Transforming Growth Factor-β as a Regulator of Site-Specific T-Cell Inflammatory Response
Dr B. R. Lúðvíksson, Department of Immunology, National University Hospital of Iceland, Reykjavík, Iceland. E-mail: email@example.com
A common immunopathological hallmark of many autoimmune inflammatory diseases is a T-cell invasion and accumulation at the inflamed tissue. Although the exact molecular and microenvironmental mechanisms governing such cellular invasion and tissue retention are not known, some key immunological principles must be at work. Transforming growth factor-β (TGF-β) is known to modulate some of these processes including homing, cellular adhesion, chemotaxis and finally T-cell activation, differentiation and apoptosis. The chronicity of such T-cell-driven inflammation probably involves an innate immunological response leading to a T-1 (Th/Tc), T-2 or T-3 (Th/Tr) T-cell adaptive immune response. Several studies suggest that the key to T-cell final destination resides on its and the antigen-presenting cell's phenotype as well as the coreceptor expression pattern and their signalling intensity. Recent observations suggest other equally important regulatory elements of T-cell inflammatory response that are sensitive to TGF-β modulation. These include: (i) the stage of T-cell activation/differentiation; (ii) the chemotactic/adhesion molecule expression pattern; and (iii) the conditioning at the immunological synapse determining their sensitivity to known regulators such as TGF-β. In this article, we focus on how the phenotype of the responding T cell and the T-cell receptor (TCR)-signalling intensity could drive the given inflammatory response. In particular, we discuss how TGF-β can influence the process of T-cell migration and activation during such site-specific inflammation.
The pathogenesis of chronic organ-specific autoimmune inflammation depends on the trafficking of lymphocytes from the site of induction to sites of inflammation. Adhesion molecule expression patterns, corresponding molecules of endothelial cells and chemotaxis of T cells are amongst the key initiating factors for the immmunopathological cascade that follows. They mediate T-cell adhesion, tissue transmigration, tissue retention and finally their dynamic recirculation through the different tissue compartments. Considerable body of evidence also suggests that naïve T cells selectively migrate into primary and secondary lymphoid organs but not into nonlymphoid organs through their expression pattern of L-selectins and CCR7 [1, 2]. In contrast, most memory T cells express low levels of these receptors but instead have upregulated other adhesion and chemokine receptors. Thus, 40% of naïve T cells are found in the lymph nodes, while activated T cells will be found more readily within the nonlymphoid compartments. However, several discrepancies from this view have emerged and have recently been reviewed elsewhere .
In order to initiate an inflammatory response, the T cell has to migrate into the tissue. It is known that even during ‘noninflammatory’ conditions it is estimated that approximately 1010 T cells are entering different tissues every 30 min. Such robust tissue migration depends on a multistep navigation of lymphocytes into the tissue that is under constant immune surveillance by immune regulators, including various regulatory cells and secreted factors. Because of the complex and pleiotropic nature of transforming growth factor-β (TGF-β) on T cells, we still do not know exactly what factors are involved during its T-cell modifying process. TGF-β has been reported to inhibit many T-cell functions, including proliferation and development of cytotoxic T cells and T helper cells of types 1 and 2 [4–9]. However, under certain conditions, TGF-β has also been found to stimulate T cells, partly by preventing apoptosis, but also by inducing proliferation [10–13]. In addition, TGF-β is important in both the generation and the action of most regulatory CD4+ and CD8+ T cells . These cells suppress immune responses of T cells and play a vital role in inhibiting autoimmune T cells. Several subsets of these regulatory cells have been described. In general, these cells can be divided into those that use immunosuppressive cytokines to exert their regulatory effects and those that use cell contact. In the former group are T regulatory (Tr1) and Th3 cells. The Tr1 cells act predominantly by secreting high quantities of interleukin-10 (IL-10) , whereas Th3 cells act by secreting predominantly TGF-β and variable amounts of IL-10 and IL-4 . CD4+ T cells constantly expressing the IL-2 receptor α chain (CD25+) have been shown to mediate their suppression through contact-dependent mechanism, at least in vitro[17, 18]. Although the function of these cells is known to be contact-dependent, they have also been reported to produce the anti-inflammatory cytokines TGF-β[19, 20], IL-10 [20–22] and IL-4 . However, the total loss of IL-10, IL-4  and TGF-β in knockout murine models has not been able to interrupt the suppressive effect of these cells. The importance of cytokines in the generation of the regulatory cells will probably be clarified in the near future as the transcription factor (Foxp3) that controls the development of these cells has recently been discovered . The regulatory role and pleiotropic activity of this system becomes especially important when a relatively small amount of microbial invasion leads to robust immune activation, making it essential that a high level of cross-regulation is at work. Indeed, it has become apparent that if certain essential regulatory elements are deleted from the genome or made dysfunctional, it can lead to catastrophic autoimmune reactions.
