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1.  The Hookworm Tissue Inhibitor of Metalloproteases (Ac-TMP-1) Modifies Dendritic Cell Function and Induces Generation of CD4 and CD8 Suppressor T Cells 
Hookworm infection is a major cause of disease burden for humans. Recent studies have described hookworm-related immunosuppression in endemic populations and animal models. A Tissue Inhibitor of Metalloproteases (Ac-TMP-1) has been identified as one of the most abundant proteins released by the adult parasite. We investigated the effect of recombinant Ac-TMP-1 on dendritic cell (DC) and T cell function. Splenic T cells from C57BL/6 mice injected with Ac-TMP-1 showed reduced proliferation to restimulation with anti CD3 or bystander antigens such as OVA. Incubation of bone marrow-derived DCs with Ac-TMP-1 decreased MHC Class I and, especially, Class II expression but increased CD86 and IL-10 expression. Co-incubation of splenic T cells with DCs pulsed with Ac-TMP-1 induced their differentiation into CD4+ and, particularly, CD8+ CD25+Foxp3+ T cells that expressed IL-10. These cells were able to suppress proliferation of naïve and activated CD4+ T cells by TGF-Β-dependent (CD4+ suppressors) or independent (CD8+ suppressors) mechanisms. Priming of DCs with non-hookworm antigens, such as OVA, did not result in the generation of suppressor T cells. These data indicate that Ac-TMP-1 initiates the development of a regulatory response through modifications in DC function and generation of suppressor T cells. This is the first report to propose a role of suppressor CD8+ T cells in gastrointestinal helminthic infections.
Author Summary
Chronic infections with helminths have been suggested to induce suppressor cells by a variety of mechanisms. The published immunological and epidemiological data on hookworm infection in humans and animal models suggest that these parasites are particularly successful in establishing chronicity and modulation. We have demonstrated that the recombinant form of a hookworm tissue inhibitor of metalloprotease (Ac-TMP-1) induces bone marrow-derived DCs to downregulate MHC Class I and II and release anti-inflammatory cytokines such as IL-10 and TGF-Β. More importantly, DCs pulsed with Ac-TMP-1 promote the development of regulatory CD4+ and, especially, CD8+ T cells from naïve T cells, which are able to suppress proliferation of naïve and activated splenic CD4+ T cells; this suppression is mediated by TGF-Β for CD4+ suppressor cells, but it is independent of the cytokine for CD8+ suppressor cells. These studies initiate the first steps to investigate the relevance and nature of suppressor T cells in hookworm infections and their mechanism of suppression.
PMCID: PMC2678263  PMID: 19468296
2.  Characterization of Protective Human CD4+CD25+ FOXP3+ Regulatory T Cells Generated with IL-2, TGF-β and Retinoic Acid 
PLoS ONE  2010;5(12):e15150.
Protective CD4+CD25+ regulatory T cells bearing the Forkhead Foxp3 transcription factor can now be divided into three subsets: Endogenous thymus-derived cells, those induced in the periphery, and another subset induced ex-vivo with pharmacological amounts of IL-2 and TGF-β. Unfortunately, endogenous CD4+CD25+ regulatory T cells are unstable and can be converted to effector cells by pro-inflammatory cytokines. Although protective Foxp3+CD4+CD25+ cells resistant to proinflammatory cytokines have been generated in mice, in humans this result has been elusive. Our objective, therefore, was to induce human naïve CD4+ cells to become stable, functional CD25+ Foxp3+ regulatory cells that were also resistant to the inhibitory effects of proinflammatory cytokines.
Methodology/Principal Findings
The addition of the vitamin A metabolite, all-trans retinoic acid (atRA) to human naïve CD4+ cells suboptimally activated with IL-2 and TGF-β enhanced and stabilized FOXP3 expression, and accelerated their maturation to protective regulatory T cells. AtRA, by itself, accelerated conversion of naïve to mature cells but did not induce FOXP3 or suppressive activity. The combination of atRA and TGF-β enabled CD4+CD45RA+ cells to express a phenotype and trafficking receptors similar to natural Tregs. AtRA/TGF-β-induced CD4+ regs were anergic and low producers of IL-2. They had potent in vitro suppressive activity and protected immunodeficient mice from a human-anti-mouse GVHD as well as expanded endogenous Tregs. However, treatment of endogenous Tregs with IL-1β and IL-6 decreased FOXP3 expression and diminished their protective effects in vivo while atRA-induced iTregs were resistant to these inhibitory effects.
We have developed a methodology that induces human CD4+ cells to rapidly become stable, fully functional suppressor cells that are also resistant to proinflammatory cytokines. This methodology offers a practical novel strategy to treat human autoimmune diseases and prevent allograft rejection without the use of agents that kill cells or interfere with signaling pathways.
PMCID: PMC3003689  PMID: 21179414
3.  Dual Roles of Immunoregulatory Cytokine TGF-β in the Pathogenesis of Autoimmunity-Mediated Organ Damage1 
Ample evidence suggests a role of TGF-β in preventing autoimmunity. Multiorgan inflammatory disease, spontaneous activation of self-reactive T cells, and autoantibody production are hallmarks of autoimmune diseases, such as lupus. These features are reminiscent of the immunopathology manifest in TGF-β1-deficient mice. In this study, we show that lupus-prone (New Zealand Black and White)F1 mice have reduced expression of TGF-β1 in lymphoid tissues, and TGF-β1 or TGF-β1-producing T cells suppress autoantibody production. In contrast, the expression of TGF-β1 protein and mRNA and TGF-β signaling proteins (TGF-β receptor type II and phosphorylated SMAD3) increases in the target organs, i.e., kidneys, of these mice as they age and develop progressive organ damage. In fact, the levels of TGF-β1 in kidney tissue and urine correlate with the extent of chronic lesions that represent local tissue fibrosis. In vivo TGF-β blockade by treatment of these mice with an anti-TGF-β Ab selectively inhibits chronic fibrotic lesions without affecting autoantibody production and the inflammatory component of tissue injury. Thus, TGF-β plays a dual, seemingly paradoxical, role in the development of organ damage in multiorgan autoimmune diseases. According to our working model, reduced TGF-β in immune cells predisposes to immune dysregulation and autoantibody production, which causes tissue inflammation that triggers the production of anti-inflammatory cytokines such as TGF-β in target organs to counter inflammation. Enhanced TGF-β in target organs, in turn, can lead to dysregulated tissue repair, progressive fibrogenesis, and eventual end-organ damage.
