The role of T helper 17 (Th17) and regulatory T cells (Treg) in human organ transplantation and autoimmune disease

Abstract

Uncommitted (naive) murine CD4⁺ T helper cells (Thp) can be induced to differentiate towards T helper 1 (Th1), Th2, Th17 and regulatory (T_reg) phenotypes according to the local cytokine milieu. This can be demonstrated most readily both in vitro and in vivo in murine CD4⁺ T cells. The presence of interleukin (IL)-12 [signalling through signal transduction and activator of transcription (STAT)-4] skews towards Th1, IL-4 (signalling through STAT-6) towards Th2, transforming growth factor (TGF)-β towards T_reg and IL-6 and TGF-β towards Th17. The committed cells are characterized by expression of specific transcription factors, T-bet for Th1, GATA-3 for Th2, forkhead box P3 (FoxP3) for T_regs and RORγt for Th17 cells. Recently, it has been demonstrated that the skewing of murine Thp towards Th17 and T_reg is mutually exclusive. Although human Thp can also be skewed towards Th1 and Th2 phenotypes there is as yet no direct evidence for the existence of discrete Th17 cells in humans nor of mutually antagonistic development of Th17 cells and T_regs. There is considerable evidence, however, both in humans and in mice for the importance of interferon (IFN)-γ and IL-17 in the development and progression of inflammatory and autoimmune diseases (AD). Unexpectedly, some models of autoimmunity thought traditionally to be solely Th1-dependent have been demonstrated subsequently to have a non-redundant requirement for Th17 cells, notably experimental allergic encephalomyelitis and collagen-induced arthritis. In contrast, T_regs have anti-inflammatory properties and can cause quiescence of autoimmune diseases and prolongation of transplant function. As a result, it can be proposed that skewing of responses towards Th17 or Th1 and away from T_reg may be responsible for the development and/or progression of AD or acute transplant rejection in humans. Blocking critical cytokines in vivo, notably IL-6, may result in a shift from a Th17 towards a regulatory phenotype and induce quiescence of AD or prevent transplant rejection. In this paper we review Th17/IL-17 and T_reg biology and expand on this hypothesis.

Introduction

Despite repertoire restriction in the thymus (‘central tolerance’) [1], the generation of a diverse T cell repertoire inevitably results in the production of T cell receptors with specificity for self-antigens [2,3]. Naive thymic emigrants therefore have the potential not only to respond to foreign antigens but also to components of self. Maturation of naive T cells depends critically on their interaction with the physicochemical environment and results in the development of cells with an effector (and memory) or regulatory function and the tolerization of autoreactive cells. It is critically important for the prevention of autoimmune diseases, therefore, that self-reactive naive T cells are not induced to mature into effector cells.

Murine experiments have demonstrated that naive CD4⁺ helper T cells (Thp) can develop into at least four types of committed helper T cells, namely T helper 1 (Th1), Th2, Th17 and regulatory T cells (T_regs) (see below). In humans, there is evidence for the existence of all but discrete Th17 cells, although helper T cells secreting interleukin (IL)-17 have clearly been described [4]. IL-17 has a proinflammatory role and has been implicated in many inflammatory conditions in humans and mice, while T_regs have an anti-inflammatory role and maintain tolerance to self-components (see below).

Naive T cells can be induced to commit to particular lineages based on mode of stimulation, antigen concentration, costimulation and cytokine milieu [5]. The pathways of differentiation towards Th1 and Th2 cells have been elucidated previously with IL-4 signalling through signal transduction and activator of transcription-6 (STAT-6) possibly the most important cytokine in inducing Th2 cell differentiation [6] and IL-12 signalling through STAT-4, the central cytokine for commitment towards a Th1 lineage [7]. Once differentiated, each lineage is characterized by its own cytokine profile (with interferon (IFN)-γ being the signature cytokine of Th1 cells and IL-4 the archetypal cytokine of Th2 cells) and transcription factors (T-bet for Th1 [8,9], GATA-3 for Th2 [10], forkhead box P3 (FoxP3) for T_regs[11,12] and RORγt for Th17 cells [13]). Although Th1 responses have been implicated in the development of autoimmune diseases (AD) [14], reduction in IFN-γ signalling in mice (using IFN-γ knock-out strains or blocking IFN-γ) paradoxically worsens susceptibility to AD, most notably experimental allergic encephalomyelitis (EAE) and collagen-induced arthritis (CIA) [15,16] in the absence of exaggerated Th2 responses, implying the involvement of other effector populations. Recent evidence suggests that naive T cells (in mice) can also be induced to differentiate along a pathway favouring development of Th17 or T_reg cells in a mutually exclusive manner [17–19]. Indeed, the Th17 population is important in mediating autoimmune diseases in animals [20,21].

