NK AND TARGET CELL BIODYNAMIC MECHANISMS

该项目的长期目标是阐明， 生理过程之间的调节和功能联系， 自然杀伤（NK）细胞和被NK细胞（靶细胞）结合的细胞， 是NK-靶细胞相互作用的生物学后果的基础。 拟议研究的直接目标是调查，在NK和 靶细胞，细胞内时间变化的多种机制 pH（pHi）和离子化Ca2+浓度（[Ca2 +] i），两种细胞 功能可能与生理相关，并决定生理 导致靶细胞溶解的过程。 为了实现这些目标，我们 建议使用从人外周血和人细胞中分离的NK细胞 细胞系，在谱系和对NK细胞介导的裂解的敏感性方面不同， i）对NK细胞的变化进行相关的时间测量， 和靶细胞[Ca2 +] i和pHi，其作为NK-1的结果而启动。 靶细胞粘附。 将通过流量进行补充测量 流式细胞术（以辨别细胞中参数变化的异质性和动力学） 大群体的NK-靶细胞缀合物）和成像细胞术（以 获得更详细和空间分辨的动力学信息， 分离的缀合物）; ii）在流式细胞仪中在 NK-靶细胞[Ca2 +] i和pHi反应模式的基础。 协会 反应模式和靶细胞溶解之间的关系将在单次 分选到琼脂糖包被的载玻片上的缀合物的细胞毒性测定; 和iii）确定对缀合相关的时间变化的影响， [Ca2+] i和pHi暴露NK和靶细胞的条件， 调节细胞溶解功能（细胞因子，生物活性单核苷酸， 烷化剂），确定的细胞跨膜离子流（HCO3，Na+， Ca~（2+）、Cl~-、H~+）和pHi（酸化、碱化）。 一体化 这些实验的结果将确定[Ca2 +] i的变化模式 以及NK-靶细胞粘附导致的pHi;这些模式如何变化 （频率、振幅、动力学）作为操纵细胞溶解的函数 活性和靶细胞特性（谱系，NK敏感性）; 模式与目标的出现和不出现相关联 细胞溶解;以及NK细胞和靶细胞控制[Ca2 +] i和 在这些不同的实验条件下的pHi波动模式。 的 因此，拟议的研究将提供更全面的看法， NK细胞和靶细胞影响免疫应答的生理过程。 这种潜在的细胞溶解相互作用的结果，这将进一步 了解NK细胞在人类重要生物过程中的作用 健康（即，肿瘤和病毒感染细胞的裂解，B的调节 胸腺和骨髓中的细胞和发育过程），从而 为健康相关的治疗性NK细胞提供合理依据 操纵

The long term goal of this project is to elucidate characteristics of, regulation of, and functional links between physiological processes in natural killer (NK) cells and cells bound by NK cells (target cells) which underlie the biological consequences of the NK-target cell interaction. The immediate goals of proposed studies are to investigate, in both NK and target cell, the multiple mechanisms of temporal changes in intracellular pH (pHi) and concentration of ionized Ca2+ ([Ca2+]i), two cellular functions likely to be associated with, and determinative of, physiological processes leading to target cell cytolysis. To accomplish these goals, we propose to use NK cells isolated from human peripheral blood and human cell lines, differing in lineage and sensitivity to NK cell-mediated lysis, as target cells to: i) make correlated temporal measurements of changes in NK and target cell [Ca2+]i and pHi which are initiated as a consequence of NK- target cell adhesion. Complementary measurements will be made by flow cytometry (to discern heterogeneity and kinetics of parameter changes in large populations of NK-target cell conjugates) and imaging cytometry (to obtain more detailed and spatially resolved kinetics information in isolated conjugates); ii) sort conjugates in the flow cytometer on the basis of NK-target cell [Ca2+]i and pHi response patterns. The association between response pattern and target cytolysis will be evaluated in single cell cytotoxicity assays of conjugates sorted onto agarose-coated slides; and iii) determine effects upon conjugation-associated temporal changes in [Ca2+]i and pHi of exposing NK and target cells to conditions which modulate cytolytic function (cytokines, bioactive mononucleotides, alkylating agents), defined cellular transmembrane ion flows (HCO3, Na+, Ca2+, Cl-, H+), and pHi (acidification, alkalinization). Integration of results form these experiments will identify patterns of change in [Ca2+]i and pHi which result from NK-target cell adhesion; how these patterns vary (frequency, amplitude, kinetics) as a function of manipulating cytolytic activity and target cell properties (lineage, NK-sensitivity); what patterns are associated with occurrence and non-occurrence of target cytolysis; and mechanisms by which NK and target cells govern [Ca2+]i and pHi fluctuation patterns under these various experimental conditions. The proposed studies will thus provide a more comprehensive view of physiological processes by which NK cells and target cells influence the outcome of this potentially cytolytic interaction which will further the understanding of the NK cell role in biological processes central to human health (i.e., lysis of tumors and virus-infected cells, regulation of B cells and developmental processes in thymus and marrow), and thereby provide a rational basis for health-related therapeutic NK cell manipulations.