CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells

A key issue in mammalian immunology is how CD4+CD25+Foxp3+ regulatory T cells (T(reg) cells) suppress immune responses. Here we show that T(reg) cells induced apoptosis of effector CD4+ T cells in vitro and in vivo in a mouse model of inflammatory bowel disease. T(reg) cells did not affect the early activation or proliferation of effector CD4+ T cells. Cytokines that signal through the common gamma-chain suppressed T(reg) cell-induced apoptosis. T(reg) cell-induced effector CD4+ T cell death required the proapoptotic protein Bim, and effector CD4+ T cells incubated with T(reg) cells showed less activation of the prosurvival kinase Akt and less phosphorylation of the proapoptotic protein Bad. Thus, cytokine deprivation-induced apoptosis is a prominent mechanism by which T(reg) cells inhibit effector T cell responses.     

Aurintricarboxylic acid promotes the conversion of naive CD4+CD25- T cells into Foxp3-expressing regulatory T cells

Naive peripheral CD4(+)CD25(-) T cells can be converted into Foxp3-expressing regulatory T cells under appropriate stimulation conditions. Considering that continuous exposure to antigens is one of the prerequisites for the differentiation and maintenance of Treg cells, we investigated whether preventing activation-induced cell death while providing continuous TCR stimulation could promote the expression of Foxp3 in murine naive CD4(+) T cells. Among the several anti-apoptotic agents tested, aurintricarboxylic acid (ATA) was found to induce the in vitro conversion of naive CD4(+) T cells into Foxp3(+) Treg cells with suppressive activity. Neutralizing studies with an antibody against transforming growth factor (TGF)-β revealed that ATA requires the presence of TGF-β to induce Foxp3 expression in naive CD4(+)CD25(-) T cells. Although ATA itself did not activate the Smad signaling pathway, it down-regulated the extracellular signal-regulated kinase and mammalian target of rapamycin signaling cascade in activated T cells. Lastly, combined exposure to ATA and TGF-β had a synergistic effect on the rate of induction and maintenance of Foxp3 expression. These results indicate that ATA could be exploited to efficiently prepare inducible regulatory T cells in vitro and may aid in more precisely identifying the specific signaling pathways that drive Foxp3 expression in T cells.     

FTY720-induced conversion of conventional Foxp3- CD4+ T cells to Foxp3+ regulatory T cells in NOD mice

Citation Sun Y, Wang W, Shan B, Di J, Chen L, Ren L, Li W, Li D‐J, Lin Y. FTY720‐induced conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in NOD mice. Am J Reprod Immunol 2011; 66: 349–362 Problem FTY720 is known as an agonist of sphingosine‐1‐phosphate (S1P) receptor, but little is known about the possibility that FTY720 induces the conversion of conventional Foxp3^?CD4⁺ T cells to Foxp3⁺ regulatory T cells in non‐obese diabetic (NOD) mice. Method of study FTY720 treatment was performed using Foxp3^?CD4⁺ T cells purified from NOD mice. Results FTY720 caused an increase in Foxp3⁺ Treg cells in lymphoid organs in NOD mice. FTY720 effectively induced Foxp3 expression in Foxp3^?CD4⁺ T cells both in vitro and in vivo, an effect that was inhibited by a TGF‐β‐neutralizing antibody or the proinflammatory cytokine IL‐6. T‐cell‐mediated embryo rejection in NOD mice was prevented upon FTY720 treatment. Conclusions The use of FTY720 along with Ag administration may represent a useful therapeutic strategy to selectively expand Ag‐specific Foxp3⁺ Tregs to intervene autoimmune and infectious diseases.     

Monocyte-derived dendritic cells from breast cancer patients are biased to induce CD4+CD25+Foxp3+ regulatory T cells

DCs orchestrate immune responses contributing to the pattern of response developed. In cancer, DCs may play a dysfunctional role in the induction of CD4(+)CD25(+)Foxp3(+) Tregs, contributing to immune evasion. We show here that Mo-DCs from breast cancer patients show an altered phenotype and induce preferentially Tregs, a phenomenon that occurred regardless of DC maturation stimulus (sCD40L, cytokine cocktail, TNF-α, and LPS). The Mo-DCs of patients induced low proliferation of allogeneic CD3(+)CD25(neg)Foxp3(neg) cells, which after becoming CD25(+), suppressed mitogen-stimulated T cells. Contrastingly, Mo-DCs from healthy donors induced a stronger proliferative response, a low frequency of CD4(+)CD25(+)Foxp3(+) with no suppressive activity. Furthermore, healthy Mo-DCs induced higher levels of IFN-γ, whereas the Mo-DCs of patients induced higher levels of bioactive TGF-β1 and IL-10 in cocultures with allogeneic T cells. Interestingly, TGF-β1 blocking with mAb in cocultures was not enough to completely revert the Mo-DCs of patients' bias toward Treg induction. Altogether, these findings should be considered in immunotherapeutic approaches for cancer based on Mo-DCs.     