Transforming growth factor-β isoforms
The TGF-β family is composed of several different isoforms of proteins that have diverse functions upon tissue development and homoeostasis. Three different isoforms are expressed in mammals – TGF-β1, -β2 and -β3 – with 70–76% sequence homology . Despite some biological similarities, the isoforms have different expression patterns and functional differences that have been confirmed in TGF-β knockout mice . Thus, TGF-β2 and TGF-β3 are important regulators of cellular differentiation and affect development and embryogenesis, whereas the effects of TGF-β1 are predominantly immunologic [4, 28, 29]. TGF-β1 is a multifunctional homodimeric protein of 25 kDa. It represents a family of structurally related polypeptides termed the transforming growth factor super family . TGF-β1 is secreted by a variety of cell types including B and T lymphocytes, macrophages, dendritic cells (DCs) and platelets. The dimerized prodomain, also called latency-associated protein, is noncovalently associated with the C-terminal portion of a larger precursor producing a latent complex of larger weight, including another glycoprotein of 125 kDa called latent TGF-β-binding protein. Latent TGF-β can be converted to active TGF-βin vitro by plasmin, cathepsin D or treatment with heat or acidic pH. Its conversion to active TGF-β at different tissue sites is highly regulated through mostly unknown mechanisms.
Transforming growth factor-β at the inflammatory site
Although TGF-β1 is well known for its immune-suppressive and anti-inflammatory properties, it is also capable of promoting inflammation. The beginning of the inflammatory process involves the removal of antigen from the tissue and transfer to the lymph node where it is presented by DCs to T cells. Recently, Randolph et al.  showed that TGF-β1 might play an important role in these early steps by inducing monocytes to express CD16, which predisposes them to become DCs. The TGF-β1-treated monocytes demonstrated increased expression of CD86, improving their survival, migration and stimulatory capacity , thus placing TGF-β1 at the very first step of the inflammatory response. TGF-β is known to induce migration of both CD4+ and CD8+ T cells, thereby possibly attracting these cells to sites of inflammation . TGF-β1 is also known to be a powerful chemoattractant for neutrophils, lymphocytes and monocytes/macrophages in the earlier stages of inflammation [33–35]. The effect of this cytokine therefore appears to change in response to the demands. It is pro-inflammatory in the early inflammatory process, which calls for effective elimination of an invading agent/repair of injury, and acts in an anti-inflammatory manner in the later steps, which calls for a different activity, as the prolonged inflammation may be harmful to the host.
