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.     

A profile of TNFR2+ regulatory T cells and CD103+ dendritic cells in the peripheral blood of patients with asthma

The interaction of tolerogenic CD103⁺ dendritic cells (DCs) with regulatory T (Tregs) cells modulates immune responses by inducing immune tolerance. Hence, we determined the proportion of these cells in the peripheral blood mononuclear cells (PBMC) of asthmatic patients. We observed lower trends of CD11b^-CD103⁺ DCs and CD86 within CD11b^-CD103⁺ DCs, while increased levels of Foxp3 expressing CD25^+/-TNFR2⁺ cells in asthmatics. There was a positive correlation in the expression of Foxp3 within CD3⁺CD4⁺CD25⁺TNFR2⁺ Tregs and CD11b^-CD103⁺ as well as the expression of CD86 within HLA-DR⁺CD11c⁺CD11b^-CD103⁺ DCs. In conclusion, we suggest that the increased levels of Tregs in blood could continuously suppress the T helper 2 (Th2) cells activation in the circulation which is also supported by the increase of anti-inflammatory cytokines IL-10 and TNF. Overall, functional immunoregulation of the regulatory cells, particularly Tregs, exhibit immune suppression and induce immune tolerance linked with the immune activation by the antigen presenting cells (APC).     

Integrated T-cell receptor and costimulatory signals determine TGF-β-dependent differentiation and maintenance of Foxp3+ regulatory T cells

Foxp3‐expressing Tregs play a non‐redundant role in protecting against immune pathologies. Foxp3⁺ Tregs can arise intra‐ and extra‐thymically, however, the signals directing their differentiation and maintenance in the periphery are not well understood. We show that stimulation of mouse naïve CD4⁺ T cells in vitro with optimal doses of anti‐CD3/anti‐CD28 resulted in high frequencies of Foxp3⁺ T cells via a TGF‐β‐dependent mechanism. Addition of TGF‐β and retinoic acid overcame the inhibition of Foxp3 expression observed during high‐strength anti‐CD3/anti‐CD28 stimulation. Reducing the strength of TCR or costimulatory signals with inhibitors of mammalian target of rapamycin (mTOR) or MEK/ERK signalling also enhanced expression of Foxp3 in a TGF‐β‐dependent manner. Addition of TGF‐β was further required to maintain Foxp3 expression in ex vivo derived Foxp3⁺ Tregs upon prolonged anti‐CD3/anti‐CD28 signalling. Thus, induction/maintenance of Foxp3 expression by TGF‐β is modulated by the integrated strength of TCR/costimulatory signals.     

CD4Foxp3 Tregs protect against innate immune cell-mediated fulminant hepatitis in mice

Foxp3⁺ Tregs play important roles in maintaining homeostasis by suppressing excessive immune responses that result in serious tissue damage; yet, it is largely unknown about the impact of Tregs on innate immune cells in hepatitis models in vivo. In this study, we examined the effect of hepatic Tregs on innate immune-mediated liver injury by using the murine model of polyI:C and d-galactosamine (d-GalN)-induced hepatitis. Administration of polyI:C/d-GalN increased the number of CD4⁺Foxp3⁺ Tregs in the liver. Depletion of Tregs leaded to higher levels of proinflammatory cytokine expression and severer liver injury, whereas adoptive transfer of Foxp3⁺ Tregs attenuated liver injury in polyI:C/d-GalN-treated mice. In addition, depletion of Tregs leaded to a reduction in TGF-β and IL-10 expression in polyI:C/d-GalN-treated mice. Both of these cytokines were important for suppression of polyI:C/d-GalN-induced liver injury. TGF-β was derived from Tregs. IL-10 was derived from active Kupffer cells, and coincubation of Kupffer cells with Tregs increased IL-10 secretion. Furthermore, TGF-β blockade abrogated Treg-mediated suppression of proinflammatory cytokine production by innate immune cell in vitro.     

Expression of IL-7 receptor in human peripheral regulatory T cells

Introduction

Regulatory T cells (Tregs, CD4 + CD25^high Foxp3⁺) play a crucial role in allergy and other inflammatory diseases. However, the isolation of viable Tregs on the basis of intracellular expression of specific Forkhead Box Protein P3 (Foxp3) is difficult. In this study we checked if the expression of IL-7 receptor (CD127) on the Tregs could be a useful marker for isolation of viable Treg Foxp3⁺ cells.

Material and methods

Twenty-five patients sensitized to grass pollen with allergic rhinitis (AR) and ten healthy subjects were included. We compared Foxp3 expression in different CD4⁺ T cell subsets by flow cytometry and we assessed the relationship between the expression of Foxp3 and CD127 within regulatory T cells.

Results

Within the CD⁴+ lymphocytes 3.68 ±2.0% showed expression of Foxp3, 51.82 ±8.03% of CD4⁺CD25^high were Foxp3 positive (Foxp3⁺), whereas 82.12 ±5.4% of CD4⁺CD25^highCD127^low were Foxp3⁺. High intracellular expression of Foxp3 correlated with low superficial CD127 expression (r = 0.42, p = 0.017). There were no significant differences regarding the analysed markers between AR patients and healthy controls.