Origin and T cell receptor diversity of Foxp3+CD4+CD25+ T cells

Foxp3(+)CD4(+)CD25(+) regulatory T cells can differentiate from Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) naive T cells. However, the impact of these two processes on size and composition of the peripheral repertoire of regulatory T cells is unclear. Here we followed the fate of individual Foxp3(+)CD4(+)CD25(+) thymocytes and T cells in vivo in T cell receptor (TCR) transgenic mice that express a restricted but polyclonal repertoire of TCRs. By utilizing high-throughput single-cell analysis, we showed that Foxp3(+)CD4(+) peripheral T cells were derived from thymic precursors that expressed a different TCRs than Foxp3(-)CD4(+) medullary thymocytes and Foxp3(-)CD4(+) T cells. Furthermore, the diversity of TCRs on Foxp3(+)CD4(+) regulatory T cells exceeded the diversity of TCRs on Foxp3(-)CD4(+) naive T cells, even in mice that lack expression of tissue-specific antigens. Our results imply that higher TCR diversity on Foxp3(+) regulatory T cells helps these cells to match the specificities of autoreactive and naive T cells.     

CD8+ Foxp3+ T cells share developmental and phenotypic features with classical CD4+ Foxp3+ regulatory T cells but lack potent suppressive activity

"Suppressor T cells" were historically defined within the CD8(+) T-cell compartment and recent studies have highlighted several naturally occurring CD8(+) Foxp3(-) Treg populations. However, the relevance of CD8(+) Foxp3(+) T cells, which represent a minor population in both thymi and secondary lymphoid organs of nonmanipulated mice, remains unclear. We here demonstrate that de novo Foxp3 induction in peripheral CD8(+) Foxp3(-) T cells is counter-regulated by DC-mediated co-stimulation via CD80/CD86. CD8(+) Foxp3(+) T cells fail to develop in TCR-transgenic mice with Rag1(-/-) background, similar to classical CD4(+) Foxp3(+) Tregs. Notably, both naturally occurring and induced CD8(+) Foxp3(+) T cells express bona fide Treg markers including CD25, GITR, CTLA4 and CD103, and show defective IFN-γ production upon restimulation when compared with their CD8(+) Foxp3(-) counterparts. However, utilizing DEREG transgenic mice for the isolation of Foxp3(+) cells by eGFP reporter expression, we demonstrate that induced CD8(+) Foxp3(+) T cells similar to activated CD8(+) Foxp3(-) T cells only mildly suppress T-cell proliferation and IFN-γ production. We therefore categorize CD8(+) Foxp3(+) T cells as a tightly controlled population sharing certain developmental and phenotypic properties with classical CD4(+) Foxp3(+) Tregs, but lacking potent suppressive activity.     

Role of activin A in the induction of Foxp3+ and Foxp3- CD4+ regulatory T cells

Regulatory T cells (Tregs) play a crucial role in the maintenance of immune homeostasis. The two best studied types of CD4(+) regulatory T cells are the Foxp3(+) Tregs and the T regulatory type 1 (Tr1) cells. CD4(+) regulatory T cells play a protective role in autoimmune disease. On the other hand, they also may have pathogenic properties in infectious diseases and carcinogenesis. Because of their potential for the therapy of various human diseases, factors responsible for expanding regulatory T cells are of interest. One of these factors, the TGFbeta family member activin A, is expressed in different inflammatory conditions, such as inflammatory bowel disease, rheumatoid arthritis, and asthma. Although activin A might have pro- and anti-inflammatory properties depending on the context of expression, this review focuses on the role of activin A for the expansion of the CD4(+) regulatory T-cell pool.     

