同种异基因移植引流淋巴结及外周血T细胞活化分析

目的:研究同种异基因移植早期局部引流淋巴结和外周血T细胞的活化情况。方法:以小鼠前臂皮下非血管化心肌移植为模型,利用流式细胞术分析前臂引流淋巴结及外周血不同T细胞亚群的CD69表达水平。结果:同种异基因移植后72h,引流淋巴结CD4⁺T细胞和CD8⁺T细胞的CD69表达百分率均明显高于移植前(P＜0.01),且CD8⁺T细胞的表达水平高于CD4⁺T细胞(P＜0.01);各组外周血CD4⁺T细胞和CD8⁺T细胞的CD69表达百分率均无显著差异;局部应用弗氏完全佐剂和全身应用CsA可分别增强和抑制同种异基因移植后CD4⁺T细胞和CD8⁺T细胞的CD69表达(P＜0.05或P＜0.01)。结论:局部引流淋巴结T细胞CD69的表达水平可及时反映受者T细胞对同种异基因抗原的识别情况。     

正常人外周血TCRVα24+ NKT细胞体外活化特性观察

目的:探讨正常人外周血自然杀伤T(NKT)细胞的数量以及体外活化后表达CD69、IFN-γ和IL-4的规律并与CD3⁺ T细胞进行比较。方法:取正常成人外周血,直接三色荧光标记后溶血获取有核细胞,或以佛波醇酯(PDB)+离子霉素(Ion)刺激并培养6h,经三色荧光标记后溶血并获取有核细胞,以流式细胞术分析NKT细胞和T细胞的数量以及表达CD69和IL-4、IFN-γ的情况。结果:NKT细胞约占外周血T淋巴细胞总数的(1.34±0.42)%(x±s);PDB+Ion活化6h后NKT细胞CD69表达率为(96.71±1.33)%,明显高于对照组(11.47±2.86)%(P＜0.05);同样条件下CD3⁺T细胞CD69表达率分别为(98.60±0.47)%和(1.07±0.45)%(P＜0.05);当莫能菌素(monensin)存在时以PDB+Ion刺激6h后,IL-4阳性NKT细胞的百分比为48.62±2.44,明显高于对照组31.57±3.31(P＜0.05);IFN-γ阳性NKT细胞百分比为46.65±11.91,也高于对照组13.45±6.29(P＜0.01)。CD3⁺T细胞在刺激后表达IL-4和IFN-γ均明显升高,但IL-4表达率远远低于NKT细胞;而且对照组CD3⁺T细胞两种细胞因子表达率都明显低于NKT细胞。结论:正常成人外周血含有少量的NKT细胞,这些细胞IL-4和IFN-γ的表达率明显高于CD3⁺T细胞,是特定微环境里的重要免疫调节细胞。     

慢性乙肝患者外周血CD4CD25FOXP3 Tregs及HBV抗原特异性CTLs的检测和分析

目的: 检测慢性乙肝(CHB)患者外周血中CD4⁺CD25⁺FOXP3⁺调节性T淋巴细胞(Treg细胞)和乙肝病毒(HBV)特异性细胞毒性T淋巴细胞(CTLs)的表达及意义。方法: 收集28例CHB患者和15例健康人外周血单个核细胞标本,运用流式细胞仪对Treg细胞亚群进行定量分析,同时采用酶联免疫斑点法检测HBV抗原特异性CTLs,并结合丙氨酸氨基转移酶(ALT)和 HBV DNA的临床情况进行分析。结果: CHB组CD4⁺CD25⁺FOXP3⁺ Treg细胞的频率显著高于健康对照组 (3.14%±0.97% vs 1.95%±0.68%,P＜0.05);HBV抗原特异性CTL斑点计数为阳性(19.28±3.85)。CHB组Treg的频率与乙肝病毒载量呈正相关(r=0.831, P＜0.01),与HBV特异性CTL斑点计数值呈负相关(r＝-0.540,P＜0.01)。结论: CHB患者外周血CD4⁺CD25⁺FOXP3^＋Treg细胞表达升高并与病毒载量相关,而与HBV反应的CTLs数量呈负相关,提示Treg细胞可通过抑制细胞免疫反应影响病毒清除。     

