Analysis Of T Cell Responses In Leishmaniasis

我们以前建立了一种皮肤利什曼原虫感染的模型，结合了自然传播的两个主要特征：将少量的后循环前鞭毛体接种到小鼠耳部真皮中。根据在β2微球蛋白KO小鼠、CD8 KO小鼠和CD8耗竭小鼠中获得的结果，该模型中的适应性免疫证实了Th1细胞的作用，此外还揭示了对CD8+T细胞的需求，这些小鼠都未能控制皮肤感染。目前正在使用表达卵清蛋白的转基因寄生虫研究感染过程中CD8+T细胞的诱导，对于这种寄生虫，有针对显性第I类限制性表位的TCR转基因T细胞。感染的树突状细胞在体内和体外以不依赖于TAP的方式有效地处理I类表位呈递给转基因T细胞，提示利什曼原虫特异性CD8+T细胞的交叉激发不一定涉及外源抗原的胞浆处理和内质网转位。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。寄生虫的存留依赖于局部IL-10的产生，IL-10 KO和IL-4/IL-10 KO小鼠以及在慢性期用抗IL-10R抗体治疗的野生型小鼠皮肤中的主要L.IL-10被证明是由CD4+CD25+免疫调节T细胞产生的，这是一种细胞系，以前被认为其主要功能仅限于调节外周耐受。由于抗IL-10受体抗体治疗而获得无菌治愈的小鼠不再对再次感染具有免疫力。采用自然攻击模型，比较编码LACK、M15和MAP抗原的质粒DNA与热灭活的前鞭毛体加重组IL-12(rIL-12)免疫的效力和持久性。虽然两种疫苗都对皮肤疾病提供了完全的保护，但这种保护在DNA疫苗接种的小鼠中持续的时间更长，并且至少部分依赖于CD8+T细胞。加入免疫刺激序列的寡脱氧核苷酸CpG作为佐剂可显著提高灭活疫苗或重组疫苗的耐受性。同样是以CD8依赖的方式。由于愈合的皮损对再感染具有终身免疫力，因此使用被称为利什曼化的毒力较强的主要利什曼原虫的疫苗接种仍然是可实现的获得性免疫效力和持久性的黄金标准，但由于接种皮损的严重性，仍存在问题。在初步研究中，加入寡脱氧核苷酸CpG免疫刺激序列，无论是否在活疫苗中添加灭活的前鞭毛体，都几乎完全减弱了皮肤病理，而不损害初次感染建立对再次感染的长期免疫的能力。人类利什曼病的临床形式从自我修复的皮肤损害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种类的不同。使用一种与自限性皮肤病相关的物种的动物模型显示，保护性免疫需要依赖CD40/CD40L、IL-12驱动的Th1反应。我们发现，与大利什曼原虫不同，引起内脏疾病的利什曼原虫(L)以及与持续性皮损和偶尔的全身疾病相关的利什曼原虫(热带利什曼原虫)不能激发人树突状细胞产生CD40L诱导的IL-12p70。所有利什曼原虫均上调CD40、CD86和HLA-DR的表面表达，CD40L诱导的IL-12p40的分泌在未感染和感染的DC中均可观察到，无论是哪种利什曼原虫。RT-PCR分析证实，异源二聚体IL-12的产生受到IL-12p35亚基转录调控的限制，尤其依赖于微生物启动信号和CD40的参与来进行高水平的诱导。利什曼原虫物种启动树突状细胞产生CD40L依赖的IL-12p70的能力的内在差异可能至少部分地解释了治愈型和非愈合型利什曼病的进化。微生物启动DC的种间差异可能与含有多态磷酸多糖分子的表达有关，因为L.主要限制性多乳糖表位缺失的L.主要突变体不再启动DC，并在正常耐药小鼠中产生非帮助性病变。

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 induction of CD8+ T cells during infection is being studied using transgenic parasites expressing ovalbumin, for which TCR transgenic T cells specific for a dominant Class I restricted eptiope are available. Infected dendritic cells efficiently process the epitope for class I presentation to the transgenic T cells in vivo and in vitro in a TAP independent manner, suggesting that cross-priming of leishmania-specific CD8+ T cells does not necessarily involve cytosolic processing and ER translocation of exogenous antigens. The conditions favoring the persistence of low numbers of parasites in the skin following healing have also been studied. The persistence of the parasite depends upon the production of IL-10 in the site, as evidenced by the complete clearance of L. major from the skin in IL-10 KO and IL-4/IL-10 KO mice and in wild type mice treated during the chronic phase with anti-IL-10R antibodies. The IL-10 was shown to be produced by CD4+CD25+ immunoregulatory T cells, a lineage of cells whose primary fucntion was previously thought to be confined to the regulation of peripheral tolerance. Mice that achieved sterile cure as a consequence of anti-IL-10 receptor antibody treatment were no longer immune to reinfection. 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 disease , this protection lasted longer in the DNA vaccinated mice, and was dependent, at least in part, on CD8+ T cells. Inclusion of oligodeoxynucleotide CpG immunostimulatory sequences as adjuvant significantly enhanced the durability of killed or recombinant vaccines. again in a CD8 dependent manner. 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, but remains problematic because of the severity of the vaccination-lesions. 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. The inter-species differences in microbial priming of DC may be related to expression of polymorphic phosphoglycan-containing molecules, since L. major mutants spefically deficient in L. major restricted polygalactose epitopes no longer prime DC and produce non-helaing lesions in normally resistant mice.