HBV转基因小鼠肝脏NKT细胞功能与PD1、CD28表达分析

目的研究HBV转基因小鼠肝脏中NKT细胞的功能与表面PD1、CD28表达的关系。方法分离小鼠肝脏、脾脏、胸腺和腹膜淋巴结单个核细胞,利用流式细胞检测技术,分别检测其淋巴细胞中NKT细胞的频率,同时检测肝脏NKT细胞PD1、CD28的表达及IFN-γ、IL-4的分泌功能,比较肝脏、脾脏、胸腺和腹膜淋巴结这几个主要免疫组织淋巴细胞中NKT细胞所占的比例,并分析肝脏NKT细胞PD1、CD28的表达与细胞功能的关系。结果与正常同品系小鼠比较,HBV转基因小鼠肝脏、脾脏、胸腺和腹膜淋巴结NKT细胞数量明显减少(P<0.05),与脾脏、胸腺和腹膜淋巴结相比,肝脏淋巴细胞中含有大量的NKT细胞;与正常同品系小鼠比较,HBV转基因小鼠肝脏NKT细胞PD1的表达明显增多(P<0.05),CD28的表达明显减少(P<0.05),肝脏NKT细胞IFN-γ、IL-4的分泌功能明显降低(P<0.05)。结论肝脏中含有大量的NKT细胞,HBV转基因小鼠肝脏NKT细胞的功能存在明显的缺陷,并提示PD1的增加和CD28的降低可能与NKT细胞功能的下调密切相关。     

微小RNA-7敲减对ConA诱导的小鼠急性肝损伤的影响

目的:研究微小RNA-7(miR-7)敲减(KD)对急性肝损伤(ALI)模型小鼠的影响。方法:野生型(WT)小鼠和miR-7KD小鼠腹腔注射30 mg/kg刀豆蛋白(ConA)建立急性肝损伤模型;48 h后,观察小鼠肝脏的形态、重量及其脏器指数变化;HE染色观察小鼠肝脏组织病理学变化;血清学方法检查血清中谷丙转氨酶(ALT)的水平;ELISA法检测血清中细胞因子IL-4和IFN-γ的水平;流式细胞术检测肝脏组织中CD4~+T细胞的比例及其相关的细胞因子IL-4和IFN-γ的表达变化。结果:与对照组相比,miR-7敲减后急性肝损伤小鼠的肝脏组织颜色变浅,重量减轻,重量指数明显增加(P0.05);HE染色显示miR-7KD小鼠血清炎症细胞浸润显著增多;血清学方法检测发现急性肝损伤小鼠血清ALT的水平明显上升(P0.05);ELISA法检测显示miR-7KD小鼠血清中IFN-γ水平明显升高(P0.01),而IL-4的表达水平则明显降低(P0.01);流式细胞术检测结果显示,miR-7KD小鼠肝脏中CD4+T细胞比例显著升高(P0.01),其相关的细胞因子IFN-γ的表达水平也显著上调(P0.01),而IL-4的水平没有发生明显变化。结论:敲减miR-7基因可明显促进ConA诱导的小鼠急性肝损伤。     

幽门螺杆菌感染小鼠胃黏膜CD8 T细胞的免疫表型及应答特征

目的探究幽门螺杆菌(Helicobacter pylori,H. pylori)感染后小鼠胃黏膜CD8 T细胞的表型特征及应答规律。方法建立H. pylori感染的小鼠模型,同时设立对照组。流式细胞仪检测胃黏膜CD8 T细胞的频率、表型、趋化因子受体及细胞因子。Real-time PCR检测胃黏膜H.pylori的定植量。结果实验组小鼠胃黏膜CD8 T细胞的频率显著高于对照组(P0.01)。胃黏膜CD8 T细胞高表达CD44、CD103、CD69,低表达CD62L、CCR7。与对照组相比,感染组胃黏膜CD8 T细胞上调表达趋化因子受体CCR3、CCR4、CCR5、CXCR5。非特异和特异刺激CD8 T细胞均分泌细胞因子IFN-γ。结论 H.pylori感染后,小鼠胃黏膜CD8 T细胞是一群通过趋化因子受体上调从而募集到胃黏膜的Trm细胞(常驻记忆型T细胞),主要通过分泌细胞因子IFN-γ进行免疫应答。     

