Analysis Of T Cell Responses In Leishmaniasis

我们最近在一个皮肤利什曼病小鼠模型中发现，CD8+T细胞在皮肤对大型利什曼原虫原发和继发感染的免疫中发挥着重要作用。在体外共培养中，感染的树突状细胞(DC)可诱导纯化的CD8+T细胞释放干扰素-γ，表明DC能有效地处理外源性递送的抗原，并由主要组织相容性复合体(MHC)I类分子递呈。在这项研究中，我们研究了OVA257-264肽(SIINFEKL)被感染的DC处理并呈递给TCR转基因OT-I CD8+T细胞的机制。在同基因小鼠耳部真皮内接种主要NT-OVA寄生虫，可特异性地诱导引流淋巴结中转移的幼稚OT-I CD8+T细胞增殖。体外共培养条件下，大鼠NT-OVA感染的DC可诱导纯化的OT-I CD8+T细胞增殖和分泌干扰素-γ。我们进一步证明，主要NT-OVA感染的DC呈递SIINFEKL不依赖于TAP1，提示利什曼原虫特异性CD8+T细胞的交叉激发不涉及外源抗原的胞浆加工和内质网转位。我们将后一种观察结果扩展到野生型L.主要抗原的交叉呈递，表明在利什曼原虫的情况下，多肽配体被产生并加载到吞噬小体的I类上。进一步研究了在耐药小鼠愈合后，有利于低数量寄生虫在皮肤中持续存在的条件，并已扩展到对正常抵抗但未能治愈某些主要乳杆菌菌株的小鼠的研究，以及对正常敏感的BALB/c小鼠的研究，这些小鼠即使在没有IL-4受体信号的情况下也无法控制某些主要乳杆菌菌株。在每一种情况下，未能从皮肤中消除原虫至少在一定程度上与CD4+CD25+调节性T细胞产生IL-10有关，这些T细胞是感染部位的宿主和/或扩张。在每种情况下，寄生虫的清除都可以通过抗IL-10R或抗CD25来实现，这也会导致该部位产生干扰素的细胞数量急剧增加。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。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。为了开发一种在保持长期保护的同时将病理降至最低的利什曼方案，将含有CpG的免疫刺激寡核苷酸(CpG ODN)与活体寄生虫共同注射。皮内感染的C57BL/6小鼠皮内感染L.main/CpG ODN后，小鼠的皮肤几乎没有损伤，并显示出对寄生虫生长的早期抑制。CpG ODN引起一过性炎症，包括早期募集和聚集产生干扰素-g？的CD4+淋巴细胞。活疫苗的减毒不会影响其提供长期免疫的能力，因为接种主要乳杆菌/CpG ODN的小鼠在长达6个月的时间内完全不会再次感染主要乳杆菌。我们的结果表明，使用CpG ODN进行免疫调节可能是提高已被广泛应用的高效活疫苗安全性的一种实用方法。

We showed recently in a murine model of cutaneous leishmaniasis combining two main features of natural transmission, low parasite dose and inoculation into a dermal site, that CD8+ T cells play an essential role in immunity to primary and secondary infection with L. major in the skin. In co-cultures in vitro, infected dendritic cells (DC) induced the release of IFN-gamma by purified primed CD8+ T cells, indicating that DC can efficiently process exogenously delivered antigen for presentation by major histocompatibility complex (MHC) class I molecules. In this study we address the mechanisms by which the OVA257-264 peptide (SIINFEKL), expressed as part of the 3'nucleotidase (NT-OVA) by transfected L. major parasites, is processed by infected DC and presented to TCR transgenic OT-I CD8+ T cells. Inoculation of L. major NT-OVA parasites in the ear dermis of congenic mice specifically induce the proliferation of transferred naive OT-I CD8+ T cells in the draining lymph nodes. In co-cultures in vitro, L. major NT-OVA-infected DC induce the proliferation of purified naive OT-I CD8+ T cells as well as IFN-gamma secretion. We further demonstrated that presentation of SIINFEKL by L. major NT-OVA-infected DC is TAP1-independent, suggesting that cross-priming of leishmania-specific CD8+ T cells does not involve cytosolic processing and ER translocation of exogenous antigens. We extended this latter observation to wild-type L. major antigen cross presentation, suggesting that in the case of Leishmania, peptide ligands are generated and loaded onto Class I in the phagosome. The conditions favoring the persistence of low numbers of parasites in the skin following healing in resistant mice has been further studied, and has been extended to studies in normally resistant mice that fail to heal certain strains of L. major, and to normally susceptible BALB/c mice that fail to control certain strains of L. major even in the absence of IL-4 receptor signalling. In each case, the failure to eliminate prasites from the skin is related, at least in part, to production of IL-10 by CD4+CD25+ regulatory T cells that home to and/or are expanded at the site of infection. Parasite clearance can in each case be achieved by treatment with anti-IL-10R or anti-CD25, which also results in a dramatic up-regulation of the number of IFN gamma producing cells in the site. Mice that achieved sterile cure as a consequence of anti-IL-10 receptor antibody treatment were no longer immune to reinfection. 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 an attempt to develop a leishmanization protocol that minimizes pathology while maintaining long term protection, live parasites were co-injected with CpG-containing immunostimulatory oligodeoxynucleotides (CpG ODNs). C57Bl/6 mice infected intradermally using L. major/CpG ODN developed little or no dermal lesions and showed an early containment of parasite growth. The CpG ODNs provoked a transient inflammation that included an early recruitment and accumulation of IFN-g?producing CD4+ lymphocytes in the site. Attenuation of the live vaccine did not compromise its ability to confer long-term immunity, as mice receiving L. major/CpG ODN were totally protected against re-infection with L. major for up to 6 months. Our results suggest that immune-modulation using CpG ODNs might be a practical approach to improving the safety of a highly effective live vaccine that has already been widely applied.