抗CD137单克隆抗体对哮喘患者外周血CD4^＋CD25^＋T淋巴细胞死亡方式的影响

目的探讨抗CD啪单克隆抗体对哮喘患者外周血CD4^＋CD25^＋T淋巴细胞的影响。方法采用密度梯度离心法及尼龙棉柱法分离16例健康志愿者（对照组）及12例哮喘患者（哮喘组）外周血T淋巴细胞,磁性细胞分离器（MACS）分离得到CD4^＋CD25^＋T淋巴细胞,分别利用电镜及流式细胞仪观察、检测抗CD137单克隆抗体干预72h的细胞自噬率、凋亡率、胀亡率及FOXp3的表达。结果抗CD137单克隆抗体干预后两组外周血CD4^＋CD25^＋T淋巴细胞自噬率及凋亡率均增加,但哮喘组均低于对照组。结论抗CD137单克隆抗体可促进CD4^＋CD25^＋T淋巴细胞凋亡和自噬。     

肺癌患者外周血CD8+CD28+、CD4+CD25+调节性T淋巴细胞水平变化及意义

目的观察肺癌患者外周血CD8+CD2+8、CD4+CD2+5调节性T淋巴细胞水平变化,并探讨其临床意义。方法采用流式细胞术检测86例肺癌(肺癌组)、65例肺良性肿瘤患者(良性组)及50例健康体检者(对照组)外周血中的CD8+CD2+8、CD4+CD2+5调节性T淋巴细胞。结果肺癌组术前CD8+CD2+8、CD4+CD2+5调节性T淋巴细胞分别为17.35%±3.03%、9.65%±2.01%,术后分别为12.97%±2.29%、12.23%±2.19%;良性组分别为12.22%±2.22%、14.91%±2.49%,对照组分别为11.81%±2.19%、15.22%±2.55%;肺癌组手术前后、肺癌组与良性组、对照组比较,P均<0.01;肺癌组术后与对照组比较,P均<0.05。外周血CD8+CD2+8、CD4+CD2+5调节性T淋巴细胞水平与肺癌TNM分期及淋巴结转移有关(P均<0.05),与病理类型无关(P>0.05)。结论肺癌患者外周血中CD8+CD2+8调节性T淋巴细胞水平降低、CD4+CD2+5调节性T淋巴细胞水平升高;外周血CD8+CD2+8、CD4+CD2+5调节性T淋巴细胞水平检测有助于肺癌的临床分期及疗效判定。     

非小细胞肺癌患者CD3+、CD4+、CD8+、CD44+表达的临床研究

目的研究非小细胞肺癌患者外周血淋巴细胞中CD3+、CD4+、CD8+、CD4+4的表达水平。方法取65例非小细胞肺癌患者及22例健康正常人外周静脉血,应用流式细胞仪检验非小细胞肺癌患者(实验组)与健康人外周血淋巴细胞中(对照组)CD3+、CD4+、CD8+、CD4+4的表达水平。结果实验组与对照组CD3+、CD3+CD4+、CD3+CD8+、CD4+4在淋巴细胞中的比例存在显著性差异(P<0.05),其中,实验组占总淋巴细胞的比例分别为48.07±10.33%、30.93±6.68%、17.13±3.37%、55.45±4.35%;对照组CD3+、CD3+CD4+、CD3+CD8+、CD4+4占总淋巴细胞的比例分别为58.83±10.88%、34.89±6.45%、23.91±4.42%、62.85±7.56%;但鳞癌与腺癌组CD4+4的表达无显著性差异(P>0.05),其中,鳞癌组CD4+4所占比例为61.32±8.06%,腺癌组为64.43±6.76%。结论非小细胞肺癌患者外周血T细胞亚群及CD4+4的表达水平较正常组均低,其表达水平与组织类型无关。     

