妊娠浓度的雌激素对诱导CD4~+CD25~-naive T细胞转化为CD4~+CD25~+Treg细胞的影响

目的 体外观察妊娠浓度的雌激素能否诱导CD4+CD25-naiveT细胞转化为CD+CD25+Treg细胞,并探讨其相关性.方法 以CD4+CD25-T细胞作为反应细胞,实验组加入妊娠水平的雌激素(E2)及CD3/CD28单抗作为刺激原培养72 h,设阴性对照组(仅加入CD3/CD28单抗)和空白对照组.72 h后检测各组中CD4+CD25+T细胞和CD4+Foxp+T细胞比例变化及Foxp3 mRNA表达.结果 1)阴性对照组CD+CD25+T细胞比例较空白对照组显著增高(P＜0.001),而实验组CD4+CD25+T细胞比例进一步升高(P＜0.001).2)阴性对照组不能诱导CD4+Foxp+T细胞比例增高,但实验组CD4+Foxp3+T细胞的比例则较其它2组均显著升高(P＜0.001).3)RT-PCR提示阴性对照组Foxp3 mRNA的表达量较空白对照组无显著差异(P＞0.05);而实验组F0xp3 mRNA的表达昔较其它2组均显著升高(P＜0.001).结论 体外淋巴细胞刺激实验提示妊娠状态下雌激素的高水平与CD4+CD25+Foxp3+Treg细胞比例的升高密切相关.     

小鼠脾细胞CD4~+ CD25~+调节性T细胞的表型特征

观察CD4+CD25+T和CD4+CD25-T细胞的表型和细胞因子的表达。自小鼠脾脏制备单个细胞悬液,分离CD4+T细胞、CD4+CD25+和CD4+CD25-T细胞,进行细胞表面标记,激活后进行细胞内细胞因子染色,利用流式细胞仪在单个细胞水平上分析细胞表面分子、转录因子和细胞因子表达之间的关系。结果:在CD4+T细胞中,约有7.8%的细胞同时表达CD25分子。与CD4+CD25-T细胞相比,CD4+CD25+T细胞CD44的表达略有增加,CD45RB的表达明显下降,CTLA-4和Foxp3明显增加。以同时表达CTLA-4和Foxp3的细胞为主,其次为单独表达Foxp3的细胞。细胞因子的研究结果表明,与CD4+CD25-T细胞相比,CD4+CD25+T细胞IL-2、IFN-γ明显减少,而只产生IL-10的细胞略有增加。CD4+CD25+调节性T细胞无论在表型、转录因子的表达以及细胞因子表达方面均于非调节性T细胞不同。     

抗ICOS抗体体外对哮喘大鼠血液和淋巴液CD4~+CD25~+Foxp3~+调节性T细胞数量及其功能影响

目的:研究抗ICOS抗体对哮喘大鼠外周血和淋巴液来源CD4+CD25+Foxp3+调节性T细胞(Treg)数量及其功能的影响。方法:抗ICOS抗体处理血液和淋巴液中单个核细胞(MNC),流式细胞仪检测MNC中CD4+CD25+Foxp3+T细胞百分率,酶联免疫吸附试验(ELISA)检测MNC培养液上清IL-10和TGF-β1含量。结果:末次激发后各个时间点收集MNC,体外培养96 h,各组淋巴液来源MNC体外培养体系中CD4+CD25+Foxp3+Treg细胞百分率均显著高于血液(P<0.05),哮喘组淋巴液和血液来源MNC体外培养体系中CD4+CD25+Foxp3+Treg细胞百分率均显著低于正常对照组(P<0.05),抗ICOS抗体组淋巴液和血液来源MNC体外培养体系中CD4+CD25+Foxp3+Treg细胞百分率显著低于哮喘组(P<0.05)。末次激发后0 h收集淋巴液来源和血液来源MNC培养上清中抗ICOS抗体组IL-10显著低于哮喘组和正常对照组(P<0.05);末次激发后不同时间点收集MNC培养上清中各组TGF-β1无明显差别。结论:用抗ICOS抗体阻断ICOS/ICOSL信号通路加重哮喘大鼠Treg细胞缺陷,并在哮喘激发早期0 h抑制血液和淋巴液来源MNC体外培养体系中CD4+CD25+Foxp3+Treg细胞分泌IL-10,但对于TGF-β1分泌无显著影响。     

