Immune Receptors on Cytotoxic Lymphocytes and Target Cells

描述（由申请人提供）：颗粒介导的CTL裂解靶细胞的显著敏感性和特异性已经得到证实，并且被认为是CTL高度选择性和快速破坏病毒感染细胞所必需的。CTL介导的靶细胞破坏的敏感性和有效性机制尚不完全清楚。最近，观察到不同CTL克隆对靶细胞裂解敏感性的变化，为研究造成这些差异的因素提供了机会，并进一步了解如何使用含有横向移动单体ICAM-1和肽- mhc （pMHC）的支持脂质双分子层来控制靶细胞裂解的敏感性来模拟靶细胞表面。我们将研究免疫突触（IS）的形成以及不同靶细胞裂解敏感性的CTL对颗粒极化和释放的模式。我们还将研究CD8和CD4共受体在CD4+和CD8+ CTL募集颗粒的不同模式中的作用。我们将利用新开发的基于半导体纳米晶体的生物传感器，称为量子点（QD），承载预定数量的具有各种活性的紧密间隔的pMHC复合物，模拟MHC在靶细胞膜上聚集，并将评估pMHC-pMHC接近对CTL激活的意义。最后，基于对病毒学突触（VS）的研究，我们提出了一种关于hiv特异性CD4+ CTL功能的新观点，其中激活的T细胞聚集在突触的中心簇中gp120-CD4相互作用。这些CD4细胞簇维持Lck的激活，表明可能与TCR细胞簇协同作用，促进细胞溶解颗粒在分泌域的聚焦。为了验证这一假设，我们将研究gp120介导的信号传导对暴露于hiv感染细胞的平面双层模型表面的CD4+ CTL的细胞溶解颗粒极化和释放模式的影响。同时，我们还将确定gp120-CD4相互作用对CD4+ CTL裂解靶细胞敏感性的影响。这些实验有望揭示CTL敏感破坏HIV感染细胞的机制，并有望更好地了解CD4+ CTL在HIV特异性免疫中的作用。进一步了解CTL根除病毒感染细胞的细胞毒性机制将有助于开发新的策略来提高细胞毒性淋巴细胞的性能，并使患者能够更好地抵抗HIV感染。

DESCRIPTION (provided by applicant): Remarkable sensitivity and specificity of granule-mediated target cell lysis by CTL is well established and is thought to be necessary for highly selective and rapid destruction of virus-infected cells by CTL. Mechanisms underlying the sensitivity and efficacy of CTL mediated destruction of target cells are not entirely clear. Recently, observed variations in the sensitivity of target cell lysis by different CTL clones offer an opportunity to investigate factors responsible for these differences and to learn more about how the sensitivity of target cell lysis is controlled using supported lipid bilayers containing laterally mobile monomeric ICAM-1 and peptide-MHC (pMHC) to model the surface of target cells. We will examine immunological synapses (IS) formation and patterns of granule polarization and release by CTL with different sensitivity of target cell lysis. We will also examine the role of CD8 and CD4 co-receptors in differential patterns of granule recruitment by CD4+ and CD8+ CTL. We will utilize newly developed biosensors, based on semiconductor nanocrystals, called quantum dots (QD), bearing pre-determined numbers of closely spaced pMHC complexes with various activities to mimic MHC clustering on target cell membranes and will evaluate the significance of the pMHC-pMHC proximity for CTL activation. Finally, we propose a novel view concerning the function of HIV-specific CD4+ CTL based on studies of virological synapse (VS), in which activated T cells cluster gp120-CD4 interactions in a central cluster of the synapse. These CD4 clusters sustain activation of Lck suggesting a possible synergy with TCR clusters that facilitates focusing of cytolytic granules in the secretory domain. To test the hypothesis we will examine the effect of gp120-mediated signaling on pattern of cytolytic granule polarization and release in CD4+ CTL exposed to planar bilayers modeling surface of HIV-infected cells. In parallel, we will also determine the impact of gp120-CD4 interactions of the sensitivity of target cell lysis by CD4+ CTL. The proposed experiments are expected to reveal mechanisms of sensitive destruction of HIV infected cells by CTL and promise to better understand the role of CD4+ CTL in HIV-specific immunity. Further understanding of the mechanisms of cytotoxicity exercised by CTL eradicating virus-infected cells will be instrumental for the development of new strategies to enhance the performance of cytotoxic lymphocytes and to enable patients to better fight HIV infection. Narrative: Using cutting edge imaging technology, we will explore molecular mechanism of granule release by virus-specific cytotoxic T lymphocytes (CTL) accounting for effective and rapid destruction of virus-infected cells. The results of proposed experiments will likely lead to the development of new approaches to enhance sensitivity and specificity of virus-specific CTL in the context of anti-viral therapy.