慢性粘膜免疫应答诱导的小鼠自身免疫综合征

空弯菌(CJ-S131)慢性感染小鼠具有SLE样自身免疫综合征。本文进一步用甲醛化空弯菌(CJ-S131菌苗)经口免疫BALB/c小鼠,每周两次,持续14周.模拟慢性感染时的慢性肠道粘膜免疫应答.发现免疫小鼠也具有SLE样自身免疫综合征,且较该菌慢性感染小鼠更显著。主要表现有:(1)全身免疫系统的淋巴组织显著增生;(2)B淋巴细胞多克隆激活;(3)多种自身抗体(包括抗ds-DNA.ss-DNA和组蛋白抗体)显著升高;(4)免疫复合物性肾小球肾炎;(5)多器官性(如肝、肠等组织)慢性炎症;(6)血管炎和血管周围炎。在体外自身免疫应答诱导实验中,免疫小鼠的脾细胞培养上清中,免疫球蛋白(总Ig)和抗DNA抗体显著高于非免疫小鼠。上述结果证实.空肠弯菌慢性感染所致的自身免疫综合征.和该菌抗原诱导的慢性肠道粘膜免疫应答有关。本实验为研究感染和自身免疫的关系提供了实用的动物模型。     

同种特异T细胞疫苗诱导移植物存活期延长的研究——Ⅱ.抗独特型T细胞免疫反应

同种特异T细胞疫苗(TCV)免疫诱导出同种免疫反应低下,同种移植物存活时间显著延长,推测其作用机制可能是同种特异TCV免疫诱导机体抗同种特异TCV(独特型)T细胞的上调.实验证实了“抗TCV-T细胞”的存在.以同种特异TCV免疫动物可诱导出对TCV特异的细胞增殖反应.TCV免疫小鼠淋巴细胞能特异杀伤TCV细胞.将TCV免疫小鼠脾细胞作为调节细胞,观察到它能显著地抑制同种MLR,表明它是一“抑制性T细胞”.这些结果提示,同种特异TCV免疫可诱导机体免疫网络中独特型-抗独特型的上调,产生了同种反应T细胞的独特型T细胞,从而保护同种移植物免受同种反应性T细胞的攻击使同种移植物存活时间显著延长.     

同种特异T细胞疫苗诱导移植物存活期延长的研究——Ⅲ.抗T细胞抗体的活性分析

本文观察了TCV免疫小鼠血清对同种抗原和非特异抗原刺激的淋巴细胞反应性的影响,结果显示,TCV免疫小鼠血清能显著抑制ConA诱导的淋巴细胞增殖和同种MLR,表明该血清中存在有抑制T细胞功能的“抑制因子”。实验还发现TCV免疫小鼠血清中存在针对多种不同抗原决定簇的抗体,包括有TCV特异的、活化T细胞的和正常淋巴细胞的。TCV吸收实验证明TCV免疫小鼠血清中能特异地与TCV结合的抗体不能与其它活化T细胞结合。经细胞膜分子SDS-PAGE和免疫印迹分析,TCV免疫小鼠血清中的抗体所识别的细胞膜抗原分子量大约为15kD,35/38kD左右。     