心理应激对正常人外周血T淋巴细胞体外活化表面分子表达的作用

目的:探讨心理应激增加对感染的易感性的细胞和分子免疫学机理。方法:采用双荧光染色流式细胞分析法对20名健康大学生志愿者(男女各半)在应激前后进行外周血淋巴细胞免疫表型及T细胞体外丝裂原刺激早期活化抗原表达分析。连续一周期末考试被设定为心理应激。结果:免疫表型分析显示,应激前后CD2、CD3、CD4、CD8、CD19、CD20、CD16、CD56等淋巴细胞的表面分子阳性的细胞的百分比的差异无统计学显著性;与应激前的结果相比,应激后的T细胞在体外培养条件下多克隆刺激剂活化后CD69的表达明显降低,植物血凝素(phytohemagglutinin,PHA)组CD69⁺CD3⁺/CD3⁺的百分率由应激前的28.1±4.1降低到应激后的17.6±3.8,佛波醇酯(phorbol 12,13-dibutyrate,PDB)组CD69⁺CD3+/CD3⁺的百分率由应激前的80.7±6.8降至应激后的65.8±7.9,而在没有刺激剂作用的条件下,T细胞CD69表达率应激前后的差别无显著。结论:应激对免疫系统的影响并不在于改变外周血淋巴细胞各亚群的比例的层面上;心理应激能降低健康人T细胞体外活化的反应性,这可能与心理应激个体对感染的易感性增加有关。     

支气管哮喘病人CD4+T细胞CD25、CD30表达状况

目的：通过观察哮喘病人外周血CD4⁺T细胞CD25、CD30表达水平，了解哮喘病人T细胞活化状态。方法：将分离出的CD4⁺T细胞分别用PPD、PHA刺激，最后用流式细胞仪检测抗原刺激前后细胞表面CD25、CD30表达水平。结果：①哮喘病人CD4⁺T细胞CD25、CD30自然表达比率均低于健康对照（P<0.05、P<0.05）。②用PHA刺激哮喘病人CD4⁺T细胞后，CD25表达水平明显高于健康对照（P<0.01），但CD30表达无差异。③PPD刺激组CD25、CD30表达与健康对照间无差异。结论：哮喘病人CD4⁺T细胞活化状态明显异常。哮喘病人的CD4⁺T细胞无刺激因素时，活化水平低下，但接受刺激后表现出高水平的活化状态。     

自然流产中外周血和蜕膜组织自然杀伤细胞亚群的比例变化

目的和方法:采用流式细胞仪检测淋巴细胞亚群法探讨自然流产与正常早孕之间外周血和蜕膜自然杀伤细胞亚群的差异。结果:外周血中自然流产组的CD56⁺的百分率较早孕组有减少的趋势,而CD56⁺CD16⁺的百分率则较早孕组显著减少,CD16⁺的百分率两组间无差异。自然流产组的蜕膜CD56⁺、CD56⁺CD16⁺、CD16⁺的百分率均明显低于早孕组。结论:蜕膜中CD56⁺NK细胞的减少可能是自然流产的原因之一,外周血中CD56⁺和CD56⁺CD16⁺NK细胞的丢失可能对自然流产的发生具有诊断价值。     

镁对哮喘患者外周血CD4+ CD25+调节性T细胞凋亡及Foxp3表达的影响

目的:观察镁对分离培养的健康人和哮喘患者外周血CD4⁺CD25⁺调节性T细胞凋亡及叉头框蛋白3(Foxp3)表达的影响。方法:经磁珠分离法分离出健康人和哮喘患者外周血CD4⁺CD25⁺T细胞,分镁剂干预组(10 mmol/L)及空白组培养72 h后,用流式细胞仪检测CD4⁺CD25⁺T细胞的凋亡率及Foxp3表达情况。结果:(1)健康人外周血CD4⁺CD25⁺T细胞的纯度为77.4％~92.3%,哮喘患者CD4⁺CD25⁺T细胞的纯度为75.2％~93.8％。(2)CD4⁺CD25⁺T细胞占外周血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%,两者没有显著差异(P＞0.05)。(3)镁(10mmol/L)可以诱导健康组及哮喘组外周血CD4⁺CD25⁺T细胞凋亡率增加(P＜0.05),但对Foxp3的表达无影响(P＞0.05)。结论:镁促进CD4⁺CD25⁺T调节细胞凋亡增加可能为其治疗支气管哮喘的作用机制之一。     

