In vivo depletion of CD4+CD25+ regulatory T cells in cats

To establish a characterized model of regulatory T cell (Treg) depletion in the cat we assessed the kinetics of depletion and rebound in peripheral and central lymphoid compartments after treatment with anti-CD25 antibody as determined by cell surface markers and FOXP3 mRNA expression. An 82% decrease in circulating CD4+CD25+ Tregs was observed by day 11 after treatment. CD4+CD25+ cells were also reduced in the thymus (69%), secondary lymphoid tissues (66%), and gut (67%). Although CD4+CD25+ cells rebound by day 35 post-treatment, FOXP3 levels remain depressed suggesting anti-CD25 antibody treatment has a sustainable diminutive effect on the Treg population. To determine whether CD25+ Treg depletion strategies also deplete activated CD25+ effector cells, cats were immunized with feline immunodeficiency virus (FIV) p24-GST recombinant protein, allowing them to develop a measurable memory response, prior to depletion with anti-CD25 antibody. Anti-FIV p24-GST effector cell activity in peripheral blood after depletion was sustained as determined by antigen-specific T cell proliferation and humoral responses against FIV p24-GST with an ELISA for antigen-specific feline IgG. Furthermore, development of an anti-mouse response in Treg-depleted cats was similar to control levels indicating the retained capacity to respond to a novel antigen. We conclude that despite alterations in CD25+ cell levels during depletion, the feline immune system remains functional. We demonstrate here a model for the study of disease pathogenesis in the context of reduced numbers of immunosuppressive CD4+CD25+ Tregs throughout the feline immune system.     

Phenotypic and functional characteristics of CD4+ CD39+ FOXP3+ and CD4+ CD39+ FOXP3neg T-cell subsets in cancer patients

Human CD4(+) CD39(+) regulatory T (Treg) cells hydrolyze exogenous adenosine triphosphate (ATP) and participate in immunosuppressive adenosine production. They contain two T-cell subsets whose role in mediating suppression is not understood. Frequencies of both CD4(+) CD39(+) subsets were evaluated in peripheral blood lymphocytes of 57 cancer patients and in tumor infiltrating lymphocytes (TILs) of 6 patients. CD4(+) CD39(+) and CD4(+) CD39(neg) T cells isolated using immunobeads and cell sorting were cultured under various conditions. Their conversion into CD39(+) FOXP3(+) CD25(+) or CD39(+) FOX(neg) CD25(neg) cells was monitored by multiparameter flow cytometry. Hydrolysis of exogenous ATP was measured in luminescence assays. Two CD4(+) CD39(+) cell subsets differing in expression of CD25, FOXP3, CTLA-4, CD121a, PD-1, latency associated peptide (LAP), glycoprotein A repetitions predominant (GARP), and the cytokine profile accumulated with equal frequencies in the blood and tumor tissues of cancer patients. The frequency of both subsets was significantly increased in cancer. CD39 expression levels correlated with the subsets' ability to hydrolyze ATP. Conventional CD4(+) CD39(neg) T cells incubated with IL-2 + TGF-β expanded to generate CD4(+) CD39(+) FOXP3(+) Treg cells, while CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset cells stimulated via the TCR and IL-2 converted to FOXP3(+) CTLA4(+) CD25(+) TGF-β-expressing Treg cells. Among CD4(+) CD39(+) Treg cells, the CD4(+) CD39(+) FOXP3(neg) CD25(neg) subset serves as a reservoir of cells able to convert to Treg cells upon activation by environmental signals.     

Developmental changes of FOXP3-expressing CD4+CD25+ regulatory T cells and their impairment in patients with FOXP3 gene mutations

FOXP3 is required for the generation and function of CD4(+)CD25(+) regulatory T (Treg) cells. To elucidate the biological role of Treg cells, we used a monoclonal anti-FOXP3 antibody to examine the frequencies of Treg cells during child development. The percentages of CD4(+)CD25(+)FOXP3(+) T cells were constant shortly from after birth through adulthood. CD4(+)CD25(+)FOXP3(+) T cells in cord blood showed the naive CD45RA(+)CD45RO(-) phenotype, whereas adult CD4(+)CD25(+)FOXP3(+) T cells expressed mostly the memory CD45RA(-)CD45RO(+) phenotype. The age-dependent dominance of memory CD4(+)CD25(+)FOXP3(+) T cells implies functional differences between naive and memory Treg cells. Notably, four patients with FOXP3 gene mutations revealed a paucity of CD4(+)CD25(+)FOXP3(+) T cells. Importantly, one patient with a frame shift mutation, who showed typical symptoms of IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked), exhibited marked T cell activation, whereas others with missense mutations, who were clinically milder, did not. This observation suggests a possible genotype-phenotype correlation in IPEX.     