小剂量氨茶碱对支气管哮喘患者外周血CD4+CD25+调节性T细胞凋亡的影响

目的 观察小剂量氨茶碱对分离培养的健康人和支气管哮喘(简称哮喘)患者外周血CD4+CD25+调节性T细胞(T regulatory cells,Treg)凋亡的影响.方法 经密度梯度离心法、尼龙棉柱法、磁珠分离法分离出健康人和哮喘患者外周血CD4+CD25+Treg,分小剂量氨茶碱(1.13 mg/L)、及空白组培养72 h后,用流式细胞仪检测凋亡率变化.结果 ①健康人外周血CD4+CD25+Treg的纯度为77.4%～92.3%,哮喘患者CD4+CD25+Treg的纯度为75.2%～93.8%.②CD4+CD25+Treg占外周血CD4+T细胞的比例在健康组为4.12%～7.98%,在哮喘组为4.51%～8.68%.两者差异无统计学意义.③小剂量氨茶碱均可以诱导健康组及哮喘组外周血CD4+CD25+Treg凋亡率增加(P<0.05).结论 小剂量氨茶碱(1.13 mg/L)可能通过促进CD4+CD25+Treg凋亡来发挥免疫调节作用.     

FOXP3在系统性红斑狼疮患者CD4+CD25+调节性T细胞中的表达

目的 研究FOXP3在系统性红斑狼疮(SLE)患者的CD4+CD25调节性T细胞中的表达.方法 流式细胞仪检测CD4+CD25+,CD4+CD25bright T细胞中FOXP3蛋白的表达.利用免疫磁珠细胞分选法(MACS)分选出CD4+CD25+调节性T细胞,实时定量聚合酶链反应(real-time PCR)检测FOXP3基因的表达.结果 以FOXP3作为CD4+CD25+调节性T细胞的主要识别标志,初发、活动性SLE患者的CD4+CD25+ FOXP3+调节性T细胞在CD4+T细胞中的比例高于健康对照组[(9.1±0.7)%VS(4.8±0.4)%,P＜0.01],与SLEDAI评分呈明显正相关.CD4+CD25+调节性T细胞中FOXP3在蛋白和mRNA水平上的表达,SLE患者与健康对照组相比无明显差异.结论 SLE患者CD4+CD25+调节性T细胞中FOXP3的表达无明显异常,其CD4+T细胞中调节性T细胞的比例高于健康对照组.提示研究这类细胞在SLE发病中的作用应更关注其功能.     

肺结核患者外周血CD4+T细胞穿孔素表达下降

目的研究活动性肺结核患者外周血单个核细胞(PBMCs)穿孔素(Perforin)的表达及临床意义。方法采集19例活动期肺结核患者和23位健康对照外周血,纯化PBMCs,用结核分枝杆菌ESAT-6和CFP-10混合性抗原肽库刺激,通过细胞表面标记和细胞内细胞因子染色技术,采用流式技术检测CD4+、CD8+T细胞穿孔素的表达。结果与对照组比较,肺结核患者PBMCs中的CD4+T、CD8+T细胞亚群分布无显著性差异,但肺结核患者PBMCs及CD4+T细胞中穿孔素的表达比例(百分率)下降(PBMCs:肺结核组19.55%±2.53%,对照组27.53%±1.90%,P<0.05;CD4+T细胞:肺结核组4.42%±0.84%,对照组8.15%±1.29%,P<0.05),而PBMCs及CD4+、CD8+T细胞中穿孔素的表达量无显著性差异。结论活动期初治肺结核CD4+T细胞群内Perforin的表达比例下降可能会使CD4+T细胞对靶细胞的细胞毒性作用(CTL)下降。     

氨茶碱对健康人外周血T细胞及CD4~+亚群凋亡的影响

目的观察氨茶碱对分离培养的健康人外周血T细胞及CD4+亚群凋亡的影响。方法密度梯度离心法及尼龙棉柱法分离健康成年人外周血T细胞,及CD8阴性选择磁性分离健康成年人外周血CD4+T细胞。分两部分进行:①外周血T细胞分对照组、氨茶碱组(0.14～18μg/ml),培养72h后用流式细胞术检测凋亡率变化;②CD4+T细胞分对照组、小剂量氨茶碱组(1.13μg/ml)培养48～72h后用流式细胞术检测凋亡率变化。结果①T细胞的分离纯度为81.3%～94.5%,CD4+T细胞的分离纯度为84%～90%;②(0.14～18μg/ml)氨茶碱共培养人外周血T细胞72h后,其中氨茶碱1.13～18μg/ml组凋亡率差异有统计学意义(P值均<0.05);氨茶碱0.14～0.56μg/ml组凋亡率与阴性对照组比较差异无统计学意义;③氨茶碱1.13μg/ml组干预后,培养CD4+T细胞48～72h,经流式细胞术检测凋亡率,与阴性对照组相比,P值均<0.05,48h的凋亡率与72h者相比,差异有统计学意义(P<0.05)。结论小剂量氨茶碱(1.13μg/ml)可诱导人外周血T细胞和CD4+T细胞凋亡率增加。     