急性心肌梗死后辅助性T细胞功能失衡的意义

目的：探讨急性心肌梗死(AMI)后辅助性T淋巴(Th)细胞功能失衡的意义。方法： 采用流式细胞分析法对33例AMI病人，22例不稳定心绞痛(UA)病人和35例AMI大鼠进行Th细胞胞内干扰素γ(IFN-γ)和白介素4(IL-4)的动态监测，同时应用RT-PCR方法检测AMI大鼠心肌组织IFN-γ和IL-4以及T细胞表面趋化因子受体CCR3、CCR5和CXCR3 mRNA的表达。结果：AMI和UA病人发病24 h内Th1型胞内IFN-γ的水平明显高于对照组。UA患者IFN-γ高表达持续时间较短，发病1周后恢复；AMI患者IFN-γ高表达持续时间较长，发病后1周甚至1月仍增高。Th2型胞内IL-4未见明显变化。AMI大鼠心梗后Th细胞胞内IFN-γ和IL-4均无明显变化，T细胞表面趋化因子受体CCR3、CCR5和CXCR3 mRNA表达亦无显著差异，但AMI 1周、2和1月末心肌组织局部IFN-γ mRAN的表达明显增加。结论： AMI后机体出现T细胞功能失衡，主要表现为Th1细胞功能亢进，可能参与AMI的发病；AMI后T细胞功能失衡可能作为自身免疫病的发病机制之一，参与了AMI后自身免疫性心肌炎的发病和自身免疫因素引起的心肌损伤和心室重塑过程。     

白细胞介素-22预处理对耐甲氧西林金黄色葡萄球菌系统性感染小鼠肝脏炎症应答的影响

目的在耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)系统性感染小鼠模型中,观察白细胞介素(interleukin)-22预处理对小鼠肝脏组织炎症应答的影响。方法于感染前24 h对Balb/c小鼠经尾静脉注射25μg重组小鼠IL-22,7×1010CFU/kg的MRSA(ATCC43300)经尾静脉注射感染小鼠,感染6 h后取外周血和肝脏组织。检测血清中谷氨酸氨基转移酶(alanine aminotransferase,ALT)和7种促炎因子[包括:IL-1β、IL-12p70、干扰素-γ(interferon-γ,IFN-γ)、IL-6、趋化因子CXCL1、IL-10、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)]的表达水平;实时定量PCR法检测小鼠肝脏中IFN-γ、IL-6、CXCL1和TNF-αm RNA的水平;对小鼠肝脏组织进行HE染色,观察肝细胞坏死和炎症细胞浸润情况,对组织切片进行病理学评分;分离小鼠肝脏内淋巴细胞(intrahepatic lymphocytes,IHLs),应用流式细胞术检测不同炎症细胞亚群的数量。结果 MRSA感染小鼠血清和肝脏中IFN-γ、IL-6、CXCL1和TNF-α的表达水平均较空白对照组显著升高(P0.05),IL-22预处理可降低MRSA感染所致的上述细胞因子升高表达(P0.05)。MRSA感染亦可导致肝脏炎症损伤,主要表现为血清ALT水平升高、肝脏坏死和炎症病理学评分升高以及肝脏内淋巴细胞和各种非特异性炎症细胞向肝脏募集浸润的数量增加。IL-22预处理可降低MRSA感染所致的肝脏炎症损伤,ALT水平下降,病理学评分降低,炎症细胞向肝脏募集浸润的数量亦减少。结论 IL-22预处理降低MRSA系统性感染小鼠肝脏的炎症反应。     

