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     

弗氏佐剂与氢氧化铝佐剂对诱导小鼠获得性免疫应答作用的比较

为研究、比较不同佐剂对诱导小鼠产生获得性免疫应答的不同作用,以卵清白蛋白(OVA)为抗原,分别混合完全弗氏佐剂(CFA)或Al(OH)3佐剂,对C57BL/6小鼠进行常规免疫,采用流式细胞技术对细胞内细胞因子IFN-γ和IL-4进行检测;ELISA方法对特异性抗OVA抗体滴度及抗体亚型进行了检测。结果显示在免疫后CFA组产生以IFN-γ为主的细胞因子而Al(OH)3组产生以IL-4为主的细胞因子;两组中均产生特异性抗OVA IgG抗体,但CFA组以IgG2a亚型为主,而Al(OH)3组则以IgG1亚型为主,不产生IgG2a亚型抗体。实验表明,经CFA加抗原免疫后机体产生的免疫应答以Th1型细胞免疫为主,抗体类型为IgG2a;而Al(OH)3佐剂则诱导机体产生Th2型细胞免疫应答,抗体类型为IgG1。     

自身反应性T细胞系免疫生物学活性研究

目的：探讨自身反应性T细胞系过继转移对受体小鼠免疫应答的影响，为在BXSB小鼠探索T细胞疫苗的可行性奠定基础。方法：应用体外扩增的自身反应性T细胞过继转移给同系小鼠体内：^3H－TdR掺入法检测细胞增殖，ELISA检测抗体细胞因子水平。结果：将自身反应性T细胞系过继转移到年轻未发病的同系小鼠体内可以诱导血清中自身抗体和外来抗原反应性抗体以及细胞因子IFN－γ水平显著升高，可以降低受体小鼠对外来抗原（OVA）体液免疫应答能力。结论：自身反应性T细胞的过继转移可以影响机体自免疫应答和对外来抗原的体液免疫应答能力。     

抗OX-40和抗IL-4抗体体外促进Th1细胞的初步探讨

将灭活的自身黑色素瘤细胞MEL 72与BCG混合免疫MEL 72黑色素瘤病人 ,10d后手术方法获取局部引流淋巴结 ,制成淋巴细胞悬液 ,以抗CD3抗体体外培养 ,ELISA方法检测细胞上清中肿瘤特异性Th1、Th2细胞因子的表达。进一步用抗OX 40抗体与CD4+ T淋巴细胞表面OX 40作用或用抗IL 4抗体阻断IL 4的分泌 ,观察肿瘤特异性免疫反应能否向Th1方向转换。结果表明 :用灭活的自体肿瘤细胞免疫机体能诱导出该肿瘤细胞特异性的Th1和Th2。应用抗OX 40抗体与抗IL 4抗体作用后 ,Th1细胞因子分泌水平增加 (TNF β和IFN γ ) ,结果提示 ,应用抗OX 40和抗IL 4抗体可诱导肿瘤特异性Th1反应。     

黄芪多糖佐剂对脾脏NK细胞及NKT细胞的作用

目的观察脾脏NK细胞及NKT细胞在黄芪多糖（APS）发挥免疫佐剂功能的作用。方法使用鸡卵白蛋白（OVA）与APS经皮下免疫C57BL/6小鼠3次。末次免疫7～14d,取血清,使用ELISA观察OVA特异性抗体的含量;分离脾脏淋巴细胞,使用流式细胞仪检测NK和NKT细胞的百分比含量;使用PMA和Ionimycin刺激淋巴细胞,使用细胞内细胞因子染色的方法检测IFN-γ和IL-4的分泌情况。结果 APS组小鼠OVA特异性抗体含量明显高于OVA组小鼠（P〈0.05）,但小鼠脾脏内NK和NKT细胞的百分比含量与OVA组和正常小鼠差别不大（P〉0.05）。经PI刺激以后,APS组小鼠脾脏内NK细胞中IL-4＋细胞明显高于OVA组和正常组（P〈0.05）,且IFN-γ＋细胞的比例明显下降（P〈0.05）;虽然经过PI刺激以后,APS组小鼠脾脏内NKT细胞中IL-4＋细胞升高不明显,但是IFN-γ＋细胞的比例明显下降（P〈0.05）。结论 APS可以通过调节NK和NKT细胞的功能来促进体液免疫应答。     

