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Characterisation of thymus-derived regulatory T cells that protect against organ-specific autoimmune disease
Microbes and Infection, 3, 2001, 905−910 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S1286457901014514/REV
Characterisation of thymus-derived regulatory T cells that protect against organ-specific autoimmune disease Leigh A. Stephens, Don Mason* Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
ABSTRACT – Immunological tolerance to self is, in part, an active process mediated by a certain population of T lymphocytes. Our own work on the origin, phenotypic characterisation and mode of action of these regulatory T cells in rats forms the main focus of this review. © 2001 Éditions scientifiques et médicales Elsevier SAS regulatory T cells / autoimmune disease / tolerance
1. Introduction Until recently it has commonly been maintained that self tolerance of T cells is attained by clonal deletion of autoreactive T cells that encounter self antigen in the thymus [1] and by the induction of peripheral nonresponsiveness (anergy) [2] or deletion [3, 4] in T cells that recognise those self antigens that are not expressed intrathymically. Additionally, there is evidence that some self antigens, for which autoreactive T cells are not deleted, do not normally evoke an immune response because these antigens are not expressed in sufficient levels on the appropriate antigen-presenting cells (APCs) (immunological ignorance) [5, 6]. However, it is now evident that the foregoing explanations for self tolerance of T cells require significant modification and extension. First, the demonstration that many self antigens, whose expression was once considered entirely extrathymic, are also expressed in the thymus (reviewed in [7]), calls into question the validity of the assumption that there are topologically two types of self antigen. Second, the finding that organspecific autoimmunity develops in rats and mice that are genetically relatively lymphopenic or are made so by a variety of experimental procedures, indicates that self tolerance is, in part, an active process mediated by a certain population of T lymphocytes [8, 9]. Our own work on the origin, phenotypic characterisation and mode of action of these regulatory T cells forms the main focus of this review. In order to put our recent data into context our earlier findings will first be briefly described.
*Correspondence and reprints. E-mail address: [email protected] (D. Mason). Microbes and Infection 2001, 905-0
2. Characterisation of regulatory T cells in the TxX rat model of organ-specific autoimmune disease Rats rendered lymphopenic by a process of adult thymectomy and split dose γ-irradiation (termed TxX rats) develop autoimmune thyroiditis and diabetes with a high incidence [10, 11]. The experimental protocol is shown in figure 1. The strains of rat used in these experiments do not suffer from any spontaneous autoimmune disease and a large series of experiments has been carried out to determine the nature of this lymphopenia-induced autoimmunity. In particular, cell transfer experiments, using nonlymphopenic rats as donors and syngeneic TxX rats as recipients, have established that autoimmunity can be prevented in all recipients by a particular subset of peripheral donor T cells. The same subset of cells can protect against both thyroiditis and diabetes, despite the differing pathogenesis of these two diseases [12]. The protective peripheral cells (Treg) were found to have the following characteristics: (1) They are CD4+TCRαβ+CD45RC–RT6+Thy1– [11]. This is the phenotype of a primed T cell. The CD4+RT6+ phenotype is common also to the regulatory T cells that protect against diabetes in the BB rat model of IDDM [13, 14]. (2) They are present in donors thymectomised several weeks earlier [11]. Thus Treg are not necessarily recent thymic migrants and can persist in the periphery. (3) They are absent in the periphery of donor animals that lack the target organ for the autoimmune response. Thus, the adoptive transfer of peripheral CD4+ lymphocytes from athyroid donors into TxX recipients fails to prevent the onset of thyroiditis [15]. However, cells from athyroid donors still prevent autoimmune diabetes in 905
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3. CD25 as a marker for regulatory T cells in rats
Figure 1. The experimental protocol for the induction of lymphopenia in rats. Rats of either the PVG.RT1c or PVG.RT1u strain are thymectomised at 3–6 weeks of age, rested for 1–2 weeks and then given repeated low-dose 137Cs γ irradiation as shown. Organ-specific autoimmunity develops in over 70% of animals within a few months of the last dose of irradiation but is never seen in unmanipulated controls. Rats of the RT1c MHC haplotype most frequently develop thyroiditis with high antithyroglobulin antibody titres, while animals of the RT1u strain develop insulin-dependent diabetes mellitus (IDDM). Subsets of lymphocytes from syngeneic donors, defined by their differential expression of surface antigens, are injected into lymphopenic recipients to assay their capacity to prevent autoimmunity.