PMCID: PMC2291535  PMID: 18209088
4.  Nasal Anti-CD3 Antibody Ameliorates Lupus by Inducing an IL-10-Secreting CD4+CD25-LAP+ Regulatory T Cell and Is Associated with Down-Regulation of IL-17+CD4+ICOS+CXCR5+ Follicular Helper T Cells1 
Lupus is an Ab-mediated autoimmune disease. One of the potential contributors to the development of systemic lupus erythematosus is a defect in naturally occurring CD4+CD25+ regulatory T cells. Thus, the generation of inducible regulatory T cells that can control autoantibody responses is a potential avenue for the treatment of systemic lupus erythematosus. We have found that nasal administration of anti-CD3 mAb attenuated lupus development as well as arrested ongoing lupus in two strains of lupus-prone mice. Nasal anti-CD3 induced a CD4+CD25-latency-associated peptide (LAP)+ regulatory T cell that secreted high levels of IL-10 and suppressed disease in vivo via IL-10- and TFG-β-dependent mechanisms. Disease suppression also occurred following adoptive transfer of CD4+CD25-LAP+ regulatory T cells from nasal anti-CD3-treated animals to lupus-prone mice. Animals treated with nasal anti-CD3 had less glomerulonephritis and diminished levels of autoantibodies as measured by both ELISA and autoantigen microarrays. Nasal anti-CD3 affected the function of CD4+ICOS+CXCR5+ follicular helper T cells that are required for autoantibody production. CD4+ICOS+CXCR5+ follicular helper T cells express high levels of IL-17 and IL-21 and these cytokines were down-regulated by nasal anti-CD3. Our results demonstrate that nasal anti-CD3 induces CD4+CD25-LAP+ regulatory T cells that suppress lupus in mice and that it is associated with down-regulation of T cell help for autoantibody production.
PMCID: PMC2753458  PMID: 18941193
5.  Donor-specific CD8+Foxp3+ T cells protect skin allografts and facilitate induction of conventional CD4+Foxp3+ regulatory T cells 
CD4+ regulatory T cells play a critical role in tolerance induction in transplantation. CD8+ suppressor T cells have also been shown to control alloimmune responses in pre-clinical and clinical models. However, the exact nature of the CD8+ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen-specific CD8+Foxp3+ T cells can be induced and significantly expanded by stimulating naive CD8+ T cells with donor dendritic cells in the presence of IL-2, TGF-β1, and retinoic acid. These CD8+Foxp3+ T cells express enhanced levels of CTLA-4, CCR4 and CD103, inhibit the up-regulation of co-stimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor-specific and contact-depended manner. Importantly, upon adoptive transfer, the induced CD8+Foxp3+ T cells protect full MHC-mismatched skin allografts. In vivo, the CD8+Foxp3+ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4+Foxp3+ T cells and concurrently suppress effector T cell expansion. We conclude that donor-specific CD8+Foxp3+ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.
PMCID: PMC3429694  PMID: 22681667
CD8 suppressor cells; Transforming growth factor-β1; Cytotoxic T lymphocytes; CD8α+CD11c+ dendritic cells; CD4 regulatory T cells; Allogeneic skin transplantation; Graft rejection
6.  CD25+CD4+ Regulatory T Cells from the Peripheral Blood of Asymptomatic HIV-infected Individuals Regulate CD4+ and CD8+ HIV-specific T Cell Immune Responses In Vitro and Are Associated with Favorable Clinical Markers of Disease Status 
Human immunodeficiency virus (HIV) disease is associated with loss of CD4+ T cells, chronic immune activation, and progressive immune dysfunction. HIV-specific responses, particularly those of CD4+ T cells, become impaired early after infection, before the loss of responses directed against other antigens; the basis for this diminution has not been elucidated fully. The potential role of CD25+CD4+ regulatory T cells (T reg cells), previously shown to inhibit immune responses directed against numerous pathogens, as suppressors of HIV-specific T cell responses was investigated. In the majority of healthy HIV-infected individuals, CD25+CD4+ T cells significantly suppressed cellular proliferation and cytokine production by CD4+ and CD8+ T cells in response to HIV antigens/peptides in vitro; these effects were cell contact dependent and IL-10 and TGF-β independent. Individuals with strong HIV-specific CD25+ T reg cell function in vitro had significantly lower levels of plasma viremia and higher CD4+: CD8+ T cell ratios than did those individuals in whom this activity could not be detected. These in vitro data suggest that CD25+CD4+ T reg cells may contribute to the diminution of HIV-specific T cell immune responses in vivo in the early stages of HIV disease.
PMCID: PMC2211981  PMID: 15280419
cytokine; proliferation; human; suppression; FoxP3
7.  The Transcription Factor T-bet Regulates Mucosal T Cell Activation in Experimental Colitis and Crohn's Disease 
The Journal of Experimental Medicine  2002;195(9):1129-1143.
