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.
