Antigen processing and presentation of recombinant Listeria proteins

描述（由申请人提供）：CD8+ T淋巴细胞是主要的免疫效应细胞，对许多细胞内病原体（包括病毒、细菌和寄生虫）以及肿瘤作出反应。为了识别感染细胞或肿瘤细胞，CD8+ T细胞必须首先通过其T细胞受体与专业抗原呈递细胞（pAPC）表面显示的肽- mhc （pMHC）复合物相互作用来启动。这可以通过自然暴露于病原体或肿瘤抗原发生，也可以通过故意将蛋白质引入pAPC的抗原加工机制，如在接种疫苗期间所做的那样。引入蛋白以引发CD8+ T细胞反应的最有效方法之一是使用活的、减毒的载体表达该蛋白，如细胞内细菌、单核增生李斯特菌。由于李斯特菌在初次感染后几分钟内进入宿主细胞的细胞质，由细菌分泌的蛋白质很容易被宿主细胞的抗原加工机制所接触，这使得李斯特菌成为引发CD8+ T细胞反应的特别有效的载体。因此，我们一直在研究重组李斯特菌蛋白在MHC I类分子上表达的加工机制。我们最近的研究发现，重组李斯特菌抗原的加工和呈现方式与内源性合成蛋白相比有显著差异。具体来说，我们发现分泌的李斯特菌蛋白产生pMHC的速率相同，独立于其来源的蛋白质的细胞半衰期。我们的假设是，这些加工和呈现的差异反映了负责内源性合成蛋白质和外源性细菌蛋白质降解的细胞机制的真正差异。我们在本申请中提出的研究将集中于确定哪些因素导致这些处理差异。我们将通过1)确定蛋白质半衰期和加工效率的差异是否取决于细胞的内源性蛋白质加工机制，2)确定李斯特菌定位在确定分泌重组蛋白的加工途径中所起的作用。我们的研究将为我们提供对I类抗原加工和呈递机制的更详细的了解。

DESCRIPTION (provided by applicant): CD8+ T lymphocytes are the primary immune effector cells that respond to many intracellular pathogens, including viruses, bacteria and parasites, as well as tumors. To recognize infected or neoplastic cells, CD8+ T cells must first be primed by interacting through their T cell receptor with peptide-MHC (pMHC) complexes displayed on the surface of professional antigen presenting cells (pAPC). This can occur either through natural exposure to the pathogen or tumor antigen or by intentionally introducing protein to the antigen processing machinery of a pAPC, as is done during vaccination. One of the most effective ways to introduce proteins for the purpose of priming a CD8+ T cell response is to express that protein using a live, attenuated vector such as the intracellular bacterium, Listeria monocytogenes. Because Listeria enters the host cell cytosol within minutes after initial infection, proteins secreted by the bacterium are readily accessible to the host cell's antigen processing machinery, making Listeria a particularly effective vector for eliciting CD8+ T cell responses. Therefore, we have been investigating the mechanisms involved in the processing of recombinant Listeria proteins for presentation on MHC class I molecules. Our recent studies have uncovered a striking difference in the way recombinant Listeria antigens are processed and presented when compared to endogenously synthesized proteins. Specifically, we have found that pMHC production from secreted Listeria proteins occurs at the same rate, independent of the cellular half-life of the protein from which it is derived. It is our hypothesis that these differences in processing and presentation reflect real differences in the cellular machinery responsible for the degradation of endogenously synthesized proteins vs. exogenously derived bacterial proteins. The studies we propose in this application will focus on determining what factors contribute these differences in processing. We will do this by 1) determining whether differences in protein half-life and processing efficiency are dependent on the endogenous protein processing machinery of the cell and 2) establishing the role Listeria localization plays in determining the processing pathway followed by secreted recombinant proteins. Our studies should provide us with a more detailed understanding of the mechanisms involved in class I antigen processing and presentation. PUBLIC HEALTH RELEVANCE: These studies should increase our understanding of the mechanisms of antigen processing and presentation of proteins expressed using an intracellular bacterial pathogen as a vector. How proteins are degraded and presented in infected cells can impact the quality and magnitude of the cellular immune response. This knowledge is critical to our understanding of how cellular immunity is elicited to antigenic proteins derived from pathogenic organisms and malignant cells. It is expected that this knowledge will aid in the design of effective vaccines.