Decidual CD4+CD25+CD127dim/- regulatory T cells in patients with unexplained recurrent spontaneous miscarriage

Objectives

To investigate the frequency and function of CD4⁺CD25⁺CD127^dim/− regulatory T (Treg) cells in decidua of patients with unexplained recurrent spontaneous miscarriage (URSM).

Study design

The decidual lymphocytes from patients who experienced URSM and normal pregnant women (controls) were collected by Ficoll density gradient centrifugation. CD4⁺CD25⁺CD127^dim/− Treg cells were isolated by magnetic cell sorting. The proportion of Treg cells and IL-10, TGF-β in Treg cells were determined by flow cytometry. Inhibitory effects of Treg cells on effecter T cells were detected with or without the presentation of anti-IL-10 antibodies and anti-TGF-β antibodies.

Results

The frequency of CD4⁺CD25⁺CD127^dim/− Treg cells was decreased in URSM decidua compared to controls (2.09% ± 0.86% vs. 2.97% ± 1.19%, p = 0.005), and the expression of IL-10 and TGF-β in Treg cells was lower in the URSM group than in the control group (p = 0.04 and p = 0.01, respectively). Furthermore, the suppressive effect of CD4⁺CD25⁺CD127^dim/− Treg cells on the proliferation of effector T cells was decreased in URSM decidua (p < 0.05). Suppression was mediated predominantly through IL-10 and TGF-β in decidua.

Conclusions

Decreased frequency and immunosuppressive capacity of CD4⁺CD25⁺CD127^dim/− Treg cells was found in URSM decidua. Treg cells inhibit proliferation of effector T cells mainly via IL-10 and TGF-β in URSM decidua.     

Adoptive transfer of CD4+CD25+ regulatory T cells for prevention and treatment of spontaneous abortion

Objectives

The purpose of this study was to examine whether adoptive transfer with in vitro expanded CD4⁺CD25⁺ regulatory T cells (Tregs) could prevent immune response-mediated spontaneous abortion in mice.

Study design

Female CBA/J mice were mated with male Balb/c as the control with normal pregnancy or with DBA/2J mice as a model of spontaneous abortion. The CBA/J mice mated with DBA/2J were treated intravenously with freshly isolated or in vitro expanded Tregs on day 1 or 4 of pregnancy, respectively. The numbers of surviving and reabsorbed fetuses in the different groups of mice were counted on day 14 of pregnancy, and the concentrations of cytokines in individual sera and the supernatants of cultured Tregs were measured by ELISA.

Results

Adoptive transfer with freshly isolated Tregs only slightly reduced the fetal resorption rate, which was not significantly different from that of the mice without Treg treatment, regardless of treatment at early stage and implementation of pregnancy. In contrast, adoptive transfer with in vitro expanded Tregs significantly reduced the fetal resorption rates, particularly for treatment at early stage of pregnancy (P < 0.05). Furthermore, adoptive transfer with in vitro expanded Tregs at early stage of pregnancy significantly increased the levels of serum IL-10, TGF-β1, and the ratios of IL-10 to IFN-γ.

Conclusions

Our data clearly indicated that adoptive transfer with in vitro expanded Tregs at early stage of pregnancy protected fetuses from spontaneous abortion by re-establishing immune tolerance in mice.     

Umbilical cord blood CD45+/CD34+ cells coexpression in preterm and full-term neonates: a pilot study

Objective.?Human umbilical cord blood (hUCB) is rich in stem cells. The CD45⁺/CD34⁺ coexpression in hUCB is a marker of hemopoietic progenitor cells. The objective of this study is to compare the coexpression of hUCB CD45⁺/CD34⁺ cells in preterm (PT) and full-term (FT) neonates.

Methods.?We studied the coexpression of hUCB CD45⁺/CD34⁺ cells in PT and FT neonates. The study included 25 PT (29–36 weeks gestation) and 25 FT (37–41) neonates delivered at Ain Shams University, Maternity Hospital. Absolute mononuclear layer cord blood CD45⁺/CD34⁺ cell count were measured by flow cytometry. Morbidity was assessed for 12 of the studied 25 PT infants, using Morbidity Assessment Index for Newborns score.

