原发性肝癌患者外周效应功能T细胞的保护意义北大核心CSCD

目的:探讨原发性肝癌(HCC)患者外周循环中T细胞的表型特点及意义。方法:收集并分离北京佑安医院肝病介入中心收治的39例HCC患者的外周血单个核细胞。合成5种肿瘤相关抗原(TAA)多肽,采用酶联免疫斑点测定法(ELSPOT)及流式细胞术分析T细胞反应及表型。结果:TAA特异性T细胞免疫反应阳性率为56.41%;阳性患者消融治疗后无复发生存期显著优于阴性患者(P<0.01),其CD4+和CD8+T细胞中TTE的百分比显著高于阴性患者[CD4:0.83%(0.34%,1.33%)vs 0.36%(0.24%,0.58%),P=0.02;CD8:7.57%(3.46%,11.29%)vs 4.76%(2.42%,6.77%),P=0.03];在阳性患者中,进展期患者CD8+PD1+和CD8+CD152+T细胞百分比显著高于早期患者[(40.65±4.39)%vs(27.66±2.64)%;(17.96±1.72)%vs(11.84±0.89%)],差异有统计学意义(P=0.01;P<0.01)。结论:HCC患者外周血中存在的TAA特异性T细胞免疫反应具有保护作用,诱导特异性效应T细胞免疫反应并阻断T细胞耗竭是HCC免疫治疗的重要方向。     

肿瘤引流淋巴结中免疫活性细胞分布的原位分析

目的:观察人乳腺癌和胃癌局部引流淋巴结(LDLN)从无转移、微转移到晚期转移过程中,免疫组织学变化及免疫活性细胞(ICC)的分布特征。方法:采用传统的病理学方法,对22例乳腺癌LDLN(71个)和7例进展期胃癌LDLN(28个)进行组织形态学分类,并以抗穿孔素、抗颗粒酶B、抗CD8、抗CD56、抗CD68、抗S-100、抗CD134及抗CD25单克隆抗体(mAb)进行催化信号放大(Catalyzedsignalamplification,CSA)免疫组化染色,检测肿瘤LNDN中ICC的分布。结果:肿瘤LDLN中以副皮质区增生和窦组织细胞增生为主,细胞毒性T淋巴细胞(CTL)及树突状细胞(DC)的数量,从无转移、微转移到晚期转移过程中有逐渐减少的趋势。在无和微转移的淋巴结内,穿孔素 、颗粒酶B 及S100 DC的数量高于晚期转移淋巴结(P<0.05);而S100 DC不仅数量减少,而且其形态也有变化,呈多角形、星形,并有胞质突起,与周围淋巴细胞接触呈活化状态的DC变为椭圆形,少有胞质突起或呈短突起的静止状态的DC。CD134 细胞及CD25 细胞的数量在晚期转移淋巴结中明显高于无和微转移淋巴结(P<0.01)。ICC在无和微转移的前哨和非前哨淋巴结中的分布无统计学意义(P>0.05)。结论:肿瘤LDLN中ICC分布的变化,提示随着肿瘤的进展,其免疫微环境向抑制机体抗肿瘤免疫的方向偏移。     

肝细胞性肝癌肿瘤浸润调节性T细胞的表型分析和功能探讨

目的通过健康志愿者和肝细胞性肝癌(hepatocellular carcinoma,HCC)患者中调节性T细胞(Tregs)的频率和表型分析,探讨肿瘤局部浸润Treg细胞的功能和局部聚集可能的机制。方法分离健康人和HCC患者外周血单个核细胞(PBMCs)及HCC患者肝组织浸润的单个核细胞,流式检测Treg细胞频率、表型及功能特征,免疫组化检测Foxp3在肝组织内的表达。结果 CD4+Foxp3+Treg细胞在HCC患者体内明显增加且主要聚集在肿瘤局部,此外,与HCC患者自身PBMCs相比,TILs中的Treg细胞表达更高频率的功能相关分子(CTLA-4、GITR等)以及趋化因子受体CCR6和CXCR3,虽然我们发现TILs与PBMCs中Treg细胞上Ki67的表达没有统计学差异但是两者均有大约15%左右的Treg细胞为Ki67阳性。结论 HCC患者体内Treg细胞显著高于健康者及血管瘤病人;HCC患者肿瘤局部的Treg细胞比外周具有更加显著的抑制功能且局部增加的Treg细胞可能是由于局部增殖和外周趋化到局部。因此,Treg细胞的增加可能与HCC的发生及发展有密切关系。     