糖皮质激素对ITP患者外周血CD4+CD25+FOXP3+Treg细胞含量及GR受体表达的影响

目的观察糖皮质激素(GC)对特发性血小板减少性紫癜(ITP)患者CD4+CD2+5FOXP3+Treg细胞含量及GR受体表达的影响。方法将ITP患者按GC治疗反应情况分为激素敏感型19例(敏感组)和激素抵抗型11例(抵抗组),另选15例健康体检者作为对照组,采用流式细胞仪检测各组外周血CD4+CD2+5FOXP3+Treg细胞含量,RT-PCR法检测GC受体亚型GRα和GRβ表达水平。结果敏感组治疗前后CD4+CD2+5FOXP3+Treg细胞含量均明显低于对照组,GRβmRNA表达水平明显高于对照组,P均<0.05;抵抗组治疗前CD4+CD2+5FOXP3+Treg细胞含量明显低于对照组,GRβmRNA表达水平明显高于对照组,P均<0.05。结论 GC治疗ITP的重要机理在于可提高患者体内CD4+CD2+5FOXP3+Treg细胞含量,ITP患者对GC治疗的敏感性与GRβ表达水平有关。     

CD14+CD16+单核细胞在妊娠糖尿病患者外周血中的表达及意义

目的探讨CD+14CD+16单核细胞在妊娠糖尿病中的表达情况及其相关意义。方法 30例妊娠糖尿病患者(GDM)及30例健康对照为研究对象,血样均取自于研究对象清晨空腹的外周血,以肝素钠抗凝。采用流式细胞术(FCM)测定外周血中CD+14CD+16的表达水平。结果 GDM患者外周血中CD+14CD+16的表达水平显著高于对照组(P<0.05)。结论 CD1+4CD1+6单核细胞增加是GDM发生机制中的重要环节。     

肺结核患者外周血CD4~+T淋巴细胞凋亡与细胞因子相关性及临床意义研究

目的探讨肺结核患者外周血CD4+T淋巴细胞凋亡与血清IL-4、IL-6、TGF-β的相关性及其临床意义。方法分离结核病患者和健康者外周血单个核细胞,标记后用流式细胞仪测定CD4+T淋巴细胞凋亡率;采用酶联免疫吸附试验测定42例肺结核患者及28例健康人群血清IL-4、IL-6、TGF-β水平。结果结核病患者外周血CD4+T淋巴细胞凋亡率(15.8820±4.6500)显著高于对照组(7.9389±4.16564)(t=7.491,P<0.01),血清IL-4水平(0.6219±0.78565)较对照组(0.2120±0.07367)显著升高(t=2.732,P<0.05)。肺结核患者中涂阳组IL-6水平(0.0602±0.01833)较涂阴组(0.0905±0.05023)显著升高(t=-2.296,P<0.05),TGF-β水平复治组(0.1128±0.04704)较初治组(0.0748±0.01659)显著升高(t=-3.019,P<0.05)。外周血CD4+T淋巴细胞凋亡率与IL-4水平呈正相关(r=0.366,P<0.05);血清IL-6的含量与TGF-β的含量呈正相关(r=0.829,P<0.01)。结论结核病患者CD4+T淋巴细胞凋亡率显著增加,可能与肺结核感染免疫有关;IL-4、IL-6和TGF-β是抗结核感染免疫的重要调节因子,其血清水平可作为监测病情活动和转归的重要指标。     

Human CD8+CD25+ thymocytes share phenotypic and functional features with CD4+CD25+ regulatory thymocytes

