超抗原SEB活化的NKT细胞亚群及耐受功能的研究

目的 研究多肽类抗原.超抗原金黄色葡萄球菌肠毒索B(staphylococcal entcrotoxin B,SEB)活化的NKT细胞亚群及耐受特征.方法 小鼠脾细胞分别与SEB和ConA体外培养,MTT方法 测定细胞增殖;特异性耐受特征的研究使用体外活化第3天的细胞,吸出上清后添加二次抗原ConA、LPS、IL-2,继续培养3 d,MTT方法测定细胞对二次抗原的应答反应能力.用流式细胞测定法解析SEB和ConA体外活化的淋巴细胞在第0、5、10和15天时的T和NKT淋巴细胞亚群.结果 SEB和ConA均诱导了小鼠淋巴细胞在体外增殖;SEB活化的淋巴细胞对ConA、LPS和IL-2的二次应答反应能力消失,细胞增殖的OD570nm值由一次应答反应的0.433±0.07分别下降到0.19±0.01、0.14 ±0.02和0.15±0.04(P＜0.01).EonA活化的淋巴细胞的二次应答反应依然存在并依赖IL-2.SEB活化的淋巴细胞是CD4+NK1.1+和CD8+NK1.1+NKT细胞,而不是CD4-CD8-NKT细胞.ConA活化的淋巴细胞是CD3+、CD4+和CD8+T淋巴细胞并包括了CD4-C08-/CD3+NK1.1+NKT细胞.结论 除了α-Calcer脂多糖类抗原能够活化NKT细胞,多肽类超抗原SEB也能活化NKT细胞并具有特异性免疫耐受功能.能够介导免疫耐受的NKT细胞亚群可能是SEB活化的CIM+、CD8+NKT细胞而不是CD4-CD8-/CD3+NK1.1+NKT细胞.     

超抗原SEB 活化的NKT 细胞亚群形态和分化途径的研究

目的:以NKT细胞形态和分化途径为焦点, 研究超抗原SEB活化的NKT 细胞亚群的特征.方法:C57BL/J小鼠脾细胞分别经SEB或者ConA诱导, 收集体外扩增10 d 和5 d的淋巴细胞以及获取正常黏附性巨噬细胞.用荧光抗体染色, 流式细胞仪测定淋巴细胞和大淋巴细胞群表面CD69的表达、 NKT细胞亚群百分数和不同NKT 细胞亚群的分化途径.倒置显微镜400倍下比较大淋巴细胞的形态.结果:SEB活化的淋巴细胞和大淋巴细胞表面CD69分子表达由正常值的0.11%分别提高到55.00%和68.95%.大细胞群中CD8+ 和TCRVB8+NKT 细胞亚群的百分数由原始的0.36%和0.81%分别提高到30.29%和31.48%.这些细胞位于流式细胞图的上部, 是大体积细胞群.显微镜下显示SEB活化的大淋巴细胞体积不仅大于ConA活化的T淋巴细胞, 而且比正常巨噬细胞大5倍以上, 是非黏附性细胞.胞内质粒松散、胞质与核之比＞1.SEB活化的CD8+ 和TCRVβ8+NKT细胞亚群均由T细胞直接分化而来.结论:SEB活化10 d的NKT细胞亚群主要是CD8+ NK1.1+和TCRVβ8+ NK1.1+的NKT细胞.这些细胞是大体积淋巴细胞, 应属于T细胞的亚群.     

NKT细胞亚群及其功能研究进展

NKT细胞是一群兼具NK细胞和T细胞特征的免疫细胞。根据其表达的TCR类型及发育是否依赖于CD1d分子,可将其分为三类:I型NKT细胞、II型NKT细胞和NKT样细胞。NKT细胞活化后,能够迅速产生IFN-γ、IL-4等多种细胞因子,并正向或负向调节免疫应答。由于NKT细胞数量较少且功能复杂,对NKT细胞亚群及其功能认识的不足成为制约其临床应用的瓶颈。本文对NKT细胞亚群及其功能进行综述,阐明了NKT细胞在免疫应答和免疫调节中发挥的重要作用,并指出某些NKT细胞亚群研究的不足。随着人们对NKT细胞亚群及其功能的深入研究,以NKT细胞为主体的免疫治疗具有巨大的临床应用前景。     