体外CDl34L单抗或CTLA4Ig对狼疮样BXSB小鼠脾细胞分泌IL-6、IFN-γ及自身抗体的影响

目的：探讨体外CD134L单抗或CTLA4Ig对狼疮样BXSB小鼠脾细胞分泌IL-6、IFN-γ及自身抗体的影响。方法：采用未经治疗的狼疮样BXSB小鼠模型，应用CDl34L单抗和／或CTLA4Ig体外特异性阻断CDl34-CDl34L或B7-CD28通路后，用MTT法测定分裂原刀豆蛋白A（ConA）诱导的脾淋巴细胞增殖反应和用ELISA方法测定ConA诱导的脾细胞培养上清液中IL-6、IFN-γ和抗ds-DNA抗体的表达水平，并与经中药狼疮方或强的松治疗的狼疮样BXSB小鼠模型进行比较。结果：（1）在单纯培养或经ConA刺激后培养，相比于正常对照鼠，狼疮样小鼠脾淋巴细胞都表现有增殖反应性的显著增高，IFN-γ、IL-6蛋白量的增高和抗ds-DNA抗体的过度分泌。（2）经强的松或中药狼疮方体内治疗后，狼疮样小鼠脾淋巴细胞体外培养的增殖反应性及IFN-γ、IL-6的分泌都受到明显抑制，抗ds-DNA抗体的产生也明显减少。（3）体外单独应用CD134L单抗或CTLA41g特异性阻断CD134-CD134L或B7-CD28共刺激信号通路，同样可以显著抑制体外培养的狼疮样小鼠脾淋巴细胞的增殖反应及IFN-γ、IL-6的分泌，并可明显减少抗ds-DNA抗体的产生，但它们的抑制作用比强的松、中药狼疮方体内治疗狼疮样小鼠时所表现出的类似效应要弱。（4）联合CD134L单抗和CTLA4Ig，则治疗作用显著提高，其抑制脾淋巴细胞的增殖反应及IFN-γ、IL-6的分泌，抗ds-DNA抗体产生的作用都优于中药狼疮方的体内治疗效果，而与强的松的体内治疗效应相当。结论：CD134-CD134L可以提供独立的、非B7-CD28依赖的，另一种驱动抗原特异性T细胞增殖的协同刺激通路。同时阻断B7与CD28、CD134L与CD134间的相互作用，使自身反应性T淋巴细胞的活化和增殖受到快速而最大限度的抑制，对治疗SLE等自身免疫性疾病可能是一种较理想的免疫干预模式。     

同种特异T细胞疫苗诱导移植物存活期延长的研究——Ⅰ.同种特异T细胞疫苗诱导的细胞免疫水平低下

在体外实验中观察了同种特异T细胞疫苗（TCV）免疫鼠T淋巴细胞对同种抗原和有丝分裂原（ConA）的反应能力。B6同种抗原特异TCV免疫的BALB/c小鼠对B6同种抗原的反应（MLR）能力明显变抑制,对无关第三者同种抗原AKR的反应能力也显著下降,表明存在抗原非特异性的抑制作用。在BALB/c同种抗原特异TCV免疫的B6小鼠中,获得一致的结果。在观察两组TCV免疫动物T细胞对Cond诱导的淋巴细胞增殖实验中,与正常小鼠 T细胞反应性比较,TCV免疫动物 T细胞的增殖能力显著下降,表现为抗原非特异作用。此与MLR中ConA-T细胞免疫动物的结果相一致。在CML实验里,同种抗原特异TCV免疫小鼠的脾细胞体外诱导同种CTL活性明显受到抑制,CTL 活性十分低下。体外实验结果表明:同种特异TCV免疫小鼠可诱发同种抗原反应和对ConA诱发的增殖反应能力显著低下,表现为抗原非特异性作用。     

组蛋白诱导抗核抗体产生及其对肾脏损害

目的 寻找系统性红斑狼疮中诱导抗核抗体（ANA）生成的真正的自身核成分免疫原。方法 从Con A活化的脾淋巴细胞中提取组蛋白免疫同系BALB／c小鼠,ELISA方法测定IgG类抗组蛋白、抗dsDNA抗体,免疫荧光法检测抗核抗体核型和免疫复合物在肾小球内的沉积,免疫印迹法测定抗可溶性核抗原抗体,考马斯亮蓝法检测尿蛋白含量,光镜下观察肾脏形态变化,电镜下观察肾小球电子致密物。结果 活性组蛋白诱导IgG类抗组蛋白、抗dsDNA等多种抗核抗体生成,且诱发同系小鼠产生SLE样肾脏病理变化。结论 活性组蛋白是诱发抗核抗体生成、造成肾损害的免疫原之一。     

