T和B记忆细胞

Ｔ和Ｂ记忆细胞是初次免疫应答后克隆消除保留下来的高亲和力细胞，它们有独特的表型，具有淋巴因子分泌、分布和迁移性以及活化的特点。短期免疫记忆要求残存抗原持续性存在，长期免疫记忆与ｂｃｌ－２表达有关。     

人B7-H4基因转染细胞的构建及其对T细胞的共刺激效应的初步研究

目的 构建含有人B7-H4基因的重组逆转录病毒载体，获得稳定表达B7-H4基因的1929细胞并初步研究其对T细胞的共信号作用及可能机制。方法从含人B7-H4基因的cDNA序列FLJ22418中，采用PCR扩增出B7-H4全长基因，双酶切装入逆转录病毒载体pEGZ-HA-Term，与辅助病毒载体脂质体法共转染包装细胞293T，用其培养上清感染1929细胞72h后，经Zeocin筛选出稳定表达B7-H4蛋白的L929细胞株；采用免疫荧光标记和流式细胞仪分析免疫分子的表达、^3H-TdR掺入检测细胞增殖以及ELISA测定细胞因子的水平。结果构建了含人B7-H4基因的重组逆转录病毒载体和获得含有B7-H4基因的重组病毒，筛选获得能稳定高表达人B7-H4蛋白的I_929转基因细胞；该转基因细胞对T细胞体外具有抑制增殖、活化和细胞因子分泌的作用。结论构建了稳定表达人B7-H4蛋白的细胞株，B7-H4分子在体外通过抑制T细胞分泌IL-2和促进其凋亡作用可显著地下调T细胞功能的效应。     

小鼠B7-H3对T细胞的体外正性协同刺激作用

小鼠B7-H3作为协同刺激分子的生物学功能至今尚未明确.本文拟通过构建小鼠B7-H3基因的真核表达载体,获取稳定表达小鼠B7-H3基因的细胞株,并进一步通过体外实验研究其对T淋巴细胞体外活化及功能的调节作用.作者运用RT-PCR技术从小鼠肝脏组织中获得全长小鼠B7-H3基因,并构建真核表达载体pIRES2-B7-H3,...     

哮喘病人B细胞体外产生IgE过程中幼稚性T细胞的影响作用

目的：探讨支气管哮喘发病机制中Ｔ细胞的调节作用。方法：分离出病人辅助性Ｔ细胞中的幼稚性（Ｎａｉｖｅ）Ｔ细胞亚群即ＣＤ４＾＋ ＣＤ４５ＲＡ＾＋Ｔ细胞亚群,并将其与各自的Ｂ淋巴细胞共同培养。同时设定非特异性刺激原ＰＷＭ（美洲商陆）刺激组在刺激组,测定培养上清液中ＩｇＥ含量。结果：非刺激组病人ＩｇＥ含量明显高于健康对照（Ｐ〈０．０１）,刺激组病人与健康对照ＩｇＥ含量无显著差别（Ｐ〉０．０５）。结论：支气     

人胎胸腺素对T细胞增殖及IL—2分泌的影响

胸腺素具有广泛的免疫调节作用。本文观察了人胎胸腺素（ＨＦＴ）在体外对人周围血Ｔ细胞的增殖及对周围血单个核细胞（ＰＢＭＣ）分泌ＩＬ－２的影响，实验结果表明：ＨＦＴ能刺激Ｔ细胞增殖，１％ＨＦＴ的作用最强，但未见ＨＦＴ与ＰＨＡ对Ｔ细胞增殖的协同促进作用；而ＨＦＴ对ＰＢＭＣ分泌ＩＬ－２的影响不明显，ＨＦＴ与ＰＨＡ共同作用的效应约有提高，亦仅相当于ＩＬ－２在１．９￣７．９ｕ／ｍｌ时的效应。     

