Activation and proliferation of T lymphocyte subpopulations in patients with brucellosis

BACKGROUND: T-cell proliferation is a standard method to evaluate cellular immune responses against intracellular infectious agents. The present study was undertaken to look for expression of an early activation marker (CD69) and proliferation using a nonradioactive method to evaluate cellular immune response against a salt-extractable antigen from Brucella melitensis 16M (RCM-BM) in patients suffering from brucellosis. METHODS: Expression of CD69 on membrane of CD4+ and CD8+ T-cells was determined by flow cytometry. Lymphoproliferation was determined by tritiated thymidine and 5-bromo-2'-deoxyuridine (BrdU) incorporation using liquid scintillation counter or flow cytometry, respectively, to evaluate DNA synthesis. RESULTS: Thirty healthy donors and 24 patients suffering from brucellosis were included in this study. In all cases, incubation with mitogen induced expression of CD69 and proliferation of both CD4+ and CD8+ T-cells. In contrast, only brucellosis patients responded with expression of CD69 and proliferation against RCM-BM antigen from Brucella melitensis (p < 0.001). CONCLUSIONS: Methods used in this study were useful to evaluate immune response against specific antigen or polyclonal stimulation. CD4+ and CD8+ T cells from patients became equally activated and proliferated in response to RCM-BM antigen. Our data suggest that both T-cell subpopulations play an important role in immune response against Brucella.     

Suppression of T-cell proliferative response in Plasmodium falciparum malaria patients--preliminary results

We report suppression of T-cell proliferative responses to P.falciparum specific antigen and mitogens. T-cells derived from malaria patients were co-cultured with P.falciparum antigen or mitogens and the T-cell activity determined by radioactive thymidine incorporation assay system. We found inhibition of T-cell responses to P.falciparum antigen in 13 out of 24 malaria patients studied. The suppression ranged from 4%-60%. Results of mitogenic responses of T-cells showed a wide variation. Suppression of concanavalin A (Con A) responses ranged from 48%-64% (4 out of 10 patients) while phytohaemagglutinin (PHA) responses varied from 4%-60% (8 out of 10 patients) and those of purified protein derivative (PPD) antigen from 12%-44% (3 out of 6 patients). Together, these preliminary results show a marked impairment in T-cell responses to parasite antigen and mitogens in P. falciparum infected patients.     

Response of IFN-gamma and IgG to ESAT-6 and 38 kDa recombinant proteins and their peptides from Mycobacterium tuberculosis in tuberculosis patients and asymptomatic household contacts may indicate possible early-stage infection in the latter

BACKGROUND: The ESAT-6 antigen from Mycobacterium tuberculosis evokes a protective immune response in murine models and is widely recognized by tuberculosis patients (TB) and healthy household contacts (HHC). However, little is known about human immune response to this antigen in populations from areas of high endemicity. This study aimed to determine the capacity of T-cells from a group of TB patients and HHC for cell proliferation and production of cytokines type Th1 or Th2 (IL-4, IL-10, and IFN-gamma) and to identify total IgG reactivity to the recombinant protein rESAT-6 and five overlapping synthetic peptides as well as to r38 kDa and two peptides. METHODS: T-cells from nine TB patients and nine HHC were stimulated with rESAT-6 and five overlapping synthetic peptides, previously selected from a set of 21 peptides and each of 16 amino acids in length (P1, P4, P6, P8, and P20). Similar experiments were carried out with r38 kDa and two peptides of 20 amino acids in length (38G and 38K). Cytokines in supernatants and total IgG from serum were determined by ELISA. RESULTS: Stimulation index (SI) was highest in HHC to rESAT-6 and peptides P1, P8, and P20. Differences in response to 38 kDa and 38G peptide between TB patients and HHC were not demonstrated. Cytokines from T-cell cultures were tested with a resulting SI=3.0. IFN-gamma was produced predominantly in HHC to rESAT-6, P8, and P20, while in TB patients production of IL-10 was detected in relation to r38 kDa. IL-4 was detected in minimal amounts in both groups. IgG from TB patients was predominantly recognized in connection with rESAT-6 and the P4 peptide, with an important response against r38 kDa detected in HHC. CONCLUSIONS: ESAT-6 recognition by HHC could indicate that these responses represent possible early-stage infections.     