外周血自然杀伤T细胞数目减少、抑制性受体上调和功能障碍可能参与手足口病重症化

目的探讨手足口病(hand foot and mouth disease,HFMD)患儿外周血自然杀伤T细胞(natural killer T cells,NKT)数目、表型及功能活性在不同病情、EV71感染中的变化及意义。方法应用流式细胞术检测39例健康儿童和55例HFMD患儿的外周NKT细胞数量、表型(NKD2A、CD94和NKG2D)及杀伤颗粒或细胞因子分泌功能(CD107a、GB或IFN-γ、IL-10)。酶联免疫吸附法检测血浆细胞因子和趋化因子(IL-2、IL-6、IL-10、TNF-α、IFN-γ和MCP-1)的水平。结果与健康儿童相比,重症(EV71~+/EV71~-)患儿NKT细胞数量及百分比均显著降低(P0.05);重症(尤其EV71~-重症)患儿NKT细胞表达抑制性NKG2A/CD94受体显著升高(P0.05);轻症患儿的NKT细胞分泌IL-10显著增多(P0.05);重症HFMD患儿血浆IL-6、TNF-α、IFN-γ和MCP-1的水平显著增高(P0.05);EV71-重症患儿IL-10水平明显增高(P0.05)。NKT细胞数目与CD107a~+NKT或GB~+NKT%呈正相关(P0.05);IL-10~+NKT%与NKG2D~+NKT%或IFN-γ~+NKT%呈正相关(P0.05)。结论随着HFMD病情进展,外周血NKT细胞数目减少,抑制性受体(NKG2A/CD94)表达上调,细胞因子分泌和细胞毒性功能障碍等,可能促进HFMD患儿免疫功能紊乱并参与HFMD重症化。     

MCMV早期感染对小鼠CD8~+ T、γδT和NK细胞功能的影响

目的通过体内和体外实验观察小鼠巨细胞病毒(murine cytomegalovirus,MCMV)早期感染对小鼠脾脏CD8~+ T、γδT和NK细胞亚群和功能变化的影响,以将感染增强免疫细胞功能的方法作为提高机体抗肿瘤免疫效应途径的理论基础。方法分别用5×10~4PFU(A组)、5×10~5PFU(B组)MCMV和无菌PBS(C组)腹腔注射小鼠,体内感染9 d;用1×10~4PFU(D组)、1×10~5PFU(E组)和1×10~6PFU(F组)MCMV感染离体小鼠脾脏淋巴细胞24 h和48 h,同时设置空白对照组(G组)。多色流式细胞术检测脾脏淋巴细胞表型和功能,ELISA检测体内感染组IFN-γ、IL-2和颗粒酶B的水平。结果与C组比较,MCMV体内感染小鼠后,MCMV特异性CTL,B组CD8~+ T细胞分泌的IFN-γ、IL-2和表达的颗粒酶B,A、B组γδT细胞表达的颗粒酶B、CD107a,B组NK细胞表达的CD107a水平均增加;A、B组未成熟NK细胞数减少,但成熟1阶段NK细胞明显增加;以上指标在A、B组间均无差异。ELISA结果提示A、B组IFN-γ、IL-2和颗粒酶B水平均较C组升高,且随病毒滴度增加而升高。与G组相比,体外感染24 h和48 h后D、E、F组CD8~+ T细胞表达的CD69,24 h后CD4~+ T细胞表达的CD69,24 h后F组CD8~+ T细胞分泌的IFN-γ,24 h和48 h后各感染组CD8~+ T细胞分泌的TNF-α以及24 h后D、E组CD4~+ T细胞分泌的TNF-α均增加,CD8~+ T细胞和CD4~+ T细胞各组间IL-2分泌和颗粒酶表达未见明显差异;与G相比,体外感染24 h和48 h后各感染组均可见NK细胞IL-2和颗粒酶B水平升高;以上表面分子、细胞因子和颗粒酶B的表达水平普遍随着病毒滴度的增加而升高。结论MCMV早期感染后CD8~+ T、γδT和NK细胞功能可能较前增强,这将为抗肿瘤免疫治疗提供理论基础。     