CD8+CD25+ cells, which expressed high levels of Foxp3, glucocorticoid-induced tumor necrosis factor receptor (GITR), CCR8, tumor necrosis factor receptor 2 (TNFR2), and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) mRNAs, were identified in the fibrous septa and medullary areas of human thymus. Activated CD8+CD25+ thymocytes did not produce cytokines, but most of them expressed surface CTLA-4 and transforming growth factor beta1 (TGF-beta1). Like CD4+CD25+, CD8+CD25+ thymocytes suppressed the proliferation of autologous CD25-T cells via a contact-dependent mechanism. The suppressive activity of CD8+CD25+ thymocytes was abrogated by a mixture of anti-CTLA-4 and anti-TGF-beta1 antibodies and it was mediated by their ability to inhibit the expression of the interleukin 2 receptor alpha chain on target T cells. These results demonstrate the existence of a subset of human CD8+CD25+ thymocytes sharing phenotype, functional features, and mechanism of action with CD4+CD25+ T regulatory cells.     

CD19+CD5+ cells as indicators of preeclampsia

Preeclampsia is a devastating pregnancy-associated disorder affecting 5% to 8% of pregnant women worldwide. It emerges as an autoimmune-driven disease, and, among others, the autoantibodies against angiotensin type 1 receptor II have been proposed to account for preeclampsia symptoms. Despite much attention focused on describing autoantibodies associated with preeclampsia, there is no clue concerning the cell population producing them. CD19(+)CD5(+) B-1a B cells constitute the main source of natural and polyreactive antibodies, which can be directed against own structures. Here, we aimed to identify the B-cell subpopulation responsible for autoantibody production during preeclampsia and to study their regulation, as well as their possible use as markers for the disease. The frequency of CD19(+)CD5(+) cells in peripheral blood of preeclamptic patients is dramatically increased compared with normal pregnant women as analyzed by flow cytometry. This seems to be driven by the high human chorionic gonadotropin levels present in the serum and placenta supernatant of preeclamptic patients versus normal pregnant women. Not only ≈95% of CD19(+)CD5(+) cells express the human chorionic gonadotropin receptor, but these cells also expand on human chorionic gonadotropin stimulation in a lymphocyte culture. Most importantly, isolated CD19(+)CD5(+) cells produce autoantibodies against angiotensin type 1 receptor II, and CD19(+)CD5(+) cells were further detected in the placenta of preeclamptic but not of normal pregnancies where barely B cells are present. Our results identify a B-cell population able to produce pregnancy-pathological autoantibodies as possible markers for preeclampsia, which opens vast diagnostic and therapeutic applications.     

Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28⁻ suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28⁻ T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28⁻ T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

Abstract

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28^? suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28^? T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28^? T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

CD11c+CD88+CD317+ myeloid cells are critical mediators of persistent CNS autoimmunity

Natalizumab, a humanized monoclonal antibody (mAb) against α4-integrin, reduces the number of dendritic cells (DC) in cerebral perivascular spaces in multiple sclerosis (MS). Selective deletion of α4-integrin in CD11c⁺ cells should curtail their migration to the central nervous system (CNS) and ameliorate experimental autoimmune encephalomyelitis (EAE). We generated CD11c.Cre^+/−ITGA4^fl/fl C57BL/6 mice to selectively delete α4-integrin in CD11c⁺ cells. Active immunization and adoptive transfer EAE models were employed and compared with WT controls. Multiparameter flow cytometry was utilized to immunophenotype leukocyte subsets. Single-cell RNA sequencing was used to profile individual cells. α4-Integrin expression by CD11c⁺ cells was significantly reduced in primary and secondary lymphoid organs in CD11c.Cre^+/−ITGA4^fl/fl mice. In active EAE, a delayed disease onset was observed in CD11c.Cre^+/−ITGA4^fl/fl mice, during which CD11c⁺CD88⁺ cells were sequestered in the blood. Upon clinical EAE onset, CD11c⁺CD88⁺ cells appeared in the CNS and expressed CD317⁺. In adoptive transfer experiments, CD11c.Cre^+/−ITGA4^fl/fl mice had ameliorated clinical disease phenotype associated with significantly diminished numbers of CNS CD11c⁺CD88⁺CD317⁺ cells. In human cerebrospinal fluid from subjects with neuroinflammation, microglia-like cells display coincident expression of ITGAX (CD11c), C5AR1 (CD88), and BST2 (CD317). In mice, we show that only activated, but not naïve microglia expressed CD11c, CD88, and CD317. Finally, anti-CD317 treatment prior to clinical EAE substantially enhanced recovery in mice.

