γ-干扰素诱导人外周血CD4~+CD25~-T细胞转化为CD4~+CD25~+调节性T细胞的可行性研究

目的研究γ干扰素(interferon-γ,IFN-γ)将人外周血CD4~+CD25~- T细胞诱导为CD4~+CD25~+调节性T细胞(CD4~+CD25~+Tregs)的可行性。方法采用免疫磁珠法分离19名健康志愿者外周血单个核细胞中的CD4~+CD25~- T细胞,在抗CD3和抗CD28抗体存在条件下给予不同水平IFN-γ刺激72 h,以流式细胞仪检测CD4~+CD25~+T细胞比例并以real time-PCR法检测其特异性表型标志FoxP3表达水平。将诱导生成的CD4~+CD25~+T细胞与自体CD4~+CD25~-T细胞共培养检测诱导生成的CD4~+CD25~+T细胞抑制CD4~+CD25~-T细胞增殖的能力。结果IFN-γ刺激后CD4~+CD25~+T细胞比例上调且其FoxP3表达较诱导前明显增高,当IFN-γ水平为20、40 ng/mL时,CD4~+CD25~+T细胞上调比例最高;40 ng/mL IFN-γ诱导的CD4~+CD25~+T细胞FoxP3表达最高,但不及CD4~+CD25~+Tregs。IFN-γ诱导所得CD4~+CD25~+T细胞可抑制自体CD4~+CD25~-T细胞增殖,40 ng/mL IFN-γ诱导的CD4~+CD25~+T细胞抑制能力与CD4~+CD25~+Tregs相当。结论 IFN-γ可诱导CD4~+CD25~-T细胞转化为CD4~+CD25~+T细胞,当IFN-γ水平为40 ng/mL时诱导率最高且诱导生成的CD4~+CD25~+T细胞表型和功能与CD4~+CD25~+Tregs相当。     

FoxP3+ CD4+ CD25+调节性T细胞在重症肌无力发病机制中的作用

目的 在细胞与分子水平检验重症肌无力(myasthenia gravis,MG)患者外周血中CD4+CD25+调节性T细胞(CD4+CD25+Tregs)的表达缺陷,探讨CD4+CD25+Tregs亚群异常与MG发病间的关系.方法 流式细胞技术检测21例MG患者(11例经胸腺切除)与20名健康对照者(healthy controls,HCs)外周血CD4+CD25+Tregs及FoxP3+CD4+CD25+Tregs含量,实时荧光定量聚合酶链反应(RT-FQ-PCR)分析MG患者与HCs外周血CD4+CD25+Tregs中FoxP3 mRNA的表达.结果 MG患者外周血CD4+CD25+ Tregs占CD4+T细胞含量与HCs比较无统计学差异(P＞0.05).MG患者外周血FoxP3+CD4+CD25+ Tregs含量及FoxP3 mRNA表达量与HCs比较均显著性降低(P＜0.05);胸腺切除的MG患者与未经胸腺切除的MG患者外周血FoxP3+CD4+CD25+ Tregs含量及FoxP3mRNA表达量无统计学差异(P＞0.05).结论 MG患者外周血CD4+CD25+ Tregs数量正常,但其表面分子FoxP3的表达下调,这种CD4+CD25+ Tregs亚群的异常发现有助于深入阐明MG的免疫发病机制.     

