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Th2 paradigm in immunity to infectious diseases
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Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases Peter McGuirk and Kingston H.G. Mills Current dogma suggests that immunity to infection is controlled by distinct type 1 (Th1) and type 2 (Th2) subpopulations of T cells discriminated on the basis of cytokine secretion and function. However, a further subtype of T cells, with immunosuppressive function and cytokine profiles distinct from either Th1 or Th2 T cells, termed regulatory T (Tr) cells has been described. Although considered to have a role in the maintenance of self-tolerance, recent studies suggest that Tr cells can be induced against bacterial, viral and parasite antigens in vivo and might prevent infection-induced immunopathology or prolong pathogen persistence by suppressing protective Th1 responses. These observations have significant implications for our understanding of the role of T cells in immunity to infectious diseases and for the development of new therapies for immune-mediated disorders.
Several subsets of regulatory T (Tr) cells with distinct phenotypes and distinct mechanisms of action have now been identified. These include type 1 Tr cells (Tr1) cells [1–6], which secrete high levels of interleukin (IL)-10 and low to moderate levels of transforming growth factor (TGF)-β, type 3 T (Th3) cells [7,8], which primarily secrete TGF-β, and CD4+CD25+ T cells, which inhibit immune responses through cell–cell contact [9] (Table 1). In addition to the various CD4+ Tr populations, recent studies have identified CD8+ Tr cells, which secrete either IL-10 or TGF-β [10,11]. Initial reports indicated that the primary function of Tr cells was in maintaining peripheral tolerance and homeostasis at mucosal surfaces and suggested that they could be directed against self-antigens [12]. However, until recently, little was known about their antigen specificities or induction in vivo, and progress on their characterization has been difficult as a result of failures to cultivate and clone Tr cells in vitro. A significant advance has been made in the field of Tr cells with the ex vivo propagation and cloning of pathogen-specific murine [4] and human [2,5] Tr1 cells during infection. Furthermore, treatment of mice with killed bacteria promotes the development of allergen-specific CD4+CD45RBlow Tr cells [13]. Peter McGuirk Kingston H.G. Mills* Immune Regulation Research Group, Department of Biochemistry, Trinity College, Dublin 2, Ireland. *e-mail: kingston.mills@ tcd.ie
Phenotype and function of Tr cells Tr1 cells
T cells, from ovalbumin (OVA) T-cell receptor (TCR)-transgenic mice, cultured with OVA and IL-10 result in the generation of T-cell clones with a unique cytokine profile distinct from that of Th0, Th1 or Th2 cells. These Tr1 cells produce IL-10 and IL-5, http://immunology.trends.com
with or without TGF-β, but with little or no IL-2, IL-4 or interferon (IFN)-γ production, and proliferate poorly following polyclonal TCR-mediated activation [1]. More recently, in vitro manipulation with immunosuppressive drugs and anti-IL-4 and antiIL-12 antibodies has facilitated the expansion of Tr1 cells that exclusively secrete IL-10 [14]. The expression of CCR5 and T1–ST2 on Tr1 cells [4], markers previously expressed preferentially on Th1 and Th2 cells, respectively, suggest that Tr1 cells represent a phenotypically distinct subtype of CD4+ T cells. Functional studies on Tr1 cells have suggested that these cells have immunosuppressive properties and have been shown to prevent the development of Th1-mediated autoimmune diseases [1]. However, Tr1 and other Tr-cell populations also suppress immune responses to pathogens, tumors and alloantigens. Co-culture of naïve CD4+ T cells and human Tr1 clones in the presence of allogenic antigen-presenting cells (APCs) results in the suppression of proliferative responses [1]. Similarly, Tr1 clones specific for filamentous haemaggultinin (FHA) from Bordetella pertussis suppress proliferation and cytokine production by a Th1 clone against an unrelated antigen, influenza virus haemagglutinin (HA) [4]. In both cases, the suppressive effects of Tr1-cell clones are reversed by neutralizing IL-10, suggesting that, regardless of their antigen specificities, Tr1 cell suppression is a bystander effect mediated through the production of IL-10. Th3 cells
Another subset of CD4+ Tr cells was identified in studies of oral tolerance. In multiple sclerosis patients, oral treatment with myelin basic protein (MBP) induces a significant increase in the frequency of MBP-specific T cells that secrete TGF-β [8]. These Th3 cells were originally generated and identified in mice orally tolerized to MBP and suppressed the induction of experimental autoimmune encephalomyelitis (EAE) by a TGF-β-dependent mechanism [7]. The in vivo significance of Th3 cells and a role for TGF-β in their induction, as well as effector function, is suggested by the recent finding that certain tumor cell lines actively produce TGF-β [15]. The induction of TGF-β secreting regulatory Th3 cells might represent a novel subversion strategy
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Table 1. Characteristics of regulatory T cells +
CD4 CD25
+
+
Tr1
Th3
CD8 Tr
Refs
Surface phenotype CD25 low CD45RB CD45RO CTLA-4 T1–ST2
+ + + +++ ?
+ + + − ++
+ ? + ++ ?
? ? + − ?
[4,9,17] [1,9,13,17] [1,9,11,28] [18,28] [4]
Cytokine secreted IL-10 TGF-β
+/− +/−
+++ +
+ +++
++ +/−
[1–6,9,11,19,57] [4,7,10,11,19,23,28,57]
Differentiation factors
?
IL-10, IFN-α
IL-4, TGF-β
?
[1,4,6,11,28]
IL-10 IL-10
TGF-β TGF-β
TGF-β, IL-10 [1–11,19,23] ? [1,4,7,9,18]
a
Suppressor mechanism in vitro Cell contact in vivo Cell contact, IL-10, TGF-β
The symbols − to + correspond to the relative surface marker expression or cytokine production by different Tr subtypes; +/− corresponds to cytokine production by the relevant cell type, reported for some but not other studies; ? corresponds to unknown or untested. Abbreviations: IFN, interferon; IL, interleukin; TGF, transforming growth factor; Th, T helper; Tr, T regulatory. a
employed by tumors to suppress the induction of protective tumor-specific cytotoxic T-lymphocyte (CTL) responses [15]. Furthermore, because TGF-β is broadly expressed and acts on multiple cell types, TGF-β-secreting Th3 cells probably have a major role in many aspects of immune regulation and T-cell homeostatis [16]. CD4+CD25+ Tr cells
Phenotypic markers, rather than cytokine profiles, have also been used to define and deplete Tr cells in vivo. The major focus has been on a population of CD4+ T cells that constitutively express the IL-2Rα (CD25). CD4+CD25+ T cells comprise ~5–10% of the peripheral T-cell pool and exhibit potent immunosuppressive abilities both in vitro and in vivo [9]. The regulatory effect of this Tr-cell subset was thought not to be mediated by cytokine production but appeared to relate to their ability to inhibit IL-2 production, by a mechanism dependent on cell–cell contact and expression of the inhibitory costimulatory molecule CTLA-4 [17,18]. However, CTLA-4 is also expressed on activated CD4+CD25− T cells, therefore, such agonistic effects of anti-CTLA-4 mAb on costimulation of effector T cells cannot be excluded. Antibody blockade of the interactions of CD80 and/or CD86 with CTLA-4 on activated T cells could inhibit the normal downregulatory effects of CTLA-4 on effector cells and thus raise the threshold that is required for CD4+CD25+ mediated suppression [19]. Cell surface TGF-β might be involved in cell-contact suppression by CD4+CD25+ Tr cells [20]. However, more recent studies demonstrating the presence of fully functional CD4+CD25+ Tr cells in both Smad3, TGF-β and TGF-β receptor-II-defective mice have cast considerable doubt over the involvement of TGF-β in CD4+CD25+ Tr-cell mediated suppression [19,21]. The conflicting data on the role of TGF-β has recently been explained by the observation that suppression by CD4+CD25+ Tr cells was independent of membranebound TGF-β, but these cells transferred suppressor http://immunology.trends.com