Results.?The absolute CD45⁺/CD34⁺ count did not differ between PT and FT infants: Z?=??0.485, p?=?0.63. There was no correlation between absolute cell count and GA (r?=?0.013, p?=?0.9) for all 50 neonates. Mode of delivery did not affect the absolute count in the PT infants: Z?=?–0.6, p?=?0.57. There was no correlation between the degree of morbidity and absolute cell count in PT neonates; r?=?0.13, p?=?0.69.

Conclusion.?The absolute cell count is not affected by gestational age and did not relate to morbidity scores in the studied PT infants. Further, wide-scale work will be needed to study CD45⁺/CD34⁺ count in hUCB in sick PT neonates.     

CD4~+CD25~+FOXP3~+调节性T淋巴细胞数量及功能变化与子宫内膜异位症患者发病相关性研究

目的:分析子宫内膜异位症(EMs)患者在位、异位内膜及外周血内CD4~+CD25~+FOXP3~+调节性T淋巴细胞(Treg细胞)的数量、分布及功能特性,探讨Treg细胞与EMs发病之间的关系。方法:收集EMs患者在位、异位内膜组织及外周血标本,并以非内异症患者作为对照,免疫组化分析在位、异位内膜内叉头状/翅膀状螺旋转录因子3(FOXP3)阳性细胞的数量及分布变化情况,并与EMs患者的r AFS临床分期进行相关性分析。流式细胞术检测Treg细胞占外周血CD4~+T淋巴细胞的百分比,及磁珠分选外周血内CD4~+CD25~+T细胞及CD4~+CD25-T细胞,3H-thymidine法测定CD4~+CD25~+T细胞免疫抑制功能变化情况。结果:EMs、非EMs患者在位内膜内平均FOXP3阳性细胞密度并无显著差异[(76.44±62.14)/mm~2vs(50.59±20.79)/mm~2;WU=152.0,P=0.20]。进一步按月经周期行亚组分析:EMs分泌期内膜内FOXP3阳性细胞密度显著高于非EMs患者[(94.84±53.91)/mm~2vs(31.37±19.02)/mm~2;MU=43.00,P=0.03]。不同r AFS期别患者卵巢异位症病灶内FOXP3阳性细胞密度并无显著差异[I~II期:(123.00±115.00)/mm~2vs III~IV期:(111.00±108.00)/mm~2;MU=139.5,P0.05]。EMs患者外周血内Treg细胞比例显著低于非EMs患者[(0.58±0.21)%vs(1.35±0.38)%,P0.001],但EMs患者外周血CD4~+CD25~+T淋巴细胞对CD4~+CD25-T淋巴细胞增殖抑制率,与非EMs相比并无显著变化[(68.43±18.15)%vs(66.37±17.78)%,P0.05]。结论:EMs患者在位内膜内Treg细胞失去正常的周期波动性,EMs患者分泌期内Treg细胞数目增加可能与内异症发病相关。EMs患者外周血内Treg细胞比例下降,但其免疫抑制功能并无明显改变。     

体外受精-胚胎移植反复失败患者外周血中CD4~+CD25~+Treg的表达

目的:探讨外周血中CD4+CD25+调节性T淋巴细胞(CD4+CD25+Treg)在体外受精-胚胎移植反复失败(RIF)发病机制中的作用。方法:用流式细胞分析技术检测12例RIF组、15例胚胎移植妊娠组、15例正常未孕组患者外周血中CD4+CD25+Treg的表达。结果:RIF组患者外周血中CD4+CD25+Treg的表达率为(0.80±0.56)%,低于正常未孕组的(4.05±0.91)%,两组差异有统计学意义(P<0.05);胚胎移植妊娠组患者外周血中CD4+CD25+Treg的表达率为(11.01±2.09)%,高于RIF组,两组差异有统计学意义(P<0.05)。结论:辅助生殖中反复植入失败可能与CD4+CD25+Treg的表达下降有关,这为免疫治疗提供了理论依据。     

