FUNCTIONAL TCR ANALYSIS OF SIV SPECIFIC CTL

SIV 特异性 CTL 的功能 TCR 分析

• 批准号: 7958642
• 负责人: Marcelo J Kuroda
• 金额: $ 6.04万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7958642
• 关键词: Affinity; Antigens; B-Lymphocytes; Binding; Biological Assay; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Fraction; Characteristics; Computer Retrieval of Information on Scientific Projects Database; Data; Epitopes; Evolution; Frequencies; Funding; Grant; Immunization; In Vitro; Infection; Institution; Monkeys; Peptide/MHC Complex; Peptides; Peripheral Blood Mononuclear Cell; Population; Primates; Probability; Process; Property; Research; Research Personnel; Resources; SIV; Source; System; T-Lymphocyte; United States National Institutes of Health; Vaccinated; Vaccination; Vaccines; cohort; cytotoxic; macrophage; monocyte; plasmid DNA; response

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We have completed the study to characterize the TCR repertoire and the functional evolution of TCRs that recognize dominant and subdominant SIV-CTL epitopes that arise during immunization. We demonstrated that vaccine-elicited epitope-specific CD8+ T lymphocytes have a clonal diversity comparable to that induced by SHIV-89.6P infection, and these clonal CD8+ T lymphocyte populations can persist. Moreover, in the vaccinated monkey cohort, the clonal make-up of an epitope-specific CD8+ T lymphocyte population was almost identical in monkeys vaccinated with plasmid DNA/rMVA and in monkeys following vaccination with rAd. We have also completed the first part of the study to investigate the mechanism of CTL immunodominance. Our data suggest that in our experimental system the peptide binding affinity, efficiency of Ag presentation or processing and T cell functional differences are not likely to be the mechanism responsible for immunodominance. However, the analysis of the TCR repertoire revealed the usage of higher numbers of TCR clones by the dominant p11C-specific CTL population. Preferential usage of specific TCRs and the in vitro functional TCR-alpha and -beta chain-pairing assay suggests that every peptide/MHC complex may only be recognized by a limited number of unique combinations of alpha and beta chain pairs. The wider array of TCR clones used by the dominant p11C-specific CTL population might be explained by the higher probability of generating those specific TCR chain pairs. Thus these data suggest that Ag-specific na¿ve T cell precursor frequency may be predetermined and that this dictates immunodominance of SIV-specific CD8+ T cell responses. To further understand the basic functional properties of antigen-specific CTL responses, the required optimal antigen concentration for efficient proliferation and the sensitivity of the specific functional cytotoxic activity among CTLs specific for different epitopes were compared in detail. In this study, we demonstrated that regardless of their immunodominance hierarchies, every CTL needed a low peptide concentration (between 0.01-1nM) for their optimal CTL expansion in vitro. Interestingly, the functional cytotoxic sensitivity of every CTL was lower than its sensitivity of in vitro proliferation. More importantly, the sensitivity of cytotoxic activity was very homogeneous among every CTLs studied. This homogeneous feature will allow us to distinguish the real epitope-specific CTL population from the cross-reactive ones. Finally, we have also demonstrated that in the in vitro CTL expansion system with fresh PBMC, monocyte/macrophages and B cells did not function as APCs. Only a small fraction of cells with the characteristics of DCs were capable of expanding those specific CTLs at low peptide concentration.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们已经完成了表征 TCR 库和 TCR 功能进化的研究，这些 TCR 识别免疫过程中出现的显性和次显性 SIV-CTL 表位。 我们证明，疫苗诱导的表位特异性 CD8+ T 淋巴细胞具有与 SHIV-89.6P 感染诱导的克隆多样性相当的克隆多样性，并且这些克隆 CD8+ T 淋巴细胞群体可以持续存在。 此外，在接种疫苗的猴子队列中，接种质粒 DNA/rMVA 的猴子和接种 rAd 后的猴子的表位特异性 CD8+ T 淋巴细胞群的克隆组成几乎相同。 我们还完成了研究CTL免疫优势机制的第一部分。我们的数据表明，在我们的实验系统中，肽结合亲和力、Ag 呈递或加工的效率以及 T 细胞功能差异不太可能是免疫优势的机制。然而，对 TCR 库的分析表明，占优势的 p11C 特异性 CTL 群体使用了更多数量的 TCR 克隆。优先使用特定 TCR 和体外功能性 TCR-α 和 -β 链配对测定表明，每种肽/MHC 复合物可能只能被有限数量的 α 和 β 链对的独特组合识别。占优势的 p11C 特异性 CTL 群体使用的 TCR 克隆范围更广，这可能是因为生成这些特定 TCR 链对的可能性更高。因此，这些数据表明Ag特异性幼稚T细胞前体频率可能是预先确定的，并且这决定了SIV特异性CD8+T细胞应答的免疫优势。为了进一步了解抗原特异性CTL反应的基本功能特性，详细比较了有效增殖所需的最佳抗原浓度以及针对不同表位的CTL之间的特定功能性细胞毒活性的敏感性。在这项研究中，我们证明，无论其免疫优势层次如何，每个 CTL 都需要低肽浓度（0.01-1nM 之间）才能实现最佳 CTL 体外扩增。 有趣的是，每个CTL的功能性细胞毒性敏感性均低于其体外增殖的敏感性。更重要的是，所研究的每个 CTL 的细胞毒活性敏感性都非常一致。这种同质特征将使我们能够区分真正的表位特异性 CTL 群体和交叉反应群体。最后，我们还证明，在使用新鲜 PBMC 的体外 CTL 扩增系统中，单核细胞/巨噬细胞和 B 细胞不能发挥 APC 的功能。只有一小部分具有 DC 特征的细胞能够在低肽浓度下扩增这些特定的 CTL。