activity to conventional CD4+ T cells, which functioned partially through soluble TGF-β [22]. In addition, dismissal of a role for IL-10 production is discordant with recent studies in mice suggesting that CD25+ cells within the CD4+CD45RBlow population are the T cells that prevent inflammatory bowel disease [11] or airway inflammation [13] by an IL-10 dependent mechanism. Furthermore, CD25+CD45RBlow cells from IL-10−/− mice are unable to protect against CD45RBhigh-mediated colitis [23,24]. Together, these data suggest that CD4+CD25+ Tr cells might use multiple, and as yet unidentified, mechanisms to mediate suppression. Considerable controversy surrounds the use of CD25 as a marker for Tr cells. Recent studies in the rat have demonstrated that, at least in the periphery, Tr cells that prevent autoimmune diabetes reside in both the CD25+ and CD25− subpopulations [25]. Furthermore, data indicating that multiple rounds of antigen-stimulation are required for the induction of functional Tr cells [4,26], suggesting that pathogenspecific Tr cells might become fully mature after repeated antigen stimulation in vivo and exert their regulatory role at the terminal stages of infection. However, the lineage relationship of CD25+CD4+ Tr cells to Tr1 and Th3 cells is not clear. Although some progress has been made in attempts to find alternative markers for Tr cells, such as the glucocorticoid-induced tumor necrosis factor receptor (GITR) [27], surface antigens that are exclusively expressed on Tr cells (if they exist) have yet to be identified, and this is an impediment to progress in understanding the relationship and function of distinct Tr cells in vivo. Role of Tr cells in infection
The demonstration of Th1 and Th2 subtypes of T cells in the 1980s provided a useful conceptual framework for understanding immunity to infectious diseases. It predicted that immunity to intracellular pathogens was mediated by Th1 cells, which activate
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Coxsackievirus B3 HIV (gp120) Measles virus (NP) Rhinovirus Respiratory syncytialvirus Cytomegalovirus Bordetella pertussis (FHA) Mycobacterium tuberculosis (lipoarabinomannan) Mycobacterium leprae Mycobacterium bovis Legionella pneumophilia Salmonella typhimurium Listeria monocytogenisis Yersinia enterocolitica Plasmodium falciparum Plasmodium berghei Leishmania donovani Schistosoma mansoni (egg glycolipid)
LPS CpG dsRNA HIV (gp120) Measles virus (NP) Rhinovirus Bordetella pertussis (FHA) Escherichia coli (LT) Vibrio cholerae (CT) Histoplasma capsulatum Mycobacterium tuberculosis Lactobacillus reuteri Plasmodium falciparum Plasmodium berghei Leishmania (phosphoglycans)
IL-12
IL-10 and TGF-β IL-10 homologs
Epstein-Barr virus Equine herpes virus Herpes saimiri virus Cytomegalovirus Orfvirus TRENDS in Immunology
Fig. 1. Pathogen evasion through modulation of cytokine production by cells of the innate immune system. Several viruses, bacteria and parasites, many of which cause chronic or persistent infection in humans and some of which are associated with immunosuppression, are capable of inhibiting interleukin (IL)-12 and/or stimulating IL-10 and transforming growth factor (TGF)-β production by macrophages or dendritic cells (DCs). For a small number of these pathogens, immunomodulatory molecules have been identified, including HIV gp120, measles virus nucleoprotein (NP), Bordetella pertussis filamentous haemagglutinin (FHA), Escherichia coli heat labile enterotoxin (LT), cholera toxin (CT), Leishmania phosphoglycans and Schistosoma mansoni egg glycolipid, which bind to cell-surface receptors (e.g. integrins and complement receptors) and either activate or inhibit signalling for cellular IL-10 or IL-12, respectively. Alternatively, certain viruses have evolved immunosuppressive strategies by encoding homologs of anti-inflammatory cytokines, including IL-10. Abbreviation: LPS, lipopolysaccharide.
macrophages and promote B cells to secrete complement-fixing and virus-neutralizing IgG2a antibodies, whereas immunity to extracellular pathogens was mediated by Th2 cells, which provide helper activity for antibody production, especially IgG1, IgE and IgA. The induction of pathogen-specific Th1 or Th2 immune responses is crucial in host protection and resolution of otherwise potentially fatal disease processes. However, the same effector mechanisms that have evolved to protect the host from invading microorganisms can induce immunemediated pathology if not properly regulated. Nevertheless, inflammatory diseases associated with the immune response to infection are relatively infrequent, suggesting that specific mechanisms exist to limit collateral damage. In the Th1/Th2 model this was explained through reciprocal regulation by cytokines secreted by the other subtype or possibly through IL-10 secreted by Th2 and some Th1 cells, via a negative feedback loop. However, there is now accumulating evidence to suggest that a novel and functionally distinct subpopulation of Tr cells has an important role in this process. The demonstration that Tr1 cells can suppress protective Th1 responses against an infectious pathogen [4] suggests that a paradigm shift is required in our understanding of the regulation of http://immunology.trends.com
T-cell responses during infection. Although it has been demonstrated that Th2 and Tr1 cells secrete IL-10, Tr1 cells do not secrete IL-4. Despite the fact that they express the CD4 marker, normally associated with helper function, Tr1 cells do not appear to act as classical T helper cells. Although Th3 cells have been shown to provide help for IgA production [28], the primary function of Tr1 cells appears to be to suppress inflammatory or Th1-type responses. Certain Tr1 cells can also suppress Th2 responses [29], therefore there could be implications for our understanding and treatment of allergic diseases. The theory that allergic inflammation could result from reduced cross-regulation by Th1-inducing pathogens might need revision in light of the observation that certain pathogens also induce Tr1 cells. The development of chronic infections and the failure of the host to clear certain pathogens in the face of detectable CTL, Th1 and antibody responses have been difficult to rationalize. Persistence of infections, such as those caused by HIV and hepatitis C virus, have been explained either on the basis of an imbalance in the Th1/Th2 profile or a failure to mount an as yet unidentified protective immune response. However, suppression of the protective immune response by anti-inflammatory cytokines or cells induced directly or indirectly by the pathogen might provide a more plausible explanation for the chronic infections caused by certain parasites, bacteria and viruses. Induction of high levels of IL-10 [30], and reciprocal inhibition of IL-12 production by innate cells, has been reported for a large number of pathogens (Fig. 1). In certain cases, pathogen-derived immunomodulatory molecules have been identified, which stimulate IL-10 production from macrophages and/or dendritic cells (DCs) [4,31–34] or inhibit IL-12 signalling pathways [35,36] (Mills et al. unpublished). These molecules could interact with pathogen recognition receptors, analogous to Toll-like receptors (TLRs), but stimulate IL-10, TGF-β or regulatory cytokines other than IL-12. Pathogen-stimulated IL-10, TGF-β or IL-10 homologs encoded by certain viruses [37], with concomitant inhibition of IL-12 by DCs, macrophages or epithelial cells, might directly prevent the induction or activation of Th1 or CTL responses, and could provide the appropriate cytokine milieu for the induction of Tr1 or Th3 cells. Thus, pathogen-stimulated IL-10 or TGF-β production by innate cells might suppress the immune response early in certain infections and this immunosuppression is sustained following the induction of Tr1 or Th3 cells. There is already evidence that IL-10-secreting CD4+ Tr1 cells might contribute to local immunosuppression during infection with B. pertussis [4] and might contribute to prolonged chronic infection in patients infected with hepatitis C virus [5]. Furthermore, stimulation with polymorphic variations of Plasmodium falciparum [38], purified protein derivative (PPD) from
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iDC LPS, CpG, dsRNA
Helminth PC, CT, LT, yeast hyphae
FHA
DC2
DCr
DC1 IL-10/ TGF-β
IL-12
IL-4 Tr Th2
Th1
IFN-γ
IL-10 and TGF-β
IL-4
Cell mediated immunity, IgG2a, inflammation
Immune regulation tolerance
Humoral immunity IgG1, IgA, IgE TRENDS in Immunology
Fig. 2. Proposed model for CD4+ T-cell differentiation and function in immunity to infection. Certain pathogen-derived immunomodulatory molecules bind to dendritic cells (DCs) or other innate cells, including macrophages, and stimulate maturation of immature DCs (iDCs) into DC1 and DC2, which direct the differentiation of Th1 and Th2 cells, respectively. Other pathogen-derived molecules might activate maturation of iDCs into DCs, which direct the induction of regulatory T (Tr) cells (designated DCr). The function of Tr cells is to suppress Th1, and in certain cases, Th2 responses, by the release of anti-inflammatory cytokines or contact-dependent mechanisms, acting directly on the T cell or the antigen presenting cell (APC). Abbreviations: CT, cholera toxin; FHA, filamentous haemagglutinin; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; LT, labile enterotoxin; TGF, transforming growth factor.