Hepatitis B surface antigen-specific CD8-positive T cell clones. Antigen specificity and interleukin-2 production.

Clones of hepatitis B surface antigen-reactive CD8+ and CD4+ T cells were obtained from peripheral blood mononuclear cells (PBM) of a hepatitis B immunized individual whose PBM proliferated when cultured with hepatitis B surface antigen (HBsAg). Lymphocytes were activated by culturing for 2 weeks with HBsAg and high concentrations of interleukin-2 (IL-2), then cloned in the presence of irradiated HBsAg-activated PBM and autologous Epstein-Barr virus (EBV)-transformed B cells, together with antigen and IL-2. All clones examined proliferated in an antigen-specific manner. Of 7 clones examined by flow cytometry, 4 were CD4+, CD8-; and 3 were CD4-, CD8+. Several clones produced IL-2 activity after stimulation with hepatitis B surface antigen. Since development of CD8+ T-cell clones specific for soluble antigens is difficult, the high frequency with which CD8+ cells were cloned in these experiments suggests that the cloning strategy employed might have general use for development of CD8+ clones. Availability of hepatitis B virus specific T cell clones of different phenotypes may help elucidate mechanisms of immunotolerance in hepatitis B infection.

     

IL-2受体与CD4阳性细胞在良恶性淋巴增生病变组织中的数量、分布及意义

对50例良恶性淋巴增生病变组织及其短期培养细胞进行免疫表型研究,重点观察白细胞介素-2受体及CD4阳性细胞的数量、分布及其变化情况。结果发现:①多种良恶性淋巴增生病变组织中均有不等量的白细胞介素-2受体及CD4抗原的表达;②部分肿瘤性及非肿瘤性CD4阳性细胞、B细胞及R-S细胞上亦有白细胞介素-2受体存在,而在CD1或CD8阳性细胞上则未见;③正常人淋巴细胞经PHA刺激并发生增殖后所形成的细胞克隆亦有较多的白细胞介素-2受体和CD4抗原表达。提示:尽管白细胞介素-2受体不是某一类细胞的特异性抗原标记,但与细胞的免疫表型有一定关系。文内还结合文献扼要讨论了白细胞介素-2、白细胞介素-2受体及辅助性T淋巴细胞在淋巴瘤发病中的作用和意义。     

肺癌患者淋巴细胞亚群CD3~+CD4~+和CD3~+CD8~+及其表面受体NKG2D、NKG2A的表达及相关分析

目的观察非小细胞肺癌患者外周血淋巴细胞亚群CD3+CD4+和CD3+CD8+及其表面受体NKG2D、NKG2A的表达水平,分析其表达水平与肺癌肿瘤免疫逃逸的相关性。方法采集34例肺癌患者外周血,按照TNM分期分为早期组(Ⅰ期和Ⅱ期)和晚期组(Ⅲ期、Ⅳ期),用流式细胞仪检测外周血,分析淋巴细胞亚群CD3+CD4+和CD3+CD8+及其表面受体NKG2D、NKG2A的表达及相关性。结果晚期组CD3+CD4+的表达水平较早期组降低,而CD3+CD8+的表达水平升高(P<0.05);晚期组肺癌患者CD3+CD4+NKG2D表达率(6.69±3.58%)高于早期组的表达率(2.38±1.13%),CD3+CD4+NKG2A表达率(2.28±0.88%)低于早期组的表达率(5.46±2.09%)(P<0.01);晚期组肺癌患者CD3+CD8+NKG2D表达率(90.57±2.49%)低于早期组的表达率(93.51±3.19%),CD3+CD8+NKG2A表达率(12.32±4.24%)高于早期组的表达率(7.15±3.30%)(P<0.01)。结论非小细胞肺癌病理分期间淋巴细胞亚群CD3+CD4+和CD3+CD8+表达水平的改变,NKG2D、NKG2A在CD3+CD4+和CD3+CD8+表面的表达失衡,可能是肿瘤免疫逃逸的机制之一。     

