Role of TGF-β in the Induction of Foxp3 Expression and T Regulatory Cell Function

Introduction  A number of studies have suggested that transforming growth factor beta (TGF-β) plays a critical role in immune suppression mediated by Foxp3⁺ regulatory T cells. TGF-β in concert with interleukin 2 is a potent induction factor for the differentiation of Foxp3⁺ Treg from naive precursors. Polyclonal TGF-β-induced Treg (iTreg) are capable of preventing the autoimmune syndrome that develops in scurfy mice that lack Foxp3⁺ Treg. Antigen-specific iTreg can be used to both prevent and treat autoimmune gastritis that is induced by transfer of naive or primed autoantigen-specific T cells. TGF-β complexed with latency-associated peptide is expressed on the surface of activated thymus-derived Treg. Coculture of activated Treg with naive responder T cells results in the de novo generation of fully functional Foxp3⁺ T cells in a contact-dependent and TGF-β-dependent manner. Conclusions and Speculations  Generation of functional Foxp3⁺ T cells via this pathway may represent a mechanism by which Treg maintain tolerance and expand their repertoire.     

T Cell Receptor-Regulated TGF-β Type I Receptor Expression Determines T Cell Quiescence and Activation

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TGF-β signaling in fibrosis

Transforming growth factor β (TGF-β) is a central mediator of fibrogenesis. TGF-β is upregulated and activated in fibrotic diseases and modulates fibroblast phenotype and function, inducing myofibroblast transdifferentiation while promoting matrix preservation. Studies in a wide range of experimental models have demonstrated the involvement of the canonical activin receptor-like kinase 5/Smad3 pathway in fibrosis. Smad-independent pathways may regulate Smad activation and, under certain conditions, may directly transduce fibrogenic signals. The profibrotic actions of TGF-β are mediated, at least in part, through induction of its downstream effector, connective tissue growth factor. In light of its essential role in the pathogenesis of fibrosis, TGF-β has emerged as an attractive therapeutic target. However, the pleiotropic and multifunctional effects of TGF-β and its role in tissue homeostasis, immunity and cell proliferation raise concerns regarding potential side effects that may be caused by TGF-β blockade. This minireview summarizes the role of TGF-β signaling pathways in the fibrotic response.     

TGF-β signaling in fibrosis

Transforming growth factor β (TGF-β) is a central mediator of fibrogenesis. TGF-β is upregulated and activated in fibrotic diseases and modulates fibroblast phenotype and function, inducing myofibroblast transdifferentiation while promoting matrix preservation. Studies in a wide range of experimental models have demonstrated the involvement of the canonical activin receptor-like kinase 5/Smad3 pathway in fibrosis. Smad-independent pathways may regulate Smad activation and, under certain conditions, may directly transduce fibrogenic signals. The profibrotic actions of TGF-β are mediated, at least in part, through induction of its downstream effector, connective tissue growth factor. In light of its essential role in the pathogenesis of fibrosis, TGF-β has emerged as an attractive therapeutic target. However, the pleiotropic and multifunctional effects of TGF-β and its role in tissue homeostasis, immunity and cell proliferation raise concerns regarding potential side effects that may be caused by TGF-β blockade. This minireview summarizes the role of TGF-β signaling pathways in the fibrotic response.     

TGF-β in transplantation tolerance

TGF-β is a cytokine required for the induction and maintenance of transplantation tolerance in animal models. TGF-β mediates anti-inflammatory effects by acting on many immune cell-types. Central for transplantation tolerance is the role for TGF-β in the induction of Foxp3 and regulatory capacity in CD4(+) T cells. Recently, however, the general anti-inflammatory role of TGF-β in CD4(+) T cell polarization was questioned by the discovery that, in the presence of inflammatory cytokines such as IL-6 or IL-1, TGF-β drives the differentiation of Th17 cells associated with transplant rejection. A better understanding of the factors determining TGF-β production and activation, Foxp3 induction and Treg stability is vital for the development of tolerogenic strategies in transplantation.     

