Deeply analyzing MHC class I-restricted peptide presentation mechanistics across alleles, pathways, and disease coupled with TCR discovery/characterization

项目摘要/摘要 ·我们的框架假设是，肽加工、HLA等位基因特异性和 多肽编辑产生了显著的“漏斗效应”，其中多肽只占很小一部分 从细胞蛋白质组和配基组获得的所有可能的多肽只占一小部分 所有来自细胞多肽组的多肽。我们的研究重点是了解差异的相互作用 每一步的序列识别最终通知所有的高度受限的子集的功能呈现 可能的蛋白质组源多肽--以及如何从配位体中识别最“可翻译”的Phla靶标。 ·我们将使用我们的等位基因特定质谱学平台发现多肽，严格测试这一假设， Artemis，从间皮蛋白和高危HPVE6/E7癌蛋白中鉴定相关的人类白细胞抗原限制性多肽。 已鉴定的多肽将得到验证，并进行生化表征。细胞表面展示的多肽阵列来自 将使用E6/E7和Mesothelin来确定多肽加工的漏斗效应的程度 多肽组合物。患者T细胞对已识别的间皮蛋白肽的反应将使用状态- 最先进的方法论。不寻常的多肽和交替的结合构象将被表征为晶体- 图形化的。我们将评估病毒免疫逃避机制、HLA-G呈递和受体相互作用， 和糖基化多肽的呈现。可交付成果包括绘制完整的间鞘蛋白和E6/E7 跨多个等位基因和呈现方式的呈现组，被列为未来临床利用的免疫系统 不是治疗的目标。 ·意义：从基础科学的角度来看，分析细胞多肽和配基是基本的 为了了解免疫系统如何应对感染和癌症，免疫逃避机制， MHC蛋白的识别规则，抗原处理/编辑的机制，以及如何定义自我。从… 从翻译的角度来看，有效的人类白细胞抗原限制性多肽代表了诊断和治疗的靶点， 通过治疗性疫苗接种、基于工程T细胞的过继免疫疗法和基于抗体的识别 识别特定的疾病相关多肽/人类白细胞抗原复合体我们的研究在以下方面推进了这两个目标 阐明了控制多肽加工和呈递的潜在分子原理。 对一组扩展的经典和非经典人类HLAI类蛋白进行了格栅研究，在这个过程中， 确定对未来临床应用有用的特定Phlas。

Project Abstract/Summary • Our framing hypothesis is that the combined constraints of peptide processing, HLA allelic specificity, and peptide editing generate a significant “funnel effect”, where the peptidome constitutes only a tiny percentage of all possible peptides obtainable from a cell’s proteome and the ligandome constitutes only a small percentage of all peptides from a cell’s peptidome. Our research focus is understanding how the interplay of differential sequence recognition at each step ultimately informs functional presentation of a highly restricted subset of all possible proteome-derived peptides – and how to identify the most “translatable” pHLA targets from ligandomes. • We will rigorously test this hypothesis using our allele-specific mass-spec platform for peptide discovery, ARTEMIS, to identify relevant, HLA-restricted peptides from Mesothelin and high-risk HPV E6/E7 oncoproteins. Identified peptides will be validated and characterized biochemically. Cell-surface displayed peptide arrays from E6/E7 and Mesothelin will be used to determine the magnitude of the “funnel effect” of peptide processing on peptidome composition. Patient T cell responses to identified Mesothelin peptides will be mapped using state- of-the-art methodologies. Unusual peptides and alternate binding conformations will be characterized crystallo- graphically. We will assess viral immunoevasion mechanisms, HLA-G presentation and receptor interactions, and the presentation of glycosylated peptides. Deliverables include mapping the full Mesothelin and E6/E7 presentomes across multiple alleles and presentation modalities, ranked for future clinical exploitation as immu- notherapy targets. • Significance: From a basic science perspective, assaying cellular peptidomes and ligandomes is fundamental for understanding how the immune system responds to infections and cancer, immunoevasion mechanisms, the recognition rules of MHC proteins, the mechanistics of antigen processing/editing, and how self is defined. From a translational perspective, validated HLA-restricted peptides represent targets for diagnosis and therapy, through therapeutic vaccination, engineered T cell-based adoptive immunotherapies, and antibody-based recog- nition of specific, disease-associated peptide/HLA complexes Our proposed studies advance both objectives by elucidating the underlying molecular principles governing peptide processing and presentation through inte- grated studies of an extended set of classical and non-classical human HLA class I proteins and, in the process, identify specific pHLAs useful for future clinical applications.