抗原85B-6kDa早期分泌靶抗原(Ag85B-ESAT-6)亚单位疫苗黏膜免疫对结核分枝杆菌的免疫应答

目的 评价抗原85B-6kDa早期分泌靶抗原融合蛋白(AE)亚单位疫苗经黏膜免疫小鼠诱导的免疫应答及其对结核分枝杆菌(MTB)感染的保护力。方法 分别用AE、 AE联合环二腺苷酸(c-di-AMP)亚单位疫苗经鼻黏膜免疫小鼠,ELISA检测抗体水平以及1型辅助T(Th1)细胞的细胞因子γ干扰素(IFN-γ)、白细胞介素2(IL-2)和Th2细胞的IL-10分泌水平,实时荧光定量PCR检测IFN-γ、 IL-2、 IL-10和肿瘤坏死因子α(TNF-α)的mRNA水平。MTB静脉感染免疫小鼠,ELISA检测感染小鼠血清抗体及脾细胞细胞因子分泌水平,HE染色分析肺病理改变,平板法计数菌落形成单位(CFU)检测脾和肺荷菌数。结果 AE和AE联合c-di-AMP经鼻黏膜免疫可诱导小鼠产生高水平的特异性抗体,促进脾细胞增殖、脾和肺脏Th1/Th2型细胞因子和TNF-α转录增加、脾Th1/Th2型细胞因子分泌增加。小鼠MTB感染后,与对照组相比,AE和AE联合c-di-AMP免疫小鼠特异性抗体水平仍升高,Th1/Th2型细胞免疫应答增强,肺组织呈炎症反应,肺、脾荷菌数显著降低,且AE联合c-di-...     

口服免疫Ag85A脂质体DNA诱导T细胞亚群应答效应研究

目的:拟采用构建的可在真核细胞内表达Ag85A基因的质粒,以阳离子脂质体为运载体,制成DNA疫苗,经口途径投予小鼠,以观察Ag85A脂质体DNA疫苗诱导免疫应答效应,为口服DNA疫苗的临床应用提供理论和实验依据.方法:ELISA方法检测Ag85A特异性抗体产生水平及血清Th1型细胞因子IFN-γ及Th2型细胞因子IL-4的分泌水平,ELISPOT技术检测口服DNA疫苗后小鼠可分泌IFN-γ和IL-4脾淋巴细胞数量.流式细胞术观察口服DNA疫苗后小鼠脾淋巴细胞CD4+T细胞及CD8+T细胞亚群的变化,从而判断口服DNA疫苗的免疫效果及脂质体是否有免疫增强作用.结果:口服自制Ag85ADNA疫苗可见血清中抗Ag85A特异性抗体的产生;下调了脾CD4+T细胞和CD8+T细胞亚群的数量;分泌IFN-γ的Th1型细胞比率和血清中的IFN-γ水平下降,而分泌IL-4的Th2型细胞比率和血清中的IL-4水平升高.口服DNA疫苗组诱导Ag85A特异性抗体产生,脂质体具有免疫佐剂作用.结论:应用阳离子脂质体为运载体,重组Ag85A DNA疫苗口服免疫C57BL/6小鼠,诱导了CD4+及CD8+T细胞亚群的下调及IFN-γ的表达减少,而玎IL-4的分泌呈增高趋势;疫苗可诱导Ag85A特异性抗体的分泌,产生了明显的体液应答.     

SARS康复者特异性细胞免疫和免疫记忆T细胞功能的研究

目的　观察完全康复期严重急性呼吸综合征 (SARS)患者外周血针对SARS冠状病毒(SARS CoV)S抗原多肽的特异性细胞免疫反应。方法　分离外周血单个核细胞 (PBMC) ,经混合S抗原多肽刺激后 ,采用酶联免疫吸附试验 (ELISA)和酶联免疫斑点试验 (ELISPOT)检测 ,分析完全康复期SARS患者抗原特异性T淋巴细胞应答反应。结果　当SARS CoV的S抗原混合多肽刺激后 ,只有SARS康复期患者的PBMC分泌大量的IFN γ。同时 ,IFN γ产生细胞的阳性率也显著高于健康对照组 (P <0 .0 5)。此外 ,当用多克隆刺激剂 (PMA +ionomycin)刺激后 ,正常人和康复期SARS患者的PBMC产生等量的IFN γ及IFN γ产生细胞 ,两者经统计学处理 ,差异无统计学意义 (P >0 .0 5)。结论SRAS CoV不仅能诱导中和抗体的产生 ,而且还可诱导抗原特异性细胞免疫应答反应 ,并可在体内长期维持免疫记忆功能。     