超抗原SEB诱导的免疫耐受与细胞因子无关

目的 研究超抗原金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B,SEB)诱导的耐受效应和耐受调节机制.方法 收集SEB活化10天的细胞,充分洗涤后做为效应细胞,分别与刀豆蛋白(ConA)、脂多糖(LPS)和白介素-2(IL-2)共同培养,用MTT方法测定细胞的耐受性应答反应.正常淋巴细胞在与ConA、LPS、IL-2共同培养的同时添加效应细胞,用MTF方法测定效应细胞的抑制性应答反应功能.流式细胞技术解析耐受性效应细胞类型.结果 效应细胞对ConA、LPS和IL-2的应答反应能力明显降低(P<0.01,n=3),但保持对ConA的应答反应能力.效应细胞抑制正常淋巴细胞与ConA、LPS和IL-2的应答反应(P<0.01,n=3),尤其抑制ConA和IL-2诱导的细胞增殖.SEB活化的效应细胞中CD8+NK1.1+、TcRVβ8+NK1.1+和CD4+NK1.1+NKT细胞以及TcRVβ8+T、CD8+T细胞数量明显增加(P<0.01和P<0.05,n=4).结论 超抗原SEB诱导的耐受性应答反应是效应细胞的直接作用,与细胞因子无关;这些效应细胞能抑制T淋巴细胞增殖,并保存识别ConA的受体功能.     

在小鼠异基因骨髓细胞移植中超抗原SEB诱导的耐受特征

目的:研究在异基因骨髓细胞移植中葡萄球菌肠毒素B(SEB)诱导的耐受强度和细胞学特征。方法:选用C57BL/J小鼠作为受体,BALB/c小鼠作为供体。所有受体小鼠在接受6×107骨髓细胞前均接受6.0Gy60Coγ射线的照射,随机分成3组:组1为只接受6.0Gy60Coγ射线照射的对照组(照射对照组,RI);组2为照射后注射生理盐水(移植对照组,Tran.);组3为照射后注射60μgSEB(SEB组)。180d后由组2和组3实验鼠获得两组C57BL/L-BALB/c嵌合体小鼠。用流式细胞术分析移植后30~180d受体小鼠体内CD4 T、CD8 T、CD3 /NK1.1 NKT淋巴细胞亚群的数量和MHCH-2Kb、H-2Kd抗原表达的百分率,用MLR方法测定嵌合体小鼠淋巴细胞对ConA和异源性抗原的反应性。结果:(1)接受大剂量骨髓细胞移植后,注射SEB和注射生理盐水的两组小鼠均可存活180d以上,SEB组小鼠呈现出BALB/c供体小鼠的颜色特征(白色);Tran.组小鼠呈现出其灰白色。(2)SEB组嵌合体小鼠对ConA的反应性明显低于RI组和Tran.组;对异源性抗原的应答高于Tran.组。(3)SEB组小鼠外周血中CD4 T细胞的数量在移植后30~60d明显下降,随后增加;CD8 T细胞的数量不变。CD3 /NK1.1 NKT细胞的数量,从接受移植后30d开始增加,随着时间的延长而递增,到180d达到5.71%。存活180d的嵌合体小鼠,体内供体小鼠特有的MHCH-2Kd抗原的表达率高达80.95%,自体MHCH-2Kb抗原表达的百分率只占1.45%。(4)与SEB组相反,Tran.组小鼠CD8 T细胞的数量持续下降;CD3 /NK1.1 NKT细胞的数量的增加只在移植后180d上升达到5.07%。结论:在异基因骨髓细胞移植中,SEB诱导的耐受性比单纯骨髓移植诱导的耐受性强。SEB诱导的耐受性与移植早期特异性CD4 T细胞数量的减少和NKT细胞数的持续增加有关。反应性的降低表现为T细胞对ConA反应性的下降。     

