卡介菌多糖核酸提高复发性生殖器疱疹患者CD8+ T细胞的细胞因子表达

目的　研究卡介菌多糖核酸 (BCG PSN)对复发性生殖器疱疹 (RGH)患者外周血CD8 T细胞IL 12、IFN γ、IL 4表达的影响。方法　应用流式细胞仪对 4 5例RGH患者和 15名健康志愿者外周血CD8 T细胞IL 12、IFN γ和IL 4的表达进行检测。结果　治疗前RGH患者外周血IFN γ和IL 12阳性CD8 T细胞百分率均显著下降 (P <0 .0 5 ) ,Tc1 Tc2平衡失调 (P <0 .0 5 )。治疗后BCG PSN组外周血IL 12和IFN γ阳性CD8 T细胞百分率显著升高 (P <0 .0 5 ) ,Tc1 Tc2恢复平衡。病例对照组IL 12、IFN γ及IL 4阳性CD8 T细胞百分率于治疗前后变化差异均无显著性 (P >0 .0 5 )。BCG PSN组与病例对照组治疗前后IL 12、IFN γ阳性CD8 T细胞百分率间差异均有显著性 (P <0 .0 5 )。BCG PSN组复发率较低 ,复发病情较轻。结论　RGH患者存在以Tc1水平低下为主的Tc1 Tc2比例失衡和IL 12表达水平低下 ;BCG PSN通过上调外周血CD8 T细胞IFN γ、IL 12的表达 ,纠正机体细胞因子失衡状态而减少疾病的复发     

在单个细胞水平上分析抗原特异性Th1/Th2细胞因子产生的关联性

目的　在单个细胞水平上 ,观察抗原特异性Th1和Th2细胞因子产生的关联性 ,为进一步阐明CD4 T细胞的分化 ,细胞因子产生的相互关系及其特征提供理论依据。方法　从OVA TCR转基因小鼠的脾和淋巴结中分离CD4 T细胞 ,在体外在抗原提呈细胞存在下 ,用卵白蛋白 (OVA)抗原多肽刺激 3d后 ,再以同样的培养条件刺激 5～ 6h ,固定细胞 ,然后进行细胞表面和细胞内细胞因子染色 ,最后利用流式细胞仪在单个细胞水平上分析Th1和Th2细胞因子产生的关联性。结果　抗原特异性CD4 T细胞经抗原再一次刺激后 ,分泌Th1(IFN γ和IL 2 )和Th2 (IL 4、IL 5和IL 10 )细胞因子。IL 12促进IFN γ的表达 ,控制Th2细胞的分化。此外 ,大多数抗原特异性CD4 T细胞只产生 1种细胞因子 ,1个细胞同时产生 2种细胞因子极少见。结论　在单个细胞水平上的研究结果表明 ,经抗原短暂刺激后 (3d) ,不同的CD4 T细胞亚群只产生 1种Th1和 或Th2细胞因子 ,同时产生两种以上者占有很低的比率     

日本血吸虫rGST-Sj32蛋白免疫小鼠感染前后细胞因子水平的变化

目的 :为进一步探讨rGST Sj32保护性免疫机制。 方法 :用rGST Sj32加弗氏完全佐剂 (FCA)皮下免疫BALB/c小鼠。于免疫前 ,攻击感染前 (第 3次免疫后 2wk)以及攻击感染后10d、30d及 4 5d ,各剖杀免疫组与对照组小鼠 5只 ,取脾细胞培养 ,对体外培养的脾细胞诱生的细胞因子进行分析。结果 :用rSj32刺激体外培养的小鼠脾细胞时 ,第 3次免疫后2wk(即攻击感染前 )剖杀小鼠的脾细胞分泌IL 4、IL 5和IFN γ的水平与佐剂对照组相比较 ,均有不同程度的升高 ,分别为 ( 10 .2 1± 3.6 5 )ng/L (P >0 .0 5 )、( 19.89± 9.5 7)ng/L (P >0 .0 5 )、( 5 .0 9± 2 .5 1) μg/L (P <0 .0 1)。攻击感染后10d、30d及 4 5d ,对照组与免疫组IFN γ的水平未见升高 ;对照组IL 4和IL 5的水平随着感染时间的延长明显升高 ,免疫组升高不如对照组明显。结论 :rGST Sj32疫苗免疫BALB/c小鼠后 ,可诱导以Th1类细胞因子为主的免疫应答 ;而攻击性感染则诱导以Th2型细胞因子为主的免疫应答     