ICAM-1 参与系统性红斑狼疮中免疫球蛋白产生的实验研究

目的:探讨ICAM-1在系统性红斑狼疮(SLE)免疫球蛋白产生中的作用。方法:流式细胞术检测分析SLE患者、健康者、anti-CD3 Ab/IFN-α刺激健康者CD4~+T细胞及Pristane诱导的狼疮样小鼠及对照鼠中CD4~+T细胞的活化表型;分选获得Pristane处理小鼠来源的CD4~+T细胞和B细胞,并于体外交叉共培养,加入ICAM-1阻断抗体或同型对照抗体,ELISA检测培养上清中IgG和IgM水平。结果:SLE患者及anti-CD3 Ab/IFN-α刺激健康者CD4~+T细胞表达HLA-DR,CD69和ICOS等分子的细胞比例明显高于对照组。Pristane诱导的狼疮样小鼠CD4~+T细胞的活化程度也显著高于对照;Pristane诱导的狼疮样小鼠血清中抗snRNP和抗dsDNA自身抗体水平显著高于对照组;Pristane处理小鼠来源的活化CD4~+T细胞与B细胞共培养可以促进B细胞产生IgM和IgG。而经抗ICAM-1抗体阻断处理的培养上清中IgM含量低于对照组,而IgG含量显著高于对照组。结论:Pristane来源的活化CD4~+T细胞可促进B细胞分化,ICAM-1分子可能在B细胞分化早期发挥作用,但不利于IgM型向IgG型的类别转换,为ICAM-1在SLE中抗体类别转化的分子机制提供新的理论依据。     

甲醛化空肠弯曲菌菌苗诱导肠道免疫应答机理的初步研究

甲醛化空弯菌CJ-S131菌苗经口免疫小鼠,有效地诱导了肠道粘膜的特异性免疫应答。应用改良的免疫荧光技术,可检测到肠粘膜表面覆有一层空弯菌特异性的抗体及固有膜内的许多特异性抗体形成细胞(SACC)。此外,在空肠和回肠粘膜上皮细胞间有CJ-S131特异性荧光细胞;Peyer氏小结生发中心内可见大小不等,处于不同发育阶段的SACC;结肠固有层内SACC成群出现。用活菌攻击免疫小鼠,其各肠段的病理学检查可见以单核细胞、淋巴细胞和浆细胞浸润为主的炎症反应。     

空弯菌抗原诱导的自身抗体产生动力学

用CJ-S131菌苗经口免疫小鼠16周,观察免疫小鼠体内的自身抗体(抗ds-DNA,ss-DNA,组蛋白和ENA抗体)产生动力学。在CJ-S131菌苗连续免疫的16周内,免疫小鼠有两个自身抗体产生峰。免疫后第6周,出现第一峰,抗组蛋白和ENA抗体的阳性率高于抗ds-DNA和ss-DNA抗体;在第16周,抗ds-DNA和ss-DNA抗体的阳性率高于抗组蛋白和ENA抗体。在正常小鼠体内,存在“自然”抗核酸抗体(抗ds-DNA和ss-DNA抗体),随年龄而增长,但个体差异较大;而抗核蛋白抗体(抗组蛋白和ENA抗体)始终测不出。上述结果提示,抗组蛋白抗体和抗ENA抗体在自身免疫病的发病机理和早期诊断中,具有潜在意义,值得进一步研究。     

T细胞疫苗的实验设想、证实及应用

把引起自身免疫性疾病的自身反应性T细胞或导致同种移植排斥反应的同种反应性T细胞看作是致病性T细胞,将它们活化并灭活后,以疫苗的形式免疫动物,诱导机体产生针对致病性T细胞或同种反应性T细胞的免疫应答,从而消除或减轻这些细胞的致病作用;表现为对自身免疫性疾病和对同种移植物排斥的防治作用。这一方法称为T细胞疫苗接种（TCV）。TCV的作用机制是上调免疫网络中的抑制性作用,产生了抗独特型T细胞,抑制了相应克隆T细胞的作用。用TCR多肽免疫替代TCV亦是有效的方法。TCV或TCR多肽免疫对防治自身免疫性疾病有潜在的应用价值,对控制同种排斥反应也是一条有效措施。     

空肠弯曲菌与可提取核抗原的分子交叉反应

取空肠弯曲菌（ＣＪ－Ｓ１３１）免疫的Ｂａｌｂ／ｃ小鼠脾细胞与骨髓瘤细胞ＳＰ２／Ｏ融合，制备出４种能与空肠弯曲菌和可提取核抗原（ＥＮＡ）起反应的单抗。以Ｈｅｐ－２细胞作基质的免疫荧光染色表明，４种单抗均呈阳性结果，证实为抗核抗体。当单抗腹水被空肠弯曲菌菌体吸收后，其抗ＥＮＡ的活性明显降低，证明这４种单抗与空肠弯曲菌ＣＪ－Ｓ１３１和ＥＮＡ存在交叉反应性。免疫印渍试验表明，４种单抗均与空肠弯曲菌ＣＪ－Ｓ１３１的４３ｋＤ外膜蛋白反应。这一结果提示：空肠弯曲菌ＣＪ－Ｓ１３１的４３ｋＤ外膜蛋白与ＥＮＡ之间可能存在相似的分子构象，这为自身免疫病发机制的分子模拟假说提供了实验的依据。     

