This study analyzes mechanisms by which interleukin 13 (IL-13) affects "infectious tolerance" in rat recipients of cardiac allografts, with emphasis on interactions between intragraft Ad-IL-13 gene transfer and systemic infusion of regulatory cells. Although exogenous viral IL-13 was modestly effective on its own, adjunctive Ad-IL-13 gene therapy and adoptive transfer of suboptimal dose of regulatory T cells exerted synergistic effects, as evidenced by long-term cardiac allograft survival in test recipients. Local IL-13 induction (determined by enzyme-linked immunosorbent assay and immunohistology) diminished intragraft apoptosis, and upregulated antiapoptotic A20 and antioxidant heme oxygenase 1 (HO-1). Ad-IL-13 plus regulatory cells synergistically diminished the frequency of cells positive by TUNEL (TdT [terminal deoxynucleotidyltransferase]-mediated dUTP nick-end labeling) assay, and enhanced cytoprotective gene expression. These findings correlated with in vitro studies in which Ad-IL-13 decreased tumor necrosis factor alpha (TNF-alpha)-mediated cytotoxicity, conferred resistance to apoptosis, and increased HO-1/A20 expression in human umbilical vein endothelial cell (HUVEC) cultures. However, inhibition of HO-1 after treatment with tin protoporphyrin reversed the immunomodulatory/antiapoptotic effects of Ad-IL-13 both in vivo (infectious transplantation tolerance), and in vitro (HUVECs). Thus, by decreasing apoptosis/TNF-alpha-mediated cytotoxicity, and by facilitating induction of antiapoptotic/antioxidant molecules in HUVECs, this study documents the cytoprotective function of Ad-IL-13 in vitro, and points toward in vivo synergy between Ad-IL-13 and regulatory cells in the infectious transplantation tolerance pathway. Results of HO-1 neutralization studies suggest that HO-1 represents one of the putative IL-13 downstream effectors.