ANALYSIS OF T CELL RESPONSES IN HUMAN 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。在这个模型中，小的、愈合的皮肤损伤的演变经历了三个不同的阶段，这是以前没有意识到的：1)最初的？沉默的？急性期，持续4-5周，有利于在没有病变形成的情况下建立真皮中寄生虫的高峰负荷；2)急性期，持续5-10周，对应于与中性粒细胞、巨噬细胞和嗜酸性粒细胞急性渗透到真皮相关的病变的发展和消退，与开始免疫和杀死部位的寄生虫相一致；和3)慢性期，持续动物的生命，在此期间，在没有明显病理的情况下，少量寄生虫持续存在于皮肤中。根据在2微球蛋白KO小鼠、CD8 KO小鼠和CD8耗竭小鼠中获得的结果，该模型中的获得性免疫证实了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。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。慢性部位的特点是真皮中CD8和CD4T淋巴细胞数量较多，能够产生针对抗原的干扰素-g。体内用抗IFNG、抗CD4或抗CD8抗体治疗后，寄生虫数量显著增加，皮肤损伤重新激活。因此，IFNG压力对于维持较低的寄生虫负担是必要的，但不足以清除它。寄生虫的持久性取决于局部IL-10的产生，证据是：a)耳朵和引流淋巴结中存在IL-10染色的细胞，b)用抗IL-10R抗体治疗后，寄生虫从皮肤上完全清除，c)在IL-10KO和IL-4/IL-10KO小鼠中，主要的L.低剂量的皮内感染模型已经扩展到热带利什曼原虫，它是人乳头状皮肤利什曼病的罪魁祸首。虽然寄生虫的生长被控制住了，但没有皮肤病理，寄生虫不会清除，寄生虫血症在动物的一生中会在皮肤上持续传播。这一平衡再次被发现被平衡的IFNG和IL-10水平所建立。在IL-10缺陷小鼠和用抗IL-10R抗体治疗的小鼠中，热带乳杆菌引起的慢性感染从皮肤中清除。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利什曼病提供了完全保护，但这种保护在DNA疫苗接种的小鼠中持续的时间更长。此外，只有DNA疫苗将急慢性阶段皮肤中的寄生虫负担降低到治愈小鼠的低水平，并且只有DNA疫苗消除了挑战小鼠作为媒介沙蝇的感染库的能力。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，与主要利什曼原虫不同，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系统性疾病相关的物种(热带利什曼原虫)不激发人类树突状细胞产生CD40L诱导的IL-12p70，这些寄生虫与DC相互作用的内在差异可能至少部分解释了利什曼病可治愈和不可愈合形式的演变。

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. In this model, the evolution of small, healing dermal lesions occurs in three distinct phases that have not been appreciated before: 1) an initial ?silent? phase, lasting 4-5 wks, favoring the establishment of the peak load of parasites in the dermis in the absence of lesion formation; 2) an acute phase, lasting 5-10 wks, corresponding to the development and resolution of a lesion that is associated with an acute infiltration of neutrophils, macrophages, and eosinophils into the dermis, and is coincident with the onset of immunity and the killing of parasites in the site; and 3) a chronic phase, lasting for the life of the animal, during which a low number of parasites persist in the skin in the absence of overt pathology. 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 ?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 low dose, intradermal infection model has been extended to L. tropica, which is responsible for anthroponotic cutaneous leishmaniasis. While the growth of the parasite is contained, there is no dermal pathology, parasite clearance does not occur, and a transmissible level of parasitemia persists in the skin for the life of the animal. This equilibrium was again found to be established by counter-balanced levels of IFNg and IL-10. The chronic infections due to L. tropica were cleared from the skin in IL-10 deficient mice, and in mice treated with anti-IL-10R antibodies. 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. 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, and these intrinsic differences in parasite interactions with DC may account, at least in part, for the evolution of healing and non-healing forms of leishmanial disease.