As a result, a novel hypothesis has been proposed [22] with regards to inflammatory and autoimmune diseases, namely that skewing of responses towards Th17 or Th1 and away from T_reg (and Th2) may be responsible for the development and progression of AD or transplant rejection in humans and that blockade of critical cytokines may result in a shift in this polarization from Th17/Th1 phenotypes towards T_reg and Th2 (i.e. that ‘regulation and ‘dysregulation’ are inducible and remediable). Our own observations suggest that human effector T cells can be identified that produce mutually exclusive IFN-γ or IL-17 profiles. Additionally, the hypothesis predicts that blockade of critical cytokines for generation of Th17 (namely IL-6) can result in remission of AD. The purpose of this review is to discuss the relevance of Th17 and T_reg in human disease pathogenesis and progression.

IL-17

First cloned in 1993 from a murine cDNA library and known originally as CTLA-8 [23,24], IL-17A is a member of a family of IL-17 cytokines (IL-17A–F [25–29]) which are structurally homologous to each other and to a gene in the herpesvirus saimiri [23,30]. It was described initially as a product of activated and memory CD4⁺ T cells [23,30,30,31] but it is now known that the production of IL-17A is more ubiquitous and has been demonstrated in γδ T cells [32], CD8⁺ memory T cells [31,33], eosinophils [34], neutrophils [31] and monocytes [35]. Nevertheless, the predominant source of IL-17A (henceforth referred to as IL-17) remains the CD4⁺ memory T cell population [4,33].

The broad cell and tissue distribution of receptors for IL-17 (of which five have been described, namely IL-17R (the dominant receptor for IL-17A), IL-17RB, IL-17RC, IL-17RD and IL-17RE) in both humans and mice [27,29,30,36–38] and the diversity of expression through alternate splicing (reviewed in Moseley et al. 2003 [39]) argues for a pleiotropic spectrum of biological activity that may extend beyond the purely immunological, with the potential to act on many different cell types. Indeed, experiments in animals suggest that, unlike other cytokines, very little redundancy exists in the IL-17 network as IL-17R-deficient mice are very susceptible to lethal bacterial infections [40] and have inhibited T cell responses [41].

Nevertheless, the predominant function of IL-17 is thought to be as a proinflammatory mediator through a variety of mechanisms as summarized in Fig. 1. Locally, IL-17 stimulates production of IL-6, nitric oxide and prostaglandin E₂ (PGE₂) [4,30,42], while synergy with other inflammatory cytokines such as IL-1β, tumour necrosis factor (TNF)-α, IFN-γ[43–45] and CD40 ligand (by increasing surface levels of CD40) [46] leads to up-regulation of gene expression and progression and amplification of local inflammation. IL-17 mediates chemotaxis of neutrophils and monocytes to sites of inflammation through the chemoattractant mediators IL-8, monocyte chemoattractant protein (MCP)-1 and growth-related protein (Gro)-α[45,47–49] while enhancing production of haematopoietic growth factors, such as granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage (GM)–CSF [26,50], which promote the growth and maturation of the recruited myeloid cells. Furthermore, IL-17 acts as a bridge between the innate and adaptive immune response by augmenting the induction of co-stimulatory molecules such as ICAM-1 by other cytokines [45,51], thereby supporting T cell activation.

Fig. 1.

Proinflammatory effects of interleukin-17.

Although much is now known regarding the biology of IL-17 in murine systems and there is compelling evidence for an important role of IL-17 in inflammatory/autoimmune conditions [52,53], attempts to overexpress IL-17A ubiquitously in mice have failed due to generalized overproduction being lethal to developing embryos [54], while overexpression of IL-17E leads to a generalized inflammatory syndrome [55]. In humans, there is also a considerable body of evidence suggesting an important role for IL-17 in the aetiopathogenesis of inflammatory and autoimmune diseases, as discussed below.

Rheumatoid arthritis (RA)

Many lines of evidence support the role of IL-17 in the pathogenesis of human RA [56]. Levels of IL-17 are elevated in the synovium of patients with RA [57,58] and synovial cultures from patients with RA spontaneously secrete IL-17 [59]; the source of this IL-17 is local production by T cells [59] and juxta-articular bone lymphocytes [60]. Pathologically, this cytokine can activate and enhance all mechanisms of tissue injury that have been described previously in rheumatoid arthritis. In particular, IL-17 can up-regulate and/or synergize with local inflammatory mediators such as IL-6 [61,62], IL-1β and TNF-α[61–63], pro-oxidants such as nitric oxide [64], as well as promoting extracellular matrix injury through stimulation of production of matrix metalloproteinases (MMP) [65,66] and inhibition of matrix repair components such as proteoglycans and collagens [67,68]. Furthermore, bone injury is enhanced [69] through promotion of osteoclastogenesis via osteoclast activating factor [57]. The combination of these factors has pathological effects on bone (resorption), extracellular matrix (degeneration), synovium (proliferation and inflammation), blood vessels (angiogenesis) and immune cells (recruitment and activation of monocytes and lymphocytes) in the rheumatoid joint. Not surprisingly, perhaps, intra-articular injection of IL-17 in normal mouse joints induces similar changes to RA [64] and excess local IL-17 (via adenovirus-mediated gene expression vectors) exacerbates significantly CIA [20].