Mycobacterium tuberculosis [39] and Onchocerca volvulus antigen (OvAg) [2] downregulate T-cell proliferative responses by the induction of anergic IL-10-producing T cells. This provides a possible explanation for the low level of responses following infection with these pathogens. In addition, the detection of viral-specific IL-10-producing CD4+ T cells from chronic retroviral-infected humans and mice suggests that certain viruses might directly subvert specific immune responses by inducing Tr1 cells [40,41]. Although the primary function of Tr1 cells in infection could prevent inflammatory pathology and the development of autoimmune diseases mediated by Th1 cells, it is also possible that pathogens that cause persistent or chronic infections have exploited these cells to counteract protective immune responses and thereby prolong their survival in the host. Indeed, the role of IL-10 in striking a balance between pathology and protection is well recognized [42]. A role for CD4+CD25+ Tr cells in inhibiting both harmful and helpful immune responses to a pathogen has recently been described in a murine model of Pneumocystis carnii infection; CD4+CD25+ Tr cells could control protective immunity and T-cell-mediated inflammatory responses [43]. Differentiation of Tr cells
Tr cells, similar to Th1 and Th2 cells, arise from naïve precursors and can be differentiated in vitro. Although there is little evidence for cytokinemediated differentiation of CD4+CD25+ Tr cells, TGF-β could be involved in the generation and expansion of human CD4+ CD25+ Tr cells [44]. http://immunology.trends.com
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In the case of murine Tr1 cells, it has been shown that IL-10 might act as a differentiation factor but not as a growth factor for these cells [4]. Furthermore, recent studies have demonstrated that human CD4+ T cells primed in the presence of IL-10 and IFN-α differentiate into Tr1 cells [6] and that culture of murine T-cell precursors with TGF-β promotes the induction of Th3 cells [28]. The question that arises is whether a distinct subtype or activation status of APCs exists, which promotes the differentiation of regulatory rather than effector T cells from naïve precursors. The production of IL-12 and IL-4 by innate cells, and activation of DC maturation, is a key process in directing naïve T cells to differentiate into Th1 and Th2 cells, respectively. Lipopolysaccharide (LPS), dsRNA and oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) promote maturation of DCs that direct naïve T cells to a Th1 subtype (termed DC1) [45,46]. By contrast, phosphorycholine-containing glycoproteins derived from nematode parasites, cholera toxin or yeast hyphae activate DCs that selectively induce Th2 cells (termed DC2) [45,47,48]. Recent studies have suggested that activation of DCs that secrete IL-10, but not IL-12, can direct naïve T cells to a Tr1 subtype [4,49]. IL-10 inhibits the stimulatory capacity of DCs through the downregulation of MHC class II molecules and the costimulatory molecules CD80 and CD86, thus preventing DC maturation. APCs from the liver [50] and Peyer’s patch [51] secrete high levels of IL-10 and selectively induce IL-10-secreting alloreactive Tr1 cells or Th2, respectively. It has also been suggested that immature DCs drive the induction of human Tr1 cells in vitro [26]. Self or foreign antigen presented by IL-10secreting APCs to naïve T cells in the absence of ‘signal 2’ might promote the differentiation of Tr cells, a strategy for the maintenance of immunological tolerance. However, the finding that the homeostasis of Tr cells is dependent on cosignalling through CD28 and CD154 [52,53] suggests that the induction of Tr cells might not simply be the result of insufficient activation, but is dependent on APCs that express CD40 and CD80 and/or CD86. Support for this hypothesis has come from recent studies showing that stimulation of DCs with tumor necrosis factor (TNF)-α was found to upregulate a range of costimulatory molecules, including CD40, CD80 and CD86 [54]. However, in contrast to LPS and anti-CD40 stimulated DCs, DCs cultured in the presence of TNF-α did not produce detectable levels of IL-12 and could promote the induction of peptidespecific IL-10-producing-Tr1 cells. Furthermore, bone-marrow derived DCs stimulated with FHA from B. pertussis results in significantly enhanced CD86 and moderately enhanced CD40 expression, but does not affect expression of CD80 or CCR5 [4]. Thus, FHA-stimulated DCs, which induce Tr1 cells, have a distinct phenotype to the Th1-driving DCs and,
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Disease
Inflammation, GvHD Th1
Tr
Therapy
Cancer, chronic infection
IFN-γ
Expand Tr
IFN-γ
Tr
Th1
IFN-γ
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Allergy Th2
Tr
IL-4
Inhibit Tr
IL-10 and TGF-β
IFN-γ
Expand Tr
IL-4
IL-10 and TGF-β
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Fig. 3. Possible disease therapies based on regulatory T (Tr)-cell manipulation in vivo. In a normal healthy individual, Tr cells maintain homeostasis and prevent inflammation at mucosal surfaces, including the gut and in the lungs, but permit Th1 and cytotoxic T-lymphocyte (CTL) responses to control infection and tumors. However, imbalances in regulatory and effector arms of the immune response are apparent in certain disease states. Tr cells might be expanded in chronic infection and possibly cancer, resulting in suppressed Th1 responses. By contrast, overactive Th1 responses contribute to the pathology in autoimmune diseases, infection-induced inflammation, graft-versushost disease (GvHD) and graft rejection, whereas Th2 cells mediate allergy. Therefore, strategies that can inhibit or expand Tr cells in vivo might offer therapeutic potential for treatment of these diseases. Abbreviations: IFN, interferon; IL, interleukin; TGF, transforming growth factor.
although they have certain characteristics of immature DCs, they are phenotypically and functionally distinct. It has also been suggested that immature DCs, such as Langerhans cells, induce T-cell anergy, whereas semi-mature DCs, with or without IL-10 production, drive the induction of Tr cells [55]. In addition, antigen presentation by murine DCs overexpressing human Serrate1 induces peripheral CD4+ T cells to become Tr cells [56], therefore, in addition to Th1-driving and Th2-driving DCs, DCs activated with an appropriate stimulus, such as that from certain pathogen-derived molecules, possibly in the environment of a mucosal surface, could selectively promote the induction of Tr cells (Fig. 2). However, much of the evidence suggests that DC1, DC2 or Tr-driving DC (designated DCr) correspond to functional subtypes of DCs, activated through distinct signals from pathogenderived molecules, rather than different DC lineages in vivo. Furthermore, the APCs and cells secreting the cytokines that drive the differentiation of Th1, Th2 or Tr cells are not confined to DCs because macrophages and other innate cells could also have a major role in this process (Fig. 2). Acknowledgements This work was supported by The Science Foundation Ireland, The Wellcome Trust and The Health Research Board of Ireland. We apologize to authors that we were unable to quote due to space limitations.
Conclusions and future perspectives
The resurgent interest in suppressor, reborn as regulatory, T cells is set to change the face of cellular immunology and will probably have a major impact on our understanding of the pathogenesis and treatment of a range of human diseases. The ability to induce memory T cells that produce suppressive http://immunology.trends.com
Definition of regulatory T (Tr) cells Immunosuppressive T cells with cytokine profiles distinct from either type 1 (Th1) or type 2 (Th2) T cells. Questions to be addressed • Can functional subtypes of Tr cells be distinguished phenotypically? • What is the lineage relationship of CD4+CD25+ cells to Tr1 and Th3 cells? • Are there distinct types of dendritic cell responsible for the induction of different Tr cells? • Do Tr cells have effector functions other than immunosuppression? • Do Tr cells have a role in limiting inflammatory pathology or in pathogen persistence? • Are Tr (rather than Th2) cells primarily responsible for controlling Th1 response in vivo, for example, in response to self-antigens, pathogens, tumors, allografts and the fetus? • Will it be possible to develop safe and effective therapies for human diseases based on specific activation or inhibition of Tr cells?
cytokines on stimulation with specific antigen in vivo has opened up the possibility of developing vaccines against autoimmune diseases and therapies to control graft rejection, graft-versus-host disease, inflammation and allergy (Fig. 3). Because Tr cells proliferate poorly in culture and would have to be prepared individually for each patient, strategies for the induction or expansion of these cells in vivo would be required. It should also be possible to design therapies for the treatment of certain chronic infections and cancers by selectively inhibiting the induction or function of Tr cells. However, skewing established T-cell responses might prove more difficult than inducing polarized responses in naïve individuals. Other major questions to be addressed are whether strategies aimed at inhibiting or expanding Tr cells will work in humans and will they be safe? Do we risk the possibility of upsetting the balance between Th1 and Th2, or effector and regulatory cells leading to inflammatory pathology, or increased susceptibility to other diseases? The demonstration of Tr1 cells that recognize the same antigens or peptides on a pathogen as Th1 cells [4,5], coupled with the suggestion that Tr1 and Th3 cells could be more prevalent at mucosal surfaces [4,7,51], also has important implications for the development of mucosal vaccines against infectious diseases. Immunization by oral or nasal routes, although attractive in terms of vaccine administration and local immunity, might favor the induction of immunosuppressive Tr cells rather than protective Th1 or Th2 cells. This requires the identification of safe but effective adjuvants that will selectively induce the appropriate T-cell subtype. Thus, manipulation of Tr cells in vivo has considerable potential in the prevention and treatment of human diseases, but further research on the molecular characteristics, induction and function of Tr cells is required before their therapeutic potential is realized.