The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-γ/interleukin (IL)-4 and transforming growth factor (TGF)-β activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L+ CD4+ T cells exacerbated colitis in reconstituted SCID mice, T-bet–deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet–deficient CD62L− CD4+ T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-β signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-γ/IL-4 and TGF-β responses and the development of chronic intestinal inflammation in T cell–mediated colitis. Furthermore, TGF-β was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-β and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell–mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.
PMCID: PMC2193714  PMID: 11994418
T-bet; GATA-3; cytokines; colitis; IFN-γ
8.  CD4+CD25+ Regulatory T Cells Can Mediate Suppressor Function in the Absence of Transforming Growth Factor β1 Production and Responsiveness 
CD4+CD25+ regulatory T cells inhibit organ-specific autoimmune diseases induced by CD4+CD25−T cells and are potent suppressors of T cell activation in vitro. Their mechanism of suppression remains unknown, but most in vitro studies suggest that it is cell contact–dependent and cytokine independent. The role of TGF-β1 in CD4+CD25+ suppressor function remains unclear. While most studies have failed to reverse suppression with anti–transforming growth factor (TGF)-β1 in vitro, one recent study has reported that CD4+CD25+ T cells express cell surface TGF-β1 and that suppression can be completely abrogated by high concentrations of anti–TGF-β suggesting that cell-associated TGF-β1 was the primary effector of CD4+CD25+-mediated suppression. Here, we have reevaluated the role of TGF-β1 in CD4+CD25+-mediated suppression. Neutralization of TGF-β1 with either monoclonal antibody (mAb) or soluble TGF-βRII-Fc did not reverse in vitro suppression mediated by resting or activated CD4+CD25+ T cells. Responder T cells from Smad3−/− or dominant-negative TGF-β type RII transgenic (DNRIITg) mice, that are both unresponsive to TGF-β1–induced growth arrest, were as susceptible to CD4+CD25+-mediated suppression as T cells from wild-type mice. Furthermore, CD4+CD25+ T cells from neonatal TGF-β1−/− mice were as suppressive as CD4+CD25+ from TGF-β1+/+ mice. Collectively, these results demonstrate that CD4+CD25+ suppressor function can occur independently of TGF-β1.
PMCID: PMC2193919  PMID: 12119348
CD4+ suppressor T cells; autoimmunity; tolerance; immunoregulation; IL-2 receptor
9.  IL-10-Producing Th1 Cells and Disease Progression Are Regulated by Distinct CD11c+ Cell Populations during Visceral Leishmaniasis 
PLoS Pathogens  2012;8(7):e1002827.
IL-10 is a critical regulatory cytokine involved in the pathogenesis of visceral leishmaniasis caused by Leishmania donovani and clinical and experimental data indicate that disease progression is associated with expanded numbers of CD4+ IFNγ+ T cells committed to IL-10 production. Here, combining conditional cell-specific depletion with adoptive transfer, we demonstrate that only conventional CD11chi DCs that produce both IL-10 and IL-27 are capable of inducing IL-10-producing Th1 cells in vivo. In contrast, CD11chi as well as CD11cint/lo cells isolated from infected mice were capable of reversing the host protective effect of diphtheria toxin-mediated CD11c+ cell depletion. This was reflected by increased splenomegaly, inhibition of NO production and increased parasite burden. Thus during chronic infection, multiple CD11c+ cell populations can actively suppress host resistance and enhance immunopathology, through mechanisms that do not necessarily involve IL-10-producing Th1 cells.
Author Summary
Dendritic cells are well known as myeloid cells that bridge innate and adaptive immunity, and play an important role in the induction of cell-mediated immunity to a variety of pathogens. However, very little is known about the function of dendritic cells after infection has become established. In this study, we have examined the role of dendritic cells during the later phases of experimental visceral leishmaniasis, caused by infection with Leishmania donovani. We show for the first time that dendritic cells are responsible for promoting the development of splenic pathology, and that their removal during established infection leads to improved host resistance. Furthermore, our studies provide the first formal evidence in vivo that dendritic cells making IL-27 can induce production of the regulatory cytokine IL-10 by effector Th1-like CD4+ T cells. Surprisingly, we also found that other populations of CD11c+ cells were able to induce pathology and suppress host resistance, yet did not stimulate IL-10 production in CD4+ T cells, suggesting that the latter T cell population may not play an essential role in disease progression. Our studies provide new insights into dendritic cell function in chronic parasite infection and suggest potential new avenues for immunotherapy against visceral leishmaniasis.
PMCID: PMC3406093  PMID: 22911108
10.  Transforming growth factor β induced FoxP3+ regulatory T cells suppress Th1 mediated experimental colitis 
Gut  2006;55(5):671-680.
Background and aims
The imbalance between effector and regulatory T cells plays a central role in the pathogenesis of inflammatory bowel diseases. In addition to the thymus, CD4+CD25+ regulatory T cells can be induced in the periphery from a population of CD25− T cells by treatment with transforming growth factor β (TGF‐β). Here, we analysed the in vivo function of TGF‐β induced regulatory T (Ti‐Treg) cells in experimental colitis.
Ti‐Treg cells were generated in cell culture in the presence or absence of TGF‐β and tested for their regulatory potential in experimental colitis using the CD4+CD62L+ T cell transfer model.
Ti‐Treg cells significantly suppressed Th1 mediated colitis on CD4+CD62L+ T cell transfer in vivo, as shown by high resolution endoscopy, histology, immunohistochemistry, and cytokine analysis. Further analysis of in vivo and in vitro expanded Ti‐Treg cells showed that exogenous interleukin 2 (IL‐2) was crucial for survival and expansion of these cells.
