The Ex Vivo Induction of Human CD103+ CD25hi Foxp3+ CD4+ and CD8+ Tregs is IL‐2 and TGF‐β1 Dependent

The expression of the integrin αE (CD103), may enhance the retention of regulatory T cells to peripheral inflammatory sites and possibly contribute to their suppressive potential. The aim of this study was to define the regulatory role of IL‐2 and TGF‐β1 on the CD103 expression and the optimal in vitro conditions for the induction/expansion of human CD4⁺ and CD8⁺ Tregs. Cord blood mononuclear cells (CBMC) were stimulated under various culture conditions, including anti‐CD3, anti‐CD28, IL‐2 and TGF‐β1. TGF‐β1 and IL‐2 were both required for optimal expression of CD103. In addition, TGF‐β1 and IL‐2 synergistically induced CD103 expression on CD8⁺ T cells, whereas, only additive induced expression was noted on CD4⁺ T cells. Surprisingly, CD103 expression was not dependent upon CD28 costimulation. IL‐2 also played a central role in CD103 expression by CD25^hi Foxp3+ Tregs. IL‐2, TGF‐β1 and anti‐CD3 defined the optimal stimulatory conditions favouring the induction/expansion of both CD4⁺ and CD8⁺ human Tregs from naive CBMC. Thus, this study provides new insights into the regulatory role of IL‐2 upon CD103 expression by human cord blood CD4⁺ and CD8⁺ T cells. Furthermore, it identifies the in vitro culture conditions driving the differentiation of the novel phenotype CD4⁺ and CD8⁺ CD103⁺ CD25^hi Foxp3+ Tregs from human CBMC.     

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.     

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.     

Cells with regulatory function of the innate and adaptive immune system in primary Sjögren's syndrome

The aim of the present study was to describe subsets of cells with regulatory properties in primary Sjögren's syndrome (pSS), and to correlate these cell populations with clinical symptoms. Among the 32 investigated patients, 23 had extraglandular manifestations (EGMs), while nine had only glandular symptoms. Twenty healthy individuals served as controls. The percentages of natural killer (NK), natural killer T cells (NK T), interleukin (IL)‐10 producing T regulatory type 1 (Tr1) cells and CD4⁺CD25⁺ regulatory T cells (T_reg) cells were determined by flow cytometry and serum cytokine levels of IL‐4, IL‐6, IL‐10, tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ were evaluated by enzyme‐linked immunosorbent assay (ELISA). Functional tests were carried out to assess the suppressor properties of T_reg cells in patients and controls. Peripheral NK, NK T and Tr1 cell percentages were elevated in pSS, while CD4⁺CD25⁺ T_reg cells showed reduced frequencies in patients compared to controls. In pSS, elevated percentages of NK T, Tr1 and CD4⁺CD25⁺ T_reg cells were observed in patients with EGMs, when compared to patients with sicca symptoms only. CD4⁺CD25⁺ T_reg cell percentages showed a negative correlation with sialometry values. The in vitro functional assay demonstrated lower suppression activity of CD4⁺CD25⁺ T_reg cells in patients compared to controls. Serum IL‐6 and TNF‐α levels were elevated, while IL‐10 was decreased in patients compared to controls. Negative correlation was found between IL‐10 levels and the percentages of Tr1 cells. Changes in the investigated subsets of regulatory cells in pSS may contribute to the development and progression of the disease.     

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.     

Induction and mechanism of action of transforming growth factor-β-secreting Th3 regulatory cells

Summary: Th3 CD4⁺ regulatory cells were identified during the course of investigating mechanisms associated with oral tolerance. Different mechanisms of tolerance are induced following oral antigen administration, including active suppression, clonal anergy and deletion. Low doses favor active suppression whereas high doses favor anergy/deletion. Th3 regulatory cells form a unique T‐cell subset which primarily secretes transforming growth factor (TGF)‐β, provides help for IgA and has suppressive properties for both Th1 and Th2 cells. Th3 type cells are distinct from the Th2 cells, as CD4⁺ TGF‐β‐secreting cells with suppressive properties have been generated from interleukin (IL)‐4‐deficient animals. In vitro differentiation of Th3 cells from Th precursors from T‐cell antigen receptor (TCR) transgenic mice is enhanced by culture with TGF‐β, IL‐4, IL‐10, and anti‐IL‐12. Th3 CD4⁺ myelin basic protein regulatory clones are structurally identical to Th1 encephalitogenic clones in TCR usage, MHC restriction and epitope recognition, but produce TGF‐β with various amounts of IL‐4 and IL‐10. Because Th3 regulatory cells are triggered in an antigen‐specific fashion but suppress in an antigen‐non‐specific fashion, they mediate “bystander suppression” when they encounter the fed autoantigen at the target organ. In vivo induction of Th3 cells and low dose oral tolerance is enhanced by oral administration of IL‐4. Anti‐CD86 but not anti‐CD80 blocks the induction of Th3 cells associated with low dose oral tolerance. Th3 regulatory cells have been described in other systems (e.g. recovery from experimental allergic encephalomyelitis) but may be preferentially generated following oral antigen administration due to the gut immunologic milieu that is rich in TGF‐β and has a unique class of dendritic cells. CD4⁺CD25⁺ regulatory T‐cell function also appears related to TGF‐β.     