蛋白激酶C抑制剂对T细胞表达IL-2及IFN-γ的影响

目的:研究蛋白激酶C(PKC)抑制剂H7和棉酚对体外活化T细胞表达白细胞介素-2(IL-2)和γ-干扰素(INF-γ)的影响。方法:在莫能菌素(monensin)存在时,以佛波醇酯(PDB)+离子霉素(I)体外活化人外周血单个核细胞(PBMC),以流式细胞术胞内细胞因子检测法分析H7和棉酚对CD3⁺T细胞表达IL-2和IFN-γ水平的影响。结果:PDB+I处理PBMC4h后,表达IL-2和IFN-γ的CD3⁺T细胞百分率分别为16.64±2.04和25.81±3.53(x±s),而对照组二者的比率为1.06±0.22及3.12±0.77(P＜0.05)。棉酚(50μmol/L)可明显抑制IL-2及IFN-γ的表达,抑制后表达率分别为2.08±0.12及9.01±1.90。H7作用比棉酚强,50μmol/L的H7抑制后的IL-2及IFN-γ阳性T细胞比率分别为0.43±0.06和2.40±0.27。结论:PKC在CD3⁺T细胞表达IL-2及IFN-γ中发挥重要作用;PKC抑制剂H7及棉酚明显抑制IL-2及IFN-γ的表达,提示H7和棉酚可通过抑制PKC活性,对依赖于T细胞功能的特异性免疫应答有调节作用。     

鼻息肉组织T淋巴细胞IFN-γ、IL-4的表达

目的:通过检测γ-干扰素(IFN-γ)、白细胞介素-4(IL-4)两种细胞因子在鼻息肉组织T淋巴细胞(CD3⁺细胞)中的表达, 探讨鼻息肉的可能发病机制。方法:采用流式细胞术检测21例鼻息肉患者鼻息肉组织、外周血中T淋巴细胞IFN-γ、IL-4的分泌情况, 并与正常人下鼻甲粘膜及外周血进行比较。 结果:在正常人下鼻甲粘膜中几乎未见CD3⁺IL-4⁺和CD3⁺IFN-γ⁺细胞。鼻息肉组织中有大量T淋巴细胞浸润, IL-4、IFN-γ的含量分别为(13.606±0.644)%、(32.938±2.477)%;患者外周血中IL-4、IFN-γ的含量分别为(6.686±0.204)%、(64.312±1.611)%, 与之相比, 病人鼻息肉组织中IL-4的含量显著高于外周血(P＜0.05)而IFN-γ的含量明显低于外周血(P＜0.05)。正常人外周血IL-4、IFN-γ的含量分别为(0.560±0.051)%, (0.246±0.020)%, 与之相比, 病人外周血中IL-4、IFN-γ的含量均显著高于正常人(P＜0.05)。结论:鼻息肉患者鼻粘膜局部免疫异常, Th细胞因子分泌优势发生改变, 造成鼻腔粘膜"微环境"改变, 可能与鼻息肉的形成有密切关系。     

2, 4, 6-三硝基苯磺酸诱导的大鼠结肠炎CD4T细胞增加

目的：探讨CD4⁺ T细胞在2, 4, 6-三硝基苯磺酸（TNBS）诱导的大鼠结肠炎发病过程中的变化。方法：采用免疫组化、流式细胞术和Western-blot方法观察TNBS诱导的结肠炎大鼠CD4⁺ T细胞的数目及其蛋白表达的变化。结果：TNBS诱导的结肠炎大鼠外周血和肠黏膜CD4⁺ T细胞数目增加,肠系膜淋巴结CD4⁺ T细胞蛋白表达升高。结论：TNBS诱导的大鼠结肠炎发病过程中CD4⁺ T细胞明显增加，与人类结肠炎具有相似之处，本模型为研究结肠炎治疗药物提供了新的作用靶点。     

Anomalies of the CD8+ T cell pool in haemochromatosis: HLA-A3-linked expansions of CD8+CD28− T cells