睾丸内胰岛移植对CD4+ CD25+调节性T细胞分布的影响

背景：CD4+ CD25+调节性T细胞是维持机体免疫耐受的重要调控者，参与了多种移植免疫耐受的诱导。 目的：拟观察小鼠睾丸内胰岛移植后CD4+ CD25+调节性T细胞分布的特点。 设计、时间及地点：观察对照动物实验，2007-04/2008-01在江西省实验动物中心完成。 材料：成年Balb/c小鼠。 方法：分离小鼠胰岛细胞，采用胰管内注射胶原酶水浴消化及Ficoll 400不连续密度梯度离心法纯化，以双硫腙染色，计算胰岛细胞纯度，以体外葡萄糖刺激胰岛素分泌试验判定胰岛细胞功能。将胰岛移植至小鼠睾丸或肾包膜下，每只移植300~400个胰岛。在胰岛移植24 h后麻醉处死小鼠，取脾脏、睾丸及淋巴结，制成细胞悬液，免疫磁珠法分离CD4+CD25+ T细胞，通过流式细胞仪分析计数。 主要观察指标：胰岛细胞的纯度及功能，CD4+ CD25+调节性T细胞的分布。 结果：纯化后每只胰腺获得(478±53)个胰岛细胞，纯度为(81.5±12.3)%，纯化后细胞形态完好，活度大于90%。睾丸内胰岛移植时，睾丸、淋巴结及脾脏中CD4+CD25+调节性T细胞均显著增多（P < 0.05~0.01）。 结论：睾丸内胰岛移植能明显上调睾丸、淋巴结及脾脏中CD4+ CD25+调节性T细胞数量。     

重症肌无力患者外周血CD4+T细胞OX40的表达及作用机制研究

目的 分析重症肌无力(MG)患者外周血CD4+T细胞协同刺激分子OX40表达及其对FoxP3+CD4+CD25+调节性T细胞(Treg)的调控作用,初步探讨OX40在MG免疫学发病中的作用机制.方法 以流式细胞技术检测42例MG患者及38名健康对照的外周血OX40+CD4+T细胞、FoxP3+CD4+CD25+Treg表达水平,比较OX40表达在MG患者不同临床疾病状态、Osserman分型、临床绝对评分、胸腺病理类型等情况下的差异,并分析OX40对FoxP3+CD4+CD25+Treg细胞的影响.结果 (1) MG患者外周血OX40+CD4+T细胞占淋巴细胞百分比高于健康对照组(P<0.01).(2)MG患者OX40+CD4+T细胞百分比在发作或加重期高于缓解期(P＜0.05);在临床绝对评分呈中、重度患者OX40+CD4+T细胞百分比高于轻度患者(均P＜0.05);Osserman Ⅱ、Ⅳ型患者OX40+CD4+T细胞百分比高于Ⅰ型患者(均P＜0.05);胸腺增生及胸腺瘤患者OX40+CD4+T细胞百分比高于胸腺正常患者(P＜0.05,P＜0.01).(3)MG患者外周血OX40+CD4+T细胞百分比与FoxP3+CD4+CD25+Treg细胞百分比呈负相关(r=-0.843,P=0.01).结论 协同刺激分子OX40参与MG发病,可能通过抑制FoxP3+CD4+CD25+Treg细胞生成发挥作用.     

同种异基因大鼠间充质干细胞移植对体内CD4+CD25+T细胞的影响**☆

背景：间充质干细胞的免疫抑制作用近年来逐渐得到证实并开始应用于临床,但相关研究成果主要来源于体外细胞实验。 目的：观察同种异基因大鼠骨髓间充质干细胞从静脉输入后对体内CD4+CD25+调节性T细胞的影响。 设计、时间及地点：以动物为对象的对照观察实验,于2008-03/09在南方医科大学珠江医院移植免疫研究所完成。 材料：Wistar大鼠6只用于制备骨髓间充质干细胞,SD大鼠20只作为输注骨髓间充质干细胞的受体鼠。 方法：从Wistar大鼠骨髓分离培养间充质干细胞,取第3~5代细胞进行实验。①体外淋巴细胞增殖实验：首先将间充质干细胞悬液从尾静脉注入SD大鼠体内10 d后脱臼处死,取大鼠脾脏制备脾淋巴细胞。实验分5组：分别为脾淋巴细胞/骨髓间充质干细胞为1∶10,1∶50,1∶100+ConA 5 μg组,脾淋巴细胞+ConA 5μg组（增殖组）,单纯淋巴细胞组(空白组),检测共培养体系中CD4+CD25+ / CD4+ T淋巴细胞比率。②体内注射骨髓间充质干细胞实验：将5×109 L-1,5×108 L-1,5×107 L-1间充质干细胞及PBS静脉输入SD大鼠体内,10 d后取受体鼠胸腺、脾脏、外周血检测CD4+CD25+ / CD4+ T淋巴细胞比率。 主要观察指标：①淋巴细胞增殖体系中CD4+CD25+/CD4+比率的变化。②SD大鼠胸腺、脾脏、外周血CD4+CD25+/CD4+比率的变化。 结果: ①体外淋巴细胞共培养体系中,1∶10组T细胞亚群CD4+ CD25+ / CD4+细胞百分率较增殖组显著升高（P < 0.01）。②体内输注间充质干细胞达5×109 L-1的受体鼠外周血和脾脏CD4+CD25+ / CD4+ T细胞比率上升,与输注PBS组相比有显著差异（P < 0.05）,而在胸腺中各组比率无明显差异。 结论：高浓度同种异基因大鼠间充质干细胞不仅可以在体外实验中增加CD4+CD25+ / CD4+ T细胞比率,静脉输入后仍具有相同作用。     