Ubiquitin-like protein MNSFβ negatively regulates T Cell function and survival

Monoclonal non-specific suppressor factor β (MNSFβ) is a ubiquitously expressed member of the ubiquitin-like family that is involved in various biological functions. Previous studies have demonstrated that MNSFβ covalently binds to intracellular pro-apoptotic protein Bcl-G and regulates apoptosis in macrophages. In this study, we demonstrate that MNSFβ negatively regulates T cell function. In murine T-helper type 2 clone, D10.G4.1 (D10) cells transfected with MNSFβ cDNA, CD3/CD28-induced ERK1/2 phosphorylation leading to IL-4 production was significantly inhibited. The formation of MNSFβ-Bcl-G complex was induced by the CD3/CD28 stimulation. Co-transfection with MNSFβ and Bcl-G greatly enhanced CD3/CD28-induced apoptosis in D10 cells. Similarly, co-over-expression of MNSFβ and Bcl-G caused a marked enhancement of apoptosis in purified splenic T cells. Interestingly, this MNSFβ adduct was also induced in T cells derived from DO11.10 mice stimulated with antigen. Collectively, CD3/CD28-inducible MNSFβ-Bcl-G complex may be involved in the regulation of T cell function and survival.     

Cross-Regulation of T Regulatory—Cell Response after Coxsackievirus B3 Infection by NKT and γδ T Cells in the Mouse

Coxsackievirus B3 (CVB3) variants H3 and H310A1 differ by a single nonconserved amino acid in the VP2 capsid region. C57Bl/6 mice infected with the H3 virus develop myocarditis correlating with activation of T cells expressing the Vγ4 T cell receptor chain. Infecting mice with H310A1 activates natural killer T (NKT; mCD1d-tetramer⁺ TCRβ⁺) cells, but not Vγ4 T cells, and fails to induce myocarditis. H310A1 infection preferentially activates M2 alternatively activated macrophage and CD4⁺FoxP3 (T regulatory) cells, whereas CD4⁺Th1 (IFN-γ⁺) cells are suppressed. By contrast, H3 virus infection activates M1 proinflammatory and CD4⁺Th1 cells, but not T regulatory cells. The M1 macrophage show significantly increased CD1d expression compared to M2 macrophage. The ability of NKT cells to suppress myocarditis was shown by adoptive transfer of purified NKT cells into H3-infected NKT knockout (Jα18 knockout) mice, which inhibited cardiac inflammation and increased T regulatory cell response. Cardiac virus titers were equivalent in all mouse strains indicating that neither Vγ4 nor NKT cells participate in control of virus infection. These data show that NKT and Vγ4 cells cross-regulate T regulatory cell responses during CVB3 infections and are the primary factor determining viral pathogenesis in this mouse model.Enteroviruses and adenoviruses cause approximately 80% of clinical viral myocarditis in all age groups.¹ Cardiac injury results from direct viral injury to infected myocytes and also from host immune responses triggered by the infection.² Host responses include: i) induction of proinflammatory cytokines [IL-6, IL-1β, and tumor necrosis factor-α (TNF-α)] that suppress myocardial cell contractility³; ii) lysis of infected cardiocytes⁴; and iii) humoral or cellular autoimmunity to heart antigens, leading to cardiocyte death or dysfunction.^5–7 T-cell depletion of mice infected with coxsackievirus B3 (CVB3) dramatically reduces animal mortality and cardiac inflammation,⁸ and heart-specific, autoimmune CD8⁺ T cells isolated from CVB3-infected mice⁹ transfer myocarditis into uninfected recipients. Furthermore, immunizing mice with cardiac myosin in adjuvant causes cardiac inflammation closely resembling the virus-induced disease.^7,10–12 Several studies demonstrate that induction of autoimmunity in myocarditis corresponds to a decrease in T regulatory cells,^13,14 and T regulatory 1 (Tr1) cells making IL-10 are the probable suppressive effectors causing myocarditis resistance in both myosin- and CVB3-induced disease.^12,15,16 Recently, studies have shown that γδ T cells activated during pathological CVB3 infections are primarily responsible for preventing T regulatory cell responses and directly kill differentiated CD4⁺CD25⁺FoxP3⁺ T regulatory cells through Fas-dependent mechanisms.^2,17Not all CVB3 variants cause myocarditis. Two CVB3 variants, H3 and H310A1, have been cloned and characterized. The H310A1 virus was isolated from the parental H3 virus using a monoclonal antibody to the viral receptor and has a single nonconserved mutation in the VP2 capsid protein in a puff region known for decay accelerating factor (DAF) binding.¹⁸ Unlike the highly myocarditic H3 virus, the H310A1 virus is amyocarditic and preferentially activates T regulatory cells¹⁶ due to an inability to stimulate γδ T cells during H310A1 virus infections.¹⁹ As shown here, although the γδ T cell response is defective in H310A1-infected mice, substantial numbers of natural killer T (NKT) cells are present in the hearts of H310A1-infected, but not H3-infected, animals. This raises the question whether NKT cells promote the generation of T regulatory cells in the myocarditis-resistant animals. This idea is supported by recent studies in which CVB3-infected mice given the NKT ligand, α-galactosylceramide (α-GalCer), develop significantly less myocarditis than untreated animals.²⁰ This study found alterations in cytokine environment in the α-GalCer–treated mice but did not investigate the role of T regulatory cells in causing the anti-inflammatory cytokine response.Although somewhat controversial, various reports indicate that NKT cells suppress autoimmunity or promote tolerance by their effect on T regulatory cell response. Interaction of antigen-presenting cells and NKT cells through CD1d during oral tolerance to nickel results in secretion of IL-4 and IL-10, and activation of T regulatory cells.^21–23 Similarly, systemic tolerance could not be established in a mouse model of anterior chamber–associated immune deviation in CD1d knockout (KO) mice unless the animals were transfused with NKT cells and CD1d⁺ antigen-presenting cells.²⁴ Other studies show that αGalCer, a well-known and specific NKT CD1d-restricted ligand, increases T regulatory cell numbers in vivo²⁵ and can suppress autoimmune diabetes in NOD mice.^26–28 Cytokines, such as transforming growth factor-β (TGF-β) and IL-10, can be produced by NKT cells,^29,30 which could affect dendritic cell cytokine (IL-10) and accessory molecule (CD40, CD80, and/or CD86) expression,^31–33 leading to T regulatory cell responses.^27,34 Here, studies show that NKT cells activated during H310A1 infection cause the increased T regulatory cell response seen in this model. These experiments show that the nature of the innate immune response following enterovirus infection is crucial in determining autoimmunity induction.     

