Activation-induced cell death (AICD) is one of the major mechanisms to eliminate unwanted and activated lymphocytes during the end of immune response to maintain lymphocyte homeostasis. The overall aim of this proposal is to understand the molecular basis whereby T cells resist to AICD during a clonal expansion phase of an immune response, but gradually become susceptible to it toward the end of the immune response. Work from several laboratories has clearly shown a prominent role for NF-kappaB/rel protein activation in promoting cell survival. We previously identified one such NF-kappaB/rel-regulated anti-apoptotic gene IEX-1 L, an unspliced variant of IEX-1 (Immediate Early responsive gene X-1). Our more recent data using IEX-1-transgenic (Tg.) mice demonstrate that IEX-1, similar to IEX-1 L, also protects T cells from AICD both in vitro and in vivo. Constitutive expression of IEX-1 in lymphocytes results in the accumulation of memory/effector-like T cells in the spleen and lymph node and increased susceptibility to a lupus-like autoimmune disease, clearly suggesting a role for IEX-1 in the survival of responding T cells. Consistent with its anti-death activity, IEX-1 is recently found to potentially bind to Bcl-xL and metallothionein (MT), a cysteine -rich molecule that scavenges hydroxyl radical. We hypothesize that high levels of IEX-1 expression during the early phase of T cell activation protect T cells from apoptosis, whereas decreased expression of IEX-1 renders them susceptible to AICD at the end of immune responses. To test this, we will use anti-sense IEX-1-containing retrovirus to inhibit IEX-1 expression, sensitizing activated T cells to apoptosis. We will also examine IEX-1 expression in antigen (Ag)-stimulated T cells in the setting of inflammation to establish a physiological relevance of our observations with IEX-1-Tg. mice.
Aim 2 will determine in vivo and in vitro effects of IEX-1 on the life span of Ag-stimulated T cells with or without expressing a truncated IkBa (N) by crossbreeding T cell receptor/IEX-l-Tg mice with IkBa (N)- mice. Finally, aim 3 will verify the interactions of IEX-1 with Bcl-xL and/or MT proteins through various biochemical approaches and correlate the interactions to the anti-death activity of IEX-1. These studies will underscore the role of IEX-1 in the regulation of T cell homeostasis and potentially identify novel targets for therapeutic intervention in autoimmune diseases.
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