人外周血NKT细胞亚群表型及生物学特征

目的:观察正常人周围血NKT细胞的频率、表型特征及功能,进一步了解NKT细胞在免疫应答中的作用。方法:分离正常成年人PBMCs,利用流式细胞术(FCM)检测TCRvβ11、CD4、CD8、CD45RA、CD62L、CCR7等表面分子的表达;PMA+Ionomycin刺激PBMCs后,检测细胞因子的产生。结果:正常成年人周围血CD3+TCRvβ11+NKT细胞的平均频率为0.35%,其范围为0.11%～1.20%。根据CD4和CD8分子的表达,可将CD3+TCRvβ11+NKT细胞分为CD4+、CD8+、CD4-CD8-三个亚群,分别为56.24%、25.82%、16.47%。此外多数CD3+TCRvβ11+NKT细胞表达CD45RA,近半数细胞表达CD62L,少数细胞表达CCR7。细胞因子表达的结果显示,经PMA和Ionomycin刺激后,近30%的CD3+TCRvβ11+NKT细胞分泌IFN-γ,6.9%的细胞分泌IL-4。CD4+NKT细胞产生IL-4的量要明显高于CD8+NKT细胞,但IFN-γ的表达二者没有明显差别。重要的是初始和记忆性NKT细胞都能产生细胞因子,以记忆性NKT细胞为主。结论:正常人周围血中CD3+TCRvβ11+NKT细胞的频率很小,但其表型复杂,产生细胞因子IFN-γ和IL-4的频率高,参与免疫调节及免疫应答的过程。     

NKT细胞：独特的T淋巴细胞亚群

NKT细胞是一种特殊类型的T淋巴细胞，同时表达NK细胞和T淋巴细胞标记，具有较为恒定的TCRα链和独特的CD1限制性，参与自身免疫、感染免疫、抗肿瘤免疫和移植免疫等多种免疫反应，被认为是连接先天性免疫和获得性免疫的桥梁。     

耐受调节性CD8+NKT细胞体外稳定性的研究

目的:探讨超抗原SEB活化并扩增的CD8+NKT细胞耐受调节功能的稳定性.方法:超抗原SEB活化并体外扩增的10 d、20d、30 d和冻存效应细胞被用于本研究.以正常C57BL/J鼠脾细胞为对照,将各个效应细胞与刺激剂刀豆蛋白(ConA)或脂多糖(LPS)共同培养72 h,测定效应细胞对刺激剂的应答反应能力.在正常小鼠淋巴细胞与上述刺激剂反应的同时添加各效应细胞,72 h后测定效应细胞抑制正常淋巴细胞对刺激剂的应答反应能力.体外扩增和冻存的效应细胞与异源鼠脾细胞做混合淋巴细胞培养,MTT法测定细胞的增殖情况.效应细胞用荧光抗体染色,用流式细胞术(FCM)解析NKT细胞亚群.结果:与正常淋巴细胞对刺激剂的应答反应能力相比,体外扩增10 d、20 d、30 d和冻存效应细胞对ConA或LPS的应答反应能力明显降低,细胞增殖的A值分别由正常值0.67和0.61分别下降至0.30和0.31,0.28和0.20,0.26和0.24,以及0.22和0.23(P＜0.05,n=3).效应细胞抑制正常淋巴细胞对上述刺激剂ConA或LPS的应答反应,分别由正常值0.67和0.61下降至0.33和0.39,0.30和0.43,0.36和0.43,以及0.26和0.29(P＜0.05,n=3).效应细胞与异源鼠脾细胞反应与对照组相比明显的降低,分别由正常值0.70下降至20d的0.42,30 d的0.42以及冻存效应细胞的0.54(P＜0.05,n=3).在这群效应细胞中,主要是CD8+NKT细胞,由原始的0.36%增加到41.59%(P＜0.05,n=3).结论:耐受调节性CD8+NKT细胞可以在体外进行传代培养,并且这些传代培养细胞的耐受调节功能依然存在.     