MicroRNA-126调控CD4~+Foxp3~+T细胞的外周诱导及功能

目的探讨Micro RNA-126对CD4~+Foxp3~+Tregs的外周诱导调控及功能的影响。方法分选Balb/c小鼠脾脏CD4~+CD25-初始T细胞,在anti-CD3/CD28的激活下,用TGF-β进行诱导培养,在第3、5天FACS检测CD4~+Foxp3~+Tregs的比例,Real-time PCR检测mi R-126的表达;采用mi R-126抑制剂抑制mi R-126后,FACS检测CD4~+Foxp3~+Tregs的比例,Real-time PCR检测mi R-126的表达;进而采用mi R-126 ASO下调CD4~+Foxp3~+Tregs中mi R-126的表达,Real-time PCR检测IL-10和TGF-β的表达,CFSE标记技术分析CD4~+Foxp3~+Tregs免疫抑制功能。结果 mi R-126在活化的Tregs中的表达高于在活化的CD4~+CD25-T细胞中的表达(P0.05);TGF-β在体外诱导生成Foxp3~+CD4~+T细胞的比例组间差异具有统计学意义(P0.05),同时在Tregs的诱导过程中,mi R-126的表达上调(P0.05);CD4~+CD25-T细胞体外瞬时转染mi R-126抑制剂,与对照组比较,Foxp3~+CD4~+T细胞的比例组间差异具有统计学意义(P0.05),mi R-126抑制剂能有效下调mi R-126的表达(P0.05);与对照组相比,mi R-126 ASO转染组Tregs中Foxp3、CTLA-4和GITR的表达均降低(P0.05),且TGF-β和IL-10的m RNA相对表达均降低(P0.05);CFSE标记细胞增殖实验显示,mi R-126 ASO Tregs组CD4~+CD25-T细胞增殖与Tcon组、Control Tregs组比较,差异具有统计学意义(P0.05)。结论下调mi R-126的表达能显著削弱CD4~+Foxp3~+Tregs的外周诱导和免疫抑制功能。     

体外DCs在抗CD45RB抗体诱导免疫耐受中的作用机制

目的:探讨树突状细胞(dendritic cells,DCs)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抗体诱导免疫耐受的机制。方法:采用DCs的常规诱导方法(rmGM-CSF、IL-4和LPS),在诱导过程中加入不同剂量的抗CD45RB抗体,成熟后利用流式细胞仪检测细胞表型、周期和吞噬能力,ELISA法检测IL-12分泌量,混合淋巴细胞培养检测DCs对T细胞增殖能力的影响。结果:DCs经抗CD45RB抗体处理后,CD11C和CD83表达升高,CD86表达下降,自身增殖和吞噬能力增强,但分泌IL-12和刺激T细胞增殖的能力明显下降。结论:耐受性树突状细胞(tolerogenic dendritic cells,tDCs)能显著抑制T细胞的增殖,它的产生是抗CD45RB抗体诱导免疫耐受的主要机制之一。     

结核病患者外周血中CD4~+ CD25~+调节性T细胞水平的检测

探讨CD4~+CD25~+调节性T细胞及其转录因子Foxp3在结核病发病机制中的作用。研究对象为肺结核患者22例(病例组)以及健康对照者23例(对照组)。采用FACS检测外周血CD4~+CD25~+调节性T细胞的百分率,采用real-time PCR检测外周血单个核细胞Foxp3mRNA的表达以及CD4~+CD25~+调节性T细胞与CD4~+T细胞、CD8~+T细胞、IFN-γ和IL-4的相关性。结核病患者外周血CD4~+CD25~+调节性T细胞占CD4~+T细胞的百分率,病例组(3.38±1.23)%高于对照组(1.97±0.62)%,两组比较差异有统计学意义(P0.05)。血清单个核细胞Foxp3 mRNA相对表达水平为134.54±6.76,高于对照组(40.98±2.34,P0.05)。CD4~+CD25~+调节性T细胞与CD4~+T细胞、CD8~+T细胞以及与IFN-γ和IL-4的表达呈负相关。结核病CD4~+CD25~+调节性T细胞数量增加、特异性转录因子Foxp3 mRNA表达上升,由此引发的免疫抑制效应可能是结核病发生发展的重要原因之一。     