HBsAg体外冲击的慢性乙肝患者树突状细胞的生物学特性及其对HBV特异性CTL的诱导作用

目的 :研究HBsAg冲击的慢性乙肝患者单核细胞来源的树突状细胞 (DCs)的功能状况及体外对HBV特异性CTL的诱导作用 ,初步探讨诱导特异性抗HBV细胞免疫的途径。方法 :分离慢性乙肝患者外周血单核细胞 ,以GM CSF +IL 4 +TNF α培养诱导DCs,加入HBsAg冲击以诱导HBV特异性DCs。采用FCM测定细胞表面免疫分子CD1a、CD83、CD86、CD80、CD4 0以及HLA DR的表达水平 ,ELISA法检测培养上清中细胞因子IL 6、IL 12的分泌含量 ,MTT法测定DC刺激同种异体淋巴细胞增殖的能力 ,LDH法检测DC诱导的患者外周血T细胞对HepG2 2 2 15 (转染HBVDNA)、HepG2肝癌细胞株及K5 6 2白血病细胞株的细胞毒作用。结果 :HBsAg冲击的DC其表达CD1a、CD83、CD86、CD80、CD4 0、HLA DR表面分子明显高于对照组 (P <0 0 1,P <0 0 5 ) ,分泌IL 12的水平也高于对照组 (P <0 0 1) ,而分泌IL 6的水平则较对照组显著降低(P <0 0 1) ;HBsAg冲击的DC刺激同种异体淋巴细胞增殖的能力明显增强 (P <0 0 5 ) ,并可有效地诱导自体CTL对转HBV基因的HepG2 2 2 15细胞高效特异性杀伤作用 (P <0 0 1)。结论 :慢性乙型肝炎患者单核细胞来源的DCs经HBsAg抗原冲击后 ,生物学活性增强 ,并且能有效地诱导对HBV特异性反应的CTL。     

质粒DNA促进HBsAg-抗HBs复合型疫苗诱生的免疫应答的研究

目的　研究HBsAg 抗HBs 质粒DNA复合型疫苗的免疫原性及其诱生细胞免疫应答的类型。方法　分别以HBsAg、HBsAg 抗 HBs(IC)、pI/AmpHBs、IC pI/AmpHBs及IC pI/Amp免疫小鼠 ,检测抗 HBs的效价 ,分析抗 HBsIgG亚类 (ELISA) ;取免疫小鼠脾细胞 ,体外抗原刺激 ,用竞争性RT PCR方法检测IFN γ及IL 4mRNA转录水平。结果　IC pI/AmpHBs诱生的抗 HBs效价明显高于IC或pI/AmpHBs单独免疫组 ,其IgG2a/IgG1比值高于IC免疫组 ,而低于pI/AmpHBs免疫组。IC pI/AmpHBs免疫小鼠脾细胞在HBsAg刺激下 ,IFN γmRNA转录水平明显高于其他免疫组 ,其IFN γmRNA的T/C(目的片段Target/竞争片段Competitor)比值为IC免疫小鼠脾细胞的 10倍 ;IC pI/AmpHBs免疫小鼠脾细胞IL 4mRNA转录水平亦高于其他免疫组 ,其IL 4mRNA的T/C比值为IC免疫小鼠脾细胞的 2倍。结论　HBsAg 抗HBs 质粒DNA复合型疫苗在增强体液免疫应答的同时可诱导脾细胞IFN γ的表达水平增高。     