调节性T细胞与肝细胞肝癌免疫的研究进展

调节性T细胞（Treg）是一类具有独特免疫调节功能的T细胞亚群,可通过多种方式抑制肿瘤免疫,在肿瘤的发生、发展过程中发挥着极为重要的作用。随着对Treg研究的不断深入,越来越多的证据表明Treg通过多种机制参与了肝癌特别是肝细胞肝癌的形成和发展,减少Treg数量能够增强患者的抗肿瘤免疫功能,是肝细胞肝癌预后的独立危险因子;反之,肝癌局部微环境的一些细胞因子能够影响Treg的表型,增强其抑制功能。     

树突状细胞引流淋巴结归巢的研究进展

树突状细胞(dendritic cells,DCs)是目前已知的体内功能最强的专职性抗原提呈细胞(professional antigen-present-ing cells,pAPC),在引发和调节机体的免疫反应中起着重要作用。树突状细胞最重要的功能是摄取、加工处理、提呈抗原,并刺激初始T细胞(naive T cells)活化、增殖,从而激发机体的免疫应答。DCs这一功能是在体内迁移过程中发挥的,其中DCs归巢至引流淋巴结被认为是激发免疫应答的关键步骤之一。本文将就近年来DCs归巢至引流淋巴结的研究进展作一综述。     

小鼠肝移植模型中调节性T细胞的数量和表型改变

目的探究小鼠肝移植模型中移植肝内免疫排斥反应程度和调节性T细胞的数量及功能随时间变化的规律,为揭示机体免疫耐受的建立机制提供理论依据。方法同基因或异基因肝移植术后3、7、14和28 d留取移植肝标本(每组每个时间点n=4),HE染色进行排斥活性指数评定(Banff评分标准),抗CD3、B220和Foxp3抗体免疫组化染色鉴定移植物内T、B淋巴细胞及调节性T细胞的浸润情况,实时荧光定量PCR检测Foxp3 mRNA表达,流式细胞术检测Treg细胞表面CLAT-4的表达。结果异基因肝移植术后2周内发生免疫排斥反应,随后逐渐好转;移植物内浸润的炎性细胞主要为T淋巴细胞,其中Foxp3 mRNA及Foxp3阳性细胞数术后异基因组(15.0±1.3)显著高于同基因组(2.7±1.0)。异基因移植肝内浸润的调节性T细胞表面CLAT-4表达(53%±3%)也显著高于同基因组(13%±2%)(P0.05)。结论异基因肝移植术后免疫排斥反应由调节性T细胞负向调节并达到免疫耐受。     

鼠巨细胞病毒对小鼠调节性T细胞分化和效应性T细胞活化的影响

目的 在整体水平探讨小鼠巨细胞病毒(MCMV)感染对小鼠调节性T细胞(Treg)亚群CD4+CD25+Foxp3+T细胞分化和CD4+CD25+Foxpp3-效应性T细胞(TE)活化的影响.方法 建立全身播散型MCMV感染小鼠模型,依据主要脏器内病毒滴度,确定感染后第28天为本模型急、慢性期界定点.42只小鼠分别于感染MCMV后第1、3、7、14、28、45和60天处死6只;另设42只小鼠作为对照.流式细胞术检测CD4+CD25+F0xp34+Treg和活化的CD4+CD25+Foxp3-TE在脾单个核细胞中所占比率变化.结果 MCMV感染急性期CD4+CD25+Foxp3+Treg比率持续降低,第28天左右降至最低值,与对照组比较差异有统计学意义(P<0.01);随后直线上升,第45天高于基线,第60天升至峰值,与对照组比较差异有统计学意义(P<0.05);CD4+CD25+Foxp3-TE在感染后第3～7天高于对照组(P<0.05),其后持续下降,在感染后第45天和第60天显著低于对照组水平(P<0.05).Treg/CD4+CD25+TE比率在感染后第3～14天显著降低(P<0.05);随后逐渐上升,在感染后第45天和第60天均显著高于对照组(P<0.05).结论 MCMV可在慢性感染期诱导CD4+CD25+Foxp3+Treg种群扩增,抑制CD4+CD25+Foxp3-Treg活化增殖,这可能是MCMV逃避特异性免疫而实现长期潜伏的机制之一.     

