ANALYSIS OF T CELL RESPONSES IN HUMAN LEISHMANIASIS

我们之前建立了一种皮肤利什曼病模型，该模型由L. major感染引起，结合了自然传播的两个主要特征；在小鼠耳真皮中接种少量的超环原毛菌。在这个模型中，小的、愈合的皮肤病变的演变发生在三个不同的阶段，这些阶段以前没有被认识到：1)初始的？阶段，持续4-5周，有利于在没有病变形成的情况下在真皮中建立寄生虫的峰值负荷；2)急性期，持续5-10周，对应于病变的发展和消退，与中性粒细胞、巨噬细胞和嗜酸性粒细胞急性浸润到真皮有关，与免疫的开始和部位寄生虫的杀死相一致；3)慢性阶段，持续动物的一生，在没有明显病理的情况下，少量寄生虫持续存在于皮肤中。该模型中的适应性免疫证实了Th1细胞的作用，此外还显示了对CD8+ T细胞的需求。2微球蛋白KO小鼠，CD8 KO小鼠和CD8缺失小鼠，每种情况下都未能控制皮肤感染。还研究了愈合后有利于皮肤中寄生虫数量持续减少的条件。慢性部位的特点是真皮中大量CD8+和CD4+ T淋巴细胞能够对抗原产生IFN g。在体内用抗ifng、抗cd4 +或抗cd8 +抗体治疗可导致寄生虫数量的急剧增加和皮肤病变的再激活。因此，IFNg压力对于维持较低的寄生虫负担是必要的，但不足以清除寄生虫。寄生虫的持续存在取决于在该部位产生IL-10，证据如下：a)在耳朵和引流淋巴结中存在IL-10染色细胞，b)抗il - 10r抗体治疗导致寄生虫从皮肤上完全清除，c) IL-10 KO和IL-4/IL-10 KO小鼠皮肤上完全清除L. major。低剂量的皮内感染模型已扩展到引起人源性皮肤利什曼病的热带乳杆菌。虽然寄生虫的生长受到控制，但没有皮肤病理，寄生虫不会清除，并且在动物的一生中，皮肤中仍存在可传播水平的寄生虫血症。这种平衡再次被发现是由IFNg和IL-10的平衡水平建立的。IL-10缺失小鼠和抗il - 10r抗体小鼠的皮肤均清除了热带乳杆菌引起的慢性感染。利用自然攻毒模型比较了用编码抗原LACK、M15和MAPS的质粒dna混合接种疫苗与热灭原毛菌加重组IL-12 （IL-12）接种疫苗的效力和持久性。虽然这两种疫苗都对皮肤利什曼病具有完全的保护作用，但在接种DNA疫苗的小鼠中，这种保护作用持续的时间更长。此外，只有DNA疫苗在急性和慢性阶段将皮肤中的寄生虫负担降低到愈合小鼠中达到的低水平，并且只有DNA疫苗消除了受激小鼠作为媒介沙蝇感染宿主的能力。人类利什曼病的临床形式从自愈的皮肤病变到通常致命的内脏疾病不等。这些不同的临床结果主要归因于引起感染的利什曼原虫种类的差异。使用与自限性皮肤疾病相关的物种L. major的动物模型显示，保护性免疫需要CD40/ cd40l依赖、il -12驱动的Th1反应。我们发现，与主要利什曼原虫（L. major）不同，导致内脏疾病的利什曼原虫（L. dononvani），以及与持续性、皮肤病变和偶尔的全系统疾病相关的利什曼原虫（L. tropica），不会为CD40L诱导的IL-12p70的产生启动人类树突状细胞，并且寄生虫与DC相互作用的这些内在差异可能至少部分地解释了利什曼原虫愈合和非愈合形式的进化。

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.