502 The infectious tolerance, as originally described by Dr. H. Waldmann's group, was induced and perpetuated in adult thymectomized (ATX) mouse recipients of skin allografts (Tx). We have recently shown, however, that cardinal features of such a classic peripheral form of tolerance may be applied to euthymic rats. Hence: (i) treatment of sensitized rats with RIB-5/2, a CD4 nondepleting mAb, abrogated accelerated (<36 h) rejection, and produced indefinite cardiac Tx survival; (ii) tolerant cells in mAb-treated hosts could disable naive or even alloimmune cells, so that they failed to trigger rejection; (iii) donor-specific and organ non-specific tolerance could be transferred by regulatory T cells into new cohorts of cardiac or renal test Tx recipients. This study was designed to expose mechanisms underlying the infectious tolerance pathway, with particular emphasis on the role of the thymus. Thymectomy in naive animals profoundly influenced the tolerogenic effects of CD4-targeted therapy. Indeed, unlike in euthymic rats, only ca. 50% of ATX+ RIB-5/2 mAb-treated hosts maintained cardiac Tx indefinitely (MST >200 days; n=7). Interestingly, ATX abrogated the ability of spleen cells from CD4 mAb-treated long-term Tx recipients to prolong test cardiac Tx survival after adoptive transfer (MST <7.0 days; n=4). Thus, thymus is critical for generation of regulatory T cells under cover of CD4 mAb therapy, and for the ability of cells to confer tolerance in an infectious manner. As thymectomy prevented the development of regulatory T cells, we then asked which mechanism may have contributed to the acquisition of"non-infectious" form of tolerance in ATX rats. We have analyzed intra-Tx cytokine gene programs by competitive template RT-PCR. Intra-Tx expression of IL-2, IL-4, and IL-10 mRNA was diminished in both euthymic and ATX + CD4 mAb-treated long-term hosts, as compared to rejecting controls. However, cardiac Tx from ATX tolerant rats showed significant IFN-γ gene expression, suggesting a less efficient downregulation of Th1-like cells in the absence of regulatory T cells. In an attempt to break tolerance, long-term recipients were challenged with exogenous rIL-2, or with alloactivated spleen cells. Indeed, this treatment recreated Tx rejection in ATX, but not in euthymic hosts. The challenge with rIL-2 was more effective than that utilizing alloimmune cells (MST = 7.0 days [n=3] and 24.7 days[n=3], respectively). Thus, a state of cytokine-responsive anergy contributes to the maintenance of cardiac Tx in ATX + CD4 mAb-treated rats. In conclusion, both central and peripheral immune mechanisms play a role in the acquisition of infectious tolerance in Tx recipients. A rather unstable level of clonal anergy may replace regulatory T cells in ATX long-term hosts. This study adds further substance to the discussion why all attempts to induce transplant tolerance in adult patients have been so far unsuccessful.