recipient animals, indicating that the absence of the thyroid does not nonspecifically interfere with the generation of Treg. Because the peripheral Treg had the phenotype of a primed cell it was anticipated that CD4+CD8– thymocytes would be less potent at preventing diabetes and thyroiditis in TxX rats. However, contrary to these expectations, this thymocyte subset was more potent than the peripheral cells in preventing autoimmunity [11, 16]. Further, CD4+CD8– thymocytes from athyroid donors could prevent autoimmune thyroiditis in TxX recipients even when, as noted above, the peripheral subset could not [15]. This finding demonstrated that the Treg present in the thymus were generated in situ and did not represent a population that had been primed in the periphery and that had then re-entered the thymus. On the basis of this finding it was concluded that, in addition to mediating its recognised functions of positive and negative selection, the thymus possessed a third function which was to generate a subset of CD4+CD8– cells that were committed to the role of preventing organ-specific autoimmune diseases [17]. A statistical analysis of the protective thymocyte population indicated that there was a fine balance between autoreactive T cells and Treg so that, in the intact animal, the latter population was in just sufficient functional excess to maintain self tolerance [16]. This result suggests that this balance is under some homeostatic control. 906
More recent experiments have further characterised the CD4+CD45RC– regulatory T cells that prevent autoimmune diabetes in rats with respect to cell surface expression of CD25 ([18] and summarized in table I). This study was prompted by the work of Sakaguchi and colleagues in which CD25 was shown to be expressed on a subset of anergic CD4+ T cells capable of preventing various organspecific autoimmune diseases from developing in neonatally thymectomised BALB/c mice or in nude mice receiving CD25-depleted T cells [19, 20]. The majority of CD25+ cells in the peripheral tissues of both mouse [19] and rat [18] have low to intermediate expression of the CD45RC isoform (CD45RB in mice), and hence constitute a subset of the cells defined previously in rats to have autoimmune disease-preventing ability. CD25 expression on T cells is also similar in rats and mice, with approximately 6–10% of CD4+ cells positive for this marker in the spleen, lymph nodes, and thymus, but with a lower frequency (3%) among CD4+ cells obtained from the thoracic duct lymph of rats [18]. It has now been shown that, compatible with the data on regulatory T cells in the mouse, CD25+ cells can prevent autoimmune diabetes in rats. However, additional experiments indicated that not all disease-preventing T cells in rats express CD25. In initial experiments using spleen and lymph nodes of rats as a source of the peripheral regulatory T cells, it was found that CD25 expression subdivided the CD4+CD45RC– cells into those with regulatory activity and those without: the CD25+ fraction protected from diabetes at a dose of 1–2 × 106, whereas the CD25– cells did not protect, even at the higher dose of 10 × 106 [18]. This suggested that CD25 was an appropriate marker for the cells with regulatory function, and that the CD45RC– subset protected from autoimmune disease simply because it contained the majority of the CD25+ cells. In support of these data, CD25 was recently shown to be expressed on CD4+ T cells that can protect against diabetes in NOD mice [21].
Table I. Summary of data on the CD25 phenotype of regulatory T cells that protect against diabetes in TxX rats. Source of Treg Spleen and lymph nodes (CD4+CD45RC–) TDL (CD4+CD45RC–) Thymus (CD4+8–) Thymectomised donor Spleen and lymph nodes (CD4+CD45RC–Thy1–)
CD25+ cells
CD25– cells
protection (2 × 106) protection (2 × 106) protection (106) protection (1.5 × 106)
no protection (107) protection (5 × 106) disease acceleration (107) protection (107)
T cells obtained from different tissues of normal syngeneic donors, as indicated in the left-hand column, were sorted according to CD25 expression and transferred into prediabetic TxX PVG.RT1u rats. The outcome of this cell transfer on the onset of diabetes is described. The dose of cells injected is indicated in parentheses. Microbes and Infection 2001, 905-0
Characterisation of thymus-derived regulatory T cells
The CD25 phenotype of the regulatory CD4+CD8– thymocytes in rats was also examined and yielded essentially the same results as described for the mouse model of gastritis, in that regulatory cells were found only in the CD25+ subset (5–10%) of CD4+CD8– thymocytes [22]. In contrast, the CD25– subset of thymocytes contained cells with autoaggressive potential as it led to a reduction in the time of disease onset in TxX recipients. Thus, the regulatory cells were already phenotypically and functionally distinct in the thymus before emigration to the periphery. These data suggested that CD25 might be a stable marker for a separate lineage of T cells that could regulate a range of different autoimmune responses and are present in similar frequencies in the thymus and periphery. However, these results had to be reconciled with the earlier observation from this laboratory in which depletion of CD25-expressing cells from the CD4+CD45RC– subset did not lead to a loss of regulatory activity, which would be expected if all regulatory cells were CD25+ [11]. One difference between these two sets of experiments was the source of regulatory T cells, which in the earlier experiments were obtained by overnight cannulation of the thoracic duct. When we repeated the experiments using regulatory T cells purified from thoracic duct lymph (TDL) the earlier result was reconfirmed: although the CD25+ cells did protect against diabetes at a reduced cell dose (2 × 106), there was also significant protection using the CD25– subset of CD45RC– cells at a higher dose (5 × 106). Unexpectedly, we also discovered that the CD25– subset of CD4+CD45RC– cells from spleen and lymph nodes were also protective if the donors had been thymectomised 4 weeks previously. The simplest interpretation of this finding is that the CD25– recent thymic emigrants, which are also present in the CD25–CD45RC– subset, can promote diabetes, and that their removal by prior thymectomy reveals a population of CD25– memory cells with the potential for preventing diabetes. The relationship between the CD25+ and CD25– regulatory T cells is yet to be established. If CD25 expression is not a stable marker for regulatory cells then both cell types may belong to the same T cell lineage. It is notable that the frequency of CD25+ regulatory T cells in TDL was lower than in lymph node and spleen, whereas the presence of CD25– Treg could be more readily demonstrated in TDL than in these lymphoid organs. While these observations are compatible with a direct lineage relationship between CD25+ and CD25– Treg, other interpretations are possible as the recirculation dynamics of Treg are not known. It has been shown in other models of autoimmunity that CD4+ T cells with the capacity to downregulate immune responses can be induced in vivo by a process of ’infectious tolerance’ [23, 24]. In this process, established regulatory T cells can induce, in the periphery, the regulatory phenotype in naive T cells exported from the thymus. The presumption (yet to be established) is that the T cells that are subject to the process of infectious tolerance arise from CD25– thymocytes. If this is so, then the CD25– Treg found in the periphery of rats may represent cells that owe their regulatory phenotype to infectious tolerance. This possibility is of interest as the specificity of the infected cells is Microbes and Infection 2001, 905-0
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not necessarily identical with that of the Treg that mediate the ’infection’ [23].