CD4+CD25+T细胞的扩增方法与临床应用

CD4+CD25+T细胞是最重要的一类调节性T细胞(Tr).体内固有CD4+CD25+T细胞的自然扩增率极低,不能满足临床治疗的需要.通过采用FoxP3基因转染技术、阻断细胞活化信号、DC诱导、加入细胞因子等方法,对CD4+CD25+T细胞的数量和功能进行扩增,使其在器官移植、自身免疫性疾病和肿瘤免疫等领域具有广泛的临床应用前景.     

浆细胞样树突状细胞与调节性T细胞在移植免疫研究中的新进展

浆细胞样树突状细胞(pDC)是一类在病毒入侵时分泌Ⅰ型IFN并介导细胞免疫应答的抗原提呈细胞,除了启动获得性免疫应答,它们在扩增调节性T细胞及介导免疫耐受方面也发挥着重要的作用。结合最新研究,此文着重介绍pDC诱导、扩增调节性T细胞及其介导免疫耐受的分子机制,并对pDC来源在介导耐受中的作用进行了介绍,其对于免疫耐受的影响与受者所处的免疫环境、免疫效应发生部位密切有关。深入研究pDC与移植免疫耐受间的密切联系将有助于探讨临床细胞免疫治疗的更多可能性。     

不同刺激诱导肺癌患者引流淋巴结细胞抗自体肿瘤的体外研究

目的：探索适合于临床大规模应用的有效刺激、活化肿瘤引流淋巴结（Tumor-draining lymph node，TDLN）细胞的方法。方法：对3种刺激剂（IL-2、IL-2＋自身肺癌细胞抗原和IL-2＋GM-CSF＋IL-4＋自身肺癌细胞抗原）诱导TDLN细胞体外抗自体肿瘤作用，通过乳酸脱氢酶法测定了CTL杀伤活性，观察了细胞形态学变化，通过流式细胞技术测定了细胞CD83的阳性表达率。结果：经MTT法检测，IL-2＋GM-CSF＋IL-4＋自身肺癌细胞抗原诱导的TDLN细胞的增殖程度明显高于IL-2＋自身肺癌细胞抗原刺激组和IL-2刺激组（P〈0．01）。IL-2＋GM-CSF＋IL-4＋自身肺癌细胞抗原诱导的肿瘤特异性CTL活性明显高于IL-2＋自身肺癌细胞抗原刺激组和IL-2刺激组，CD83阳性表达细胞率也明显高于IL-2＋自身肺癌细胞抗原刺激组和IL-2刺激组。结论：用IL-2＋GM-CSF＋IL-4＋自身肺癌细胞抗原刺激和活化TDLN，优于单独用IL-2刺激或IL-2＋自身肺癌细胞抗原刺激，该作用可能与其诱导TDLN细胞产生DC数量的增加有关。     

Induction of antigen-specific regulatory T cells in the liver-draining celiac lymph node following oral antigen administration

Regulatory T cells are induced by oral administration of an antigen, but the physiological requirements and localization of the inductive sites are largely unknown. Using an adoptive transfer system of cells transgenic for ovalbumin T-cell receptor (OVA TCR tg), we found that antigen-specific CD4+ T cells were activated in the liver-draining celiac lymph node (CLN) shortly after ovalbumin feeding, and that a significantly higher proportion of the T cells in the CLN developed into the putative regulatory phenotype [co-expressing CD25 with the glucocortico-induced tumour necrosis factor (TNF) receptor family related gene (GITR), cytotoxic T-lymphocyte antigen (CTLA)-4 and CD103] than in Peyer's patches, the mesenteric and peripheral lymph nodes and the spleen. In addition, a particularly high level of expression of CD103 on the OVA-specific T cells in the CLN may favour homing to the epithelium of the intestine. While equally suppressive, OVA tg T cells isolated from the CLN of OVA-fed DO11.10 mice were less dependent on transforming growth factor (TGF)-beta for suppression than cells isolated from the peripheral and mesenteric lymph nodes, which indicates the involvement of an additional suppressive mechanism. The expression of FoxP3 was not up-regulated in any of the lymph node compartments studied. Our phenotypic and functional findings suggest that the induction of regulatory T cells in the CLN may be relevant in the control of the immune response to dietary antigens.     