T细胞亚群和免疫耐受

无论采取什么样的方法，我们在耐受诱导研究中的最终目标大部分是建立在对T细胞的调控上，涉及到TCR、共刺激分子、T细胞生长因子及某些T细胞亚群的诱导增生等方面。这些靶T细胞的活化、无能、凋亡或存活均与耐受状态的建立存在某种关联。本文将对近年来T细胞亚群与免疫耐受关系的研究作一综述。     

超抗原对NK细胞亚群凋亡诱导配体表达与功能的调节

目的 研究凋亡诱导配体分子在超抗原活化NK细胞CD56^bright出亚群和CD56^dim亚群上的表达规律和功能。方法以超抗原金黄色葡萄球菌肠毒素A/B（SEA/B）活化PBMC为模型,应用双重免疫荧光染色和流式细胞术分析,观察不同活化模型中凋亡诱导配体分子在NK细胞CD56^bright和CD56^dim亚群表达的变化;采用^51Cr释放实验及抗体阻断实验,观察NK细胞亚群的功能与凋亡诱导配体分子表达的关系;利用激光共聚焦显微镜,观察凋亡诱导配体分子在NK细胞杀伤相的分布。结果当超抗原浓度为0．1μg／mL时,TRAIL和TNF在NK细胞上的表达率及NK细胞杀伤活性于培养第2天时达到高峰,FasL在NK细胞上表达未见明显变化。TRAIL和TNF分子聚集于NK细胞与靶细胞的接触部位,FasL分布未见明显变化。结论超抗原SEA和SEB促进TRAIL和TNF在CD56^dim亚群表达,TRAIL和TNF可能参与NK细胞免疫突触的形成。     

Study of the NKT cell subsets with superantigen SEB-activated tolerance

AIM: To study the NKT cell subsets and their differentiation. METHODS: Splenic lymphocytes from C57BL/J mice that had received SEB treatment were collected as effector cells on the 10(th) day. The cells were cultured in medium containing ConA, LPS and IL-2 for 3 days and measured their response to mitogens and cytokine. The inhibitory action of the effector cells was examined. The effector cells were cultured with normal lymphocytes and above mitogens or cytokine for 3 day. The cells proliferation was assessed with MTT method.The NKT cell subsets among these effector cells with the tolerance function were analyzed and their differentiation sources and correlation of functions were detected by flow cytometry. RESULTS: The response of SEB-activated effector cells to ConA, LPS and IL-2 was significantly decreased compared with that of normal lymphocytes. The A values of cell proliferation were decreased from 0.80+/-0.04, 0.60+/-0.03 and 0.55+/-0.07 in control groups to 0.60+/-0.05, 0.30+/-0.05 and 0.27+/-0.04 in effector groups, respectively (P<0.01, n=3).The inhibitory ability of effectors cells against the response of normal lymphocytes to ConA, LPS and IL-2 were clearly observed. They inhibited the response of normal lymphocytes to several mitogens and cytokine. And the A values of cell proliferation were decreased to 0.26+/-0.02, 0.48+/-0.04 and 0.34+/-0.02, respectively (P<0.01, n=3). The CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRV8(+)NK1.1(+) NKT cell subsets among SEB-activated effector cells with tolerance function were significantly increased and shown that they come from T cell population. And the CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells by ConA or SEB-activated were shown coming from NK cell population. CONCLUSION: The effector cells with tolerance function activated by superantigen SEB relate to CD4(+)NK1.1(+), CD8(+)NK1.1(+), TcRVbeta8(+)NK1.1(+) NKT cell subsets. The NKT cell subsets come from T cells. The CD4(-)CD8(-)/NK1.1(+)CD3(+)NKT cells differentiating from NK cells are not involved in the regulation of tolerance.     