异基因造血干细胞移植中CD4+CD25+调节性T细胞与急性移植物抗宿主病的相关性

CD4+CD25+调节性T细胞是调节性T细胞的一个重要亚群,在诱导移植耐受方面起着重要作用。 目的：综述CD4+CD25+调节性T细胞的特性、免疫调节作用机制及其与急性移植物抗宿主病的相关性。 方法：应用计算机检索PubMed、Medline数据库1990-01/2010-02,维普数据库2000-01/2010-02以及运用Google网络数据库有关CD4+CD25+调节性T细胞及其他与急性移植物抗宿主病相关性研究的文献。 结果与结论：CD4+CD25+调节性T细胞在异基因造血干细胞移植后产生的急性移植物抗宿主病中起着重要作用,不仅能有效预防和治疗急性移植物抗宿主病,同时还能保留移植物抗白血病效应作用。随着研究的深入,发现CD4+CD25+调节性T细胞很有希望成为急性移植物抗宿主病早期风险预测的一项重要实验诊断指标,并且有可能通过调控移植后患者体内CD4+CD25+调节性T细胞水平来预防和控制急性移植物抗宿主病的发生。但是,要将CD4+CD25+调节性T细胞应用于临床尚有问题有待解决,例如CD4+CD25+调节性T细胞细胞表面特异性标志物是什么,如何提高CD4+CD25+调节性T细胞在人体内的活性以及怎样充分发挥其免疫抑制功能等。     

肾移植患者外周血中的CD4+ CD25+ CD127low/-调节性T细胞

背景：越来越多的实验在分析免疫耐受标志,以期能够更好地辅助患者进行移植后免疫抑制治疗。 目的：分析肾移植后患者外周血中CD4+ CD25+ CD127low/-调节性T细胞在肾移植免疫耐受中的作用。 方法：采集62例肾移植后患者(急性排斥反应组22例,移植稳定组40例)及20例健康对照者的外周抗凝血,经免疫染色,应用流式细胞仪分析CD4+ CD25+ CD127low/-调节性T细胞所占CD4+ T细胞百分含量,同时采用ELISA方法检测患者血清中白细胞介素2和白细胞介素10的质量浓度。 结果与结论：移植稳定组中CD4+ CD25+ CD127low/-调节性T细胞所占CD4+ T细胞百分含量显著高于健康对照组和急性反应排斥组(P < 0.01);CD4+ CD25+ CD127low/-调节性T细胞百分含量与白细胞介素2呈显著负相关(P < 0.05),与白细胞介素10呈显著正相关(P < 0.01)。提示CD4+ CD25+ CD127low/-调节性T细胞在肾移植后免疫耐受的机制中发挥了一定作用。     

