Analysis Of T Cell Responses In Leishmaniasis

利什曼病 T 细胞反应分析

• 批准号: 6985685
• 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/6985685
• 关键词: CpG islands; Leishmania major; Primates; active immunization; cellular immunity; cytotoxic T lymphocyte; dendritic cells; enzyme linked immunosorbent assay; flow cytometry; helper T lymphocyte; human tissue; immune response; immunomodulators; interleukin 10; laboratory mouse; leishmaniasis; leukocyte activation /transformation; nonhuman therapy evaluation; polymerase chain reaction; protozoal vaccine; tissue /cell culture

Despite the fact that intracellular pathogens that are confined within host cell phagosomes are able to induce CD8+ T cells in vitro and in vivo, and in the case of Leishmania this response is required for protection, the mechanisms controlling the cross-presentation of phagosome-derived antigens are poorly understood.To facilitate the study of cross-presentation of Leishmania antigens, we engineered L. major parasites to express a fragment of ovalbumin containing the SIINFEKL epitope recognized by TCR transgenic OT-I CD8+ T cells, as part of a parasite 3? nucleotidase protein. In vivo and in vitro, both secreted and cytosolic forms of NT-OVA were efficiently cross-presented to CD8+ OT-I T cells. The presentation of the SIINFEKL peptide by L. major NT-OVA-infected DC in vitro or by APCs involved in cross-priming in vivo, was not affected by the absence of TAP1, suggesting that the conventional MHC class I processing pathway is not used for this particular epitope. When we extended these studies to the cross-presentation of wild-type L. major antigens recognized by a polyclonal population of in vivo primed CD8+ T cells, a dispensable role for TAP1 was still observed. Furthermore, TAP1 deficient mice infected with a low dose of L. major in the ear were able to control infection as efficiently as wild type mice. In parallel studies, we observed that cross presentation of OVA secreted by transfected Toxoplasma gondii was strictly TAP dependent in vitro and in vivo. Thus in the case of Leishmania, exogenous antigen processing and presentation does not appear to follow a phagosome-to-cytosol pathway as it has been reported for latex beads and other intracellular microorganisms. A role for lysosomal hydrolases in the processing of Leishmania antigens was indicated by the strong inhibition of cross-priming observed using leupeptin treated DC. The L. major murine model has been useful to emphasize the importance of Th1/Th2 balance in resistance or susceptibility to Leishmania infection. However, Th2 polarization, as occurs during L. major infection in BALB/c mice, has failed to account for severe or non-cure forms of leishmaniasis in humans. The role of other disease promoting factors might be more easily studied in mouse strains that do not maintain a confounding, strong Th2 bias during infection. We have conducted a series of experiments in C57BL/6 mice involving an L. major strain, Seidman (Sd), that in contrast to the majority of L. major isolates, produces non-healing lesions in these mice. In B6 mice that developed non-healing dermal lesions, a polarized IFN-g response, with no detectable IL-4, was measured in the CD4+ T cells from the lesion and draining node, and the magnitude of this response even exceeded that seen in the mice infected with a healing strain of L. major. Thus Th1 response defects and Th2 immune deviation cannot explain the failure of these mice to control infection. A role for CD4+CD25+ regulatory T cells was indicated by the results of CD25 depletion experiments, which increased parasite killing in the site. The treatment of mice with IL-10R antibody had an even more pronounced effect than CD25+ depletion on promoting healing and parasite clearance. Thus Treg and IL-10 are essential susceptibility factors in this model, and their effects seem not so much to inhibit Th1 priming as to suppress the expression of effector activities in the inflammatory site. We observed in our studies of Sd infection in B6 mice that despite their inability to heal their primary site of infection, when the animals were re-challenged in the contra-lateral ear, they were completely protected. Thus the Treg and other suppressive mechanisms that function within the primary inflammatory site to prevent effective clearance by Th1 cells do not inhibit the expression of Th1 effector activity in a naive rechallenge site. We have argued that the activation of Treg during infection provides a benefit to the host by not only controlling the severity of inflammation, but by maintaining a chronic infection the Treg will also maintain an effector memory pool necessary for resistance to reinfection. Since individuals with healed lesions and presumed persistent infection 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 leishmanization protocols that minimize pathology while maintaining long term protection, live parasites have been co-injected with TLR agonists, either CpG ODNs or imiguimod, into Rhesus monkeys. The CpG ODN (D class) provoked a transient inflammation but reduced the size of the cutaneous lesion that developed. Thus, 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.