Accumulating body of evidence suggests that it is important to make a distinction not only between the different stages of the inflammatory response but also between the different phenotypes and effector functions of the responding T cells. Today, surprisingly little is known about their different TGF-β responsiveness and chemotactic propensities. This is particularly important as polarized effector T cells are known to play a central role in the progression of diseases with striking polarized Th1 versus Th2 pathological features. Defective responses to biological immune modifiers like TGF-β and IL-10 have also been implicated in the pathogenesis of some of these diseases, including Crohn's disease, Wegener's granulomatosis, giant cell arteritis, multiple sclerosis, rheumatoid arthritis (RA) and insulin-dependent diabetes mellitus [36–39]. The pleiotropic effects of TGF-β are well documented in recent studies on fibroblasts cultured from RA and osteoarthritis (OA) synovium. Expression of IL-1β, tumour necrosis factor-α (TNF-α), IL-8, macrophage inflammatory protein-1α (MIP-1α) and metalloproteinase-1 (MMP-1) mRNA was found to be increased in RA/OA by TGF-β1 treatment. In addition, DNA-binding activities of the transcription factors nuclear factor-κβ (NF-κβ) and activator protein-1 (AP-1) were shown to increase by TGF-β1 as well . TGF-β has also been shown to induce accumulation of neutrophils and exacerbate arthritic response in an animal model of RA . Its pathognomonic role at the inflammatory site has also been suggested by TGF-β's ability to induce vascular endothelial growth factor expression, consequently contributing directly to angiogenesis in the inflamed tissue . In RA, MMPs are thought to play an important pathogenic role in joint destruction. In this context, TGF-β is known to regulate their production and activity . Another common denominator for these diseases is a Th1-like cytokine expression pattern. Thus, as TGF-β seems to be a powerful negative modulator of the primary response and differentiation of Th1 T cells, it is conceivable that its anti-inflammatory effects are at their height during the early inflammatory phase of these diseases. However, at later stages, its trophic stromal effects become more apparent through tissue-remodelling mechanisms.
Not only the microenvironment but also the differential stage of the inflammatory cell is crucial for the immunological response. This has particularly become apparent through different TGF-β receptor expression pattern of T cells. There are three major TGF-β receptors: TGF-β RI, RII and RIII. TGF-β RII is the only one of these that can both bind free TGF-β and has an intracellular domain . Cottrez and Groux  recently demonstrated that naïve and resting Th1/Th2 cells constitutively express the TGF-βII receptor and are susceptible to TGF-β, whereas activated/memory T cells downregulate this receptor, losing their TGF-β responsiveness. In addition, it was demonstrated that IL-10 upregulates the expression of the TGF-β RII receptor and therefore activated/memory T cells could regain their TGF-β susceptibility in the presence of IL-10 . Cellular senescence also affects the outcome of TGF-β immunomodulation. TGF-β-responsive RA/OA synovial fibroblasts lose their pro-inflammatory responses to TGF-β1 after the tenth to fifteenth passages . Together, these studies suggest that the modulatory effect of TGF-β at the inflammatory site depends on the maturational and differential stage of the responding cell.
Transforming growth factor-β signalling at the inflammatory site
It has been speculated that variable intracellular signalling events could attribute to the many different functional effects of TGF-β. The effect of TGF-β is mediated through the TGF-β type I and II receptors that have serine/threonine kinase domains. Binding of the ligand causes the assembly of a receptor complex that phosphorylates proteins of the Smad family. The phosphorylated Smad2 or -3 proteins are released from the anchoring protein, SARA, and subsequently bind the co-Smad, which enables them to travel as a complex to the nucleus . The final signalling outcome depends on which partner proteins interact with the Smad complex in the nucleus and on whether these act as transcriptional, costimulatory or coinhibitory factors. The available partner proteins are specific to a particular cell type and particular conditions. Thus, the conditioning at the immunological synapse will predetermine which transcriptional factors will bind to the Smad complex and dictate its outcome within the cell. The pleiotropic effects of TGF-β may also be explained by the possibility that mitogen-activated protein kinase pathways could influence its signalling intensity and pattern. Thus, it has been shown that growth factors can downregulate TGF-β receptor expression through their Ras-MEK-extracellular signal-regulated kinase pathway . In addition, it has been demonstrated that various cytokines may also interfere with TGF-β signalling; for example, the cytokine pathways MAP kinase kinase 4 (MKK4)/Jun N-terminal kinase (JNK) and MKK3/p38 enhance the activity of Jun and activating transcription factor 2 (ATF2) transcription factors. Activated Jun could then, through interaction with AP-1-binding site, cooperate with Smads through direct physical contact in the nucleus . Finally, a recent study indicates a synergistic activation of AP-1 by TGF-β and lipopolysaccharide (LPS), possibly causing combined effects of three signalling processes: LPS/JNK/AP-1, TGF-β/Smads and the complex of AP-1 with Smads . Reciprocally, cytokine signalling could also be modified by TGF-β. These complex interactions of signalling during T-cell activation could then direct its final response pattern, given the immediate microenvironmental milieu at the inflammatory site.
