流式细胞术检测表达FoxP3的CD4+CD25+调节性T细胞

目的 探讨转录因子FoxP3在外周血CD4 CD25 调节性T细胞(Treg)内的表达特性.方法 取正常人外周血单个核细胞(PBMC),经固定/透膜处理进行细胞内染色,用流式细胞术(FCM)检测T细胞内FoxP3.以抗人CD3/CD28诱导T细胞活化,再用定量PCR及FCM术分别检测FoxP3 mRNA及蛋白质表达水平.结果 FoxP3主要表达于CD4 T细胞,尤其是CD25高表达的CD4 T细胞,达97%左右;中等程度表达CD25的细胞中也含有少量FoxP3阳性细胞.T细胞活化后,表达FoxP3的CD4 CD25 T细胞比例显著增高,FCM检测结果与定量PCR结果一致.结论 FoxP3不仅参与维持Treg细胞的功能,也与T细胞的活化有关.FCM检测细胞核内表达的FoxP3分子为研究Treg细胞提供了一种快速、简便的方法.     

动脉粥样硬化患者单个核细胞Siglec-1在促进CD4+和CD8+T淋巴细胞增殖和分泌细胞因子中的作用

目的 探讨动脉粥样硬化(AS)患者单个核细胞(PBMC)唾液酸黏附素(Siglec-1)在刺激白细胞分化抗原(CD)4+和CD8+T淋巴细胞活化增殖中的作用.方法 实验研究.用磁珠分离长征医院18例急性冠状动脉综合征(ACS)和41例稳定型心绞痛(SA)患者及32名健康对照者CD14阳性PBMC后,用不同浓度α-干扰素(IFN-α,0、2、5、10 ng/ml)刺激Siglec-1高表达,或用小干扰RNA或抗Siglec-1单抗靶向抑制Siglec-1表达,再与1名第三方健康献血者CD4 +/CD8+T淋巴细胞共培养5d.然后,将实验分为11组:健康人CD14(1组),健康人CD14+ IFN-α5 ng/ml(2组),健康人CD14+ IFN-Ω 5 ng/ml+ anti-Siglec-1 2 μg/ml(3组),ACS CD14(4组),ACS CD14+ siRNA干扰对照组(Mock,5组),ACS CD14+ siRNA 679 40 nmol/L(6组),ACS CD14+ anti-Siglec-1 2μg/ml(7组),SACD14(8组),SA CD14+ Mock(9组),SA CD14+ siRNA 679 40 nmol/L(10组),SA CD14+ anti-Siglec-12 μg/ml(11组);每组测定10份标本.用CCK-8活细胞计数试剂盒检测共培养T淋巴细胞增殖,用ELISA检测共培养T淋巴细胞分泌白细胞介素(IL)-2、IL-10、IL-12、γ干扰素(IFN-γ).测定各组PBMC刺激T淋巴细胞分泌细胞因子的计量数据用中位数(四分位数)表示,采用非参数秩和检验.结果 靶向阻断Siglec-1后(6组),PBMC刺激CD4+T淋巴细胞及CD8+T淋巴细胞的增殖能力减弱,PBMC刺激CD4+T淋巴细胞分泌IL-2、IL-12、IFN-γ分别为67.00(62.50～ 87.30)、0.86(0 ～1.63)、47.82(37.60 ～ 56.67) pg/ml,且分泌能力减弱;IL-10为56.00(46.25 ～67.40) pg/ml,且分泌能力增强;未处理组(4组)上述细胞因子分别为213.70(187.50 ～ 275.30)、6.87 (4.90 ～8.93)、114.90(89.50～167.40)、21.08(15.70～33.20) pg/ml,二者差异有统计学意义(U值分别为8.50、17.00、8.50、87.50,P均＜0.05).当健康对照组单核细胞经IFN-α刺激上调Siglec-1表达后(2组),刺激CD4+T淋巴细胞分泌IL-2、IL-12、IFN-γ分别为220.44(174.30 ～ 312.30)、7.90 (6.540 ～10.40)、143.75(78.20～210.00) pg/ml,且能力增强;IL-10为21.95(16.30 ～25.00) pg/ml,而能力减弱,与1组比较,差异有统计学意义(U值分别为89.50、98.00、100.00、0,P均＜0.05).抑制或增强Siglec-1对CD8+T淋巴细胞分泌的上述细胞因子无影响(P均＞0.05).结论 IFN-α可刺激Siglec-1表达增加,Siglec-1可通过促进CD4+/CD8+T淋巴细胞增殖或CD4+T淋巴细胞分泌Th1型细胞因子参与AS发病过程.     

