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‐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.     

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.     

T cell Suppression In Vitro During Toxoplasma gondii Infection is the Result of IL‐2 Competition Between Tregs and T cells Leading to Death of Proliferating T cells

A reduced proliferation to T cell mitogens is observed in vitro in murine cells isolated during the acute phase of Toxoplasma gondii infection. Foxp3⁺ regulatory T cells (Tregs) mediate this suppression, which is interleukin (IL)‐2 dependent. In this work, we analysed the mechanism of this Treg‐mediated suppression. We found that removal of antigen‐presenting cells (APC) from spleen cells from infected mice did not modify suppression but further elimination of Tregs led to a restored proliferation, demonstrating that Tregs mediate suppression in the absence of APC. Production of IL‐2 by T cells from infected animals was abolished but partially restored when Tregs were removed. However, IL‐2 levels and T cell proliferation were restored when Tregs and T cells were separated by transwells, indicating that Tregs require close proximity with T cells to induce suppression. Tregs from infected mice were able to reduce proliferation of CTLL‐2 cells in the classical IL‐2 bioassay, strongly suggesting that Tregs compete with T cells for IL‐2. We found that T cells from infected mice died after a few rounds of division in vitro, but addition of recombinant IL‐2 or removal of Tregs abolished this effect. Our results showed that suppression of T cell proliferation during acute Toxoplasma gondii infection is the result of death of proliferating T cells by Treg‐mediated IL‐2 competition. Thus, immunosuppression is due to death of proliferating T cells as a consequence of low IL‐2 availability.     

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.     

IFN‐γ‐STAT1 signal regulates the differentiation of inducible Treg: Potential role for ROS‐mediated apoptosis

Regulatory CD4⁺ T cells are important for the homeostasis of immune cells, and their absence correlates with autoimmune disorders. However, how the immune system regulates Treg homeostasis remains unclear. We found that IFN‐γ‐deficient‐mice had more forkhead box P3 (FOXP3⁺) cells than WT mice in all secondary lymphoid organs except the thymus. However, T‐bet‐ or IL‐4Rα‐deficient mice did not show a similar increase. In vitro differentiation studies showed that conversion of naïve T cells into FOXP3⁺ cells (neo‐generated inducible Treg (iTreg)) by TGF‐β was significantly inhibited by IFN‐γ in a STAT‐1‐dependent manner. Moreover, an in vivo adoptive transfer study showed that inhibition of FOXP3⁺ iTreg generation by IFN‐γ was a T‐cell autocrine effect. This inhibitory effect of IFN‐γ on iTreg generation was significantly abrogated after N‐acetyl‐L ‐cysteine treatment both in vitro and in vivo, indicating that IFN‐γ regulation of iTreg generation is dependent on ROS‐mediated apoptosis. Therefore, our results suggest that autocrine IFN‐γ can negatively regulate the neo‐generation of FOXP3⁺ iTreg through ROS‐mediated apoptosis in the periphery.     

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.     

Protease‐activated receptor‐2 signalling by tissue factor on dendritic cells suppresses antigen‐specific CD4+ T‐cell priming

The precise function of tissue factor (TF) expressed by dendritic cells (DC) is uncertain. As well as initiating thrombin generation it can signal through protease‐activated receptor 2 (PAR‐2) when complexed with factor VIIa. We investigated the expression and function of TF on mouse bone marrow (BM) ‐derived DC; 20% of BM‐derived DC expressed TF, which did not vary after incubation with lipopolysaccharide (LPS) or dexamethasone (DEX). However, the pro‐coagulant activity of DEX‐treated DC in recalcified plasma was 30‐fold less than LPS‐treated DC. In antigen‐specific and allogeneic T‐cell culture experiments, the TF on DEX‐treated DC provided a signal through PAR‐2, which contributed to the reduced ability of these cells to stimulate CD4⁺ T‐cell proliferation and cytokine production. In vivo, an inhibitory anti‐TF antibody and a PAR‐2 antagonist enhanced antigen‐specific priming in two models where antigen was given without adjuvant, with an effect approximately 50% that seen with LPS, suggesting that a similar mechanism was operational physiologically. These data suggest a novel TF and PAR‐2‐dependent mechanism on DEX‐DC in vitro and unprimed DC in vivo that contributes to the low immunogenicity of these cells. Targeting this pathway has the potential to influence antigen‐specific CD4⁺ T‐cell activation.     