TGF-β superfamily co-receptors in cancer

Transforming growth factor-β (TGF-β) superfamily signaling via their cognate receptors is frequently modified by TGF-β superfamily co-receptors. Signaling through SMAD-mediated pathways may be enhanced or depressed depending on the specific co-receptor and cell context. This dynamic effect on signaling is further modified by the release of many of the co-receptors from the membrane to generate soluble forms that are often antagonistic to the membrane-bound receptors. The co-receptors discussed here include TβRIII (betaglycan), endoglin, BAMBI, CD109, SCUBE proteins, neuropilins, Cripto-1, MuSK, and RGMs. Dysregulation of these co-receptors can lead to altered TGF-β superfamily signaling that contributes to the pathophysiology of many cancers through regulation of growth, metastatic potential, and the tumor microenvironment. Here we describe the role of several TGF-β superfamily co-receptors on TGF-β superfamily signaling and the impact on cellular and physiological functions with a particular focus on cancer, including a discussion on recent pharmacological advances and potential clinical applications targeting these co-receptors.     

TGF-β and BMP7 interactions in tumour progression and bone metastasis

The skeleton is the second most frequent site of metastasis. However, only a restricted number of solid cancers, especially those of the breast and prostate, are responsible for the majority of the bone metastases. Metastatic bone disease is a major cause of morbidity, characterised by severe pain and high incidence of skeletal and haematopoietic complications (fractures, spinal cord compression and bone marrow aplasia) requiring hospitalisation. Despite the frequency of skeletal metastases, the molecular mechanisms for their propensity to colonise bone are poorly understood and treatment options are often unsatisfactory. TGF-β and the signalling pathway it controls appears to play major roles in the pathogenesis of many carcinomas, both in their early stages, when TGF-β acts to arrest growth of many cell types, and later in cancer progression when it contributes, paradoxically, to the phenotype of tumour invasiveness. Here we discuss some novel insights of the TGF-β superfamily—including BMPs and their antagonists—in the formation of bone metastasis. Increasing evidence suggests that the TGF-β superfamily is involved in bone homing, tumour dormancy, and development of micrometastases into overt bone metastases. The established role of TGF-β/BMPs and their antagonists in epithelial plasticity during embryonic development closely resembles neoplastic processes at the primary site as well as in (bone) metastasis. For instance, the tumour-stroma interactions occurring in the tissue of cancer origin, including epithelium-to-mesenchyme transition (EMT), bear similarities with the role of bone matrix-derived TGF-β in skeletal metastasis formation.     

