<h3>Purpose</h3> CD4<sup>+</sup>CD25<sup>high</sup>Foxp3<sup>+</sup> regulatory T cells (Treg) can establish immunologic tolerance in animal models but are hampered by immunosuppressive drugs. IL-2-anti-IL-2 complexes (IL-2c) promote Treg expansion in vivo even with concomitant calcineurin inhibitor administration. Here, we combined recipient IL-2c therapy with pre-transplant Treg administration during ex vivo lung perfusion (EVLP), to test the hypothesis that this strategy would facilitate expansion and function of the Tregs within the graft. <h3>Methods</h3> We performed rat orthotopic left lung transplantation (OLT) experiments using Fischer 344 (F344, RT-1<sup>lv1</sup>) donors and Wistar Kyoto (WKy, RT-1<sup>l</sup>) recipients. Expanded WKy Tregs were administered to the F344 donor lungs during EVLP. Some animals received IL-2c (30 ug recombinant mouse IL-2 and 150 ug anti-mouse IL-2, given i.p. once on day 0) and/or Tacrolimus (Tac, 0.1mg/kg/day, 7 days). Treatment groups were: no treatment (n=7), Tac (n=5), IL-2c/Tac (n=4), Treg (n=6), and Treg/IL-2c/Tac (n=5). Necropsy was performed 7 days post-transplant. <h3>Results</h3> Graft CD25<sup>high</sup>Foxp3<sup>+</sup> content increased as a result of Treg therapy (p<0.001, Fig 1). ISHLT A and B grades were attenuated in the IL-2c/Tac and Treg/IL-2c/Tac groups (p<0.001, Fig 2-3). Graft Treg content and Treg-to-effector T cell ratio (Treg/Teff) at day 7 was increased by Treg/IL-2c/Tac (p<0.001, p=0.025, Fig 4). <h3>Conclusion</h3> Treg therapy increased intragraft Treg content, and IL-2c/Tac further increased intragraft Tregs 7 days after transplantation, with reductions observed in A and B rejection scores. The combination of graft-directed Treg cell therapy with a Treg-permissive immunosuppressive strategy may be a viable therapeutic approach for effective pre-transplant graft immunomodulation in lung transplantation.