IL-2协同抗CD45RB抗体调节Treg/Th17细胞的比例并延长小鼠移植皮肤存活时间

目的:研究IL-2在抗CD45RB抗体诱导免疫耐受中对Treg/Th17细胞分化的影响,进一步阐明抗CD45RB抗体诱导免疫耐受的机制。方法:用免疫磁珠分选C57BL/6小鼠脾脏中的CD4+T细胞,在抗CD45RB抗体与IL-2的作用下培养72小时后,流式检测Treg/Th17细胞的变化。以BALB/c小鼠为供体,C57BL/6小鼠为受体建立同种异基因皮肤移植模型,分别给予抗CD45RB抗体及IL-2等治疗,术后1、3、5、7、9天取受体鼠脾细胞,动态检测Treg/Th17细胞的变化;术后第9天取移植皮肤HE染色观察炎性细胞的浸润情况;观察并记录移植皮肤的存活时间。结果:CD4+T细胞在IL-2联合抗CD45RB抗体的作用下培养72小时后,Treg比例升高,Th17细胞比例下降;IL-2联合抗CD45RB抗体治疗后明显延长小鼠移植皮肤的存活时间。结论:IL-2可以明显增强抗CD45RB抗体诱导免疫耐受的形成,使Treg细胞上调,下调Th17细胞,有利于免疫耐受的形成。     

静脉输注过氧化氢溶液上调小鼠脾脏和外周血CD4+Foxo3+调节性T细胞

背景：现已证实,天然生成的调节性T细胞在维护机体免疫稳态、抑制炎性反应、抗移植排斥、抑制抗肿瘤免疫应答以及防治自身免疫病中都有重要的功能和作用。 目的：探讨静脉输注过氧化氢对BALB/c小鼠CD4⁺CD25⁺Foxo3⁺ 调节性T细胞的影响。 方法：取BALB/c小鼠12只,随机分为两组。H₂O₂处理组小鼠尾静脉注射含有H₂O₂的PBS;PBS组小鼠静脉注射无菌PBS。分别在注射2周后处死小鼠,采集小鼠外周血、脾脏和胸腺,采用流式细胞仪检测其中CD4⁺CD25⁺ Foxp3⁺T细胞比例。 结果与结论：H₂O₂处理组小鼠外周血和脾脏CD4⁺Foxp3⁺ T细胞比例、CD4⁺Foxp3⁺/CD4⁺细胞比例均高于对照组小鼠(P < 0.05)。提示静脉注射H₂O₂能上调小鼠外周血和脾脏内CD4⁺Foxp3⁺调节性T细胞的比例。     

抑制NF-κB活性诱导同种异体小鼠心脏移植耐受的早期作用

目的 研究调节性T细胞(Tr)和Th17细胞在特异性NF-κB抑制诱导同种异体小鼠心脏移植耐受的早期作用机制.方法 以BALB/c小鼠为供体,C57BL/6小鼠(对照组)和IκBα△N-Tg小鼠(实验组)分别作为受体建立同种异体腹部异位心脏移植模型;流式细胞术分别检测两组受鼠脾脏内Tr在移植前以及移植术后7 d、30 d和100 d的变化规律,以及Th17细胞在移植术后5 d时的变化;Western blot检测两组心脏移植物内IL-17在移植后3 d和5 d的表达.结果 实验组心脏移植物存活时间均大于100 d,HE染色未见心脏移植物内有明显淋巴细胞浸润;实验组Tr比例在移植后7 d和30 d时明显升高(21.23±3.95,23.17±4.11 vs 11.64±1.96,P0.05),而对照组Tr在移植前后则无明显变化;与对照组相比,实验组Th17细胞及移植物内IL-17表达在移植术后5 d时均明显下降.结论 特异性受体T细胞NF-κB功能缺陷可以打破受体内Tr/Th17细胞平衡,在移植术后早期促进T细胞向Tr分化而抑制向Th17细胞分化,从而阻止急性排斥反应发生,诱导耐受.     