4. The mechanism of action of regulatory T cells The way that regulatory T cells prevent autoimmunity is not well understood. In vitro experiments in mice have shown that Treg are nonresponsive to polyclonal stimulation (characterised by poor proliferation and lack of IL-2 production), and can suppress the mitogen-induced proliferation of other T cells via an undefined mechanism requiring direct cell–cell contact between the two cell types [25–27]. Although the CD4+CD25+ regulatory cells required specific antigen to mediate their suppressive effect, the effector mechanism itself was not antigenspecific in vitro [26, 28]. Modulation of APC function by regulatory T cells is one potential mechanism for their suppressive effect, and in a recent report it was shown that CD4+CD25+ cells downregulated B7-1 and B7-2 costimulatory molecules on APCs [29]. However, there is good evidence that direct T–T interactions may be more important, as the suppression still occurred even when fixed APCs were used in cultures [26] or when the regulatory T cells and the ones that they controlled were responding to different APCs in the same culture [28]. No role for TGF-β or other soluble mediators could be found in these experiments. In contrast, in vivo experiments, using the protocol illustrated in figure 1, designed to evaluate the role of IL-4 and TGF-β in the protective mechanism of Treg against thyroiditis, showed that both cytokines played essential roles [12]. Similarly, in vivo experiments in the mouse model of inflammatory bowel disease reveal an important role for TGF-β (and IL-10) in the action of CD4+CD45RBlo regulatory T cells [30, 31]. However, in these experiments IL-4 was shown not to be required for regulation. Currently this apparent conflict between the in vitro and in vivo studies is not understood, but it may be that more quantitative experiments will resolve the difficulty. The in vivo experiments in rats could show a role for TGF-β and IL-4 only when the number of regulatory T cells used to control the lymphopenia-induced autoimmunity was reduced to a just sufficient minimum [12]. At higher cell doses the injection of neutralising mAbs to these cytokines did not abrogate the protection from autoimmunity provided by the injected regulatory cells. While it may be that the neutralisation of the cytokines at the higher doses of Treg was inadequate to inhibit their biological action, it may instead be that cytokines are involved in the prevention of autoimmunity only when the number of regulatory T cells is limiting. It remains to be determined whether synergy can be demonstrated in vitro between Treg and TGF-β in the prevention of T-cell activation. IL-2 is probably also involved as a growth factor for Treg and this reactivity may be the basis of the homeostatic mechanism mentioned above, as suggested by the absence of CD25+ regulatory T cells in IL-2 knockout mice [32], in addition to the effect of exogenous IL-2 on in vitro cultures of CD25+ cells, which transiently overcomes their anergic 907
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Figure 2. Homeostatic control of the regulatory T cell subset. As described in the text, there is good evidence for a fine balance between the size of the autoreactive T cell population and the regulatory one that controls it. This figure illustrates a possible negative-feedback mechanism that maintains the appropriate balance between the two cell types. Recognition of the autoantigen stimulates the autoreactive T cells to produce IL-2 which in turn causes expansion of the regulatory population until it is sufficiently large to control the autoreactive one. Currently there are insufficient data to determine whether the homeostatic mechanism operates intrathymically or in the periphery. However, the survival of the regulatory cells requires the persistence of the relevant autoantigen in the periphery and for this reason the site at which homeostasis is established has tentatively been placed in the figure.
and suppressive action. If this is so, then IL-2 secreted by autoreactive T cells may expand the numbers of Treg to the level where further activation of autoreactive cells is controlled (figure 2). This hypothesis provides a possible explanation for the failure, already mentioned, to detect regulatory T cells for the prevention of autoimmune thyroiditis in the periphery of athyroid rats.
5. Summary Figure 3 summarises some of the principle features of regulatory T cells in rats. The work of others in mice, together with our own unpublished studies in humans, provides compelling evidence that essentially the same cells exist in all three species. CD4+CD25+ cells have now been found to suppress a range of autoimmune diseases in rats and mice and exist in human PBL and thymus in 908
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Figure 3. Schematic illustration of the production, differentiation and action of regulatory T cells. There are two subsets of mature CD4+CD8– thymocytes in rats. Both types are CD45RC– CD62L+ TCRαβ+ [37]. However, the minor subset (about 7% of the total) expresses CD25 and contains regulatory T cells [18]. The CD25– subpopulation is the precursor for the peripheral CD4+ T cells that mediate immune responses to both foreign antigens and to at least some self antigens. In the periphery the regulatory T cells are CD45RC– while the potentially immunoreactive subset becomes CD45RC+ (mature, resting naive cells). On activation the naive cells become CD45RC– and may express the IL-2 receptor (including CD25). Relatively little is known of the mode or site of action of regulatory cells. For a discussion on this point see [17]. Not shown in the figure is the lineage of the CD25– regulatory T cells that are found in the periphery but are not, as far as can be ascertained, in the thymus. The possible origin of these cells is discussed in the text. similar frequencies as rodents ([18] and unpublished). Furthermore, the CD4+CD25+ cells present in human thymus have the same anergic and suppressive activity in vitro as the corresponding cells in mice (Stephens L.A., Mottet C., Mason D., Powrie F., Human CD4+CD25+ thymocytes and peripheral T cells have immune suppressive activity in vitro, Eur. J. Immunol. 31 (2001), 1247–1254). However, we have shown in rats that CD25 is not an exclusive marker for regulatory cells in the periphery. Thus, activated immunologically competent CD4+ cells also express CD25, while some regulatory T cells are CD25–. Our studies also show that caution must be taken when concluding that any subset does not contain regulatory cells, as the presence of autoreactive cells in a particular cell subset can mask the presence of regulatory cells if these are present in low numbers. The precise mechanism used by regulatory T cells to suppress immune responses in vitro awaits further characterisation, as does its relevance to the mechanism of in vivo suppression. Clearly, a failure of development or activity of regulatory T cells is an intriguing candidate Microbes and Infection 2001, 905-0
Characterisation of thymus-derived regulatory T cells
mechanism that may contribute to the aetiology of autoimmune disease, and deserves further examination.