抑郁症中糖皮质激素受体与CD4+CD25+调节性T细胞的关系及其所致免疫失衡机制的初步探讨

目的 研究抑郁症患者外周血细胞因子白介素-10(IL-10)、转化生长因子β(TG-β)的变化、CD4+CD25+调节性T细胞(Treg)的数量及其特征性标志叉头样转录因子P3(Foxp3)的表达与糖皮质激素受体(GR)的表达之间的关系,探讨抑郁症患者免疫失衡的可能机制.方法 纳入36例抑郁症患者和36名正常对照,根据Hamilton抑郁量表总分将患者划分为轻、中和重不同抑郁程度组;利用ELISA方法测定受试者外周血血清细胞因子IL-10、TGF-β浓度;逆转录-聚合酶链反应(RT-PCR)检测GR的α及β两种亚型(GRα、GRβ)、Foxp3 mRNA表达水平;免疫磁珠分离CD4+ CD25+ Treg,共聚焦显微镜观察Foxp3与GR在CD4+CD 25+ Treg上的共表达.结果 与正常对照组比较,患者组血清IL-10、TGF-β的水平降低(P＜0.05),外周血CD4+ CD25+ Treg数量及在CD4+T细胞中的比例明显少于对照组(P＜0.01),且重度抑郁组上述指标明显低于中度和轻度抑郁组(P＜0.01).抑郁各组单个核细胞GRamRNA和FoxP3 mRNA表达水平随抑郁程度而降低(P＜0.01),GRβmRNA却在重度抑郁组表达增加.共聚焦显微镜下可观察到重度抑郁患者CD4+CD25+Treg上GR与Foxp3表达显著减少.结论 糖皮质激素受体可能通过影响调节性T细胞的功能和数量在抑郁症患者免疫失衡的病理生理机制中发挥着重要的作用.     

脑梗死大鼠CD4~+CD25~+调节性T细胞的作用

目的研究脑梗死大鼠血中CD4+CD25+调节性T细胞的变化及作用。方法大鼠40只,随机分为5组,线栓法制作大脑中动脉闭塞模型后分别检测24h、72h、1周、2周时血中CD4+CD25+调节性T细胞的表达。结果不同时间水平之间CD4+CD25+调节性T细胞的表达有显著性差异(P0.05),CD4+CD25+调节性T细胞的表达MCAO组明显高于假手术组(P0.01)。时间与分组间有交互效应(P0.01),但趋势不同。结论 CD4+CD25+调节性对脑梗死有潜在的脑保护作用。     

载脂蛋白E拟肽对实验性自身免疫性脑脊髓炎小鼠脑脊髓CD4+、CD8+T淋巴细胞表达的影响

目的 探讨载脂蛋白(Apo)E拟肽对实验性自身免疫性脑脊髓炎(EAE)小鼠脑脊髓CD4+、CD8+T淋巴细胞表达的影响.方法 40只C57BL/6J雌性小鼠随机分成EAE组、EAE治疗组、正常对照组、正常治疗组;采用髓鞘少突胶质细胞糖蛋白制备的完全抗原诱导EAE模型小鼠.免疫诱导次日,EAE治疗组和正常治疗组小鼠每隔2d皮下注射ApoE拟肽,EAE组和正常对照组小鼠皮下注射等量的生理盐水.免疫诱导后各组每日进行神经功能缺损评分(NDS);35 d后用免疫组化检测各组小鼠脑脊髓CD4+T细胞、CD8+T细胞的表达.结果 EAE治疗组NDS的峰值及终末评分显著低于EAE组(均P＜0.05).与正常对照组及正常治疗组比较,EAE组大脑、脑干和脊髓中CD4+T细胞数明显增高,大脑CD8+T细胞数明显增高(均P＜0.05).EAE治疗组小鼠大脑、脑干、脊髓组织CD4+T细胞表达显著低于EAE组(均P＜0.05);两组间CD8+T细胞表达水平的差异无统计学意义.结论 ApoE拟肽可抑制CD4+T细胞的表达,减轻免疫炎症反应,对EAE小鼠有神经保护作用;而对CD8+T细胞的表达无明显影响.     

