Anti-CD4 monoclonal antibody-induced allograft tolerance in rats despite persistence of donor-reactive T cells

Although CD4-targeted therapy abrogates acute rejection and may induce permanent graft acceptance in rodents, little is known about the mechanisms of longterm graft survival in these models. Recently, we have shown that treatment with a nondepleting anti-CD4 monoclonal antibody (mAb) (RIB-5/2) induces longterm survival of renal, heart, and skin allografts in strong major histocompatibility complex I/II incompatible rat strains. Here, we demonstrate that the development of major histocompatibility complex-specific and tissue-nonspecific tolerance rather than graft adaptation is responsible for long-term anti-CD4 mAb-induced transplant survival, Donor-specific but not third-party heart and pancreatic islet grafts were accepted permanently 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 mixed lymphocyte reaction was largely unaffected. The reverse transcriptase-polymerase chain reaction analyses of long-term renal allografts before and after donor-specific antigen challenge revealed no changes in CD3 mRNA level, but showed up-regulation of CD25, interleukin (IL) 2, interferon (IFN) gamma, 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 IFN-gamma declined rapidly, whereas IL-4 persisted, resulting in a reversal of IFN-gamma/IL-4 ratio. 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 T helper (Th) 1/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.