6. Concluding remarks: the need for an active mechanism of T-cell tolerance Clonal deletion as the mechanism of self/nonself discrimination among T cells is conceptually attractive since with such a mechanism no perturbation of the immune system should give rise to autoimmunity. Consequently, given the ease with which autoreactive T cells can be demonstrated in the periphery, even for self antigens such as insulin that are expressed intrathymically, one may ask why deletion is so relatively inefficient. At least two interrelated possibilities may be considered. First, the finite number of MHC molecules on an APC in the thymus sets a limit on the number of self peptides that it can present. Elution of peptides from cell-bound MHC molecules, derived from cell lines, indicate that an APC has approximately 103 different peptides associated with its MHC molecules [33, 34]. Those that are sufficiently frequent to be identified are all self peptides or derive from endogenous viruses and some of these self peptides are presented at a very high copy number (such that less than ten different peptides account for 70% of the MHC molecules available to bind peptide). This implies that other self peptides must be presented at a much lower copy number. If these figures apply to the APCs in the thymus that mediate negative selection, then competition for MHC binding may impose a limit on the number of different self peptides that are presented at a high enough level to ensure deletion of low-affinity autoreactive T cells. Consequently, the peripheral T-cell repertoire will be profoundly tolerant for abundant self antigens but contain some cells that react, possibly with relatively low affinity, with self antigens presented in the thymus at low levels. In principle the inadequacy of presentation of self antigens in the thymus may be overcome if medullary APCs are heterogeneous, with different cells dedicated to the presentation of different self peptides. While there is some evidence for such specialisation [35], there is, as mentioned above, a second limitation on clonal deletion as a means of achieving self tolerance. A quantitative analysis of the demands made upon the peripheral T-cell repertoire for the recognition of foreign antigens indicates the nature of this limitation. A crucial parameter in the functioning of the T-cell repertoire is the probability (here referred to as P) that a given T cell will recognise a randomly chosen foreign peptide appropriately presented in the context of self-MHC. Published data, reviewed in [36], indicate that, for CD4+ T cells P ∼ 10–4. (Given that a mouse has approximately 108 naive CD4+ T cells about 104 of these will respond to a single foreign peptide. Conversely, about 104 T cells are required to interrogate an antigen-presenting cell before one is found to react with a particular peptide). This value of P imposes a severe limit on the number of self peptides for which tolerance can be achieved by clonal deletion. Evidently, if this number significantly exceeds 104 then few thymocytes will survive negative selection. Microbes and Infection 2001, 905-0
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With approximately 105 genes in the mammalian genome the potential number of self peptides is likely much greater than 104. The preceding discussion indicates that intrathymic clonal deletion gives rise to a peripheral T-cell repertoire that is very effectively deleted of T cells that respond to abundant self peptides, but that contains autoreactive T cells for less abundant ones. As the studies on regulatory T cells indicate, tolerance to these latter self antigens, particularly those associated with endocrine secretions, requires a dominant, cell-mediated control mechanism that is provided by a specialised subset of CD4+ T cells that become committed intrathymically for their regulatory role.
Acknowledgments Our work was supported by grants from the British Diabetic Association and Medical Research Council, UK.
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[11] Fowell D., Mason D., Evidence that the T cell repertoire of normal rats contains cells with the potential to cause diabetes. Characterization of the CD4+ T cell subset that inhibits this autoimmune potential, J. Exp. Med. 177 (1993) 627–636. [12] Seddon B., Mason D., Regulatory T cells in the control of autoimmunity: the essential role of transforming growth factor beta and interleukin 4 in the prevention of autoimmune thyroiditis in rats by peripheral CD4(+)CD45RCcells and CD4(+)CD8(-) thymocytes, J. Exp. Med. 189 (1999) 279–288. [13] Rossini A.A., Mordes J.P., Greiner D.L., The pathogenesis of autoimmune diabetes mellitus, Curr. Opin. Immunol. 2 (1989) 598–603. [14] Whalen B.J., Greiner D.L., Mordes J.P., Rossini A.A., Adoptive transfer of autoimmune diabetes mellitus to athymic rats: synergy of CD4+ and CD8+ T cells and prevention by RT6+ T cells, J. Autoimmun. 7 (1994) 819–831. [15] Seddon B., Mason D., Peripheral autoantigen induces regulatory T cells that prevent autoimmunity, J. Exp. Med. 189 (1999) 877–882. [16] Saoudi A., Seddon B., Fowell D., Mason D., The thymus contains a high frequency of cells that prevent autoimmune diabetes on transfer into prediabetic recipients, J. Exp. Med. 184 (1996) 2393–2398. [17] Seddon B., Mason D., The third function of the thymus, Immunol. Today 21 (2000) 95–99. [18] Stephens L.A., Mason D., CD25 is a marker for CD4+ thymocytes that prevent autoimmune diabetes in rats, but peripheral T cells with this function are found in both CD25+ and CD25– subpopulations, J. Immunol. 165 (2000) 3105–3110. [19] Sakaguchi S., Sakaguchi N., Asano M., Itoh M., Toda M., Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases, J. Immunol. 