T cell responses are controlled by intracellular signals emanating from ligation of cell surface receptors. The concept of two signals being required for T cell activation is well established. Signal two is generally regarded as that brought about by ligation of CD28 with B7-1 or B7-2. However, more recent studies have suggested that other cell surface receptors are involved in costimulating T cells. We have extensively studied a member of the TNFR family, namely OX40 (CD134), whose expression is induced on T cells 1-3 days after the initial activation event. We have demonstrated that OX40 costimulation prolongs clonal expansion of CD4 cells, enhances T cell differentiation and cytokine secretion, and suppresses T cell apoptosis. Thus, OX40 is responsible for the high frequency of effector T cells that develop in primary immune responses, and for the survival of a high frequency of memory T cells over time. In the present application, we seek to extend our functional data and define molecularly the mode of action of OX40. We will further elucidate how OX40 suppresses cell death through regulating the pro- and anti-apoptotic members of the Bcl-2 family, and how OX40 modulates survivin and p27kip, two molecules involved in regulating cell division that we have identified as targets of OX40. Expression studies will be complemented where appropriate with retroviral transduction of OX40 knockout T cells with Bcl-xL, Bcl-2, survivin, and dominant negative p27, in order to restore survival, division, and cytokine production of OX40- /o T cells. We will investigate control of OX40-induced responses by TRAF2 and 5, initially with expression studies of wt T cells responding to agonist anti-OX40 or OX40L-expressing APCs, and then with studies of T cells from TRAF2 dn Tg mice and TRAF5 knockout mice. We will then investigate signaling pathways controlled by PI3k/Akt and PKC0 leading to JNK and NF-v-,B activation, that are induced by OX40, and confirm these pathways with retroviral transduction of dominant negative constructs. In each case functional responses will be directly correlated with the particular intracellular pathway that is engaged by OX40 signals. These studies are tremendously important in defining the action of OX40 and will therefore help in defining which molecular pathways are necessary and sufficient for promoting division, survival, and cytokine _production by primary T cells.
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