组蛋白去乙酰化酶抑制剂对急性移植物抗宿主病模型小鼠CD4~+CD25~+Foxp3~+细胞的影响

背景:防治移植物抗宿主病,提高移植存活率是异基因造血干细胞移植急待解决的核心问题;因此寻找新的免疫抑制剂成为必要。近期的研究发现组蛋白去乙酰化酶抑制剂具有免疫调节作用。目的:通过建立小鼠急性移植物抗宿主病模型,观察组蛋白去乙酰化酶抑制剂SAHA对小鼠急性移植物抗宿主病的影响及免疫调节作用。方法:选择C57BL/6(H-2b)→BALB/C(H-2d)作为完全异基因移植供、受体建立移植物抗宿主病小鼠模型,移植后3,5,7,9,11 d,治疗组小鼠腹腔注射组蛋白去乙酰化酶抑制剂SAHA 35 mg/(kg·次)(0.2 m L),对照组小鼠腹腔注射灭菌水0.2 m L/次,通过流式细胞术、实时定量PCR、病理学等方法,比较两组小鼠的急性移植物抗宿主病临床表现、生存时间及CD4+CD25+Foxp3+细胞百分率。结果与结论:治疗组较对照组小鼠出现急性移植物抗宿主病时间推迟,程度减轻、生存时间延长。治疗组生存率显著高于对照组(P0.05)。移植后治疗组小鼠外周血CD4+CD25+Foxp3+细胞阳性百分率随时间延长呈逐渐递增趋势;反之对照组小鼠外周血CD4+CD25+Foxp3+细胞阳性百分率随时间延长呈逐渐递减趋势(P0.05)。提示SAHA可延迟急性移植物抗宿主病的发生,减轻急性移植物抗宿主病的严重程度,以上作用可能是通过提升CD4+CD25+Foxp3+细胞的比例来实现的。     

哮喘大鼠淋巴液与血液CD4~+CD25~+Treg及IL-10、TGF-β水平比较和地塞米松干预的影响

目的:观察CD4+CD25+Foxp3+调节性T细胞、IL-10、TGF-β在哮喘大鼠淋巴液与血液中的水平,以及地塞米松干预的影响,探讨其与哮喘发病的关系。方法:建立大鼠哮喘模型,收集激发后0、24、48 h淋巴液、血液中淋巴细胞,采用流式细胞术(FCM)检测淋巴液及血液中CD4+CD25+Foxp3+T细胞百分率;采用ELISA方法检测血浆及淋巴液中IL-10、TGF-β水平。结果:哮喘组淋巴液和血液的CD4+CD25+Foxp3+T细胞百分率水平、血浆IL-10和TGF-β浓度在各时间点均低于对照组和治疗组(P0.05);哮喘组淋巴液中CD4+CD25+Foxp3+T细胞百分率水平、IL-10浓度在不同时间点均高于血液水平(P0.05),而TGF-β浓度则低于血液水平(P0.05);治疗组淋巴液和血液中CD4+CD25+Foxp3+T细胞百分率水平、血浆IL-10和TGF-β浓度与对照组相比无显著性差异(P0.05)。结论:哮喘大鼠淋巴液中存在CD4+CD25+Foxp3+调节性T细胞数量以及功能失调,且淋巴液中CD4+CD25+Foxp3+调节性T细胞水平显著高于其血液水平,地塞米松可能通过影响CD4+CD25+Foxp3+调节性T细胞数量以及功能而发挥治疗作用。     

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。     

Lewis肺癌细胞通过TLR9对CD4~+CD25~+Treg细胞影响的研究

目的:本研究以Lewis肺癌细胞为研究对象,探讨肿瘤细胞通过TLRs对CD4+CD25+Treg细胞的影响。方法:我们采用流式细胞术检测了Lewis肺癌细胞与脾淋巴细胞共培养系统中CD4+CD25+Treg细胞数量变化;通过RT-PCR方法检测了共培养对Foxp3和TLR1-9mRNA表达的影响;采用TLR9受体阻断剂氯喹阻断Lewis肺癌TLR9的表达。结果:与对照组相比,共培养组CD4+CD25+Treg细胞数量及Foxp3 mRNA表达均明显增高(P0.05);Lewis肺癌细胞与淋巴细胞共培养后可影响多种TLRs表达,其中TLR9 mRNA表达与对照组相比明显增高(P0.05),阻断Lewis肺癌细胞TLR9可明显降低CD4+CD25+Treg细胞数量及Foxp3 mRNA表达(P0.05)。结论:Lewis肺癌细胞可通过TLR9促进CD4+CD25+Treg细胞产生及功能增强,参与诱导肿瘤的免疫耐受,从而促进肿瘤的发生和发展。     