Many of these effects of IL-17 on synovium and bone can be antagonized in vitro by treatment with IL-4, IL-13 or anti-IL-17 blocking antibody. These interventions can reduce IL-17-driven production of inflammatory factors such as leukaemia inhibitory factor [61], CCL20/MIP3α[70] as well as decreasing matrix metalloproteinase (MMP) production and increasing tissue inhibitors of MMPs [67]. Some of the effects of IL-17 on articular cartilage can be attenuated by this approach in vivo[64], while blockade of IL-17 abrogates completely the spontaneous development of inflammatory arthritis in IL-1R antagonist-deficient mice [71], and mice lacking IL-17 are highly resistant to CIA [72].

Respiratory diseases

The importance of IL-17 to airways immunity is highlighted by the susceptibility of IL-17R knock-out mice to fatal pulmonary infections [40], which correlates with impaired neutrophil mobilization and bacterial clearance [48,73]. Similarly, the pulmonary response to local introduction of Escherichia coli endotoxin requires the presence of IL-17 for neutrophil accumulation in the bronchoalveolar space [47,73]. In mice and rats, there is evidence that activated lymphocytes from lung tissues can produce substantial amounts of IL-17 [47,74] and that the effect of IL-17 on the respiratory epithelium is to produce chemokines that favour a neutophilic infiltrate [47,75] and that increase neutrophil activity in vivo (as measured by myeloperoxidase and elastase release) [54,73].

Given these findings, it is perhaps not surprising that exaggerated IL-17 responses are implicated in the pathogenesis of inflammatory airways diseases. Human respiratory epithelial cells (and even some nonepithelial cells [34,75]), in a similar manner to murine ones, are responsive to IL-17 and can be stimulated to produce the same chemoattractant molecules [49,50,75–77], although there is now evidence supporting the notion that, under normal physiological conditions, the human bronchoalveolar space releases only very low amounts of IL-17 [78]. Severe respiratory inflammation precipitated by exposure to organic dust in humans is characterized by a marked increase in IL-17 levels in the broncheoalveolar lavage and a 50-fold increase in neutrophil recruitment to the lung [78]. Similarly, there are suggestions that in pulmonary asthma there is not only an increase in the number of IL-17-producing cells (T lymphocytes and eosinophils in broncheoalveolar lavage) in comparison to healthy controls both locally and within the circulation [34], but also higher levels of intracellular IL-17 in IL-17-producing cells than their healthy counterparts [34], although not all studies agree on this [79].

Pathologically, IL-17 is likely to exert its effects through exaggerated physiological mechanisms (highlighted above), namely synergy with other cytokines and the recruitment of neutrophils to lung tissues. Indeed, studies in allergen-sensitized mice suggest that neutrophil accumulation in the lungs following encounter with allergen is orchestrated by IL-17 transcription [80]. This latter mechanism may be critically important as neutrophilic infiltrates and neutrophil enzymatic activity correlate with the degree of bronchial hyperreactivity in patients with asthma [81]. IL-17-induced IL-6 release may have dual pathological significance, as IL-6 promotes neutrophil elastase release [82] (elastase activity is thought to be a key mediator in the pathogenesis of chronic airway diseases [83,84] and reciprocally controls activity of neutrophil IL-6 [85]) and is one of the mechanisms by which IL-17 stimulates release of mucin by respiratory epithelial cells [86]. Furthermore, IL-6 is important in the generation of IL-17-producing cells (mouse data, see below). Also, many of the effects of IL-17 on lung tissues can be antagonized by glucorticoids [34,75], which form the mainstay of treatment for inflammatory and allergic pulmonary diseases in humans.

Despite the evidence in favour of a significant role for IL-17 in inflammatory respiratory diseases in humans, most of the evidence is circumstantial and there is no direct evidence that this cytokine is the causative mediator or the central participant (i.e. that production of IL-17 is the trigger that precipitates these pulmonary diseases) and it may be that an elevated IL-17 response is simply part of the generalized inflammatory milieu. Certainly more evidence regarding the role of the cytokine in lung pathology is required.