人脐带血与周围血中NKT细胞频率、亚群、表型及功能的比较

目的比较足月产新生儿脐带血和正常人周围血NKT细胞的频率、亚群、表型特征及功能。方法分离足月产新生儿脐带血CBMCs和正常成年人周围血PBMCs,流式细胞术检测TCRvβ11、CD4、CD8、CD45RA、CD62L、CCR7等表面分子的表达及细胞因子IL-4和IFN-γ的产生。结果 CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞的平均频率分别为0.35%和0.33%,二者无显著差别。CBMCs中CD4+NKT细胞频率(67.39%)高于PBMCs中CD4+NKT细胞频率(54.08%),但CD8+NKT细胞,PBMCs明显高于CBMCs(22.35%对5.86%),CD4-CD8-NKT二者相差无统计学意义。CBMCs中CD3+TCRvβ11+CD45RA+NKT细胞的频率(88.37%)高于PBMCs(61.32%)。CBMCs中CD62L(56.66%对49.60%)和CCR7(22.64%对20.03%)的比例稍高于PBMCs,但其差别无显著性。未刺激CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞均不产生IL-4和IFN-γ,PMA+Ionomycin刺激后,CBMCs中CD3+TCRvβ11+NKT细胞仍不能产生IL-4和IFN-γ,但PBMCs中CD3+TCRvβ11+NKT细胞却有6.74%产生IL-4、26.96%产生IFN-γ、1.26%同时分泌IL-4和IFN-γ,产生细胞因子的CD3+TCRvβ11+NKT细胞主要为记忆性NKT细胞。结论 CBMCs和PBMCs中CD3+TCRvβ11+NKT细胞的亚群、表型和功能均存在一定差别,记忆性NKT细胞可能与其再次免疫应答相关。     

移植物抗宿主病小鼠肝脏及异体CD8+ T细胞趋化因子和趋化因子受体表达分析

为探讨趋化因子及其受体介导的效应性细胞迁移在移植物抗宿主病(graft-versus-host disease,GVHD)发生发展中的重要作用,采用次要组织相容性抗原异配的GVHD小鼠模型,应用流式细胞分选术(FACS)、实时荧光定量聚合酶链反应等方法,分析GVHD靶器官和异体CD8+T细胞趋化因子及受体的表达。肝脏高表达干扰素诱导蛋白-10(IP-10)、干扰素诱导的T细胞趋化因子(ITAC)、γ干扰素诱生单核因子(MIG)、巨噬细胞炎症蛋白(MIP)-1α/β等趋化因子。异体CD8+T细胞上则高表达CXC趋化因子受体(CXCR)3和CC趋化因子受体(CCR)5。随着肝脏趋化因子的表达增加,异体CD8+T细胞迁移浸润的数量也增高。因而趋化因子及异体CD8+T细胞上趋化因子受体的特异对应关系,促使大量异体CD8+T细胞迁移浸润并造成肝脏损伤。     

化学修饰的糖脂4′″-dh-iGb3对NKT细胞分泌Th1/Th2型细胞因子的影响

为研究两种i Gb3类似物(化学修饰的糖脂4′″-dh-i Gb3和4-HO-i Gb3)对NKT细胞Th1/Th2型细胞因子分泌的影响。采用流式细胞术检测经腹腔注射两种i Gb3类似物后C57BL/6小鼠脾脏NKT细胞数量的变化以及NKT细胞胞内IFN-γ和IL-4的表达水平;用Real-ti me PCR方法检测体外培养的脾脏淋巴细胞与i Gb3类似物共孵育后IFN-γ和IL-4的mRNA表达水平,并用ELISA方法检测孵育上清中IFN-γ和IL-4的含量。结果显示:与i Gb3组相比,经两种i Gb3类似物体内刺激后脾脏NKT细胞的数量都没有显著性变化。糖脂4-dh-i Gb3能够较i Gb3更强地诱导脾脏NKT细胞胞内IFN-γ的表达,也能够上调体外培养的脾脏淋巴细胞IFN-γ的mRNA水平及IFN-γ的分泌,而IL-4在所检测的各个水平上都没有显著性变化。提示化学修饰的糖脂4′″-dh-i Gb3能够诱导C57BL/6鼠脾脏NKT细胞Th1型细胞因子的分泌,而并不显著影响Th2型细胞因子的分泌,从而诱导Th1/Th2型细胞因子平衡向Th1方向偏移。     

Blockade of IL-33 ameliorates Con A-induced hepatic injury by reducing NKT cell activation and IFN-γ production in mice