淋巴细胞CD3~+、CD4~+、CD8~+亚群检测在早发型新生儿败血症诊断中的价值

目的探讨早发型新生儿败血症诊断中,淋巴细胞CD3~+、CD4~+、CD8~+百分比检测的价值。方法将2014年1月至2015年6月出生,7d内发病,疑似感染的患儿纳入本研究,入院24h内采集静脉血,检测CD3~+、CD4~+、CD8~+淋巴细胞百分比,C反应蛋白(CRP)及血常规,在抗菌药物使用前做血培养。将其中血培养证实为早发型新生儿败血症的足月新生儿作为败血症组,血培养阴性的作为局部感染组。同时选择同期因高胆红素血症(排除感染等因素所致)而住院的足月新生儿作为非感染组。采用流式细胞仪对上述患儿标本进行检测,观察并比较3组间淋巴细胞亚群CD3~+、CD4~+、CD8~+百分比。结果败血症组CD3~+[(40.3±10.6)%],CD4~+[(28.6±11.2)%],CD8~+[(10.8±2.6)%]低于局部感染组的CD3~+[(64.8±9.8)%],CD4~+[(48.9±10.2)%],CD8~+[(17.6±5.6)%]和非感染组的CD3~+[(62.6±11.6)%]、CD4~+[(46.4±13.6)%]、CD8~+[(16.5±7.3)%],差异有统计学意义(P0.05),局部感染组的CD3~+、CD4~+、CD8~+与非感染组之间比较,差异无统计学意义(P0.05)。结论CD3~+、CD4~+、CD8~+百分比可以作为诊断早发型新生儿败血症的指标。     

实时定量PCR检测原发性胆汁性肝硬化单核细胞中Siglec-1 mRNA的表达

目的检测Sigtec-1（sialic add-binding immtmoglobulin-like lectins,唾液酸结合的免疫球蛋白样凝集素,CD169）在原发性胆汁性肝硬化（primary biliary cirrhosis,PBC）患者外周血单核细胞上的mRNA表达水平,并探讨其在原发性胆汁性肝硬化发生发展中的作用。方法实时荧光相对定量PCR方法检测30例原发性胆汁性肝硬化患者及30例健康对照者、30例肝炎后肝硬化对照者外周血单核细胞中Siglec-1mRNA的含量;生化常规测定所有人选者血清GGT、ALP指标水平。结果原发性胆汁性肝硬化组Siglec-1mRNA的相对表达量为健康对照组的3．42倍,差异具有统计学意义（P〈0．01）。PBC患者Siglec-1mRNA增高与GGT（r=0．482,P〈0．01）和ALP（r=0．365,P〈0．05）密切相关。结论原发性胆汁性肝硬化患者单核细胞中mRNA含量显著增加,说明原发性胆汁性肝硬化患者外周血单核细胞已经发生巨噬细胞化,单核巨噬细胞介导的免疫炎症反应在原发性胆汁性肝硬化发生发展过程中起重要作用。     

