Antigen export in M. tuberculosis evasion of CD4 T cells

最近一项临床试验的结果表明，开发有效的结核病疫苗的一个主要障碍是 其中疫苗诱导了多功能T细胞反应，但它并没有预防结核病。这一障碍也是 在动物模型中显示，因为现有的结核病疫苗诱导T细胞反应，但对 细菌的负担。这些结果表明，抗原特异性T细胞是由感染或接种疫苗产生的 它们可以通过体外再刺激激活，但在感染部位不能有效激活。 该项目的长期目标是通过以下方式指导有效结核病疫苗的开发：1)表征 限制抗原特异性CD4T细胞识别结核分枝杆菌感染能力的主要机制 细胞并在感染部位被激活；以及2)开发克服或绕过这一点的解决方案 机制。我们发现，结核分枝杆菌感染细胞对CD4T细胞的激活能力很差，原因是 多个分泌的细菌抗原从MHC II类抗原加工中分流出来 呈现途径，并从感染细胞输出。这种新的机制，我们称之为抗原 出口，涉及细胞内的囊泡运输，需要微管导向的分子马达， 激动素-2。当我们耗尽结核分枝杆菌感染细胞的激动素2，从而阻止抗原输出时，我们发现 感染细胞增加MHC II类抗原提呈和CD4T细胞激活，导致改善 胞内结核分枝杆菌的控制。这表明CD4T细胞具有发挥有效作用的潜力。 抗分枝杆菌活性，但它们的活性受到感染细胞抗原提呈差的限制，而且 抗原呈递是次要的抗原输出。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，包括抗原输出小泡(AEV)的萌发 来自吞噬小体，AEV的细胞内靶向，AEV膜与质膜的融合 将抗原释放到细胞外空间。我们的目标是确定可以作为靶点的宿主分子 阻止抗原输出并使抗原特异性的CD4T细胞在结核病中更有效的药物。通知结核病 疫苗设计，我们假设非分泌(和非输出)抗原比 结核疫苗中的分泌性抗原，因为分泌性抗原是从受感染的细胞输出的，使 被感染的细胞很难被针对这些抗原的CD4T细胞识别。来检验这一假设 针对非分泌性抗原的CD4T细胞比针对非分泌性抗原的CD4T细胞更有效 分泌型抗原，我们对免疫优势分泌型结核分枝杆菌抗原(Ag85B)进行了修饰，使其成为 不分泌，我们将确定表达非分泌-非出口Ag85B的细菌是否更好 体内受CD4T细胞控制。总之，我们提议的研究将指导人们努力使自然发生 而疫苗诱导的CD4T细胞在结核病中更有效，并有助于解决全球性的 肺结核。

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.