Chemotaxis and transforming growth factor-β
Chemokine receptors as well as adhesion molecules play an important role in the recruitment and selective transfer of T cells to secondary lymphoid organs or sites of inflammation. It has become apparent that as T cells develop into Th1 or Th2 effector cells, they may also acquire different migratory capacities through expression of specific chemokine receptors as well as adhesion molecules. This provides the immune system with the tools to respond in the most specific and effective way when needed. Recently, Sallusto et al.  stimulated cord blood T cells under Th1- or Th2-polarizing conditions, with and without the presence of TGF-β1. They demonstrated that polarized T cells expressed distinctive chemokine receptor profiles. The chemokine receptors CCR3 and CCR4 were mainly expressed by the Th2 subset, while CCR5 and CXCR3 were preferentially found on the Th0 and Th1 subsets. While TGF-β1 prevented the development of the Th2 subset and the CCR3 expression, it induced the expression of CCR4. TGF-β1 also induced CCR7 expression, which enables the seminaïve T cells to migrate to secondary organs where the cognate chemokine MIP-3β is expressed. The authors suggest that the main effect of TGF-β1 is to prevent the T cells from reaching maturity and inducing the expression of CCR7, thereby ‘sending’ the T cells off to secondary lymph nodes to complete the maturation/education . This opinion is supported by the work of Franitza et al.  who demonstrated that pretreatment with TGF-β1 caused an increased homing of naïve human T cells towards the spleen of irradiated NOD/SCID mice. TGF-β1 induced the expression of CXCR4 by naïve T cells and thereby induced their migration to the spleen, which is rich in CXCR4 ligand stromal cell-derived factor-1α (SDF-1α) . Franitza and colleagues  also observed that TGF-β1 had the same effect in vitro, in a three-dimensional gel system. Pretreatment of naïve human T cells with TGF-β1 induced the expression of CXCR4, thereby causing a significantly elevated chemotactic response towards SDF-1α. However, this effect was dose and time dependent. Interestingly, TGF-β1 did not alter the response towards MIP-3β, which is a CCR7-specific T-cell chemoattractant, which is in disagreement with the findings of Sallusto et al. . Also, naïve and memory T cells responded differently to TGF-β1 pretreatment, as only the naïve T cells responded to TGF-β1, while memory T cells were unaffected. As these cell populations expressed similar levels of TGF-β receptors, the probable explanation lies in a differential expression of a downstream signalling molecule .
Although the selective migration of T cells may be of great importance, the active retention of T cells may be equally significant. TGF-β1 may play an important role in the sustained inflammatory process observed in several autoimmune diseases by contributing to active detainment of T cells within the inflamed tissue.
The chronic inflammation in RA synovium may partly be sustained by the influence of TGF-β1. It induces the expression of the chemokine receptor CXCR4 by activated synovial T cells, which is normally expressed by unprimed T cells and lost after repeated antigenic stimulations [48, 50]. The CXCR4 ligand, SDF-1α, is expressed by the synovial endothelial cells. The activated T cells are therefore retained within the synovium. Furthermore, under normal circumstances, the highly differentiated T cells would undergo activation-induced cell death, but TGF-β1 prevents this, consequently sustaining a state of chronic inflammation .
TGF-β1 has been shown to modulate chemokine and chemokine receptor expression both in peripheral systems and within the central nervous system. It upregulates CXCR3 and CXCR4 on human natural killer cells . Similarly, it induces CXCR1 expression on rat microglial cells both at the mRNA and at the protein level . However, the function of its receptiveness is inhibited in the TGF-β1-pretreated cells. In addition, TGF-β1 has been shown to enhance astrocyte expression of CCR1 and chemotaxis to its ligand MIP-1α.