RANTES和MIP-1α基因对HIV-1核酸疫苗诱导免疫应答的影响

目的　研究RANTES(活化T细胞调节的、正常T细胞表达和分泌的分子 )、MIP 1α(巨噬细胞炎症蛋白 1α)基因对HIV 1核酸疫苗诱导免疫应答的影响 ,以探求HIV 1核酸疫苗的新策略。方法　pCI neoGAG联合RANTES、MIP 1α基因或者pCI neoGAG单独免疫BALB c小鼠 ,采用ELISA检测免疫小鼠的特异性抗体和IFN γ水平 ,以MTT比色法检测免疫小鼠脾淋巴细胞的增殖 ,用乳酸脱氢酶 (LDH)试验检测小鼠特异性细胞毒性T淋巴细胞 (CTL)的应答。结果　与pCI neoGAG免疫组比较 ,pCI neoGAG联合RANTES、MIP基因免疫组小鼠血清的抗HIV 1p2 4抗体滴度升高 ,IFN γ升高 ,小鼠的脾淋巴细胞增殖实验刺激指数 (SI)以及特异性CTL活性均高 ,差异都有显著性 (P <0 .0 1)。结论　RANTES、MIP 1α基因联合HIV 1核酸疫苗免疫小鼠 ,可能增强特异性TH1细胞和CTL反应 ,RANTES、MIP 1α基因对体液免疫有加强作用。因此 ,RANTES、MIP 1α基因对于HIV 1核酸疫苗是具有较好应用前景的免疫佐剂。     

树突状细胞免疫受体是治疗哮喘的新靶点

目的探讨树突状细胞(dendritic cells,DCs)表面表达的Ⅱ型膜蛋白——树突状细胞免疫受体(dendritic cells immunal receptor,Dcir)在哮喘中的作用。方法使用卵清蛋白(OV-albumin,OVA)同时诱导Dcir-/-小鼠和野生型小鼠发生哮喘,通过对比2种小鼠模型气管中嗜酸性粒细胞的浸润、血清中抗体的产生、肺的病理表现和淋巴结细胞中Th2细胞因子的释放,来阐明Dcir在哮喘中的作用。为了证明Dcir在哮喘中的作用是通过DC细胞介导的免疫应答而非直接影响T细胞免疫实现的,我们使用含有OVA特异性T细胞受体的转基因小鼠DO11.10与DO11.10×Dcir-/-小鼠进行对比;并通过体外实验验证了Dcir缺失对na觙ve T细胞产生Th2细胞因子以及conventional DC(cDC)和plasmacytoid DC(pDC)增殖速度的影响。结果在OVA诱导的小鼠哮喘模型中,Dcir-/-小鼠气管内嗜酸性粒细胞的浸润较野生型小鼠亢进;Dcir-/-小鼠血清中抗体的产生较野生型小鼠增加;肺部的病理结果显示,相对于野生型小鼠,Dcir-/-小鼠的肺部有更多的浸润细胞、黏液分泌和更为严重的气管纤维化;Dcir-/-小鼠淋巴结细胞释放的Th2细胞因子较野生型小鼠升高。在诱导DO11.10和DO11.10×Dcir-/-小鼠的哮喘模型时,两者的各项指标之间没有明显差异,说明Dcir参与的哮喘恶性化是通过DC细胞介导的免疫应答实现的。在DC细胞与抗原和na觙ve T细胞共同培养的过程中,Dcir-/-小鼠来源的DC细胞诱导了更多的Th2细胞产生。在将骨髓细胞(BMC)诱导为cDC和pDC的体外实验中,Dcir-/-小鼠对GM-CSF的信号和Flt-3 ligand的信号感受性均增强,从而诱导出了比野生型小鼠更多的cDC和pDC。结论 Dcir-/-小鼠增加了OVA诱导的哮喘模型的恶性度。Dcir-/-小鼠对哮喘模型恶性度的增加是由过度增殖的DC细胞介导的免疫应答而并非直接影响T细胞的感受性。在哮喘模型中Dcir通过抑制树突状细胞对GM-CSF和对Flt-3 ligand的信号感受性,从而降低哮喘模型中DC细胞的分化增殖能力和Th2免疫应答。Dcir的配体有可能作为一种治疗哮喘的新药物获得应用。     