Review
References 1 Groux, H. et al. (1997) A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389, 737–742 2 Doetze, A. et al. (2000) Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by Th3/Tr1-type cytokines IL-10 and transforming growth factor-β but not by a T(h)1 to T(h)2 shift. Int. Immunol. 12, 623–630 3 Cavani, A. et al. (2000) Human CD4+ T lymphocytes with remarkable regulatory functions on dendritic cells and nickel-specific Th1 immune responses. J. Invest. Dermatol. 114, 295–302 4 McGuirk, P. et al. (2002) Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195, 221–231 5 MacDonald, A.J. et al. (2002) CD4 T helper type 1 and regulatory T cells induced against the same epitopes on the core protein in hepatitis C virusinfected persons. J. Infect. Dis. 185, 720–727 6 Levings, M.K. et al. (2001) IFN-α and IL-10 induce the differentiation of human type 1 T regulatory cells. J. Immunol. 166, 5530–5539 7 Chen, Y. et al. (1994) Regulatory T-cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265, 1237–1240 8 Fukaura, H. et al. (1996) Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-β1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J. Clin. Invest. 98, 70–77 9 Shevach, E.M. (2000) Regulatory T cells in autoimmmunity. Annu. Rev. Immunol. 18, 423–449 10 Garba, M.L. et al. (2002) HIV antigens can induce TGF-β1-producing immunoregulatory CD8+ T cells. J. Immunol. 168, 2247–2254 11 Gilliet, M. and Liu, Y.J. (2002) Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells. J. Exp. Med.195, 695–704 12 Sakaguchi, S. (2000) Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101, 455–458 13 Zuany-Amorim, C. et al. (2002) Suppression of airway eosinophilia by killed Mycobacterium vaccae-induced allergen-specific regulatory T cells. Nat. Med. 8, 625–629 14 Barrat, F.J. et al. (2002) In vitro generation of interleukin 10-producing regulatory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2inducing cytokines. J. Exp. Med. 195, 603–616 15 Seo, N. et al. (2001) Interleukin-10 expressed at early tumour sites induces subsequent generation of CD4+ T-regulatory cells and systemic collapse of antitumour immunity. Immunology 103, 449–457 16 Gorelik, L. and Flavell, R.A. (2002) Transforming growth factor-β in T-cell biology. Nat. Immunol. Rev. 2, 46–53 17 Thornton, A.M. and Shevach, E.M. (2000) Suppressor effector function of CD4+CD25+ immunoregulatory Tcells is antigen nonspecific. J. Immunol.164, 183–190 18 Read, S. et al. (2000) Cytotoxic T lymphocyteassociated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192, 295–302 19 Shevach, E.M. (2002) CD4+CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2, 389–400 20 Nakamura, K. et al. (2001) Cell contact-dependent immunosuppression by CD4+CD25+ regulatory http://immunology.trends.com
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T cells is mediated by cell surface-bound transforming growth factor β. J. Exp. Med. 194, 629–644 Lucas, P.J. et al. (2000) Disruption of T-cell homeostasis in mice expressing a T-cell-specific dominant negative transforming growth factor β II receptor. J. Exp. Med. 191, 1187–1196 Jonuleit, H. et al. (2002) Infectious tolerance: human CD25+ regulatory T cells convey suppressor activity to conventional CD4+ T helper cells. J. Exp. Med. 196, 255–260 Asseman, C. et al. (1999) An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190, 995–1004 Singh, B. et al. (2001) Control of intestinal inflammation by regulatory T cells. Immunol. Rev. 182, 190–200 Stephens, L.A. and Mason, D. (2000) 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, 3105–3110 Jonuleit, H. et al. (2000) Induction of interleukin 10-producing, nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192, 1213–1222 Shimizu, J. et al.(2002) Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3, 135–142 Weiner, H.L. (2001) Induction and mechanism of action of transforming growth factor-β-secreting Th3 regulatory cells. Immunol. Rev. 182, 207–214 Cottrez, F. et al. (2000) T regulatory cells 1 inhibit a Th2-specific response in vivo. J. Immunol. 165, 4848–4853 Redpath, S. et al. (2001) Hijacking and exploitation of IL-10 by intracellular pathogens. Trends Microbiol. 9, 86–92 Taoufik, Y. et al. (1997) Human immunodeficiency virus gp120 inhibits interleukin-12 secretion by human monocytes: an indirect interleukin-10mediated effect. Blood 89, 2842–2848 Marie, J.C. et al. (2001) Mechanism of measles virus-induced suppression of inflammatory immune responses. Immunity 14, 69–79 Van der Kleij, D. et al. (2002) Triggering of innate immune responses by schistosome egg glycolipids and their carbohydrate epitope GalNAc β 1-4(Fuc α 1-2Fuc α 1-3)GlcNAc. J. Infect. Dis. 185, 531–539 Barnes, P.F. et al. (1992) Cytokine production induced by Mycobacterium tuberculosis lipoarabinomannan. Relationship to chemical structure. J. Immunol. 149, 541–547 Feng, G.J. et al. (1999) Extracellular signalrelated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase. J. Immunol. 163, 6403–6412 Ryan, E.J. et al. (2001) Immunomodulators and delivery systems for vaccination by mucosal routes. Trends Biotechnol. 19, 293–304 Fickenscher, H. et al. (2002) The interleukin-10 family of cytokines. Trends Immunol. 23, 89–96 Plebanski, M. et al. (1999) Interleukin 10-mediated immunosuppression by a variant CD4 T-cell epitope of Plasmodium falciparum. Immunity 10, 651–660 Boussiotis, V.A. et al. (2000) IL-10-producing T cells suppress immune responses in anergic tuberculosis
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patients. J. Clin. Invest. 105, 1317–1325 40 Ostrowski, M.A. et al. (2001) Quantitative and qualitative assessment of human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cell immunity to gag in HIV-1-infected individuals with differential disease progression: reciprocal interferon-γ and interleukin-10 responses. J. Infect. Dis. 184, 1268–1278 41 Iwashiro, M. et al. (2001) Immunosuppression by CD4+ regulatory T cells induced by chronic retroviral infection. Proc. Natl. Acad. Sci. U. S. A. 98, 9226–9230 42 Moore, K.W. et al. (2001) Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19, 683–765 43 Hori, S. et al. (2002) CD25+CD4+ regulatory T cells suppress CD4+ T-cell-mediated pulmonary hyperinflammation driven by Pneumocystis carinii in immunodeficient mice. Eur. J. Immunol. 32, 1282–1291 44 Yamagiwa, S. et al. (2001) A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. J. Immunol. 166, 7282–7289 45 Moser, M. and Murphy, K.M. (2000) Dendritic cell regulation of Th1-Th2 development. Nat. Immunol. 1, 199–205 46 Hemmi, H. et al. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 47 d’Ostiani, C.F. et al. (2000) Dendritic cells discriminate between yeasts and hyphae of the fungus Candida albicans. Implications for initiation of T helper cell immunity in vitro and in vivo. J. Exp. Med. 191, 1661–1674 48 Gagliardi, M.C. et al. (2000) Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming. Eur. J. Immunol. 30, 2394–2403 49 Akbari, O. et al. (2001) Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat. Immunol. 2, 725–731 50 Khanna, A. et al. (2000) Effects of liver-derived dendritic cell progenitors on Th1- and Th2-like cytokine responses in vitro and in vivo. J. Immunol. 164, 1346–1354 51 Iwasaki, A. and Kelsall, B.L. (1999) Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin-10 and induce the differentiation of T helper type 2 cells. J. Exp. Med. 190, 229–239 52 Kumanogoh, A. et al. (2001) Increased T-cell autoreactivity in the absence of CD40–CD40 ligand interactions: a role of CD40 in regulatory T cell development. J. Immunol. 166, 353–360 53 Salomon, B. et al. (2000) B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12, 431–440 54 Menges, M. et al. (2002) Repetitive injections of dendritic cells matured with tumor necrosis factor α induce antigen-specific protection of mice from autoimmunity. J. Exp. Med. 195, 15–21 55 Lutz, M.B. and Schuler, G. Immature, semimature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol. (in press) 56 Hoyne, G.F. et al. (2000) Serrate1-induced notch signalling regulates the decision between immunity and tolerance made by peripheral CD4+ T cells. Int. Immunol. 12, 177–185 57 Levings, M.K. et al. (2001) Human CD25+CD4+ T regulatory cells suppress naïve and memory T-cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193, 1295–1301
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Pathogen-specific regulatory T cells provoke a shift in the Th1/Th2 paradigm in immunity to infectious diseases Peter McGuirk and Kingston H.G. Mills Current dogma suggests that immunity to infection is controlled by distinct type 1 (Th1) and type 2 (Th2) subpopulations of T cells discriminated on the basis of cytokine secretion and function. However, a further subtype of T cells, with immunosuppressive function and cytokine profiles distinct from either Th1 or Th2 T cells, termed regulatory T (Tr) cells has been described. Although considered to have a role in the maintenance of self-tolerance, recent studies suggest that Tr cells can be induced against bacterial, viral and parasite antigens in vivo and might prevent infection-induced immunopathology or prolong pathogen persistence by suppressing protective Th1 responses. These observations have significant implications for our understanding of the role of T cells in immunity to infectious diseases and for the development of new therapies for immune-mediated disorders.
