Interleukin‐10‐secreting type 1 regulatory T cells in rodents and humans

Summary: Interleukin‐10 (IL‐10)‐secreting T regulatory type 1 (Tr1) cells are defined by their specific cytokine production profile, which includes the secretion of high levels of IL‐10 and transforming growth factor‐β(TGF‐β), and by their ability to suppress antigen‐specific effector T‐cell responses via a cytokine‐dependent mechanism. In contrast to the naturally occurring CD4⁺CD25⁺ T regulatory cells (Tregs) that emerge directly from the thymus, Tr1 cells are induced by antigen stimulation via an IL‐10‐dependent process in vitro and in vivo. Specialized IL‐10‐producing dendritic cells, such as those in an immature state or those modulated by tolerogenic stimuli, play a key role in this process. We propose to use the term Tr1 cells for all IL‐10‐producing T‐cell populations that are induced by IL‐10 and have regulatory activity. The full biological characterization of Tr1 cells has been hampered by the difficulty in generating these cells in vitro and by the lack of specific marker molecules. However, it is clear that Tr1 cells play a key role in regulating adaptive immune responses both in mice and in humans. Further work to delineate the specific molecular signature of Tr1 cells, to determine their relationship with CD4⁺CD25⁺ Tregs, and to elucidate their respective role in maintaining peripheral tolerance is crucial to advance our knowledge on this Treg subset. Furthermore, results from clinical protocols using Tr1 cells to modulate immune responses in vivo in autoimmunity, transplantation, and chronic inflammatory diseases will undoubtedly prove the biological relevance of these cells in immunotolerance.     

Dendritic cells expand antigen-specific Foxp3+CD25+CD4+ regulatory T cells including suppressors of alloreactivity

Summary: Thymic derived naturally occurring CD25⁺CD4⁺ T regulatory cells (Tregs) suppress immune responses, including transplantation. Here we discuss the capacity of dendritic cells (DCs) to expand antigen‐specific Tregs, particularly polyclonal Tregs directed to alloantigens. Initial studies have shown that mature DCs are specialized antigen‐presenting cells (APCs) for expanding antigen‐specific CD25⁺ CD4⁺ Tregs from TCR transgenic mice. When triggered by specific antigen, these Tregs act back on immature DCs to block the upregulation of CD80 and CD86 costimulatory molecules. More recently, DCs have been used to expand alloantigen‐specific CD25⁺CD4⁺ Tregs from the polyclonal repertoire in the presence of interleukin‐2 (IL‐2). Allogeneic DCs are much more effective than allogeneic spleen cells for expanding CD25⁺CD4⁺ Tregs. The DC‐expanded Tregs continue to express high levels of Foxp3, even without supplemental IL‐2, whereas spleen cells poorly sustain Foxp3 expression. When suppressive activity is tested, relatively small numbers of DC‐expanded CD25⁺CD4⁺ Tregs exert antigen‐specific suppression in the mixed leukocyte reaction (MLR), blocking immune responses to the original stimulating strain 10 times more effectively than to third party stimulating cells. DC‐expanded Tregs also retard graft versus host disease (GVHD) across full major histocompatibility complex (MHC) barriers. In vitro and in vivo, the alloantigen‐specific CD25⁺CD4⁺ Tregs are much more effective suppressors of transplantation reactions than polyclonal populations. We suggest that the expansion of Tregs from a polyclonal repertoire via antigen‐presenting DCs will provide a means for antigen‐specific control of unwanted immune reactions.     

Making regulatory T cells with defined antigen specificity: role in autoimmunity and cancer

Summary: There is increasing evidence that agonist ligand presentation either intrathymically or extrathymically plays a crucial if not essential role in the generation of regulatory T cells (Tregs). Thus, it is possible to induce Tregs of any desired specificity in vivo. The same goal can be achieved in vitro by expanding antigen‐specific CD4⁺ T cells and retrovirally transducing them. In contrast, in vitro expansion of Tregs is limited to antigens that have resulted in Treg generation in vivo. Antigen‐specific Tregs can be used in cellular therapy with the goal to prevent autoimmune disease or even to interfere with established autoimmunity. The latter requires that the Tregs can suppress effector cells that have already caused harm, which is possible because of the antigen‐dependent homing properties of Tregs, i.e. these cells can accumulate in antigen‐draining lymph nodes and exit into inflamed tissue. Generally, the in vivo interference is dependent on cytokines such as transforming growth factor‐β and interleukin‐10 that were dispensable in in vivo analysis of immunosuppression. The precise mechanisms of suppression remain enigmatic, however, but may be further elucidated by the molecular analysis of suppressed versus non‐suppressed T cells.     