IL-18基因修饰增强肿瘤抗原多肽致敏的树突状细胞体内诱导的抗肿瘤免疫反应

目的 :研究肿瘤抗原多肽致敏的白细胞介素 18(IL 18)基因修饰的树突状细胞体内诱导的抗肿瘤免疫反应。方法 :①以Lewis 3LL肺癌细胞特异性抗原肽mut1冲击致敏IL 18基因修饰的骨髓来源的树突状细胞 (DC IL 18 mut1) ,每次用其 1× 10 5 只皮下免疫小鼠 2次 ,然后测定脾细胞的NK活性及CTL杀伤活性 ;②以DC IL 18 mut1每次 2× 10 5 只皮下免疫 1次 ,然后再以 5× 10 53LL细胞攻击 ,在诱导及效应阶段分别以单抗阻断不同免疫成份 ,观察肿瘤的生长。结果 :以DC IL 18 mut1皮下免疫后可诱导出比DC mut1等免疫组更高水平的 3LL肺癌细胞特异性CTL ,并使NK活性明显增加 ;单抗体内阻断实验提示在DC IL 18 mut1免疫诱导阶段 ,CD4 + T细胞和抗原共刺激分子、IFN γ均起到重要作用 ,而效应阶段CD8+ T、IFN γ、NK起作用 ,而CD4 + T则是非必需的。结论 :DC IL 18 mut1皮下免疫后可诱导高水平的抗肿瘤免疫活性 ,其机理与抗原有效提呈、特异性CTL诱导、NK活性增加以及CD4 + 、CD8+ T、NK细胞、IFN γ参与密切相关。     

系统性红斑狼疮患者外周血CD4~+T细胞细胞因子的检测

本文通过检测系统性红斑狼疮 (SLE )患者外周血中Th1和Th2分泌IFN γ、IL 2、IL 4细胞因子的表达情况 ,探讨Th1/Th2细胞因子水平与SLE的发病关系。采用流式细胞分析法对 4 1例SLE患者和 15例健康志愿者外周血中细胞因子(IFN γ、IL 2、IL 4 )在辅助性T细胞 (CD4 )内的表达进行检测。结果活动期SLE患者Th1细胞因子的表达率较正常人明显下降 (P <0 0 1)。SLE患者Th1细胞因子 (IFN γ、IL 2 )的表达与SLEDAI之间具有明显的负相关性 (P <0 0 5 )。活动期SLE患者Th1细胞因子的表达明显下降 ,SLE患者Th1细胞因子水平与SLEDAI之间呈负相关性     

抗CD28单抗对T细胞体外抗瘤作用的影响

采用健康人外周血T淋巴细胞分别与BEL 740 2人肝癌细胞和抗CD2 8混合培养 ,检测淋巴细胞活化增殖能力、CTL细胞的杀伤活性、培养上清IFN γ和TNF α的水平。结果发现 ,在BEL 740 2刺激细胞存在时 ,抗CD2 8促进增殖的最佳浓度为 6 μg/ml,而单一抗CD2 8不能刺激T细胞增殖。BEL 740 2细胞和抗CD2 8共刺激后IFN γ和TNF α分泌均比单纯BEL 740 2高 ,同时其诱生的CTL细胞杀伤BEL 740 2的能力也强于对照组 ,其差异均具有显著性 (P <0 0 1)。上述结果提示 ,抗CD2 8单抗共刺激诱导CD4+ 和CD8+ T细胞参与抗肿瘤免疫 ,使IFN γ、TNF α的分泌增多 ,增强了体外T细胞抗瘤作用。     

T细胞疫苗抑制自身免疫性甲状腺炎的发生

目的　研究T细胞疫苗 (TCV)在小鼠实验性自身免疫性甲状腺炎 (EAT)发生中的阻断作用及可能机制。方法　磁珠分离CD4 T细胞 ,体外活化 ,戊二醛处理获得T细胞疫苗 ,体内接种EAT小鼠。通过EAT评价、细胞因子测定及细胞增殖试验了解TCV对EAT的阻断作用 ;流式细胞术测定小鼠CD4 CD2 5 T细胞百分率。结果　TCV接种后小鼠体内自身抗体水平明显下降 ,甲状腺无明显病理变化 ,IL 2和IFN γ水平以及Tg刺激的淋巴细胞增殖能力均显著降低 ,同时CD4 CD2 5 T细胞百分率较EAT组有明显增高。结论　TCV接种能明显抑制小鼠EAT的发生 ,可能与机体内CD4 CD2 5 Treg细胞的增加有关     