B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用

 目的: 探讨B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用。方法: 抗CD45RB抗体对BALB/c裸鼠进行预处理后制备脾脏单细胞悬液,与BALB/c小鼠T淋巴细胞和C57BL/6小鼠脾细胞混合培养,流式细胞术分析Th1、Th2、Treg和Tm淋巴细胞。以B6.μMT^-/-小鼠为受体、BALB/c小鼠为供体建立皮肤移植模型,移植后向受体鼠腹腔注射抗CD45RB单抗,监测脾淋巴细胞CD3⁺CD45RB^hi细胞比例。在混合淋巴培养过程中加入抗CD45RB单抗,分离B细胞,建立以BALB/c小鼠为供体、B6.μMT^-/-小鼠为受体的心脏移植模型,通过尾静脉注射B细胞给B6.μMT^-/-小鼠,观察受体鼠生存期和B细胞分布。结果: 在裸鼠体内用抗CD45RB抗体处理过的B淋巴细胞,与T淋巴细胞混合培养时,可使Treg和Th2淋巴细胞比例明显升高,Th1淋巴细胞的比例明显下降,Tm细胞无明显变化。在体内B淋巴细胞缺失的情况下,抗CD45RB抗体依然能够降低T细胞表面CD45RB的表达,与对照组B淋巴细胞存在组相比,抗CD45RB抗体对T淋巴细胞表面CD45RB下调更为快速,但最终CD3⁺CD45RB^hi T细胞比例无明显变化。体外抗CD45RB抗体处理过的B淋巴细胞可以延长受体鼠的生存时间。B6.μMT^-/-鼠在接受抗CD45RB抗体处理的B细胞并进行同种异体心脏移植后,B细胞可向胸腺迁移。结论: 在抗CD45RB抗体诱导的免疫耐受中,B淋巴细胞可能通过介导各T淋巴细胞亚群比例发挥着重要作用,且在中枢耐受中也起到一定作用,但是仅靠B淋巴细胞无法形成完全耐受。     

Role of mRNA stability in the different patterns of cytokine production by CD4+ cells from young and old mice.

CD4+ cells from young (3 months) and old (19 months) mice were stimulated by plate-bound anti-CD3 monoclonal antibody (mAb) alone or also by soluble anti-CD28 mAb. Supernatants were analysed by enzyme-linked immunosorbent assay (ELISA) to determine cytokine concentrations. Total RNA was extracted from cells, reverse transcribed and the cDNA amplified by polymerase chain reaction (PCR) to evaluate the amount of specific mRNA. The results indicate that anti-CD3 alone is not sufficient to induce interleukin-2 (IL-2) production in CD4+ cells from both young and old mice. However, anti-CD28, together with anti-CD3 mAb, induces a much higher production of IL-2 in CD4+ cells from young as compared with old mice. Conversely, interferon-gamma (IFN-gamma) production is also induced by anti-CD3 alone and is higher in CD4+ cells from old as compared with young mice. Upon addition of anti-CD28 mAb, IFN-gamma production increases in both groups, but it remains much higher in old than in young mice. Also the production of IL-4 and IL-10 is induced by anti-CD3 mAb but it is increased by the addition of anti-CD28 mAb. CD4+ cells from old mice produce more IL-4 and IL-10 as compared with cells from young mice. The amounts of cytokine specific mRNA in CD4+ cells from young and old mice parallel the cytokine levels in culture supernatants. Results on the mRNA turnover indicate that when CD4+ cells are stimulated by anti-CD3 or costimulated also by anti-CD28 mAb, the IFN-gamma, IL-4 and IL-10 specific mRNAs are more stable in old than in young mice, suggesting that mRNA stability has a relevant role in the different patterns of cytokine production.     