Our data suggest that regulatory Ti‐Treg cells expand by TGF‐β and exogenous IL‐2 derived from effector T cells at the site of inflammation. In addition to Tr1 and thymic CD4+CD25+ T cells, peripheral Ti‐Treg cells emerge as a class of regulatory T cells with therapeutic potential in T cell mediated chronic intestinal inflammation.
PMCID: PMC1856126  PMID: 16162681
transforming growth factor β; FoxP3+; T cells; experimental colitis
11.  Vaccination Without Auto-antigen Protects Against Collagen II-Induced Arthritis via Immune Deviation and Regulatory T Cells 1 
Anti-inflammation immunotherapy has been successfully applied for the treatment of autoimmune diseases. Mucosal vaccines against autoimmune disorders are beneficial by influencing the regulatory compartment of gut and systemic adaptive immune systems. A Salmonella vector expressing colonization factor antigen I (CFA/I), shown to behave as an anti-inflammatory vaccine, stimulates the production of CD4+ CD25+ T cells and regulatory cytokines. In this work, we queried whether Salmonella-CFA/I can protect DBA/1 mice from collagen-induced arthritis (CIA). The incidence of arthritis and cartilage loss in vaccinated DBA/1 mice was remarkably lower when compared to unprotected mice. Clinical findings were accompanied by the suppression of inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-27. Vaccination evoked a multi-tier response consisting of IL-4 producing Th2 cells, an increased production of TGF-β by CD4+ T cells, and suppression of collagen II (CII)-specific CD4+ T cell proliferation. To assess the contribution of Salmonella-CFA/I-primed CD4+ T cells, adoptive transfer studies with total CD4+, CD4+CD25−, or CD4+ CD25+ T cells were performed 15 days post-challenge. Mice receiving either subset showed reduced disease incidence and low clinical scores; however, mice receiving total CD4+ T cells showed delayed disease onset by 10 days with reduced clinical scores, reduced IL-17 and IL-27, but enhanced IL-4, IL-10, IL-13, and TGF-β. Inhibition of TGF-β or IL-4 compromised protective immunity. These data show that Salmonella-CFA/I vaccination of DBA/1 mice protects against CIA by stimulating TGF-β- and IL-4-producing regulatory CD4+ T cells.
PMCID: PMC2599931  PMID: 18684965
Th1/Th2 cells; tolerance; vaccination; mucosa
12.  B Cell-Derived Interleukin 10 Does Not Regulate Spontaneous Systemic Autoimmunity in MRL.Faslpr mice 
B cells contribute to the pathogenesis of chronic autoimmune disorders like systemic lupus erythematosus (SLE) via multiple effector functions. However, B cells are also implicated in regulating SLE and other autoimmune syndromes via release of IL-10. B cells secreting IL-10 have been termed “Breg” and have been proposed as a separate subset of cells, a concept that remains controversial. The balance between pro- and anti-inflammatory effects could determine the success of B cell targeted therapies for autoimmune disorders and it is therefore pivotal to understand the significance of B cell-secreted IL-10 in spontaneous autoimmunity. By lineage specific deletion of Il10 from B cells we demonstrate that B cell-derived IL-10 is ineffective in suppressing the spontaneous activation of self-reactive B and T cells during lupus. Correspondingly, severity of organ disease and survival rates in mice harboring Il10 deficient B cells are unaltered. Genetic marking of cells that transcribe Il10 illustrates that the pool of IL-10 competent cells is dominated by CD4 T cells and macrophages. IL-10 competent cells of the B lineage are rare in vivo and among them short-lived plasmablasts have the highest frequency, suggesting an activation rather than lineage-driven phenotype. Putative Breg phenotypic subsets such as CD1dhiCD5+ and CD21hiCD23hi B cells are not enriched in Il10 transcription. These genetic studies demonstrate that in a spontaneous model of murine lupus, IL-10 dependent B cell regulation does not restrain disease and thus the pathogenic effects of B cells are not detectably counterbalanced by their IL-10 dependent regulatory functions.
PMCID: PMC3253138  PMID: 22156495
13.  Suppression of Glomerulonephritis in NZB/NZW Lupus Prone Mice by Adoptive Transfer of Ex Vivo Expanded Regulatory T Cells 
PLoS ONE  2009;4(6):e6031.
Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause characterized by expansion of autoreactive lymphocytes. Regulatory T cells (Tregs) are a component of the normal immune system and contribute to the maintenance of peripheral tolerance. Treg abnormalities have been associated with several autoimmune diseases and there is interest in the role of Tregs in SLE. We previously demonstrated that transfer of expanded CD4+CD25+CD62LHI Tregs slows the development of lupus in (NZBxNZW)F1 (B/W) mice. However in the absence of Treg specific surface antigens, cell purification remains a compromise between the breadth and purity of the population isolated. Importantly, purified populations always contain Foxp3− effector T cells (Teffs) that theoretically could exacerbate autoimmunity in the recipient. Here we explore the impact of transferring the more comprehensive, but less pure Treg subset defined by CD4+CD25+ expression on development of murine lupus. All cells were FACS sorted and expanded prior to adoptive transfer. Development of proteinuria and survival were measured. We found that exogenous expansion of CD4+CD25+ cells produced a population containing 70–85% CD4+Foxp3+Tregs. Expanded Tregs had higher CTLA-4 and Foxp3 expression, increased in vitro suppression capacity, and prolonged in vivo survival as compared to freshly isolated cells. Adoptive transfer of expanded CD4+CD25+ Tregs inhibited the onset of glomerulonephritis and prolonged survival in mice. Importantly the population of Teff contained within the adoptively transferred cells had reduced survival and proliferation capacity as compared to either co-transferred Tregs or transferred Teffs expanded in the absence of Tregs. These studies demonstrate that adoptive transfer of expanded CD4+CD25+Foxp3+Tregs has the capacity to inhibit the onset of murine lupus and that this capacity is significant despite transfer of co-cultured Teff cells. These data indicate that when co-expanded with regulatory T cells, exogenously activated Teffs from autoimmune patients may not pose a significant risk of promoting disease.