Preliminary study on HBsAg-specific T cell clones

Clones of HBsAg-reactive CD8+ and CD4+ T cells were obtained from PBM of a hepatitis B immunized individual whose PBM proliferated when cultured with HBsAg. Lymphocytes were activated by culturing for 2 weeks with HBsAg and high concentrations of IL-2, then cloned in the presence of irradiated HBsAg-activated PBM and autologous EBV-transformed B cells, together with antigen and IL-2. All clones examined exhibited proliferation in an antigen-specific manner. Of 7 clones examined by flow cytometry, 4 were CD4+, CD8-; and 3 were CD4-, CD8+. Several clones produced IL-2 activity after stimulation with HBsAg. Since development of CD8+ T-cell clones specific for soluble antigens has been difficult, the high frequency with which CD8+ cells were cloned in these experiments suggests that the cloning strategy employed may have general use for development of CD8+ clones, Availability of HBV-specific T cell clones of different phenotype may help elucidate the mechanisms of immunotolerance in HB infection.     

抗原刺激后小鼠CD4~＋ T细胞IL-10受体Ⅰ表达情况的研究

目的：检测抗原刺激后小鼠CD4＋T淋巴细胞表面白细胞介素10受体Ⅰ（interleutin-10 receptorⅠ,IL-10R1）表达变化情况。方法：取C57BL/6小鼠脾细胞,溶血后采用尼龙毛去除B淋巴细胞后,流式细胞仪无菌分选CD4＋T淋巴细胞,利用anti-CD3和anti-CD28多克隆抗体刺激分选后细胞,分别在第0、12、24、48和72 h应用流式细胞术检测IL-10R1表达情况。结果：0 h时小鼠CD4＋T淋巴细胞不表达IL-10R1,随着刺激时间的延长IL-10R1表达增加,24 h时达到最高,随后表达水平开始下降,72 h时表达情况与0 h基本相同。结论：白细胞介素10（interleutin-10,IL-10）对小鼠CD4＋T淋巴细胞的调节可能是在IL-10R1短暂表达的72 h内。     

艾灸对宫颈癌放疗患者外周血T细胞亚群的影响

目的：探索艾灸对宫颈癌放疗患者T细胞免疫系统的作用。方法：采用单克隆抗体（MCAb)，借助APAAP桥联酶标法，检测2组宫颈癌放疗患者外周血T细胞亚群的数量。结果：艾灸治疗2个月后宫颈癌放疗患者外周血CD3^ 、CD4^ T细胞数量较艾灸前有明显增加（P＜0．01），而CD8^ T细胞数量下降，差异显著（P＜0．05）；空白对照组CD3^ 、CD4^ T细胞数量下降，但尚无显著性差异（P＞0．05）。同时分别观察了CD4^ ／CD8^ T细胞的比值，艾灸治疗组明显上升（P＜0．05），而空白组有下降趋势，但无显著性差异。结论：艾灸对宫颈癌放疗患者外周血T细胞有保护作用及免疫调节作用。     

Pregnancy estrogen drives the changes of T-lymphocyte subsets and cytokines and prolongs the survival of H-Y skin graft in murine model