T regulatory cells lacking CD25 are increased in MS during relapse

Dysregulation of inflammatory responses is considered to be a key element in autoreactive immune responses. T regulatory cells (Tregs) are important to maintain self-tolerance and the role of CD4⁺CD25⁺FoxP3⁺ Tregs in autoimmunity has been extensively investigated. Recently, it was shown that Tregs in systemic lupus erythematosus lacked CD25 but were biologically functional. These data warrants for further investigation of CD25^? Tregs in human autoimmunity. We analyzed relapsing–remitting multiple sclerosis (MS) patients by multicolor flow cytometry for the expression of CD3, CD4, IL2R (CD25), FoxP3, and the IL7R (CD127). Further, the level of Tregs was compared in remitting and relapsing patients and correlated with disease duration. Patients in relapse exhibited higher levels of FoxP3-positive Tregs lacking CD25 compared to healthy controls (p < 0.05), indicating that Tregs attempt to restrain immune activity during relapse. The proportion of Tregs tended to be decreased with disease duration, while CD25⁺CD4⁺ and CD25⁺CD8⁺ effector T-cell proportions were elevated and positively correlated with overall disease duration (p < 0.05). In conclusion, while MS patients in remission have normal levels of Tregs of different phenotype, relapsing patients show an increased proportion of systemic CD25^? FoxP3⁺ Tregs. With time, the proportion of Tregs decrease while effector T cells expand.     

Interferon-alpha regulates the dynamic balance between human activated regulatory and effector T cells: implications for antiviral and autoimmune responses

An adequate effector response against pathogens and its subsequent inactivation after pathogen clearance are critical for the maintenance of immune homeostasis. This process involves an initial phase of T‐cell effector (Teff) activation followed by the expansion of regulatory T cells (Tregs), a unique cell population that limits Teff functions. However, significant questions remain unanswered about the mechanisms that regulate the balance between these cell populations. Using an in vitro system to mimic T‐cell activation in human peripheral blood mononuclear cells (PBMC), we analysed the patterns of Treg and Teff activation, with special attention to the role of type I interferon (IFN‐I). Interestingly, we found that IFN‐α, either exogenously added or endogenously induced, suppressed the generation of CD4⁺ FoxP3^HI IFN‐γ^Neg activated Tregs (aTregs) while simultaneously promoting propagation of CD4⁺ FoxP3^Low/Neg IFN‐γ^Pos activated Teffs (aTeffs). We also showed that IFN‐α‐mediated inhibition of interleukin (IL)‐2 production may play an essential role in IFN‐α‐induced suppression of aTregs. In order to test our findings in a disease state with chronically elevated IFN‐α, we investigated systemic lupus erythematosus (SLE). Plasma from patients with SLE was found to contain IFN‐I activity that suppressed aTreg generation. Furthermore, anti‐CD3 activated SLE PBMCs exhibited preferential expansion of aTeffs with a very limited increase in aTreg numbers. Together, these observations support a model whereby a transient production of IFN‐α (such as is seen in an early antiviral response) may promote CD4 effector functions by delaying aTreg generation, but a chronic elevation of IFN‐α may tip the aTeff:aTreg balance towards aTeffs and autoimmunity.     

The lifestyle of naturally occurring CD4+CD25+Foxp3+ regulatory T cells

Summary: Numerous studies over the past 10 years have demonstrated the importance of naturally occurring CD4⁺CD25⁺Foxp3⁺ regulatory T cells (nTregs) in immune regulation. We analyzed the mechanism of action of nTregs in a well‐characterized model of autoimmune gastritis and demonstrated that nTregs act at an early stage of disease progression to inhibit the differentiation of naïve T cells to pathogenic T‐helper 1 effectors. The effects of nTregs in this model are not antigen‐specific but are mediated by activation of the nTregs by ubiquitous self‐peptide major histocompatibility complex class II complexes together with cytokines released by activated effector cells. Studies in vitro confirmed that some nTregs exist in an activated state in vivo and can be activated to exert non‐specific suppressor effector function by stimulation with interleukin‐2 in the absence of engagement of their T‐cell receptor. Natural Tregs can differentiate in vitro to exhibit potent granzyme B‐dependent, partially perforin‐independent cytotoxic cells that are capable of specifically killing antigen‐presenting B cells. Natural Treg‐mediated killing of antigen‐presenting cells may represent one pathway by which they can induce long‐lasting suppression of autoimmune disease.     