Naive Tumour‐Specific CD4+ T Cells were Efficiently Primed in Acute Lymphoblastic Leukaemia

The recognition and neutralization of tumour cells is one of the big challenges in immunity. The immune system has to recognize syngeneic tumour cells and has to be primed and respond in an adequate manner. Priming of a leukaemia‐specific immune response is a crucial step in tumour immunology that can mislead to tumour tolerance either by T cell ignorance, deletion or Treg induction. To resemble the situation of acute lymphoblastic leukaemia (ALL) in patients, we used the murine BALB/c model with syngeneic BM185 tumour cells. We established a tumour cell line that expresses the neo‐antigen ovalbumin (BM185‐OVA/GFP) to allow the application of T cell receptor transgenic, antigen‐specific CD4⁺ T cells. Here, we demonstrate that effective anti‐ALL immunity can be established by in vivo priming of CD4⁺ T cells that is sufficient to differentiate into effector cells. Yet they failed to control tumour alone, but initiated a Th1 response. An efficient tumour clearance was dependent on both antigen‐specific CD4⁺ T cells and CD8⁺ effector T cells from the endogenous repertoire. The tolerogeneic milieu was characterized by increased Tregs numbers and elevated IL‐10 level. Tregs hamper effective antitumour immune response, but their depletion did not result in reduced tumour growth. In contrast, neutralization of IL‐10 improved median mouse survival. Future therapies should focus on establishing a strong CD4+ T cells response, either by adjuvant or by adoptive transfer.     

Ex‐vivo tolerogenic F4/80+ antigen‐presenting cells (APC) induce efferent CD8+ regulatory T cell‐dependent suppression of experimental autoimmune uveitis

It is known that inoculation of antigen into the anterior chamber (a.c.) of a mouse eye induces a.c.‐associated immune deviation (ACAID), which is mediated in part by antigen‐specific local and peripheral tolerance to the inciting antigen. ACAID can also be induced in vivo by intravenous (i.v.) inoculation of ex‐vivo‐generated tolerogenic antigen‐presenting cells (TolAPC). The purpose of this study was to test if in‐vitro‐generated retinal antigen‐pulsed TolAPC suppressed established experimental autoimmune uveitis (EAU). Retinal antigen‐pulsed TolAPC were injected i.v. into mice 7 days post‐induction of EAU. We observed that retinal antigen‐pulsed TolAPC suppressed the incidence and severity of the clinical expression of EAU and reduced the expression of associated inflammatory cytokines. Moreover, extract of whole retina efficiently replaced interphotoreceptor retinoid‐binding protein (IRBP) in the preparation of TolAPC used to induce tolerance in EAU mice. Finally, the suppression of EAU could be transferred to a new set of EAU mice with CD8⁺ but not with CD4⁺regulatory T cells (T_reg). Retinal antigen‐pulsed TolAPC suppressed ongoing EAU by inducing CD8⁺ T_reg cells that, in turn, suppressed the effector activity of the IRBP‐specific T cells and altered the clinical symptoms of autoimmune inflammation in the eye. The ability to use retinal extract for the antigen raises the possibility that retinal extract could be used to produce autologous TolAPC and then used as therapy in human uveitis.     