155 (1995) 1151–1164. [20] Asano M., Toda M., Sakaguchi N., Sakaguchi S., Autoimmune disease as a consequence of developmental abnormality of a T cell subpopulation, J. Exp. Med. 184 (1996) 387–396. [21] Salomon B., Lenschow D.J., Rhee L., Ashourian N., Singh B., Sharpe A., Bluestone J.A., B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes, Immunity 12 (2000) 431–440. [22] Itoh M., Takahashi T., Sakaguchi N., Kuniyasu Y., Shimizu J., Otsuka F., Sakaguchi S., Thymus and autoimmunity: production of CD25+CD4+ naturally anergic and suppressive T cells as a key function of the thymus in maintaining immunologic self-tolerance, J. Immunol. 162 (1999) 5317–5326. [23] Waldmann H., Cobbold S., How do monoclonal antibodies induce tolerance? A role for infectious tolerance? Annu. Rev. Immunol. 16 (1998) 619–644. [24] Modigliani Y., Coutinho A., Pereira P., Le Douarin N., Thomas-Vaslin V., Burlen-Defranoux O., Salaun J., Bandeira A., Establishment of tissue-specific tolerance is driven by regulatory T cells selected by thymic epithelium, Eur. J. Immunol. 26 (1996) 1807–1815. 910
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[25] Thornton A.M., Shevach E.M., CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production, J. Exp. Med. 188 (1998) 287–296. [26] Takahashi T., Kuniyasu Y., Toda M., Sakaguchi N., Itoh M., Iwata M., Shimizu J., Sakaguchi S., Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state, Int. Immunol. 10 (1998) 1969–1980. [27] Read S., Mauze S., Asseman C., Bean A., Coffman R., Powrie F., CD38+ CD45RB(low) CD4+ T cells: a population of T cells with immune regulatory activities in vitro, Eur. J. Immunol. 28 (1998) 3435–3447. [28] Thornton A.M., Shevach E.M., Suppressor effector function of CD4+CD25+ immunoregulatory T cells is antigen nonspecific, J. Immunol. 164 (2000) 183–190. [29] Cederbom L., Hall H., Ivars F., CD4+CD25+ regulatory T cells down-regulate co-stimulatory molecules on antigenpresenting cells, Eur. J. Immunol. 30 (2000) 1538–1543. [30] Powrie F., Carlino J., Leach M.W., Mauze S., Coffman R.L., A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediated colitis by CD45RB(low) CD4+ T cells, J. Exp. Med. 183 (1996) 2669–2674. [31] Asseman C., Mauze S., Leach M.W., Coffman R.L., Powrie F., An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation, J. Exp. Med. 190 (1999) 995–1004. [32] Papiernik M., de Moraes M.L., Pontoux C., Vasseur F., Penit C., Regulatory CD4 T cells: expression of IL-2R alpha chain, resistance to clonal deletion and IL-2 dependency, Int. Immunol. 10 (1998) 371–378. [33] Hunt D.F., Michel H., Dickinson T.A., Shabanowitz J., Cox A.L., Sakaguchi K., Appella E., Grey H.M., Sette A., Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad, Science 256 (1992) 1817–1820. [34] Rudensky A., Preston-Hurlburt P., Hong S.C., Barlow A., Janeway C.A., Sequence analysis of peptides bound to MHC class II molecules, Nature 353 (1991) 622–627. [35] Smith K.M., Olson D.C., Hirose R., Hanahan D., Pancreatic gene expression in rare cells of thymic medulla: evidence for functional contribution to T cell tolerance, Int. Immunol. 9 (1997) 1355–1365. [36] Mason D., A very high level of crossreactivity is an essential feature of the T-cell receptor, Immunol. Today 19 (1998) 395–404. [37] Seddon B., Saoudi A., Nicholson M., Mason D., CD4+CD8– thymocytes that express L-selectin protect rats from diabetes upon adoptive transfer, Eur. J. Immunol. 26 (1996) 2702–2708.
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Characterisation of thymus-derived regulatory T cells that protect against organ-specific autoimmune disease Leigh A. Stephens, Don Mason* Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK
ABSTRACT – Immunological tolerance to self is, in part, an active process mediated by a certain population of T lymphocytes. Our own work on the origin, phenotypic characterisation and mode of action of these regulatory T cells in rats forms the main focus of this review. © 2001 Éditions scientifiques et médicales Elsevier SAS regulatory T cells / autoimmune disease / tolerance
1. Introduction Until recently it has commonly been maintained that self tolerance of T cells is attained by clonal deletion of autoreactive T cells that encounter self antigen in the thymus [1] and by the induction of peripheral nonresponsiveness (anergy) [2] or deletion [3, 4] in T cells that recognise those self antigens that are not expressed intrathymically. Additionally, there is evidence that some self antigens, for which autoreactive T cells are not deleted, do not normally evoke an immune response because these antigens are not expressed in sufficient levels on the appropriate antigen-presenting cells (APCs) (immunological ignorance) [5, 6]. However, it is now evident that the foregoing explanations for self tolerance of T cells require significant modification and extension. First, the demonstration that many self antigens, whose expression was once considered entirely extrathymic, are also expressed in the thymus (reviewed in [7]), calls into question the validity of the assumption that there are topologically two types of self antigen. Second, the finding that organspecific autoimmunity develops in rats and mice that are genetically relatively lymphopenic or are made so by a variety of experimental procedures, indicates that self tolerance is, in part, an active process mediated by a certain population of T lymphocytes [8, 9]. Our own work on the origin, phenotypic characterisation and mode of action of these regulatory T cells forms the main focus of this review. In order to put our recent data into context our earlier findings will first be briefly described.