PMCID: PMC2696596  PMID: 19551149
14.  Mutual antagonism of TGF-beta and Interleukin-2 in cell survival and lineage commitment of induced regulatory T cells 
Cell Death and Differentiation  2012;19(8):1277-1287.
Transforming growth factor beta (TGF-β)- and Interleukin-2 (IL-2)-mediated signaling enables the generation and expansion of induced regulatory T (iTreg) cells that carry high hopes for the treatment of chronic inflammatory and autoimmune diseases. Knowledge about factors stabilizing their lineage commitment and lifespan, however, is limited. Here, we investigated the behavior of iTreg cells, derived from apoptosis-defective mouse mutants, during activated cell autonomous cell death, triggered by cytokine-deprivation, or activation-induced cell death (AICD) after restimulation of the T-cell receptor, and compared these responses with those of effector T cells. We observed that iTreg cells were much more sensitive to IL-2-deprivation but poorly susceptible to AICD. In fact, when apoptosis was compromised, T-cell receptor (TCR)-religation resulted in methylation-independent, ERK- and PI3K/mTOR-mediated loss of Foxp3 expression, impaired suppressive capacity and effector cytokine production. Although iTreg cells prevented colitis induction they rapidly lost Foxp3-GFP expression and gained ability to produce effector cytokines thereby imposing Th1 cell fate on resident effector cells. Surprisingly, iTreg cell conversion itself was limited by TGF-β-mediated Bim/Bcl2L11-dependent apoptosis. Hence, the very same cytokine that drives the generation of iTreg cells can trigger their demise. Our results provide novel insights in iTreg cell biology that will assist optimization of iTreg-based therapy.
PMCID: PMC3392638  PMID: 22322859
activation-induced cell death; activated cell autonomous cell death; TGF-β; regulatory T cells
15.  Modulation of autoimmune diseases by interleukin (IL)-17 producing regulatory T helper (Th17) cells 
Following the discovery of interleukin (IL)-17 producing T helper (Th17) cells as a distinct lineage of CD4+ T helper cells it became clear that these cells play an important role in the host defense against extracellular fungal and bacterial pathogens and participate in the pathogenesis of multiple inflammatory and autoimmune disorders. Depending on the microenvironment, Th17 cells can alter their differentiation programme ultimately giving rise to either protective or pro-inflammatory pathogenic cells. We found that besides the conventional in vitro protocol for Th17 differentiation by transforming growth factor-beta (TGF-β) plus IL-6 cytokines, a combination of IL-23 plus IL-6 can also induce Th17 cells. The Th17 cells induced by IL-23 plus IL-6 (termed as effector Th17, Teff17 cells) are pathogenic upon adoptive transfer into non-obese diabetic (NOD) mice contributing to the development of type 1 diabetes (T1D) while cells induced by TGF-β plus IL-6 (termed as regulatory T cells, Treg17 cells) are non pathogenic and regulatory, and suppressed the pathogenic T cells in T1D. These cells differentially expressed a number of cytokines where Teff17 cells exhibited an increase in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-22 whereas Treg17 cells demonstrated increased expression of IL-21 and immunosuppressive cytokine IL-10. Differentiation of Th17 cells is controlled by a transcription factor, RORγT although these cells also express variable levels of T-bet and FoxP3 transcription factors. This points to a dual functional role of Th17 subsets in autoimmune diseases particularly T1D. We suggest that similar to conventional regulatory T cells (Treg), induction of regulatory Treg17 cells could play an important role in modulating and preventing certain autoimmune diseases.
PMCID: PMC3928692  PMID: 24434314
Autoimmunity; cytokines; immunoregulation; helper T cell plasticity; regulatory T cells; Th17 cells; Type 1 diabetes
16.  Toxoplasma gondii Infection Induces Suppression in a Mouse Model of Allergic Airway Inflammation 
PLoS ONE  2012;7(8):e43420.
Allergic asthma is an inflammatory disorder characterized by infiltration of the airway wall with inflammatory cells driven mostly by activation of Th2-lymphocytes, eosinophils and mast cells. There is a link between increased allergy and a reduction of some infections in Western countries. Epidemiological data also show that respiratory allergy is less frequent in people exposed to orofecal and foodborne microbes such as Toxoplasma gondii. We previously showed that both acute and chronic parasite T. gondii infection substantially blocked development of airway inflammation in adult BALB/c mice. Based on the high levels of IFN-γ along with the reduction of Th2 phenotype, we hypothesized that the protective effect might be related to the strong Th1 immune response elicited against the parasite. However, other mechanisms could also be implicated. The possibility that regulatory T cells inhibit allergic diseases has received growing support from both animal and human studies. Here we investigated the cellular mechanisms involved in T. gondii induced protection against allergy. Our results show for the first time that thoracic lymph node cells from mice sensitized during chronic T. gondii infection have suppressor activity. Suppression was detected both in vitro, on allergen specific T cell proliferation and in vivo, on allergic lung inflammation after adoptive transference from infected/sensitized mice to previously sensitized animals. This ability was found to be contact- independent and correlated with high levels of TGF-β and CD4+FoxP3+ cells.
PMCID: PMC3429463  PMID: 22952678
17.  Conversion of Peripheral CD4+CD25− Naive T Cells to CD4+CD25+ Regulatory T Cells by TGF-β Induction of Transcription Factor Foxp3 
The Journal of Experimental Medicine  2003;198(12):1875-1886.