Several subsets of regulatory T (Tr) cells with distinct phenotypes and distinct mechanisms of action have now been identified. These include type 1 Tr cells (Tr1) cells [1–6], which secrete high levels of interleukin (IL)-10 and low to moderate levels of transforming growth factor (TGF)-β, type 3 T (Th3) cells [7,8], which primarily secrete TGF-β, and CD4+CD25+ T cells, which inhibit immune responses through cell–cell contact [9] (Table 1). In addition to the various CD4+ Tr populations, recent studies have identified CD8+ Tr cells, which secrete either IL-10 or TGF-β [10,11]. Initial reports indicated that the primary function of Tr cells was in maintaining peripheral tolerance and homeostasis at mucosal surfaces and suggested that they could be directed against self-antigens [12]. However, until recently, little was known about their antigen specificities or induction in vivo, and progress on their characterization has been difficult as a result of failures to cultivate and clone Tr cells in vitro. A significant advance has been made in the field of Tr cells with the ex vivo propagation and cloning of pathogen-specific murine [4] and human [2,5] Tr1 cells during infection. Furthermore, treatment of mice with killed bacteria promotes the development of allergen-specific CD4+CD45RBlow Tr cells [13]. Peter McGuirk Kingston H.G. Mills* Immune Regulation Research Group, Department of Biochemistry, Trinity College, Dublin 2, Ireland. *e-mail: kingston.mills@ tcd.ie
Phenotype and function of Tr cells Tr1 cells
T cells, from ovalbumin (OVA) T-cell receptor (TCR)-transgenic mice, cultured with OVA and IL-10 result in the generation of T-cell clones with a unique cytokine profile distinct from that of Th0, Th1 or Th2 cells. These Tr1 cells produce IL-10 and IL-5, http://immunology.trends.com
with or without TGF-β, but with little or no IL-2, IL-4 or interferon (IFN)-γ production, and proliferate poorly following polyclonal TCR-mediated activation [1]. More recently, in vitro manipulation with immunosuppressive drugs and anti-IL-4 and antiIL-12 antibodies has facilitated the expansion of Tr1 cells that exclusively secrete IL-10 [14]. The expression of CCR5 and T1–ST2 on Tr1 cells [4], markers previously expressed preferentially on Th1 and Th2 cells, respectively, suggest that Tr1 cells represent a phenotypically distinct subtype of CD4+ T cells. Functional studies on Tr1 cells have suggested that these cells have immunosuppressive properties and have been shown to prevent the development of Th1-mediated autoimmune diseases [1]. However, Tr1 and other Tr-cell populations also suppress immune responses to pathogens, tumors and alloantigens. Co-culture of naïve CD4+ T cells and human Tr1 clones in the presence of allogenic antigen-presenting cells (APCs) results in the suppression of proliferative responses [1]. Similarly, Tr1 clones specific for filamentous haemaggultinin (FHA) from Bordetella pertussis suppress proliferation and cytokine production by a Th1 clone against an unrelated antigen, influenza virus haemagglutinin (HA) [4]. In both cases, the suppressive effects of Tr1-cell clones are reversed by neutralizing IL-10, suggesting that, regardless of their antigen specificities, Tr1 cell suppression is a bystander effect mediated through the production of IL-10. Th3 cells
Another subset of CD4+ Tr cells was identified in studies of oral tolerance. In multiple sclerosis patients, oral treatment with myelin basic protein (MBP) induces a significant increase in the frequency of MBP-specific T cells that secrete TGF-β [8]. These Th3 cells were originally generated and identified in mice orally tolerized to MBP and suppressed the induction of experimental autoimmune encephalomyelitis (EAE) by a TGF-β-dependent mechanism [7]. The in vivo significance of Th3 cells and a role for TGF-β in their induction, as well as effector function, is suggested by the recent finding that certain tumor cell lines actively produce TGF-β [15]. The induction of TGF-β secreting regulatory Th3 cells might represent a novel subversion strategy
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Table 1. Characteristics of regulatory T cells +
CD4 CD25
+
+
Tr1
Th3
CD8 Tr
Refs
Surface phenotype CD25 low CD45RB CD45RO CTLA-4 T1–ST2
+ + + +++ ?
+ + + − ++
+ ? + ++ ?
? ? + − ?
[4,9,17] [1,9,13,17] [1,9,11,28] [18,28] [4]
Cytokine secreted IL-10 TGF-β
+/− +/−
+++ +
+ +++
++ +/−
[1–6,9,11,19,57] [4,7,10,11,19,23,28,57]
Differentiation factors
?
IL-10, IFN-α
IL-4, TGF-β
?
[1,4,6,11,28]
IL-10 IL-10
TGF-β TGF-β
TGF-β, IL-10 [1–11,19,23] ? [1,4,7,9,18]
a
Suppressor mechanism in vitro Cell contact in vivo Cell contact, IL-10, TGF-β
The symbols − to + correspond to the relative surface marker expression or cytokine production by different Tr subtypes; +/− corresponds to cytokine production by the relevant cell type, reported for some but not other studies; ? corresponds to unknown or untested. Abbreviations: IFN, interferon; IL, interleukin; TGF, transforming growth factor; Th, T helper; Tr, T regulatory. a
employed by tumors to suppress the induction of protective tumor-specific cytotoxic T-lymphocyte (CTL) responses [15]. Furthermore, because TGF-β is broadly expressed and acts on multiple cell types, TGF-β-secreting Th3 cells probably have a major role in many aspects of immune regulation and T-cell homeostatis [16]. CD4+CD25+ Tr cells
Phenotypic markers, rather than cytokine profiles, have also been used to define and deplete Tr cells in vivo. The major focus has been on a population of CD4+ T cells that constitutively express the IL-2Rα (CD25). CD4+CD25+ T cells comprise ~5–10% of the peripheral T-cell pool and exhibit potent immunosuppressive abilities both in vitro and in vivo [9]. The regulatory effect of this Tr-cell subset was thought not to be mediated by cytokine production but appeared to relate to their ability to inhibit IL-2 production, by a mechanism dependent on cell–cell contact and expression of the inhibitory costimulatory molecule CTLA-4 [17,18]. However, CTLA-4 is also expressed on activated CD4+CD25− T cells, therefore, such agonistic effects of anti-CTLA-4 mAb on costimulation of effector T cells cannot be excluded. Antibody blockade of the interactions of CD80 and/or CD86 with CTLA-4 on activated T cells could inhibit the normal downregulatory effects of CTLA-4 on effector cells and thus raise the threshold that is required for CD4+CD25+ mediated suppression [19]. Cell surface TGF-β might be involved in cell-contact suppression by CD4+CD25+ Tr cells [20]. However, more recent studies demonstrating the presence of fully functional CD4+CD25+ Tr cells in both Smad3, TGF-β and TGF-β receptor-II-defective mice have cast considerable doubt over the involvement of TGF-β in CD4+CD25+ Tr-cell mediated suppression [19,21]. The conflicting data on the role of TGF-β has recently been explained by the observation that suppression by CD4+CD25+ Tr cells was independent of membranebound TGF-β, but these cells transferred suppressor http://immunology.trends.com
activity to conventional CD4+ T cells, which functioned partially through soluble TGF-β [22]. In addition, dismissal of a role for IL-10 production is discordant with recent studies in mice suggesting that CD25+ cells within the CD4+CD45RBlow population are the T cells that prevent inflammatory bowel disease [11] or airway inflammation [13] by an IL-10 dependent mechanism. Furthermore, CD25+CD45RBlow cells from IL-10−/− mice are unable to protect against CD45RBhigh-mediated colitis [23,24]. Together, these data suggest that CD4+CD25+ Tr cells might use multiple, and as yet unidentified, mechanisms to mediate suppression. Considerable controversy surrounds the use of CD25 as a marker for Tr cells. Recent studies in the rat have demonstrated that, at least in the periphery, Tr cells that prevent autoimmune diabetes reside in both the CD25+ and CD25− subpopulations [25]. Furthermore, data indicating that multiple rounds of antigen-stimulation are required for the induction of functional Tr cells [4,26], suggesting that pathogenspecific Tr cells might become fully mature after repeated antigen stimulation in vivo and exert their regulatory role at the terminal stages of infection. However, the lineage relationship of CD25+CD4+ Tr cells to Tr1 and Th3 cells is not clear. Although some progress has been made in attempts to find alternative markers for Tr cells, such as the glucocorticoid-induced tumor necrosis factor receptor (GITR) [27], surface antigens that are exclusively expressed on Tr cells (if they exist) have yet to be identified, and this is an impediment to progress in understanding the relationship and function of distinct Tr cells in vivo. Role of Tr cells in infection
The demonstration of Th1 and Th2 subtypes of T cells in the 1980s provided a useful conceptual framework for understanding immunity to infectious diseases. It predicted that immunity to intracellular pathogens was mediated by Th1 cells, which activate
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Coxsackievirus B3 HIV (gp120) Measles virus (NP) Rhinovirus Respiratory syncytialvirus Cytomegalovirus Bordetella pertussis (FHA) Mycobacterium tuberculosis (lipoarabinomannan) Mycobacterium leprae Mycobacterium bovis Legionella pneumophilia Salmonella typhimurium Listeria monocytogenisis Yersinia enterocolitica Plasmodium falciparum Plasmodium berghei Leishmania donovani Schistosoma mansoni (egg glycolipid)
LPS CpG dsRNA HIV (gp120) Measles virus (NP) Rhinovirus Bordetella pertussis (FHA) Escherichia coli (LT) Vibrio cholerae (CT) Histoplasma capsulatum Mycobacterium tuberculosis Lactobacillus reuteri Plasmodium falciparum Plasmodium berghei Leishmania (phosphoglycans)
IL-12
IL-10 and TGF-β IL-10 homologs
Epstein-Barr virus Equine herpes virus Herpes saimiri virus Cytomegalovirus Orfvirus TRENDS in Immunology
Fig. 1. Pathogen evasion through modulation of cytokine production by cells of the innate immune system. Several viruses, bacteria and parasites, many of which cause chronic or persistent infection in humans and some of which are associated with immunosuppression, are capable of inhibiting interleukin (IL)-12 and/or stimulating IL-10 and transforming growth factor (TGF)-β production by macrophages or dendritic cells (DCs). For a small number of these pathogens, immunomodulatory molecules have been identified, including HIV gp120, measles virus nucleoprotein (NP), Bordetella pertussis filamentous haemagglutinin (FHA), Escherichia coli heat labile enterotoxin (LT), cholera toxin (CT), Leishmania phosphoglycans and Schistosoma mansoni egg glycolipid, which bind to cell-surface receptors (e.g. integrins and complement receptors) and either activate or inhibit signalling for cellular IL-10 or IL-12, respectively. Alternatively, certain viruses have evolved immunosuppressive strategies by encoding homologs of anti-inflammatory cytokines, including IL-10. Abbreviation: LPS, lipopolysaccharide.
