The role of intestinal lymph DCs in intiating immune responses

DC可以被认为是免疫系统的侦察兵。它们的功能是穿过身体的组织，并在适当的时候启动适应性免疫反应以攻击入侵的病原体。为了执行这一功能，它们不断地从组织通过淋巴迁移到淋巴结，在那里它们能够与循环淋巴细胞相互作用。在大鼠中，我们能够以最小的体外操作收集从肠迁移的DC。这是通过首先去除肠系膜淋巴结（MLN）并允许传入和传出淋巴管愈合来实现的。以这种方式，通常会被捕获在淋巴结中的DC能够继续通过淋巴，并且可以通过将套管放置到主要淋巴管之一胸导管中来收获。胸导管插管术是一种历史悠久的技术，已被证明在体内定义DC的功能方面非常有用，使我们能够表明DC对于启动对引入肠道的抗原的免疫应答以及防止对无害的自身抗原的破坏性应答都是至关重要的。令我们惊讶的是，我们发现一种化合物（TLR 7/8配体，小分子R-848）增加淋巴中迁移的DC的数量，并激活淋巴结中的DC，当两者混合并喂给动物时，不能刺激针对蛋白质（卵清蛋白）的免疫应答。相反，饲喂E.大肠杆菌热不稳定肠毒素（Etx）与卵白蛋白混合刺激强烈的抗卵白蛋白免疫应答，而不增加迁移的DC的数量或引起淋巴结DC的活化状态的可检测的变化。随后的实验表明，增加淋巴中携带卵清蛋白的DC的数量可能是对任何免疫刺激的结果至关重要的许多因素之一。我们计划操纵纯化的DC，以确定启动免疫反应最重要的DC特性。在确定了体内免疫原性DC的最重要特征后，我们将能够开发使用培养的DC筛选潜在免疫原性化合物的方法。另外两个最近的观察结果为研究肠淋巴的新途径提供了基础。第一个是发现淋巴DC不像以前理解的那样分为两个，而是三个不同的子集。根据CD 11b/c和CD 32的表达，可以将先前鉴定的CD 172 + DC分为两组。CD 172 + CD 11b/chigh DC在肠淋巴中数量最多，而CD 172 + CD 11b/clow DC能够分泌免疫刺激性细胞因子IL-12。因为我们能够在迁移性DC到达MLN之前收集它们，所以本申请中描述的实验将使我们能够定义这3个子集迁移性DC的不同功能特性。我们还观察到胸导管淋巴液含有许多直径在50- 200 nm之间的膜结合小泡。这些囊泡类似于外泌体，外泌体是由许多细胞类型在体外产生的囊泡，包括DC。外泌体在体外与抗原呈递细胞混合时能够刺激或抑制免疫反应，并且正在癌症免疫疗法的临床试验中进行测试。然而，外来体以前没有在淋巴中被鉴定，它们的生理重要性尚不清楚。因此，我们将从肠淋巴液中纯化和表征外泌体。我们还将研究它们是否参与免疫应答的控制，无论是在稳态还是在口服佐剂后。我们相信这些实验将促进对肠道DC在口服疫苗接种中作用的理解，并有助于开发新一代口服佐剂。

DCs may be thought of as the scouts of the immune system. Their function is to pass through the tissues of the body and, when appropriate, initiate adaptive immune responses to attack invading pathogens. In order to perform this function, they are constantly migrating from tissues, via the lymph, to lymph nodes where they are able to interact with circulating lymphocytes. In rats, we are able to collect the DCs migrating from the intestine with minimal in vitro manipulation. This is achieved by first removing the mesenteric lymph nodes (MLN) and allowing the afferent and efferent lymphatics to heal. In this way, DCs that would normally be trapped in the lymph nodes are able to continue through the lymph and can be harvested by placing a cannula into one of the major lymphatic vessels, the thoracic duct. Thoracic duct cannulation is a long-established technique that has proven extremely useful in defining the functions of DCs in vivo, enabling us to show that DCs are critical both for initiating immune responses to antigens introduced into the intestine, and in preventing damaging responses against harmless self antigens. We have found, to our surprise, that a compound (a TLR7/8 ligand, the small-molecule R-848) that increases the number of migrating DCs in lymph, and activates DCs in lymph nodes, is not able to stimulate an immune response against a protein (ovalbumin) when the two are mixed and fed to animals. Conversely, feeding E. coli heat labile enterotoxin (Etx) mixed with ovalbumin stimulates a strong anti-ovalbumin immune response without increasing the number of migrating DCs or causing detectable changes in the activation state of lymph node DCs. Subsequent experiments have suggested that increasing the number of DCs carrying ovalbumin in the lymph is likely to be one of a number of factors critical to the outcome of any immune stimulation. We plan to manipulate purified DCs to define the DC characteristics most important for initiating immune responses. Having identified the most important characteristics of immunogenic DCs in vivo, we will be able to develop methods for using cultured DCs to screen potential immunogenic compounds. Two other recent observations have provided the basis for new avenues of research into intestinal lymph. The first is the discovery that lymph DCs fall not into two, as previously understood, but three distinct subsets. The previously-identified CD172+ DCs can be separated into two groups based on their expression of both CD11b/c and CD32. The CD172+ CD11b/chigh DCs are the most numerous in intestinal lymph, while CD172+ CD11b/clow DCs are able to secrete IL-12, an immunostimulatory cytokine. Because we are able to collect migratory DCs before they reach the MLN, the experiments described in this application will enable us to define the different functional properties of these 3 subsets migratory DCs. We have also observed that thoracic duct lymph contains many membrane-bound vesicles between 50-200nm in diameter. These vesicles resemble exosomes, vesicles that are generated by a number of cell types in vitro, including DCs. Exosomes, when mixed with antigen-presenting cells in vitro are able to stimulate or suppress immune responses, and are being tested in clinical trials for cancer immunotherapy. However, exosomes have not previously been identified in lymph and their physiological importance is unclear. We will therefore purify and characterize the exosomes from intestinal lymph. We will also investigate whether they are involved in the control of immune responses, either in he steady state or after the administration of oral adjuvants. We believe that these experiments will advance understanding of the role of intestinal DCs in oral vaccination, and help with the development of a new generation of oral adjuvants.