甲基转移酶抑制剂延长小鼠心脏移植物存活期的作用研究

探讨甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷调控体内幼稚T细胞转化对同种心脏移植物存活期的影响及其机制。将C57BL/6小鼠分为实验与对照2组,实验组每日腹腔注射5-氮杂-2′-脱氧胞苷(0.25mg/kg体质量),对照组接受等体积的二甲亚砜腹腔注射。然后,提取小鼠脾细胞,抗CD3与抗CD28抗体协同共刺激后,检测CD4+Foxp3+T细胞占CD4+T细胞比例。以BALB/c小鼠为供鼠,两组C57BL/6小鼠为受鼠,建立腹部异位心脏移植模型,监测两组移植心存活时间,检测受鼠脾细胞中CD4+T细胞凋亡率,观察移植心脏组织病理学变化。结果显示,实验组移植前脾细胞中CD4+Foxp3+T细胞占CD4+T细胞比例为(22.7±2.6)%,明显高于对照组的(12.6±1.1)%,差异有统计学意义(P0.05);实验组移植物存活(22.8±2.8)d,明显长于对照组(8±1.8)d,P0.05);实验组移植后脾细胞中CD4+T细胞凋亡率(11.5±1.22)%,高于对照组的(6.6±0.91)%,差异有统计学意义(P0.05)。实验组移植物淋巴细胞浸润数量少于对照组。以上结果提示甲基转移酶抑制剂5-氮杂-2′-脱氧胞苷受体腹腔注射可延长同种移植心脏存活期,其机制可能与调控幼稚T细胞转化为CD4+Foxp3+T细胞,诱导CD4+T细胞凋亡有关。     

建立小鼠腹腔心脏移植模型的方法改进

背景:小鼠心脏移植模型目前有颈部移植和腹腔移植。由于颈部空间狭窄,颈总动脉相对细小,移植心脏易于周围组织粘连,限制移植心脏搏动,不利于移植物长期观察。腹主动脉相对较粗,易于吻合,且长期血管通常率高,能够对移植心脏长期观察进行慢性移植物血管病变研究。目的:对小鼠腹部心脏移植的技术方法进行改进,为进行移植免疫学研究提供动物模型。方法:采用供心主动脉与受体腹主动脉、供心肺动脉与受体下腔静脉端侧吻合方法对小鼠进行腹部心脏移植,按随机数字表法将小鼠分为3组,同系移植组为同种同基因C57→C57,同种移植组为同种异基因Babl/c→C57,抗CD45RB mAb组为抗CD45RB mAb 200μg腹腔注射Babl/c→C57。以供心搏动有力,且小鼠存活72h以上视为移植成功。设移植后40d为观察终点。结果与结论:共实施手术36例次,受体存活30只,成功率为83.3%。其中受体准备时间为(15±2)min,供心摘取时间为(8±1)min,血管吻合时间为(33±2)min。抗CD45RB mAb腹腔注射组小鼠移植心脏存活时间较同种移植组明显延长(P=0.001)。提示所建立的小鼠心脏移植模型稳定可靠,可用于移植免疫学方面的研究。抗CD45RB mAb可明显延长移植心脏存活时间。     

血红素加氧酶1在间充质干细胞上调哮喘患者外周血调节性T细胞中的作用

背景：上调CD4⁺CD25⁺CD127^low/-调节性T细胞是目前治疗哮喘的新靶点。骨髓间充质干细胞在体外可上调正常人外周血的调节性T细胞,但机制尚未明确。 目的：观察血红素加氧酶1对间充质干细胞上调哮喘患者外周血CD4⁺CD25⁺CD127^low/-调节性T细胞的影响。 方法：分别加入0,15,30,45,60 μmol/L氯化高铁血红素和0,5,10,15,20 μmol/L锌-原卟啉对骨髓间充质干细胞进行预处理,荧光定量PCR检测各组间充质干细胞中血红素加氧酶1 mRNA的表达量。密度梯度离心法分离哮喘急性发作期患者和健康对照者的外周血单个核细胞,分别与经氯化高铁血红素预处理、经锌-原卟啉预处理、不经预处理的间充质干细胞共孵育,加入植物血凝素反应72 h,流式细胞仪检测各组CD4⁺CD25⁺CD127^low/-调节性T细胞占CD4⁺T细胞的比例。 结果与结论：人骨髓间充质干细胞中有血红素加氧酶1表达,其表达在体外可被诱导和抑制。间充质干细胞中血红素加氧酶1 mRNA的表达量随氯化高铁血红素浓度增加而增加(P < 0.05),随锌-原卟啉浓度增加而减少(P < 0.05),具有剂量依赖性。间充质干细胞在体外可提高哮喘患者和健康对照者CD4⁺CD25⁺CD127^low/-调节性T细胞的水平(P < 0.01),诱导间充质干细胞中血红素加氧酶1的表达可使共孵育后CD4⁺CD25⁺CD127^low/-调节性T细胞水平提高(P < 0.01),抑制间充质干细胞中血红素加氧酶1的表达可使共孵育后CD4⁺CD25⁺CD127^low/-调节性T细胞的水平降低(P < 0.01),但仍高于对照组(P < 0.01)。提示骨髓间充质干细胞部分通过血红素加氧酶1上调哮喘患者外周血CD4⁺CD25⁺CD127^low/-调节性T细胞。     