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.     

Transforming growth factor beta 1 plays an important role in inducing CD4+CD25+forhead box P3+ regulatory T cells by mast cells

The role of mast cells (MCs) in the generation of adaptive immune responses especially in the transplant immune responses is far from being resolved. It is reported that mast cells are essential intermediaries in regulatory T cell (T_reg) transplant tolerance, but the mechanism has not been clarified. To investigate whether bone marrow‐derived mast cells (BMMCs) can induce T_regs by expressing transforming growth factor beta 1 (TGF‐β1) in vitro, bone marrow cells obtained from C57BL/6 (H‐2^b) mice were cultured with interleukin (IL)‐3 (10 ng/ml) and stem cell factor (SCF) (10 ng/ml) for 4 weeks. The purity of BMMCs was measured by flow cytometry. The BMMCs were then co‐cultured with C57BL/6 T cells at ratios of 1:2, 1:1 and 2:1. Anti‐CD3, anti‐CD28 and IL‐2 were administered into the co‐culture system with (experiment groups) or without (control groups) TGF‐β1 neutralizing antibody. The percentages of CD4⁺CD25⁺forkhead box P3 (FoxP3)⁺ T_regs in the co‐cultured system were analysed by flow cytometry on day 5. The T_reg percentages were significantly higher in all the experiment groups compared to the control groups. These changes were deduced by applying TGF‐β1 neutralizing antibody into the co‐culture system. Our results indicated that the CD4⁺ T cells can be induced into CD4⁺CD25⁺FoxP3⁺ T cells by BMMCs via TGF‐β1.     

DX5+CD4+ T cells modulate cytokine production by CD4+ T cells towards IL‐10 via the production of IL‐4

CD4⁺ Th cells play a critical role in orchestrating the adaptive immune response. Uncontrolled Th1 responses are implicated in the pathogenesis of autoimmune diseases. T cells with immune‐modulatory properties are beneficial for inhibiting such inflammatory responses. Previously we demonstrated that repetitive injections of immature DC induce expansion of DX5⁺CD4⁺ T cells, which upon adoptive transfer show potent regulatory properties in murine collagen‐induced arthritis as well as in delayed‐hypersensitivity models. However, their regulatory mechanism remains to be defined. Here, we analyzed the effect of DX5⁺CD4⁺ T cells on other CD4⁺ T cells in vitro. Although proliferation of naïve CD4⁺ T cells upon antigenic triggering was not altered in the presence of DX5⁺CD4⁺ T cells, there was a striking difference in cytokine production. In the presence of DX5⁺CD4⁺ T cells, an IL‐10‐producing CD4⁺ T‐cell response was induced instead of a predominant IFN‐γ‐producing Th1 response. This modulation did not require cell–cell contact. Instead, IL‐4 produced by DX5⁺CD4⁺ T cells was primarily involved in the inhibition of IFN‐γ and promotion of IL‐10 production by CD4⁺ T cells. Together, our data indicate that DX5⁺CD4⁺ T cells modulate the outcome of Th‐responses by diverting Th1‐induction into Th responses characterized by the production of IL‐10.     

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.     

Interleukin‐17 Facilitates the Immune Suppressor Capacity of High‐Grade Glioma‐Derived CD4 (+) CD25 (+) Foxp3 (+) T Cells Via Releasing Transforming Growth Factor Beta

High‐grade glioma is a malignant tumour; the pathogenesis is to be further investigated. Interleukin (IL)‐17 is an inflammatory cytokine. Chronic inflammation is a pathological feature of cancer. This study aimed to characterize the glioma‐derived IL‐17⁺ regulatory T cells (Treg). In this study, single cells were isolated from surgically removed high‐grade glioma tissue and examined by flow cytometry. The immune suppressor effect of IL‐17⁺ Tregs on CD8⁺ T cells was assessed in vitro. The results showed that abundant IL‐17⁺ Tregs were found in high‐grade glioma tissue. The immune suppressor molecule, transforming growth factor (TGF)‐beta, was detected in the IL‐17⁺ Tregs. The proliferation of CD8⁺ T cells was suppressed by culturing with the IL‐17⁺ Tregs, which was partially abrogated by neutralizing antibodies of either TGF‐beta or IL‐17 and completely abrogated by neutralizing antibodies against both TGF‐beta and IL‐17. In conclusion, IL‐17⁺ Tregs exist in the high‐grade glioma tissue; this subset of T cells can suppress CD8⁺ T cell activities via releasing TGF‐beta and IL‐17.     