牛膝多糖体外诱导人T细胞表达IFN-γ和IL-4蛋白的机制探讨

目的 :探讨牛膝多糖免疫调节作用的机制。方法 :使用牛膝多糖在不同浓度或在不同时间内对人T细胞进行体外诱导培养 ,用ELISA方法测定培养上清液的IFN γ及IL 4的蛋白表达情况。结果 :①人T细胞受不同浓度ABPS刺激时 ,IFN γ分泌水平随ABPS浓度递增 ,在 4 0 0 μg ml时 ,IFN γ表达量最高 (P <0 0 5 ) ,而IL 4的分泌始终处于低水平 (P >0 0 5 )。②人T细胞在ABPS刺激不同时间后 ,IFN γ分泌水平逐步增高 ,在 4 8小时时与其他各时间点比较 ,有非常显著的差异 (P <0 0 0 1) ,而IL 4的分泌始终处于低水平 (P >0 0 5 )。结论 :①ABPS能诱导人T细胞分泌IFN γ ,该作用呈时间、剂量依赖性变化 ,而抑制IL 4的分泌。②在蛋白表达水平上证实ABPS能够促进Th1类细胞因子的分泌 ,而抑制Th2类细胞因子的分泌。     

A critical analysis of the T cell hybrid technique

The T cell hybridization technique can be used to prepare continuous cell lines which express the antigen specificity and function of T cells within the milieu of a proliferating lymphoma. Technical details for the preparation and maintenance, selection and analysis of T cell hybrids are defined. Techniques for cloning of hybrid cells and production of hybrid-derived tumors are also presented. Parameters influencing the hybridization frequency and the production of functional hybrids are discussed. A variety of T cell subsets, including suppressor cells and delayed-type hypersensitivity effector cells as well as T cells maintained on TCGF, are excellent sources of primary parents for hybridization. When BW 5147 is used as the T lymphomas parent in these experiments, the resulting hybridization frequencies range between 10 and 434 X 10(-7). We have had moderate success using YAC-1; however, additional lines such as L5178Y, BALENTL 5, EL4 BU and S491TB.2 have proved ineffective as sources of T lymphoma parents. The technique of T cell hybridization is evaluation in terms of retention of differentiated functions and the stability and growth characteristics of the resultant hybrids.     

Thymic Hormone-Induced Accelerated Development of Antibody Responsiveness to Sheep Red Blood Cells in the Newborn Mouse*

ABSTRACT: The influence of thymic preparations on the kinetics of the development of PFC response to sheep erythrocytes was tested in mice 0 to 4 weeks old. Soluble thymic factor (STF) or thymic epithelial culture supernatant (TES) was prepared from the thymuses of C57B1/6 mice. STF was injected to gravid mice on day 13 or on day 18 of gestation. A marked acceleration in the appearance of PFC to SRBC was noted only in the offspring from those mothers having received STF on day 18. The ability to generate anti-SRBC response in vitro by splenocytes from 1-to 4-week-old mice was significantly improved in the presence of TES. The accelerated appearance of the response to a T-dependent antigen is attributed to increase in the ratio of T helper/T suppressor cells resulting from differentiation and/or clonal expansion under the influence of STF crossing of the placental barrier in the materno-fetal direction.     

类风湿关节炎前后肠道T淋巴细胞的分布变化

目的探讨在类风湿关节炎条件下大鼠小肠(十二指肠、空肠、回肠)上皮内T淋巴细胞(intraepithelial lymphocyte,IEL)、固有层T淋巴细胞(lamina propria lymphocyte,LPL)的变化规律。方法通过免疫组织化学染色方法,观察正常组和致病组肠管黏膜的T淋巴细胞的变化规律,然后分别对IEL和LPL计数,并进行统计学分析。结果大鼠致病组肠道各部分(十二指肠、空肠、回肠)上皮层和固有层可见T淋巴细胞数量增多。结论通过免疫组织化学方法可见在类风湿关节炎条件下,肠道黏膜的上皮层和固有层均可见T淋巴细胞的分布增多,肠道各段的IEL、LPL可能在黏膜免疫应答中起着关键的生物学作用。     