IL-15对异基因抗原刺激下人CD8+T 细胞增殖分化影响的初步研究

目的：探讨IL-15对异基因抗原刺激下人CD8+T细胞增殖分化的影响。方法：在IL-15存在的条件下,将从健康志愿者外周血中新鲜分离的CD8+T细胞与HLA-A,-B,-DR完全错配的异体抗原提呈细胞（APCs）进行混合淋巴细胞培养（MLRs）。经9 d培养后,对诱导后产生的CD28-CD8+和CD28+CD8+T细胞亚群进行细胞毒功能测定和表型检测,并对CD8+T细胞增殖分化过程中CD28分子的动态变化规律进行监测。结果：在IL-15诱导及异基因抗原刺激下,CD8+T细胞增殖分化后产生的CD28+CD8+T细胞具有细胞毒作用,与外周血新鲜分离的类似细胞相比,其高表达了FasL、颗粒霉素-B和穿孔素;而CD28-CD8+抑制性T细胞不具有细胞毒作用,与外周血新鲜分离的类似细胞相比,低表达了FasL、颗粒霉素-B和穿孔素。在异基因抗原提呈细胞刺激下,IL-15可诱导CD8+T细胞增殖分化过程中CD28-/CD28+细胞数比例升高（从0.24到1.01）,并可诱导CD28+T细胞上的CD28分子丢失。结论：IL-15通过调节CD28分子的表达影响异基因抗原对CD8+T细胞的增殖分化。     

人T细胞抗原受体基因的命名

<正>人T细胞抗原受体(TCR)是由二硫键连接的二条多肽链(αβ或γδ)经组成的异二聚体,属lg基因超家族,每条链具有与lg分子相似的V区、D区(仅对β和δ链)J区和C区.根据TCR的不同,可将T细胞分为αβ~+T细胞和γδ~+T细胞.人外周血中95%的T细胞为αβ~+TCR,其配体是MHC-抗原肽复合物;而γδ~+TCR一般仅表达于CD4~-CD8T细胞,这些γδ~+T细胞识别的抗原结构目前尚不清楚,但在机体抗感染免疫和抗肿瘤免疫中发挥着重要的作用.从胸腺内的发育过程来看,αβ~+T细胞和γδ~+T细胞可能来源于同一祖先,而且γδTCR的表达要早于αβTCR,分别相当于妊娠第九周和第十周.     

TPA和IFN-γ活化角朊细胞对T淋巴细胞增殖和细胞因子分泌的作用

角朊细胞激活对T淋巴细胞增殖和细胞因子分泌的作用。TPA和IFN γ联合作用 ,激活角朊细胞表达表面分子 ,FACS检测MHCI、II类分子和CD80、CD86分子水平 ,RT PCR检测B7 H1共刺激分子表达。由激活角朊细胞提供T淋巴细胞激活的第二信号 ;绵羊红细胞花环沉淀分离纯化T细胞 ,抗CD3单抗提供第一信号 ,建立体外混合培养系统 ,3 H TdR渗入法检测T细胞增殖 ,ELISA检测培养上清细胞因子浓度。结果显示 ,TPA和IFN γ联合作用可诱导角朊细胞表达B7 H1共刺激分子 ,并上调其MHCII类分子。以B7 H1为第二信号 ,可以协同抗CD3单抗对T细胞的增殖作用 ,并呈现独特的细胞因子分泌格局。因此角朊细胞激活后表达B7 H1共刺激分子 ,此第二信号可刺激T淋巴细胞增殖 ,对其功能分化具有调节作用     

动脉粥样硬化中的T细胞和B细胞的研究进展

动脉粥样硬化(atherosclerosis,AS)是在动脉壁上形成的纤维脂肪病变,由它引起的心血管疾病(cardiovascular disease,CVD)是造成全世界范围内死亡的第一大病因。该疾病的主要特征是脂蛋白在内皮下的累积导致血管壁中持续发生的炎症反应,因此多种固有免疫细胞和适应性免疫细胞都参与了该疾病的发...     

Low-dose UVB radiation perturbs the functional expression of B7.1 and B7.2 co-stimulatory molecules on human Langerhans cells

In previous studies, we have shown that ultraviolet (UV) B radiation perturbs the APC function of Langerhans cells (LC) by interfering with as-yet unidentified co-stimulatory signals. Recently, B7.1 and B7.2 on APC were shown to deliver important co-stimulatory signals through interaction with their counterreceptors CD28 and CTLA-4 on T cells. To determine whether UVB affects the functional expression of B7.1 or B7.2 on LC, B7.1 and B7.2 expression was studied on human LC by multiparameter flow cytometry. Little, if any, B7.1 or B7.2 was detected on LC freshly isolated from skin. However, following 48 h of tissue culture, expression of both B7.1 and B7.2 were markedly up-regulated. To test whether these molecules were functional, primary mixed epidermal cell leukocyte reactions (MECLR) were performed. Blocking monoclonal antibody (mAb) to B7.1 or B7.2 both inhibited the MECLR, with anti-B7.2 being much more effective than anti-B7.1. UVB radiation dose-dependently (100–200 J/m²) suppressed the culture-induced up-regulation of B7.1 and B7.2 on LC. Since LC exposed to the same UVB flux (UVB-LC) failed to stimulate alloreactive T cells in a MECLR, we questioned whether this was related to their inability to provide B7 co-stimulation. Indeed, when effective B7-CD28 signaling was ascertained by adding submitogenic doses of exogenous anti-CD28 mAb to UVB-LC, the proliferative response of alloreactive T cells was restored. We conclude that the suppressive effects of low-dose UVB radiation on the APC function of LC are, at least in part, due to an inhibition of functional B7.1 and B7.2 expression.     