影响空弯菌抗原诱导自身免疫应答的因素

利用慢性粘膜免疫应答小鼠模型,研究在空弯菌抗原(CJ-S131菌苗)诱导下,不同品系、性别的小鼠的自身免疫应答。经肠道,持续用CJ-S131菌苗免疫16周后,用ELISA或CELISA检测小鼠体内的抗ds-DNA,ss-DNA,组蛋白,ENA和胸腺细胞抗体。在五个品系小鼠中,四个品系小鼠(BALB/C,ICR,KM和SMMC/B)均有一种或多种被测的自身抗体升高。其中SMMC/B和KM小鼠的EI值较高,BALB/C和ICR小鼠次之。C57BL/6和对照组无显著性差异,提示对空弯菌抗原诱导的自身免疫应答不敏感。在遗传背景的制约下,性别也影响自身抗体的产生,有三种可能:(1)自身抗体升高主要见于雌性小鼠,如雌性KM小鼠被测的各种自身抗体均有升高;(2)雌性,雄性小鼠均有一种以上自身抗体升高,如SMMC/B和BALB/C小鼠;(3)雌、雄性小鼠的自身抗体均无升高,如C 57BL/6。本文结果表明,空弯菌苗诱导的自身抗体种类和水平,受小鼠的遗传背景和性别两个因素的相互制约和影响。     

甲醛化空肠弯曲菌(CJ-S_(131))与佐剂诱导的自身免疫动物模型

甲醛化CJ-S_(131)辅以佐剂免疫小鼠,可诱导自身免疫反应。模型鼠出现的T、B细胞功能改变,血清出现高水平的自身抗体,在肝、肾、肠等组织出现的炎症性病理损伤,均类似于CJ-S_(121)慢性感染的小鼠。本模型优点在于实验周期短,用菌体抗原免疫小鼠代替复杂的感染过程,而诱导自身免疫反应。本模型可用于诸如免疫调节剂的筛选,自身免疫的分子机理等自身免疫反应的实验研究。     

空肠弯曲菌慢性感染诱致自身免疫反应的机理初探

用空肠弯曲菌CJ—S131株感染昆明种小鼠3个月,观察到:(1)感染鼠血清中,出现高滴度的抗ds—DNA和ss—DNA抗体;(2)Con A诱导的抑制细胞功能降低和TH/Ts细胞比值增加;(3)PFC形成和LPS诱致的淋巴细胞转化作用增强:(4)DTH和Con A及PHA诱致的淋巴细胞转化作用增强。提示,CJ—S131感染小鼠使其T_S细胞功能降低,导致T_H细胞功能偏亢,B细胞功能亢进,这可能与自身免疫反应的产生有关。     

The Mechanisms and Applications of T Cell Vaccination for Autoimmune Diseases: a Comprehensive Review

Autoimmune diseases (ADs) are a spectrum of diseases originating from loss of immunologic self-tolerance and T cell abnormal autoreactivity, causing organ damage and death. However, the pathogenic mechanism of ADs remains unclear. The current treatments of ADs include nonsteroidal anti-inflammatory drugs (NSAIDS), antimalarials, corticosteroids, immunosuppressive drugs, and biological therapies. With the need to prevent side effects resulting from current treatments and acquire better clinical remission, developing a novel pharmaceutical treatment is extremely urgent. The concept of T cell vaccination (TCV) has been raised as the finding that immunization with attenuated autoreactive T cells is capable of inducing T cell-dependent inhibition of autoimmune responses. TCV may act as an approach to control unwanted adaptive immune response through eliminating the autoreactive T cells. Over the past decades, the effect of TCV has been justified in several animal models of autoimmune diseases including experimental autoimmune encephalomyelitis (EAE), murine autoimmune diabetes in nonobese diabetic (NOD) mice, collagen-induced arthritis (CIA), and so on. Meanwhile, clinical trials of TCV have confirmed the safety and efficacy in corresponding autoimmune diseases ranging from multiple sclerosis (MS) to systemic lupus erythematosus (SLE). This review aims to summarize the ongoing experimental and clinical trials and elucidate possible molecule mechanisms of TCV.     