CD52 is a novel costimulatory molecule for induction of CD4+ regulatory T cells

We previously reported that 4C8 monoclonal antibody (mAb) provides a costimulatory signal to human CD4+ T cells and consequently induces regulatory T (Treg) cells, which are hypo-responsive and suppress the polyclonal response of bystander CD4+ cells in a contact-dependent manner. In this study, we identified the antigen of 4C8 mAb as CD52. Costimulation with Campath-1H, a humanized anti-CD52 mAb, also induced Treg cells. Anti-CD52-induced Treg cells suppressed the proliferation of both CD4+ and CD8+ T cells provided with polyclonal or allogeneic stimulation. When Treg cells were induced from Staphylococcal enterotoxin B (SEB) treated cells, they suppressed the response to SEB more efficiently than that to another superantigen, SEA. Furthermore, anti-CD52-induced Treg cells could be expanded by culture with IL-2 followed by CD52-costimulation, and co-injection of expanded Treg cells suppressed lethal xenogeneic graft versus host disease (GvHD) reactions in SCID mice caused by human peripheral blood mononuclear cells (PBMCs).     

三种荧光色素标记的CD4抗体在流式细胞术研究T辅助细胞中的有效性评价

探讨三种荧光色素标记的CD4抗体在流式细胞术检测培养刺激前后的T 辅助(Th)细胞中的有效性,从而确定Th细胞研究时优选且有效的CD4抗体.将佛波脂(PMA)和离子霉素(Ion)培养刺激前后的正常人外周血单个核细胞(PBMC)分别用CD3-APC和CD8-PerCP确定Th细胞(CD3+CD8- T细胞)百分率,用CD...     

Clonal growth of carp (Cyprinus carpio) T cells in vitro

Carp kidney leukocytes co-cultured with a supporting cell layer resulted in the rapid proliferation of various types of leukocytes including immature leukocytes. Expressions of marker genes for multiple blood cell lineages were observed in the primary culture. However, after several passages, the proliferating cells expressed only T cell and macrophage marker genes.Further RT-PCR analysis revealed that the proliferating cells expressed TCR constant regions (Cα, Cβ, Cγ, Cδ), CD3γ/δ and CD4 (CD4L-1), but did not express CD8α and CD8β. Additionally, in situ hybridization analysis showed that the majority of proliferating cells expressed Cα, Cβ, Cγ, Cδ and CD4. Moreover, 5′-RACE sequences of TCR variable regions (Vα, Vβ, Vγ, Vδ) revealed that the proliferating cells contained a polyclonal T cell repertoire, and most of the Vα and Vβ sequences were functional, but the Vγ and Vδ sequences were non-functional with frame shifts and stop codons. Taken together, these results indicate that the proliferating cells after serial passages predominantly contained CD4+ CD8− αβT cells that simultaneously co-expressed non-functional γδTCR. To obtain CD4+ αβT cell (helper T cell) clones, single cells were picked up from the bulk culture, seeded into each well of 96-well plates and cultured in the presence of supporting cells and conditioned media. T cell colonies formed from single cells after 2-3 weeks. These colony cells expressed Cα, Cβ, Cδ and CD4, and weakly expressed Cγ, but did not express CD8α, CD8β and CD4L-2. Taken together, these results indicate that these clonal T cells resemble a subpopulation of mammalian CD4+ helper T cells.     

Induction of contact-dependent CD8(+) regulatory T cells through stimulation with staphylococcal and streptococcal superantigens