The present study consists of a phenotypic and functional characterization of peripheral blood T lymphocytes in a group of 21 patients with hereditary haemochromatosis (HH), an MHC class I-linked genetic disease resulting in iron overload, and a group of 30 healthy individuals, both HLA-phenotyped. The HH patients studied showed an increased percentage of CD8⁺ CD28⁻ T cells with a corresponding reduction in the percentage of CD8⁺ CD28⁺ T cells in peripheral blood relative to healthy blood donors. No anomalies of CD28 expression were found in the CD4⁺ subset. The presence of the HLA-A3 antigen but not age accounted for these imbalances. Thus, an apparent failure of the CD8⁺ CD28⁺ T cell population ‘to expand’, coinciding with an ‘expansion’ of CD8⁺ CD28⁻ T cells in peripheral blood of HLA-A3⁺ but not HLA-A3⁻ HH patients was observed when compared with the respective HLA-A3-matched control group. A significantly higher percentage of HLA-DR⁺ but not CD45RO⁺ cells was also found within the peripheral CD8⁺ T cell subset in HH patients relative to controls. Phytohaemagglutinin (PHA) stimulation of peripheral blood mononuclear cells (PBMC) for 5 days showed: (i) that CD8⁺ CD28⁺ T cells both in controls and HH were able to expand in vitro; (ii) that CD8⁺ CD28⁻ T cells decreased markedly after activation in controls but not in HH patients. Moreover, functional studies showed that CD8⁺ cytotoxic T lymphocytes (CTL) from HH patients exhibited a diminished cytotoxic activity (approx. two-fold) in standard ⁵¹Cr-release assays when compared with CD8⁺ CTL from healthy controls. The present results provide additional evidence for the existence of phenotypic and functional anomalies of the peripheral CD8⁺ T cell pool that may underlie the clinical heterogeneity of this iron overload disease. They are of particular relevance given the recent discovery of a novel mutated MHC class I-like gene in HH.     

Large numbers of dysfunctional CD8+ T lymphocytes bearing receptors for a single dominant CMV epitope in the very old

Longitudinal studies suggest that a set of immune parameters including high percentages of peripheral CD8⁺, CD28^–, CD57⁺ T lymphocytes, low CD4 and B cell counts, and poor T cell proliferative responses to mitogens is associated with decreased remaining longevity in the free-living very elderly (>85 years). This combination of immune parameters was also significantly associated with an inverted CD4/CD8 ratio and cytomegalovirus seropositivity. Here, using tetramer technology, we show markedly increased numbers of CD8⁺ T cells bearing receptors for one single CMV epitope in the very elderly. Moreover, the fraction of these tetramer-reactive cells secreting interferon- after specific antigenic stimulation was significantly lower in the old than in the young, as was the percentage of CD28-positive cells in this population. Therefore, we conclude that marked expansions of CMV-specific CD8⁺ T cells have occurred and that the obsession of a large fraction of the entire CD8⁺ T cell subset with one single viral epitope may contribute to the increased incidence of infectious disease in the elderly by shrinking the T cell repertoire available for responses to other antigens.     

Increase of CD57+ T cells in knee joints and adjacent bone marrow of rheumatoid arthritis (RA) patients: implication for an anti-inflammatory role

The distribution of CD57⁺ T and CD56⁺ T cells in patients with RA was examined. In control osteoarthritis patients, these cells exist as a minor population in the peripheral blood. Our data show that in patients with RA, CD57⁺ T cell levels are elevated in peripheral blood, knee joint fluid, knee synovial membrane and bone marrow (BM), compared with peripheral blood of controls. CD57⁺ T cells are especially high in knee joint fluid and joint-adjacent BM, while CD56⁺ T cells show no such increase. CD57⁺ T cells contain a major population of CD8⁺ cells and higher proportions of CD4^?8^? cells and γδ T cells than do CD57^?T cells. CD57⁺T cells in peripheral blood and joint fluid increase with the duration of disease. Erythrocyte sedimentation rate (ESR) is inversely correlated with the proportion of CD57⁺T cells in the joint fluid. Although RA frequently occurrs in patients with CD3⁺57⁺ cell leukaemia, and some CD57⁺T cells are likely to be involved in the onset of RA, we suggest that CD57⁺T cells may rather suppress inflammation of RA, and other cellular components (e. g. granulocytes) may govern the severity of the inflammation of RA. These CD57⁺ T cells are probably generated extrathymically in the adjacent BM or joint space.     