日本血吸虫感染BALB／c小鼠脾脏细胞学的变化

目的观察BALB／c小鼠感染日本血吸虫（Schistosoma japonicum，$）6．8周后脾脏细胞学的改变情况。方法用日本血吸虫尾蚴腹贴法建立日本血吸虫感染的小鼠模型。6～8周后观察肝脏、脾脏大小．称重：做脾脏淋巴细胞计数；用流式细胞仪检测脾脏淋巴细胞的大小、密度和T、B细胞分类以及Th细胞亚群睛况：用酶联免疫吸附实验（enzyme linked immunosorbent assay，ELISA）检测血清中可溶性虫卵抗原（soluble egg antigen，SEA）所诱导的特异性抗体IgG的产生情况。结果日本血吸虫感染BALB／c小鼠6～8周后，脾脏体积明显增大，重量增加，细胞数量明显增多。流式细胞仪检测发现脾脏中出现一群体积明显增大的细胞群，该群细胞表达少量的CD4、CD8、CD19和DX－5分子；Th1／Th2轴发生偏移，Th17细胞数量增多；ELISA结果表明血清中SEA特异性抗体IgG水平明显升高。结论日本血吸虫感染的BALB／c小鼠脾脏出现明显的细胞学改变，尤其是Th1细胞数目下降，浆细胞、Th2细胞及Th17细胞数目增多，为研究日本血吸虫感染后期虫卵肉芽肿形成的免疫病理机制奠定了基础。     

CD4~+CD25~+CD127~-T细胞在抗CD45RB抗体诱导免疫耐受中的作用

目的:研究CD4+CD25+CD127-T细胞在抗CD45RB抗体诱导的免疫耐受中所发挥的作用,从而阐明抗CD45RB抗体诱导免疫耐受作用的机制。方法:体内实验建立小鼠异位心脏移植模型,观察抗CD45RB抗体对移植物生存期的影响。体外实验观察抗CD45RB抗体对T细胞增殖抑制能力和对CD4+CD25+CD127-T细胞生成的影响。流式细胞术检测外周血和混合细胞中CD4+CD25+CD127-T细胞百分率,ELISA法检测血清和培养液中IL-2和IL-10含量,Real-TimePCR法检测脾脏和混合细胞中Foxp3基因的表达,移植心脏病理学观察。结果:抗CD45RB抗体显著延长移植物存活时间(P0.01),对ConA刺激引起的T淋巴细胞增殖具有明显的抑制能力(P0.05)。与对照组相比,实验组CD4+CD25+CD127-T细胞百分率和Foxp3 mRNA表达量均明显增加(P0.05),实验组IL-2水平较对照组明显降低(P0.05),但IL-10含量较对照组明显升高(P0.05)。病理结果显示对照组移植心脏出现典型细胞免疫性损伤病理改变,而实验组中几乎无炎性细胞浸润现象。结论:抗CD45RB抗体能显著延长移植物存活时间,其诱导免疫耐受机制与上调CD4+CD25+CD127-T细胞百分率和增加Foxp3 mRNA表达量有关。     