The bacterial superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes are potent stimulators of polyclonal T-cell proliferation. They are the causes of toxic shock syndrome but also induce CD25⁺ FOXP3⁺ regulatory cells in the CD4 compartment. Several studies have recently described different forms of antigen-induced regulatory CD8⁺ T cells in the context of inflammatory diseases and chronic viral infections. In this paper we show that bacterial superantigens are potent inducers of human regulatory CD8⁺ T cells. We used four prototypic superantigens of S. aureus (toxic shock syndrome toxin-1 and staphylococcal enterotoxin A) and Str. pyogenes (streptococcal pyrogenic exotoxins A and K/L). At concentrations below 1 ng/ml each toxin triggers concentration-dependent T-cell receptor Vβ-specific expression of CD25 and FOXP3 on CD8⁺ T cells. This effect is independent of CD4⁺ T-cell help but requires antigen-presenting cells for maximum effect. The cells also express the activation/regulatory markers cytotoxic T-lymphocyte antigen-4 and glucocorticoid-induced tumour necrosis factor receptor-related protein and skin homing adhesins CD103 and cutaneous lymphocyte-associated antigen. Superantigen-induced CD25⁺ FOXP3⁺ CD8⁺ T cells were as potent as freshly prepared naturally occurring CD4⁺ regulatory T cells in suppressing proliferation of CD4⁺ CD25⁻ T cells in response to anti-CD3 stimulation. Although superantigen-induced CD8⁺ CD25⁺ FOXP3⁺ express interleukin-10 and interferon-γ their suppressive function is cell contact dependent. Our findings indicate that regulatory CD8⁺ T cells may be a feature of acute bacterial infections contributing to immune evasion by the microbe and disease pathogenesis. The presence and magnitude of regulatory CD8⁺ T-cell responses may represent a novel biomarker in such infections. Superantigen-induced regulatory CD8⁺ T cells also have therapeutic potential.     

A persistent T cell expansion in the peripheral blood of a normal adult male: a new clinical entity?

A dramatic and persistent T cell expansion in a healthy adult male was initially identified, using anti-T cell receptor for antigen (TCR)-specific MoAbs. The expanded T cells were found to be expressing TCR containing V alpha 12.1 and V beta 5.2, and they composed approximately one third of all the CD8+ T cells. The cells were shown to be not only non-activated (HLA-DR-, IL-2R-) but also of 'virgin' cell type (CD45RA+/CD45RO-) and they persisted over the observation period of more than one and a half years. Various T and B cell markers, and all other laboratory and physical parameters analysed, were normal. The expanded CD8+ T cells were further characterized by polymerase chain reaction (PCR) amplification, using V beta- and C beta-specific primers, followed by hybridization with J beta-specific probes. Close to 90% of the V alpha 12.1+ V beta 5.2+ T cells were found to utilize the J beta 2.5 gene segment, thus strongly suggesting the expanded T cells to be monoclonal. The condition may constitute a T cell counterpart to 'monoclonal gammopathy of undetermined significance' (MGUS), and by analogy we suggest it should be designated 'monoclonal T cell expansion of undetermined significance' (MTUS).     

Analysis of clones derived from human CD7+CD4-CD8-CD3- thymocytes.

The differentiation of human thymocyte precursors was studied by analysis of clonal progeny of CD4-CD8-CD3- (triple negative or TN) thymocytes. Using a culture system of phytohemagglutinin, IL-2, and irradiated allogeneic lymphoid feeder cells, we found that 48% of clones (104 total) derived from TN thymocyte suspensions were TCR gamma delta cells, 12% of clones were TCR alpha beta cells, and 34% were CD16+CD3- cells. Importantly, 6% of clones were novel subsets of CD4+CD8-CD3- or CD4-CD8+CD3- thymocytes. The majority of TCR alpha beta, TCR gamma delta, and CD16+CD3- clones expressed low levels of CD4. Molecular analysis of freshly isolated TN- thymocytes prior to in vitro culture demonstrated that up to 40% of cells had TCR gamma, delta, and beta gene rearrangements, but were negative in indirect immunofluorescence assays for cytoplasmic TCR delta and beta. These data provide evidence at the clonal level for the presence of precursors of the TCR alpha beta and TCR gamma delta lineages in the human TN thymocyte pool. Moreover, a substantial proportion of freshly isolated human TN thymocytes had already undergone TCR gene rearrangement prior to in vitro culture. Whether these precursors of the TCR alpha beta and TCR gamma delta lineages mature from cells already containing TCR gene rearrangements into sTCR+ cells or differentiate in vitro from cells with TCR genes in germline configuration remains to be determined. Nonetheless, these data demonstrate that the predominant clone types that grow out of human TN thymocytes in vitro are TCR gamma delta and NK cells.