Background Estrogen as well as CD4＋Foxp3＋ regulatory T cells were shown to have a protective role not only in maintaining maternal-fetal tolerance but also against autoimmune diseases. We aimed to investigate whether the pregnancy levels of estrogen are enough to induce transplant tolerance as to maintain fetal-maternal tolerance. Methods We established H-Y skin graft transplantation in C57BL/6 ovariectomized mice that reconstituted with estrogen. Subsequently, consecutive daily estrogen injection was administrated. Tregs and the cytokines in the peripheral blood were detected by flow cytometry and ELISA pre- and post-transplant. Results The results indicated that pregnancy levels of estrogen could promote Tregs in secondary lymphoid organs and peripheral blood （P 〈0.05） but not thymus （P 〉0.05）. The estrogen-treated recipients accepted H-Y skin grafts for more than 35 days （median survival time （MST）： （44.0_＋1.2） days） compared with estrogen-untreated mice （MST： （23.0_＋1.6） days） （P 〈0.05）. It was also observed that estrogen up-regulated the expression of Foxp3, but did not affect CD3＋CD8＋ effector T-cells in non-transplant mice. While in the presence of H-Y antigens, the expression of Foxp3 was more significant and CD3＋CD8＋ effector T cells were decreased significantly （P 〈0.05）. Meanwhile, the up-regulated IL-10 and IL-4, and down-regulated IFN-v could be observed （P 〈0.05）. Conclusions Pregnancy levels of estrogen may promote the conversion of peripheral Tregs in secondary lymphoid organs, but show no effect on the natural Tregs production, differentiation and maturity in central lymphoid organs. Furthermore, pregnancy levels of estrogen could significantly prolong the survivals of H-Y skin grafts by the expansion of TreQs, suppression of CD3＋CD8＋ effector T-cells and immune shift towards Th2 cvtokines.     

5名电离辐射事故患者外周血T细胞T细胞抗原受体、T细胞分化抗原决定簇－3效应的研究

文中报道了５名事故性急性骨髓型放射病患者照后２．５和３．５年外周血Ｔ淋巴细胞Ｔ细胞受体（ＴＣＲ）基因，ＴＣＲ、Ｔ细胞分化抗原决定簇－３（ＣＤ_３）表达与ＴＣＲ／ＣＤ_３复合物功能的辐射效应．发现５名患者于照后２．５年，２名（５．２Ｇｙ和２．４Ｇｙ，５５岁）于照后３．５年外周血Ｔ细胞应答抗ＣＤ３单抗刺激而增殖的能力尚未完全恢复；经同时用ＩＬ－２和抗ＣＤ_３单抗刺激，增殖能力比单用抗ＣＤ_３单抗刺激有所增强；后２名的外周血ＴＣＲ、ＣＤ_３阳性细胞百分率一直低于正常对照和其他患者；并见一患者出现ＤＮＡ重排杂交带型．本文并从ＴＣＲ／ＣＤ_３在介导Ｔ细胞抗原刺激反应中的作用，电离辐射对ＴＣＲ／ＣＤ_３复合物的影响，后果和意义等方面进行了讨论．     

The effect of Helicobacter pylori infection on expression of macrophage migration inhibitory factor by T cells and macrophages in gastric mucosa

Numerousstudieshaverecentlyfoundthatmacrophagemigrationinhibitoryfactor(MIF)playsapivotalroleininflammatoryandimmune mediated diseasessuchasendotoxemia,septicshock,delayed type hypersensitivityreactions,chroniccrescentic glomerulonephritis,andrheumatoidarthritis.112However,itsmolecularfunctionandrolein gastrointestinaldiseaseshasnotbeenfullystudiedand thuswarrantsacomprehensiveinvestigation.Helicobacterpylorus(H.pylori)isbelievedtoplayan importantroleinthedevelopmentofgastricinflammationandul…     