CD40 is necessary for activation of naïve T cells by a dendritic cell line in vivo but not in vitro

The importance of CD40–CD40L interactions during CD4⁺ T‐cell activation has been extensively investigated over the years; however, it still remains questionable whether the interaction is a prerequisite for dendritic cell (DC)‐mediated antigen‐specific priming in vivo. Naïve CD4⁺ T cells require two signals for proper activation and induction of differentiation: signal 1 is provided by peptide antigens in the context of the major histocompatibility complex (MHC) class II, while signal 2 is delivered by costimulatory molecules such as CD80 or CD86 present on the antigen‐presenting cell (APC). It is well known that the expression of CD80/CD86 is upregulated after interaction between CD40 on APCs and CD40L expressed by at least partly activated T cells. We used a DC line, JawsII, to compare the importance of CD40 expression and downstream signalling in vitro and in vivo. JawsII cells represent pre‐immature bone marrow‐derived DCs expressing low levels of MHC molecules, low levels of B7 molecules and no CD40. We have previously shown that JawsII cells, despite the lack of CD40 expression, are capable of priming naïve allogeneic T cells in vitro. In correlation with the current literature, we present data showing that constitutive expression of CD40 significantly increases the priming capacity of JawsII cells in vitro. In addition, we show that CD40 expression is required for JawsII cell‐dependent T‐cell priming in vivo.     

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.     

The effect of age on the cognate function of CD4+ T cells

Summary: With increasing age, the ability to produce protective antibodies in response to immunization declines, resulting in reduced efficacy of vaccination. We have examined how reductions in CD4⁺ T‐cell function contribute to reduced humoral responses, using a model that allows us to compare identical numbers of antigen‐specific naive T cells from young and aged T‐cell receptor transgenic mice. Naive cells from aged mice exhibit reduced responses, both in vitro and in vivo. In vitro, responses of aged T cells can be enhanced by addition of interleukin (IL)‐2. In vivo, using an adoptive transfer model with young hosts, naive cells from aged mice exhibit significant reductions in cognate helper function, leading to reduced B‐cell expansion and differentiation. These age‐related defects could be overcome by prior in vitro T helper 2 effector generation with aged T cells. This improvement in cognate function of the aged effectors may be related to the enhancement of CD154 expression, which occurs on aged T cells in the presence of exogenous IL‐2. We also found no difference in B‐cell expansion and differentiation when young cells were transferred to young or aged hosts. Our results indicate that age‐related reductions in humoral responses are mainly due to defects in the cognate helper function of naive CD4⁺ T cells from aged individuals.     

Bystander stimulation of activated CD4+ T cells of unrelated specificity following a booster vaccination with tetanus toxoid

Antigen‐specific CD4⁺ T cells are central to natural and vaccine‐induced immunity. An ongoing antigen‐specific T‐cell response can, however, influence surrounding T cells with unrelated antigen specificities. We previously observed this bystander effect in healthy human subjects following recall vaccination with tetanus toxoid (TT). Since this interplay could be important for maintenance of memory, we have moved to a mouse model for further analysis. We investigated whether boosting memory CD4⁺ T cells against TT in vivo would influence injected CD4⁺ TCR transgenic T cells (OT‐II) specific for an unrelated OVA peptide. If OT‐II cells were pre‐activated with OVA peptide in vitro, these cells showed a bystander proliferative response during the ongoing parallel TT‐specific response. Bystander proliferation was dependent on boosting of the TT‐specific memory response in the recipients, with no effect in naive mice. Bystander stimulation was also proportional to the strength of the TT‐specific memory T‐cell response. T cells activated in vitro displayed functional receptors for IL‐2 and IL‐7, suggesting these as potential mediators. This crosstalk between a stimulated CD4⁺ memory T‐cell response and CD4⁺ T cells activated by an unrelated antigen could be important in human subjects continually buffeted by environmental antigens.     