IL-33, a recently described member of the IL-1 family, has been identified as a cytokine endowed with pro-Th2 type functions. To date, there are only limited data on its role in physiological and pathological hepatic immune responses. In this study, we examined the role of IL-33 in immune-mediated liver injury by exploring the model of concanavalin A (Con A)-induced hepatitis. We observed that the level of IL-33 expression in the liver was dramatically increased at 12?h after Con A injection. Meanwhile, ST2L, the receptor of IL-33, was significantly up-regulated in lymphocytes including T and natural killer T (NKT) cells, especially in NKT cells. Moreover, administration of recombinant IL-33 exacerbated Con A-induced hepatitis, while pretreatment of IL-33-blocking antibody or psST2-Fc plasmids showed a protective effect probably by inhibiting the activation of late stage of T cells and NKT cells and also decreasing the production of the cytokine IFN-??. Furthermore, depletion of NKT cells abolished the protective effect of IL-33-blocking antibody, and IL-33 failed to exacerbate Con A-induced hepatitis in IFN-??^?/? mice. These data suggested the critical roles of NKT cells and IFN-?? in the involvement of IL-33 in Con A-induced hepatitis. Blockade of IL-33 may represent a novel therapeutic strategy through IL-33/ST2L signal to prevent immune-mediated liver injury.     

IL-18 time-dependently modulates Th1/Th2 cytokine production by ligand-activated NKT cells

While IL-18 synergizes with IL-12 to induce a Th1 immune response, it also promotes a Th2 response. Here we investigate the modulatory role of IL-18 on the Th1/Th2 cytokine response. The injection of alpha-galactosylceramide (alpha-GalCer), a ligand for NKT cells, elevated mouse serum levels of both IFN-gamma and IL-4. When the mice were treated 2 h before alpha-GalCer challenge with IL-18, IFN-gamma production but not IL-4 production was remarkably up-regulated. In contrast, pretreatment with IL-18 6 h before the challenge enhanced IL-4 production. However, this IL-18-enhanced IL-4 production was not elicited in mice injected with anti-CD3 Ab. Liver mononuclear cells (MNC) produced a similar cytokine production pattern when MNC from mice treated with IL-18 either 2 h or 6 h before challenge were stimulated with alpha-GalCer in vitro. Expression of SOCS1 and SOCS3 was notably up-regulated in the liver MNC from mice pretreated 6 h before with IL-18; in particular, SOCS3 expression was confined to the liver NKT cells. Inhibition of SOCS3 by RNA interference up-regulated the phosphorylation of STAT3 and suppressed in vitro IL-4 production by IL-18-primed liver MNC stimulated with alpha-GalCer, but it did not affect IFN-gamma production. These results suggest that IL-18 time-dependently modulates Th1/Th2 cytokine production in ligand-activated NKT cells by regulating/inducing SOCS3 expression.     

腺病毒载体介导的LacZ基因在神经前体细胞的转染和表达

为了观察含报告基因 L ac Z的重组 5型腺病毒载体 ( Ad5CMVLac Z)在神经前体细胞转染和表达的量效关系并探讨用该载体构建基因修饰细胞的可能性 ,本研究用不同滴度 ( 1× 10 3～ 1× 10 1 0 PFU/ml)的 Ad5CMVLac Z转染体外培养的 SD胎鼠(胎龄 12 d)海马神经前体细胞 ,用β-半乳糖苷酶 (β-gal)免疫组化反应检测转染效率。结果显示 :当病毒滴度为 1× 10 7时 ,转染率约为 5 0 % ,当滴度增加到 1× 10 1 0时 ,转染率达 10 0 %。Ad5CMVL ac Z在神经前体细胞的转染率具有滴度依赖性的量效关系。表明高滴度的 Ad5CMVLac Z成功地转染了大部分神经前体细胞 ,Lac Z基因也得到充分表达。提示神经前体细胞是该载体转染的适宜靶细胞 ,并有可能通过该载体转导目的基因 ,构建出基因修饰细胞     

Some characteristics of IL-5-producing T cells in mouse liver induced by Schistosoma japonicum infection