macrophages and promote B cells to secrete complement-fixing and virus-neutralizing IgG2a antibodies, whereas immunity to extracellular pathogens was mediated by Th2 cells, which provide helper activity for antibody production, especially IgG1, IgE and IgA. The induction of pathogen-specific Th1 or Th2 immune responses is crucial in host protection and resolution of otherwise potentially fatal disease processes. However, the same effector mechanisms that have evolved to protect the host from invading microorganisms can induce immunemediated pathology if not properly regulated. Nevertheless, inflammatory diseases associated with the immune response to infection are relatively infrequent, suggesting that specific mechanisms exist to limit collateral damage. In the Th1/Th2 model this was explained through reciprocal regulation by cytokines secreted by the other subtype or possibly through IL-10 secreted by Th2 and some Th1 cells, via a negative feedback loop. However, there is now accumulating evidence to suggest that a novel and functionally distinct subpopulation of Tr cells has an important role in this process. The demonstration that Tr1 cells can suppress protective Th1 responses against an infectious pathogen [4] suggests that a paradigm shift is required in our understanding of the regulation of http://immunology.trends.com
T-cell responses during infection. Although it has been demonstrated that Th2 and Tr1 cells secrete IL-10, Tr1 cells do not secrete IL-4. Despite the fact that they express the CD4 marker, normally associated with helper function, Tr1 cells do not appear to act as classical T helper cells. Although Th3 cells have been shown to provide help for IgA production [28], the primary function of Tr1 cells appears to be to suppress inflammatory or Th1-type responses. Certain Tr1 cells can also suppress Th2 responses [29], therefore there could be implications for our understanding and treatment of allergic diseases. The theory that allergic inflammation could result from reduced cross-regulation by Th1-inducing pathogens might need revision in light of the observation that certain pathogens also induce Tr1 cells. The development of chronic infections and the failure of the host to clear certain pathogens in the face of detectable CTL, Th1 and antibody responses have been difficult to rationalize. Persistence of infections, such as those caused by HIV and hepatitis C virus, have been explained either on the basis of an imbalance in the Th1/Th2 profile or a failure to mount an as yet unidentified protective immune response. However, suppression of the protective immune response by anti-inflammatory cytokines or cells induced directly or indirectly by the pathogen might provide a more plausible explanation for the chronic infections caused by certain parasites, bacteria and viruses. Induction of high levels of IL-10 [30], and reciprocal inhibition of IL-12 production by innate cells, has been reported for a large number of pathogens (Fig. 1). In certain cases, pathogen-derived immunomodulatory molecules have been identified, which stimulate IL-10 production from macrophages and/or dendritic cells (DCs) [4,31–34] or inhibit IL-12 signalling pathways [35,36] (Mills et al. unpublished). These molecules could interact with pathogen recognition receptors, analogous to Toll-like receptors (TLRs), but stimulate IL-10, TGF-β or regulatory cytokines other than IL-12. Pathogen-stimulated IL-10, TGF-β or IL-10 homologs encoded by certain viruses [37], with concomitant inhibition of IL-12 by DCs, macrophages or epithelial cells, might directly prevent the induction or activation of Th1 or CTL responses, and could provide the appropriate cytokine milieu for the induction of Tr1 or Th3 cells. Thus, pathogen-stimulated IL-10 or TGF-β production by innate cells might suppress the immune response early in certain infections and this immunosuppression is sustained following the induction of Tr1 or Th3 cells. There is already evidence that IL-10-secreting CD4+ Tr1 cells might contribute to local immunosuppression during infection with B. pertussis [4] and might contribute to prolonged chronic infection in patients infected with hepatitis C virus [5]. Furthermore, stimulation with polymorphic variations of Plasmodium falciparum [38], purified protein derivative (PPD) from
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iDC LPS, CpG, dsRNA
Helminth PC, CT, LT, yeast hyphae
FHA
DC2
DCr
DC1 IL-10/ TGF-β
IL-12
IL-4 Tr Th2
Th1
IFN-γ
IL-10 and TGF-β
IL-4
Cell mediated immunity, IgG2a, inflammation
Immune regulation tolerance
Humoral immunity IgG1, IgA, IgE TRENDS in Immunology
Fig. 2. Proposed model for CD4+ T-cell differentiation and function in immunity to infection. Certain pathogen-derived immunomodulatory molecules bind to dendritic cells (DCs) or other innate cells, including macrophages, and stimulate maturation of immature DCs (iDCs) into DC1 and DC2, which direct the differentiation of Th1 and Th2 cells, respectively. Other pathogen-derived molecules might activate maturation of iDCs into DCs, which direct the induction of regulatory T (Tr) cells (designated DCr). The function of Tr cells is to suppress Th1, and in certain cases, Th2 responses, by the release of anti-inflammatory cytokines or contact-dependent mechanisms, acting directly on the T cell or the antigen presenting cell (APC). Abbreviations: CT, cholera toxin; FHA, filamentous haemagglutinin; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; LT, labile enterotoxin; TGF, transforming growth factor.