Targeting CD45RB alters T cell migration and delays viral clearance

CD45 is a receptor tyrosine phosphatase essential for TCR signaling. One isoform, CD45RB, is down-regulated in memory cells and targeting CD45RB with a specific antibody has been shown to inhibit graft rejection. Its role in immunity to infection, however, has not been tested. Here, we report the effect of anti-CD45RB antibody treatment on the induction of anti-influenza CD8+ T cells and viral clearance. Anti-CD45RB-treated mice had delayed pulmonary viral clearance compared with untreated mice whose infection was completely cleared by day 8 post-infection. In anti-CD45RB-treated mice, the total CD4+ and CD8+ T cell numbers in both the lungs and mediastinal nodes were substantially reduced at days 5 and 8; this effect was less marked for the spleen. CD8+ T cells specific for influenza virus were also reduced compared with the control group in all three organs at day 8. By day 11, when both treated and control groups showed no virus remaining in the lungs, specific CD8+ T cell numbers were at similar low levels. Homing to lymph nodes and lung of dye-labeled T cells was greatly inhibited (by >80%) by anti-CD45RB treatment. This reduced homing corresponded with reduced CD62L and beta1-integrin expression in both uninfected and infected mice. Since CD62L plays a critical role in homing lymphocytes to lymph nodes, and high levels of CD62L and alpha4beta1-integrin are expressed by lymphocytes that home to bronchus-associated lymphoid tissue, we suggest that reduced expression of these molecules is a key explanation for the delay in immune responses.     

Regulation of murine chronic colitis by CD4+CD25- programmed death-1+ T cells

Naturally arising CD4(+)CD25(+) regulatory T (T(R)) cells are engaged in the maintenance of self tolerance and prevention of autoimmune diseases. However, accumulating evidence suggests that a fraction of peripheral CD4(+)CD25(-) T cells also possesses regulatory activity. Programmed death-1 (PD-1) is a new member of the CD28/CTLA-4 family, which has been implicated in the maintenance of peripheral self tolerance. Here, we identified a subpopulation of CD4(+)CD25(-)PD-1(+) T cells in the spleen of naive mice that constitutively expressed CTLA-4 and FoxP3 and was hypoproliferative in response to anti-CD3 antibody stimulation in vitro. However, the CD4(+)CD25(-)PD-1(+) T cells uniquely produced large amounts of IL-4 and IL-10 in response to anti-CD3 and anti-CD28 mAb stimulation, unlike the CD4(+)CD25(+) T(R) cells. The CD4(+)CD25(-)PD-1(+) T cells exhibited a suppressor activity against the proliferation of anti-CD3 antibody-stimulated CD4(+)CD25(-)PD-1(-) T cells in vitro, which was partially abrogated by anti-CTLA-4 mAb, but not by anti-IL-10 or anti-PD-1 mAb. Remarkably, the CD4(+)CD25(-)PD-1(+) T cells inhibited the development of colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells into C.B17-scid/scid mice, albeit to a lesser extent than CD4(+)CD25(+) T(R) cells, in a CTLA-4-dependent manner. These results indicate that the CD4(+)CD25(-)PD-1(+) T cells contain substantial amounts of T(R) cells that are involved in the maintenance of peripheral tolerance.     

Identical expression of CD45R isoforms by CD45RC+ 'revertant' memory and CD45RC+ naive CD4 T cells.

Naive and memory CD4 T cells are frequently defined by exon-specific monoclonal antibodies (mAb) which stain (or not) high- or low-molecular-weight (MW) isoforms of the leucocyte common antigen CD45. The link between isoform and the naive/memory designation is complicated by the fact that CD4 T cells with a 'memory' phenotype (CD45RA-, RB-, RC-, or CD45RO+) may revert ('revertants') and re-express the high mw isoform (CD45RA+, RB+, RC+). Isoform expression also changes during normal T-cell development. Furthermore, the picture may be incomplete since an exon-specific mAb will not detect all possible isoforms on a cell. We have used molecular techniques to determine whether revertant CD4 memory T cells were different from naive T cells with respect to CD45R isoform expression. Using the anti-CD45RC mAb OX22 to purify rat lymphocyte subsets, CD45R isoform expression was examined at the mRNA level in CD4 T cells at different stages of development and compared with that of B cells and unseparated lymphocytes. B cells contained abundant message for the highest MW 3-exon isoform ABC, the 2-exon isoforms AB and BC, and the null isoform O. Both immature CD45RC- (i.e. CD4+8- 'single positive' thymocytes, and peripheral Thy-1+ recent thymic emigrants) and mature CD45RC- 'antigen-experienced' CD4 T cells had message for single-exons B, possibly C and for the O exon. In contrast, CD45RC+ CD4 T cells contained mRNA coding for ABC (low level), AB, BC, B, C (low level) and O (low level). Importantly, there was no difference between CD45RC+ T cells that had not seen antigen ('truly native') and CD45RC+ antigen-experienced revertant memory T cells. This observation has implications for understanding long-term immunological memory.     