PACAP对创伤性脑损伤大鼠CD4+/CD8+T细胞水平的影响

目的 观察垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase activiting polypeptide,PACAP)对大鼠创伤性脑损伤(traumatic brain injury,TBI)的影响及对血液和脾脏CI4+、CD8+T细胞数量的影响.方法 所有实验均在在江苏省麻醉学重点实验室内完成.将雄性SD大鼠随机(随机数字法)分为假手术组(n=6)、生理盐水+ TBI组(n=6)、PACAP+TBI组(n=6),采用大鼠创伤性脑损伤模型,于创伤前20min侧脑室微量注射PACAP 1μg/5μl,创伤后24h取材,采用HE染色方法观察脑组织损伤程度,采用流式细胞术检测各组大鼠血液及脾脏中CI4+、CD8+T细胞的数量.实验结果采用完全随机设计方差分析法.结果 与假手术组比较,TBI大鼠创伤周围皮层神经元水肿、坏死,海马神经元排列紊乱,血液和脾脏CI4+T细胞的数量减少(P=0.000,P =0.005),CD8+T细胞的数量增加(P=0.01);侧脑室内微量注射PACAP后,能明显减轻TBI大鼠皮层和海马的神经元损伤,增加血液和脾脏CD4+T细胞的数量(P=0.019,P=0.839),降低CD8+T细胞的数量.结论 侧脑室微量注射PACAP能减轻大鼠创伤性脑损伤,可能与其对T细胞影响有关.     

流式细胞术检测外周血CD14+细胞的活化程度

目的　利用流式细胞术检测外周血CD14+细胞活化程度。方法　应用流式细胞术分别检测了 2 5份 ( 7份肝素抗凝的正常对照标本、7例肝素抗凝、11例枸橼酸钠抗凝的慢性活动性乙型肝炎标本 )外周血 (PBMC)CD14+细胞的侧向角光散射 (颗粒度 )SSC值、细胞活化相关表面抗原 (CD6 9)表达率。结果　肝素抗凝HBV组CD14+细胞颗粒度值 [( 85 3 1± 2 46 3)道 ]高于肝素抗凝正常对照组CD14+细胞颗粒度值 [( 474 5± 47 9)道 ](P≤ 0 0 5 ) ;肝素抗凝HBV组CD14+细胞颗粒度值 [( 85 3 1±2 46 3)道 ]高于枸橼酸钠抗凝组 [( 5 2 0 1± 10 5 6 )道 ](P≤ 0 0 5 ) ;肝素抗凝HBV组CD6 9在CD14+细胞的表达率 ( 2 2 71± 13 5 7) %明显高于肝素抗凝正常对照组CD6 9在CD14+细胞的表达率 ( 7 18±4 2 7) % (P≤ 0 0 5 ) ;肝素抗凝HBV组CD6 9在CD14+细胞的表达率 ( 2 2 71± 13 5 7) %明显高于枸橼酸钠抗凝HBV组 ( 3 93± 3 94) % (P≤ 0 0 1)。结论　采用流式细胞术检测细胞颗粒度、活化抗原表达率 ,是一种灵敏、快速、客观的评价外周血CD14+细胞活化程度的方法 ;使用不同抗凝剂肝素或枸橼酸钠对外周血CD14+细胞颗粒度、活化抗原表达率有影响 ,肝素抗凝血用来检测CD14+细胞活化程度效果更好。     

用流式细胞术结合单克隆抗体CD71,Ki-67和PCNA研究儿童急性淋巴细胞白血病的细胞增殖(英文)

为研究儿童急性淋巴细胞白血病的细胞增殖动力学,我们用流式细胞术结合单克隆抗体CD71,Ki-67和增殖性细胞核抗原(PCNA)检测了21例初诊急性淋巴细胞白血病(ALL)儿童骨髓细胞的增殖性抗原表达情况。结果表明:白血病细胞的CD71标记指数(LI)与正常细胞相比无显著差异,且个体间异质性较大;白血病细胞的Ki-67和PCNA的LI明显高于正常细胞,每例标本的PCNA LI均高于Ki-67LI,两者间有明显相关关系。本实验结果提示白血病细胞的增殖活力明显高于正常细胞,单克隆抗体Ki-67和PCNA是检测白血病细胞增殖活力的有效生物学指标,且PCNA的敏感性高于Ki-67。     

3例新型冠状病毒肺炎患者外周血CD4~+T淋巴细胞和CD4~+/CD8~+比值变化

正新型冠状病毒肺炎原名为新型冠状病毒感染的肺炎,是2019年底开始在国内流行的按甲类传染病管理的乙类呼吸道传染病,主要通过呼吸道飞沫和密切接触传播。2020年1月30日世界卫生组织(WHO)将新型冠状病毒肺炎疫情列为国际关注的突发卫生公共事件~([1])。新型冠状病毒肺炎病原体是一种人类从前没有发现的新型冠状病毒~([2]),患者CT影像学检查结果表现出病毒性肺炎特征,患者咽拭子标本新型冠状病毒核酸检测阳性,外周血白细胞计数正     