The role of physiological self-antigen in the acquisition and maintenance of regulatory T-cell function

Summary: The CD4⁺CD25⁺ regulatory T cells (Tregs) are efficient regulators of autoimmunity, but the mechanism remains elusive. We summarize recent data for the conclusion that disease‐specific Tregs respond to tissue antigens to maintain physiological tolerance and prevent autoimmunity. First, polyclonal Tregs from antigen‐positive donors suppress autoimmune ovarian disease (AOD) or experimental autoimmune prostatitis in day 3 thymectomized (d3tx) mice more efficiently than Tregs from antigen‐negative donors. Second, Tregs of antigen‐negative adult mice respond to cognate antigen in vivo and rapidly gain disease‐specific Treg function. Third, in d3tx female recipients devoid of neonatal ovarian antigens, only female Tregs suppressed AOD; the male Tregs gain AOD‐suppressing function by responding to the ovarian antigen in the recipients and mask the supremacy of female Tregs in AOD suppression. Fourth, when Tregs completely suppress AOD, the ovary‐draining lymph node is the only location with evidence of profound and persistent (but reversible) host T‐cell suppression. Fifth, from these nodes, highly potent AOD‐suppressing Tregs are retrievable. We conclude that self‐tolerance involves the continuous priming of Tregs by autoantigens, and in autoimmune disease suppression, the effector T‐cell response is continuously negated by potent disease‐specific Tregs that accumulate at the site of autoantigen presentation.     

Infectious tolerance and the long-term acceptance of transplanted tissue

Summary: Short courses of antibody treatment aimed at blocking the coreceptors CD4 and CD8 and/or costimulatory molecules such as CD40L are able to bring about long‐term acceptance and tolerance of allogeneic transplants. This tolerant state is operational, in that potential effector cells remain but are tightly regulated through the induction of antigen‐specific CD4⁺ regulatory T cells (Tregs). CD4⁺CD25⁺FoxP3⁺ Tregs appear to play a prominent role, although other categories of Tregs have been documented. Transforming growth factor β (TGFβ) has been found to play a major role in the induction of the tolerant state with therapeutic antibodies as well as promoting the induction of FoxP3⁺ T cells from naïve populations. The observation that Tregs can be found in tolerated grafts has led to the idea that they may interact with the grafted tissue to establish a state of acquired privilege symmetrical with a similar privileged microenvironment around antigen‐presenting cells in lymphoid tissues. Dampening of aggressive immune responses by Tregs allows antigen to persist and be presented in an innocuous way to promote tolerance in new cohorts of T cells throughout the life of the tolerated graft. Regulation may operate at many stages of an immune response, even as a censor at the terminal differentiation stages of effector function.     

Interleukin 10 and dendritic cells are the main suppression mediators of regulatory T cells in human neurocysticercosis

Neurocysticercosis is caused by the establishment of Taenia solium cysticerci in the central nervous system. It is considered that, during co‐evolution, the parasite developed strategies to modulate the host's immune response. The action mechanisms of regulatory T cells in controlling the immune response in neurocysticercosis are studied in this work. Higher blood levels of regulatory T cells with CD4⁺CD45RO⁺forkhead box protein 3 (FoxP3)^high and CD4⁺CD25^highFoxP3⁺CD95^high phenotype and of non‐regulatory CD4⁺CD45RO⁺FoxP3^med T cells were found in neurocysticercosis patients with respect to controls. Interestingly, regulatory T cells express higher levels of cytotoxic T lymphocyte antigen 4 (CTLA‐4), lymphocyte‐activation gene 3 (LAG‐3), programmed death 1 (PD‐1) and glucocorticoid‐induced tumour necrosis factor receptor (GITR), suggesting a cell‐to‐cell contact mechanism with dendritic cells. Furthermore, higher IL‐10 and regulatory T cell type 1 (Tr1) levels were found in neurocysticercosis patients’ peripheral blood, suggesting that the action mechanism of regulatory T cells involves the release of immunomodulatory cytokines. No evidence was found of the regulatory T cell role in inhibiting the proliferative response. Suppressive regulatory T cells from neurocysticercosis patients correlated negatively with late activated lymphocytes (CD4⁺CD38⁺). Our results suggest that, during neurocysticercosis, regulatory T cells could control the immune response, probably by a cell‐to‐cell contact with dendritic cells and interleukin (IL)‐10 release by Tr1, to create an immunomodulatory environment that may favour the development of T. solium cysticerci and their permanence in the central nervous system.     

