Enhancing Immunotherapeutic Value of Activated NK Cells

本研究的主要目的是确定被细胞因子激活的人NK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细胞，当它们的激活受体交联时，显示出广泛的凋亡潜力。为了研究这个？激活诱导细胞死亡？在NK细胞中进一步，我们从早期的微阵列研究中得到了启发。我们的基因芯片数据显示，IL-2刺激培养的人NK细胞后，抗凋亡分子TOSO在IL-2培养的细胞中下调了5倍。TOSO也被称为Fas凋亡抑制分子3 (FAIM3)，是免疫球蛋白基因超家族的成员，它通过在FADD的c端结合并阻断细胞死亡途径的进一步激活来抑制Fas介导的凋亡。TOSO主要在某些种类的免疫细胞中表达。然而，TOSO在NK细胞活化的生理背景下的作用及其功能尚不清楚。这促使我们研究TOSO在活化诱导NK细胞死亡中的作用。我们还证实了TOSO水平下调的事实，并使用实时PCR测定了TOSO表达的相对水平。此外，我们发现人NK细胞在IL-2刺激下表达高水平的死亡受体Fas。为了研究TOSO在这一现象中的作用，我们正在克隆人类TOSO基因并在原代人NK细胞中过表达。我们还计划建立一个稳定表达TOSO的细胞系模型。

The main objective of this study is to determine the mechanism by which human NK cells activated with cytokines undergo apoptosis when they encounter activating ligands on target cells. We observed that human primary NK 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 anti-apoptotic 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 by binding to FADD at its C-terminus and blocking further activation of the cell death pathway. TOSO is expressed mainly in certain classes of immune cells. 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. We also confirmed the fact that TOSO levels were down regulated and quantitated the relative levels of TOSO expression using real time PCR assays. Further, we found that human NK cells when stimulated with IL-2 expressed high levels of the death receptor Fas. In order to study the involvement of TOSO in this phenomenon, we are in the process of cloning the human TOSO gene and over expressing it in primary human NK cells. We are also planning to establish a cell line model for stable expression of TOSO.