尽管限制在宿主细胞吞噬体内的细胞内病原体能够在体外和体内诱导 CD8+ T 细胞，并且就利什曼原虫而言，这种反应是保护所必需的，但控制吞噬体来源的交叉呈递的机制为了促进利什曼原虫抗原交叉呈递的研究，我们对大型利什曼原虫寄生虫进行了改造，使其表达含有识别的 SIINFEKL 表位的卵清蛋白片段由 TCR 转基因 OT-I CD8+ T 细胞作为寄生虫的一部分 3？核苷酸酶蛋白。在体内和体外，NT-OVA 的分泌形式和胞质形式均有效地交叉呈递给 CD8+ OT-I T 细胞。 SIINFEKL 肽在体外由 L. Major NT-OVA 感染的 DC 或由参与体内交叉引发的 APC 呈递，不受 TAP1 缺失的影响，表明不使用传统的 MHC I 类加工途径对于这个特定的表位。当我们将这些研究扩展到由体内引发的 CD8+ T 细胞多克隆群体识别的野生型大利斯特氏菌抗原的交叉呈递时，仍然观察到 TAP1 的可有可无的作用。此外，在耳朵中感染低剂量硕大利斯特菌的 TAP1 缺陷小鼠能够像野生型小鼠一样有效地控制感染。在平行研究中，我们观察到转染的弓形虫分泌的 OVA 的交叉呈递在体外和体内均严格依赖于 TAP。因此，在利什曼原虫的情况下，外源抗原的加工和呈递似乎并不遵循吞噬体到胞质溶胶的途径，正如乳胶珠和其他细胞内微生物所报道的那样。使用亮肽素处理的 DC 观察到的交叉引发的强烈抑制表明了溶酶体水解酶在利什曼原虫抗原加工中的作用。大利什曼原虫鼠模型有助于强调 Th1/Th2 平衡在抵抗或易感利什曼原虫感染方面的重要性。然而，在 BALB/c 小鼠中发生重大利什曼病期间发生的 Th2 极化未能解释人类严重或无法治愈的利什曼病。其他疾病促进因素的作用可能更容易在感染期间不保持混杂的强 Th2 偏好的小鼠品系中进行研究。我们在 C57BL/6 小鼠中进行了一系列实验，涉及主要利斯特氏菌菌株 Seidman (Sd)，与大多数主要利斯特氏菌分离株相比，该菌株在这些小鼠中产生不愈合的损伤。在出现不愈合真皮损伤的 B6 小鼠中，在来自损伤和引流结的 CD4+ T 细胞中测量到了极化的 IFN-g 反应，没有检测到 IL-4，这种反应的强度甚至超过了在感染了 L. Major 治愈菌株的小鼠。因此，Th1 反应缺陷和 Th2 免疫偏差不能解释这些小鼠未能控制感染。 CD25 耗竭实验的结果表明了 CD4+CD25+ 调节性 T 细胞的作用，这增加了该部位的寄生虫杀伤力。用 IL-10R 抗体治疗小鼠在促进愈合和寄生虫清除方面比 CD25+ 消除具有更显着的效果。因此，Treg 和 IL-10 是该模型中重要的易感因子，它们的作用似乎与其说是抑制 Th1 启动，不如说是抑制炎症部位效应活性的表达。我们在 B6 小鼠 Sd 感染的研究中观察到，尽管它们无法治愈其主要感染部位，但当这些动物的对侧耳朵再次受到攻击时，它们受到了完全的保护。因此，在原发性炎症位点内发挥作用以防止 Th1 细胞有效清除的 Treg 和其他抑制机制不会抑制初始再攻击位点中 Th1 效应子活性的表达。我们认为，感染期间 Treg 的激活不仅可以控制炎症的严重程度，而且通过维持慢性感染，Treg 还将维持抵抗再感染所需的效应记忆库，从而为宿主带来益处。由于病变已治愈且假定持续感染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，但仍然存在问题，因为疫苗接种损伤的严重程度。为了开发利什曼化方案，在保持长期保护的同时最大限度地减少病理，将活寄生虫与 TLR 激动剂（CpG ODN 或咪吉莫特）共同注射到恒河猴中。 CpG ODN（D 类）会引起短暂的炎症，但会缩小所形成的皮肤病变的大小。因此，使用 CpG ODN 进行免疫调节可能是提高已广泛应用的高效活疫苗安全性的实用方法。