Thymic production of human FOXP3(+) regulatory T cells is stable but does not correlate with peripheral FOXP3 expression

In humans functionally mature FOXP3⁺ regulatory T (Treg) cells can be found already in the fetus, but the kinetics of their maturation is still unknown. Here, we show that from birth to until 10 years of age the thymic production of FOXP3⁺ Treg cells is very stable and correlates with T-lymphopoiesis in general. The level of FOXP3 expression in the blood was also very stable, even when children and adults were compared, but there was no correlation between thymic and peripheral FOXP3 levels. Analysis of the cell cycle-associated marker Ki67 showed that a substantial fraction of peripheral FOXP3⁺ cells is dividing. This characteristic was obtained in the periphery, since it was not observed in thymic CD4⁺ FOXP3⁺ cells. These data suggest that the thymic output of human Treg cells is intrinsically stable, while in the periphery the increased rate of proliferation severs the connection between production and homeostatic maintenance of the FOXP3⁺ Treg cell population.     

卡介苗刺激后CD8~+T细胞活化、增殖及其调节

目的:探讨卡介苗(BCG)刺激后,结核菌素试验阳性(PPD+)正常人外周血单个核细胞(PBMCs)中CD8+T细胞的活化、增殖、细胞因子产生及调节性T细胞(Treg)对其调节作用。方法:体外用BCG刺激PPD+正常人PBMCs,检测CD8+T细胞的细胞因子产生、活化和增殖。纯化后获得调节性T细胞(Treg)和CD25-细胞,检测Treg对CD8+T细胞增殖的调节作用。结果:BCG诱导CD8+T细胞表达CD69和CD25等活化分子。在低剂量IL-2存在的条件下,BCG诱导CD8+T细胞发生增殖,且增殖的CD8+T细胞大部分表达Granzyme-B。体外BCG短期刺激PBMCs后,CD8+T细胞几乎不产生IFN-γ、IL-2和TNF-α。此外,调节性T细胞抑制CD8+T细胞增殖。结论:BCG诱导CD8+T细胞活化、增殖和表达颗粒酶,Treg抑制CD8+T细胞增殖。     

老年初诊急性髓细胞白血病患者CD4+CD25+调节性T细胞的表达

背景：研究证实,很多恶性肿瘤患者体内CD4⁺CD25⁺调节性T细胞存在高表达,近期也有研究发现,急性髓细胞白血病患者外周血CD4⁺CD25⁺调节性T细胞同样表现出高比例表达。 目的：分析老年初诊急性髓细胞白血病患者CD4⁺CD25⁺调节性T细胞的表达特点。 方法：纳入初诊急性髓细胞白血病患者92例,将年龄在60岁以下者设为中青年组(n=22),年龄在60岁以上者设为老年观察组(n=70)。在老年观察组中,32例经规范化疗后完全缓解,设为完全缓解组;将余下38例设为老年组,依据FAB分型标准,分为M2 6例、M3 19例、M4 7例、M5 6例。另选择同期体检健康人群42名作为正常对照组。抽取受试者外周静脉血,检测CD4⁺CD25⁺调节性T细胞表达情况。 结果与结论：老年组、完全缓解组CD4⁺CD25^highFOXP3⁺调节性T细胞比例高于正常对照组(P < 0.01),并且老年组CD4⁺CD25^high FOXP3⁺调节性T细胞比例高于完全缓解组(P < 0.01)。老年组、完全缓解组CD4⁺FOXP3⁺T细胞比例高于正常对照组(P < 0.01),并且老年组CD4⁺ FOXP3⁺T细胞比例高于完全缓解组(P < 0.01)。老年组CD4⁺CD25^high FOXP3⁺调节性T细胞与CD4⁺ FOXP3⁺T细胞比例高于中青年组(P < 0.01)。老年组不同分型间CD4⁺CD25^high FOXP3⁺调节性T细胞和CD4⁺ FOXP3⁺T细胞比例比较差异均无显著性意义(P > 0.05)。Pearson相关性检验结果显示,老年初诊急性髓细胞白血病患者外周血CD4⁺CD25^high FOXP3⁺调节性T细胞比例和CD4⁺ FOXP3⁺T细胞比例呈正相关(r=0.87,P=0.019)。表明老年初诊急性髓细胞白血病患者CD4⁺CD25⁺调节性T细胞比例高于健康人群和中青年急性髓细胞白血病患者。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