T cell-mediated oral tolerance is intact in germ-free mice

Commensal enteric bacteria stimulate innate immune cells and increase numbers of lamina propria and mesenteric lymph node (MLN) T and B lymphocytes. However, the influence of luminal bacteria on acquired immune function is not understood fully. We investigated the effects of intestinal bacterial colonization on T cell tolerogenic responses to oral antigen compared to systemic immunization. Lymphocytes specific for ovalbumin-T cell receptor (OVA-TCR Tg(+)) were transplanted into germ-free (GF) or specific pathogen-free (SPF) BALB/c mice. Recipient mice were fed OVA or immunized subcutaneously with OVA peptide (323-339) in complete Freund's adjuvant (CFA). Although the efficiency of transfer was less in GF recipients, similar proportions of cells from draining peripheral lymph node (LN) or MLN were proliferating 3-4 days later in vivo in GF and SPF mice. In separate experiments, mice were fed tolerogenic doses of OVA and then challenged with an immunogenic dose of OVA 4 days later. Ten days after immunization, lymphocytes were restimulated with OVA in vitro to assess antigen-specific proliferative responses. At both high and low doses of OVA, cells from both SPF and GF mice fed OVA prior to immunization had decreased proliferation compared to cells from control SPF or GF mice. In addition, secretion of interferon (IFN)-gamma and interleukin (IL)-10 by OVA-TCR Tg(+) lymphocytes was reduced in both SPF and GF mice fed OVA compared to control SPF or GF mice. Unlike previous reports indicating defective humoral responses to oral antigen in GF mice, our results indicate that commensal enteric bacteria do not enhance the induction of acquired, antigen-specific T cell tolerance to oral OVA.     

Balance of Th1/Th2 Cytokines Associated with the Preventive Effect of Incomplete Freund's Adjuvant on the Development of Adjuvant Arthritis in LEW Rats

Complete Freund's adjuvant (CFA) could induce adjuvant arthritis (AA) in LEW rats and incomplete Freund's adjuvant (IFA) could induce oil induced arthritis (OIA) in DA but not in LEW rats. Lymph node cells (LNCs) from these AA and OIA rats showed increased mRNA expression of IFN-gamma, IL-2 and TNF-alpha but not IL-4. LNCs from IFA immunized LEW rats showed increased expression of IL-4, reduced expression of IFN-gamma and TNF-alpha and no IL-2, in contrast to IFA immunized DA rats. The pretreatment of IFA before CFA challenge could completely prevent AA in LEW rats and their LNCs showed increased expression of IL-4 and IFN-gamma but not IL-2 and TNF-alpha. In F1 (LEW x DA) rats, IFA could not induce OIA but the pretreatment of IFA before CFA challenge could induce very mild AA with 80% incidence, LNCs showing an elevated expression of all the above cytokines. These findings suggest that increased Th1 cytokine expression is associated with disease development and that increased IL-4 expression or the balance of Th2 over Th1 cytokine expression plays an important regulatory role in disease development.     

Effect of rolipram, a phosphodiesterase IV inhibitor, on allergic footpad swelling using various adjuvants in mice

We studied the effect of rolipram, a phosphodiesterase (PDE) IV inhibitor, on allergic footpad swelling in mice. For this study, varying adjuvants including complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and Imject Alum (Alum) were used because the extent of antigen-specifically induced T helper type 1 (Th1) and Th2 responses had been shown to depend on adjuvants used. To induce allergic footpad swelling, we immunized mice with ovalbumin (OVA) emulsified in either CFA or IFA, dissolved in Alum or in phosphate-buffered saline (PBS) as a control (day 0), followed by subcutaneous injection of the antigen into footpads on day 21. Rolipram was given orally to the animals daily from days 0-20. Results showed that treatment with rolipram was followed by an increase in early swelling at 0.5 h and a decrease in late swelling at 6 and 24 h in the CFA group. In the IFA group, rolipram significantly enhanced swelling at, but not after, 30 min. In the Alum and the PBS groups, the PDE inhibitor failed to affect the OVA-specific footpad reaction at all times examined. Treatment of the CFA and IFA groups with rolipram significantly inhibited the production of the Th1 antibody anti-OVA immunoglobulin G2a (IgG2a), and the drug enhanced Th2 cell-dependent anti-OVA IgE production. In both groups, rolipram also enhanced the secretion of Th2 cytokines including interleukin-4 (IL-4) and IL-10. These findings suggest that rolipram may facilitate early allergic footpad swelling mediated by Th2 immune responses, while the late phase of swelling associated with Th1 responses may be attenuated by the PDE IV inhibitor.     