Schistosome infection could cause significant liver damage in animal; Th2 cells play an important role in the progress of this disease. In our study, C57BL/6 mice were infected by Schistosoma japonicum and lymphocytes were isolated from the liver to detect some characteristics of interleukin-5 (IL-5)-producing T cells by different methods. The results revealed that S. japonicum infection could induce a large amount of IL-5 in mouse liver T cells by the means of fluorescent bead immunoassay and RT-PCR. Although, mouse liver contained many T cell subsets, such as Th cells, Tc cells, NKT cells, and γδ T cells. Fluorescence activated cell sorting results indicated that Th cells were the main source of IL-5 in the T cell population after phorbol 12-myristate 13-acetate and ionomycin stimulation. Moreover, the percentage of IL-5-producing Th cells continued to increase from 4 to 8 weeks after S. japonicum infection, which differed from the changes of IFN-γ⁺ Th1 cells, IL-4⁺ Th2 cells, and IL-17A⁺ Th17 cells during S. japonicum infection. Additionally, cytokines co-expression results demonstrated that 36.2 % of IL-5⁺ Th cells could express IL-4, and 10 % of it could produce IFN-γ or IL-17A. Collectively, these findings implied that IL-5-producing Th cells posses some properties which differ from other cytokines secreting Th cells.     

Abrogation of spontaneous liver tolerance during immune response to Candida albicans: contribution of NKT and hepatic mononuclear cells

Hepatic mononuclear cells (HMC) are a heterogeneous population with innate immune properties involved in the response to several pathogens. Herein, during the primary infection with Candida albicans, we observed dynamic changes in CD3+, NK+ and NKT+ intrahepatic lymphoid subsets and a significant increase in the absolute number of antigen-presenting cells (APC). The liver tolerogenic microenvironment sustained by higher levels of IL-10, transforming growth factor-β and IL-4 was severely modified upon the robust IFN-γ production after the fungal colonization. NKT cells purified from infected animals released significant amounts of IFN-γ and the production of this cytokine was exacerbated after a second contact with the fungus. Interestingly, C. albicans per se was unable to activate tolerogenic NKT cells from naive animals. In vitro experiments performed with HMC cells depleted of the CD11b/c+ population revealed that in the absence of APC, NKT cells are unable to produce IFN-γ in response to C. albicans. Our findings constitute the first evidence that this innate lymphocyte population is involved in the pathogenesis of C. albicans infection.     

Enhancement of the synthetic ligand-mediated function of liver NK1.1Ag+ T cells in mice by interleukin-12 pretreatment

We previously reported that mouse NK1.1 Ag+ T (NKT) cells activated by interleukin-12 (IL-12) act as anti-tumour/anti-metastatic effectors. However, IL-12 reportedly induces a rapid disappearance of liver NKT cells by activation-induced apoptosis. In the present study, however, we show that injection of IL-12 into mice merely down-regulates the NK1.1 expression of liver NKT cells and Vbeta8+ intermediate T-cell receptor cells and CD1d/alpha-galactosylceramide (alpha-GalCer)-tetramer reactive cells in the liver remained and did not decrease. Furthermore, when IL-12-pretreated (24 hr before) mice were injected with alpha-GalCer, not only serum interferon-gamma but also serum IL-4 concentrations increased several-fold in comparison to the control alpha-GalCer-injected mice. However, IL-12 pretreatment markedly up-regulated serum ALT levels and Fas-ligand expression on NKT cells after alpha-GalCer injection in middle-aged mice only. Consistently, the liver mononuclear cells (MNC) from IL-12-pretreated mice stimulated with alpha-GalCer in vitro produced much greater amounts of interferon-gamma and IL-4, and also showed a more potent cytotoxicity against tumour targets than those from mice pretreated with phosphate-buffered saline. Liver MNC from middle-aged mice, but not from young mice pretreated with IL-12, also showed increased cytotoxicity following in vitro alpha-GalCer stimulation against cultured hepatocytes. Furthermore, IL-12 treatment of middle-aged mice enhanced tumour necrosis factor receptor 1 mRNA expression in liver Vbeta8+ T cells, and in vitro experiments also revealed that IL-12 pretreatment of liver MNC from middle-aged mice enhanced their tumour necrosis factor-alpha production after alpha-GalCer stimulation. Synthetic ligand-mediated functions of NKT cells, including IL-4 production, are thus enhanced by IL-12 pretreatment.     