Aging and human CD4 regulatory T cells

Alterations in immunity that occur with aging likely contribute to the development of infection, malignancy and inflammatory diseases. Naturally occurring CD4⁺ regulatory T cells (Treg) expressing high levels of CD25 and forkhead box P3 (FOXP3) are essential for regulating immune responses. Here we investigated the effect of aging on the number, phenotypes and function of CD4⁺ Treg in humans. The frequency and phenotypic characteristics of CD4⁺, FOXP3⁺ T cells as well as their capacity to suppress inflammatory cytokine production and proliferation of CD4⁺, CD25⁻ T cells (target cells) were comparable in young (age ≤40) and elderly (age ≥65) individuals. However, when CD4⁺, FOXP3⁺ Treg and CD4⁺, CD25⁻ T cells were co-cultured at a ratio of 1:1, the production of anti-inflammatory cytokine IL-10 from CD4⁺, CD25⁻ T cells was more potently suppressed in the elderly than in the young. This finding was not due to changes in CTLA-4 expression or apoptosis of CD4⁺, FOXP3⁺ Treg and CD4⁺, CD25⁻ T cells. Taken together, our observations suggest that aging may affect the capacity of CD4⁺, FOXP3⁺ T cells in regulating IL-10 production from target CD4⁺ T cells in humans although their other cellular characteristics remain unchanged.     

A CFSE based assay for measuring CD4+CD25+ regulatory T cell mediated suppression of auto-antigen specific and polyclonal T cell responses

CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as suppressors of immune mediated reactions. The analysis of Treg function in patients with autoimmune, allergic or oncogenic diseases has emerged over the past years. In the present study we describe a CFSE based protocol to measure Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in coculture experiments by means of a CFSE based and a classical [(3)H]thymidine incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic stimulation, data obtained by the CFSE assay allowed the detection of a significant Treg suppression whereas this was clearly underestimated using the [(3)H]thymidine assay. In addition, an indirect CFSE based method was developed to analyze antigen specific responses of total CD4(+) T cells and Treg depleted CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show an impaired suppression of myelin reactive T cells when compared to healthy controls. Moreover, this experimental setup permits the measurement of cytokine production of the antigen proliferated CFSE(low) T cells by additional flow cytometric analyses. In conclusion, the described CFSE based Treg suppression assay is a valuable tool to study suppressor T cells in (auto)immune disorders.     

CD4+ CD25+调节性T细胞在HIV/AIDS患者中的作用及其与HIV病毒载量的相关性研究

目的 研究HIV感染者／AIDS患者外周血CD4^＋ CD25^＋ 调节性T细胞（CD4^＋ CD25^＋ regulatory Tcell，Treg）频率、功能及其临床意义。方法 选择31例HIV感染者／AIDS患者和30例健康对照者，采用流式细胞仪检测各组外周血Treg的表型和频率。采取MACS磁珠分选CD4^＋CD25^＋T细胞，利用[^3H]胸腺嘧啶掺入法检测CD4^＋ CD25^＋T细胞在特异性HIV抗原刺激下对CD4^＋ CD25-T细胞的增殖影响。结果HIV／AIDS患者组与正常对照组相比较，外周血CD4^＋ CD25^＋ T细胞频率在统计学上差异无统计学意义。与正常对照组比较，HIV感染者外周血CD4^＋ CD25^＋ T细胞频率升高，差异有统计学意义（P〈0.01）；与正常对照组比较，AIDS患者者外周血CD4^＋ CD25^＋ T细胞频率降低，差异有统计学意义（P〈0．0001）。HIV RNA病毒载量与患者外周血CD4^＋ CD25^＋ T细胞数量呈正相关性（P〈0．01）。CD4^＋ CD25^＋ T细胞具有抑制HIV特异性的CD4^＋ CD25^- T细胞的增殖作用。结论HIV感染者／AIDS患者的细胞免疫功能紊乱，CD4^＋ CD25^＋ T细胞能抑制HIV感染者／AIDS患者的HIV特异性细胞免疫反应，促进HIV病毒复制，与形成持续HIV感染有关。     

The frequency of regulatory CD3+CD8+CD28- CD25+ T lymphocytes in human peripheral blood increases with age

