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

项目摘要 在开发巨细胞病毒（CMV）作为诱导效应分化的新疫苗平台的过程中， T细胞免疫，我们观察到恒河猴CMV（RhCMV）表达猴免疫缺陷（SIV）抗原， 引发免疫反应，控制并最终清除高致病性SIV。令人惊讶的是，保护 仅在遗传修饰的载体中观察到，所述载体引发受非多态性、高度多态性限制的CD 8 + T细胞。 保守的MHC-E而不是经典的MHC-I。因此，靶向HLA-E呈递的HIV肽代表了一种新的治疗方法。 一种新的、意想不到的、非传统的艾滋病疫苗开发方法，最近已经进入了 临床阶段。然而，我们对导致这种疾病的分子机制只有非常有限的了解。 CMV是唯一能够引发对任何抗原的这些非常规应答的疫苗载体， 慢病毒感染的细胞易受T细胞控制。我们发现MHC-E呈现多种抗原， 这是出乎意料的，因为MHC-E主要结合九聚体VMAPRTL（L，I，V，F）L（VL 9）自身肽 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是如何装载 在体外未感染的细胞或感染HIV或CMV的细胞中的多种非规范肽，并鉴定 RhCMV感染的细胞和MHC-E限制性CD 8 + T细胞启动所需的分子机制 vivo.这些目标将由一个具有相关经验的国际调查小组来完成。 目的1是使用一组独特的MHC-E/肽特异性试剂来监测MHC-E肽负载， 在抑制特定的细胞蛋白和途径后，通过骨髓和HIV感染的细胞呈递。蜂窝 将从初步CRISPR/cas9筛选的命中中或基于它们在CRISPR/cas9筛选中的已知功能来选择靶标。 囊泡运输或经典MHC分子的肽装载。在目标2中，我们将研究广泛的 在CMV感染的细胞中观察到的细胞内囊泡结构的重组有助于 具有非典型肽的MHC-E。特别强调的是病毒microRNA的作用， 通过靶向囊泡分选蛋白来促进囊泡运输，并且这似乎是MHC-E限制性T细胞 CMV感染细胞的刺激。病毒microRNA的作用以及所选的诱导宿主途径 将在目标3中检查这些T细胞的体内分布。这将通过产生重组RhCMV来实现 缺乏microRNA或表达靶向宿主基因的小发夹RNA。由于最近的研究结果表明， 启动MHC-E限制性T细胞需要感染表达micro-RNA 142的骨髓细胞，我们将鉴定 通过结合复杂的成像和空间分析， 转录组学技术。我们希望这项研究的结果将影响基础和翻译 免疫学，包括新疫苗和免疫治疗剂的设计。

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.