Antigen export in M. tuberculosis evasion of CD4 T cells

最近一项临床试验的结果说明了开发有效结核疫苗的一个主要障碍， 疫苗诱导多功能T细胞反应，但它不能预防结核病。这一障碍也是 在动物模型中说明，因为现有的TB疫苗诱导T细胞应答，但对 细菌负荷这些结果表明，抗原特异性T细胞是通过感染或接种疫苗产生的 并且它们可以通过离体再刺激而被激活，但是它们在感染部位不能被有效地激活。 该项目的长期目标是通过以下方式指导有效结核疫苗的开发：1）表征 这是限制抗原特异性CD 4 T细胞识别M的能力的主要机制。结核病感染者 细胞并在感染部位被激活;以及2）开发克服或绕过 机制我们发现M。结核病感染的细胞激活CD 4 T细胞很差， 多种分泌的细菌抗原从MHC II类抗原加工中分流， 这些蛋白质通过递呈途径从受感染的细胞输出。我们称之为抗原的新机制 输出，涉及胞内囊泡运输，并需要微管定向分子马达， 驱动蛋白-2。当我们耗尽M。结核感染的细胞驱动蛋白2，从而阻止抗原输出，我们发现， 通过感染的细胞增加MHC II类抗原呈递和CD 4 T细胞活化，导致改善的 胞内M.结核这表明，CD 4 T细胞具有发挥有效作用的潜力。 它们具有抗分枝杆菌活性，但它们的活化受到感染细胞的抗原呈递差和抗分枝杆菌活性差的限制。 抗原呈递是抗原输出的第二步。该项目将扩展这些发现，并描述 抗原输出所需的其他细胞机制，包括抗原输出囊泡（AEV）的出芽 从吞噬体，AEV的细胞内靶向，以及AEV膜与质膜的融合， 将抗原释放到细胞外空间。我们的目标是鉴定出可以被靶向的宿主分子， 阻断抗原输出并使抗原特异性CD 4 T细胞在结核病中更有效的药物。告知结核病 在疫苗设计中，我们假设非分泌（和非输出）抗原比 结核病疫苗中的分泌抗原，因为分泌抗原是从受感染的细胞中输出的， 感染的细胞缺乏被那些抗原特异性的CD 4 T细胞识别的靶标。为了验证这个假设， 针对非分泌性抗原的CD 4 T细胞比针对非分泌性抗原的CD 4 T细胞更有效。 分泌抗原，我们已经修改了免疫显性分泌M。结核抗原（Ag 85 B），使其 不分泌，我们将确定表达非分泌非输出Ag 85 B的细菌是否更好 在体内由CD 4 T细胞控制。总之，我们提出的研究将指导努力，使自然发生的 而疫苗诱导的CD 4 T细胞对结核病更有效，有助于解决全球结核病问题。 结核

A major obstacle to developing an efficacious TB vaccine is illustrated by the results of a recent clinical trial in which the vaccine induced polyfunctional T cell responses, yet it did not prevent TB. This obstacle is also illustrated in animal models, as existing TB vaccines induce T cell responses, but have only modest effects on bacterial burdens. These results suggest that antigen-specific T cells are generated by infection or vaccination and they can be activated by ex vivo restimulation, but they are not activated effectively at the site of infection. The long term objective of this project is to guide development of efficacious TB vaccines by: 1) characterizing a major mechanism that limits the ability of antigen-specific CD4 T cells to recognize M. tuberculosis-infected cells and become activated at the site of infection; and 2) developing solutions that overcome or bypass that mechanism. We have discovered that M. tuberculosis-infected cells activate CD4 T cells poorly because multiple secreted bacterial antigens are shunted away from the MHC class II antigen processing and presentation pathway and are exported from the infected cells. This novel mechanism, which we term antigen export, involves intracellular vesicular transport, and requires the microtubule-directed molecular motor, kinesin-2. When we deplete M. tuberculosis-infected cells of kinesin 2 and thus block antigen export, we find increased MHC class II antigen presentation and CD4 T cell activation by infected cells, resulting in improved control of intracellular M. tuberculosis. This indicates that CD4 T cells have the potential to exert effective antimycobacterial activity, but their activation is limited by poor antigen presentation by infected cells, and poor antigen presentation is secondary to antigen export. This project will extend these findings and characterize the other cellular mechanisms required for antigen export, including budding of antigen export vesicles (AEV) from phagosomes, intracellular targeting of AEV, and fusion of AEV membranes with the plasma membrane for release of antigens to the extracellular space. Our goal is to identify host molecules that can be targeted with drugs to block antigen export and make antigen-specific CD4 T cells more effective in TB. To inform TB vaccine design, we hypothesize that nonsecreted (and nonexported) antigens are more desirable than secreted antigens in TB vaccines, since secreted antigens are exported from infected cells, making the infected cells poor targets for recognition by CD4 T cells specific for those antigens. To test the hypothesis that CD4 T cells directed against a nonsecreted antigen are more efficacious than those directed against a secreted antigen, we have modified an immunodominant secreted M. tuberculosis antigen (Ag85B) so that it is not secreted, and we will determine whether bacteria that express nonsecreted-nonexported Ag85B are better controlled by CD4 T cells in vivo. Together, our proposed studies will guide efforts to make naturally-occurring and vaccine-induced CD4 T cells more effective in TB, and contribute to solving the problem of global tuberculosis.