Aging is commonly associated with immune deficiency and dysregulation. The aging of the immune system involves a progressive reduction in na?ve T cell output associated with thymic involution and peripheral expansion of oligoclonal memory T cells. We have investigated frequency, phenotype, and function of CD3+CD8+CD28(-)CD25+ T cells in healthy volunteers over a wide age range. We demonstrate that the frequency of CD3+CD8+CD28(-)CD25+ T cells in healthy volunteers increases with age. Peripheral CD3+CD8+CD28(-)CD25+ T cells share phenotypic and functional features with CD3+CD4+CD25+ regulatory T cells (Tregs): In particular, they strongly express CTLA-4 and forkhead box P3. We observed that in vitro, functional titration assays of CD3+CD8+CD28(-)CD25+ T cells show equivalent regulatory function in young and elderly donors, with suppression of proliferation and cytokine production in response to polyclonal T cell stimulation. Finally, CD3+CD8+CD28(-)CD25+ T cells seem to specifically express the CD122 receptor. Altogether, these observations demonstrate an increase in peripheral blood CD8+ Tregs associated with aging.     

Regulatory activity of human CD4 CD25 T cells depends on allergen concentration, type of allergen and atopy status of the donor

Regulatory CD4+ CD25+ FoxP3-positive T cells (Treg) are functional in most atopic patients with allergic rhinitis and are able to inhibit T helper type 1 (Th1) and Th2 cytokine production of CD4+ CD25- T cells. This study was designed to analyse the following additional aspects: influence of allergen concentration, influence of the type of allergen, and influence of the atopy status of the donor on the strength of the regulatory activity. CD4+ CD25- T cells from healthy non-atopic controls or from grass-pollen-allergic or wasp-venom-allergic donors were stimulated alone or in the presence of Treg with autologous mature monocyte-derived dendritic cells which were pulsed with different concentrations of the respective allergens. Treg from grass-pollen-allergic donors failed to inhibit proliferation but not cytokine production of CD4+ CD25- T cells at high antigen doses while Treg from non-atopic donors did not fail at these allergen concentrations. Proliferative responses and cytokine production of CD4+ CD25- T cells from most of the examined wasp-venom-allergic patients were not inhibited at any concentration of wasp venom. The use of wasp venom- or phospholipase A2-pulsed dendritic cells for stimulation of CD4+ CD25- T cells from donors who were not allergic to wasp stings only resulted in an inhibited proliferation and Th2 cytokine production by Treg at 10-fold lower than the optimal concentration, while interferon-gamma production was inhibited at all concentrations investigated. These data demonstrate that in allergic diseases the function of Treg is dependent on the concentration and the type of the respective allergen with different thresholds for individual allergens and patients.     

Staphylococcus aureus convert neonatal conventional CD4+ T cells into FOXP3+ CD25+ CD127low T cells via the PD‐1/PD‐L1 axis

The gut microbiota provides an important stimulus for the induction of regulatory T (Treg) cells in mice, whether this applies to newborn children is unknown. In Swedish children, Staphylococcus aureus has become a common early colonizer of the gut. Here, we sought to study the effects of bacterial stimulation on neonatal CD4⁺ T cells for the induction of CD25⁺ CD127^low Treg cells in vitro. The proportion of circulating CD25⁺ CD127^low Treg cells and their expression of FOXP3, Helios and CTLA‐4 was examined in newborns and adults. To evaluate if commensal gut bacteria could induce Treg cells, CellTrace violet‐stained non‐Treg cells from cord or peripheral blood from adults were co‐cultured with autologous CD25⁺ CD127^low Treg cells and remaining mononuclear cells and stimulated with S. aureus. Newborns had a significantly lower proportion of CD25⁺ CD127^low Treg cells than adults, but these cells were Helios⁺ and CTLA‐4⁺ to a higher extent than in adults. FOXP3⁺ CD25⁺ CD127^low T cells were induced mainly in neonatal CellTrace‐stained non‐Treg cells after stimulation with S. aureus. In cell cultures from adults, S. aureus induced CD25⁺ CD127^low T cells only if sorted naive CD45RA⁺ non‐Treg cells were used, but these cells expressed less FOXP3 than those induced from newborns. Sorted neonatal CD25⁺ CD127^low T cells from S. aureus‐stimulated cultures were still suppressive. Finally, blocking PD‐L1 during stimulation reduced the induction of FOXP3⁺ CD25⁺ CD127^low T cells. These results suggest that newborns have a higher proportion of circulating thymically derived Helios⁺ Treg cells than adults and that S. aureus possess an ability to convert neonatal conventional CD4⁺ T cells into FOXP3⁺ CD25⁺ CD127^low Treg cells via the PD‐1/PD‐L1 axis.     