Allograft rejection

Rejection of transplanted tissues involves interplay between mechanisms that maintain tolerance to the graft and factors that promote rejection. While immunological factors are important for both, the process of rejection is very much an inflammatory one and, as a consequence, the production of many proinflammatory cytokines, such as IFN-γ, IL-2, IL-6 and IL-15, locally from infiltrating lymphocytes and resident cells [e.g. proximal renal tubular epithelium (PTEC)], is increased during acute renal graft rejection [87–89]. Studies in acute rat rejection models have also identified an elevation in IL-17 mRNA (in the renal allograft) and IL-17 protein (in infiltrating mononuclear cells) as early as day 2 post-transplant [90]. Similarly, IL-17 protein is elevated in human renal allografts during borderline (subclinical) rejection together with detectable IL-17 mRNA in the urinary MNC sediment of these patients; in control (non-rejecting) patients, IL-17 is not detectable in either the biopsy sample nor the urinary sediment [90]. These findings have also been described previously [91].

IL-17 induces IL-6, IL-8, MCP-1 and complement component C3 but not regulated upon activated normal T cell exposed expressed and secreted (RANTES) nor other complement components [91] by PTECs [90–92] via the src/mitogen-activated protein kinase (MAPK) pathways [92]. An additional mechanism, through synergy with CD40-ligand and proceeding via nuclear factor (NF)-κB activation, has also been described [46].

In human lung organ transplantation, IL-17 has also been reported as being elevated during acute rejection [93], while rat models have demonstrated that collagen type V-specific lymphocytes can mediate lung allograft rejection and express IL-17 locally at the site of rejection [94]. In cardiac allograft models, antagonism of the IL-17 network (via expression of an IL-17R-immunoglobin fusion protein) can reduce intragraft production of inflammatory cytokines (namely IFN-γ) and prolong graft survival [95]. This approach, however, is more successful at preventing acute, rather than chronic, vascular rejection [96] and may indicate a more important role for IL-17 in mediating early rather than late cardiac rejection. There may, in addition, be a role for IL-17 in inducing the maturation of alloreactive dendritic cells [97].

Systemic lupus erythematosis (SLE) and other conditions

IL-17 has been implicated in a variety of other chronic human diseases, largely through demonstrations that the cytokine is overexpressed in these conditions. Examples include SLE [98], psoriasis [99,100], multiple sclerosis [101], systemic sclerosis [102] and chronic inflammatory bowel disease [103]. As this evidence is largely circumstantial, the exact role of IL-17 in these conditions is unclear.

With regard to infectious agents, it is possible that IL-17 has a role to play as a virulence factor, particularly given its homology to the gene in herpesvirus saimiri [23,30], a virus that is tropic for T cells [104]. While expression of murine IL-17 gene in a vaccinia virus does increase significantly its virulence [105], IL-17 knock-out strains of herpesvirus saimiri have unaltered pathogenicity [106], therefore the role of IL-17 in viral infections is unclear. Nevertheless, certain bacterial infections, notably Helicobacter pylori[107], Bacteroides fragilis[108] and periodontitis [109] are associated with particularly high levels of IL-17.

Similarly, there are suggestions that IL-17 may be involved in tumorigenesis, as IL-17 can both promote (e.g. human cervical cancer cell lines transplanted into nude mice) [110,111] and inhibit growth (e.g. haematopoietic tumours in immunocompetent mice) [112,113] of tumours in experimental animals. Although the role of IL-17 in tumour biology is unclear and evidence remains conflicting, IL-17 can be detected in some (such as ovarian, skin and prostatic cancers) [114–116] but not all (e.g. acute myeloid leukaemia) [117] human tumours and one emerging theme is that, when present, it promotes angiogenesis [111,114], for example through the up-regulation of angiogenic factors such as CXCL1, CXCL5, CXCL6 and CXCL8 [118], thereby facilitating tumour growth and invasion. The alternative explanation is the possibility that vascular tumours may be better able to recruit activated/memory T cells, some of which will be IL-17 producers and, by virtue of being highly vascular, may portend a poorer prognosis.

In either event, there is now considerable evidence that IL-17 is involved in the aetiopathogenesis of many human disorders. Whether IL-17 is a key causative factor or is involved in the amplification of inflammatory responses has yet to be elucidated.

Regulatory T cells

A number of cell types with immunoregulatory capacity have been described in the literature. These include IL-10-secreting Tr1 cells [119], transforming growth factor (TGF)-β-secreting Th3 cells [120], Qa-1 restricted CD8⁺ cells [121], CD8⁺ CD28^– T cells [122], CD8⁺ CD122⁺ T cells [123], γδ T cell receptor (TCR) T cells [124], natural killer (NK) cells [125,126], dendritic cells [127], apoptotic neutrophils [128], CD8⁺ CD28^– cells [129–132], CD3⁺ CD4^– CD8^– cells [133,134] and naturally occurring CD4⁺ CD25⁺ T cells [135]. Given that both human and murine knock-outs for CD4⁺ CD25⁺ cells develop severe autoimmune diseases [135–138], the focus of attention in the literature has been mainly on these regulatory T cells (referred to as T_regs in this paper).

