional theory reduced self-tolerance to negative selection and microbial defense to positive selection, while the MM96 solution was the precise opposite: positive selection of autoreactivities for self-tolerance (Treg) and negative selection (of Treg) for ridding responses. In contrast, it would now appear that positive and negative selection of autoreactive T cells are both necessary to establish either self-tolerance or competence to eliminate microbes, two processes that actually reinforce each other in the maintenance of self-integrity. Second, V region recognition has generally been held responsible for specific discrimination between what should be either tolerated or eliminated from the organism. In contrast again, it would now seem that both processes of self-tolerance and microbial defense (self/non-self discrimination) also operate on the basis of evolutionarily ancient, germ-line-encoded innate, nonspecific receptors (Medzhitov and Janeway 2000) capable of a coarse level of self/non-self discrimination (Coutinho 1975). It could thus be interesting to revisit notions of cooperativity between V-regions and such mitogen receptors, both in single cell functions (Coutinho et al. 1974) and in the system’s evolution (Coutinho 1975, 1980) as well. After all, major transitions in evolution were cooperative (Maynard-Smith and Szathmary 1995).

Despite great interest in CD4 CD25 suppressor T cells, many of the fundamental properties of these cells remain enigmatic. This is in part due to experimental limitations inherent to the study of polyclonal suppressor T cells, and the extensive use of in vitro assays. This review article intends to outline recent advances in our understanding of the biology of suppressor T cells that have emerged from the analysis of T cell receptor (TCR) transgenic models. Several laboratories have taken advantage of model systems in which suppressor T cells of defined antigen-specificity are naturally selected in order to characterize the selection and behavior of these cells in vivo. In addition to providing valuable insights into the mechanism of differentiation of suppressor T cells, these systems now offer new possibilities for understanding the mode of action of suppressor T cells. For example, adoptive transfer of small numbers of ex vivo isolated TCR transgenic suppressor T cells allows for the visualization of the fate of such cells when confronted with cognate antigen in a quasi-normal, nonlymphopenic environment. Characteristic features of the currently available TCR transgenic models of suppressor T cells will be highlighted, and particular issues pertaining to the differentiation, function, and homeostasis of this T cell subset that have emerged from these models will be discussed.

Suppressor T cells were first described in the early 1970s, but since the hypothetical soluble suppressor factor could not be identified on a molecular level and since appropriate cellular markers were lacking, the suppressor T cell concept vanished for a long time. The discovery by Sakaguchi and co-workers, that the adoptive transfer of CD25 CD4-depleted T cells induced several organ-specific autoimmune diseases in immunodeficient recipients, put the suppressor T cell model back into the focus of many immunologists. CD25 CD4 T cells were named regulatory T cells (Treg) and since then have been intensively characterized by many groups. It has now been well documented in a variety of models that CD25 CD4 Tregs, in addition to cell-intrinsic peripheral tolerance mechanisms such as anergy induction and peripheral deletion, play indispensable roles in the maintenance of natural self-tolerance, in averting autoimmune responses as well as in controlling inflammatory reactions. However, a number of fundamental questions concerning their origin, mechanism of action, and the sites of suppression remain elusive and are currently a matter of debate. Notably, the potential heterogeneity of Tregs with respect to phenotype and function deserves attention and is a major issue discussed in this review.

The balance between immunity and tolerance is important to maintain immune homeostasis. Several mechanisms are in place to ensure that the immune response is controlled, such as T cell anergy, apoptosis and immune ignorance. A fourth mechanism of peripheral tolerance is the active suppression by regulatory or suppressor T cells. The existence of suppressor T cells was first described in the early 1970s, but these cells became discredited in the 1980s. The work of Shimon Sakaguchi and others, however, has brought these cells back into the limelight and nowadays research into regulatory/suppressor T cells is a very active field of immunology. Different types of regulatory T cells have been described, including CD4CD25 T cells that constitutively express CTLA-4, GITR and Foxp3, TGF-β producing Th3 cells, IL-10 producing Tr1 cells, and CD8CD28 T cells. This review will focus on the generation and function of CD4CD25 regulatory T cells. CD4CD25 regulatory cells were originally described as thymus-derived anergic/suppressive T cells. Recent papers, however, indicate that these cells might also be generated in the periphery. CD4CD25 regulatory T cells can be activated by self- antigens and non-self-antigens, and once activated can suppress T cells in an antigen nonspecific manner. Interestingly, the suppressive effects of these cells are not restricted to the adaptive immune system (T and B cells) but can also affect the activation and function of innate immune cells (monocytes, macrophages, dendritic cells). These features make the CD4CD25 regulatory T cell subset an interesting target for immunotherapy of chronic inflammatory or autoimmune diseases.