耐受性CD8~+ NKT细胞体外增殖能力的鉴定

目的:利用CFSE标记细胞,流式细胞术(FCM)检测法,解析超抗原SEB活化的耐受性CD8+ NKT细胞在体外增殖的情况。方法:利用CFSE标记新鲜分离的C57BL/J鼠脾细胞,分别与ConA和LPS共同培养3d,收集细胞进行荧光染色并用FCM解析细胞表面CD69分子的表达率和增殖能力。CFSE标记的鼠脾细胞与SEB共培养5d和10d后,荧光染色并用FCM解析细胞表面CD69的百分数和增殖能力。SEB活化的第10天细胞经CFSE标记后在IL-2的协同作用下继续培养10d,荧光染色,FCM解析这群细胞的增殖能力、活性分子CD69的表达率和NKT细胞亚群的变化情况。结果:ConA、LPS和SEB三者均可以刺激小鼠脾细胞增殖。ConA和LPS在3d内可以使细胞增殖3代,且CD69的表达率为74.19%和41.56%;SEB在5d和10d内分别可以使细胞增殖5代和7代,细胞表面CD69的表达率为32.09%和48.66%。SEB活化的10d细胞可以在IL-2的协同下继续传代培养10d,可以增殖7代;这群细胞中CD8+ NKT细胞亚群,由原始的0.36%增加到38.58%;细胞表面CD69分子由正常值的0.11%提高到83.74%。结论:超抗原SEB活化的CD8+ NKT细胞可以在体外进行增殖培养,且这些细胞是活性化的细胞。利用CFSE标记细胞,FCM可以检测耐受性CD8+ NKT细胞在体外的增殖水平。     

NKT cell subsets in infection and inflammation

We recently identified two stably expressed cell surface markers, IL-18R and ST2L, which are selectively expressed on T1/NK1 and T2/NK2 cells, respectively. Here we use these molecules in direct ex vivo analysis of PBMCs from patients with AIDS, psoriasis (PS) atherosclerosis and to show the importance of these markers as determinants of the functional dichotomy of lymphocyte subsets, in particular NKT. In a cohort of 22 HIV patients made up of a mixture of long term non-progressors, seroconvertors, progressors and asymptomatics, we found a clear NKT1 to NKT2 shift (P=0.001) in the HIV-infected individuals. We also show a predominance of NKT2 cells over NKT1 cells in the PBMCs of patients with mild to moderate PS (N=13, P=0.005) but not in atopic dermatitis or healthy controls. However, in patients (N=6) requiring surgery for aneurysm, a predominance of Type 1 (IL-18R(+)) NKT lymphocytes over NKT2 was detected among infiltrating lymphocytes isolated from atherosclerotic plaques. Our data therefore demonstrate that ST2L and IL-18R could serve as important determinants of the immune status of human diseases.     

NKT cell ligand alpha-galactosylceramide blocks the induction of oral tolerance by triggering dendritic cell maturation

NKT cells play contradictory roles in vivo, both regulating autoimmunity and activating immunity to intracellular pathogens and tumors. In this study, we studied the effect of NKT cell activation on the induction of systemic tolerance by oral administration of antigen. Administration of alpha-galactosylceramide (alphaGC) at the time of oral ovalbumin (OVA) feeding completely blocked the OVA-specific tolerance induced by both high- and low-dose regimens in BALB/c mice. In the mesenteric lymph nodes (MLN) of alphaGC-treated mice, the proliferation of OVA-specific T cells was greater than that seen in the MLN of vehicle-treated mice in vivo. The administration of alphaGC triggered the full maturation of mesenteric dendritic cells (DC), which were in turn responsible for the enhanced division of OVA-specific T cells in vitro. To further determine whether the costimulation provided by DC in alphaGC-treated mice was responsible for the reversal of oral tolerance in vivo, mice were given alphaGC together with anti-CD80 and anti-CD86 blocking Ab. OVA-specific systemic tolerance was restored in mice given the blocking Ab, even when they simultaneously received alphaGC. Therefore, oral tolerance can be reversed via costimulation by DC that have been triggered to fully mature by the administration of alphaGC.     

Differentiation of human dendritic cell subsets for immune tolerance induction

Objectives

Since no further progress was achieved, in order to improve the long-term organ transplantation outcome, the immune tolerance appears as an interesting therapeutic goal. Dendritic cells (DCs) are specialized cells participating in the homeostasis of the immune response. Moreover, subsets of DCs, identified in humans, appear to have their respective competences in immune response modulation. Our objective is to purify from PBMC or to differentiate DC subsets from monocytes using several strategies and evaluate their IL10 secretion.