The enigma of CD57+CD28– T cell expansion—anergy or activation?

Expansion of a CD57⁺CD8^– T lymphocyte subset has been reported in HIV and human cytomegalovirus (HCMV) infection. Almost all of these T cells lack CD28 expression. While CD28^– cells are often associated with anergy, some authors believe their expansion in HIV infection precipitates immunodeficiency. We studied 15 randomly chosen patients with immune activation and observed that CD57⁺CD28^– T cell expansion may occur in various conditions and to the same degree as in HIV infection without resulting in immunodeficiency. Triple colour flow cytometry also revealed that the CD57 and CD28 antigens are coexpressed in only 3% of CD8⁺ T cells, irrespective of the underlying condition, so that almost all CD57⁺CD8⁺ cells are always CD28^–. Analysis of Fas (CD95) expression with respect to CD28 expression on CD4⁺ and CD8⁺ T cells from 10 additional patients indicated no increased commitment to apoptosis in CD28^– T cells. Semiquantitative polymerase chain reaction (PCR) comparing CD28⁺ and CD28^–CD8⁺ T cells with respect to cytokine gene expression (tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), IL-1β) in five renal transplant patients with expansion of the CD57⁺ subset detected no cytokine gene expression deficit in CD28^– T cells. A direct association of increased proportions of CD57⁺CD28^–CD8⁺ T cells with immunodeficiency/anergy is disputed.     

Costimulatory molecules in Wegener's granulomatosis (WG): lack of expression of CD28 and preferential up-regulation of its ligands B7-1 (CD80) and B7-2 (CD86) on T cells

T cells are most likely to play an important role in the pathogenesis of WG, and recently a predominant Th1 pattern of immune response has been demonstrated in granulomatous inflammation. Since the expression of costimulatory molecules has a significant impact on the cytokine profile and proliferation response of T cells, the goal of this study was to characterize the expression of costimulatory molecules (CD28, CTLA-4 (CD152), B7-1 (CD80), B7-2 (CD86)) on T cells, monocytes and B cells in WG, and to correlate the findings with clinical parameters such as disease activity, extent and therapy. WG patients (n = 24) and healthy controls (HC; n = 17) were examined for the expression of costimulatory molecules by fluorescence-activated cell sorter analysis, both in whole peripheral blood and after in vitro activation of T cells and antigen-presenting cells. Results were correlated with clinical data. The expression of CD28 on CD4⁺ and CD8⁺ cells was significantly lower in WG than in HC (CD28⁺ 81.4% in WG versus 97.9% of CD4⁺ cells (P < 0.0001); CD28⁺ 44.6% in WG versus 68.5% of CD8⁺ cells (P < 0.00001)), both in peripheral blood and after in vitro activation. A lower percentage of monocytes was B7-2⁺ in WG than in HC in peripheral blood, whereas no significant differences in the expression of B7-1 and B7-2 were observed after in vitro stimulation of monocytes and B cells. After in vitro activation a significantly higher percentage of B7-1⁺ and B7-2⁺ T cells was seen in WG. There was no significant difference in the CTLA-4 expression pattern between WG and HC. The percentage of CD28⁺ lymphocytes correlated negatively with the Disease Extent Index cumulated over the course of disease (r = ?0.46, P = 0.03), indicating a more severe manifestation in patients with lower CD28 expression. Correlations with other clinical parameters such as activity or therapy were not seen. WG patients show a lack of CD28 expression on T cells and an unusual up-regulation of its ligands B7-1 and B7-2 on T cells after in vitro activation as well as a lower expression of B7-2 on freshly isolated monocytes compared with HC. These features might promote the Th1 cytokine pattern and thereby contribute to persistently high levels of immune activation in WG.     