Mycobacterium tuberculosis [39] and Onchocerca volvulus antigen (OvAg) [2] downregulate T-cell proliferative responses by the induction of anergic IL-10-producing T cells. This provides a possible explanation for the low level of responses following infection with these pathogens. In addition, the detection of viral-specific IL-10-producing CD4+ T cells from chronic retroviral-infected humans and mice suggests that certain viruses might directly subvert specific immune responses by inducing Tr1 cells [40,41]. Although the primary function of Tr1 cells in infection could prevent inflammatory pathology and the development of autoimmune diseases mediated by Th1 cells, it is also possible that pathogens that cause persistent or chronic infections have exploited these cells to counteract protective immune responses and thereby prolong their survival in the host. Indeed, the role of IL-10 in striking a balance between pathology and protection is well recognized [42]. A role for CD4+CD25+ Tr cells in inhibiting both harmful and helpful immune responses to a pathogen has recently been described in a murine model of Pneumocystis carnii infection; CD4+CD25+ Tr cells could control protective immunity and T-cell-mediated inflammatory responses [43]. Differentiation of Tr cells
Tr cells, similar to Th1 and Th2 cells, arise from naïve precursors and can be differentiated in vitro. Although there is little evidence for cytokinemediated differentiation of CD4+CD25+ Tr cells, TGF-β could be involved in the generation and expansion of human CD4+ CD25+ Tr cells [44]. http://immunology.trends.com
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In the case of murine Tr1 cells, it has been shown that IL-10 might act as a differentiation factor but not as a growth factor for these cells [4]. Furthermore, recent studies have demonstrated that human CD4+ T cells primed in the presence of IL-10 and IFN-α differentiate into Tr1 cells [6] and that culture of murine T-cell precursors with TGF-β promotes the induction of Th3 cells [28]. The question that arises is whether a distinct subtype or activation status of APCs exists, which promotes the differentiation of regulatory rather than effector T cells from naïve precursors. The production of IL-12 and IL-4 by innate cells, and activation of DC maturation, is a key process in directing naïve T cells to differentiate into Th1 and Th2 cells, respectively. Lipopolysaccharide (LPS), dsRNA and oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG ODN) promote maturation of DCs that direct naïve T cells to a Th1 subtype (termed DC1) [45,46]. By contrast, phosphorycholine-containing glycoproteins derived from nematode parasites, cholera toxin or yeast hyphae activate DCs that selectively induce Th2 cells (termed DC2) [45,47,48]. Recent studies have suggested that activation of DCs that secrete IL-10, but not IL-12, can direct naïve T cells to a Tr1 subtype [4,49]. IL-10 inhibits the stimulatory capacity of DCs through the downregulation of MHC class II molecules and the costimulatory molecules CD80 and CD86, thus preventing DC maturation. APCs from the liver [50] and Peyer’s patch [51] secrete high levels of IL-10 and selectively induce IL-10-secreting alloreactive Tr1 cells or Th2, respectively. It has also been suggested that immature DCs drive the induction of human Tr1 cells in vitro [26]. Self or foreign antigen presented by IL-10secreting APCs to naïve T cells in the absence of ‘signal 2’ might promote the differentiation of Tr cells, a strategy for the maintenance of immunological tolerance. However, the finding that the homeostasis of Tr cells is dependent on cosignalling through CD28 and CD154 [52,53] suggests that the induction of Tr cells might not simply be the result of insufficient activation, but is dependent on APCs that express CD40 and CD80 and/or CD86. Support for this hypothesis has come from recent studies showing that stimulation of DCs with tumor necrosis factor (TNF)-α was found to upregulate a range of costimulatory molecules, including CD40, CD80 and CD86 [54]. However, in contrast to LPS and anti-CD40 stimulated DCs, DCs cultured in the presence of TNF-α did not produce detectable levels of IL-12 and could promote the induction of peptidespecific IL-10-producing-Tr1 cells. Furthermore, bone-marrow derived DCs stimulated with FHA from B. pertussis results in significantly enhanced CD86 and moderately enhanced CD40 expression, but does not affect expression of CD80 or CCR5 [4]. Thus, FHA-stimulated DCs, which induce Tr1 cells, have a distinct phenotype to the Th1-driving DCs and,
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Disease
Inflammation, GvHD Th1
Tr
Therapy
Cancer, chronic infection
IFN-γ
Expand Tr
IFN-γ
Tr
Th1
IFN-γ
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Allergy Th2
Tr
IL-4
Inhibit Tr
IL-10 and TGF-β
IFN-γ
Expand Tr
IL-4
IL-10 and TGF-β
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Fig. 3. Possible disease therapies based on regulatory T (Tr)-cell manipulation in vivo. In a normal healthy individual, Tr cells maintain homeostasis and prevent inflammation at mucosal surfaces, including the gut and in the lungs, but permit Th1 and cytotoxic T-lymphocyte (CTL) responses to control infection and tumors. However, imbalances in regulatory and effector arms of the immune response are apparent in certain disease states. Tr cells might be expanded in chronic infection and possibly cancer, resulting in suppressed Th1 responses. By contrast, overactive Th1 responses contribute to the pathology in autoimmune diseases, infection-induced inflammation, graft-versushost disease (GvHD) and graft rejection, whereas Th2 cells mediate allergy. Therefore, strategies that can inhibit or expand Tr cells in vivo might offer therapeutic potential for treatment of these diseases. Abbreviations: IFN, interferon; IL, interleukin; TGF, transforming growth factor.
although they have certain characteristics of immature DCs, they are phenotypically and functionally distinct. It has also been suggested that immature DCs, such as Langerhans cells, induce T-cell anergy, whereas semi-mature DCs, with or without IL-10 production, drive the induction of Tr cells [55]. In addition, antigen presentation by murine DCs overexpressing human Serrate1 induces peripheral CD4+ T cells to become Tr cells [56], therefore, in addition to Th1-driving and Th2-driving DCs, DCs activated with an appropriate stimulus, such as that from certain pathogen-derived molecules, possibly in the environment of a mucosal surface, could selectively promote the induction of Tr cells (Fig. 2). However, much of the evidence suggests that DC1, DC2 or Tr-driving DC (designated DCr) correspond to functional subtypes of DCs, activated through distinct signals from pathogenderived molecules, rather than different DC lineages in vivo. Furthermore, the APCs and cells secreting the cytokines that drive the differentiation of Th1, Th2 or Tr cells are not confined to DCs because macrophages and other innate cells could also have a major role in this process (Fig. 2). Acknowledgements This work was supported by The Science Foundation Ireland, The Wellcome Trust and The Health Research Board of Ireland. We apologize to authors that we were unable to quote due to space limitations.
Conclusions and future perspectives
The resurgent interest in suppressor, reborn as regulatory, T cells is set to change the face of cellular immunology and will probably have a major impact on our understanding of the pathogenesis and treatment of a range of human diseases. The ability to induce memory T cells that produce suppressive http://immunology.trends.com
Definition of regulatory T (Tr) cells Immunosuppressive T cells with cytokine profiles distinct from either type 1 (Th1) or type 2 (Th2) T cells. Questions to be addressed • Can functional subtypes of Tr cells be distinguished phenotypically? • What is the lineage relationship of CD4+CD25+ cells to Tr1 and Th3 cells? • Are there distinct types of dendritic cell responsible for the induction of different Tr cells? • Do Tr cells have effector functions other than immunosuppression? • Do Tr cells have a role in limiting inflammatory pathology or in pathogen persistence? • Are Tr (rather than Th2) cells primarily responsible for controlling Th1 response in vivo, for example, in response to self-antigens, pathogens, tumors, allografts and the fetus? • Will it be possible to develop safe and effective therapies for human diseases based on specific activation or inhibition of Tr cells?
cytokines on stimulation with specific antigen in vivo has opened up the possibility of developing vaccines against autoimmune diseases and therapies to control graft rejection, graft-versus-host disease, inflammation and allergy (Fig. 3). Because Tr cells proliferate poorly in culture and would have to be prepared individually for each patient, strategies for the induction or expansion of these cells in vivo would be required. It should also be possible to design therapies for the treatment of certain chronic infections and cancers by selectively inhibiting the induction or function of Tr cells. However, skewing established T-cell responses might prove more difficult than inducing polarized responses in naïve individuals. Other major questions to be addressed are whether strategies aimed at inhibiting or expanding Tr cells will work in humans and will they be safe? Do we risk the possibility of upsetting the balance between Th1 and Th2, or effector and regulatory cells leading to inflammatory pathology, or increased susceptibility to other diseases? The demonstration of Tr1 cells that recognize the same antigens or peptides on a pathogen as Th1 cells [4,5], coupled with the suggestion that Tr1 and Th3 cells could be more prevalent at mucosal surfaces [4,7,51], also has important implications for the development of mucosal vaccines against infectious diseases. Immunization by oral or nasal routes, although attractive in terms of vaccine administration and local immunity, might favor the induction of immunosuppressive Tr cells rather than protective Th1 or Th2 cells. This requires the identification of safe but effective adjuvants that will selectively induce the appropriate T-cell subtype. Thus, manipulation of Tr cells in vivo has considerable potential in the prevention and treatment of human diseases, but further research on the molecular characteristics, induction and function of Tr cells is required before their therapeutic potential is realized.