A Role for Trichosanthin in the Expansion of CD4+CD25+ Regulatory T Cells

CD4⁺CD25⁺ regulatory T cells (Tregs) are critical for the peripheral immune tolerance. Understanding the signals for the generation of Tregs is important for the clinical immunotherapy, but only limited progress has been made on obtaining enough peripheral Tregs. The aim of this study was to evaluate the role of trichosanthin (Tk) extracted from Chinese medicinal herb Trichosanthes kirilowi on the function of Tregs in vitro and in vivo. We reported here that Tk is needed for the expansion of freshly isolated CD4⁺CD25⁺Tregs (nTregs) into Tk‐expanded CD4⁺CD25⁺Tregs (Tk‐Tregs) through up‐regulating CD25 and Foxp3 expression. The dose–response analyses indicated that 100 ng/ml Tk was the most appropriate dose. The result of real‐time PCR showed that Tk‐Tregs expressed 1.5‐fold higher levels of Foxp3 than those observed in nTregs. Tk ‐ Tregs markedly suppressed activation of effector T cells at a suppressor/responder ratio of 1:1, 1:2, 1:4, 1:8 or 1:16, and their effect was dose dependent. Moreover, Tk‐Tregs secreted more immunosuppressive cytokines interleukin (IL)‐10 and transforming growth factor (TGF)‐β1 after stimulating with antigen and antigen‐presenting cells (APC). Transwell experiments showed that not only cell‐to‐cell contact but also soluble cytokines were involved in suppressive mechanism of Tk‐Tregs. And Tk‐Tregs were more efficient in suppressing CD25^?T cell response to specific antigen than to irrelative antigen. Most importantly, it was revealed for the first time that Tk‐Tregs could prolong the survival duration of mice with acute graft‐versus‐host disease (aGVHD). In conclusion, the study suggests a possible therapeutic potential of Tk‐Tregs for clinical treatment on aGVHD.     

Targeting cytokines secreted by CD4+CD25highCD127low regulatory T cells inhibits ovarian cancer progression

Epithelial ovarian cancer (EOC) is one of the major malignant cancers with high rates of early metastasis in which regulatory T cells (Tregs) play an important role. Tregs suppress immune responses and promote the development of tumours in patients with EOC. However, the underlying mechanisms remain unclear. In this study, we found higher levels of CD4⁺CD25^highCD127^low Tregs in patients with EOC than in patients with benign ovarian tumours and healthy donors. The immune inhibitory effect of Tregs functions by maintaining high levels of immunosuppressive cytokines in EOC. The high levels of Tregs and related cytokines (TGF‐β1 or IL‐10) were associated with lymphatic metastasis and FIGO stages of patients with EOC. Expression of matrix metalloproteinase (MMP)‐2 and tissue inhibitors of metalloproteinase (TIMP)‐2 in EOC cell lines were significantly regulated in the coculture experiment with CD4⁺CD25^highCD127^low Tregs sorted from EOC patients. Levels of MMP‐2 and TIMP‐2 conversely changed after blocking IL‐10R and TGF‐β1R in EOC cells. The invasion ability of EOC cells was also significantly downregulated in this process. The metastasis of EOC cells was correlated with the levels of TGF‐β1 or IL‐10. These findings suggested that immunosuppressive cytokines secreted by CD4⁺ Tregs could be a novel target for inhibiting EOC progression.     

Human adipose‐tissue derived mesenchymal stem cells induce functional de‐novo regulatory T cells with methylated FOXP3 gene DNA

Due to their immunomodulatory properties, mesenchymal stem cells (MSC) are interesting candidates for cellular therapy for autoimmune disorders, graft‐versus‐host disease and allograft rejection. MSC inhibit the proliferation of effector T cells and induce T cells with a regulatory phenotype. So far it is unknown whether human MSC‐induced CD4⁺CD25⁺CD127^–forkhead box P3 (FoxP3)⁺ T cells are functional and whether they originate from effector T cells or represent expanded natural regulatory T cells (nT_reg). Perirenal adipose‐tissue derived MSC (ASC) obtained from kidney donors induced a 2·1‐fold increase in the percentage of CD25⁺CD127^–FoxP3⁺ cells within the CD4⁺ T cell population from allostimulated CD25^–/dim cells. Interleukin (IL)‐2 receptor blocking prevented this induction. The ASC‐induced T cells (iT_reg) inhibited effector cell proliferation as effectively as nT_reg. The vast majority of cells within the iTreg fraction had a methylated FOXP3 gene Treg‐specific demethylated region (TSDR) indicating that they were not of nT_reg origin. In conclusion, ASC induce T_reg from effector T cells. These iT_reg have immunosuppressive capacities comparable to those of nT_reg. Their induction is IL‐2 pathway‐dependent. The dual effect of MSC of inhibiting immune cell proliferation while generating de‐novo immunosuppressive cells emphasizes their potential as cellular immunotherapeutic agent.     

