Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells.
Direct interrogation of viral peptides presented by the class I HLA of HIV-infected T cells.
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直接询问 HIV 感染 T 细胞的 I 类 HLA 呈递的病毒肽。
DOI：
10.1128/jvi.01914-14
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发表时间：
2014
影响因子：
5.4
通讯作者：
Brander
中科院分区：
文献类型：
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作者：
Yaciuk,JaneC;Skaley,Matthew;Bardet,Wilfried;Schafer,Fredda;Mojsilovic,Danijela;Cate,Steven;Stewart,ChristopherJ;McMurtrey,Curtis;Jackson,KennethW;Buchli,Rico;Olvera,Alex;Cedeño,Samandhy;Plana,Montserrat;Mothe,Beatriz;Brander
CD 8+细胞毒性T淋巴细胞（CTL）表位的鉴定传统上依赖于测试重叠肽库与T细胞的体外反应性。在这里，我们追求深度配体测序（DLS）作为直接鉴定那些配体的替代方法，这些配体是由感染细胞的I类人白细胞抗原（HLA）呈递给CTL的表位。可溶性I类HLA-A*11：01（sHLA）从HIV-1 NL 4 -3感染的人CD 4 +SUP-T1细胞中收集。将从感染细胞收获的HLA-A*11：01免疫亲和纯化并酸煮以从肽配体释放重链和轻链，然后通过尺寸排阻过滤回收。首先通过高pH高压液相色谱法对配体进行分馏，然后在低pH下通过纳米液相色谱法（nano-LC）-质谱法（MS）进行分离。选择约1000万个离子通过串联质谱法（MS/MS）进行测序。用PEAKS软件确定HLA-A*11：01配体序列，并通过与合成肽产生的光谱进行比较来确认。DLS鉴定了42种由HLA-A*11：01呈递的病毒配体，其中37种以前未检测到。这些数据表明，（i）HIV-1 Gag和Nef被广泛采样，（ii）配体长度变异是普遍的，特别是在Gag和Nef热点内，其中配体序列重叠，（iii）非规范配体是T细胞反应性的，和（iv）HIV-1配体是来自新生合成，而不是内吞采样。下一代免疫疗法必须考虑这些新生的HIV-1配体长度变异和CTL反应性表位可能在CD 4 +T细胞感染过程中缺失的发现，以增强T细胞免疫力。重要信息由洛斯阿拉莫斯国家实验室（LANL）编目的HIV-1表位在疫苗试验中取得了有限的成功。由于受感染细胞的HLA以前没有评估过HIV-1配体，因此本文的目的是直接表征标记受感染细胞的病毒配体。从HIV-1感染的CD 4 +T细胞中回收HLA呈递的肽，并通过质谱DLS对肽货物进行询问，表明典型和非典型病毒配体由HLA有效呈递，并被人CTL靶向。Nef和Gag配体主导感染细胞的抗原谱，这主要是由于从这些病毒蛋白内的选择热点进行的广泛配体采样。此外，HIV-1配体通常比预期的更长，并且这些长度变体具有相当的抗原性。这些发现强调了基于HLA的HIV-1配体呈递给CTL的观点提供了以前未实现的信息，这些信息可能会增强免疫疗法和疫苗的开发。
Identification of CD8+cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cellsin vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)–mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived fromde novosynthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+T cells into strategies designed to enhance T cell immunity.IMPORTANCEHIV-1 epitopes catalogued by the Los Alamos National Laboratory (LANL) have yielded limited success in vaccine trials. Because the HLA of infected cells have not previously been assessed for HIV-1 ligands, the objective here was to directly characterize the viral ligands that mark infected cells. Recovery of HLA-presented peptides from HIV-1-infected CD4+T cells and interrogation of the peptide cargo by mass spectrometric DLS show that typical and atypical viral ligands are efficiently presented by HLA and targeted by human CTLs. Nef and Gag ligands dominate the infected cell's antigenic profile, largely due to extensive ligand sampling from select hot spots within these viral proteins. Also, HIV-1 ligands are often longer than expected, and these length variants are quite antigenic. These findings emphasize that an HLA-based view of HIV-1 ligand presentation to CTLs provides previously unrealized information that may enhance the development of immune therapies and vaccines.