转录因子IRF8抑制Th17细胞分化并减轻T细胞免疫介导的小鼠结肠炎

 目的：转录因子干扰素调节因子（interferon regulatory factor, IRF）家族与Th17的发育密切相关,近年来发现Th17细胞在炎症性肠病的发病中发挥重要作用,本研究探讨IRF8对Th17发育及T细胞转染免疫介导的小鼠实验性肠炎的影响。方法：(1)采用流式细胞术分选野生型（WT）或IRF8全基因敲除(IRF8 -/-)小鼠脾脏和淋巴结的naive CD4 +T细胞(CD4 + CD62L +CD44 low),在Th1、Th2或Th17极化的条件下培养,采用流式细胞术检测Th1、Th2和Th17的比例。(2)建立实验性肠炎模型：采用免疫磁珠法分选WT或IRF8 -/-小鼠中的脾脏和淋巴结中CD4 +CD25 +Treg,WT小鼠的CD4 + CD45RB hi T细胞单独或者分别联合WT或IRF8 -/-小鼠的CD4 +CD25 +Treg腹腔注射给RAG1 -/-小鼠;WT或IRF8 -/-小鼠的naive CD4 + CD45RB hi T细胞腹腔注射给RAG1 -/-小鼠;观察上述小鼠每周体重的变化,第5周时处死小鼠,进行结肠炎病理评分和肠系膜淋巴结T淋巴细胞亚群检测。结果：(1)IRF8 -/-较WT的naive CD4 +T细胞在极化条件下向Th17细胞分化更明显(P<001),而对Th1和Th2细胞的分化无影响(P>0.05)。(2)CD4 + CD45RB hi T细胞转染给RAG1 -/-小鼠,IRF8 -/-较WT供体鼠引起的RAG1 -/-小鼠体重显著降低(P<0.05),结肠炎评分显著增高（P<0.05）,且肠系膜淋巴结中IL-17 +CD4 +细胞比例明显增高(P<0.01),而 IFN-γ +CD4 + 和 Foxp3 +CD4 +细胞比例无影响(P>0.05);IRF8 -/-小鼠的CD4 +CD25 +Treg对WT小鼠CD4 + CD45RB hi T细胞转染给RAG1 -/-小鼠诱发的免疫介导的结肠炎显示出正常的免疫抑制作用。结论：转录因子IRF8基因敲除促进CD4 +T细胞向Th17细胞分化,促进转染naive CD4 +T细胞诱导的实验性结肠炎的发生,IRF8基因敲除小鼠Treg细胞免疫抑制功能正常。     

NOD-SCID小鼠移植人体胃癌实验案例分析

目的：鉴定小鼠胃癌移植模型中生成的肿瘤类型及病理特征。方法：采用HE染色法对肿瘤病理组织学特征进行观察，用免疫组织化学的方法分析肿瘤中CD43、CD45RB、CD57、CK等标志物的表达及小鼠脾脏中CD43、CD57的表达以诊断肿瘤类型及来源。结果：经组织学观察，小鼠皮下肿瘤组织与人胃癌组织结构形态并不相符。经免疫组织化学分析，小鼠肿瘤抗人体的CD43、CD45RB、CD57均为阳性，提示肿瘤细胞中由大量T淋巴细胞、B淋巴细胞和NK细胞组成；但小鼠脾脏内T、B淋巴细胞和NK细胞均为阴性。结论：小鼠皮下肿瘤为淋巴瘤，来源于移植的胃癌组织中带有的淋巴组织。     

Suppression of allergic inflammation by allergen-DNA-modified dendritic cells depends on the induction of Foxp3+ Regulatory T cells

CD4⁺CD25⁺Foxp3⁺Regulatory T cells (Tregs) play important roles in regulating allergic inflammation. To analyse if allergen-DNA-modified dendritic cells (DC) can suppress allergic responses and what roles Treg cells play in DC-based allergen-specific immunotherapy. Immature DC were transfected with retrovirus encoding Der p2 DNA, and administered to mice that sensitized and challenged with Der p2 protein. After Treg cells were depleted with anti-CD25 mAb, mice were re-challenged to observe the airway inflammation, and Treg cells in spleen CD4⁺ T cells. And responses of spleen CD4⁺ T cells to Der p2 were determined. Co-culture of naïve CD4⁺ T cells with allergen-modified DC induced Foxp3+ Tregs. Sensitized and challenged mice developed allergic airway inflammation and Th2 responses, and decreased Foxp3⁺ Tregs. Treatment with allergen-modified-DC suppressed airway inflammation and Th2 responses, and increased IL-10 and IFN-γ production and Foxp3⁺ Tregs significantly; and eliminated the responses of CD4⁺ T cells to allergen. Administration of anit-CD25 mAb eliminated all the effects of modified-DC except for the increasing of IFN-γ. Allergen-modified DC can induce immune tolerance to allergens and reverse the established Th2 responses induced by allergen, with dependence on the induction of Foxp3⁺ Tregs.     