CD28 signals the differential control of regulatory T cells and effector T cells

One possible means of driving antigen‐specific immune suppression is to expand or induce antigen‐specific FoxP3‐expressing Treg cells. One way of activating and expanding these specialized cells, both in vitro and in vivo, is by strong costimulation via CD28 with an agonistic anti‐CD28 monoclonal antibody, called anti‐CD28 superagonist (CD28SA). However, CD28SA also strongly activates conventional T (Tconv) cells to secrete proinflammatory cytokines and, under certain conditions, causes serious cytokine release syndrome. In this issue of European Journal of Immunology, Tabares et al. [Eur. J. Immunol. 2014. 44: 1225–1236] address how CD28SA can be used for the differential control of human Treg and Tconv cells to suppress immune responses without serious adverse effects. They show that, depending on the dose of the antibody or by comedication of cortico‐steroid, the selective expansion of Treg cells can be achieved without significantly activating Tconv cells to produce inflammatory cytokines. This difference in CD28 signal sensitivity between the two populations can be exploited for better control of immune responses.     

Regulatory CD4+CD25+ T cells and macrophages: communication between two regulators of effector T cells

Regulatory T cells (Tregs) play an essential role in the induction and maintenance of peripheral tolerance as well as prevention of autoimmunity by limiting the strength of the immune response of effector T cells. Macrophages, a heterogeneous population of phagocytes and professional antigen presenting cells (APCs), can also exert suppressive effects on effector T cells to keep the peripheral balance of immunity. The bi-directional interactions of dendritic cells (DCs) and Tregs have been cell studied. However, much less is known about the reciprocal interaction between macrophages and Tregs. In this review, we will discuss recent observations regarding the interplay of these two regulators of immunity. Received 8 December 2006; returned for revision 17 January 2008; received from final revision 9 June 2008; accepted by G. Wallace 10 July 2008     

Imbalanced expression of functional surface molecules in regulatory and effector T cells in systemic lupus erythematosus

Regulatory T (TREG) cells play an important role in maintaining immune tolerance and avoiding autoimmunity. We analyzed the expression of membrane molecules in TREG and effector T cells in systemic lupus erythematosus (SLE). TREG and effector T cells were analyzed for the expression of CTLA-4, PD1, CD28, CD95, GITR, HLA-DR, OX40, CD40L, and CD45RO in 26 patients with active disease, 31 with inactive disease, and 26 healthy controls. TREG cells were defined as CD25^+/highCD127^Ø/lowFoxP3⁺, and effector T cells were defined as CD25⁺CD127⁺FoxP3^Ø. The ratio of TREG to effector T cells expressing GITR, PD1, HLA-DR, OX40, CD40L, and CD45RO was determined in the three groups. The frequency of TREG cells was similar in patients with SLE and controls. However, SLE patients had a decreased frequency of CTLA-4⁺TREG and CD28⁺TREG cells and an increased frequency of CD40L⁺TREG cells. There was a decrease in the TREG/effector-T ratio for GITR⁺, HLA-DR⁺, OX40⁺, and CD45RO⁺ cells, and an increased ratio of TREG/effector-T CD40L⁺ cells in patients with SLE. In addition, CD40L⁺TREG cell frequency correlated with the SLE disease activity index (P=0.0163). In conclusion, our findings showed several abnormalities in the expression of functionally critical surface molecules in TREG and effector T cells in SLE that may be relevant to the pathogenesis of this disease.     

Regulatory T cells in thymic epithelium-induced tolerance. I. Suppression of mature peripheral non-tolerant T cells

Athymic mice grafted at birth with allogeneic thymic epithelium (TE) display life-long tolerance to tissue grafts of the TE donor strain, in spite of harboring peripheral T cells capable of rejecting those grafts. Tolerance is maintained in these chimeras by TE-specific regulatory CD4 T cells. We presently address the quantification and the mechanisms of this dominant tolerance process. C57BL/6 mice containing variable but defined numbers of peripheral, resident T cells received cell transfers of graded numbers of peripheral T cells from B6(BALB E10) chimeras (C57BL/6 nude mice grafted with TE from 10-day-old BALB/c embryos), resulting in a series of animals containing a wide range of donor (tolerant) versus host (non-tolerant) T cell chimerism. Increasing the relative representation of donor T cells results in a progressive delay in the rejection of BALB/c skin grafts, life-long tolerance being achieved at a ratio of tolerant and non-tolerant T cell populations of 1. In recipients displaying full tolerance, graftreactive non-tolerant T cells were not deleted, anergized or committed to noninflammatory functions. Thus, sorted host T cells from tolerant recipients readily rejected BALB/c skin grafts upon transfer to immunodeficient animals. Finally, measurements of “helper” and inflammatory activities, as well as interleukin-4 and interferon-γ production, failed to discriminate between T cell populations from tolerant and non-tolerant animals after specific in vitro stimulation. We conclude that: (a) TE-selected regulatory T cells can suppress, in a quantitative manner, in vivo T cell responses against major and minor histocompatibility antigens expressed by the TE and, (b) this suppressive activity neither inactivates mature non-tolerant T cells, nor does it seem to drive their differentiation along noninflammatory pathways.     