泼尼松治疗原发性肾病综合征对患儿CD4+CD25+调节性T细胞的影响分析

目的　观察原发性肾病综合征（PNS）患儿泼尼松治疗前后CD4+CD25+ 调节性T细胞（CD4+CD25+ Tr）的变化,阐明肾上腺糖皮质激素治疗儿童PNS的免疫机制。方法　选择2004—2007年在深圳市儿童医院住院治疗的初发PNS患儿42例,其中激素敏感型32例,激素耐药型10例。同期25例健康体检儿童作为对照组。流式细胞术检测泼尼松治疗前后外周血CD3+CD4+CD8-、CD3+CD4-CD8+、CD4+CD25+Tr的比例,荧光定量聚合酶链反应（Real-time PCR）检测泼尼松治疗前后外周血单个核细胞（PBMC）叉头型基因P3（Foxp3）、细胞毒性T淋巴细胞相关抗原4（CTLA-4）和糖皮质激素诱导的肿瘤坏死因子受体 （GITR）基因mRNA的表达。结果 　与对照组比较,PNS患儿外周血CD3+CD4+CD8- T细胞、CD3+CD4-CD8+ T细胞、CD4+CD25+ Tr比例无明显改变（P > 0.05）。激素敏感型PNS患儿CD4+CD25+Tr比例在泼尼松治疗后明显增高,差异有统计学意义（P < 0.01）;激素耐药型PNS患儿CD4+CD25+Tr比例在泼尼松治疗前后无明显改变（P > 0.05）。激素敏感型PNS患儿在泼尼松治疗后PBMC细胞Foxp3、CTLA-4和GITR基因mRNA的表达明显增高;而激素耐药型PNS患儿泼尼松治疗前后Foxp3、CTLA-4基因表达无明显改变,仅GITR表达明显增高。 结论 泼尼松等肾上腺糖皮质激素类药物可通过上调激素敏感型PNS患儿CD4+CD25+调节性T细胞的表达发挥免疫治疗作用。     

原因不明复发性流产与蜕膜CD4~+ CD25~+ T调节细胞和CD8~+ CD28~- T细胞的关系

目的:探讨原因不明复发性流产(unexplained recurrent spontaneous abortion,URSA)与蜕膜CD4+ CD25+ T细胞和CD8+ CD28-T细胞的关系。方法:采用流式细胞四色荧光法,检测原因不明复发性流产17例(URSA组)和正常早孕人流20例(对照组)的蜕膜CD4+ CD25+ T细胞及其FoxP3(+)表达,CD8+ CD28- T细胞及其表面CD95、CD95L表达。结果:两组蜕膜中CD4+ CD25+ T细胞比例无明显差异(P>0.05),URSA组蜕膜CD4+ CD25+ T细胞中FoxP3阳性率明显低于对照组(P<0.0001)。两组蜕膜CD8+ CD28- T细胞比例及其细胞表面CD95和CD95L的阳性表达率均无明显差异(P>0.05);结论:蜕膜CD4+ CD25+ FoxP3(+)T调节细胞明显减少,是导致URSA患者母胎界面免疫耐受异常的重要原因。     

卵巢早衰患者外周血CD4~+CD25~+Treg细胞的变化及意义

目的:探讨CD4+CD25+调节性T细胞(即CD4+CD25+Treg细胞)在卵巢早衰发病机制中的作用。方法:流式细胞仪定量检测卵巢早衰(premature ovarian failure,POF)患者、卵巢储备功能下降(diminished ovarian reserve,DOR)患者及健康对照组外周血CD4+T、CD8+T细胞及CD4+CD25+Treg细胞数量,应用3H-thymidine掺入法测定POF患者及对照组外周血CD4+CD25+Treg细胞对效应性T细胞的增殖抑制功能。结果:与对照组相比,POF患者及DOR患者CD4+CD25+Treg细胞比例降低(P<0.01)、POF患者CD4+T/CD8+T细胞比值增高(P<0.05),DOR患者CD4+T/CD8+T细胞比值无明显变化(P>0.05);POF患者免疫抑制功能无明显降低(P>0.05)。结论:CD4+CD25+Treg细胞比例降低与T细胞亚群失衡可能是POF的发病机制。     