不同结核病患者CD4^＋CD25^＋Foxp3^＋调节性T细胞表达的变化

目的分析不同结核病患者体内CD4^＋CD25^＋Foxp3^＋调节性T细胞（Tr）表达的变化,探讨其在结核病免疫中的作用。方法对33例结核病患者以及同期30例健康体检者运用流式细胞术检测其外周血CD4^＋CD25“。“和CIM^＋CD25^＋Foxp3^＋Tr表达情况。结果结核组CD4^＋CD25^high和CD4^＋CD25^＋Foxp3^＋Tr检测结果分别为（8．84±2．55）％、（6．30±1．38）％,高于对照组（7．09±1．09）％、（5．22±0．64）％,差别有统计学意义（t=3．57,4．01,P〈0．01）;痰涂阳患者CD4^＋CD25^high和CD4^＋CD25^＋Foxp3^＋Tr检测结果分别为（10．52±3．27）％、（7．18±1．77）％,高于痰涂阴患者（8．21±1．94）％、（5．97±1．06）％,两组问差别均具有统计学意义（t=2．51,2．42,P〈0．05）;初治患者和复治患者间CD4^＋CD25^high和CD4^＋CD25^＋Foxp3^＋检测结果无统计学意义（t=0．03,0．02,P〉0．05）。结论结核病患者体内CD4^＋CD25^high和CD4^＋CD25^＋Foxp3^＋Tr检测结果高于健康人群,提示患者体内存在免疫系统抑制状态。     

不同结核病患者CD4+CD25+Foxp3+调节性T细胞表达的变化

Objective To investigate the expression of CD4 + CD25 + Foxp3 + regulatory T cell in patients with tuberculosis, and to discover its role in the immune response to mycobacterium tuberculosis. Methods Thirty-three patients with tuberculosis and 30 healthy controls were selected who were consulted in our hospital. Patients were classified by their chemotherapy and smear sputum and CD4 + CD25 + Foxp3 + regulatory T cell were detected by flow cytometry. Results Expression of CD4 + CD25high and CD4+ CD25 + Foxp3 + regulatory T cell in experimental group were ( 8.84 ± 2.55 ) % and (6.30 ± 1.38 ) % respectively, which were significantly higher than they were in control group (t = 3.57,4. 01, P ＜ 0. 01 ). The expression of CD4 + CD25high and CD4+ CD25 + Foxp3 + regulatory T cell in patients with positive smear sputum were also significant higher than that in patients with negative smear sputum ( t = 2. 51,2. 42,P ＜ 0.05). No significance was founded between the first-visit group and revisit group ( t = 0. 03, 0. 02, P ＞ 0.05 ). Conclusions CD4 + CD25high and CD4 + CD25 + Foxp3 + regulatory T cell in patients with tuberculosis were higher than healthy control, which may result in immune suppression.     

Control of Foxp3+ CD25+CD4+ regulatory cell activation and function by dendritic cells

Naturally occurring CD4+CD25+ regulatory T (TR) cells play crucial roles in normal immunohomeostasis. CD4+CD25+ TR cells exhibit a number of interesting in vitro properties including a 'default state' of profound anergy refractory to conventional T cell stimuli. We investigated the in vitro activation requirements of CD4+CD25+ TR cells using bone marrow-derived DC, which as professional antigen presenting cells (APC) can support the activation of normal naive T cells. Comparison of different APC types revealed that LPS-matured DC were by far the most effective at breaking CD4+CD25+ TR cell anergy and triggering proliferation, and importantly their IL-2 production. Examination of Foxp3, a key control gene for CD4+CD25+ TR cells, showed this to be stably expressed even during active proliferation. Although CD4+CD25+ TR cell proliferation was equivalent to that of CD25- cells their IL-2 production was considerably less. Use of IL-2-/- mice demonstrated that the DC stimulatory ability was not dependent on IL-2 production; nor did IL-15 appear crucial but was, at least in part, related to costimulation. DC also blocked normal CD4+CD25+ TR cell-mediated suppression partially via IL-6 secretion. DC therefore possess novel mechanisms to control the suppressive ability, expansion and/or differentiation of CD4+CD25+ TR cells in vivo.     

