Enhancing Immunotherapeutic Value of Lymphocytes by Controlling Apoptosis

本研究的主要目的是确定被细胞因子激活的NK和T细胞在遇到靶细胞上的激活配体时发生凋亡的机制。我们观察到，在体外用细胞因子IL-2和IL-15刺激的人原代NK和T细胞，当它们的激活受体发生交叉连接时，显示出广泛的凋亡潜力。为了进一步研究这种激活诱导的NK细胞死亡，我们借鉴了我们早期的基因芯片研究。我们的基因芯片数据显示，在IL-2刺激的人NK细胞培养中，抗凋亡分子TOSO的表达下调了5倍。TOSO又称Fas凋亡抑制分子3(FAIM3)，是免疫球蛋白基因超家族的成员，具有抑制Fas介导的细胞凋亡的作用。Toso主要由淋巴细胞表达。然而，Toso在NK细胞激活的生理背景中的作用及其功能还不是很清楚。这促使我们研究Toso在激活诱导NK细胞以及T和B细胞中的细胞死亡中的作用。我们用实时定量聚合酶链式反应和细胞表面单抗染色定量了Toso的相对表达水平。我们发现Toso的表达与抵抗Fas介导的细胞死亡有关，相反，Toso表达的下调与对Fas介导的激活诱导的细胞死亡(AICD)的易感性有关。不出所料，我们发现在IL-2刺激下，人类NK细胞表达高水平的Fas死亡受体。我们证实了转Toso基因的Jurkat T细胞从对AICD敏感转变为耐药。为了研究Toso基因在这一现象中的作用，我们在原代培养的人NK细胞中过表达了人Toso基因。我们还计划建立稳定表达Toso的细胞系模型。我们还培育了Toso转基因小鼠，并获得了用于这些研究的Toso基因敲除小鼠。

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.