原因不明复发性流产主动免疫治疗前后CD4+ CD25+调节性T细胞表达频率的研究

目的探讨CD4^＋CD25^＋调节性T细胞（Tr）在原因不明复发性流产（URSA）患者主动免疫治疗中的作用。方法上海交通大学医学院附属仁济医院对2004-03—2005-05就诊于门诊的18例URSA患者，采用双荧光标记流式细胞分析技术，检测其淋巴细胞主动免疫治疗前后外周血CD4^＋CD25^＋Tr细胞表达频率的变化。结果主动免疫治疗后URSA组外周血中CD4^＋CD25^＋种Tr较治疗前明显增加（P〈0．05），CD4^＋CD25^＋较治疗前降低（P〈0．05）。主动免疫治疗后，妊娠成功的妇女CD4^＋CD25^brightTr显著多于妊娠失败者。结论主动免疫治疗可提高外周血CD4^＋CD25^brightTr数量，CD4^＋CD25^brightTr可能是调控母胎界面局部免疫耐受形成的一个重要因素，有可能成为URSA患者主动免疫治疗的监测指标之一。     

原因不明复发性流产患者蜕膜CD4~+CD25~+CD127~(dim/-) Treg细胞的表达频率和功能研究

目的:探讨原因不明复发性流产(URSA)患者母胎界面免疫耐受环境的变化。方法:留取URSA患者21例(URSA组)和30例正常早孕人流妇女(对照组)蜕膜组织,制备成单个核细胞悬液,流式细胞术分析CD4+CD25+CD127dim/-Treg细胞的表达频率;取两组蜕膜单个核细胞悬液各5例,用免疫磁珠分离法分离出CD4+CD25+CD127dim/-Treg细胞,流式细胞术分析CD4+CD25+CD127dim/-Treg细胞内IL-10和TGF-β的表达水平;体外增殖抑制试验分别检测用抗IL-10抗体和抗TGF-β抗体处理的CD4+CD25+CD127dim/-Treg细胞,和未用抗体处理的CD4+CD25+CD127dim/-Treg细胞对自身效应T细胞增殖的抑制作用。结果:URSA组蜕膜CD4+CD25+CD127dim/-Treg细胞的表达频率明显低于对照组(P=0.005);URSA组蜕膜CD4+CD25+CD127dim/-Treg细胞内IL-10和TGF-β表达频率均较对照组明显降低(P=0.04,P=0.01);URSA组CD4+CD25+CD127dim/-Treg细胞对自体效应性T细胞的增殖抑制作用显著低于对照组(P0.05),且抗IL-10抗体和抗TGF-β抗体可部分阻断蜕膜CD4+CD25+CD127dim/-Treg细胞的免疫抑制功能(P均0.05)。结论:URSA患者蜕膜CD4+CD25+CD127dim/-Treg细胞不仅表达频率降低,而且免疫抑制功能也减弱,且这种免疫抑制功能的减弱主要受IL-10和TGF-β介导,揭示CD4+CD25+CD127dim/-Treg细胞对URSA患者蜕膜局部免疫耐受环境产生影响。     

Longitudinal study of CD4+ cell counts in HIV-negative pregnant patients

Objective.?To evaluate the absolute CD4⁺, CD8⁺, and lymphocyte cell counts and percentages from the first trimester through 6–12 weeks post-delivery in normal human immunodeficiency virus (HIV)-negative pregnant patients.

Methods.?A longitudinal laboratory analysis was performed during pregnancy that involved 51 HIV-negative subjects with blood analysis obtained in all trimesters, at delivery, and 6–12 weeks post-delivery. Twenty-five HIV-negative non-pregnant controls were also evaluated. Blood was analysed for absolute CD4⁺, CD8⁺, and lymphocyte cell counts and percentages. Means, standard deviations, trends, and differences were examined.

Results.?The mean white blood cell (WBC) count is elevated above the non-pregnant state and this parameter increases through the pregnancy up to and including parturition. The mean absolute lymphocyte cell count, lymphocyte percentage, and absolute CD4⁺ cell count are significantly lower during pregnancy and the progression through pregnancy appears U-shaped. The mean absolute CD8⁺ cell count is not significantly different. The CD4⁺ and CD8⁺ percentages are higher during pregnancy and this elevation persists into the 6–12 week post-delivery time period. A 3-digit drop in CD4⁺ percentage is common during pregnancy between blood draws; whereas, a 30% decrease or more in absolute CD4⁺ cell count is rare.

Conclusions.?By longitudinal analysis, pregnancy appears to significantly elevate the mean values of the WBC count, CD4⁺ percentage, and CD8⁺ percentage, but significantly decreases the absolute lymphocyte count, lymphocyte percentage, and absolute CD4⁺ cell count when compared to non-pregnant controls. The mean absolute CD8⁺ cell count appears to be unaffected.     