The role of TGF-β has been addressed in allergic inflammation. In combination with IL-13, eotaxin expression was significantly increased in fibroblasts cultured from asthmatic patients, thereby promoting the influx of eosinophils during an immune response . In addition, IL-13 has been shown to induce the expression of monocyte chemoattractant protein (MCP)-1, -2, -3 and -5, MIP-1α and eotaxin in mice. Furthermore, in CCR2–/–mice (the receptor for MCP-1, -2, -3, -4 and -5), IL-13-induced pulmonary inflammation was markedly decreased. This coincided with the downregulation of bioactive TGF-β1 in the bronchial lavage fluid , suggesting a significant role of IL-13/TGF-β in the induction of CC chemokines and the MCP-CCR2 signalling events. However, TGF-β1 was not able to induce the CC chemokine MCP-1 or RANTES on normal, OA- or RA-cultured synovial fibroblasts . Other studies on human primary epithelial cultures from asthmatic patients have shown that IL-4, IL-13 and allergen enhanced the release of TGF-α, but TGF-α is the ligand for epidermal growth factor receptor that stimulates fibroblast proliferation and goblet cell differentiation. These observations promote a link between TGF-α and airway remodelling in asthma. In addition, physical factors such as stress within the human type II alveolar epithelial cells have been found to induce IL-8 and TGF-β release from those cells . Together, the above studies place TGF-β as a pro-inflammatory modulator in the pathogenesis of the characteristic Th2–immunopathological pathway of asthma. This observation is in striking contrast to its presumptive negative immunomodulating role in diseases with a Th1-like cytokine pattern as described before.
To address the issue of redundancy and specificity of chemokines and their receptors in lymphocyte biology, Rabin et al.  evaluated the expression pattern of the CC chemokine receptors CCR1, -2, -3 and -5 and CXCR3 and CXCR4 on naïve or memory CD4+ and CD8+ human T cells. T-Cell activation through the T-cell receptor (TCR) induced the most significant expression of CXCR3 including on naïve T cells, whereas other CC chemokine receptor upregulation was limited to memory subsets. The authors concluded that the expression of individual chemokine receptors and their ligand function depended upon the T-cell stage of activation/differentiation . Such response pattern would reflect on T-cell subset-specific and activation state-specific response patterns at the immunological synapse. A link between TCR signalling and chemokine signalling has also been observed. Stimulation of T cells has been associated with increased chemotaxis towards MIP-1α, MIP-1β and interferon-γ (IFN-γ)-inducible protein-10 . Also, stimulation of naïve T cells inhibited their migration towards RANTES and MCP-1 [61, 62]. In addition, anti-CD3-induced activation of T cells inhibited their response to vasoactive intestinal peptide .
Recently, it has been demonstrated that anti-CD3- induced stimulation of T cells inhibited their migration towards SDF-1α. This was associated with the downregulation of CXCR4 as well as TCR signalling. Furthermore, preincubation of T cells with SDF-1α resulted in decreased phosphorylation of ZAP-70, SLP-76 and pp36 . The above data not only suggest that an important interaction between chemokine and TCR signalling exists but also that the differentiation and the activation state of T-cell response determine their response to chemokines.
Site-specific T-cell invasion
The best-studied trafficking molecules on T cells are the mucosal homing integrin α4β7 and the cutaneous lymphocyte-associated antigen (CLA). Their ligands are MAdCAM-1 (mucosal address in cell adhesion molecule-1), found on gastrointestinal endothelial cells, and E-selectin, found on skin venule endothelium. α4 (CD49d) shares β-chain with αE and is widely distributed on T cells and B cells. The principal mucosal homing receptor α4β7 consists of α4 (CD49d) and the β-chain, β7, which is shared with the mucosal adhesion/homing molecule αEβ7. One of the foremost functions of α4β7 is to mediate adhesion to MAdCAM-1, one of the key elements in lymphocyte homing to Peyer's patches .