应用免疫荧光搭桥法检测小鼠肠粘膜内特异性抗体形成细胞

肠粘膜固有膜内特异性抗体形成细胞(ACC)的检测是研究肠道粘膜免疫应答机理的重要指标之一,组织免疫荧光技术是检测粘膜固有膜内特异性ACC的主要方法。目前,在粘膜免疫应答机理的研究中,主要     

T-cell vaccination in multiple sclerosis

T cells that are autoreactive against myelin antigens play a pivotal role in the pathogenesis of multiple sclerosis (MS). The concept of T cell vaccination (TCV) has been developed to generate an immune response against these autoreactive pathogenic T cells. Immunologic data accumulated so far demonstrates depletion of T cells reactive against immunodominant myelin peptides after immunization in the animal model of experimental autoimmune encephalomyelitis, as well as in vaccinated MS patients. Clinical trials have confirmed the safety and efficacy of TCV in a small number of immunized MS patients. TCV resulted in reduced relapse rates and slowed the progression of neurological disability and MRI brain lesion load. Recently, there have been several double-blind, placebo-controlled studies initiated to evaluate the role of TCV in MS. Specifically, it is important to examine the effect of early TCV, given after the first episode suggestive of the disease, in order to prevent the process of epitope spreading.     

T cell vaccination in multiple sclerosis patients with autologous CSF-derived activated T cells: results from a pilot study

Myelin-reactive T cells are considered to play an essential role in the pathogenesis of multiple sclerosis (MS), an autoimmune disease of the central nervous system. We have previously studied the effects of T cell vaccination (TCV), a procedure by which MS patients are immunized with attenuated autologous myelin basic protein (MBP)-reactive T cell clones. Because several myelin antigens are described as potential autoantigens for MS, T cell vaccines incorporating a broad panel of antimyelin reactivities may have therapeutic effects. Previous reports have shown an accumulation of activated T cells recognizing multiple myelin antigens in the cerebrospinal fluid (CSF) of MS patients. We conducted a pilot clinical trial of TCV with activated CD4+ T cells derived from CSF in five MS patients (four RR, one CP) to study safety, feasibility and immune effects of TCV. CSF lymphocytes were cultured in the presence of rIL-2 and depleted for CD8 cells. After 5-8 weeks CSF T cell lines (TCL) were almost pure TCR alpha beta+CD4+ cells of the Th1/Th0 type. The TCL showed reactivity to MBP, MOG and/or PLP as tested by Elispot and had a restricted clonality. Three immunizations with irradiated CSF vaccines (10 million cells) were administered with an interval of 2 months. The vaccinations were tolerated well and no toxicity or adverse effects were reported. The data from this small open-label study cannot be used to support efficacy. However, all patients remained clinically stable or had reduced EDSS with no relapses during or after the treatment. Proliferative responses against the CSF vaccine were observed in 3/5 patients. Anti-ergotypic responses were observed in all patients. Anti-MBP/PLP/MOG reactivities remained low or were reduced in all patients. Based on these encouraging results, we recently initiated a double-blind placebo-controlled trial with 60 MS patients to study the effects of TCV with CSF-derived vaccines in early RR MS patients.     

T-cell vaccination in multiple sclerosis: update on clinical application and mode of action

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). Autoreactive T cells specific for myelin antigens are considered to play a prominent role in the initiation of the local inflammatory response, ultimately leading to myelin damage. Several studies indicate that autoreactive T cells are not completely deleted in the thymus, but are part of the normal T cell repertoire. Accidentally activated autoreactive T cells, however, may not automatically lead to autoimmune disease. Several reports support the existence of peripheral regulatory networks that prevent the activation and expansion of pathogenic T cells. Anti-idiotypic and anti-ergotypic T cells are part of this regulatory network and are thought to control autoreactive T cells by recognition of certain clonotypic and ergotypic determinants. These clonotypic networks may not function properly in patients with MS. Immunization with attenuated autoreactive T cells, termed T cell vaccination (TCV), may enhance or restore the regulatory networks to specifically suppress the autoreactive T cells as shown in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model for MS. In the past decade, TCV has been tested for MS in several clinical trails. This review summarizes these clinical trails and updates our current knowledge on the mode of action of T cell vaccination.