复发性流产外周IL-10~+Tim-3~+ T细胞降调节

目的探讨外周血CD3+T细胞中T细胞免疫球蛋白黏蛋白-3(Tim-3)及程序性死亡因子-1(PD-1)联合细胞因子在复发性流产(RSA)中的诊断价值。方法用流式细胞术检测19例RSA患者及17例正常早孕者外周血CD3~+T细胞表面Tim-3及PD-1含量,以及破细胞膜后细胞内因子干扰素(IFN)-γ和白介素(IL)-10在Tim-3~+PD-1~+、Tim-3~-PD-1~-、Tim-3~-PD-1~+和Tim-3~+PD-1~-4群细胞内的表达情况。结果 RSA患者Tim-3~+PD-1~+T细胞比例(0.57%±0.26%)明显低于早孕组(1.24%±0.77%)(P0.001)。RSA组4群T细胞中IL-10阳性细胞所占比例分别为33.55%±16.27%、0.92%±0.88%、1.61%±1.35%、16.36%±13.98%;早孕组4群T细胞中IL-10阳性细胞占比分别为45.92%±17.89%、0.49%±0.27%、0.92%±0.68%、33.43%±16.98%。RSA组和正常组Tim-3~+PD-1~+T细胞群中IL-10的含量均显著高于其他3群(P0.05);RSA组Tim-3~+PD-1~+和Tim-3~+PD-1~-T细胞中IL-10阳性细胞含量(33.55%±16.27%,16.36%±13.98%)显著低于正常早孕组(45.92%±17.89%,33.43%±16.98%)(P0.05,P0.01)。而IFN-γ在RSA组和正常组4群细胞中的表达无统计学差异。结论 RSA患者IL-10~+Tim-3~+T细胞显著降低,可作为判断RSA的新的参考指标。     

Expression and significance of dendritic cells and Th17/Treg in serum and placental tissues of patients with intrahepatic cholestasis of pregnancy

Purpose: Dendritic cells (DCs) are involved in immune system, which can also regulate the differentiation of T helper 17 (Th17) and regulatory T cells (Treg). DCs and Th17/Treg participate in preeclampsia and recurrent spontaneous abortion (RSA), but there is still lack of research in intrahepatic cholestasis of pregnancy (ICP). The aim was to evaluate the expression and significance of CD83⁺DCs, CD1a⁺DCs, interleukin-17 (IL-17) and IL-35 in serum and placental tissues of patients with ICP.

Methods: Thirty cases of mild ICP, 25 cases of severe ICP were selected, and 30 cases of normal pregnant women were selected as control group. Enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) were used to detect the expression of CD83⁺DCs, CD1a⁺DCs, IL-17 and IL-35 in serum and placenta tissues, respectively.

Results: There were more CD83⁺DCs, IL-17 expressed in placenta from women with ICP than in normal pregnancies, while the number of decidual CD1a⁺DCs, IL-35 was significantly lower in ICP than in normal pregnant women. The comparison within three groups had statistical difference (p?+DCs and CD1a⁺DCs levels had no significance. IL-17 was higher in ICP, while IL-35 was lower.

Conclusions: DCs are involved in damaging the maternal–fetal immune tolerance by changing the phenotype and mature state, which may affect the differentiation of Th17/Treg to cause ICP.     