In vitro, T_regs have the ability to inhibit proliferation and production of cytokines by responder (CD4⁺ CD25^– and CD8⁺) T cells [139–141] to polyclonal stimuli, as well as to down-modulate the responses of CD8⁺ T cells, NK cells and CD4⁺ cells to specific antigens [139,142]. These predicates translate in vivo to a greater number of functions other than the maintenance of tolerance to self-components (i.e. prevention of autoimmune disease) [143] and include control of allergic diseases [144] and regulation of responses to microbial pathogens [145,146], as well as the ability to prevent transplant rejection [147] and to maintain gastrointestinal tolerance [148] and maternal tolerance to semiallogeneic fetal antigens [149]. Indeed, donor-specific T_regs can prevent allograft rejection in some models of murine transplant tolerance [150–152] through a predominant effect on the indirect alloresponse [153].

Although mutations in Foxp3, a forkhead-winged-helix transcription factor, are responsible for the loss of T_reg function in both mice [137] and humans [154] and overexpression of Foxp3 in mouse cells leads to development of a T_reg phenotype [154–156] and can act as a phenotypic marker [12,157], FoxP3 expression may not be an ideal marker for T_regs in humans [158] as FoxP3 is induced during TCR stimulation [159] (in much the same manner as CD25), and there is some debate as to whether the induced CD4⁺CD25⁺FoxP3⁺ population is suppressive or anergic [159,160]. Recent evidence has also implicated the IL-7 receptor (CD127) as a possible biomarker of T_regs in humans as the combination of CD4 and CD25 together with low expression of CD127 identifies a group of peripheral blood T cells, which are highly suppressive in functional assays and the highest expressors of FoxP3 [161].

Regulatory T cells function in an antigen-presenting cell (APC)-independent (at least in vitro) [162] and antigen-non-specific manner [141,163]. However, they do respond to their cognate antigen [164–166] and, while anergic in vitro[139], T_regs can proliferate extensively in response to antigen in vivo[167,168]. Although the exact mechanism by which T_regs exert their effect is unknown, it is believed that their suppressive function is contact-dependent on the basis of transwell experiments, where suppression could be abrogated via separation of T_regs and effector T cells by a semipermeable membrane [141,163,169], and demonstrations that signalling through the T cell receptor is critical to their function [170,171]. These observations are divergent with in vivo data showing an important role for TGF-β and IL-10 production as mediators of T_reg activity [172,173], and do not exclude the possibility that T_reg function may involve soluble mediators acting at very short distances from the cell or bound to the cell surface [174,175]. Despite suggestions that T_regs influence cells by direct contact, these cell-to-cell interactions are poorly mapped. Indeed, there is evidence that some of the activity of T_regs progresses through intermediaries, such as NK T [176] and mast cells [177], and that their effect on target T effector cells includes an arrest in cell cycle progression caused by uncoupling of IL-2 signalling [178,179]. Recently, a role for IFN-γ in the regulatory function of T_regs has also been proposed based on the up-regulation of IFN-γ mRNA in alloantigen-reactive T_regs in vivo hours after encounter with antigen and failure of skin graft tolerance in the presence of IFN-γ neutralization [180].

T_regs express constitutively CTLA-4 (cytotoxic T lymphocyte antigen-4) and there are suggestions that signalling through this pathway may be important, for their function as antibodies (or Fab) to CTLA-4 can inhibit T_reg-mediated suppression [181–183]. However, CTLA-4 is also inducibly expressed on CD4⁺ CD25^– cells [184] and therefore these observations may be the result of CTLA-4 antibodies acting on effector rather than regulatory cells and could explain why the initial reports have been so difficult to reproduce in mice [141] and humans [140,140,185]. For a review on CTLA-4 in T_reg biology, please see Sansom et al. [186].

Development and persistence of T_regs and IL-17-producing cells

T_regs

It is now clear that CD4⁺ CD25⁺ T_regs can be derived from two sources, namely those developing within the thymus (whose contribution may therefore diminish with age) and those generated in the periphery. Thymically derived T_regs are thought to originate at the transition between the double-positive and single-positive stages following encounter between thymocytes that bear high affinity TCRs for self-peptide with their cognate antigens [165,187], but this assertion has been challenged recently by observations that thymic commitment to a T_reg phenotype may occur at an earlier developmental stage [188]. The autoreactive T_reg repertoire may be entrained by deletion following interaction with endogenous superantigens and APCs of both thymic and bone marrow origin [164], but the peripheral T_reg repertoire retains a higher frequency of autospecific than alloreactive cells [164]. How T_reg precursors commit to a T_reg lineage in the thymus is unknown, but recent evidence points (in mice) to an interaction with a gene locus intimately linked with the MHC [189] (characterization of this locus and the genes involved is awaited) and may involve an important role for IL-2 signalling [190] and/or CD28 engagement [191].