Cytotoxic T cell immunity to human cytomegalovirus glycoprotein B

Human cytomegalovirus (HCMV) is associated with significant morbidity and mortality following immunosuppression and in pregnancy. HCMV infection may be accompanied by acute disease but persists asymptomatically. Cytotoxic T lymphocytes (CTL) appear to be an important immune effector mechanism in maintaining the normal host-virus equilibrium. Glycoprotein B may be an important target for future subunit vaccines as it has been found to elicit both neutralising antibody and CTL responses. We therefore studied the ability of normal asymptomatic HCMV-seropositive individuals and women throughout pregnancy to determine the presence of HCMV and gB-specific CTL responses. CTL effector cells were induced by stimulation of peripheral blood mononuclear cells (PBMC) with AD169 HCMV-infected cells and gB-specific CTL were identified using chromium labeled, vac.gB-infected cells. In 7 HCMV-seropositive individuals, HCMV-specific CTL were identified. Three of the 7 individuals which lysed HCMV-infected cells lysed vac.gB-infected B cells. However, vac.gB-infected autologous fibroblasts, which only present MHC class I, were not killed. Using MHC class I single allele targets, no specific lytic response was observed, suggesting a MHC class II restricted CTL response. Flow cytometric analysis showed the gB-specific effector cell phenotype to be CD3+, CD4+, CD8−. In conclusion, a gB-specific CTL lytic response was identified in seropositive individuals which in most cases was MHC class II-restricted. © 1996 Wiley-Liss, Inc.     

CCK-8对内毒素血症小鼠腹腔巨噬细胞B7.1和B7.2表达及协同刺激功能的影响

目的： 探讨八肽胆囊收缩素（CCK-8）对内毒素血症小鼠腹腔巨噬细胞B7.1和B7.2表达及其协同刺激活性的影响。方法: 将BALB/c小鼠随机分组（n=4）,分别腹腔注射生理盐水（0.2-0.3 mL/mouse），LPS（100 μg/mouse）和/或CCK-8 (5 nmol/mouse)及CR1409（100 μg/mouse）、CR2945（100 μg/mouse）。12 h后收集并纯化腹腔巨噬细胞。采用流式细胞术分析细胞表面B7.1和B7.2含量的变化。用免疫磁珠从小鼠脾细胞分离CD4+T细胞，按4∶1数量比与上述处理的腹腔巨噬细胞共同体外培养，同时加入ConA 5 mg/L，采用［³H］掺入法测定CD4+T细胞增殖，反映巨噬细胞的协同刺激活性。结果: 整体应用CCK-8作用小鼠腹腔巨噬细胞，与对照组相比，CCK-8可上调静息小鼠腹腔巨噬细胞B7.2的表达，而对B7.1的表达则无影响；并且CCK-8使CD4+T细胞［³H］-TdR的掺入率升高，即促进其增殖，增强巨噬细胞的协同刺激活性。CCK-8降低LPS活化的内毒素血症小鼠腹腔巨噬细胞B7.1和B7.2的表达并且降低CD4+T细胞的［³H］-TdR掺入率，即抑制其增殖，抑制其协同刺激活性。CR1409及CR2945均能逆转CCK-8的上述作用，且CR1409的作用较CR2945更明显。结论: CCK-8通过上调巨噬细胞B7.2表达而增强其协同刺激活性；并且降低LPS活化的内毒素血症小鼠腹腔巨噬细胞B7.1和B7.2的表达，抑制其协同刺激活性。该作用由CCK1R及CCK2R共同介导，其中CCK1R起主要介导作用。     

Comparison of CD28-B7.1 and B7.2 functional interaction in resting human T cells: Phosphatidylinositol 3-kinase association to CD28 and cytokine production

