Analysis Of T Cell Responses In Human Leishmaniasis

人类利什曼病 T 细胞反应分析

• 批准号: 6503187
• 负责人: David Sacks
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6503187
• 关键词: Macaca mulatta; Psychodidae; T lymphocyte; antigen antibody reaction; cellular immunity; dendritic cells; enzyme linked immunosorbent assay; flow cytometry; host organism interaction; human tissue; interferon gamma; interleukin 12; laboratory mouse; leishmaniasis; microarray technology; microorganism immunology; nonhuman therapy evaluation; protozoal vaccine; tissue /cell culture; tumor necrosis factor alpha; vaccine development; vector vaccine

We previously established a model of cutaneous leishmaniasis due to L. major infection combining two main features of natural transmission; inoculation of a low number of metacyclic promastigotes into the mouse ear dermis. Adaptive immunity in this model confirmed a role for Th1 cells, and in addition revealed a requirement for CD8+ T cells, based on the results obtained in beta 2 microglobulin KO mice, CD8 KO mice, and CD8 depleted mice, which in each case failed to control infection in the skin. The conditions favoring the persistence of low numbers of parasites in the skin following healing have also been studied. The chronic site is characterized by a high number of both CD8+ and CD4+ T lymphocytes in the dermis that are able to produce IFN g in response to the antigen. In vivo treatment with either anti-IFNg, anti-CD4+, or anti-CD8+ antibodies resulted in a dramatic increase in parasite numbers and reactivation of dermal lesions. Thus IFNg pressure is necessary to maintain a low parasite burden but not sufficient to clear it. The persistence of the parasite depends upon the production of IL-10 in the site, as evidenced by a) the presence of IL-10 staining cells in the ear and draining lymph node, b) treatment with anti-IL-10R antibodies resulted in complete clearance of parasites from the skin, and c) the complete clearance of L. major from the skin in IL-10 KO and IL-4/IL-10 KO mice. The natural challenge model was used to compare the potency and durability of vaccination with a cocktail of plasmid DNAs encoding the antigens LACK, M15, and MAPS, with that of heat killed promastigotes plus recombinant IL-12 (rIL-12). While both vaccines conferred complete protection against dermal leishmaniasis , this protection lasted longer in the DNA vaccinated mice. Furthermore, only the DNA vaccine reduced the parasitic burden in the skin during the acute and chronic stages to the low levels achieved in healed mice, and only the DNA vaccine eliminated the capacity of challenged mice to serve as infection reservoirs for vector sand flies. Since individuals with healed lesions have life long immunity to reinfection, vaccination using virulent L. major promastigotes, termed leishmanization, remains the gold standard in terms of the potency and durability of acquired immunity that can be achieved. Can the same level of immunity be induced and sustained using live attenuated L. major? In preliminary studies, inclusion of oligodeoxynucleotide CpG immunostimulatory sequences, with or without addition of killed promastigotes to the live inoculum, has achieved virtually complete attenuation of dermal pathology without compromising the ability of the primary infection to establish long lived immunity to reinfection.The clinical forms of leishmaniasis in humans range from self-healing cutaneous lesions to often fatal visceral disease. These diverse clinical outcomes are attributed primarily to differences in the Leishmania species initiating the infections. Animal models using a species associated with self-limiting cutaneous disease, L. major, have revealed that protective immunity requires CD40/CD40L-dependent, IL-12-driven Th1 responses. We have found that in contrast to L. major, Leishmania species responsible for visceral disease (L dononvani), as well as species associated with persistent, cutaneous lesions and occasional systemic disease (L. tropica), do not prime human dendritic cells for CD40L induced IL-12p70 production. All Leishmania species upregulated surface expression of CD40, CD86 and HLA-DR, and appreciable CD40L-induced IL-12p40 secretion was observed in uninfected as well as infected DCs, regardless of species. RT-PCR analysis confirmed that the production of heterodimeric IL-12 was limited by transcriptional regulation of the IL-12p35 subunit, which was especially dependent on both a microbial priming signal and CD40 engagement for it high level induction. The intrinsic differences in the ability of Leishmania species to prime dendritic cells for CD40L dependent IL-12p70 production may account, at least in part, for the evolution of healing and non-healing forms of leishmanial disease. We have also initiated studies using Affymetrix Gene Chip analysis to compare parasite-induced gene expression patterns in human DCs. Hierarchical cluster analysis has revealed that while the majority of genes regulated by exposure to Leishmania are common between the species, a number of clusters are L. major or L. donovani specific.