Adults rendered temporarily lymphopenic have a greater propensity to develop autoimmune diseases [192,193]. Although this may reflect loss of a significant proportion of thymically derived T_regs (which are hard to regenerate given age-related thymic atrophy [194,195]) leading to loss of self-tolerance, one cannot ignore the fact that not everyone who is made lymphopenic develops autoimmune disease. One possibility is that the important determinant for maintenance of tolerance to self-components may be the relative frequency of effector cells to T_regs, as some chemotherapy agents have an equal effect on both [196]. However, depletion of CD25⁺ T cells from mice [197] or the adoptive transfer of naive T cells into lymphopenic recipients [198] is not sufficient for the development of autoimmune phenomena. The second possibility is that T_regs are generated in the periphery, an attractive notion that is supported by data showing reconstitution of the CD4⁺ CD25⁺ T_reg population through conversion of CD4⁺ CD25^– T cells [199,200]. Although this is not a robust phenomenon [12,157], there are suggestions that discrepancies may be the result of competition between CD4⁺ CD25^– and CD4⁺ CD25⁺ T cells (i.e. that the rate of conversion is related to the relative frequency of the two cell types) [201] and that the number of T_regs may be linked to the availability of IL-2 (and, by inference, the number of IL-2-producing effector cells) [202]. Studies of human T_reg populations have shown these populations to be highly proliferative and senescent in vivo with very short telomeres [203], which is consistent with their memory/CD45RO⁺ phenotype [140]. Their susceptibility to apoptosis and short telomeres (with low telomerase activity) means it is unlikely that they are capable of self-renewal; the more likely explanation is that T_regs are generated in the periphery.

Other studies have corroborated the importance of IL-2 for the development of T_regs[204] and are summarized in the review by Malek et al. [205]. Indeed, in an animal model of graft-versus-host disease (GvHD) where autoreactive T cells from donors deficient in T_regs (DO11·10 Rag^–/– animals) are infused into athymic antigen-expressing lymphopenic recipients (sOVA transgenic thymectomized, lethally irradiated mice reconstituted with Rag^–/– bone marrow), development and recovery from disease is reliant upon generation of antigen-specific effector cells followed by de novo generation of peripheral T_regs in a concomitant, IL-2-dependent manner [206]. The added implication of these findings is that both functional effector cells and T_regs can develop in parallel from the same population of T cells in response to a single antigen in the periphery.

The mechanism(s) by which T_regs are generated in the periphery are unknown. However, there are indications that, in the same manner as with Th1/Th2 cell polarization, the antigenic stimulus (linked to the amount of antigen present as well as the strength of interaction) may determine the commitment to a T_reg phenotype (low doses/weaker stimulus result in more T_reg generation) [207,208]. The mode of antigen encounter, namely through (immature or suboptimally activated) dendritic cell presentation, also seems important for the conversion of naive T cells to a T_reg phenotype [208,209], as may interactions with anti-inflammatory molecules such as thrombospondin-1 [210]. The demonstration that TGF-β-deficient mice have reduced numbers of peripheral [211,212] but not thymic T_regs[213] and that TGF-β assists the conversion of naive CD4⁺ CD25^– T cells into T_regs both in vivo[209] and in vitro[214,215] argue in favour of the importance of this cytokine in the generation and maintenance of the peripheral T_reg pool. The potential importance of these observations to human disorders will be discussed in the context of Th17 development below.