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells mediate immunological self‐tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF‐β. In this study, we show that the co‐culture of naive T cells from C57BL/6 mice with allogeneic antigen‐presenting cells (APCs) from BALB/c mice in the presence of TGF‐β, RA, and IL‐2 resulted in a striking enrichment of Foxp3⁺ T cells. These RA in vitro‐induced regulatory T (RA‐iTreg) cells did not secrete Th1‐, Th2‐, or Th17‐related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg‐cell suppressive potential. Accordingly, these RA‐iTreg cells suppressed T‐cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA‐iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA‐iTreg cells showed alloantigen‐specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic‐specific Treg cells may be generated using TGF‐β, RA, and IL‐2. Thus, RA‐iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.     

Ligation of TLR7 on CD19+CD1dhi B cells suppresses allergic lung inflammation via regulatory T cells

B cells have been described as having the capacity to regulate cellular immune responses and suppress inflammatory processes. One such regulatory B‐cell population is defined as IL‐10‐producing CD19⁺CD1d^hi cells. Previous work has identified an expansion of these cells in mice infected with the helminth, Schistosoma mansoni. Here, microarray analysis of CD19⁺CD1d^hi B cells from mice infected with S. mansoni demonstrated significantly increased Tlr7 expression, while CD19⁺CD1d^hi B cells from uninfected mice also demonstrated elevated Tlr7 expression. Using IL‐10 reporter, Il10^?/? and Tlr7^?/‐ mice, we formally demonstrate that TLR7 ligation of CD19⁺CD1d^hi B cells increases their capacity to produce IL‐10. In a mouse model of allergic lung inflammation, the adoptive transfer of TLR7‐elicited CD19⁺CD1d^hi B cells reduced airway inflammation and associated airway hyperresponsiveness. Using DEREG mice to deplete FoxP3⁺ T regulatory cells in allergen‐sensitized mice, we show that that TLR7‐elicited CD19⁺CD1d^hi B cells suppress airway hyperresponsiveness via a T regulatory cell dependent mechanism. These studies identify that TLR7 stimulation leads to the expansion of IL‐10‐producing CD19⁺CD1d^hi B cells, which can suppress allergic lung inflammation via T regulatory cells.     

Regulatory T cells in autoimmune neuroinflammation

Regulatory T cells are the central element for the maintenance of peripheral tolerance. Several subtypes of regulatory T (Treg) cells have been described, and most of them belong to the CD4⁺ T-helper (Th) cell lineage. These specific subtypes can be discriminated according to phenotype and function. Forkhead box protein 3 (FoxP3)-expressing natural Treg cells (Tregs) and IL-10-producing, T-regulatory type 1 cells (Tr1) are the best-studied types of CD4⁺ regulatory T cells in humans and experimental animal models. It was shown that they play a crucial role during autoimmune neuroinflammation. Both cells types seem to be particularly important for multiple sclerosis (MS). Here, we discuss the role of CD4⁺ regulatory T cells in autoimmune neuroinflammation with an emphasis on Tregs and Tr1 cells in MS.     

Tracking antigen‐specific CD4+ T cells throughout the course of chronic Leishmania major infection in resistant mice

Primary Leishmania major infection typically produces cutaneous lesions that not only heal but also harbor persistent parasites. While the opposing roles of CD4⁺ T‐cell‐derived IFN‐γ and IL‐10 in promoting parasite killing and persistence have been well established, how these responses develop from naïve precursors has not been directly monitored throughout the course of infection. We used peptide:Major Histocompatibility Complex class II (pMHCII) tetramers to investigate the endogenous, parasite‐specific primary CD4⁺ T‐cell response to L. major in mice resistant to infection. Maximal frequencies of IFN‐γ⁺ CD4⁺ T cells were observed in the spleen and infected ears within a month after infection and were maintained into the chronic phase. In contrast, peak frequencies of IL‐10⁺ CD4⁺ T cells emerged within 2 weeks of infection, persisted into the chronic phase, and accumulated in the infected ears but not the spleen, via a process that depended on local antigen presentation. T helper type‐1 (Th1) cells, not Foxp3⁺ regulatory T cells, were the chief producers of IL‐10 and were not exhausted. Therefore, tracking antigen‐specific CD4⁺ T cells revealed that IL‐10 production by Th1 cells is not due to persistent T‐cell antigen receptor stimulation, but rather driven by early antigen encounter at the site of infection.     

Regulatory T cells in the periphery

Summary: Recognition of a systemic antigen by CD4⁺ T cells in a lymphopenic host leads to the sequential generation of pathogenic effector cells and protective CD25⁺ forkhead box protein (Foxp3⁺) regulatory T cells (Tregs) in the periphery. Such an experimental model is potentially valuable for defining the stimuli that determine the balance of effector and regulatory T cells. Our studies have shown that interleukin‐2 (IL‐2) enhances the development of effector cells and is essential for the peripheral generation of regulatory cells. Other models of peripheral Treg generation suggest that the concentration of antigen, the nature of the antigen‐presenting cells, and cytokines such as transforming growth factor‐β and IL‐10 may all influence the peripheral generation of Tregs.