*Correspondence and reprints. E-mail address: [email protected] (D. Mason). Microbes and Infection 2001, 905-0
2. Characterisation of regulatory T cells in the TxX rat model of organ-specific autoimmune disease Rats rendered lymphopenic by a process of adult thymectomy and split dose γ-irradiation (termed TxX rats) develop autoimmune thyroiditis and diabetes with a high incidence [10, 11]. The experimental protocol is shown in figure 1. The strains of rat used in these experiments do not suffer from any spontaneous autoimmune disease and a large series of experiments has been carried out to determine the nature of this lymphopenia-induced autoimmunity. In particular, cell transfer experiments, using nonlymphopenic rats as donors and syngeneic TxX rats as recipients, have established that autoimmunity can be prevented in all recipients by a particular subset of peripheral donor T cells. The same subset of cells can protect against both thyroiditis and diabetes, despite the differing pathogenesis of these two diseases [12]. The protective peripheral cells (Treg) were found to have the following characteristics: (1) They are CD4+TCRαβ+CD45RC–RT6+Thy1– [11]. This is the phenotype of a primed T cell. The CD4+RT6+ phenotype is common also to the regulatory T cells that protect against diabetes in the BB rat model of IDDM [13, 14]. (2) They are present in donors thymectomised several weeks earlier [11]. Thus Treg are not necessarily recent thymic migrants and can persist in the periphery. (3) They are absent in the periphery of donor animals that lack the target organ for the autoimmune response. Thus, the adoptive transfer of peripheral CD4+ lymphocytes from athyroid donors into TxX recipients fails to prevent the onset of thyroiditis [15]. However, cells from athyroid donors still prevent autoimmune diabetes in 905
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3. CD25 as a marker for regulatory T cells in rats
Figure 1. The experimental protocol for the induction of lymphopenia in rats. Rats of either the PVG.RT1c or PVG.RT1u strain are thymectomised at 3–6 weeks of age, rested for 1–2 weeks and then given repeated low-dose 137Cs γ irradiation as shown. Organ-specific autoimmunity develops in over 70% of animals within a few months of the last dose of irradiation but is never seen in unmanipulated controls. Rats of the RT1c MHC haplotype most frequently develop thyroiditis with high antithyroglobulin antibody titres, while animals of the RT1u strain develop insulin-dependent diabetes mellitus (IDDM). Subsets of lymphocytes from syngeneic donors, defined by their differential expression of surface antigens, are injected into lymphopenic recipients to assay their capacity to prevent autoimmunity.
recipient animals, indicating that the absence of the thyroid does not nonspecifically interfere with the generation of Treg. Because the peripheral Treg had the phenotype of a primed cell it was anticipated that CD4+CD8– thymocytes would be less potent at preventing diabetes and thyroiditis in TxX rats. However, contrary to these expectations, this thymocyte subset was more potent than the peripheral cells in preventing autoimmunity [11, 16]. Further, CD4+CD8– thymocytes from athyroid donors could prevent autoimmune thyroiditis in TxX recipients even when, as noted above, the peripheral subset could not [15]. This finding demonstrated that the Treg present in the thymus were generated in situ and did not represent a population that had been primed in the periphery and that had then re-entered the thymus. On the basis of this finding it was concluded that, in addition to mediating its recognised functions of positive and negative selection, the thymus possessed a third function which was to generate a subset of CD4+CD8– cells that were committed to the role of preventing organ-specific autoimmune diseases [17]. A statistical analysis of the protective thymocyte population indicated that there was a fine balance between autoreactive T cells and Treg so that, in the intact animal, the latter population was in just sufficient functional excess to maintain self tolerance [16]. This result suggests that this balance is under some homeostatic control. 906
More recent experiments have further characterised the CD4+CD45RC– regulatory T cells that prevent autoimmune diabetes in rats with respect to cell surface expression of CD25 ([18] and summarized in table I). This study was prompted by the work of Sakaguchi and colleagues in which CD25 was shown to be expressed on a subset of anergic CD4+ T cells capable of preventing various organspecific autoimmune diseases from developing in neonatally thymectomised BALB/c mice or in nude mice receiving CD25-depleted T cells [19, 20]. The majority of CD25+ cells in the peripheral tissues of both mouse [19] and rat [18] have low to intermediate expression of the CD45RC isoform (CD45RB in mice), and hence constitute a subset of the cells defined previously in rats to have autoimmune disease-preventing ability. CD25 expression on T cells is also similar in rats and mice, with approximately 6–10% of CD4+ cells positive for this marker in the spleen, lymph nodes, and thymus, but with a lower frequency (3%) among CD4+ cells obtained from the thoracic duct lymph of rats [18]. It has now been shown that, compatible with the data on regulatory T cells in the mouse, CD25+ cells can prevent autoimmune diabetes in rats. However, additional experiments indicated that not all disease-preventing T cells in rats express CD25. In initial experiments using spleen and lymph nodes of rats as a source of the peripheral regulatory T cells, it was found that CD25 expression subdivided the CD4+CD45RC– cells into those with regulatory activity and those without: the CD25+ fraction protected from diabetes at a dose of 1–2 × 106, whereas the CD25– cells did not protect, even at the higher dose of 10 × 106 [18]. This suggested that CD25 was an appropriate marker for the cells with regulatory function, and that the CD45RC– subset protected from autoimmune disease simply because it contained the majority of the CD25+ cells. In support of these data, CD25 was recently shown to be expressed on CD4+ T cells that can protect against diabetes in NOD mice [21].