各类胸腹水中淋巴细胞亚群分布水平的研究

目的 了解各类胸腹水中淋巴细胞分布水平及其对胸腹水性质鉴别诊断的意义.方法 采用流式细胞术测定结核性（30例）和癌性患者（31例）外周血及胸腹水中T淋巴细胞亚群分布,ELISA法检测辅助T淋巴细胞（Th）1和Th2类细胞因子γ-干扰素（IFN-γ）、IL-12和IL-4的含量.结果 结核性和癌性胸腹水中CD3＋CD4＋T淋巴细胞含量[（73±6）%与（67±20）%]均高于外周血[（51±19）%与（48±14）%]（均P＜0.05）.尽管结核性胸腹水中CD3＋T淋巴细胞数量高于癌性胸腹水（P＜0.05）,但两者CD3＋CD4^＋和CD3＋CD8^＋T淋巴细胞亚群分布差异无统计学意义（P＞0.05）.结核性胸腹水中Th1与Th2类细胞因子比值（IFN-γ/IL-4与IL-12/IL-4）为54±24与82±19,明显高于癌性胸腹水的8±6与19±10（t值分别为10.34和16.28,均P＜0.01）.结论 结核性和癌性胸腹水中均存在CD3^＋CD＋T淋巴细胞聚集,仅靠CD3^＋CD4^＋和（或）CD3^＋CD8^＋淋巴细胞亚群分析不足以鉴别炎性与恶性胸腹水,而Th1、Th2类细胞因子的测定有助于两者的鉴别诊断.     

杜克雷嗜血杆菌与人类树突状细胞和巨噬细胞体外的相互作用

为探讨抗原呈递细胞 (APCs)在软下疳发病机制中的作用 ,将树突状细胞 (DCs)和巨噬细胞 (MQs)与流感嗜血杆菌、杜克雷嗜血杆菌及其纯化抗原脂寡糖 (LOS)、细胞致死性肿胀毒素 (HdCDT)共育 ,测定它们的吞噬活性及6、2 4h时前炎症细胞因子TNF α、IL 1 β、IL 6、IL 8的分泌情况 ;将抗原作用后的细胞与自体CD4 + T淋巴细胞共育 ,测定T淋巴细胞增生及 48h细胞上清液中的细胞因子IFN γ、IL 4、IL 1 3的水平。结果 :约 6%～ 2 7%的DCs和 1 4%～ 2 6%的MQs吞噬杜克雷嗜血杆菌的不同菌株及无荚膜流感嗜血杆菌、大肠杆菌。杜克雷菌的不同菌株诱导DCs、MQs分泌大量的TNF α、IL 6、IL 8(P <0 .0 5 ) ;LOS诱导的前炎症细胞因子水平比细菌低 5 0 %～ 67% ;HdCDT不诱导任何前炎症细胞因子的产生。 2种细菌作用后的DCs、MQs能激活T淋巴细胞产生高水平IFN γ ,并诱导产生相似的T淋巴细胞增生 ,HdCDT无此作用。提示 :杜克雷菌能影响APCs活化T细胞的能力 ,并有助于Th1型反应。     

白细胞介素15的研究进展

细胞因子在调节免疫反应中发挥着重要作用,白细胞介素(IL)2常作为免疫佐剂来增强免疫反应。IL-15是一种新近发现的细胞因子,具有与IL-2类似的作用,如激活T细胞、B细胞和自然杀伤细胞,并可介导这些细胞的增殖和存活。IL-15具备IL-2所不具备的优势,如IL-15能激活、维持和扩增CD8+记忆性T细胞,不激活调节性T淋巴细胞(Tregs),并解除Tregs对T细胞的抑制作用,而Tregs对免疫的各个方面都有负调节作用,显示了IL-15在免疫调节中的优势。     

MIF和COX-2在实验性IgA肾病肾组织中的表达

目的探讨巨噬细胞移动抑制因子(MIF)和环氧化酶2(COX-2)在实验性IgA肾病肾组织中的表达及其相关性。方法应用葡聚糖G200和大肠杆菌外膜总蛋白(OMPs)提取物分别免疫小鼠,诱导IgA肾病模型,设立葡聚糖组和大肠杆菌外膜蛋白组,同时建立空白对照组。用免疫组化方法检测MIF和COX-2在实验性IgA肾病肾组织中的表达与分布。结果MIF和COX-2在葡聚糖组和大肠杆菌外膜蛋白组肾小管上皮细胞中的表达显著上调,在两组的表达显著高于空白对照组(P<0.01)。MIF和COX-2在实验性IgA肾病肾组织的表达与肾组织损伤的程度呈正相关。结论MIF和COX-2在IgA肾病肾组织的表达比较高,提示MIF和COX-2在IgA肾病的发生、发展中发挥一定的作用。     