Antigen inhibition of collagen-induced arthritis is associated with up-regulation of IL-4 mRNA and induction of Ox40 on T cells in draining lymph nodes

The addition of a foreign antigen to an inoculum completely inhibits the development of collagen-induced arthritis (CIA). However, the mechanism of this phenomenon, antigen -inhibition, is incompletely understood. Previous studies have demonstrated that the inhibition of arthritis is not mediated through suppression of the antibody response to cartilage antigens. In this paper we investigated cytokine mRNA levels in lymph nodes cells recovered 3, 7 or 16 days from animals immunized with either collagen II in IFA or OVA + collagen II in IFA. At day 7, but not at other time-points, IL-4 mRNA was up-regulated in the lymph nodes of OVA-inhibited non-arthritic animals compared to control animals which all developed arthritis. No significant differences between the two groups could be detected when expression of IFN-gamma, IL-2, TNF-alpha, IL-1beta or IL-10 mRNA was analysed. Flow cytometry analysis of draining lymph node cells demonstrated that the T cell marker Ox40 was up-regulated in the OVA-inhibited group. Our results indicate that the complete inhibition of CIA caused by addition of OVA to the collagen II inoculum is due to the presence of a TH2 environment resulting from an increased production of IL-4 mRNA and a parallel increase in Ox40+ T cells.     

Activation of natural killer T cells by α-carba-GalCer (RCAI-56), a novel synthetic glycolipid ligand, suppresses murine collagen-induced arthritis

Alpha-carba-GalCer (RCAI-56), a novel synthetic analogue of α-galactosylceramide (α-GalCer), stimulates invariant natural killer T (NK T) cells to produce interferon (IFN)-γ. IFN-γ exhibits immunoregulatory properties in autoimmune diseases by suppressing T helper (Th)-17 cell differentiation and inducing regulatory T cells and apoptosis of autoreactive T cells. Here, we investigated the protective effects of α-carba-GalCer on collagen-induced arthritis (CIA) in mice. First, we confirmed that α-carba-GalCer selectively induced IFN-γ in CIA-susceptible DBA/1 mice in vivo. Then, DBA/1 mice were immunized with bovine type II collagen (CII) and α-carba-GalCer. The incidence and clinical score of CIA were significantly lower in α-carba-GalCer-treated mice. Anti-IFN-γ antibodies abolished the beneficial effects of α-carba-GalCer, suggesting that α-carba-GalCer ameliorated CIA in an IFN-γ-dependent manner. Treatment with α-carba-GalCer reduced anti-CII antibody production [immunoglobulin (Ig)G and IgG2a] and CII-reactive interleukin (IL)-17 production by draining lymph node (DLN) cells, did not induce apoptosis or regulatory T cells, and significantly increased the ratio of the percentage of IFN-γ-producing T cells to IL-17-producing T cells (Th1/Th17 ratio). Moreover, the gene expression levels of IL-6 and IL-23p19, Th17-related cytokines, were reduced significantly in mice treated with α-carba-GalCer. In addition, we observed higher IFN-γ production by NK T cells in α-carba-GalCer-treated mice in the initial phase of CIA. These findings indicate that α-carba-GalCer polarizes the T cell response toward Th1 and suppresses Th17 differentiation or activation, suggesting that α-carba-GalCer, a novel NK T cell ligand, can potentially provide protection against Th17-mediated autoimmune arthritis by enhancing the Th1 response.     

Early simultaneous production of intranodal CD4 Th2 effectors and recirculating rapidly responding central‐memory‐like CD4 T cells

This study characterizes the diversity of CD4 Th cells produced during a Th2 response in vivo. Kinetics of effector and memory cell differentiation by mouse OVA‐specific CD4 T cells was followed during primary responses to alum‐precipitated OVA. The complexity of the CD4 T response was assessed in nodes draining and distant from the site of immunization for phenotype, location and function. By 3 days IL‐4‐producing effector CD4 T cells developed in the draining node and a proportion of the responding cells had migrated to B‐cell follicles, while yet others had left the draining node. Some of these early migrant cells were recirculating cells with a central memory phenotype. These had divided four or more times in the draining node before migrating to distant nodes not exposed to antigen. We questioned the responsiveness of these early central‐memory‐like cells on secondary antigen challenge at sites distant to the primary immunization. They re‐entered cell cycle and migrated to B‐cell follicles, much more rapidly than naïve CD4 T cells and could still be induced to produce IL‐4. Their production and survival were independent of the starting frequency of antigen‐specific CD4 T cells. Thus intranodal effector cells and recirculating, rapidly responding central‐memory‐like cells emerged simultaneously from the third day of a primary Th2 response.     