Table I. Summary of data on the CD25 phenotype of regulatory T cells that protect against diabetes in TxX rats. Source of Treg Spleen and lymph nodes (CD4+CD45RC–) TDL (CD4+CD45RC–) Thymus (CD4+8–) Thymectomised donor Spleen and lymph nodes (CD4+CD45RC–Thy1–)
CD25+ cells
CD25– cells
protection (2 × 106) protection (2 × 106) protection (106) protection (1.5 × 106)
no protection (107) protection (5 × 106) disease acceleration (107) protection (107)
T cells obtained from different tissues of normal syngeneic donors, as indicated in the left-hand column, were sorted according to CD25 expression and transferred into prediabetic TxX PVG.RT1u rats. The outcome of this cell transfer on the onset of diabetes is described. The dose of cells injected is indicated in parentheses. Microbes and Infection 2001, 905-0
Characterisation of thymus-derived regulatory T cells
The CD25 phenotype of the regulatory CD4+CD8– thymocytes in rats was also examined and yielded essentially the same results as described for the mouse model of gastritis, in that regulatory cells were found only in the CD25+ subset (5–10%) of CD4+CD8– thymocytes [22]. In contrast, the CD25– subset of thymocytes contained cells with autoaggressive potential as it led to a reduction in the time of disease onset in TxX recipients. Thus, the regulatory cells were already phenotypically and functionally distinct in the thymus before emigration to the periphery. These data suggested that CD25 might be a stable marker for a separate lineage of T cells that could regulate a range of different autoimmune responses and are present in similar frequencies in the thymus and periphery. However, these results had to be reconciled with the earlier observation from this laboratory in which depletion of CD25-expressing cells from the CD4+CD45RC– subset did not lead to a loss of regulatory activity, which would be expected if all regulatory cells were CD25+ [11]. One difference between these two sets of experiments was the source of regulatory T cells, which in the earlier experiments were obtained by overnight cannulation of the thoracic duct. When we repeated the experiments using regulatory T cells purified from thoracic duct lymph (TDL) the earlier result was reconfirmed: although the CD25+ cells did protect against diabetes at a reduced cell dose (2 × 106), there was also significant protection using the CD25– subset of CD45RC– cells at a higher dose (5 × 106). Unexpectedly, we also discovered that the CD25– subset of CD4+CD45RC– cells from spleen and lymph nodes were also protective if the donors had been thymectomised 4 weeks previously. The simplest interpretation of this finding is that the CD25– recent thymic emigrants, which are also present in the CD25–CD45RC– subset, can promote diabetes, and that their removal by prior thymectomy reveals a population of CD25– memory cells with the potential for preventing diabetes. The relationship between the CD25+ and CD25– regulatory T cells is yet to be established. If CD25 expression is not a stable marker for regulatory cells then both cell types may belong to the same T cell lineage. It is notable that the frequency of CD25+ regulatory T cells in TDL was lower than in lymph node and spleen, whereas the presence of CD25– Treg could be more readily demonstrated in TDL than in these lymphoid organs. While these observations are compatible with a direct lineage relationship between CD25+ and CD25– Treg, other interpretations are possible as the recirculation dynamics of Treg are not known. It has been shown in other models of autoimmunity that CD4+ T cells with the capacity to downregulate immune responses can be induced in vivo by a process of ’infectious tolerance’ [23, 24]. In this process, established regulatory T cells can induce, in the periphery, the regulatory phenotype in naive T cells exported from the thymus. The presumption (yet to be established) is that the T cells that are subject to the process of infectious tolerance arise from CD25– thymocytes. If this is so, then the CD25– Treg found in the periphery of rats may represent cells that owe their regulatory phenotype to infectious tolerance. This possibility is of interest as the specificity of the infected cells is Microbes and Infection 2001, 905-0
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not necessarily identical with that of the Treg that mediate the ’infection’ [23].
4. The mechanism of action of regulatory T cells The way that regulatory T cells prevent autoimmunity is not well understood. In vitro experiments in mice have shown that Treg are nonresponsive to polyclonal stimulation (characterised by poor proliferation and lack of IL-2 production), and can suppress the mitogen-induced proliferation of other T cells via an undefined mechanism requiring direct cell–cell contact between the two cell types [25–27]. Although the CD4+CD25+ regulatory cells required specific antigen to mediate their suppressive effect, the effector mechanism itself was not antigenspecific in vitro [26, 28]. Modulation of APC function by regulatory T cells is one potential mechanism for their suppressive effect, and in a recent report it was shown that CD4+CD25+ cells downregulated B7-1 and B7-2 costimulatory molecules on APCs [29]. However, there is good evidence that direct T–T interactions may be more important, as the suppression still occurred even when fixed APCs were used in cultures [26] or when the regulatory T cells and the ones that they controlled were responding to different APCs in the same culture [28]. No role for TGF-β or other soluble mediators could be found in these experiments. In contrast, in vivo experiments, using the protocol illustrated in figure 1, designed to evaluate the role of IL-4 and TGF-β in the protective mechanism of Treg against thyroiditis, showed that both cytokines played essential roles [12]. Similarly, in vivo experiments in the mouse model of inflammatory bowel disease reveal an important role for TGF-β (and IL-10) in the action of CD4+CD45RBlo regulatory T cells [30, 31]. However, in these experiments IL-4 was shown not to be required for regulation. Currently this apparent conflict between the in vitro and in vivo studies is not understood, but it may be that more quantitative experiments will resolve the difficulty. The in vivo experiments in rats could show a role for TGF-β and IL-4 only when the number of regulatory T cells used to control the lymphopenia-induced autoimmunity was reduced to a just sufficient minimum [12]. At higher cell doses the injection of neutralising mAbs to these cytokines did not abrogate the protection from autoimmunity provided by the injected regulatory cells. While it may be that the neutralisation of the cytokines at the higher doses of Treg was inadequate to inhibit their biological action, it may instead be that cytokines are involved in the prevention of autoimmunity only when the number of regulatory T cells is limiting. It remains to be determined whether synergy can be demonstrated in vitro between Treg and TGF-β in the prevention of T-cell activation. IL-2 is probably also involved as a growth factor for Treg and this reactivity may be the basis of the homeostatic mechanism mentioned above, as suggested by the absence of CD25+ regulatory T cells in IL-2 knockout mice [32], in addition to the effect of exogenous IL-2 on in vitro cultures of CD25+ cells, which transiently overcomes their anergic 907
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Figure 2. Homeostatic control of the regulatory T cell subset. As described in the text, there is good evidence for a fine balance between the size of the autoreactive T cell population and the regulatory one that controls it. This figure illustrates a possible negative-feedback mechanism that maintains the appropriate balance between the two cell types. Recognition of the autoantigen stimulates the autoreactive T cells to produce IL-2 which in turn causes expansion of the regulatory population until it is sufficiently large to control the autoreactive one. Currently there are insufficient data to determine whether the homeostatic mechanism operates intrathymically or in the periphery. However, the survival of the regulatory cells requires the persistence of the relevant autoantigen in the periphery and for this reason the site at which homeostasis is established has tentatively been placed in the figure.
and suppressive action. If this is so, then IL-2 secreted by autoreactive T cells may expand the numbers of Treg to the level where further activation of autoreactive cells is controlled (figure 2). This hypothesis provides a possible explanation for the failure, already mentioned, to detect regulatory T cells for the prevention of autoimmune thyroiditis in the periphery of athyroid rats.