CD4+T-bet+, CD4+pSTAT3+ and CD8+T-bet+ T cells accumulate in peripheral blood during NZB treatment

Circulating T cells and monocytes expressing T-bet, pSTAT1 and pSTAT3 increase in relapsing-remitting multiple sclerosis (RRMS) during relapse. Natalizumab (NZB) is an effective drug in RRMS, but exacerbation of the disease after its discontinuation has been described in some patients. The aim of this research was to study the effect of NZB treatment on circulating lymphomonocyte subpopulations expressing T-bet, pSTAT1, pSTAT3 and CD4+CD25+Foxp3+ regulatory T cells. Flow cytometry was used to evaluate the percentages of circulating CD4+ and CD8+ T cells, CD14+ monocytes and B cells expressing T-bet, pSTAT1, and pSTAT3, and CD4+CD25+Foxp3+ regulatory T cells from RRMS patients before and after 6-12 NZB infusions. In NZB-treated RRMS patients, the percentages of CD4+pSTAT1+ and CD8+pSTAT1+ T cells, CD14+pSTAT1+ monocytes, CD4+T-bet+, CD8+T-bet+ and CD4+pSTAT3+ T cells and CD14+pSTAT3+ monocytes increased after 12 drug infusions and were similar to those observed in untreated relapsing RRMS patients. Otherwise in vitro NZB exposure of peripheral blood mononuclear cells from untreated RRMS patients and controls had no effect. It was concluded that NZB treatment determines an accumulation of CD4+pSTAT1+, CD8+pSTAT1+, CD4+T-bet+, CD8+T-bet+ and CD4+STAT3+ T cells in peripheral blood that may account for the exacerbation of the disease observed in some patients after the discontinuation of the drug.     

Decrease of CD4+ CD45+ T-cells in chronic-progressive multiple sclerosis

Summary Circulating lymphocyte subpopulations defined by anti-CD45 and other more common T-cell-specific monoclonal antibodies were analysed in 77 patients with multiple sclerosis and 38 healthy controls. A selective decrease of CD4+ CD45+ cell percentages and absolute numbers in chronic-progressive patients was found; in 13 out of 26 patients this subpopulation was less than 11% CD4+ CD45+ cells. Similarly, the whole CD45+ cell subset, as well as CD45+ cells expressed as percentages of CD4+ cells, were significantly reduced in chronic-progressive multiple sclerosis. CD4+ CD45+ cells, commonly termed inducer of suppression T-lymphocytes, did not correlate with percentages or numbers of CD8+ cells. It is concluded that suppressor inducer T-cells act on the CD8+ subset function rather than reducing CD8+ cell numbers. Since CD4+ CD45+ cells represent an early stage of lymphocyte maturation (naive T-cells), an under-representation of this subpopulation in active multiple sclerosis might reflect an increased conversion of naive cells into memory cells. This concept may be relevant for a better understanding of the disease pathogenesis.     

Temporal kinetics of CD8+CD28+ and CD8+CD28− T lymphocytes in the injured rat spinal cord

This study aims to explore the temporal changes of cytotoxic CD8⁺CD28⁺ and regulatory CD8⁺ CD28⁻ T‐cell subsets in the lesion microenvironment after spinal cord injury (SCI) in rats, by combination of immunohistochemistry (IHC) and flow cytometry (FCM). In the sham‐opened spinal cord, few CD8⁺ T cells were found. After SCI, the CD8⁺ T cells were detected at one day post‐injury (dpi), then markedly increased and were significantly higher at 3, 7, and 14 dpi compared with one dpi (p < 0.01), the highest being seven dpi. In CD8⁺ T cells, more than 90% were CD28⁺, and there were only small part of CD28⁻ ( < 10%). After 14 days, the infiltrated CD8⁺ T cells were significantly decreased, and few could be found in good condition at 21 and 28 dpi. Annexin V and propidium iodide (PI) staining showed that the percentages of apoptotic/necrotic CD8⁺ cells at 14 dpi and 21 dpi were significantly higher than those of the other early time‐points (p < 0.01). These results indicate that CD8⁺ T cells could rapidly infiltrate into the injured spinal cords and survive two weeks, however, cytotoxic CD8⁺ T cells were dominant. Therefore, two weeks after injury might be the “time window” for treating SCI by prolonging survival times and increasing the fraction of CD8⁺ regulatory T‐cells. © 2016 Wiley Periodicals, Inc.     