Changes of CD4+CD25+Foxp3+ regulatory T cells in aged Balb/c mice

A progressive decline in the integrity of the immune system is one of the physiologic changes during aging. The frequency of autoimmune diseases or immune disorders increases in the aging population, but the state of regulatory T (Treg) cells in aged individuals has not been well determined. In the present study, we investigated the levels, phenotypes, and function of CD4(+)CD25(+) Treg cells in Balb/c mice, which were older than 20 months. Significantly enhanced percentages of CD4(+)CD25(+) Treg cells in the periphery (blood, spleen, and lymph nodes) of the aged mice were observed. These Treg cells showed modified Vbeta family distribution, reduced levels of CD45 receptor B and CD62 ligand molecules, as well as normal levels of forkhead box p3. However, when the inhibiting function of Treg cells was assayed in the in vitro assays and in a delayed-type hypersensitivity (DTH) model, CD4(+)CD25(+) Treg cells of aged mice displayed significantly lower inhibiting ability on alloantigen-induced DTH reaction or cytokine productions (IL-2 and IFN-gamma) but not cell proliferation of effector T cells, as compared with CD4(+)CD25(+) Treg cells of young mice. In addition, the percentages of CD4(+)CD8(-)CD25(+) Treg cells in the thymi of aged mice increased significantly, but their total cell numbers decreased markedly in these mice. Our present studies indicated collectively that the percentages, phenotypes, the size of TCR repertoire, and function of CD4(+)CD25(+) Treg cells were altered significantly with aging in mice. The functional defects of CD4(+)CD25(+) Treg cells may shed light on the role of CD4(+)CD25(+) Treg cells in the increased sensitivity to autoimmune diseases of aged populations.     

CD4+CD25+ but not CD4+Foxp3+ T cells as a regulatory subset in primary biliary cirrhosis

Increasing evidence indicates a role for regulatory T cells (Tregs) in the immune response and in autoimmune diseases, but the role of Tregs and cytokines in autoimmune hepatic diseases remains largely unclear and controversial, especially in patients with primary biliary cirrhosis (PBC). This study was undertaken to investigate Tregs and different cytokines in the liver and peripheral blood of PBC patients. We found that these patients demonstrated a reduction of CD4⁺CD25⁺ T cells but elevated CD4⁺Foxp3⁺ T cells in peripheral blood mononuclear cells (PBMCs) and CD4⁺ T cells. The percentage of CD4⁺CD25⁺ T cells in PBMCs was negatively correlated with elevated plasma interferon (IFN)-γ levels. A liver-specific analysis showed that the frequency of Foxp3⁺ Tregs, transforming growth factor (TGF)-β1 and IFN-γ were increased in PBC patients. Our findings suggest that an imbalance between CD4⁺CD25⁺ Tregs and cytotoxic cytokines plays a crucial role in the pathogenesis of PBC while the role of Foxp3 needs further investigation.     

Alterations of peripheral CD4+CD25+Foxp3+ T regulatory cells in mice with STZ-induced diabetes

Complications arising from abnormal immune responses are the major causes of mortality and morbidity in diabetic patients. CD4+CD25+T regulatory cells (Tregs) play pivotal roles in controlling immune homeostasis, immunity and tolerance. The effect of hyperglycemia on CD4+CD25+Tregs has not yet been addressed. Here we used streptozotocin (STZ)-induced diabetic mice to study the effects of long-term hyperglycemia on CD4+CD25+Tregs in vivo. Four months after the onset of diabetes, the frequency of CD4+CD25+Foxp3+ T regulatory cells was significantly elevated in the spleen, peripheral blood lymphocytes (PBLs), peripheral lymph nodes (pLNs) and mesenteric LNs (mLNs). CD4+CD25+Tregs obtained from mice with diabetes displayed defective immunosuppressive functions and an activated/memory phenotype. Insulin administration rescued these changes in the CD4+CD25+ Tregs of diabetic mice. The percentage of thymic CD4+CD25+ naturally occurring Tregs (nTregs) and peripheral CD4+Helios+Foxp3+ nTregs were markedly enhanced in diabetic mice, indicating that thymic output contributed to the increased frequency of peripheral CD4+CD25+Tregs in diabetic mice. In an in vitro assay in which Tregs were induced from CD4+CD25- T cells by transforming growth factor (TGF)-β, high glucose enhanced the efficiency of CD4+CD25+Foxp3+ inducible Tregs (iTregs) induction. In addition, CD4+CD25- T cells from diabetic mice were more susceptible to CD4+CD25+Foxp3+ iTreg differentiation than those cells from control mice. These data, together with the enhanced frequency of CD4+Helios-Foxp3+ iTregs in the periphery of mice with diabetes, indicate that enhanced CD4+CD25+Foxp3+ iTreg induction also contributes to a peripheral increase iCD4+CD25+Tregs in diabetic mice. Our data show that hyperglycemia may alter the frequency of CD4+CD25+Foxp3+ Tregs in mice, which may result in late-state immune dysfunction in patients with diabetes.     