Even though dendritic cells (DCs) are well known for their capacity to induce immune responses, recent results show that they are also involved in the induction of tolerance. These two contrary effects of otherwise homologous DCs on a developing immune response maybe explained by different DC developmental stages, i.e., different subsets of DCs may exist and/or different spatial distribution of DCs in the body might influence their function. However, independently from the subtype(s), it is obvious that the ability of DCs to act in a tolerogenic fashion depends on the maturation status, since immature DCs are prone to induce regulatory T cells and hence promote tolerance, whereas mature DCs stimulate effector T cells, facilitating immunity. The means by which DCs convey tolerance are not entirely clear yet, but secretion of suppressive cytokines such as IL-10 and induction of regulatory lymphocytes are involved. In this review we focus on the interaction between DCs and T cells and highlight some mechanisms in the decision-making process of whether immunity or tolerance is induced.

Autoimmune gastritis (AIG) is an experimental model that closely resembles human autoimmune gastritis, the underlying pathology of pernicious anemia. Pathogenic CD4 T cells are reactive to the parietal cell autoantigen, H/K ATPase, and are controlled by CD4CD25 T cells in an immunosuppressive cytokine-independent manner. Comparison of CD4CD25 T cell-mediated suppression in other autoimmune models shows inconsistencies with respect to requirements of cytokines for immunosuppression. More recent data, however, indicate that the evidence for requirement of IL-10 and TGF- could be due to the complex nature of the T cells causing the disease as well as the role of induced regulatory T cell populations. AIG provides a well-defined model that may allow for better analysis of CD4CD25 T cell in vivo biology. Evidence from this model indicates that immune responses must be initiated and then CD4CD25 T cells are recruited to control the quality of the immune response.

Induction and maintenance of peripheral tolerance are important mechanisms to maintain the balance of the immune system. Growing evidence indicates that dysregulation of mucosal T cell responses may lead to loss of tolerance to commensal flora and to the development of inflammatory bowel diseases (IBD). Many studies suggest that active suppression of enteroantigen reactive cells mediated by regulatory T cells contributes to the maintenance of natural intestinal immune homeostasis. The use of the multiple animal models has not only improved our understanding of IBD, but also contributed to new suggestions of treatment strategies involving the use of regulatory T cells. The present review summarizes our current knowledge of regulatory T cells and their involvement in experimental IBD. The well-characterized SCID T cell transfer model and the naturally occurring regulatory CD4CD25 T cells are highlighted.

Discovery of the CD4CD25 T cells has stemmed from investigation of the AOD in the d3tx mice. Besides CD4CD25 T cell depletion, d3tx disease induction requires effector T cell activation prompted by lymphopenia. This is supported by other neonatal AOD models in which T cell-mediated injury has been found to be triggered by immune complex or Ag immunization. In addition, there is growing evidence that support a state of neonatal propensity to autoimmunity, which depends on concomitant endogenous antigenic stimulation, concomitant nematode infection, resistance to CD4CD25 T cell regulation, and participation of the neonatal innate system. The suppression of d3tx disease by polyclonal CD4CD25 T cells appears to be dependent on endogenous Ag and the persistence of regulatory T cells. Thus, suppression of AOD occurs in the ovarian LN, and AOD emerges upon ablation of the input regulatory T cells; and in AIP, the hormone-induced expression of prostate Ag in the CD4CD25 T cell donors rapidly enhances the capacity to suppress disease over Ag negative donors. Finally, genetic analysis of AOD and its component phenotypes has uncovered seven loci. As the general themes that emerged, significant epistatic interactions among the loci play a role in controlling disease susceptibility, the majority of the loci are linked to susceptibility loci of other autoimmune diseases, and the genetic intervals encompass candidate genes that are differentially expressed between CD4CD25 T cells and other T cells. The candidate genes include , TNFR superfamily genes, or , an oocyte auto Ag that reacts with autoantibody in sera of d3tx mice.