CD4+CD25+ regulatory T cells (Treg) are instrumental in the maintenance of immunological tolerance. One critical question is whether Treg can only be generated in the thymus or can differentiate from peripheral CD4+CD25− naive T cells. In this paper, we present novel evidence that conversion of naive peripheral CD4+CD25− T cells into anergic/suppressor cells that are CD25+, CD45RB−/low and intracellular CTLA-4+ can be achieved through costimulation with T cell receptors (TCRs) and transforming growth factor β (TGF-β). Although transcription factor Foxp3 has been shown recently to be associated with the development of Treg, the physiological inducers for Foxp3 gene expression remain a mystery. TGF-β induced Foxp3 gene expression in TCR-challenged CD4+CD25− naive T cells, which mediated their transition toward a regulatory T cell phenotype with potent immunosuppressive potential. These converted anergic/suppressor cells are not only unresponsive to TCR stimulation and produce neither T helper cell 1 nor T helper cell 2 cytokines but they also express TGF-β and inhibit normal T cell proliferation in vitro. More importantly, in an ovalbumin peptide TCR transgenic adoptive transfer model, TGF-β–converted transgenic CD4+CD25+ suppressor cells proliferated in response to immunization and inhibited antigen-specific naive CD4+ T cell expansion in vivo. Finally, in a murine asthma model, coadministration of these TGF-β–induced suppressor T cells prevented house dust mite–induced allergic pathogenesis in lungs.
PMCID: PMC2194145  PMID: 14676299
anergy; IL-10; OVA TCR transgenic; house dust mite; asthma
18.  The role of transforming growth factor beta in the generation of suppression: an interaction between CD8+ T and NK cells 
The Journal of Experimental Medicine  1994;180(5):1937-1942.
CD8+ T cells have suppressor effector functions, but the mechanisms involved in the generation of this activity are poorly understood. We report that natural killer (NK) cells have an important role in the acquisition of this function. CD8+ cells induce NK cells to produce transforming growth factor-beta (TGF-beta) which, in turn, stimulates CD8+ T cells to become suppressors of antibody production. Using a monocyte-dependent and -independent method to induce antibody production, we first observed that the addition of NK cells to CD8+ cells was required for optimal suppression. Next, we determined that the interaction of CD8+ T cells with NK cells resulted in a striking increase NK cell TGF-beta mRNA and its production. This cytokine appeared to be involved in the induction of T suppressor cell activity since: (a) anti-TGF-beta 1 completely abrogated the suppression of immunoglobulin G synthesis; (b) TGF-beta 1 could substitute for NK cells in inducing CD8+ T cells to develop suppressor activity; and (c) a short exposure of T cells to TGF-beta 1 in the absence of B cells was sufficient for the generation of suppressor activity by CD8+ T cells. Interferon gamma did not have this property. These studies provide strong evidence that in addition to its suppressive properties, TGF- beta is involved in the generation of CD8+ T suppressor effector cells. Because NK cell function is decreased in many autoimmune diseases, these cells may fail to interact properly with these individuals' CD8+ cells in generating suppressors of aggressive anti-self responses.
PMCID: PMC2191718  PMID: 7964469
19.  A Novel Probiotic Mixture Exerts a Therapeutic Effect on Experimental Autoimmune Encephalomyelitis Mediated by IL-10 Producing Regulatory T Cells 
PLoS ONE  2010;5(2):e9009.
Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS.
Methodology/Principal Findings
The preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4+CD25+Foxp3+ regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-β1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4+CD25+ Tregs are involved in the suppressive effect induced by the lactobacilli mixture.
Our data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.
PMCID: PMC2814855  PMID: 20126401
20.  BAG-1 suppresses expression of the key regulatory cytokine transforming growth factor beta [TGF-β1] in colorectal tumour cells 
Oncogene  2012;32(38):10.1038/onc.2012.480.
As colorectal cancer remains the second highest cause of cancer-related deaths in much of the industrialised world, identifying novel strategies to prevent colorectal tumour development remains an important challenge. BAG-1 is a multi-functional protein, the expression of which is up-regulated at relatively early stages in colorectal tumorigenesis. Importantly, BAG-1 is thought to enhance colorectal tumour progression through promoting tumour cell survival. Here we report for the first time a novel role for BAG-1, establishing it as a suppressor of transforming growth factor beta [TGF-β1] expression in colorectal tumour cells. Microarray analysis first highlighted the possibility that BAG-1 may regulate TGF-β1 expression, a key cytokine in normal colonic tissue homeostasis. Q-RT-PCR and ELISA demonstrated TGFB1 mRNA and protein expression to be significantly increased when BAG1 levels were reduced by siRNA; additionally, induction of BAG-1L caused suppression of TGFB1 mRNA in colorectal tumour cells. Using reporter and ChIP assays, a direct association of BAG-1 with the TGFB1 gene regulatory region was identified. Immunohistochemistry and Weiser fraction data indicated levels of BAG-1 and TGF-β1 are inversely correlated in the normal colonic epithelium in vivo, consistent with a role for BAG-1-mediated repression of TGF-β1 production. In vitro studies showed that the change in TGF-β1 production following manipulation of BAG-1 is functionally relevant; through induction of anchorage-independent growth in TGF-β1 dependent NRK fibroblasts and regulation of SMAD2 phosphorylation in TGF-β1 sensitive adenoma cells. Taken together, this study identifies the anti-apoptotic protein BAG-1 as a suppressor of the inhibitory growth factor TGF-β1, suggesting that high expression of BAG-1 can impact on a number of the hallmarks of cancer, of potential importance in promoting the early stages of colorectal tumorigenesis. Establishing BAG-1 as a repressor of TGF-β1 has important biological implications, and highlights a new role for BAG-1 in colorectal tumorigenesis.