5. Summary Figure 3 summarises some of the principle features of regulatory T cells in rats. The work of others in mice, together with our own unpublished studies in humans, provides compelling evidence that essentially the same cells exist in all three species. CD4+CD25+ cells have now been found to suppress a range of autoimmune diseases in rats and mice and exist in human PBL and thymus in 908
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Figure 3. Schematic illustration of the production, differentiation and action of regulatory T cells. There are two subsets of mature CD4+CD8– thymocytes in rats. Both types are CD45RC– CD62L+ TCRαβ+ [37]. However, the minor subset (about 7% of the total) expresses CD25 and contains regulatory T cells [18]. The CD25– subpopulation is the precursor for the peripheral CD4+ T cells that mediate immune responses to both foreign antigens and to at least some self antigens. In the periphery the regulatory T cells are CD45RC– while the potentially immunoreactive subset becomes CD45RC+ (mature, resting naive cells). On activation the naive cells become CD45RC– and may express the IL-2 receptor (including CD25). Relatively little is known of the mode or site of action of regulatory cells. For a discussion on this point see [17]. Not shown in the figure is the lineage of the CD25– regulatory T cells that are found in the periphery but are not, as far as can be ascertained, in the thymus. The possible origin of these cells is discussed in the text. similar frequencies as rodents ([18] and unpublished). Furthermore, the CD4+CD25+ cells present in human thymus have the same anergic and suppressive activity in vitro as the corresponding cells in mice (Stephens L.A., Mottet C., Mason D., Powrie F., Human CD4+CD25+ thymocytes and peripheral T cells have immune suppressive activity in vitro, Eur. J. Immunol. 31 (2001), 1247–1254). However, we have shown in rats that CD25 is not an exclusive marker for regulatory cells in the periphery. Thus, activated immunologically competent CD4+ cells also express CD25, while some regulatory T cells are CD25–. Our studies also show that caution must be taken when concluding that any subset does not contain regulatory cells, as the presence of autoreactive cells in a particular cell subset can mask the presence of regulatory cells if these are present in low numbers. The precise mechanism used by regulatory T cells to suppress immune responses in vitro awaits further characterisation, as does its relevance to the mechanism of in vivo suppression. Clearly, a failure of development or activity of regulatory T cells is an intriguing candidate Microbes and Infection 2001, 905-0
Characterisation of thymus-derived regulatory T cells
mechanism that may contribute to the aetiology of autoimmune disease, and deserves further examination.
6. Concluding remarks: the need for an active mechanism of T-cell tolerance Clonal deletion as the mechanism of self/nonself discrimination among T cells is conceptually attractive since with such a mechanism no perturbation of the immune system should give rise to autoimmunity. Consequently, given the ease with which autoreactive T cells can be demonstrated in the periphery, even for self antigens such as insulin that are expressed intrathymically, one may ask why deletion is so relatively inefficient. At least two interrelated possibilities may be considered. First, the finite number of MHC molecules on an APC in the thymus sets a limit on the number of self peptides that it can present. Elution of peptides from cell-bound MHC molecules, derived from cell lines, indicate that an APC has approximately 103 different peptides associated with its MHC molecules [33, 34]. Those that are sufficiently frequent to be identified are all self peptides or derive from endogenous viruses and some of these self peptides are presented at a very high copy number (such that less than ten different peptides account for 70% of the MHC molecules available to bind peptide). This implies that other self peptides must be presented at a much lower copy number. If these figures apply to the APCs in the thymus that mediate negative selection, then competition for MHC binding may impose a limit on the number of different self peptides that are presented at a high enough level to ensure deletion of low-affinity autoreactive T cells. Consequently, the peripheral T-cell repertoire will be profoundly tolerant for abundant self antigens but contain some cells that react, possibly with relatively low affinity, with self antigens presented in the thymus at low levels. In principle the inadequacy of presentation of self antigens in the thymus may be overcome if medullary APCs are heterogeneous, with different cells dedicated to the presentation of different self peptides. While there is some evidence for such specialisation [35], there is, as mentioned above, a second limitation on clonal deletion as a means of achieving self tolerance. A quantitative analysis of the demands made upon the peripheral T-cell repertoire for the recognition of foreign antigens indicates the nature of this limitation. A crucial parameter in the functioning of the T-cell repertoire is the probability (here referred to as P) that a given T cell will recognise a randomly chosen foreign peptide appropriately presented in the context of self-MHC. Published data, reviewed in [36], indicate that, for CD4+ T cells P ∼ 10–4. (Given that a mouse has approximately 108 naive CD4+ T cells about 104 of these will respond to a single foreign peptide. Conversely, about 104 T cells are required to interrogate an antigen-presenting cell before one is found to react with a particular peptide). This value of P imposes a severe limit on the number of self peptides for which tolerance can be achieved by clonal deletion. Evidently, if this number significantly exceeds 104 then few thymocytes will survive negative selection. Microbes and Infection 2001, 905-0
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With approximately 105 genes in the mammalian genome the potential number of self peptides is likely much greater than 104. The preceding discussion indicates that intrathymic clonal deletion gives rise to a peripheral T-cell repertoire that is very effectively deleted of T cells that respond to abundant self peptides, but that contains autoreactive T cells for less abundant ones. As the studies on regulatory T cells indicate, tolerance to these latter self antigens, particularly those associated with endocrine secretions, requires a dominant, cell-mediated control mechanism that is provided by a specialised subset of CD4+ T cells that become committed intrathymically for their regulatory role.
Acknowledgments Our work was supported by grants from the British Diabetic Association and Medical Research Council, UK.
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