我们之前建立了由大型利什曼病感染引起的皮肤利什曼病模型，结合了自然传播的两个主要特征；将少量后循环前鞭毛体接种到小鼠耳真皮中。该模型中的适应性免疫证实了 Th1 细胞的作用，此外根据在 β2 微球蛋白 KO 小鼠、CD8 KO 小鼠和 CD8 耗尽小鼠中获得的结果，还揭示了对 CD8+ T 细胞的需求，这些小鼠在每种情况下均未能控制皮肤感染。还研究了有利于愈合后皮肤中少量寄生虫持续存在的条件。慢性部位的特征是真皮中存在大量 CD8+ 和 CD4+ T 淋巴细胞，能够响应抗原产生 IFN g。使用抗 IFNg、抗 CD4+ 或抗 CD8+ 抗体进行体内治疗会导致寄生虫数量急​​剧增加以及真皮损伤的重新激活。因此，IFNg 压力对于维持低寄生虫负荷是必要的，但不足以清除它。寄生虫的持久性取决于该部位 IL-10 的产生，a) 耳朵和引流淋巴结中存在 IL-10 染色细胞，b) 用抗 IL-10R 抗体治疗导致完全清除皮肤上的寄生虫，以及 c) 在 IL-10 KO 和 IL-4/IL-10 KO 小鼠中完全清除皮肤上的 L. Major。使用自然攻击模型来比较编码抗原 LACK、M15 和 MAPS 的质粒 DNA 混合物与热灭活前鞭毛体加重组 IL-12 (rIL-12) 的疫苗接种的效力和持久性。虽然这两种疫苗都能提供针对皮肤利什曼病的完全保护，但这种保护作用在接种 DNA 疫苗的小鼠中持续时间更长。此外，只有 DNA 疫苗才能将急性和慢性阶段的皮肤寄生虫负担降低到治愈小鼠所达到的低水平，并且只有 DNA 疫苗才能消除受攻击小鼠作为媒介白蛉感染储存库的能力。由于病变愈合的个体对再感染具有终生免疫力，因此使用有毒力的大型利什曼原鞭毛体进行疫苗接种（称为利什曼化）仍然是获得性免疫的效力和持久性方面的黄金标准。使用减毒活的硕大利斯特菌能否诱导并维持相同水平的免疫力？在初步研究中，包含寡脱氧核苷酸 CpG 免疫刺激序列，无论是否在活接种物中添加杀死的前鞭毛体，已经实现了皮肤病理学的几乎完全减弱，而不损害原发感染建立长期免疫再感染的能力。人类利什曼病的危害范围从自愈性皮肤损伤到往往致命的内脏疾病。这些不同的临床结果主要归因于引发感染的利什曼原虫物种的差异。使用与自限性皮肤病相关的物种 L. Major 进行的动物模型表明，保护性免疫需要 CD40/CD40L 依赖性、IL-12 驱动的 Th1 反应。我们发现，与硕大利什曼原虫不同，导致内脏疾病的利什曼原虫 (L. dononvani) 以及与持续性皮肤病变和偶发全身性疾病相关的利什曼原虫 (热带利什曼原虫) 不会为人类树突细胞提供 CD40L诱导 IL-12p70 产生。所有利什曼原虫物种均上调 CD40、CD86 和 HLA-DR 的表面表达，并且在未感染和感染的 DC 中观察到明显的 CD40L 诱导的 IL-12p40 分泌，无论物种如何。 RT-PCR 分析证实，异二聚体 IL-12 的产生受到 IL-12p35 亚基转录调控的限制，其高水平诱导尤其依赖于微生物启动信号和 CD40 参与。利什曼原虫物种启动树突细胞产生 CD40L 依赖性 IL-12p70 的能力的内在差异可能至少部分解释了利什曼病的治愈和非治愈形式的进化。我们还启动了使用 Affymetrix 基因芯片分析来比较人类 DC 中寄生虫诱导的基因表达模式的研究。层次聚类分析表明，虽然大多数受利什曼原虫暴露调节的基因在物种之间是常见的，但许多基因簇是利什曼原虫或利什曼原虫特有的。