Site-specific accumulation of recently activated CD4+ Foxp3+ regulatory T cells following adoptive transfer

CD4(+) Foxp3(+) regulatory T (Treg) cells are required for the maintenance of self-tolerance, as demonstrated by profound autoimmunity in mice and humans with inactivating Foxp3 mutations. Recent studies demonstrate that Treg cells are anatomically compartmentalized within secondary lymphoid organs based on their TCR repertoire and specific organ-protective function; however, whether this reflects differential homing or in situ selection is not known. Here, using Foxp3-GFP reporter mice, we have examined the ability of polyclonal Treg cells from cervical LNs to return to their site-of-origin following adoptive transfer to nonlymphopenic congenic recipients. We find that bulk cervical LN Treg cells do not home directly to cervical LNs but rather accumulate site specifically over time following transfer. Site-specific enrichment is both more rapid and more pronounced among a population of recently activated (CD69(+) ) Treg cells. These data suggest that compartmentalization of Treg cells within secondary lymphoid organs may be governed by antigen recognition and implicate CD69 as a potential marker of recently activated Treg cells recognizing locally expressed antigens.     

小鼠CD4~+ CD25~+ Foxp3~+调节性T细胞体外扩增

目的:本研究旨在探讨CD4+CD25+Foxp3+调节性T细胞体外扩增的方法。方法:采用磁珠分选小鼠CD4+T细胞,αCD3单克隆抗体包被24孔板,加入αCD28单克隆抗体、雷帕霉素、rhIL-2,培养3周后,流式细胞仪测定培养细胞中CD4+CD25+T细胞的含量,实时定量PCR检测CD4+CD25+T细胞Foxp3 mRNA的表达;单向混合淋巴细胞反应和增殖抑制试验测定扩增的CD4+CD25+T细胞的增殖及其抑制功能;ELISA检测培养上清中IL-10和TGF-β1的含量。结果:小鼠CD4+T细胞培养3周后,CD4+CD25+T细胞达(76.05±2.73)%,高于未加雷帕霉素组(52.17±1.36)%(P<0.001),磁珠分选的CD4+CD25+T细胞Foxp3 mRNA的表达是未加雷帕霉素组的5倍(P<0.001),增殖能力是未加雷帕霉素组的0.29倍(P<0.001),对CD4+T细胞增殖抑制能力是未加雷帕霉素组的3.6倍(P<0.001),培养上清中IL-10和TGF-β1分别是对照组的1.8倍和1.6倍(P<0.001)。结论:小鼠CD4+T细胞在含有1μg/ml的αCD28、rhIL-2 100 U/ml和终浓度为10 nmol/L雷帕霉素的培养体系中培养3周后能有效扩增CD4+CD25+Foxp3+调节性T细胞。     

CD4~+Foxp3~+调节性T细胞在恶性血液病中的变化及机制研究

目的:分析CD4+Foxp3+调节性T细胞(CD4+Foxp3+Treg)在恶性血液病患者外周血的比例变化,探讨CD4+Foxp3+Treg参与恶性血液病发病的可能机制。方法:用流式细胞仪检测急性白血病、淋巴瘤患者及健康对照外周血CD4+Foxp3+Treg细胞的比例;然后用小鼠的淋巴瘤细胞EL-4和红白血病瘤细胞FBL3的培养上清液与C57BL/6小鼠脾细胞共同培养72小时,RT-PCR检测Foxp3 mRNA的表达。结果:恶性血液病患者外周血CD4+Foxp3+Treg数量显著高于正常对照(14.9±2.92)%、(5.68±1.21)%,P<0.001。小鼠EL-4和FBL3细胞上清液均能够使小鼠脾细胞Foxp3 mRNA表达水平明显增高。结论:恶性血液病患者CD4+Foxp3+Treg比例增高可能导致抗肿瘤免疫功能低下,此外肿瘤细胞分泌的可溶性物质使Foxp3表达增高,增强了CD4+Foxp3+Treg细胞的抑制功能,使肿瘤易于生长和转移。