CTLA-4 up-regulation plays a role in tolerance mediated by CD45

Cytolytic T lymphocyte-associated antigen 4 (CTLA-4) is a critical down-regulatory molecule in T cells that plays a major role in peripheral tolerance. Although the CD45 protein tyrosine phosphatase is a potent immunomodulatory target, the mechanisms by which antibody against CD45RB isoforms (anti-CD45RB) induces allograft tolerance remain unclear. We show here that anti-CD45RB treatment alters CD45 isoform expression on T cells, which is associated with rapid up-regulation of CTLA-4 expression. These effects appear specific and occur without up-regulation of other activation markers. Administration of a blocking monoclonal antibody to CTLA-4 at the time of transplantation prevents anti-CD45RB therapy from prolonging islet allograft survival. In addition, treatment with cyclosporin A blocks anti-CD45RB-induced CTLA-4 expression and promotes acute rejection. These data suggest that anti-CD45RB acts through mechanisms that include CTLA-4 up-regulation and demonstrate a link between CD45 and CTLA-4 that depends on calcineurin-mediated signaling. They demonstrate also that CTLA-4 expression may be specifically targeted to enhance allograft acceptance.     

Blockade of CD70-CD27 Interaction Inhibits Induction of Allergic Lung Inflammation in Mice

The interaction between the TNF receptor family member CD27 and its ligand CD70 provides a costimulatory signal for T-cell activation. In this study, we investigated the effects of neutralizing anti-CD70 monoclonal antibody (mAb) in a murine model of allergic lung inflammation to determine whether CD27 contributes to the development of pathogenic Th2 cells and pulmonary inflammation. BALB/c mice were immunized by an injection of ovalbumin (OVA) with alum adjuvant and challenged with aerosolized OVA in PBS. Some groups of mice were treated with anti-CD70 mAb or control rat IgG during the induction or effector phase. The administration of anti-CD70 mAb during the induction phase, but not the effector phase, reduced eosinophil infiltration in lung tissue compared with control IgG-treated mice. Treatment with anti-CD70 mAb also resulted in the decreased production of Th2 cytokines (IL-4, IL-5, and IL-13) in the bronchoalveolar lavage fluid and draining lymph node cell cultures. We further revealed that antigen-specific CD4 T cells were separated into CD27(+) and CD27(-) populations in the lymph nodes of OVA-immunized DO11.10/Rag-2(-/-) mice. The CD27(+) CD4 T cells produced a high concentration of IFN-γ, representing Th1 cells. In contrast, CD27(-) CD4 T cells produced high concentrations of IL-4, IL-5, and IL-13, representing Th2 cells. Moreover, the population of CD27(-) Th2 cells was significantly reduced by the anti-CD70 mAb treatment. These results indicate an important role for CD27 in the development of pathogenic Th2 cells in a murine model of allergic lung inflammation.     

Functional Changes of Human Peripheral B-Lymphocytes in Pre-Eclampsia

Problem  The aim of our study was to investigate the functional changes of human peripheral B-lymphocytes in healthy and pre-eclamptic pregnancies.
Method of study  Twenty patients with pre-eclampsia and 15 healthy third-trimester pregnant women were recruited in this study. Peripheral blood mononuclear cells (PBMCs) were isolated and directly stained with fluorescein isothiocyanate (FITC)-labeled anti-CD27 monoclonal antibody (mAb) and phycoerythrin (PE)-labeled anti-CD38 mAb. The percentages of the individual B-cell subsets were estimated out of total lymphocytes by flow cytometric analysis. Additionally, the enriched PBMCs were cultured with or without the stimulation of pokeweed mitogen (PWM) for 5 days. Then morphologic observation of plasma cells was analysed by Wright-Giemsa stain, and antibody-producing cells were detected by enzyme-linked immunospot assay.
Results  The percentage of CD27⁻CD38⁻ naïve B-cells and CD27⁻CD38⁺ plasma cells did not differ between study groups ( P  >   0.05). The percentage of CD27⁺CD38⁻ memory B-cells and CD27⁺CD38⁺ plasma cell pre-cursors increased in pre-eclamptic women compared with the controls ( P  <   0.05). Irrespective of whether the PBMCs were stimulated with or w/o PWM in vitro , the mean percentages of generated plasma cells were significantly higher in pre-eclamptic group than in the controls ( P  <   0.05). There were more antibody-producing cells in pre-eclamptic women following the activation of PWM than those in the controls ( P <  0.01).
Conclusion  Our findings implicate that the functional changes of human circulating B-cells might contribute to the etiology of pre-eclampsia.     