Autoimmune disorders of the central nervous system (CNS), including multiple sclerosis (MS), are thought to be mediated by aberrant adaptive immune responses against self-antigens. In experimental autoimmune encephalomyelitis (EAE), a model of MS, activated myelin-reactive CD4⁺ T helper cells are the main drivers of disease activity (1). In active EAE, dendritic cells (DC) are professional antigen-presenting cells (APC) at the inoculation site, namely in draining lymph nodes and other secondary lymphoid organs, where they present myelin autoantigen to naïve CD4⁺ T lymphocytes (2). Myeloid APC within the CNS are essential for the reactivation and retention of these autoreactive CD4⁺ T cells, and for perpetuation of disease activity. Specifically, myeloid cells within cerebral perivascular spaces previously considered DC based solely on their expression of CD11c are sufficient to permit EAE (3). Myeloid APC, including DC, use α4-integrin to gain access to sites of ongoing inflammation. We previously showed that the number of DC was significantly reduced in cerebral perivascular spaces in autopsy material of an MS patient treated with natalizumab, a humanized monoclonal antibody (mAb) against α4-integrin (4). Conceivably, antagonizing or diminishing the function of α4-integrin selectively in myeloid cells should ameliorate EAE through an impaired reactivation of CNS-specific CD4⁺ T cells and a reduction of direct inflammatory effects exerted by these cells.To further understand the role of CD11c⁺ cells within the CNS during CD4⁺ T cell-mediated CNS autoimmunity, we generated and characterized CD11c.Cre^+/−ITGA4^fl/fl mice, which lack α4-integrin expression in CD11c⁺ cells. We identified CD11c⁺CD88⁺CD317⁺ as mediators of persistent clinical EAE, and propagators of inflammation within the CNS.     

Precommitment of CD4+CD8+ thymocytes to either CD4 or CD8 lineages.

CD4+ and CD8+ mature T cells arise from CD4+CD8+ precursors in the thymus. During this process, cells expressing T-cell receptors (TCRs) reactive with self major histocompatibility complex (MHC) class I or II molecules are positively selected to the CD8 or CD4 lineage, respectively. It is controversial whether lineage commitment of CD4+CD8+ thymocytes is controlled directly by TCR specificity for MHC (instructional model) or, alternatively, by processes that operate independently of TCR specificity (stochastic model). We show here that CD4+CD8+ thymocytes bearing a MHC class I-restricted transgenic TCR can be subject to two alternative developmental fates. One population of CD4+CD8+ cells is positively selected by MHC class I molecules to the CD8 lineage as expected, whereas the other CD4+CD8+ population rearranges endogenous TCR genes and is positively selected by MHC class II molecules to the CD4 lineage. Blocking TCR-MHC class II interactions in vivo does not interfere with the generation of CD4+CD8+ cells expressing endogenous TCRs but does prevent their subsequent maturation to CD4+ cells. These data support a version of the stochastic model in which CD4+CD8+ thymocytes are precommitted to the CD4 or CD8 lineage independently of TCR specificity for MHC and prior to positive selection.     

CD25+CD4+ T cells contribute to the control of memory CD8+ T cells

Previously we demonstrated that IL-15 and IL-2 control the number of memory CD8+ T cells in mice. IL-15 induces, and IL-2 suppresses the division of these cells. Here we show that CD25+CD4+ regulatory T cells play an important role in the IL-2-mediated control of memory phenotype CD8+ T cell number. In animals, the numbers of CD25+CD4+ T cells were inversely correlated with the numbers of memory phenotype CD8+ T cells with age. Treatment with anti-IL-2 caused CD25+CD4+ T cells to disappear and, concurrently, increased the numbers of memory phenotype CD8+ T cells. This increase in the numbers of CD8+ memory phenotype T cells was not manifest in animals lacking CD4+ cells. Importantly, adoptive transfer of CD25+CD4+ T cells significantly reduced division of memory phenotype CD8+ T cells. Thus, we conclude that CD25+CD4+ T cells are involved in the IL-2-mediated inhibition of memory CD8+ T cell division and that IL-2 controls memory phenotype CD8+ T cell numbers at least in part through maintenance of the CD25+CD4+ T cell population.