AGE-ASSOCIATED ALTERATIONS IN HUMAN NK CELL SYSTEM

IL2缺乏和对IL2的反应性降低是导致 高龄时T细胞免疫力下降。然而，目前还不清楚是否 自然杀伤(NK)细胞，非特异性抗肿瘤效应和 抗病毒免疫在衰老过程中也会受到影响。凭借以下优势 他们分泌细胞因子的能力，如干扰素-γ(干扰素-γ) 迅速地，NK细胞可能在早期阶段发挥重要作用。 感染/恶性肿瘤/免疫调节。我们观察到了一种衰老-- 纯化的人NK细胞体外敏感性的相关下降 LL2，即释放干扰素-γ或LAK活性。我们的长期目标是 为了研究细胞和分子机制参与的 IL-2诱导的细胞因子分泌功能优先下降 NK细胞在衰老过程中的作用。 我们推测，在衰老过程中，可能会有一个频率降低 IL-2应答、分泌干扰素-γ的NK细胞和/或IL-2缺陷- IL-2受体水平上的NK细胞相互作用或表达 IL-2受体γ链或干扰素-γ基因，导致干扰素- 每个细胞的伽马分泌。 为了验证这一假设，排序后的IL2释放的LFN-伽马量 将测量激活的NK亚群。CD56(亮)和CD56(暗)， 细胞代表两个不同的NK亚群。干扰素-γ的产生频率 健康的年轻人和老年人的NK细胞中的分泌物将是 由Elispot定量。流式细胞术定量分析 CD56(明亮的)(不成熟的)和CD56(暗的)(成熟的)NK亚群 搞定了。为了研究潜在缺陷的IL2-NK细胞相互作用， α(CD25)和β(CD122)LL2受体链的表达 (流式细胞术)及其对IL-2的亲和力或每NK细胞密度 (与(125)I-L12的平衡结合研究)将被测量。统治 在IL2的延迟移位之外，内化的动力学 将遵循单元格表面边界L12。找出潜在的缺陷 在干扰素-γ基因的表达中，LL2细胞中的干扰素-γ的mRNA水平 激活的、分选的NK细胞将通过定量RT-PCR进行分析 使用聚合酶链式反应模拟。IL2R-Gamma的mRNA水平也将通过 聚合酶链式反应。因为与衰老相关的T和B细胞DNA合成下降 我们以前注意到，IL-2诱导的NK细胞的增殖能力 将会被评估。这些结果将在理论上对 恶性肿瘤、感染与免疫调节及其治疗应用 在免疫药理学干预和细胞因子基因治疗中， 尤其是在老年患者中。

IL2 deficiency and reduced responsiveness to IL2 contribute to the diminished T-cell immunity at advanced age. However, it is not clear if natural killer (NK) cells, the non-specific effectors of anti-tumor and anti-viral immunity are also affected during senescence. By virtue of their ability to secrete cytokines, e.g. interferon gamma (IFN-gamma) rapidly, NK cells may play an important role in the early phases of infection/ malignancy/immunoregulation. We have observed a senescence- related decline in in vitro sensitivity of purified human NK cells to lL2, i.e., release of IFN-gamma or LAK activity. Our long term goal is to investigate the cellular and molecular mechanisms involved in the preferential decline in IL2 inducible cytokine secretory functions of NK cells during senescence. We hypothesize that during senescence, there may be a reduced frequency of IL2 responding, IFN-gamma secreting NK cells and/or a defect in IL2- NK cell interaction at the level of IL2 receptors or the expression of IL2 receptor gamma chain or IFN-gamma genes, resulting in impaired IFN- gamma secretion per cell. To test this hypothesis, the amount of lFN-gamma released by sorted, IL2 activated NK subsets will be measured. The CD56(bright) and CD56(dim), cells represent the two distinct NK subsets. The frequency of IFN-gamma secretors among NK cells from healthy young and elderly will be quantitated by ELISPOT. Quantitative flow cytometric analysis of CD56(bright) ("immature") and CD56(dim) ("mature") NK subsets will be done. To investigate a potentially defective IL2-NK cell interaction, the expression of alpha (CD25) and beta (CD122) lL2 receptor chains (flow cytometry) and their affinity to IL2 or density per NK cell (equilibrium binding studies with (125)I-lL2) will be measured. To rule out a delayed translocation of IL2, the kinetics of internalization of cell surface bound lL2 will be followed. To locate a potential defect in IFN-gamma gene expression, the level of IFN-gamma mRNA in lL2 activated, sorted NK cells will be analyzed by quantitative RT-PCR by using PCR MIMICS. The level of IL2R-gamma mRNA will also be measured by PCR. Since a senescence-related decline in T- & B-cell DNA synthesis was noted by us before, the proliferative potential of IL2 induced NK cells will be assessed. These results will have theoretical implications in malignancy, infection and immune regulation and therapeutic applications in immunopharmacological interventions and cytokine gene therapies, especially in elderly patients.