IL-17-producing cells

In mice, a discrete population of CD4⁺ helper T cells has been described as the predominant source of IL-17. These cells have been named Th17 cells. The initial basis of this nomenclature is the dichotomous effects of IL-12 and IL-23 (both members of the same family of IL-12 cytokines, sharing a common IL-12p40 subunit but differing second subunits, IL-12p35 and IL-12p19, respectively [216]) on the cytokine profile of CD4 cells. While IL-12 (signalling through signal transducer and activator of transcription, STAT-4) had been known to allow lineage commitment towards a Th1 phenotype producing IFN-γ as its signature cytokine (via the transcription factor T-bet[8,9]) [5,7,217] and inflammatory diseases had been viewed along the lines of a Th1/Th2 paradigm (e.g. IL-12p40 neutralization in mice ameliorates inflammatory diseases [218,219]), mice deficient in IFN-γ or IFN-γ signalling remained, paradoxically, susceptible to development of EAE and CIA [15,16]. Furthermore, IL-12p40 (lacking both IL-12 and IL-23) and IL-12p19 (lacking IL-23)-deficient mice were protected against EAE and CIA whereas IL-12p35 (IL-12)-deficient strains remained susceptible [220,221] suggesting that IL-23 rather than IL-12 was important in mediating the pathogenesis of these conditions. Shortly after these observations, it was reported that IL-23 stimulates production of IL-17 from a population of memory (but not naive) CD4⁺ T cells in a manner that does not exhibit elevation of IFN-γ[21,222] and that IL-17 is linked to the inflammation seen in CIA and EAE [20,21,72]. Further evidence that IL-17 was derived in mice from a discrete population of Th cells that were distinct from the Th1 lineage, termed Th17 cells, were provided by publications showing resistance of Th1 and Th2 cells in vitro to proliferation or production of IL-17 following stimulation with IL-23 and that development of IL-17-producing cells was inhibited by the presence of IFN-γ and/or IL-4 in the culture supernatant [223,224]. It has been proposed that production of IL-12 has greater importance for systemic responses and immunity to intracellular pathogens [225] while IL-23, produced from activated human macrophages and dendritic cells (DC) [226], has a more important role for mediating mucosal immune pathology through the promotion of Th1 and Th17 cytokine profiles, respectively [227].

It is now known that IL-6 [17–19] (see below), and not IL-23 [17,18], is critical for the induction of Th17 lineage commitment (which is supported by the fact that the IL-23 receptor is expressed exclusively on activated and memory T cells [222,228]), while IL-23 seems to be important for the selective expansion of these cells and production of IL-17 [229]. Indeed, other cytokines including IL-2, IL-15, IL-18 and IL-21 can also stimulate IL-17 production from (activated) human T cells and peripheral blood mononuclear cells (PBMC) [226,229], while IL-12 potently inhibits it [229]. Development of Th17 cells is dependent upon correct co-stimulation (ICOS and CD28 [224]) and the absence of IFN-γ and IL-4, both of which are inhibitory [223]. Furthermore, Th17 lineage differentiation can be inhibited by the Th1-specific transcription factor T-bet in the context of IL-4 blockade [230] and is characterized by the expression of the orphan nuclear receptor RORγT[13]. Because IL-12 specifies lineage commitment to Th1 and has a stimulatory effect on IFN-γ secretion by Th1 cells [229], IL-12 may play a critically important role as a regulator of the balance between Th1 and Th17 responses. This assertion is supported by in vivo mouse data in which IL-23 and IL-12 had divergent pro- and anti-inflammatory roles in a model of collagen-induced arthritis [221]. It is important to state that, despite these observations, the description of discrete Th17 cells is mouse-specific and to date no committed Th17 cells have been demonstrated in humans.

Figure 2 shows the cytokine network that is thought to be important in the development and expansion of Th17 cells and the dichotomous Th1/Th17 cytokine profile engendered by these cytokines. Three recent papers have shed some light on the mechanisms by which naive precursor T cells commit to a Th17 phenotype in mice [17–19]. The first, a publication by Veldhoen et al., showed that naive CD4⁺ T cells could be skewed towards a Th17 phenotype in the presence of dendritic cells and T_regs in an inflammatory milieu (lipopolysaccharide stimulation) [18]. Absence of T_regs leads to Th1 differentiation, presumably through interaction of naive T cells with DCs producing IL-12. In the presence of T_regs and DC, the important drivers of Th17 differentiation were T_reg-derived TGF-β and DC-derived IL-6, although both TNF-α and IL-1β (both DC-derived) also augmented the commitment to Th17. In this series of experiments, the IL-17-producing cells did not express T-bet or GATA-3 and addition of IL-12 and IL-4 or IL-18 inhibited Th17 development, but the most important determinant of commitment to a Th17 lineage was the presence of TGF-β, without requirement for cell-to-cell contact. These data were corroborated by Bettelli et al., who demonstrated using cells from a Foxp3-GFP knock-in mouse strain that differentiation towards T_reg and Th17 phenotypes were mutually exclusive − activation of naive precursor cells using anti-CD3 in the presence of TGF-β lead to production of green fluorescent protein (GFP)⁺ cells (i.e. T_regs) as per previous observations, but activation in the presence of IL-6 in addition to TGF-β completely abrogated this and led to development of Th17 cells (that were GFP^–). The differentiated cells were, respectively, functionally suppressive and inflammatory and development of the Th17 phenotype was independent of IL-23 [17]. The third paper, by Mangan et al. published simultaneously [19], showed that addition of TGF-β to naive CD4⁺ T cells resulted in the development of Th17 cells, an effect which was augmented in the presence of neutralizing antibodies to Th1 and Th2 polarizing cytokines (IL-4 and IFN-γ) or the use of CD4 cells from IFN-γ deficient animals [19]. Furthermore, they demonstrated that TGF-β up-regulated expression of the IL-23 receptor (which may explain the responsiveness of the Th17 population to IL-23). As before, supplementation of the culture conditions with exogenous IL-6 resulted in loss of all Foxp3⁺ cells, while blockade of IL-6 enhanced T_reg development. Again, these findings point to mutually exclusive pathways for Th17 and T_reg development based on the availability of TGF-β and IL-6. A schematic for naive T cell commitment is represented in Fig. 3. It should be noted, once again, that these data are derived from mice and whether this pathway exists in humans has not been determined.