巨噬细胞移动抑制因子介导MPP+/MPTP诱导的小胶质细胞NLRP3炎症小体的激活

目的 探讨巨噬细胞移动抑制因子（MIF）/κb（NF-κB）对1-甲基-4-苯基吡啶离子（MPP+）/1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）激活小胶质细胞NLRP3炎症小体的影响和机制,进而对神经元的影响。方法 慢病毒MIF-shRNA感染Bv-2细 胞,敲低MIF表达。Western blot检测MPP+干预的Bv-2 NLRP3和MIF表达水平、转染病毒后细胞NLRP3、p65、caspase-1表达水平及细胞核、浆蛋白p65表达水平。ELISA检测细胞培养上清液IL-1β、IL-18表达水平。将细胞培养上清液作为条件培养基培养MN9D细胞,Western blot检测TH蛋白表达水平。C57BL/6小鼠腹腔注射MPTP构建PD小鼠模型,向中脑黑质致密部立体定位注射腺相关病毒MIF-shRNA（AAV-MIF-shRNA）敲低MIF表达。对小鼠进行旷场实验、爬杆实验、悬挂实验行为学评估,组织免疫组化检测小鼠多巴胺能神经元细胞数目和小胶质细胞活化情况,Western blot 检测小鼠黑质 MIF、NLRP3及TH表达情况。结果 感染病毒的细胞MIF mRNA（P<0.001）和MIF蛋白（P=0.014）明显降低。Western blot显示,0.2 mmol MPP+使Bv-2细胞NLRP3（P=0.012）和MIF（P=0.019）表达增高。与MPP组比较,MIF-shRNA组NLRP3（P=0.042） 和caspase-1（P=0.003）表达减少,细胞总蛋白中p65表达没有差异（P=0.978）。ELISA检测细胞上清液发现MIF-shRNA组较MPP组IL-1β（P<0.001）、IL-18（P=0.002）水平降低。与MPP组比较,MIF-shRNA组核蛋白p65表达降低（P=0.016）,浆蛋白p65表达升高（P<0.001）。相较于MPP+的条件培养基,MN9D细胞在MIF-shRNA条件培养基中TH（P=0.01）表达增加。与MPTP 组比较,注射 MIF-shRNA 的小鼠爬杆实验（P=0.024）和旷场实验（P=0.026）评分显著降低,悬挂实验评分显著升高（P=0.001）。组织免疫组化结果显示,相较于MPTP组,AAV-MIF-shRNA组小鼠TH阳性神经元细胞数量增多（P=0.004）,小胶质细胞数量减少（P=0.049）。MIF（P=0.033）、NLRP3表达减少（P=0.045）,TH蛋白明显增多（P=0.043）。结论 抑制MIF表达可以减少MPP+/MPTP干预所引起的小胶质细胞NLRP3炎症小体表达和炎症因子释放,减轻小胶质细胞激活,减轻炎症反应,改善MPTP引起的黑质多巴胺能神经元损伤,在帕金森病神经炎症方面具有保护作用。     

CD4+CD25+调节性T细胞在过敏性哮喘和无症状的过敏者中表达的差异

目的:比较过敏性哮喘患者、无症状的过敏者和不过敏的健康人外周血CD4+CD25+调节性T细胞(Treg)数量的差异及对特异性过敏原刺激的反应.方法:选取急性发作期的尘螨过敏性哮喘患者20例,无症状的尘螨过敏者24例,不过敏的健康人22例,分离外周血单个核细胞(PBMC)并进行CFSE染色,分别在不刺激和1mg/L尘螨抗...     