CD28 is a 44-kDa homodimer present on T cells providing an important costimulatory signal for T cell proliferation, cytokine production and cytokine receptor expression. CD28 activation is mediated by interaction with its counter-receptors, B7.1/CD80 and B7.2/B70/CD86. The biochemical basis of these costimulatory signals are still poorly understood, particularly in resting T cells. However, various biochemical pathways such as tyrosine phosphorylation, phospholipase C, sphingomyelinase and phosphatidylinositol 3-kinase (PI3-K) activation have been reported to play a role in CD28 signaling in tumor T cell lines and CD28-transfected cells or pre-activated T cells. In addition, recent reports propose that CD28-B7.1 and B7.2 interaction could be involved in the production of Th1 and Th2 cytokines, respectively, but the putative biochemical basis for these different functions is still unknown. We have analyzed the functional and molecular consequences of CD28 activation by B7.1 and B7.2 in human resting T cells. We demonstrate in this report that both CD28-B7.1 and CD28-B7.2 interactions induce the association of PI3-K to CD28 in the CD4 subpopulation, whereas it was barely detectable in CD8 cells. This association involves the binding of the src homology domain 2 (SH2) of p85 to tyrosine-phosphorylated CD28 and does not require pre-activation by CD3-T cell receptor. Worthmannin, a specific inhibitor of PI3-K enzymatic activity within the nanomolar range also inhibits the interleukin-2 production induced by costimulation mediated by either the B7.1- and B7.2-transfected cells or CD28 monoclonal antibodies. The only slight difference between B7.1 and B7.2 costimulation is the IC₅₀ of wortmannin being 25 and 110 nM, respectively, which could suggest differences in their activation of the T cell PI3-K.     

T cell activation by antigens on human melanoma cells co-stimulation by B7-1 is neither sufficient nor necessary to stimulate IL-2 secretion by melanoma-specific T cell clones in vitro

B7-1 expression, induced by transfection in poorly Immunogenicmurine tumours, was shown to elicit a T cell-mediated rejectionof these tumours and further active immunity against the nontransfectedtumour. We therefore asked to what level similarly induced expressionof B7 on human melanoma cells would affect the antigen-dependentresponses of tumour-specific T cell clones in vitro. Data presentedshow that B7-1 expression by melanoma lines: (i) significantlyinduced, or improved, an IL-2-dependent proliferative responseof such clones to the antigen; (ii) increased the amount ofIL-2 produced by two clones in response to the parental non-transfectedtumour cells;and (iii) increased the TNF responses of all theCD4⁺ clones. However, despite these clear co-stimulatory effectson antigen-induced responses of all T cell clones, which demonstratedaneffective interaction of the B7-1 transfected molecule withone or the other of its counter-receptors expressed on T cellclones, B7 co-stimulation did not correct the defect of IL-2secretion exhibited by many of these clones in response to invitro antigen presentation by melanoma cells. We further showthat defective IL-2 secretion in response to melanoma antigenswas not due to a T cell clone refractoriness induced by theculture, since one of these clones could be induced to secreteIL-2 by an antigen-expressing melanoma line, upon increasedlymphocyte function associated antigen-3expression induced bygene transfectlon. Together these data suggest that defectiveIL-2 secretion by many tumour-infiltrating lymphocytes clonesin response to antigen presentation by melanoma cells in vitrois not exclusively due to the inability of these cells to providean appropriate co-stimulation through the B7-1 molecule.     

转染人BTLA基因细胞株的建立及其生物学功能的初步研究

目的:构建B、T淋巴细胞衰减子(BTLA)基因转染的细胞作为免疫原,并探讨该基因转染细胞在体外的生物学功能。方法:通过RT-PCR法从经PHA活化的人外周血T细胞中克隆出人BTLA编码区全长基因,经EcoR I和BamHI双酶切后插入逆转录病毒载体pEGZ-Term中构建成重组载体pEGZ-Term/BTLA。用脂质体法以重组逆转录病毒载体转染293T细胞,并用Zeocin抗生素进行长期筛选;流式细胞术分析BTLA在基因转染细胞膜上的表达;通过MTT法和流式细胞术探讨基因转染细胞在体外对T淋巴细胞增殖与活化的影响。结果:流式细胞术检测表明BTLA基因转染的293T细胞膜上能稳定地高表达人BTLA蛋白。BTLA基因转染的293T细胞和T细胞体外共培养显示,与未转染的293T细胞相比,该基因转染的细胞能部分地抑制抗人CD3单克隆抗体(mAb)刺激的T细胞增殖;流式细胞术和ELISA法分析揭示,该基因转染的细胞能够下调T细胞表面活化标志CD25的表达并降低IFN-γ和IL-10的分泌。结论:获得稳定高表达人BTLA基因转染的细胞株。该细胞株在体外对抗人CD3 mAb刺激的T细胞的增殖与活化具有部分地抑制作用。     