吡格列酮对尿毒症ApoE~(-/-)小鼠调节性和效应T细胞平衡的影响

目的:观察吡格列酮对体外培养的有或无斑块特异性抗原氧化低密度脂蛋白(ox LDL)刺激的尿毒症Apo E-/-小鼠调节性和效应T细胞(Treg/Teff)水平和功能相关因子的影响及可能机制。方法:通过两步外科手术法建立尿毒症Apo E-/-小鼠的动物模型,以不同浓度吡格列酮(2μmol/L和20μmol/L)及PPARγ拮抗剂GW9662(5μmol/L)对有或无ox LDL(2 mg/L)刺激的尿毒症模型鼠脾细胞作用12 h后,流式细胞术检测CD4+CD25+Foxp3+Treg及IFN-γ+CD4+Teff细胞水平,实时荧光定量PCR检测Foxp3及IFNγ的mRNA表达。结果:ox LDL体外诱导尿毒症Apo E-/-小鼠脾细胞Treg/Teff失衡。吡格列酮上调ox LDL抑制下的Treg及Foxp3的表达,此作用不能被GW9662拮抗;下调有或无ox LDL刺激下Teff及IFNγ的表达,此作用可被GW9662拮抗。结论:oxLDL体外诱导尿毒症模型鼠Treg/Teff失衡,吡格列酮通过PPARγ非依赖/依赖机制调整Treg/Teff失衡。     

调节性T细胞及其CD45亚群在肝移植免疫耐受中的相关研究

调节性T细胞(Tr)作为一种具有免疫抑制功能的细胞在器官移植免疫耐受中发挥了重要作用.它通过细胞接触抑制排斥反应发生、诱导移植免疫耐受,并且这种免疫抑制具有免疫过继作用.其中CD45亚群被认为是Tr中抑制功能较强的一群细胞,在移植免疫耐受中发挥着重要作用.而肝脏作为一种免疫特惠器官,与其他器官移植相比更易产生免疫耐受....     

Prolonged survival effects induced by immature dendritic cells and regulatory T cells in a rat liver transplantation model

BackgroundDendritic cells (DCs) and regulatory T (Treg) cells are crucial for inducing immune tolerance. However, the suppressive function of infused Treg cells and immature DCs (imDCs) following solid organ transplantation remains unclear.MethodsImDCs derived from DA-donor rats and Treg cells isolated from spleens of Lewis rats were prepared. A heterotopic liver transplantation model was established to examine the immune tolerance effects of infusion of Treg-imDCs, imDCs and Treg cells individually. Th1/Th2 cytokines and TRAL were detected by ELISA. The overall rejection grade was assessed and the rejection activity index (RAI) was calculated. TUNEL-positive lymphocytes were detected in the portal area in liver sections.ResultsThe infusion of Treg-imDCs was more effective than imDCs or Treg cells individually. Moreover, the expression of IL-10 and TGF-β1 was significantly up-regulated, and IL-12 expression was significantly down-regulated, especially in the Treg-imDCs group. The percentage of TUNEL-positive cells was significantly higher in the Treg cells and imDCs groups. The RAI values in Treg-imDCs group on days 3 and 7 were lower than control, imDCs and Treg cells groups individually (p < 0.05). Both TBIL and ALT levels in the Treg-imDCs and imDCs groups were significantly lower than those of the control and Treg cells groups, and serum TRAL levels increased significantly 10 days after transplantation in the imDC and Treg-imDC groups compared with the control and Treg cells groups (P < 0.001).ConclusionThese data demonstrated that infusion of Treg cells and/or imDCs induces alloantigen tolerance and prolongs liver allograft survival. The infusion of Treg-imDCs was more effective than imDCs or Treg cells individually. ImDCs synergize with Treg cells in inducing and maintaining the feedback loop between imDCs and Treg cells in vivo.