PMCID: PMC3880928  PMID: 23108401
BAG-1; TGF-β1; colorectal cancer; adenoma; transcriptional repression
21.  Commitment to the Regulatory T Cell Lineage Requires CARMA1 in the Thymus but Not in the Periphery 
PLoS Biology  2009;7(3):e1000051.
Regulatory T (Treg) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4+ T cells in a cytokine dependent manner. Treg cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice. Here we show that mice with a Carma1 point mutation lack thymic CD4+Foxp3+ Treg cells and demonstrate a cell-intrinsic requirement for CARMA1 in thymic Foxp3 induction. However, peripheral Carma1-deficient Treg cells could be generated and expanded in vitro in response to the cytokines transforming growth factor beta (TGFβ) and interleukin-2 (IL-2). In vivo, a small peripheral Treg pool existed that was enriched at mucosal sites and could expand systemically after infection with mouse cytomegalovirus (MCMV). Our data provide genetic evidence for two distinct mechanisms controlling regulatory T cell lineage commitment. Furthermore, we show that peripheral Treg cells are a dynamic population that may expand to limit immunopathology or promote chronic infection.
Author Summary
In mammals, CD4+ T cells are essential for controlling infections, but have the potential to attack host tissues as well, resulting in autoimmune disease. A subset of CD4+ T cells, regulatory T cells (Treg)—identified by the expression of the forkhead transcription factor Foxp3—serve to prevent immunopathology by dampening immune responses. These cells are unique among CD4+ T cell subsets, as only the Treg lineage can develop in both the thymus and periphery. Using a genetic approach, we identified a mutation in the gene Carma1, a key component of T and B cell signaling, which in mice distinguishes Treg cells derived from the periphery from thymic-derived regulatory T cells. The mutation caused an absence of thymic Treg cells. However, a small population of Treg cells was observed in the spleen, lymph nodes, and colon of Carma1-mutant mice that expanded after viral infection, suggesting that peripheral development of Treg cells could still occur. Indeed, Carma1-mutant CD4+ T cells could be converted into the Treg lineage in vitro. Our results demonstrate an organ-specific requirement for the CARMA1 signaling pathway that developing thymocytes need in order to become Treg cells, but that naïve CD4+ T cells can bypass in the periphery. This dichotomy suggests that Treg cells of thymic or peripheral origin may have different specificities or functions in vivo.
The organ-specific requirement for CARMA1-dependent signaling in the thymus suggests that regulatory T cells of thymic or peripheral origin may have different roles in vivo.
PMCID: PMC2650725  PMID: 19260764
22.  TGF-β signaling is required for the function of insulin-reactive T regulatory cells 
Journal of Clinical Investigation  2006;116(5):1360-1370.
We have previously isolated insulin-reactive Tregs from diabetic NOD mice designated 2H6, from which TCR transgenic mice were generated. The T cells from these 2H6 transgenic mice recognize insulin but have suppressive properties in vitro. They protect NOD mice in vivo from spontaneous development of diabetes and adoptive transfer of disease caused by polyclonal diabetogenic spleen cells as well as the highly diabetogenic monoclonal BDC2.5 TCR transgenic T cells that recognize an islet granule antigen. Using cells from both NOD and BDC2.5 mice that express a dominant-negative TGF-β receptor type II (TGF-βDNRII), we show that 2H6 T cells protected from disease by producing TGF-β and that the ability of the target diabetogenic T cells to respond to TGF-β was crucial. We further demonstrate that TGF-β signaling in 2H6 cells was important for their protective properties, as 2H6 cells were unable to protect from adoptive transfer–induced diabetes if they were unable to respond to TGF-β. Thus, our data demonstrate that insulin-specific regulatory cells protect from diabetes by virtue of their production of TGF-β1 that acts in an autocrine manner to maintain their regulatory function and acts in a paracrine manner on the target cells.
PMCID: PMC1451206  PMID: 16670772
23.  Following EAU Recovery There Is an Associated MC5r-Dependent APC Induction of Regulatory Immunity in the Spleen 
In the spleens of mice recovered from experimental autoimmune uveitis is a melanocortin 5 receptor (MC5r)-dependent tolerogenic antigen-presenting cell (APC). Reestablishment of the mechanisms of immunomodulation in the uveitic ocular microenvironment generates a novel MC5r-dependent APC that induces, and stands ready to reactivate, protective ocular autoantigen-specific regulatory T cells.
IRBPp-specific regulatory immunity is found in the spleens of mice recovered from experimental autoimmune uveoretinitis (EAU). Induction of this regulatory immunity is dependent on the expression of the melanocortin 5 receptor (MC5r). Therefore, the authors investigated whether dependence on the expression of MC5r was with the T cells or with the APCs mediating protective regulatory immunity in the EAU-recovered mouse spleen.
Wild-type and MC5r−/− mice were immunized to induce EAU. The IRBPp-stimulated T-cell response in spleens of wild-type and MC5r−/− mice were compared for surface markers and cytokine production. Spleen APC were isolated and used to stimulate cytokine production and regulatory activity in IRBP-specific T cells from wild-type or MC5r−/− mice assayed in culture by ELISA, by flow cytometry, and in vivo by adoptive transfer into EAU mice.
IRBPp-specific CD25+CD4+ T cells from spleens of EAU-recovered wild-type mice express a Treg cell phenotype of FoxP3 and TGF-β compared with the effector T-cell phenotype of IFN-γ and IL-17 production in EAU-recovered MC5r−/− mice. APCs from the spleens of wild-type mice recovering from EAU promoted regulatory T-cell activation in IRBP-specific effector T cells from the spleens of EAU-recovering MC5r−/− mice. Spleen APCs from EAU-recovering wild-type, but not MC5r−/−, mice induced TGF-β expression by primed IRBP-specific effector T cells.
