Xenotransplantation using porcine organs may resolve a chronic shortage of donor organs for clinical transplantation if significant immunological barriers can be overcome. To determine the potential role of T lymphocytes in Xenograft (Xg) rejection, we transplanted transgenic hCD46 porcine hearts heterotopically into baboon recipients. Methods: Recipients were treated to deplete anti-Gal antibody with a non-antigenic α-Gal polyethylene glycol polymer (TPC) (n=2), TPC plus rituximab (anti-CD20) (n=1) or were untreated (n=1). None of the recipients received T-cell immunosuppression. Results: All Xgs failed within 7 days and showed evidence of a mixed humoral and cellular rejection process. Cellular infiltration consisting primarily of CD4+ T cells and few CD8+ T cells. Proliferation and cytotoxicity assays showed sensitization of CD4+ and CD8+ T cells that reacted with porcine IFN-γ (pIFN-γ)-stimulated porcine aortic endothelial cells (PAEC). The CD4+ lymphocytes displayed greater cytotoxicity than CD8+ cells. An increased frequency of PAEC-specific interleukin (IL) 2 and IFN-γ-secreting T cells was observed, suggesting a Th1 cytokine bias. An increase in the percentage of circulating CD4+CD28- cells was observed at the time of rejection and over 50% of the CD4+ cells recovered from residual pig tissue at necropsy lacked CD28 expression. Conclusions: These findings show that lymphocytes are efficiently stimulated by PAEC antigens and can mediate direct tissue destruction. These studies (1) provide an insight into the potential of cellular-mediated cardiac Xg rejection, (2) show for the first time the induction of cytotoxic pig-specific CD4+CD28- lymphocytes and (3) provide a rational basis for determining different modes of immunosuppression to treat Xg rejection. © Blackwell Munksgaard, 2006.