Fig. 2.

Model of mouse helper T cell (Th) commitment to Th1, Th17 and T regulatory cell (T_reg) phenotypes following encounter with antigen. Production of transforming growth factor (TGF)-β by naturally occurring T_regs leads to lineage commitment of precursor helper T cells (Thp) towards T_reg phenotypes. Stimulation of dendritic cells (DC) by microbial antigens causes production of interleukin (IL)-6, IL-23 and/or IL-12. Predominant production of IL-12 promotes commitment of Thp to a Th1 phenotype while IL-6, in combination with T_reg-derived TGF-β promotes skewing of Thp towards a Th17 phenotype. IL-23 produced by DCs causes proliferation and cytokine production by Th17 cells.

Fig. 3.

T helper cell commitment towards specific lineages in mice. T helper cell precursors (Thp) can be skewed towards mutually exclusive Th1, Th2, Th17 and T regulatory cell (T_reg) phenotypes on the basis of the cytokine environment. Presence of interleukin (IL)-12 promotes skewing towards Th1 commitment by signalling through signal transduction and activator of transcription (STAT)-4. Th1 cells are characterized by expression of T-bet and produce interferon-γ and tumour necrosis factor-α. Th2 cell commitment is promoted by IL-4 via STAT-6 signalling. Th2 committed cells are characterized by expression of GATA-3. Development of T_reg and Th17 phenotypes both require the presence of transforming growth factor-β but the presence of IL-6 preferentially skews the response towards a Th17 phenotype. T_regs are characterized in mice by expression of Foxp3.

A model for the regulation of T cell polarity in humans

Although the majority of the data concerning commitment to T cell lineages has been derived from mice, there is a clear difference in lineage differentiation based on the mode of stimulation and the cytokine milieu. The presence of discrete IL-17-producing cells in humans has yet to be confirmed; however, IL-17 is likely to be an important cytokine in the mediation of many inflammatory diseases and allograft rejection in humans. As such, one can propose a hypothesis with regard to the pathogenesis of autoimmune/inflammatory diseases and allograft rejection in humans that is based on extrapolations of the mouse data on the assumption that human cells exhibit discrete IL-17-producing populations and can be skewed towards different lineages.

Specifically, the observation that many inflammatory or autoimmune diseases present clinically as episodes of inflammation (flares), with periods of quiescence in between these episodes, argues for the presence of intervening periods of ‘equilibrium’ where the immune system displays tolerance to self-components (i.e. that proinflammatory components are ‘regulated’). During acute flares, a state of ‘disequilibrium’ ensues in which immune responses against self-components are dysregulated. The possibility arises that during these episodes Th cell phenotypes become skewed towards proinflamatory lineages (Th17 and Th1) (or that there is enhanced survival of these lineages) and away from anti-inflammatory phenotypes (T_reg) on the basis of the local cytokine environment and DC populations. The mechanism of such a change could either be loss of skewing towards T_reg phenotypes (with default towards Th1/Th17) or a primary shift towards the proinflammatory pathways. The central cytokine in this pathway, on the basis of the mouse data, may be IL-6, which is known to be elevated in most inflammatory conditions. Presumably, the balance is redressed during the recovery from flares and the equilibrium re-establishes itself. The part played by anti-inflammatory/immunosuppressive drugs in the resolution phase and the effects of these drugs on specific subsets of T cells is not known at present.

Conclusions

Interleukin 17 is a pleiotropic cytokine with multiple proinflammatory functions that is likely to be involved in either the causation or progression of inflammatory diseases and transplant rejection in humans. Regulatory T cells are an anti-inflammatory lineage of T cells that are derived naturally from the thymus and also generated in the periphery on the basis of the local environment. It is possible that acute flares of autoimmune diseases or acute episodes of transplant rejection may be explained by a change in the relative dominance of these pathways in the periphery, either through preferential differentiation towards proinflammatory lineages or enhanced survival of these phenotypes. A change in the local polarizing conditions may be important in the skewing of these responses.