Novel B and T cell epitopes of chicken ovomucoid (Gal d 1) induce T cell secretion of IL-6, IL-13, and IFN-γ

BACKGROUND: Chicken ovomucoid (OM, Gal d 1) has an important role in the pathogenesis of IgE-mediated allergic reactions to hen's egg white. OBJECTIVES: The purpose of this study was to clarify the mechanisms of T cell recognition of ovomucoid using intact OM and chemically modified, characterized and homogeneous solid phase synthetic peptides covering the whole molecule. METHODS: Eighteen overlapping peptides were prepared by solid phase F-moc polyamide peptide synthesis (SPPS), characterized and high-pressure liquid chromatography (HPLC) purified. The peptides, together with intact, denatured and oxidized OM, were used to stimulate patient-derived cell cultures for mapping T cell epitopes. Proliferation responses, T cell phenotype and cytokine secretion using peripheral blood mononuclear cells (PBMC) from eight individuals and T cell lines (TCL) derived from six hen's egg-allergic patients, were examined. In addition, intact, denatured, oxidized and deglycosylated OM, as well as the peptides solely or with their keyhole limpet haemocyanin (KLH) complexes, were tested. For locating IgE and IgG B cell epitopes, seven egg-allergic patient sera and three OM-polyclonal sera were used. Healthy non-allergic individuals were included as controls. RESULTS: Seven peptides were recognized by specific IgE, while OM-specific TCL recognized 10 peptides. Six of the OM peptides were commonly recognized both by patient S-IgE and blood-derived TCL. Among those, one novel epitope, peptide OM 61-74, had the ability to bind IgE. Another peptide, OM 101-114, was recognized by IgE and IgG sera, but not by any of the TCLs. In contrast, the peptides OM 41-56, OM 71-84, OM 131-144 and OM 171-186 were exclusively T cell epitopes with no affinity to specific antibodies. Abundant TCL secretion of IFN-gamma, IL-6, IL-4, IL-13, IL-10 and TNF-alpha in response to OM stimulation indicates the contribution of Th2 as well as Th1/Th0 CD4+ cell subsets. For allergic patients moderate amounts of IFN-gamma, IL-13, and high amounts of IL-6, were secreted in response to TCL stimulation by OM peptides. High amounts of IL-6 were secreted in response to all molecular forms of OM (intact-, modified-OM and the peptides 71-84 and 51-64) when TCLs from two non-allergic donors were used. CONCLUSIONS: One novel B cell epitope (OM 61-74) and 10 T cell epitopes have been identified. The most reactive epitopes of the OM molecule comprise the motifs 1-14 to 71-84, the overlapping peptide-pairs OM 121-134 and OM 131-144 and peptides OM 161-174 and 171-186. Peptides OM 1-14 and 171-186 are the only ones capable of inducing IL-4 secretion. Only one peptide (OM 11-24) induces IL-10 secretion. Those peptides recognized as both T and B cell epitopes or only T cell epitopes, have the potential to induce T cell secretion of moderate to high amounts of IL-13, IFN-gamma and particularly IL-6.     

Inhibitory effect of IL-4 on the sepharose-CD3-induced proliferation of the CD4CD45RO human T cell subset

CD45R monoclonal antibodies are able to distinguish two different subsets of the CD4 human T cells. This phenotypic split is accompanied by functional diversity. In this report we have analyzed the capabilities of CD45R subsets of CD4 human T cells to use interleukin 2 (IL-2) and IL-4 as growth factors. We have found that both cell subsets are able to proliferate after stimulation with Sepharose-CD3 in the presence of externally added IL-2 or IL-4. However, the response to IL-4 of CD4CD45RO cells was comparatively lower than the response of CD4CD45RA cells. Both cell subsets showed a good response to Sepharose-CD3 plus adherent cells (AC), but when IL-4 was present in the culture only the CD4CD45RA cells showed an enhancement in the Sepharose-CD3-induced proliferation, while proliferation of the CD4CD45RO T cell subset was inhibited. Similar effects were seen, however, in the response to CD4CD45RA or CD4CD45RO cells to Sepharose-CD3 plus IL-2. Although the precise mechanism of the inhibitory effect of IL-4 is not known, the results obtained suggest that IL-4 could interfere in some way with the signalling of IL-2 to the proliferation of the CD4CD45RO T cell subset.