A limited role of IL-1 in immune-enhancement by adjuvants.

Generation of interleukin-1 (IL-1) in the draining lymph nodes after injection of complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA) and alum was studied in line with IL-1 mRNA expression (cytoplasmic slot blot analysis) and IL-1 beta antigen detection (ELISA and immunohistochemistry) in rabbits. The expression of IL-1 beta mRNA was marked from 6 to 96 hr, with a maximum at around 24 hr post-injection of CFA, while injection of the other two adjuvants elicited only a moderate or negligible response. On the other hand, IL-1 alpha mRNA expression was almost negligible during the entire 8-week observation period after injection of the above three adjuvants. Generation of IL-1 beta antigen in the draining lymph nodes after CFA injection paralleled the expression of IL-1 beta mRNA. Immunohistochemistry revealed that cells containing IL-1 beta resided in the medullary sinuses, marginal sinuses and para-cortical area, but not in the follicles. Despite marked generation of IL-1 beta in CFA-treated draining lymph nodes, the primary antibody response (IgG) to ovalbumin differed only slightly between the three animal groups that were immunized with the antigen incorporated in CFA, IFA and alum. Further, rIL-1 beta did not significantly enhance the immune response when it was entrapped together with the antigen in IFA and alum. IL-1 beta enhanced the immune response only when it was injected with antigen without adjuvant. Thus, IL-1 seemed to play only a limited role, if any, in the augmentation of the primary immune response by the above-mentioned adjuvants.     

抗原特异性初始CD4+T细胞的体内分化及特性

为了探讨抗原特异性CD4+T细胞在体内的分裂、表型、Th1细胞因子的产生和组织器官的分布。将CFSE标记的抗原特异性初始CD4+T细胞静脉被动输给小鼠后,进行免疫,3d后处死小鼠取其脾脏、淋巴结和肺组织,分离单个核细胞,利用流式细胞计数仪在单个细胞水平上,观察细胞的分裂、表型、Th1细胞因子的产生和组织分布。结果显示在没有抗原刺激的情况下,未见初始CD4+T细胞分裂,其主要分布于淋巴结和脾脏。当受到抗原刺激后,CD4+T细胞分裂1～5次,主要分布于脾脏和肺组织,CD25的表达增加,CD62L的表达随着细胞分裂次数的增加而减少。IL-12促进CD25的表达和细胞的分裂。促进Th1细胞的分化和IFN-γ的表达。研究的结果提示,在体内,当CD4+T细胞活化后,主要分布于脾和非淋巴组织发挥其免疫效应。     

Selective suppression of antigen-specific Th2 cells by continuous micro-dose oral tolerance

The effect on antigen (Ag)-specific Th2 response as well as IgE production of continuous oral administration of micro-doses of Ag was investigated. Transgenic (Tg) mice carrying the α β-T cell receptor (TCR) genes specific for ovalbumin (OVA) peptide fragment 323 – 339 were continuously fed with micro-doses of OVA (100 μg/day) for 14 days. Mice were first immunized by OVA in alum and pertussis toxin 7 days before the oral feeding and given a second immunization 1 day after the oral treatment. This feeding regimen tolerized Th2 but not Th1 responses as shown by decrease of Ag-driven cell proliferation and cytokine secretion of IL- 4 but not of IL-2 or IFN-γ as well as by the absence of Ag-specific antibody production of IgE and IgG1, but not of IgG2a or total IgG. Numbers of clonotype-specific TCR-high CD4-positive T cells in peripheral lymphoid tissues markedly decreased in the orally treated group but not in the control group. However, total numbers of CD4-positive T cells in thymus, spleen and lymph nodes were not affected by the oral treatment, indicating that tolerance induction in Th2 cells was mainly due to the down-regulation of TCR and not clonal deletion. The population of antigen-presenting cells expressing B7-2 (CD86) Ag on the surface was decreased in the spleen of the mice which underwent the feeding regimen. The present results suggest that Ag-specific low responsiveness in Th2 cells, which resulted in suppres sion of the Ag-specific IgE production, can be achieved by continuous feeding with microdoses of Ag.