The relative contribution of antibodies, CD4+ and CD8+ T cells to sporozoite-induced protection against malaria.

Protective immunity against Plasmodium yoelii, induced by sporozoite immunization, was investigated using a quantitative method based on the measurement of plasmodial ribosomal RNA in the liver of sporozoite-challenged mice. The relative importance of the different immune mechanisms induced by sporozoite immunization was determined by evaluating quantitatively the anti-parasite activity of antibodies, CD4+ and CD8+ T cells. The role of antibodies was determined by passive transfer of immune sera to naive mice. The transfer to mice of sera obtained after a single immunizing dose reduced the liver stages by 47%. The respective contribution of CD4+ and CD8+ T-cell subsets was determined in B10 (H-2b) mice, treated with a monoclonal antibody (mAb) which inhibits B-cell maturation, and subsequently immunized once with irradiated sporozoites. These mice produced low levels of anti-sporozoite antibodies, but were capable of inhibiting the development of liver stages as efficiently as non-manipulated immunized mice. Administration of either anti-CD4 or anti-CD8 mAb to these mice, did not significantly decrease their capacity to inhibit the development of liver stages. We only observed a significant loss of immunity when the mice were depleted in vivo of both CD4+ and CD8+ T cells. In contrast to earlier studies, we found that the induction of protective immunity is not a phenomenon restricted to a few strains of mice having a particular genetic make-up. The apparent non-responsiveness observed in some strains of mice can be overcome by using larger immunizing doses.     

过继转输TSA诱导的CD4^＋CD25^＋调节性T细胞对不明原因复发性流产的治疗作用

目的观察过继转输TSA诱导的CD4^＋CD25^＋调节性T细胞（CD4＋CD25＋Treg）对不明原因流产的作用机制及妊娠预后的影响。方法以雌性CBA/J×雄性BALB/c为正常妊娠模型,以雌性CBA/J×雄性DBA/2J为自然流产模型,使用免疫磁珠方法分选雌性CBA/J小鼠脾脏CD4^＋CD25^＋Treg细胞,并使用流式细胞术检测分选纯度。采用TSA对流产孕鼠外周CD4^＋/CD25^-T细胞Foxp3基因特定位点进行表观修饰,以实现Foxp3稳定、持久的表达,并将CD4^＋Treg分别转输至流产模型孕4d（着床期）的雌性CBA/J孕鼠,于孕14d分别观察宿主孕鼠的胚胎吸收率。结果与对照组比较,过继转输TSA诱导的CD4^＋CD25^＋Treg细胞的宿主孕鼠的胚胎吸收率（11.27%）显著下降。结论孕早期过继转输TSA诱导的CD4^＋CD25^＋Treg细胞疗法能诱导宿主母胎免疫耐受,有利于妊娠的维持。     

Enhancement of activation-induced cell death by fibronectin in murine CD4+ CD8+ thymocytes.

Development of T cells in the thymus is achieved through the interactions of thymocytes with their microenvironments. This study focused on the function of fibronectin (FN), a major extracellular matrix molecule in the thymus, in the cell death induced by activation via the T-cell antigen receptor. FN alone did not increase cell death in murine thymocytes above the baseline level, but it significantly enhanced the cell death induced by fixed anti-CD3 monoclonal antibody (mAb), especially when a high concentration of anti-CD3 mAb was used. DNA fragmentation increased in parallel with cell death, indicating that cell death was a result of the apoptosis. Fluorescence-activated cell sorter (FACS) analysis revealed that the activation-induced cell death (AICD) caused by anti-CD3 mAb alone, or by a combination of anti-CD3 mAb and FN, occurred selectively in CD4+ CD8+ thymocytes. Very late activation antigen (VLA)-4 and VLA-5 are two major ligands to FN on thymocytes. The expression of both ligands was investigated at different stages of thymocyte development. VLA-4 was predominantly expressed at the CD4- CD8- stage, and thereafter the expression was reduced, whereas VLA-5 was constantly expressed during maturation. Furthermore, the enhancing effect by FN was inhibited in the presence of the Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) peptide but not in the presence of the connecting segment-1 (CS-1) peptide, suggesting that enhancement of AICD observed in CD4+ CD8+ thymocytes is mediated through VLA-5.