T Cell Clonal Dynamics Determined by High-Resolution TCR-β Sequencing in Recipients after Allogeneic Hematopoietic Cell Transplantation

Delayed reconstitution of the immune system is a long-recognized complication after allogeneic hematopoietic cell transplantation (HCT). Specifically, loss of T cell diversity has been thought to contribute to infectious complications, graft-versus-host disease (GVHD), and disease relapse. We performed serial high-resolution next-generation sequencing of T cell receptor (TCR)-β in 99 related or unrelated donor (57 unrelated, 42 related) allogeneic HCT recipients (55 with reduced-intensity conditioning, 44 with myeloablative conditioning) during the first 3 months after HCT using the immunoSEQ Assay. We measured T cell fraction, clonality (1- Peilou's evenness) and Daley-Smith richness from recipient samples at multiple time points. In agreement with previous studies, we found that although absolute T cell numbers recover relatively quickly after HCT, T cell repertoire diversity remains diminished. Restricted diversity was associated with conditioning intensity, use of antithymocyte globulin, and donor type. Increased number of expanded clones compared to donor T cell clones at day +30 was associated with the incidence of acute GVHD (hazard ratio [HR], 1.11; P = .00005). Even after exclusion of the 12 patients who developed acute GVHD before day +30, the association between acute GVHD and increased clonal expansion at day +30 remained (HR, 1.098; P = .041), indicating that increased clonal T cell expansion preceded the development of acute GVHD. Our results highlight T cell clonal expansion as a potential novel biomarker for acute GVHD that warrants further study.     

TGF-β and inflammatory blood markers in prediction of intraperitoneal adhesions

Purpose

Intraperitoneal adhesions (IA) develop as a consequence of the healing process in peritoneum injured during surgeries. IA might be formed after all types of surgical interventions regardless the surgical approach with a higher incidence in obese individuals. Here we determine the diagnostic power of TGF-β and blood inflammatory parameters in the prediction of IA in obese patients undergoing second surgical intervention.

TGF-β signalling in tumour associated macrophages

Tumour associated macrophages (TAM) represent an important component of tumour stroma. They develop under the influence of tumour microenvironment where transforming growth factor (TGF)β is frequently present. Activities of TAM regulated by TGFβ stimulate proliferation of tumour cells and lead to tumour immune escape. Despite high importance of TGFβ-induction of TAM activities till now our understanding of the mechanism of this induction is limited. We have previously developed a model of type 2 macrophages (M2) resembling certain properties of TAM. We established that in M2 TGFβRII is regulated on the level of subcellular sorting by glucocorticoids. Further studies revealed that in M2 with high levels of TGFβRII on the surface TGFβ activates not only its canonical Smad2/3-mediated signaling, but also Smad1/5-mediated signaling, what is rather typical for bone morphogenetic protein (BMP) stimulation. Complexity of macrophage populations, however, allows assumption that TGFβ signalling may function in different ways depending on the functional state of the cell. To understand the peculiarities of TGFβ signalling in human TAMs experimental systems using primary cells have to be developed and used together with the modern mathematical modelling approaches.     

Diverse roles of TGF-β receptor II in renal fibrosis and inflammation in vivo and in vitro

