FUNCTIONAL TCR ANALYSIS OF SIV SPECIFIC CTL

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 我们已经完成了这项研究，以表征TCR库和TCR的功能进化，识别免疫过程中出现的显性和亚显性SIV-CTL表位。 我们证明了疫苗诱导的表位特异性CD 8 + 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CD 8 + T淋巴细胞群体可以持续存在。 此外，在接种疫苗的猴队列中，表位特异性CD 8 + T淋巴细胞群体的克隆组成在用质粒DNA/rMVA接种疫苗的猴和用rAd接种疫苗后的猴中几乎相同。 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呈递或加工效率和T细胞功能差异不太可能是免疫优势的机制。然而，TCR库的分析揭示了占优势的p11 C特异性CTL群体使用更高数量的TCR克隆。特异性TCR的优先使用和体外功能性TCR-α和-β链配对测定表明，每种肽/MHC复合物只能被有限数量的α和α β链对的独特组合识别。优势p11 C特异性CTL群体所使用的TCR克隆的更广泛的阵列可以通过产生那些特异性TCR链对的更高概率来解释。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，这决定了SIV特异性CD 8 + T细胞应答的免疫优势。为了进一步了解抗原特异性CTL应答的基本功能特性，详细比较了有效增殖所需的最佳抗原浓度和对不同表位特异性CTL之间的特异性功能性细胞毒活性的敏感性。在这项研究中，我们证明，无论其免疫优势等级如何，每个CTL都需要低肽浓度（0.01- 1 nM之间）才能在体外进行最佳CTL扩增。 有趣的是，每个CTL的功能性细胞毒性敏感性低于其体外增殖的敏感性。更重要的是，细胞毒活性的敏感性是非常均匀的每一个研究的CTL。这种同质性特征将使我们能够区分真实的表位特异性CTL群体与交叉反应性CTL群体。最后，我们还证明了在体外CTL扩增系统与新鲜的PBMC，单核细胞/巨噬细胞和B细胞没有作为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异性CTL。

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 alpha 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.