HIV-Specific IL-2+ and/or IFN-γ+ CD8+ T Cell Reponses during Chronic HIV-1 Infection in Former Blood Donors

Objective Conflicting data have been generated from previous studies to determine which kind of relationship exists between HIV-1 specific CD8 T-cell responses and HIV-1 viral load or CD4 count over the course of infection.In this study,153 HIV-1 infected LTNPs were enrolled to investigate the role of HIV-1 specific CD8 T-cell responses in chronic HIV-1 infection among HIV-1 infected former blood donors.Methods The patients were stratified into three groups according to CD4 count:CD4≥500 cells/μL; 350 cells/μL≤CD4<500 cells/μL; CD4<350 cells/μL.PBMCs were isolated from the patients' anticoagulated blood samples.IL-2 and IFN-γ secretions of CD 8 T cells against 17 HIV-1 consensus B full peptide pools were analyzed by using ICS assay.Results An overall inverse correlation were observed between CD4 count and plasma viral load.Although no significant difference was observed during the comparisons of frequency/breadth of HIV-1 specific CD8 T cell responses,CD4 count stratification analysis showed that different correlation pattern existed in three strata: as for patients whose CD4 counts were less than 350 cells/μL,no significant correlations were identified between frequency/breadth of HIV-1 specific CD8 T cell responses and CD4 count/viral load; as for patients whose CD4 counts ranged from 350 cells/μL to 500 cells/μL,significant correlation was only observed between the response breadth of IL-2+IFN-γ+CD8 T cells and CD4 count; however,as for patients whose CD4 counts were more than 500 cells/μL,direct correlations were identified between IL-2+IFN-γ+/IL-2+/IFN-γ+CD8 T cells and viral load or CD4 count.Conclusions Universal consistent inverse correlation was only indentified between CD4 count and viral load.The relationship between HIV-1 specific CD8 T cell responses and CD4 count/viral load varied in different CD4 strata,which showed that better preserved CD4 T cells were correlated with better CD8 T cell functions.     

Immunogenicity of interleukin 12 and DNA vaccine prime-BCG boost against Mycobacterium tuberculosis

OBJECTIVE: To investigate the immunogenicity of vaccine strategies about human interleukin 12 associated with combined DNA (Ag85A and ESAT-6) prime-BCG boost. METHODS: BALB/c mice were divided into PBS negative control and 4 immunity groups: BCG group, DNA/BCG group, DNA + IL-12/BCG group and DNA/BCG + IL-12 group. All mice received three immunizations at 2-week interval. Specific IgG antibody in serum of mice was determined with indirect ELISA in 4, 6, 8 weeks respectively after final vaccination. The splenic lymphocytes of mice were separated and stimulated with PPD to measure their proliferation by flow cytometry, and to evaluate the production of interferon-gamma (IFN-gamma) in cell suspensions of spleen cells by ELISA. The levels of CD4+ and CD8+ T-cell on surface of spleens lymphocyte were determined by flow cytometry. RESULTS: PPD could stimulate specific IgG responses in 4 immunity groups, and the average valences of 4 groups are 1:80, 1:120,1:160,1:160; the splenic lymphocyte proliferation reactions and IFN-gamma production were detectable in 4 immunity groups, and the most significant response occurred in 12 weeks. DNA + IL-12/BCG group and DNA/ BCG+IL-12 group induced higher production than BCG group and DNA/BCG group (P < 0.05), and the effects between DNA + IL-12/BCG group and DNA/BCG + IL-12 group had little difference. The numbers of CD4+ and CD8+ T-cell in 4 immunity groups were much higher than PBS group (P < 0.05), and DNA+IL-12/BCG group and DNA/BCG+IL-12 group were detected much more CD4+ and CD8+ T-cell than BCG group and DNA/BCG group (P < 0.05). The level of T-cell between DNA + IL-12/BCG group and DNA/BCG + IL-12 group had little difference. CONCLUSION: Interleukin 12 associated with the strategy of priming with the combined DNA vaccines and boosting with attenuated M. bovis vaccine (BCG) could induce much stronger specific cellular immunity compared with simple DNA/BCG or attenuated M. bovis vaccine alone.