The integrin αΕβ7 (CD103) was discovered in the late 1980s . In healthy humans and mice, its expression is almost exclusively limited to T cells and DCs within the mucosal system, especially on intraepithelial lymphocytes (IELs) in the gut . We have also observed its limited expression on naïve SP thymic CD8+ T cells . However, during an inflammatory response, its expression is increased at other sites. This has been documented in psoriasis, RA and interstitial lung diseases [68–70]. In addition, its expression is increased on CD8+ T cells in patients with Sjögren's syndrome . The only known ligand for αΕβ7 is E-cadherin that is expressed on the basolateral site of epithelial cells and adherence junctions . The function of αΕβ7 on T cells serves to retain them at the inflammatory site. This has been suggested in mice, both in inflammatory models and in αE–/– mice [39, 73]. Expression of αΕβ7 has been demonstrated by Northern blot analysis in various human tissues including gut, lung, thymus and spleen. The expressional regulation of αΕβ7 is under intense research. TGF-β is one of the best-studied enhancers of its expression. Recently, it has been shown that TGF-β induces αΕ mRNA expression at the level of transcription in human and mouse lymphoma cell line . IELs express high levels of αΕβ7 . It has been demonstrated that IELs migrating into the intestinal epithelium express much higher levels of αΕβ7 when compared with α4β7 . In addition, within the epithelial compartment, αΕβ7 expression was further enhanced through a TGF-β-dependent pathway [13, 66, 76]. The role of TGF-β in transplant rejection has also been evaluated. During the inflammatory process of tubulitis, there was a clear association between the expression of αΕβ7 on infiltrating CD8+ T cells and the inflammation severity. This coincided with the increased amount of TGF-β at the inflammatory site . A similar connection between TGF-β and αΕβ7 expression on infiltrating CD8+ T cells has been shown within inflamed salivary gland tissue from patients with Sjögren's syndrome . It is currently unclear what factors are involved in skewing αΕβ7 expression and/or sensitivity towards TGF-β on CD8+ T cells.
Spatial compartmentalization of T cells is thought to be essential for their immune surveillance and participation in the pathogenesis of inflammatory skin diseases. Not only is this thought to be guided by their different expressional and response pattern to cytokines and chemokines but also by distinct sets of adhesion molecules. Glycoproteins bearing the Sialyl–Lewis X moiety function as E-selectin ligands, including CLA, are thought to be involved in tissue-specific localization of cutaneous T cells . Very little is known about the epidermal localization of T cells. Some results suggest that intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 interactions play a vital role during this process, whereas their participation has been doubtful in others. In addition, a recently identified glycoprotein lymphocyte endothelial–epithelial cell adhesion molecule may be important in epidermal T-cell localization . The expression of αΕβ7 has been implicated in T-cell epidermal localization. Indeed, its expression has been found to be increased in common inflammatory skin disorders such as lichen planus and atopic dermatitis [80, 81]. However, it is interesting to note that our own and others' results indicate that only a minority of CD8+ peripheral T cells coexpress CLA . Within psoriatic skin lesions, TGF-β has been found to be focally upregulated, and TGF-β1-responsive elements have been identified in the 5′ proximal promoter regions of both the β7- and the αE-encoding gene regions [69, 83]. Furthermore, TGF-β1 might downregulate α4, thus increasing the availability of the β7 integrin to form a functional heterodimer with αE on infiltrating lymphocytes .