多器官功能障碍综合征患者外周血CD3+、CD4+、CD8+T淋巴细胞的变化

目的 观察多器官功能障碍综合征(MODS)患者外周血CD3+、CD4+、CD8+ T淋巴细胞的变化,并探讨其临床意义.方法 采用流式细胞仪检测35例MODS患者(MODS组)及20例健康体检者(健康对照组)外周血CD3+、CD4+、CD8+ T淋巴细胞计数.结果 MODS患者CD3+、CD4+ T淋巴细胞计数明显低于健康对照组(P均<0.01),两组CD8+ T淋巴细胞计数比较差异无统计学意义(P>0.05),故MODS组CD4+/CD8+ T淋巴细胞计数比值明显低于健康对照组(P<0.01).结论 MODS患者T淋巴细胞亚群发生变化,提示免疫失衡是MODS发生的重要因素.     

急性白血病患者外周血CD4~+CD25~+Foxp3~+调节性T细胞检测及其对细胞凋亡的作用

目的研究CD4~+CD25~+Foxp3~+调节性T细胞在急性白血病患者外周血中的检测,并探讨其对细胞凋亡的作用。方法将65例急性白血病患者(包括急性淋巴细胞性白血病33例和急性髓系白血病32例)分为未缓解组(28例)和缓解组(37例),25例健康志愿者作为对照组。根据是否合并感染又分为合并感染组(39例)和未合并感染组(26例)。采用细胞内染色的流式细胞术及荧光定量PCR的方法,分别在蛋白质和mRNA水平检测Foxp3表达,并与正常对照组进行比较。同时,利用荧光定量PCR的方法的检测凋亡相关基因Bcl-2、Bax、P53的表达。结果与正常对照相比,急性白血病患者(未缓解组和缓解组)外周血中CD4~+CD25~+Foxp3~+比例明显提高,且治疗缓解后CD4~+CD25~+Foxp3~+表达下调(P0.05)。凋亡相关基因Bcl-2表达上调,Bax、P53表达下调。结论急性白血病患者外周血CD4~+CD25~+Foxp3~+调节性T细胞明显升高,并且,影响凋亡相关基因Bcl-2、Bax、P53的表达,提示CD4~+CD25~+Foxp3~+调节性T细胞可能通过细胞凋亡信号通路影响急性白血病的发展。     

CD4~+/CD8~+ T细胞在维持性血液透析患者血管钙化发生中的作用

目的探讨维持性血液透析(maintenance hemodialysis,MHD)患者CD4+T、CD8+T细胞表达变化及其在腹主动脉血管钙化和不良心血管事件中的作用。方法选择30例血液透析方式和透析器均相同的MHD患者为试验组,以侧腹部X射线平片评价腹主动脉钙化情况,再分为钙化组(男8例,女7例)和非钙化组(男7例,女8例),选择同期健康体检者10例为对照组(男女各5例)。分别用流式细胞技术检测两组患者静脉血淋巴细胞亚群表达,免疫比浊法检测血超敏C反应蛋白(sCRP)。以t检验和卡方检验进行数据统计学分析。结果 MHD两组患者年龄、血压、血钙、血磷、血sCRP和血清甲状旁腺素(iPTH)以及CD3+T细胞和CD4+CD3+T细胞百分数差异均无统计学意义(P>0.05)。钙化组CD8+CD3+T细胞百分数为(23.54±4.62)%,较非钙化组的(39.22±7.21)%明显下降(P<0.01);钙化组CD4+/CD8+T细胞比值为(1.81±0.30),较非钙化组的(1.26±0.39)上升(P<0.01),其中CD4+/CD8+T细胞比值在1.5以上及1.5以下血管钙化发生率分别是73%和20%,差异有统计学意义(P<0.01),但CD4+/CD8+T细胞比值在1.5以上和以下其不良心血管事件发生率差异均无统计学意义(P>0.05)。结论 MHD患者存在CD4+/CD8+T细胞表达变化,CD4+/CD8+T细胞参与血管钙化的发生,但CD4+/CD8+T细胞在血管钙化及不良心血管事件发生中的作用需要进一步研究。     

Clonal analysis of CD4+/CD8+ T cells in a patient with aplastic anemia.