Specificity of regulatory CD4+CD25+ T cells for self-T cell receptor determinants

Although the phenotypic and regulatory properties of the CD4(+)CD25(+) T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4(+)CD25(+) Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline-encoded CDR3beta sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR-reactive T cells required cell-cell contact and involved CTLA-4, GITR, IL-10, and IL-17. Thus, the T-T regulatory network includes Treg cells with specificity directed toward self-TCR determinants.     

Increase in peripheral CD4 bright+ CD8 dull+ T cells in Parkinson disease

BACKGROUND: Immune abnormalities are known to be involved in the pathogenesis of sporadic Parkinson disease. OBJECTIVE: To examine whether abnormalities in peripheral lymphocytes exist in Parkinson disease. METHODS: Immune mediators, including CD1a, CD3, CD4, CD8, CD45RO, and Fas (CD95), were examined in peripheral lymphocytes of patients by 3-color flow cytometry. RESULTS: Patients with Parkinson disease displayed a significantly greater population of circulating CD3+ CD4 bright+ CD8 dull+ lymphocytes than age-matched control subjects (P =.005) and patients with cerebrovascular disease (P =.002). The increase in these cells appeared to continue for at least 17 months. These T cells also expressed CD45RO and Fas, markers for activated T cells, while CD1a, a marker for thymic T cells, was negative, suggesting that these cells are mature T cells with immune activities. CONCLUSIONS: As CD4+ CD8+ T cells are known to increase after some specific viral infections, the continuous increase in CD4 bright+ CD8 dull+ T cells shown here may indicate postinfectious immune abnormalities that are possibly associated with the pathogenesis of this slowly progressive, multifactorial neurodegenerative disease.     

CD26 + CD4 + T cell counts and attack risk in interferon-treated multiple sclerosis

Biomarkers that allow the identification of patients with multiple sclerosis (MS) with an insufficient response to immunomodulatory treatment would be desirable, as currently available treatments are only incompletely efficacious. Previous studies have shown that the expression of CD25, CD26 and CCR5 on T cells is altered in patients with active MS. We studied the expression of these molecules by flow cytometry in patients followed for six months during immunomodulatory treatment. In interferon (IFN)-beta-treated patients, we found that the hazard ratio for developing an attack was 28 in patients with CD26 + CD4 + T cell counts above median, and this risk was independent of the risk conferred by neutralizing anti-IFN-beta antibodies. CD26 + CD4 + T cell counts may identify patients with MS at increased risk of attack during treatment with IFN-beta.     

Acute aerobic exercise in humans increases cytokine expression in CD27− but not CD27+ CD8+ T-cells

Exercise alters the percentage of CD8⁺ T-cells in the bloodstream expressing type I and type II cytokines. It is unknown if this reflects a change in cytokine expression within individual cells, or whether these observations result from the exercise-induced shift in the proportions of early/intermediate (CD27⁺) and late (CD27⁻) differentiated cells, which have vastly different cytokine profiles. 16 males cycled for 60 min at 95% maximal steady state. Mononuclear cells isolated from blood collected before, immediately after, and 1 h after exercise were cultured overnight with and without phytohaemagglutinin stimulation. CD8⁺ T-cells were assessed for differentiation markers and intracellular cytokine expression by flow cytometry. The numbers and percentage of CD27⁻CD8⁺ T-cells increased immediately after exercise and fell below pre-exercise values 1 h later. At 1 h after exercise, an increased number and percentage of CD8⁺ T-cells expressing IL-2, IFN-γ, TNF-α, IL-6, IL-4, and IL-10 was observed in both stimulated and unstimulated cells. The cytokine response to exercise was confined to CD27⁻CD8⁺ T-cells, although cytokine expression among CD8⁺ T-cells was highest when the proportion of CD27⁻CD8⁺ T-cells was lowest. Moreover, the cytokine response to exercise could be predicted by the number of late cells in resting blood: cytokine expression was highest among those with low resting proportions of late cells. We conclude that exercise-induced changes in the percentage of CD8⁺ T-cells expressing cytokines are not due to proportional shifts in early/intermediate and late differentiated T-cells. Exercise may prime late-differentiated blood CD8⁺ T-cells to initiate effector functions in preparation for their extravasation into the tissues.