Dependence on MC5r expression is with an APC that promotes or selectively activates IRBP-specific FoxP3+ TGF-β+ CD25+CD4+ Treg cells in the spleens of EAU-recovered mice.
PMCID: PMC3231841  PMID: 21989727
24.  Passively Administered Pooled Human Immunoglobulins Exert IL-10 Dependent Anti-Inflammatory Effects that Protect against Fatal HSV Encephalitis 
PLoS Pathogens  2011;7(6):e1002071.
HSV-1 is the leading cause of sporadic encephalitis in humans. HSV infection of susceptible 129S6 mice results in fatal encephalitis (HSE) caused by massive inflammatory brainstem lesions comprising monocytes and neutrophils. During infection with pathogenic microorganisms or autoimmune disease, IgGs induce proinflammatory responses and recruit innate effector cells. In contrast, high dose intravenous immunoglobulins (IVIG) are an effective treatment for various autoimmune and inflammatory diseases because of potent anti-inflammatory effects stemming in part from sialylated IgGs (sIgG) present at 1–3% in IVIG. We investigated the ability of IVIG to prevent fatal HSE when given 24 h post infection. We discovered a novel anti-inflammatory pathway mediated by low-dose IVIG that protected 129S6 mice from fatal HSE by modulating CNS inflammation independently of HSV specific antibodies or sIgG. IVIG suppressed CNS infiltration by pathogenic CD11b+ Ly6Chigh monocytes and inhibited their spontaneous degranulation in vitro. FcγRIIb expression was required for IVIG mediated suppression of CNS infiltration by CD45+ Ly6Clow monocytes but not for inhibiting development of Ly6Chigh monocytes. IVIG increased accumulation of T cells in the CNS, and the non-sIgG fraction induced a dramatic expansion of FoxP3+ CD4+ T regulatory cells (Tregs) and FoxP3− ICOS+ CD4+ T cells in peripheral lymphoid organs. Tregs purified from HSV infected IVIG treated, but not control, mice protected adoptively transferred mice from fatal HSE. IL-10, produced by the ICOS+ CD4+ T cells that accumulated in the CNS of IVIG treated, but not control mice, was essential for induction of protective anti-inflammatory responses. Our results significantly enhance understanding of IVIG's anti-inflammatory and immunomodulatory capabilities by revealing a novel sIgG independent anti-inflammatory pathway responsible for induction of regulatory T cells that secrete the immunosuppressive cytokine IL-10 and further reveal the therapeutic potential of IVIG for treating viral induced inflammatory diseases.
Author Summary
We show that fatal HSV encephalitis (HSE) is caused by excessive brainstem inflammation. Once brainstem inflammation is initiated, antiviral drugs that inhibit only viral replication are ineffective in protecting against fatal HSE. Infusion of high doses of pooled human IgG (IVIG) is an effective anti-inflammatory treatment for various autoimmune diseases. One anti-inflammatory mechanism depends on sialylated IgGs (sIgG) present in limiting amounts (1–3%) in IVIG, hence the need for high doses of IVIG. We discovered a novel anti-inflammatory pathway mediated by low doses of IVIG independent of sIgG that prevented fatal HSE by suppressing CNS inflammation. The non-sIgG fraction of IVIG induced regulatory CD4+ T cells that produced the immunosuppressive cytokine IL-10 in the brainstem. Importantly, we show that IL-10 is critical for suppressing the generation of pathogenic inflammatory macrophages. Thus, IVIG has a remarkable ability to balance the host inflammatory responses to virus infection and thereby promotes virus clearance without bystander damage to the CNS, accounting for survival of all infected mice. Overall, our results provide important new insights in understanding IVIG's anti-inflammatory activity and further reveal its potential for use in treatment of viral inflammatory diseases.
PMCID: PMC3107211  PMID: 21655109
25.  Generation of human regulatory T cells de novo with suppressive function prevent xenogeneic graft versus host disease 
International immunopharmacology  2010;11(5):630-637.
Treatment with rapamycin (RAPA) favorably affects regulatory T cells (Treg) in vivo, and RAPA induces the de novo expression of FOXP3 in murine alloantigen-specific T cells. Whether RAPA acts independently or with transforming growth factor beta (TGF-β) to produce ex vivo-induced Treg generation is unknown. Naïve CD4+ T cells isolated from peripheral blood mononuclear cells were stimulated with anti-CD3/CD28 coated beads in the presence of IL-2 for 5 to 7 days. Ten ng/ml of TGF-β (1 to 100 ng/mL RAPA) was added to some of the cultures. The phenotypes were analyzed with flow cytometry. The conditioned cells were cocultured with CFSE-labeled T cells in different ratios for 5 days. CFSE dilution indicating T response cell proliferation was analyzed by flow cytometry. Xenogeneic graft-versus-host disease (x-GVHD) was induced by transplanting human peripheral blood mononuclear cells into RAG2−/− γc−/− mice exposed to total body irradiation, and various factors in the subjects were subsequently compared. CD4 cells induced by rapamycin and TGF-β (CD4RAPA/TGF-β) expressed the natural Treg phenotypes and trafficking receptors, and no significant cytotoxicity was observed. CD4RAPA/TGF-β was anergic and demonstrated potent suppressive activity in vitro. Although the transfer of human peripheral blood mononuclear cells into RAG2−/− γc−/− mice caused x-GVHD, the cotransfer of CD4RAPA/TGF-β decreased human cell engraftment and extended survival in mice. RAPA plus TGF-β induces human naïve T cells to become suppressor cells, a novel strategy for treating human autoimmune diseases and preventing allograft rejection.
PMCID: PMC3099130  PMID: 21147213
Regulatory T cells; TGF-β; Rapamycin; Graft-versus host disease; Human

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