CD8+ T Cell Subsets in Aging

Infections are a major cause of illness and death amongst elderly people. Peripheral blood CD8⁺ T lymphocytes -which play a crucial role in host defence against viral infections-, are divided in subsets based upon the expression of several cell and activation markers. Since in senescence changes in peripheral blood CD8⁺ T lymphocyte compartment have been described, studies were performed to determine whether in aging there are variations in the peripheral blood CD8⁺CD38⁺, CD8⁺CD57⁺, CD8⁺HLA-DR⁺, CD8⁺CD45RA⁺ and CD8⁺CD45RO⁺ cell subset. A decrease in the CD8⁺CD45RA⁺ lymphocytes was observed, indicating that variations in the CD8⁺ compartment can take place with ageing.     

CD28 and CD57 define four populations with distinct phenotypic properties within human CD8+ T cells

After repeated antigen exposure, both memory and terminally differentiated cells can be generated within CD8⁺ T cells. Although, during their differentiation, activated CD8⁺ T cells may first lose CD28, and CD28⁻ cells may eventually express CD57 as a subsequent step, a population of CD28⁺CD57⁺(DP) CD8⁺ T cells can be identified in the peripheral blood. How this population is distinct from CD28⁻CD57⁻(DN) CD8⁺ T cells, and from the better characterized non-activated/early-activated CD28⁺CD57⁻ and senescent-like CD28⁻CD57⁺ CD8⁺ T cell subsets is currently unknown. Here, RNA expression of the four CD8⁺ T cell subsets isolated from human PBMCs was analyzed using microarrays. DN cells were more similar to “early” highly differentiated cells, with decreased TNF and IFN-γ production, impaired DNA damage response and apoptosis. Conversely, increased apoptosis and expression of cytokines, co-inhibitory, and chemokine receptors were found in DP cells. Higher levels of DP CD8⁺ T cells were observed 7 days after Hepatitis B vaccination, and decreased levels of DP cells were found in rheumatoid arthritis patients. More DP and DN CD8⁺ T cells were present in the bone marrow, in comparison with PBMCs. In summary, our results indicate that DP and DN cells are distinct CD8⁺ T cell subsets displaying defined properties.     

A novel peripheral CD4+CD8+ T cell population: Inheritance of CD8α expression on CD4+ T cells

In this study we show the inheritance of a CD4⁺CD8⁺ peripheral T cell population in the H.B15 chicken strain. A large proportion of αβ T cells in peripheral blood (20–40%), spleen (10–20%) and intestinal epithelium (5–10%) co-express CD4 and CD8α, but not CD8β. CD4⁺ CD8αα cells are functionally normal T cells, since they proliferate in response to mitogens and signals delivered via the αβT cell receptor as well as via the CD28 co-receptor. These cells induce in vivo a graft versus host-reaction, providing further evidence for their function as CD4⁺ T cells. The CD4⁺CD8αα T cell population was found in 75% of the first progeny and in 100% of further progenies, demonstrating that co-expression of CD4 and CD8 on peripheral T cells is an inherited phenomenon. In addition, cross-breeding data suggest a dominant Mendelian form of inheritance. The hereditary expression of CD8α on peripheral CD4⁺ T cells in chicken provides a unique model in which to study the regulation of CD4 and CD8 expression.     

Demonstration of identical expanded clones within both CD8+ CD28+ and CD8+ CD28− T cell subsets in HIV type 1-infected individuals

In HIV-1-infected individuals, the CD8⁺ CD28⁻ T cell subset is considerably expanded and is frequently the largest subset of T cells found in peripheral blood. It has been assumed, but not proven, that CD8⁺ CD28⁻ T cells derive from CD8⁺ CD28⁺ T cells in vivo. To further study the ontogeny of CD8⁺ CD28⁻ T cells, we have performed analyses of the complementarity determining region 3 (CDR3) of the TCRB of CD8⁺ CD28⁺ and CD8⁺ CD28⁻ T cells from the peripheral blood of HIV-1-infected individuals. When cells from the same individual were compared, expanded peaks in CDR3 length analysis within a given BV family were frequently observed at the same location in both CD8⁺ subsets ( p < 0.001). Sequencing of cDNA corresponding to dominant peaks revealed the presence of identical expanded CD8⁺ T cell clones within both the CD28⁺ and CD28⁻ subsets on eight of nine attempts. Our results show that CD8⁺ CD28⁺ and CD8⁺ CD28⁻ T cells are phenotypic variants of the same lineage, most likely evolving from CD8⁺ CD28⁺ to end-stage CD8⁺ CD28⁻ T cells.