原因不明复发性流产患者外周血及蜕膜组织中CD+4CD+25调节性T细胞比例的变化

目的 探讨原因不明复发性流产(URSA)患者外周血及蜕膜组织中CD+4CD+25调节性T(Tr)细胞比例的变化.方法 采用双荧光标记流式细胞分析技术检测25例URSA患者(流产组)、34例正常早孕妇女(正常妊娠组)和22例正常非孕妇女(正常非孕组)外周血及蜕膜组织中CD+4CD+25Tr细胞的比例.结果 (1)流产组和正常妊娠组妇女外周血CD+4 CDbright25T细胞的比例[分别为(1.55±0.77)%、(2.65±1.10)%]均显著高于正常非孕组妇女[(0.39±0.14)%],分别比较,差异均有统计学意义(P<0.05);流产组妇女外周血CD+4 CDbright25T细胞的比例显著低于正常妊娠组妇女,两组比较,差异有统计学意义(P<0.05).(2)流产组妇女蜕膜组织中CD+4 CDbright25T细胞比例[(0.59±0.23)%]显著低于正常妊娠组妇女[(1.24±0.55)%],两组比较,差异有统计学意义(P<0.01).流产组妇女蜕膜组织中CD+4CDdim25T细胞比例[(4.23±1.52)%]与正常妊娠组[(3.75±1.88)%]比较,差异无统计学意义(P>0.05).(3)正常妊娠组妇女蜕膜组织中CD+4CDbright25T细胞占CD+4T细胞的比例(CD+4CDbright25/CD+4)为(13.10±10.25)%,显著高于外周血[(5.59±2.62)%],两者比较,差异有统计学意义(P<0.05);流产组患者蜕膜组织中CD+4CDbright25/CD+4比例[(5.16±2.83)%]与外周血[(4.64±2.07)%]比较,差异无统计学意义(P>0.05).结论 CD+4CD+25Tr细胞数量在早孕期显著升高,参与了正常妊娠的维持,有可能是调控母-胎界面局部免疫耐受形成的一个重要因素;CD+4 CD+25Tr细胞数量的减少可能与URSA的发生有关.     

再生障碍性贫血患儿CD+4CD+25T细胞及TGF β1Flt 3L水平变化的意义

目的评价免疫调节T细胞和细胞因子在再生障碍性贫血(再障)细胞免疫功能紊乱中的作用。 方法2004 02—2005 06中山大学第二附属医院采用流式细胞术检测27例特发性再障患儿骨髓及外周血淋巴细胞亚群和CD+4CD+25T细胞水平，ELISA检测骨髓转化生长因子(TGF β1)和Flt 3L水平，并与正常儿童对照。 结果与对照组比较，初诊再障患儿外周血和骨髓CD+8T细胞均显著增高(P<0.05)，重型再障(SAA)组伴外周血CD-3CD+56NK细胞及骨髓B细胞显著下降(P<0.05)。初诊SAA组骨髓CD+4CD+25T细胞\[(7.5±3.4)%\]显著高于对照组\[(4.3±0.9)%，P<0.05\]，初诊SAA组及轻型再障(MAA)组骨髓CD+4CD+25/CD+4比值分别为(28.9±11.1)%和(28.2±9.4)%，均显著高于对照组\[(17.4±0.9)%，P均<0.05\]，骨髓TGF β1分别为(2.2±1.7)μg/L和(2.0±0.6)μg/L，均较对照组［(4.4±0.9)μg/L］显著降低(分别为P<0.01、P<0.05)，而Flt 3L水平分别为(1031.1±321.8)ng/L和(694.7±424.7)ng/L，均较对照组［(63.0±37.5)ng/L］显著增高(P均<0.01)。缓解期SAA儿童除外周血CD+8T细胞仍较对照组显著增高外，其余上述指标均接近正常水平。相关分析显示，骨髓CD+4CD+25T细胞与CD+3CD+4T细胞呈显著正相关(r分别为0.495、0.540,P<0.01)；Flt 3L与骨髓CD+3、CD+4、CD+8T细胞及CD+4CD+25T细胞均呈显著正相关(r分别为0.732、0.542、0.688、0.405,P分别<0.01、0.01、0.01、0.05)，而TGF β1与骨髓CD+8T细胞和Flt 3L水平呈显著负相关(r分别为-0.431、-0.482,P分别<0.05、<0.01)。 结论儿童再障发病与CD+4CD+25T细胞数量缺乏无关，骨髓TGF β1水平显著降低和Flt 3L水平显著增高可能在再障儿童T淋巴细胞数量增多和功能紊乱中起重要作用。     

宫颈癌患者外周血TSCM与Treg的分布特征及临床意义

目的:探讨宫颈癌患者外周血中干细胞样记忆性T细胞(TSCM)与调节性T细胞(Treg)的分布特点及临床价值.方法:选取2019年10月至2020年3月在徐州医科大学附属医院住院的宫颈癌、高级别上皮内瘤变、低级别上皮内瘤变各30例为观察组,同期15例宫颈炎患者作为对照组.采集外周静脉血2ml,流式细胞学方法检测TSCM与...