Conclusions

Regulatory T cells may be purified from the fresh peripheral blood as viable regulatory Foxp3 bright cells using CD4, high expression of CD25 and low expression of CD127 antigen.     

Recirculating IL-1R2+ Tregs fine-tune intrathymic Treg development under inflammatory conditions

The vast majority of Foxp3⁺ regulatory T cells (Tregs) are generated in the thymus, and several factors, such as cytokines and unique thymic antigen-presenting cells, are known to contribute to the development of these thymus-derived Tregs (tTregs). Here, we report the existence of a specific subset of Foxp3⁺ Tregs within the thymus that is characterized by the expression of IL-1R2, which is a decoy receptor for the inflammatory cytokine IL-1. Detailed flow cytometric analysis of the thymocytes from Foxp3^hCD2xRAG1^GFP reporter mice revealed that the IL-1R2⁺ Tregs are mainly RAG1^GFP– and CCR6⁺CCR7^–, demonstrating that these Tregs are recirculating cells entering the thymus from the periphery and that they have an activated phenotype. In the spleen, the majority of IL-1R2⁺ Tregs express neuropilin-1 (Nrp-1) and Helios, suggesting a thymic origin for these Tregs. Interestingly, among all tissues studied, the highest frequency of IL-1R2⁺ Tregs was observed in the thymus, indicating preferential recruitment of this Treg subset by the thymus. Using fetal thymic organ cultures (FTOCs), we demonstrated that increased concentrations of exogenous IL-1β blocked intrathymic Treg development, resulting in a decreased frequency of CD25⁺Foxp3⁺ tTregs and an accumulation of CD25⁺Foxp3⁻ Treg precursors. Interestingly, the addition of IL-1R2⁺ Tregs, but not IL-1R2⁻ Tregs, to reaggregated thymic organ cultures (RTOCs) abrogated the IL-1β-mediated blockade, demonstrating that these recirculating IL-1R2⁺ Tregs can quench IL-1 signaling in the thymus and thereby maintain thymic Treg development even under inflammatory conditions.     

Identification of a CD4+ T cell line with Treg-like activity

Regulatory T cells (Tregs) suppress adaptive immunity and inflammation. Although they play a role in suppressing anti-tumor responses, development of therapeutics that target Tregs is limited by their low abundance, heterogeneity, and lack of specific cell surface markers. We isolated human PBMC-derived CD4⁺ CD25^high Foxp3⁺ Tregs and demonstrate they suppress stimulated CD4⁺ PBMCs in a cell contact-dependent manner. Because it is not possible to functionally characterize cells after intracellular Foxp3 staining, we identified a human T cell line, MoT, as a model of human Foxp3⁺ Tregs. Unlike Jurkat T cells, MoT cells share common surface markers consistent with human PBMC-derived Tregs such as: CD4, CD25, GITR, LAG-3, PD-L1, CCR4. PBMC-derived Tregs and MoT cells, but not Jurkat cells, inhibited proliferation of human CD4⁺ PBMCs in a ratio-dependent manner. Transwell membrane separation prevented suppression of stimulated CD4⁺ PBMC proliferation by MoT cells and Tregs, suggesting cell–cell contact is required for suppressive activity. Blocking antibodies against PD-L1, LAG-3, GITR, CCR4, HLA-DR, or CTLA-4 did not reverse the suppressive activity. We show that human PBMC-derived Tregs and MoT cells suppress stimulated CD4⁺ PBMCs in a cell contact-dependent manner, suggesting that a Foxp3⁺ Treg population suppresses immune responses by an uncharacterized cell contact-dependent mechanism.     

Role of CD4+CD25+ Regulatory T Cells in Melatonin‐Mediated Inhibition of Murine Gastric Cancer Cell Growth In Vivo and In Vitro

Melatonin is an important immune modulator with antitumor functions, and increased CD4⁺CD25⁺ regulatory T cells (Tregs) have been observed in tumor tissues of patients and animal models with gastric cancer. However, the relationship between melatonin and Tregs remains unclear. To explore this potential connection, we performed an in vivo study by inoculating the murine foregastric carcinoma (MFC) cell line in mice and then treated them with different doses of melatonin (0, 25, 50, and 100 mg/kg, i.p.) for 1 week. The results showed that melatonin could reduce the tumor tissue and decrease Tregs numbers and Forkhead box p3 (Foxp3) expression in the tumor tissue. An in vitro study was also performed to test the effects of purified Tregs on melatonin‐mediated inhibition of MFC cells. The cell cultures were divided into three groups: 1) MFC+ Tregs; 2) MFC only; and 3) MFC+CD4⁺CD25^? T cells. After treatment with different concentrations of melatonin (0, 2, 4, 6, 8, and 10 mM) for 24 h, a dose‐dependent apoptosis and cell cycle arrest at the G2/M phase was detected in melatonin‐treated MFC at melatonin concentration higher than 4 mM. There were no significant differences in the rates of apoptosis and cell cycle distributions of MFC among the three groups. In conclusion, the antigastric cancer effect of melatonin is associated with downregulation of CD4⁺CD25⁺ Tregs and its Foxp3 expression in the tumor tissue. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Oestrogen-mediated protection of experimental autoimmune encephalomyelitis in the absence of Foxp3+ regulatory T cells implicates compensatory pathways including regulatory B cells

