Inhibition of recombinant (r) adenovirus (Ad)-induced, NFkappaB-dependent dendritic cell (DC) maturation by NFkappaB decoy oligodeoxyribonucleotides (ODN) permits functional expression of a potent immunosuppressive transgene product (CTLA4Ig). These cells traffic to T cell areas of host secondary lymphoid tissue and markedly prolong organ allograft survival in the absence of systemic levels of CTLA4Ig. We hypothesize that these gene engineered DC can promote transplant tolerance by mediating apoptotic death of allospecific T cells. We have four Specific Aims: Aim I will analyze the impact of the stably immature, gene-engineered DC on alloreactive T cell apoptosis. We will test whether gene-engineered myeloid DC (MDC) and "lymphoid-related" DC (LDC), which differ in their interactions with T cells, have equal potential to mediate apoptosis of alloactivated T cells, whether Th1 and Th2 cells are equally susceptible, and the extent to which memory CD4 + T cells are also killed. In Aim II, we shall ascertain the death regulatory pathways and type of apoptosis induced by these gene engineered DC. We shall examine the contribution of the Fas:FasL and TRAIL:TRAILR pathways, key T cell survival factors (Bcl-2/BClxL) and the role of passive versus active cell death. In Aim III, we will evaluate the fate and function of the gene engineered DC, and their capacity to delete alloreactive T cells in vivo, by quantitative assessment of both the proliferation and apoptosis of CD4 + and CD8 + T cells. Mice with defective passive or active T cell death pathways will be used to evaluate the requirement of these pathways for T cell deletion. We shall also determine whether Ag-specific T cells are deleted selectively. In AIM IV, we will determine the ability of the gene engineered DC to promote donor-specific transplant tolerance and its dependence on T cell apoptosis. We will evaluate host immunocompetence and anti-donor reactivity including graft integrity, at times remote from administration of the gene engineered DC. The results will provide new insight into the potential of gene engineered DC for therapy of organ transplant rejection.