TGF-β1 binds receptor II (TβRII) to exert its biological activities but its functional importance in kidney diseases remains largely unclear. In the present study, we hypothesized that TβRII may function to initiate the downstream TGF-β signalling and determine the diverse role of TGF-β1 in kidney injury. The hypothesis was examined in a model of unilateral ureteral obstructive (UUO) nephropathy and in kidney fibroblasts and tubular epithelial cells in which the TβRII was deleted conditionally. We found that disruption of TβRII inhibited severe tubulointerstitial fibrosis in the UUO kidney, which was associated with the impairment of TGF-β/Smad3 signalling, but not with the ERK/p38 MAP kinase pathway. In contrast, deletion of TβRII enhanced NF-κB signalling and renal inflammation including up-regulation of Il-1β and Tnfα in the UUO kidney. Similarly, in vitro disruption of TβRII from kidney fibroblasts or tubular epithelial cells inhibited TGF-β1-induced Smad signalling and fibrosis but impaired the anti-inflammatory effect of TGF-β1 on IL-1β-stimulated NF-κB activation and pro-inflammatory cytokine expression. In conclusion, TβRII plays an important but diverse role in regulating renal fibrosis and inflammation. Impaired TGF-β/Smad3, but not the non-canonical TGF-β signalling pathway, may be a key mechanism by which disruption of TβRII protects against renal fibrosis. In addition, deletion of TβRII also enhances NF-κB signalling along with up-regulation of renal pro-inflammatory cytokines, which may be associated with the impairment of anti-inflammatory properties of TGF-β1.     

Thymopoiesis, Regulatory T Cells, and TCRVβ Expression in Thymoma With and Without Myasthenia Gravis, and Modulatory Effects of Steroid Therapy

We analyzed thymocyte and thymic regulatory T cell (CD4SPCD25⁺Foxp3⁺cells, Treg) development in thymoma with and without myasthenia gravis (MG, MG-thymoma, non-MG-thymoma) and in MG-associated non-neoplastic thymus (MG-NNT). An increased number of immature CD4⁺CD8⁻CD3⁻ thymocytes through the CD4⁺CD8⁺ to CD4⁺CD8⁻ transition and an abnormal T cell receptor Vβ (TCRVβ) development through the CD4⁺CD8⁺ to CD4⁻CD8⁺ transition were seen both in MG-and non-MG-thymomas. Terminal thymopoiesis, i.e., CD45RA⁺ cells within the CD4⁺CD8⁻CD3⁺ and CD8⁺CD4⁻CD3⁺ subsets, was skewed towards the CD4⁺ compartment in MG-thymoma and CD8⁺ compartment in non-MG-thymoma, but thymic export was increased only in the latter in keeping with the hypothesis that CD8⁺ lymphocytes may play a role in the initial stages of autosensitization and in disagreement with the relevance of an increased output of CD4⁺ T lymphocytes in paraneoplastic MG. Treg level in normal thymus and MG-NNT and both MG- and non-MG-thymoma was similar, and TCRVβ development in Treg cells was slightly altered in thymoma but irrespective of MG presence. Thus, the relevance of a defective Treg development in MG context remains to be established. Most alterations in thymopoiesis were corrected by therapeutic corticosteroid administration, and the effects of steroid administration may be mediated by thymic microenvironment.     

γδ T Cells in Host Defense and Epithelial Cell Biology

Recent studies have demonstrated increased numbers of γδ T cells in a variety of human infectious as well as noninfectious diseases. In some cases γδ T cells could be shown to destroy infected or transformed cells. Advances in the identification of ligands recognized by γδ T cells and the development of animal model systems to study these cellsin vivoshould overcome some of the major obstacles currently preventing a better understanding of γδ T cell function in immune responses. As we gain this knowledge it may become possible to design therapeutic strategies exploiting unique properties of γδ T cells to promote more effective immunity.     

Regional Variation in the Lamina Propria T Cell Receptor Vβ Repertoire in Normal Human Colon

Lamina propria (LP) T cell populations in the normal human colon contain oligoclonal expansions, but their distribution has not been well studied. We analyzed T cell receptor (TCR) β-chain (Vβ) variable region expression in CD4⁺and CD8⁺peripheral blood T cells and LP T cells from separated colonic segments in 13 subjects. CD4⁺and CD8⁺Vβ subset expansions were found in the LP of most individuals, and remarkable differences in CD4⁺and CD8⁺TCR repertoires were apparent between colon and blood as well as between colon segments within each individual. The presence of such T cell expansions in colon therefore cannot be used to infer immunopathology. In addition, CD8⁺Vβ expansions seen in peripheral blood T cells, which have been previously shown to be clonal in origin, were also often expanded in LP T cells of the same subject. These results suggest that LP CD8⁺T cell stimulation may contribute to CD8⁺peripheral blood T cell expansions.