INTRODUCTION: Blockade of the CD40-CD154 costimulatory pathway can prolong allograft survival, but does not prevent the development of transplant arteriosclerosis in several models. In this study, we investigated the mechanisms of CD40-CD154-independent transplant arteriosclerosis in major histocompatibility complex (MHC)-class I-mismatched aortic allografts. METHODS: MHC class I-mismatched CBK (H2k+Kb) donor aortas were transplanted into CBA (H2k) recipients who can only recognize the graft through CD8+ T cells and CD4+ T cells responding to the class I MHC mismatch through the indirect pathway of allorecognition. Recipients were treated with anti-CD154 antibody (MR1) alone or in combination with anti-CD8 (YTS169) or anti-interleukin (IL)-4 (11B11) antibodies. Grafts were analyzed by histology on days 30 and 60 and for intragraft mRNA expression on day 14 after transplantation. RESULTS: Repeated treatment with anti-CD154 alone or in combination with anti-CD8 antibody did not prevent intimal proliferation compared with untreated controls (65%+/-6%, 62%+/-9%, and 71%+/-7% luminal occlusion, respectively, 60 days after transplantation). In both treatment groups, the expression of IL-4, IL-5, and eotaxin was increased compared with control grafts, and an eosinophilic infiltration was observed. Neutralizing IL-4 in combination with CD40-CD154 blockade and CD8+ T-cell depletion abrogated transplant arteriosclerosis (9%+/-4% luminal occlusion 60 days after transplantation). CONCLUSION: Prolonged treatment with anti-CD154 was not able to prevent the development of transplant arteriosclerosis in MHC class I-mismatched aortic allografts, in the presence or absence of CD8+ T cells. This CD40-CD154 pathway resistant transplant arteriosclerosis was mediated by IL-4, because neutralizing IL-4 in addition to CD40-CD154 costimulation blockade and CD8+ T-cell depletion prevented its development.