The roles for cytokines in the generation and maintenance of regulatory T cells

Summary: As an essential mechanism for self‐tolerance, immune suppression has attracted much attention since the discovery of suppressor T cells, now called regulatory T cells (Tregs), in the 1990s. Different types of Tregs have been described based on distinct expression patterns of surface markers and cytokines. Cytokines are not only essential for function but also important for the generation of Tregs. Interleukin‐2 (IL‐2), transforming growth factor‐β, IL‐10, and other immunoregulatory molecules have been shown to control the generation of Tregs. The presence of other types of cells, in particular antigen‐presenting cells (APCs), is critical for the generation of Tregs. Cytokines can serve as either initiators or intermediates for the interactions between APCs and Tregs. This review discusses our current knowledge of how cytokines regulate the generation and maintenance of Tregs.     

Circulating Toll‐like receptor (TLR) 2, TLR4, and regulatory T cells in patients with chronic hepatitis C

Wang J‐P, Zhang Y, Wei X, Li J, Nan X‐P, Yu H‐T, Li Y, Wang P‐Z, Bai X‐F. Circulating Toll‐like receptor (TLR) 2, TLR4, and regulatory T cells in patients with chronic hepatitis C. APMIS 2010; 118: 261–70. The mechanism of hepatitis C virus (HCV) involvement in innate immune responses and immune modulation has not been well characterized. In the present work, we studied Toll‐like receptor (TLR) 2 and TLR4, which were recently recognized as the important components of innate immunity, as well as CD4⁺ CD25⁺ CD127^low/? regulatory T cells (Tregs), which actively suppress pathological and physiological immune response during HCV infection. The study involved 31 chronic hepatitis C patients and 20 healthy controls. TLR2 and TLR4 expression in peripheral blood monocytes and the number of Tregs were examined by flow cytometric analysis. Overexpression of TLR2 and TLR4 was found in chronic hepatitis C patients as compared with controls. Furthermore, increased cytokine production, including that of β‐interferon, tumor necrosis factor‐α, interleukin (IL)‐6, and IL‐8, was observed in peripheral blood mononuclear cells from chronic hepatitis C patients after challenge with TLR2 and TLR4 agonists. The number of Tregs was significantly higher in chronic hepatitis C patients and the increased Tregs were associated with HCV genotype 1b. In vitro studies demonstrated that circulating Tregs suppress T‐cell responses in chronic hepatitis C patients. Significant correlations were found between the viral load and Treg number and between TLR2 and TLR4 level in chronic hepatitis C patients. Taken together with other published data, these results suggest that TLR2, TLR4, and Tregs correlate closely with chronic HCV infection.     

CD4+CD25+ T cell depletion impairs tolerance induction in a murine model of asthma

Background Regulatory T cells (Tregs) are key players in controlling the development of airway inflammation. However, their role in the mechanisms leading to tolerance in established allergic asthma is unclear. Objective To examine the role of Tregs in tolerance induction in a murine model of asthma. Methods Ovalbumin (OVA) sensitized asthmatic mice were depleted or not of CD25⁺ T cells by anti‐CD25 PC61 monoclonal antibody (mAb) before intranasal treatment (INT) with OVA, then challenged with OVA aerosol. To further evaluate the respective regulatory activity of CD4⁺CD25⁺ and CD4⁺CD25^? T cells, both T cell subsets were transferred from tolerized or non‐tolerized animals to asthmatic recipients. Bronchoalveolar lavage fluid (BALF), T cell proliferation and cytokine secretion were examined. Results Intranasal treatment with OVA led to increased levels of IL‐10, TGF‐β and IL‐17 in lung homogenates, inhibition of eosinophil recruitment into the BALF and antigen specific T cell hyporesponsiveness. CD4⁺CD25⁺Foxp3⁺ T cells were markedly upregulated in lungs and suppressed in vitro and in vivo OVA‐specific T cell responses. Depletion of CD25⁺ cells before OVA INT severely hampered tolerance induction as indicated by a strong recruitment of eosinophils into BALF and a vigorous T cell response to OVA upon challenge. However, the transfer of CD4⁺CD25^? T cells not only suppressed antigen specific T cell responsiveness but also significantly reduced eosinophil recruitment as opposed to CD4⁺CD25⁺ T cells. As compared with control mice, a significantly higher proportion of CD4⁺CD25^? T cells from OVA treated mice expressed mTGF‐β. Conclusion Both CD4⁺CD25⁺ and CD4⁺CD25^? T cells appear to be essential to tolerance induction. The relationship between both subsets and the mechanisms of their regulatory activity will have to be further analyzed.