Role of antigen processing HIV CTL immunodominance

描述(由申请人提供)：细胞毒性T淋巴细胞(CTL)在HIV感染的免疫控制中是必不可少的，免疫优势的CTL反应已被表征为许多HLA等位基因。然而，某些表位相对免疫优势的原因尚未确定，形成免疫优势表位的抗原处理步骤也尚不清楚。此外，还没有研究研究HIV-1感染者体内积累的人类白细胞抗原限制性突变如何影响表位的产生。我们的长期目标是了解管理特定HIV抗原的降解和呈递以及通过突变导致的抗原呈递受损的规则。在初步研究中，我们已经确定了主要HIV表位侧翼区域中的等位基因特异性突变，这些突变会损害CTL对抗原的处理和识别。我们已经证明，人类白细胞抗原限制性突变可以影响内质网(ER)中较长的多肽前体的修剪，从而改变I类抗原表位的相对可用性。我们假设，抗原处理影响表位之间观察到的层级，表位侧翼区域的特定HLA相关突变改变了处理的不同步骤，并作为免疫逃逸的机制。这项提案将侧重于详细分析可能导致病毒逃逸的抗原处理途径的变化。其具体目的包括：1.确定CD8 T细胞表位的胞浆处理过程，重点研究人类白细胞抗原A3/A11限制性反应；2.确定内质网中多肽的N-糖基化在多大程度上是病毒逃避CTL反应的潜在机制。这些目标基于一个共同的实验设计。我们将跟踪在抗原提呈细胞中对感兴趣的HIV表位的完整处理，或者用亚细胞部分重建这一过程。我们将分析蛋白酶体和其他多肽酶对胞浆中HIV多肽的降解，内质网内N-延伸多肽的移位和N-延伸多肽的修剪和潜在的N-糖基化，多肽在MHC-I分子上的负载及其随后呈现给CTL。鉴于HIV的极端变异率和持续的免疫选择压力，这些研究是确定抗原处理在免疫逃逸中的作用的关键的第一步，这不仅对于理解HIV的免疫发病机制，而且对于设计候选疫苗都是重要的。

DESCRIPTION (provided by applicant): Cytotoxic T-lymphocytes (CTL) are essential for immune control in HIV infection, and immunodominant CTL responses have been characterized for many HLA alleles. However the reasons for relative immunodominance of certain epitopes have not been defined, and the steps in antigen processing that shape an immunodominant epitope are not known. Moreover no studies have examined how HLA-restricted mutations that accumulate in HIV-1 infected people affect the generation of epitopes. Our long-term goal is to understand the rules that govern degradation and presentation of specific HIV antigens and the impairment of antigen presentation through mutations. In preliminary studies, we have identified allele-specific mutations in the flanking regions of dominant HIV epitopes that impair antigen processing and recognition by CTL. We have shown that an HLA-restricted mutation can affect trimming of longer peptide precursors in the endoplasmic reticulum (ER), thereby altering the relative availability of epitopes for class I loading. We hypothesize that antigen processing influences the hierarchy observed among epitopes and that specific HLA-associated mutations in flanking regions of epitopes alter distinct steps of the processing and serve as a mechanism for immune escape. This proposal will focus on a detailed analysis of changes in antigen processing pathways that may lead to viral escape. The specific aims include: 1. To define cytosolic processing ofCD8 T cell epitopes, focusing on HLA-A3/A11-restricted responses for initial studies; 2. To determine the extent to which N-glycosylation of peptides in the ER serves as a potential mechanism of viral escape from CTL responses. These goals are based on a common experimental design. We will follow the complete processing of HIV epitopes of interest in antigen presenting cells or reconstruct this process with subcellular fractions. We will analyze the degradation of HIV polypeptides in the cytosol by the proteasome and other peptidases, the translocation into the ER and the trimming and potential N-glycosylation of N-extended peptides in the ER lumen, the loading of peptides onto MHC-I molecules and their subsequent presentation to CTL. Given the extreme mutation rate of HIV and the persistence of immune selection pressure, these studies represent a critical first step in determining the role of antigen processing in immune escape, which will be important not only for the understanding of HIV immunopathogenesis but also for the design of candidate vaccines.