Oestrogen (17β‐oestradiol, E₂) is a highly effective treatment for experimental autoimmune encephalomyelitis (EAE) that may potentiate Foxp3⁺ regulatory T (Treg) cells, which in turn limit the expansion of encephalitogenic T‐cell specificities. To determine if Treg cells constitute the major non‐redundant protective pathway for E₂, we evaluated E₂ protection of EAE after targeted deletion of Foxp3 expression in Foxp3‐DTR mice. Unexpectedly, E₂‐treated Foxp3‐deficient mice were completely protected against clinical and histological myelin oligodendrocyte glycoprotein (MOG)‐35‐55 peptide‐induced EAE before succumbing to diphtheria toxin‐induced mortality. This finding indicated the presence of alternative E₂‐dependent EAE‐protective pathways that could compensate for the lack of Treg cells. Further investigation revealed that E₂ treatment inhibited proliferation and expression of CCL2 and CXCL2, but enhanced secretion of interleukin‐10 (IL‐10) and IL‐13 by MOG‐35‐55‐specific spleen cells. These changes occurred concomitantly with increased expression of several chemokines and receptors, including CXCL13 and CXCR5, and the negative co‐activation molecules, PD‐L1 and B7.2, by B cells and dendritic cells. Furthermore, E₂ treatment resulted in higher percentages of spleen and lymph node T cells expressing IL‐17, interferon‐γ and tumour necrosis factor‐α, but with lower expression of CCR6, suggesting sequestration of MOG‐35‐55 peptide‐specific T cells in peripheral immune organs. Taken together, these data suggest that E₂‐induced mechanisms that provide protection against EAE in the absence of Foxp3⁺ Treg cells include induction of regulatory B cells and peripheral sequestration of encephalitogenic T cells.     

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.     

Natural and inducible Tregs in swine: Helios expression and functional properties

Within the population of regulatory T cells (Tregs) natural Tregs (nTregs) and inducible Tregs (iTregs) can be distinguished. Although information about Tregs in swine exists, porcine iTregs were not under investigation yet. In this study, Foxp3⁺ iTregs were generated from CD4⁺Foxp3⁻ T cells by in vitro stimulation in the presence of IL-2 and TGF-β. In comparison to ex vivo Tregs these iTregs had a similar suppressive capacity on the proliferation of CD3-stimulated PBMC, caused higher levels of IL-10 in PBMC/Treg co-cultures, but did not suppress IFN-γ levels. The Ikaros family member Helios is currently discussed to distinguish iTregs and nTregs or to serve as an activation marker of Tregs. In this study, we demonstrate the cross-reactivity of an anti-mouse/human Helios mAb with porcine Helios. Flow cytometric analyses with this antibody showed that porcine iTregs do not express Helios after in vitro iTreg induction. Nevertheless, thymic Foxp3⁺ T cells, which arise at the CD4/CD8α single-positive stage of T-cell development and are defined as nTregs, entirely expressed Helios. Although this might suggest the suitability of Helios as an nTreg–iTreg differentiation marker we also found that Helios⁻ Tregs displayed a phenotype of naive CD4⁺ T cells in vivo. Since iTregs are by definition activated/differentiated Tregs, this finding precludes that all Helios⁻ Tregs are iTregs and thus also the use of Helios as a selection marker for porcine nTregs. Furthermore, Helios⁺ Tregs displayed a more differentiated phenotype indicating that Helios might rather serve as a Treg activation/differentiation marker.     

The importance of regulatory T-cell heterogeneity in maintaining self-tolerance

CD4⁺ Forkhead box protein 3 (Foxp3)⁺ regulatory T cells (Tregs) are the major cell type that mediates dominant tolerance in the periphery. Over the past decade, extensive study of Tregs has revealed that these cells express substantial heterogeneity to maintain tolerance and regulate immune responses. Tregs possess heterogeneity with respect to their origin and processes for development, functional activity, migratory pattern, and activation status. Some of the same environmental cues and molecular pathways utilized to generate specialized T-effector cells are also integrated by Tregs to colocalize and fine-tune suppressive mechanisms to optimally regulate and restrain distinctive self and antigen-specific T-cell responses. Here, we review our current understanding and significance of Treg heterogeneity in maintaining peripheral immune tolerance. We also highlight recent work from our laboratory that has studied the extent phenotypically distinct Treg subsets are related to each other and expand in an ordered fashion to give rise to highly activated short-lived Klrg1⁺ suppressor cells to optimize immune regulation and maintain homeostasis of the Treg compartment.