Review
References 1 Groux, H. et al. (1997) A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389, 737–742 2 Doetze, A. et al. (2000) Antigen-specific cellular hyporesponsiveness in a chronic human helminth infection is mediated by Th3/Tr1-type cytokines IL-10 and transforming growth factor-β but not by a T(h)1 to T(h)2 shift. Int. Immunol. 12, 623–630 3 Cavani, A. et al. (2000) Human CD4+ T lymphocytes with remarkable regulatory functions on dendritic cells and nickel-specific Th1 immune responses. J. Invest. Dermatol. 114, 295–302 4 McGuirk, P. et al. (2002) Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J. Exp. Med. 195, 221–231 5 MacDonald, A.J. et al. (2002) CD4 T helper type 1 and regulatory T cells induced against the same epitopes on the core protein in hepatitis C virusinfected persons. J. Infect. Dis. 185, 720–727 6 Levings, M.K. et al. (2001) IFN-α and IL-10 induce the differentiation of human type 1 T regulatory cells. J. Immunol. 166, 5530–5539 7 Chen, Y. et al. (1994) Regulatory T-cell clones induced by oral tolerance: suppression of autoimmune encephalomyelitis. Science 265, 1237–1240 8 Fukaura, H. et al. (1996) Induction of circulating myelin basic protein and proteolipid protein-specific transforming growth factor-β1-secreting Th3 T cells by oral administration of myelin in multiple sclerosis patients. J. Clin. Invest. 98, 70–77 9 Shevach, E.M. (2000) Regulatory T cells in autoimmmunity. Annu. Rev. Immunol. 18, 423–449 10 Garba, M.L. et al. (2002) HIV antigens can induce TGF-β1-producing immunoregulatory CD8+ T cells. J. Immunol. 168, 2247–2254 11 Gilliet, M. and Liu, Y.J. (2002) Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells. J. Exp. Med.195, 695–704 12 Sakaguchi, S. (2000) Regulatory T cells: key controllers of immunologic self-tolerance. Cell 101, 455–458 13 Zuany-Amorim, C. et al. (2002) Suppression of airway eosinophilia by killed Mycobacterium vaccae-induced allergen-specific regulatory T cells. Nat. Med. 8, 625–629 14 Barrat, F.J. et al. (2002) In vitro generation of interleukin 10-producing regulatory CD4+ T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2inducing cytokines. J. Exp. Med. 195, 603–616 15 Seo, N. et al. (2001) Interleukin-10 expressed at early tumour sites induces subsequent generation of CD4+ T-regulatory cells and systemic collapse of antitumour immunity. Immunology 103, 449–457 16 Gorelik, L. and Flavell, R.A. (2002) Transforming growth factor-β in T-cell biology. Nat. Immunol. Rev. 2, 46–53 17 Thornton, A.M. and Shevach, E.M. (2000) Suppressor effector function of CD4+CD25+ immunoregulatory Tcells is antigen nonspecific. J. Immunol.164, 183–190 18 Read, S. et al. (2000) Cytotoxic T lymphocyteassociated antigen 4 plays an essential role in the function of CD25+CD4+ regulatory cells that control intestinal inflammation. J. Exp. Med. 192, 295–302 19 Shevach, E.M. (2002) CD4+CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2, 389–400 20 Nakamura, K. et al. (2001) Cell contact-dependent immunosuppression by CD4+CD25+ regulatory http://immunology.trends.com
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T cells is mediated by cell surface-bound transforming growth factor β. J. Exp. Med. 194, 629–644 Lucas, P.J. et al. (2000) Disruption of T-cell homeostasis in mice expressing a T-cell-specific dominant negative transforming growth factor β II receptor. J. Exp. Med. 191, 1187–1196 Jonuleit, H. et al. (2002) Infectious tolerance: human CD25+ regulatory T cells convey suppressor activity to conventional CD4+ T helper cells. J. Exp. Med. 196, 255–260 Asseman, C. et al. (1999) An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J. Exp. Med. 190, 995–1004 Singh, B. et al. (2001) Control of intestinal inflammation by regulatory T cells. Immunol. Rev. 182, 190–200 Stephens, L.A. and Mason, D. (2000) 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, 3105–3110 Jonuleit, H. et al. (2000) Induction of interleukin 10-producing, nonproliferating CD4+ T cells with regulatory properties by repetitive stimulation with allogeneic immature human dendritic cells. J. Exp. Med. 192, 1213–1222 Shimizu, J. et al.(2002) Stimulation of CD25+CD4+ regulatory T cells through GITR breaks immunological self-tolerance. Nat. Immunol. 3, 135–142 Weiner, H.L. (2001) Induction and mechanism of action of transforming growth factor-β-secreting Th3 regulatory cells. Immunol. Rev. 182, 207–214 Cottrez, F. et al. (2000) T regulatory cells 1 inhibit a Th2-specific response in vivo. J. Immunol. 165, 4848–4853 Redpath, S. et al. (2001) Hijacking and exploitation of IL-10 by intracellular pathogens. Trends Microbiol. 9, 86–92 Taoufik, Y. et al. (1997) Human immunodeficiency virus gp120 inhibits interleukin-12 secretion by human monocytes: an indirect interleukin-10mediated effect. Blood 89, 2842–2848 Marie, J.C. et al. (2001) Mechanism of measles virus-induced suppression of inflammatory immune responses. Immunity 14, 69–79 Van der Kleij, D. et al. (2002) Triggering of innate immune responses by schistosome egg glycolipids and their carbohydrate epitope GalNAc β 1-4(Fuc α 1-2Fuc α 1-3)GlcNAc. J. Infect. Dis. 185, 531–539 Barnes, P.F. et al. (1992) Cytokine production induced by Mycobacterium tuberculosis lipoarabinomannan. Relationship to chemical structure. J. Immunol. 149, 541–547 Feng, G.J. et al. (1999) Extracellular signalrelated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases differentially regulate the lipopolysaccharide-mediated induction of inducible nitric oxide synthase and IL-12 in macrophages: Leishmania phosphoglycans subvert macrophage IL-12 production by targeting ERK MAP kinase. J. Immunol. 163, 6403–6412 Ryan, E.J. et al. (2001) Immunomodulators and delivery systems for vaccination by mucosal routes. Trends Biotechnol. 19, 293–304 Fickenscher, H. et al. (2002) The interleukin-10 family of cytokines. Trends Immunol. 23, 89–96 Plebanski, M. et al. (1999) Interleukin 10-mediated immunosuppression by a variant CD4 T-cell epitope of Plasmodium falciparum. Immunity 10, 651–660 Boussiotis, V.A. et al. (2000) IL-10-producing T cells suppress immune responses in anergic tuberculosis
455
patients. J. Clin. Invest. 105, 1317–1325 40 Ostrowski, M.A. et al. (2001) Quantitative and qualitative assessment of human immunodeficiency virus type 1 (HIV-1)-specific CD4+ T cell immunity to gag in HIV-1-infected individuals with differential disease progression: reciprocal interferon-γ and interleukin-10 responses. J. Infect. Dis. 184, 1268–1278 41 Iwashiro, M. et al. (2001) Immunosuppression by CD4+ regulatory T cells induced by chronic retroviral infection. Proc. Natl. Acad. Sci. U. S. A. 98, 9226–9230 42 Moore, K.W. et al. (2001) Interleukin-10 and the interleukin-10 receptor. Annu. Rev. Immunol. 19, 683–765 43 Hori, S. et al. (2002) CD25+CD4+ regulatory T cells suppress CD4+ T-cell-mediated pulmonary hyperinflammation driven by Pneumocystis carinii in immunodeficient mice. Eur. J. Immunol. 32, 1282–1291 44 Yamagiwa, S. et al. (2001) A role for TGF-β in the generation and expansion of CD4+CD25+ regulatory T cells from human peripheral blood. J. Immunol. 166, 7282–7289 45 Moser, M. and Murphy, K.M. (2000) Dendritic cell regulation of Th1-Th2 development. Nat. Immunol. 1, 199–205 46 Hemmi, H. et al. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 47 d’Ostiani, C.F. et al. (2000) Dendritic cells discriminate between yeasts and hyphae of the fungus Candida albicans. Implications for initiation of T helper cell immunity in vitro and in vivo. J. Exp. Med. 191, 1661–1674 48 Gagliardi, M.C. et al. (2000) Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming. Eur. J. Immunol. 30, 2394–2403 49 Akbari, O. et al. (2001) Pulmonary dendritic cells producing IL-10 mediate tolerance induced by respiratory exposure to antigen. Nat. Immunol. 2, 725–731 50 Khanna, A. et al. (2000) Effects of liver-derived dendritic cell progenitors on Th1- and Th2-like cytokine responses in vitro and in vivo. J. Immunol. 164, 1346–1354 51 Iwasaki, A. and Kelsall, B.L. (1999) Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin-10 and induce the differentiation of T helper type 2 cells. J. Exp. Med. 190, 229–239 52 Kumanogoh, A. et al. (2001) Increased T-cell autoreactivity in the absence of CD40–CD40 ligand interactions: a role of CD40 in regulatory T cell development. J. Immunol. 166, 353–360 53 Salomon, B. et al. (2000) B7/CD28 costimulation is essential for the homeostasis of the CD4+CD25+ immunoregulatory T cells that control autoimmune diabetes. Immunity 12, 431–440 54 Menges, M. et al. (2002) Repetitive injections of dendritic cells matured with tumor necrosis factor α induce antigen-specific protection of mice from autoimmunity. J. Exp. Med. 195, 15–21 55 Lutz, M.B. and Schuler, G. Immature, semimature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol. (in press) 56 Hoyne, G.F. et al. (2000) Serrate1-induced notch signalling regulates the decision between immunity and tolerance made by peripheral CD4+ T cells. Int. Immunol. 12, 177–185 57 Levings, M.K. et al. (2001) Human CD25+CD4+ T regulatory cells suppress naïve and memory T-cell proliferation and can be expanded in vitro without loss of function. J. Exp. Med. 193, 1295–1301