Tolerance and tolerance induction particularly in mucosal immunity have been shown in several different animal models to be mediated in part by TGF-β. How TGF-β is mediating its immunomodulating effect in various autoimmune models is unclear, and human trials directed against various autoimmune diseases using tolerance induction as a therapeutic option have been disappointing. One possible explanation for this, which resides on our hypothesis presented here in this review, is that TGF-β-induced modulation is driven by the functional phenotype and the maturational stage of the responding cell. Therefore, its overall effect is a composite of its effects on several different cellular functions that each have a varying and possible opposing impacts on any given immune response. An approach to analyse the specific effect of TGF-β on T cells has been to develop murine models where the TGF-β type II receptor is made nonfunctional in T cells. This has been performed in several studies recently reviewed by Gorelik and Flavell . Together, these studies have demonstrated that TGF-β is vital for T-cell homoeostasis, as the altered cells become ‘spontaneously’ activated and become effector T cells with resulting multifocal inflammation. Another example of lost tolerance, where regulation of TGF-β is disturbed, is in IL-10-deficient mice, which develop inflammatory bowel disease (IBD) . As IL-10 is known to upregulate the TGF-β receptor, it is possible that insufficient TGF-β receptors are expressed, leading to lost tolerance towards nonpathogenic antigens. The same loss of tolerance appears to be present in human IBD. T cells from the mucosa of IBD patients have been shown to be incapable of receiving TGF-β signals, as they overexpress Smad7, an inhibitor of TGF-β intracellular signal . As TGF-β deficiency appears to be most harmful in locations with high antigenic load (lungs and colon), these studies indicate that TGF-β plays a key role in inducing tolerance towards nonpathogenic antigens.
T-Cell phenotype and transforming growth factor-β responsiveness
TGF-β affects T-cell proliferation via its effects on IL-2R expression and signalling function [4, 87, 88]. TGF-β also affects the differentiation [89–91] and apoptosis of T cells . Finally, antigen-presenting cell (APC) function is also modulated by TGF-β[93–96]. These considerations make it evident that further analyses of TGF-β function are best carried out by isolating the target cell. When studying TGF-βin vitro, it is of main importance to use target cells with a defined phenotype (CD4+ versus CD8+ Th1 versus Th2) and a defined maturational stage (naïve cell versus memory cell) stimulated under conditions that allow definition of indirect effects of TGF-β (i.e. effects on APCs) versus direct effects of TGF-β on T cells  (Table 1). The effects of TGF-β1 on murine naïve CD4+ T cells undergoing primary and secondary stimulation with antigen plus APCs or anti-CD3/anti-CD28, under neutral Th1 or Th2 conditions, have been evaluated . Several new insights concerning TGF-β1 function emerged from this analysis. The first is that TGF-β1 is profoundly inhibitory of the differentiation and expansion of both Th1 and Th2 T cells in mice, if it is present during primary stimulation. The second is that the presence of TGF-β1 during priming of T cells results in T cells with severely impaired secondary immune responses, even if TGF-β1 is not present during secondary stimulation; thus, TGF-β1 appears to have a long-lasting or ‘imprinting’ effect. The final observation is that while TGF-β1 had a direct suppressive effect on memory Th1 T-cell cytokine production via its ability to downregulate IL-12Rβ2 chain expression, it had no effect on or even enhanced memory Th2 T-cell cytokine production. These results suggest that the counter-regulatory effects of TGF-β1 on established Th1 and Th2 T-cell-mediated autoimmune states is likely to be very different.