Regulatory T cell therapy for the induction of clinical organ transplantation tolerance

The pursuit of transplantation tolerance is the holygrail in clinical organ transplantation. It has been established that regulatory T cells (Tregs) can confer donor-specific tolerance in mouse models of transplantation. However, this is crucially dependent on the strain combination, the organ transplanted and most importantly, the ratio of Tregs to alloreactive effector T cells. The ex vivo expansion of Tregs is one solution to increase the number of alloantigen specific cells capable of suppressing the alloresponse. Indeed, ex vivo expanded, alloantigen specific murine Tregs are shown to preferentially migrate to, and proliferate in, the graft and draining lymph node. In human transplantation it has been proposed that depletion of the majority of direct pathway alloreactive T cells will be required to tip the balance in favour of regulation. Ex vivo expansion of alloantigen specific, indirect pathway human Tregs, which can cross regulate the residual direct pathway has been established. Rapid expansion of these cells is possible, whilst they retain antigen specificity, suppressive properties and favourable homing markers. Furthermore, considerable progress has been made to define which immunosuppressive drugs favour the expansion and function of Tregs. Currently a series of clinical trials of adoptive Treg therapy in combination with depletion of alloreactive T cells and short term immunosuppression are underway for human transplantation with the aim of minimizing immunosuppressive drugs and completely withdrawal.     

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.     

Regulatory T-cell physiology and application to treat autoimmunity

Summary: Endowed with the ability to actively suppress an immune response, regulatory T cells (Tregs) hold the promise of halting ongoing pathogenic autoimmunity and restoring self‐tolerance in patients suffering from autoimmune diseases. Through many in vitro and in vivo studies, we have learned that Tregs can function in the lymph nodes as well as in the peripheral tissues. In vivo, Tregs act through dendritic cells to limit autoreactive T‐cell activation, thus preventing their differentiation and acquisition of effector functions. By limiting the supply of activated pathogenic cells, Tregs prevent or slow down the progression of autoimmune diseases. However, this protective mechanism appears insufficient in autoimmune individuals, likely because of a shortage of Tregs cells and/or the development and accumulation of Treg‐resistant pathogenic T cells over the long disease course. Thus, restoration of self‐tolerance in these patients will likely require purging of pathogenic T cells along with infusion of Tregs with increased ability to control ongoing tissue injury. In this review, we highlight advances in dissecting Treg function in vivo in autoimmune settings and summarize multiple studies that have overcome the limitations of the low abundance of Tregs and their hypoproliferative phenotype to develop Treg‐based therapies.     

Analysis of miR‐146a and miR‐142‐3p as Potential Markers of Freshly Isolated or In Vitro‐Expanded Human Treg cells

Regulatory CD4⁺ T cells (Tregs) are pivotal for prevention of autoimmunity. The use of Tregs is therefore of increasing interest in in vitro drug screening assays as well as for a cytotherapy per se against autoimmune disorders. For both purposes, in vitro expansion of peripheral blood Tregs is necessary and there is an increasing need to identify novel markers that can discriminate natural thymic‐derived Tregs (tTregs) from other T cell subsets, and ideally, such markers should be stably expressed during in vitro expansion procedures. We screened for novel miRNAs differentially expressed in tTregs and identified miR‐146a and 142‐3p as possible candidates. We analysed freshly isolated naïve and activated tTregs and non‐Treg subsets after or prior to in vitro expansion. We observed a tTreg‐specific profile of these miRNAs together with FOXP3 and Helios in freshly isolated tTregs, but observed a decline in the same markers in activated tTregs as opposed to naïve tTregs. In vitro‐expanded Tregs could be identified based on FOXP3 expression, but with loss of a discriminate profile for miRNA candidates and a decline in FOXP3 when activated tTregs were expanded. Our data demonstrate miR‐146a and 142‐3p as potential miRNA markers for discrimination between non‐Treg cells and tTregs, but these miRNAs are not stable markers for in vitro‐expanded Treg cells. In addition, the loss of FOXP3 in expansion of activated tTregs has implication for in vitro use of this cell subset in immunopharmacological assays and cytotherapy as FOXP3 is pivotal for suppressive function.     

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.