C57BL/6小鼠不同组织器官中NKT细胞的比较

目的比较C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结中NKT细胞的含量、亚型和功能的特点。方法分离正常C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结的淋巴细胞,利用细胞表面分子染色的方法,观察不同组织器官中CD3+NK1.1+NKT细胞及其亚型的含量;淋巴细胞经过PMA和离子霉素刺激后,应用细胞内细胞因子染色的方法,通过流式细胞仪观察NKT细胞IFN-γ、IL-4、IL-9和IL-17的产生情况。结果肝脏中NKT细胞的含量为(25.2±12)%,显著高于肺脏、脾脏和肠系膜淋巴结。肝脏、脾脏和肠系膜淋巴结中NKT细胞以CD4+细胞亚群为主,而肺脏中NKT细胞以CD4-CD8-亚群为主,同时肠系膜淋巴结的NKT细胞中存在CD4+CD8+亚群。不同组织器官中NKT细胞IFN-γ、IL-4、IL-9和IL-17产生的能力有差别。结论 C57BL/6小鼠肝脏、肺脏、脾脏和肠系膜淋巴结的NKT细胞在含量、表型和功能方面可能存在明显的差异。     

NKT cell activation by Leishmania mexicana LPG: Description of a novel pathway

NKT cells have been associated with protection against Leishmania donovani, yet their role in infections with Leishmania mexicana has not been addressed, nor has the activation pathway been defined after stimulation with Leishmania mexicana lipophosphoglycan (LPG). We analyzed the activation of NKT cells and their cytokine production in response to Leishmania mexicana LPG. Additionally we compared NKT-cell numbers and cytokine profile in lymph nodes of skin lesions induced by Leishmania mexicana in BALB/c and C57BL/6 mice. We show that LPG activates NKT cells primarily through the indirect pathway, initiating with TLR2 stimulation of dendritic cells (DC), thereby enhancing TLR2, MHC II, and CD86 expressions and IL-12p70 production. This leads to IFN-γ production by NKT cells. C57BL/6 mice showed enhanced DC activation, which correlated with augmented IFN-γ production by NKT cells. Additionally, infected C57BL/6 mice showed elevated percentages of NKT cells with higher IFN-γ and IL-4 production in lymph nodes.We conclude that the response of NKT cells towards Leishmania mexicana LPG initiates with the indirect activation, after binding of LPG to TLR2 in DC. This indirect activation pathway enables NKT cells to produce IFN-γ during the innate phase of Leishmania infection, the magnitude of which differs between mouse strains.     

The role of CD1d-restricted NK T lymphocytes in the immune response to oral infection with Salmonella typhimurium

CD1d-restricted natural killer T (NKT) cells belong to the innate-like lymphocytes which respond rapidly to stress and infectious challenge. We have studied murine CD1d-restricted NKT cells in the early immune response to virulent Salmonella enterica serovar Typhimurium after oral infection. In the liver and spleen, neutrophil and macrophage numbers had increased several-fold by day 5 post-infection, while the frequency of B and T lymphocytes decreased. These cellular changes occurred independently of CD1d-restricted NKT cells, and further, CD1d-restricted T cells did not influence the bacterial load. However, in CD1d(+) mice NK1.1(+) T cells and invariant CD1d-restricted T cells were activated by the infection, as demonstrated by an increase in size, up-regulation of CD69 and production of IFN-gamma. The NK1.1 antigen was down-modulated on these cells during the course of infection, while TCR levels were unaffected. While dendritic cells (DC) up-regulated CD1d-levels upon 24 h of in vitro exposure to the bacteria, increased CD1d expression was not evident on DC in vivo during infection. Furthermore, in vitro re-stimulation of CD1d-restricted T cells isolated from infected mice demonstrated a significant skewing of the cytokine profile, with suppressed IL-4 and increased IFN-gamma production.