Enhancing Immunotherapeutic Value of Lymphocytes by Controlling Apoptosis

通过控制细胞凋亡增强淋巴细胞的免疫治疗价值

• 批准号: 7732615
• 负责人: John E Coligan
• 金额: $ 35.44万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732615
• 关键词: Apoptosis; Apoptotic; B-Lymphocytes; Biological Assay; Cell Death; Cell Line; Cell surface; Cells; Cessation of life; Cultured Cells; Data; Down-Regulation; Effectiveness; Genes; Growth; Half-Life; Human; Immunoglobulin Genes; Immunotherapeutic agent; In Vitro; Interleukin-15; Interleukin-2; Knockout Mice; Ligands; Lymphocyte; Mediating; Modeling; Molecular; NK Cell Activation; Natural Killer Cells; Physiological; Polymerase Chain Reaction; Predisposition; Process; Property; Receptor Activation; Relative (related person); Resistance; Role; Staining method; Stains; T-Lymphocyte; Therapeutic; Time; Transgenic Mice; Transplantation; base; crosslink; cytokine; cytokine therapy; in vivo; interest; member; programs; receptor

The main objective of this study is to determine the mechanism by which NK and T cells activated with cytokines undergo apoptosis when they encounter activating ligands on target cells. We observed that human primary NK and T cells that were stimulated in vitro with the cytokines IL-2 and IL-15 showed extensive potential to undergo apoptosis when their activation receptors were cross linked. In order to study this activation induced cell death in NK cells further, we drew incite from our earlier microarray study. Our microarray data on genes that are modulated upon stimulation of human NK cells in culture with IL-2 showed that the antiapoptotic molecule, TOSO was down regulated in cells cultured with IL-2 by 5 fold. TOSO, also called as Fas Apoptotic Inhibitory Molecule 3 (FAIM3) is a member of immunoglobulin gene superfamily and it inhibits Fas mediated apoptosis. TOSO is expressed mainly by lymphocytes. However, the role of TOSO in the physiological context of NK cell activation and their function is not well known. This prompted us to look into the role of TOSO in activation induced cell death in NK cells, as well as in T and B cells. We quantitated the relative levels of TOSO expression using real time PCR assays and by cell surface staining using a Mab. We found Toso expression correlated with resistance to Fas mediated cell death and conversely, the down-regulation of TOSO expression correlated with susceptibility to Fas- mediated activaton induced cell death (AICD). As expected, we found that human NK cells when stimulated with IL-2 expressed high levels of the Fas death receptor . We verified that Jurkat T cells transfected with TOSO change from AICD susceptible to being resistant. In order to study the involvement of TOSO in this phenomenon, we are in the process of over expressing the human TOSO gene in primary human NK cells. We are also planning to establish a cell line model for stable expression of TOSO. We have also generated TOSO transgenic mice and obtained TOSO knockout mice for these studies.

这项研究的主要目的是确定NK和T细胞在遇到靶细胞上激活配体时通过细胞因子激活凋亡的机制。我们观察到，在体外与细胞因子IL-2和IL-15体外刺激的人类原代NK和T细胞在交叉链接激活受体时表现出广泛的发生凋亡的潜力。为了研究这种激活诱导NK细胞中的细胞死亡，我们从较早的微阵列研究中汲取了煽动。我们对使用IL-2培养的人NK细胞调节基因的微阵列数据表明，在用IL-2培养的细胞中调节抗凋亡分子，TOSO下调了TOSO。 TOSO，也称为FAS凋亡抑制分子3（FAIM3）是免疫球蛋白基因超家族的成员，它抑制了FAS介导的凋亡。 TOSO主要用淋巴细胞表达。然而，TOSO在NK细胞激活及其功能的生理环境中的作用尚不清楚。这促使我们研究了TOSO在活化诱导的NK细胞以及T和B细胞中的作用。我们使用实时PCR分析和使用MAB的细胞表面染色来定量TOSO表达的相对水平。我们发现TOSO表达与对FAS介导的细胞死亡的抗性相关，相反，TOSO表达的下调与FAS介导的Activaton诱导细胞死亡（AICD）的敏感性相关。 正如预期的那样，我们发现用IL-2刺激时的人NK细胞表达高水平的FAS死亡受体。我们验证了用TOSO从AICD转移的Jurkat T细胞易受抗性的变化。为了研究TOSO在这种现象中的参与，我们正在过度表达原代人NK细胞中人类TOSO基因。我们还计划建立一个细胞系模型，以稳定地表达TOSO。我们还产生了TOSO转基因小鼠，并为这些研究获得了TOSO敲除小鼠。