CD4 T cell activation by myelin oligodendrocyte glycoprotein is suppressed by adult but not cord blood CD25+ T cells

Regulatory T cells expressing CD25 have been shown to protect rodents from organ-specific autoimmune diseases. Similar CD25+ cells with a memory phenotype exerting suppressive function after polyclonal or allogeneic stimulation are also present in adult human blood. We demonstrate that adult human CD25+ cells regulate the response to myelin oligodendrocyte glycoprotein (MOG), as depletion of CD25(+) cells increases responses of PBMC and the addition of purified CD25+ cells suppresses MOG-specific proliferation and IFN-gamma production of CD4(+)CD25(-) T cells. In contrast, cord blood CD25+ cells do not inhibit responses to self antigens, and only a small subpopulation of cord CD25+ cells expresses the typical phenotype of adult regulatory T cells (CD45RA(-) and GITR(+)) enabling suppression of polyclonal responses. We conclude that activation of self-reactive T cells in normal healthy individuals is prevented by the presence of self-antigen-specific CD25+ regulatory T cells and that the majority of these cells mature after birth.     

CD4~+CD25~+FOXP3~+调节性T细胞及其亚型在结直肠癌组织中分布和临床意义的研究

目的:探讨CD4~+Cff25~+FOXP3~+调节性T细胞(Treg)及其亚型在结直肠癌组织(colorectal carcinoma,CRC)的分布和临床病理特征的关系.方法:收集42例CRC新鲜手术标本,应用冰冻切片、免疫组织化学SP法检测肿瘤组织和癌旁组织中FOXP3~+阳性细胞数,分析其与临床病理特征的相关性;应用双重免疫组化检测FOXP3和ICOS表达,分析FOXP3~+ICOS~+Treg和FOXP3~+ICOS-Treg两种细胞亚型分布情况.结果:CRC患者肿瘤组织中FOXP3分布显著多于癌旁组织,差异显著(P＜0.001);中低分化组Treg细胞数明显高于高分化组(P＜0.01);淋巴结转移组Treg细胞数明显高于无淋巴结转移组(P＜0.05);无远处转移组Treg细胞数明显高于有远处转移组(P＜0.05);FOXP3+ICOS+Treg细胞亚群在CRC组织中高表达,与癌旁组织相比差异显著(P＜0.01).结论:CRC的发生发展与其癌组织局部微环境中Treg数量变化相关,Treg在CRC组织过量表达可能是导致肿瘤免疫逃逸的重要因素之一;抑制CRC微环境中浸润性Treg细胞功能可提高肿瘤局部免疫治疗效果.     

CD4+ CD25+调节性T细胞AICD机制的研究

目的探讨CD4^＋CD25^＋调节性T细胞活化诱导的细胞死亡（AICD）发生的机制。方法CD4^＋CD25^＋T细胞以磁性细胞分离器（MACS）从BALB／c小鼠或DO11．10小鼠的静息T细胞分离纯化。体外细胞增殖抑制实验证实其免疫调节作用。CD4^＋CD25^＋T细胞的AICD以CD3／CD28单克隆抗体活化或以特异性OVA323-339肽、抗原提呈细胞活化等两种方法获得。CD4^＋CD25^＋T细胞凋亡相关基因的表达通过实时定量PCR检测。流式细胞仪检测细胞的凋亡率。进一步观察FasL中和抗体、TRAIL中和抗体及caspase抑制剂zVAD-fmk对CD4^＋CD25^＋T细胞凋亡的影响。结果MACS成功分离CD4^＋CD25^＋T细胞，纯度可达98％，该细胞可特异性表达Foxp3基因，能明显抑制效应性T细胞的体外增殖。CD3／CD28抗体以及OVA特异性抗原活化8d的CD4^＋CD25^＋调节性T细胞AICD达39％～45％。活化前后的CD4^＋CD25^＋调节性T细胞死亡受体家族表达发生明显变化；FasL、TRAIL中和抗体及zVAD-fmk可明显抑制CD4^＋CD25^＋调节性T细胞的凋亡。结论FasL／Fas及其他凋亡相关分子可能参与了CD4^＋CD25^＋调节性T细胞的凋亡。