Non-canonical epitope presentation and antigen processing by MHC-E

项目摘要 在巨细胞病毒(CMV)作为一种新的疫苗平台诱导效应分化的过程中 T细胞免疫，我们观察到表达猴免疫缺陷(SIV)抗原的恒河猴CMV(RhCMV) 激发免疫反应，控制并最终清除高致病性SIV。然而，令人惊讶的是，保护 仅在诱导CD8+T细胞的转基因载体中观察到受非多态、高度 保守的MHC-E而不是经典的MHC-I。因此，以人类白细胞抗原-E呈递的HIV多肽为靶标代表了一种 新的、意想不到的和非常规的艾滋病疫苗开发方法最近进入了 临床阶段。然而，我们对导致这种现象的分子机制的了解非常有限。 CMV是唯一一种能够引起对任何抗原的非常规反应的疫苗载体 慢病毒感染的细胞易受T细胞控制。我们的发现MHC-E呈现各种各样的抗原 是意想不到的，因为MHC-E主要结合非单体VMAPRTL(L，I，V，F)L(VL9)自体多肽 包含在MHC-I的可切割前导序列中。在这里，我们的目标是阐明MHC-E是如何加载的 在未感染细胞或在体外感染HIV或CMV的细胞中的各种非典型多肽，并鉴定 人巨细胞病毒感染细胞及其MHC-E限制性CD8+T细胞启动的分子机制 活着。这些目标将由一个具有相关经验的国际调查小组来实现。 目的1是使用一套独特的MHC-E/多肽特异性试剂来监测MHC-E多肽的载量和 髓系细胞和HIV感染细胞抑制特定细胞蛋白和途径的递呈。蜂窝 目标将从CRISPR/CAS9初步屏幕的点击中选择，或基于它们在 经典MHC分子的囊泡运输或多肽负载。在目标2中，我们将调查广泛的 在感染CMV的细胞中观察到的细胞内泡结构的重组有助于负载 MHC-E与非标准多肽结合。一个特别的重点将是病毒microRNA的作用，它可以重定向 靶向囊泡分离蛋白的囊泡运输及其对MHC-E限制性T细胞的影响 巨细胞病毒感染细胞的刺激。病毒microRNAs的作用以及选择的宿主诱导途径 体内的T细胞将在AIM 3中进行检测。这将通过产生重组的RhCMV来完成 缺少microRNA或表达宿主基因靶向的小发夹RNA。因为最近的结果表明 MHC-E限制性T细胞的启动需要感染表达microRNA142的髓系细胞，我们将鉴定 通过将复杂的成像和空间相结合，感染的细胞类型和特征邻近的T细胞 转录切割技术。我们预计这项研究的结果将对基础和翻译产生影响 免疫学，包括新型疫苗和免疫疗法的设计。

Project Summary In the course of developing cytomegalovirus (CMV) as a new vaccine platform for eliciting effector differentiated T cell immunity, we observed that rhesus CMV (RhCMV) expressing simian immunodeficiency (SIV) antigens elicit immune responses that control and ultimately clear highly pathogenic SIV. Surprisingly however, protection was only observed with genetically modified vectors eliciting CD8+ T cells restricted by non-polymorphic, highly conserved MHC-E instead of classical MHC-I. Targeting HIV peptides presented by HLA-E thus represents a novel, unexpected and unconventional approach to AIDS vaccine development that has recently entered the clinical phase. However, we have only a very limited understanding of the molecular mechanisms that render CMV the only vaccine vector capable of eliciting these unconventional responses to any antigen and that render lentivirus-infected cells vulnerable to T cell control. Our findings that MHC-E presents a wide variety of antigens is unexpected because MHC-E predominantly binds the nonameric VMAPRTL(L,I,V,F)L (VL9) self-peptide contained in the cleavable leader sequence of MHC-I. Here, our goal is to elucidate how MHC-E is loaded with diverse non-canonical peptides in uninfected cells or in cells infected with HIV or CMV in vitro and to identify the RhCMV-infected cells and molecular mechanisms required for the priming of MHC-E restricted CD8+ T cells in vivo. These objectives will be accomplished by an international team of investigators with relevant experience. Aim 1 is to use a unique set of MHC-E/peptide specific reagents to monitor MHC-E peptide loading and presentation by myeloid and HIV-infected cells upon inhibiting specific cellular proteins and pathways. Cellular targets will be selected from hits of preliminary CRISPR/cas9 screens or based on their known function in vesicular traffic or peptide loading of classical MHC molecules. In aim 2, we will investigate how the extensive reorganization of intracellular vesicular structures observed in CMV-infected cells contributes to the loading of MHC-E with non-canonical peptides. A particular emphasis will be on the role of viral microRNAs which redirect vesicular traffic by targeting vesicular sorting proteins and which seem to be required for MHC-E restricted T cell stimulation by CMV-infected cells. The role of viral microRNAs as well as selected host pathways for the induction of these T cells in vivo will be examined in aim 3. This will be accomplished by generating recombinant RhCMV lacking microRNAs or expressing host gene-targeting small hairpin RNAs. Since recent results suggest that priming of MHC-E restricted T cells requires infection of myeloid cells expressing micro-RNA142, we will identify the infected cell type and characterize neighboring T cells by combining sophisticated imaging and spatial transcriptomic techniques. We expect that the results of this research will impact basic and translational immunology including the design of novel vaccines and immunotherapeutics.