T cell clones were established from peripheral blood of a patient with severe aplastic anemia. 8 of 18 individual clonal T cell populations stably coexpressed CD4 and CD8 molecules, a phenotype characteristic for thymocytes and a minor subpopulation of circulating T lymphocytes. Analysis of T cell receptor genes revealed identical rearrangements of T cell receptor beta chain genes, suggesting clonality of these T cells. CD4+/CD8+ T cells clones were found to be efficiently cytotoxic towards autologous lymphoblasts. Autocytotoxicity could be blocked by a CD3 MAb, a MAb specific for monomorphic MHC class II determinants, and particularly, by an MHC-DP-specific MAb, suggesting specificity for autologous DP molecules. Perhaps more important, CD4+/CD8+ T cell clones inhibited differentiation of autologous progenitor enriched bone marrow cells in vitro by a direct cell-mediated mechanism. These data suggest that circulating cytotoxic CD4+/CD8+ T cell clones specific for autologous MHC-DP determinants may be involved in hematopoietic failure in some cases of aplastic anemia.     

Loss of HIV-1-specific CD8+ T cell proliferation after acute HIV-1 infection and restoration by vaccine-induced HIV-1-specific CD4+ T cells

Virus-specific CD8(+) T cells are associated with declining viremia in acute human immunodeficiency virus (HIV)1 infection, but do not correlate with control of viremia in chronic infection, suggesting a progressive functional defect not measured by interferon gamma assays presently used. Here, we demonstrate that HIV-1-specific CD8(+) T cells proliferate rapidly upon encounter with cognate antigen in acute infection, but lose this capacity with ongoing viral replication. This functional defect can be induced in vitro by depletion of CD4(+) T cells or addition of interleukin 2-neutralizing antibodies, and can be corrected in chronic infection in vitro by addition of autologous CD4(+) T cells isolated during acute infection and in vivo by vaccine-mediated induction of HIV-1-specific CD4(+) T helper cell responses. These data demonstrate a loss of HIV-1-specific CD8(+) T cell function that not only correlates with progressive infection, but also can be restored in chronic infection by augmentation of HIV-1-specific T helper cell function. This identification of a reversible defect in cell-mediated immunity in chronic HIV-1 infection has important implications for immunotherapeutic interventions.     

Complementary dendritic cell-activating function of CD8+ and CD4+ T cells: helper role of CD8+ T cells in the development of T helper type 1 responses

Dendritic cells (DCs) activated by CD40L-expressing CD4+ T cells act as mediators of "T helper (Th)" signals for CD8+ T lymphocytes, inducing their cytotoxic function and supporting their long-term activity. Here, we show that the optimal activation of DCs, their ability to produce high levels of bioactive interleukin (IL)-12p70 and to induce Th1-type CD4+ T cells, is supported by the complementary DC-activating signals from both CD4+ and CD8+ T cells. Cord blood- or peripheral blood-isolated naive CD8+ T cells do not express CD40L, but, in contrast to naive CD4+ T cells, they are efficient producers of IFN-gamma at the earliest stages of the interaction with DCs. Naive CD8+ T cells cooperate with CD40L-expressing naive CD4+ T cells in the induction of IL-12p70 in DCs, promoting the development of primary Th1-type CD4+ T cell responses. Moreover, the recognition of major histocompatibility complex class I-presented epitopes by antigen-specific CD8+ T cells results in the TNF-alpha- and IFN-gamma-dependent increase in the activation level of DCs and in the induction of type-1 polarized mature DCs capable of producing high levels of IL-12p70 upon a subsequent CD40 ligation. The ability of class I-restricted CD8+ T cells to coactivate and polarize DCs may support the induction of Th1-type responses against class I-presented epitopes of intracellular pathogens and contact allergens, and may have therapeutical implications in cancer and chronic infections.     

Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells

Active suppression of tumor-specific T lymphocytes can limit the efficacy of immune surveillance and immunotherapy. While tumor-recruited CD11b+ myeloid cells are known mediators of tumor-associated immune dysfunction, the true nature of these suppressive cells and the fine biochemical pathways governing their immunosuppressive activity remain elusive. Here we describe a population of circulating CD11b+IL-4 receptor alpha+ (CD11b+IL-4Ralpha+), inflammatory-type monocytes that is elicited by growing tumors and activated by IFN-gamma released from T lymphocytes. CD11b+IL-4Ralpha+ cells produced IL-13 and IFN-gamma and integrated the downstream signals of these cytokines to trigger the molecular pathways suppressing antigen-activated CD8+ T lymphocytes. Analogous immunosuppressive circuits were active in CD11b+ cells present within the tumor microenvironment. These suppressor cells challenge the current idea that tumor-conditioned immunosuppressive monocytes/macrophages are alternatively activated. Moreover, our data show how the inflammatory response elicited by tumors had detrimental effects on the adaptive immune system and suggest novel approaches for the treatment of tumor-induced immune dysfunctions.     

CD4+CD25+ T cells regulate virus-specific primary and memory CD8+ T cell responses

Naturally occurring CD4+CD25+ regulatory T cells appear important to prevent activation of autoreactive T cells. This article demonstrates that the magnitude of a CD8+ T cell-mediated immune response to an acute viral infection is also subject to control by CD4+CD25+ T regulatory cells (Treg). Accordingly, if natural Treg were depleted with specific anti-CD25 antibody before infection with HSV, the resultant CD8+ T cell response to the immunodominant peptide SSIEFARL was significantly enhanced. This was shown by several in vitro measures of CD8+ T cell reactivity and by assays that directly determine CD8+ T cell function, such as proliferation and cytotoxicity in vivo. The enhanced responsiveness in CD25-depleted animals was between three- and fourfold with the effect evident both in the acute and memory phases of the immune response. Surprisingly, HSV infection resulted in enhanced Treg function with such cells able to suppress CD8+ T cell responses to both viral and unrelated antigens. Our results are discussed both in term of how viral infection might temporarily diminish immunity to other infectious agents and their application to vaccines. Thus, controlling suppressor effects at the time of vaccination could result in more effective immunity.     

Antigen-dependent proliferation of CD4+ CD25+ regulatory T cells in vivo

The failure of CD25+ regulatory T cells (Tregs) to proliferate after T cell receptor (TCR) stimulation in vitro has lead to their classification as naturally anergic. Here we use Tregs expressing a transgenic TCR to show that despite anergy in vitro, Tregs proliferate in response to immunization in vivo. Tregs also proliferate and accumulate locally in response to transgenically expressed tissue antigen whereas their CD25- counterparts are depleted at such sites. Collectively, these data suggest that the anergic state that characterizes CD25+ Tregs in vitro may not accurately reflect their responsiveness in vivo. These observations support a model in which Treg population dynamics are shaped by the local antigenic environment.     

慢性乙肝患者外周血CD4+细胞HLA-DR、CD8+细胞CD28表达的检测及其意义

目的 检测乙肝患者外周血CD4^＋淋巴细胞表面HLA-DR和CD8^＋淋巴细胞表面CD28的表达情况,评价乙肝患者的细胞免疫状态。方法 荧光抗体CD4-PECY5、HLA-DR-FITC和CD8-PE、CD28-FITC标记淋巴细胞,流式细胞仪分别测定患者CD4^＋淋巴细胞表面HLA-DR和CD8^＋淋巴细胞表面CD28表达的百分率,并与HBV-DNA结果比较。结果 ①与正常对照组比较,乙肝患者组外周血CD4^＋淋巴细胞表面HLA-DR的表达显著升高（P〈0.001）,CD8^＋淋巴细胞表面CD28的表达显著降低（P〈0.01）;②乙肝患者DNA阳性（〉4000拷贝/ml）与阴性（〈4000拷贝/ml）组CD4^＋淋巴细胞表面HLA-DR和CD8^＋淋巴细胞表面CD28的表达比较均无显著性差异（P〉0.1）。结论 乙肝患者外周血CD4^＋细胞的免疫活化增强,CD8^＋细胞与抗原递呈细胞结合的作用减弱;淋巴细胞的活化情况与病毒复制情况及含量多少无关。