Preferential dependence of autoantibody production in murine lupus on CD86 costimulatory molecule

Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80 (B7, B7.1)/CD86 (B70, B7.2), seems an attractive means to induce antigenspecific peripheral tolerance in organ transplantation and autoimmune disease. Recently, diversities between CD80 and CD86 in expression, regulation, and function have been reported in certain cell populations and murine experimental disease models. To investigate the possible differential role of CD80 and CD86 in the development of lupus, we treated lupus-prone NZB/WF1 mice with specific monoclonal antibodies (mAb) against CD80, CD86, or both. The treatment with a combination of anti-CD80 and CD86 mAb before the onset of lupus completely prevented autoantibody production and nephritis, and prolonged survival. Interestingly, we found that anti-CD86 mAb alone, but not anti-CD80 mAb, efficiently inhibited autoantibody production. Subclass study on IgG antidouble-stranded (ds) DNA antibody revealed that the treatment with anti-CD86 mAb almost completely inhibited both IgG1 and IgG2b, but not IgG2a production. The incomplete reduction of IgG2a anti-dsDNA antibody by anti-CD86 mAb was compensated by the addition of anti-CD80 mAb. A significant reduction of mRNA for interleukin (IL)-2, interferon-γ, IL-4 and IL-6 was observed in mice treated with a combination of anti-CD80 and CD86 mAb or anti-CD86 mAb alone. Treatment with both mAb after the onset of lupus resulted in a significantly prolonged survival with reduction of autoantibody production. These results suggest that CD86 plays a more critical role in autoantibody production, and CD86, but not CD80, contributes to Th2-mediated Ig production. However, the blockade of both CD80 and CD86 are required for preventing the development and progression of lupus.     

Long-lasting unresponsiveness to polyclonal T cell-binding immunoglobulins

We have shown that mice after a single injection of anti-T cell antibody followed by multiple injections of a second xeno-, allo- or syngeneic anti-T cell antibody differing from the former in species origin developed specific, long-lasting tolerance to the second antibody. To characterize the mechanism of this anti-antibody unresponsiveness and the modalities accompanying the preinjection step, we injected mice with anti-pan T, anti-CD4 or anti-CD8 monoclonal antibodies, followed by multiple injections of polyclonal rabbit anti-mouse thymocyte globulin (RbATG). Our observations indicate that: (i) Depletion of CD4+ cells is the most important factor for tolerance induction to subsequently injected RbATG. Non-depleting mAb were less effective, and antibody-induced CD4 modulation or blockade were inconsequential. (ii) The prevention of anti-antibody responses involves specific B cell tolerance, as shown by suppression of anti-RbATG but not anti-bovine serum albumin (BSA) antibodies after challenge of tolerant mice with RbATG-BSA conjugates. (iii) Suppression of anti-antibody responses involves T cell unresponsiveness rather marginally, as demonstrated by in vitro spleen cell restimulation with RbATG and in vivo antibody response to RbATG-fluorescein isothiocyanate hapten conjugate. (iv) Analysis of isotypes of anti-RbATG anti-bodies does not suggest alterations in Th1/Th2 tuning as being responsible for this kind of tolerance. (v) Over 150-day survival of fully allogeneic skin grafts (CBA-to-C57BL/6) was observed in mice preinjected with anti pan-T or anti-CD4 mAb followed by RbATG. Mice treated with anti-CD4 mAb alone rejected allografts within 21 days and induced anti-antibodies. Taken together, our experiments suggest B cell tolerance as main mechanism in this type of acquired long-term humoral unresponsiveness to foreign, polyclonal, T cell-binding immunoglobulins.