Table 1. The effect of transforming growth factor-β (TGF-β) on naïve versus differentiated T cells
| ||↑α4β7||↑αEβ7|| ||↑CCR4|
|Differentiation/effector function||↓Th1||↓CTL function||↓Th1||↑→Th2|
| ||↓T-bet|| ||↓IL-12Rβ2||↑→IL-4|
| ||↓IFN-γ|| ||↓IFN-γ|| |
| ||↓Th2|| || || |
| ||↓GATA-3|| || || |
| ||↓IL-4|| || || |
| ||↑Th3/Tr|| || || |
| ||↑Foxp3?||↑CD8+ Tr|| || |
| ||↑CD25|| || || |
| ||↑CTLA-4|| || || |
| ||↑TGF-β|| || || |
| ||↑IL-10|| || || |
|Expansion||↓Proliferation||↓Proliferation||Without IL-2|| |
| ||↓IL-2||↓IL-2||↓Proliferation |
| || || ||With IL-2 |
Transforming growth factor-β and the T-cell receptor-signalling intensity
The effect of cellular activation and its microenvironment has been shown for T cells activated in mesenteric lymph nodes. After recirculation, such cells are preferentially found within the tissues that are drained by them, such as Peyer's patches and the lamina propria of the gut . Interestingly, their proliferation was driven by site-specific IL-4 and TGF-β secretion . Therefore, not only was their fate being predetermined by their original site of stimulation but also their secondary responsiveness was being predetermined by locally produced cytokines. In this context, it has similarly been demonstrated that TGF-β can inhibit the regulatory process of activation-induced cell death of CD4+ T cells. However, TGF-β1 can downregulate the expression of Fas ligand through c-myc downregulation, both of which are important for the process of activation-induced cell death. However, other studies have not been able to demonstrate such an effect. It has also been suggested that not only the signalling intensity but also the level of costimulation will direct the responsiveness of T cells to TGF-β. Thus, while TGF-β inhibited the proliferation of murine CD4+ when they were only stimulated through the TCR, it induced their proliferation in the presence of anti-CD28, which was independent of IL-2 . In addition, in these studies the presence of anti-CD28 TGF-β1 prevented apoptosis of naïve CD4+ murine T cells . The importance of the TCR-signalling intensity has been demonstrated in studies using an altered peptide ligand model, where nonproductive priming of naïve CD4+ murine T cells resulted in TGF-β1-producing T cells during secondary stimulation . In addition, several studies have demonstrated that the avidity and affinity of the major histocompatibility complex (MHC)–TCR interaction directs the Th differentiation into either Th1 or Th2. Although results vary, probably more studies support the theory that high strength of stimulation favours Th1 development, whereas low strength favours Th2 development . The duration of T-cell stimulation has also been shown to direct the T-cell response. Long-term cultures particularly favour the development of Th1 cells , thus suggesting that the fate of naïve peripheral CD4+ T cells is dependent upon the affinity and density of the TCR–ligand interaction and their on/off duration. Our more recent studies on human peripheral blood lymphocytes have added some new insights into this mechanism . In these studies, we have observed that the driving force behind TGF-β seems to be maintaining the homoeostasis of controlled immune response. Thus, its most powerful negative inhibitory function seems to be provoked during enhanced T-cell response after high-dose stimulation that is driven directly through the TCR. Furthermore, our preliminary data suggest that preactivated peripheral blood lymphocytes are more susceptible to such TGF-β1-mediated inhibitory mechanisms. Whether that is because of increased expression of the TGF-β1 receptor or increased transcription of Smads involved in intracellular TGF-β1 signalling remains to be investigated. Recent evidence has emerged suggesting that certain subpopulation of T regulatory cells, CD4+/CD25+, might be one of the key players in tolerance induction. The strength of TCR signalling also seems to be an important modulating factor for these cells. Therefore, their immunomodulating effector function may be more pronounced on CD4+/CD25+ T cells after weak TCR stimulation compared with strong TCR stimulation . In this context, it is interesting to note that ligation of the TCR complex with anti-CD3 induced phosphorylation of Smad2 in Jurkat T-cell line through an MEK-dependent intracellular pathway , thereby directly connecting the TGF-β and the TCR-signalling pathway within the cell, suggesting that Smad2 may function both in TGF-β- and in TCR-mediated signal transduction in T cells. It is of interest to evaluate whether the response to TGF-β is confined to CD25+ naïve peripheral blood T cells. It is also of crucial importance to further delineate which cell populations and under which conditions have the highest sensitivity towards the immunoregulatory role of TGF-β1. Such line of investigation has important implications for the understanding of immune regulation in normal, autoimmune and allergic states. We believe that such studies could provide new insights into possible therapeutic targets in various autoimmune diseases, where T cells have been found to play a significant immunopathogenic role.
This work was supported by the Landspitali, University Hospital science fund and the Research fund of the University of Iceland. We would also like to thank Ingileif Jónsdóttir for her helpful comments on the manuscript.