DESCRIPTION: The generation of T cell mediated immunity is often impaired in cancer patients. Our studies have shown that T cells from cancer patients display increased susceptibility to activation induced cell death (AICD) and also exhibit a defect in NFKB activation. These alterations may contribute to the immune dysfunction observed in T cells from these patients. Gangliosides, which are known to be overexpressed in certain tumor types, may be responsible for the suppression of NFKB and for the increased sensitivity of patient T cells to apoptosis. We and others have found that select gangliosides, including GD3, can sensitize normal human T cells to activation induced cell death (AICD). Although GD3 alone is not sufficient to induce apoptosis, it does inhibit expression of the anti-apoptotic genes clAP-1, cIAP-2 and TRAF-2. GD3 also inhibits NFkB activation, which appears to be due to stimulus-dependent degradation of Re1A . The induction of apoptosis and suppression of NFKB activity are caspase dependent, since both events are blocked by the caspase inhibitors Z-VAD-fmk and DEVD-frnk. Based on our findings, we hypothesize that immunity to tumors is diminished in cancer patients due to an imbalance between the intracellular levels of pro- versus anti-apoptotic proteins in T cells. Gangliosides including GD3 contribute to this imbalance by suppressing anti-apoptotic gene expression (cIAP/TRAF). The reduced expression of cIAPs/TRAFs allows increased caspase activity, leading to stimulus dependent RelA degradation, further depressing anti-apoptotic gene expression and inducing apoptosis. Aim 1 will define how GD3 regulates anti-apoptotic gene expression in T cells, and will assess the role of those molecules in protecting cells from NFKB inhibition and apoptosis. Initial experiments will determine the extent to which GD3 inhibits the anti-apoptotic gene expression that normally protects T cells from apoptosis. Experiments will also determine the mechanism by which GD3 alters the steady state levels of antiapoptotic genes, including clAPs, and TRAFs. Studies will also test the impact that overexpression of the antiapoptotic genes (cIAPs/TRAFs) has on RelA degradation, caspase activation, and sensitivity to AICD in GD3-treated T cells. Aim 2 will determine the mechanism by which GD3 inhibits NFKB in T cells, and the relationship of that inhibition to anti-apoptotic gene expression and to AICD. The role of GD3 in modulating caspase activity in T cells will be assessed by identifying which caspases are activated in response to GD3, T cell activation or both. The role these activated caspases play in degradation of ReIA will then be assessed by blocking select caspase function. In a final set of experiments, ReIA will be overexpressed to determine its protective effect on both GD3-inhibited antiapoptotic gene expression and in rendering cells resistant to AICD. These studies will provide insight into how gangliosides inhibit NFKB and survival gene expression, leading to increased T cell sensitivity to apoptosis.