Introduction: Although CD4-targeted therapy abrogates acute rejection, and may induce permanent graft acceptance in rodents, little is known about the mechanisms of long-term graft survival in these models. Recently, we have shown that treatment with a nondepleting anti-CD4 mAb (RIB-52) induces long-term survival of renal, heart and skin allografts in strong MHC I/II incompatible rat strains.Results: Here, we demonstrate that the development of MHC-specific and tissue-nonspecific tolerance rather than graft adaption is responsible for long-term anti-CD4 mAb-induced transplant survival. Donor-specific but not third-party heart and pancreatic islet grafts were permanently accepted without adjunctive therapy in long-term kidney allograft recipients, and infusion of naive or alloimmune splenocytes failed to break the tolerant state. Interestingly, alloreactive T cells were not depleted in these long-term survivors, as ex vivo donor-specific MLC reactivity was largely unaffected. Cytokine expression studies at mRNA (quantitative RT-PCR) and protein (immunohistology) level demonstrated a strong inhibition of CD25, IL-2, IL-10, TNFα, and IFNγ in anti-CD4 mAb treated rats (<15% of untreated controls) at day 2-5 post-transplant which further decreased during 300 day follow-up. By contrast, IL-4 expression was only moderately (30-50% of controls) inhibited. In fact, intragraft IFN g/IL-4 mRNA ratio dropped from 0.4 at day 5 to 0.07 at day 100 (2.3 at day 5 in controls). The RT-PCR analyses of long-term renal allografts before and after donor-specific antigen challenge (heart grafting at day 100) revealed no changes in CD3 mRNA level, but upregulation of CD25, IL-2, IFNγ, IL-4 and IL-10 mRNA in the early phase, suggesting the presence of alloreactive T cells in tolerant rats. At later time points, the expression of IFNγ declined rapidly, whereas IL-4 persisted, resulting in a reversal of IFNγ/IL-4 ratio (day 100: 0.07, day 105: 1.1, day 200: 0.04).Conclusion: Our data demonstrate the stability of anti-CD4 mAb induced tolerance despite persistence of alloreactive T cells, suggesting the role of active tolerance-maintaining mechanisms. The Th1/Th2 shift may be involved in this regulatory process, as anti-CD4 mAb prevents acute graft-deteriorating rejection by effectively blocking Th1 responses, and well-functioning grafts may tolerize themselves by inducing regulatory cells.