O111* Aims: Cynomolgus monkey renal allograft recipients treated with a chimerism-inducing regimen with perioperative CD154 blockade were studied long-term (>5 years) to establish reliable tolerance assays that are capable of predicting the long-term outcome. Methods: Cynomolgus monkeys in Group A were treated with a conditioning regimen consisting of low dose total body irradiation (TBI 1.5 Gy x2), thymic irradiation (TI 7 Gy), anti-thymocyte globulin (ATG), perioperative anti-CD154 monoclonal antibody (mAb) treatment, combined donor bone marrow (DBM) and kidney transplants on day 0 and one month course of cyclosporine (CyA). No immunosuppression was given after day 28. Control Groups include Group B: donor splenocyte infusion instead of DBM, Group C: no donor cell infusion, or Group D: no TI. Results: All eight recipients in Group A developed multilineage chimerism up to day 100 and 7/8 survived long-term after discontinuation of immunosuppression (>1850, >1323, 837, 755, 401, 373, 206, 58 days). Four of seven long-term survivors did not developed any evidence of rejection with the longest survival exceeding five years. Two of four long-term recipients accepted donor skin graft indefinitely while rejecting third-party skin graft within 10 days. However, 3/8 recipients in Group A eventually developed chronic rejection. All recipients in control groups B and C did not develop chimerism and lost their kidney allograft function either by acute or chronic rejection (CR) (292, 274, 250, 113, 93, 51 days). 2/3 recipients in Group D developed chimerism and one of them survived long-term without rejection (289, 71, 44). Anti-donor alloantibody (ADA), measured by flow cytometry and C4d staining of the graft biopsy were significantly relevant to CR. All six recipients that developed ADA eventually developed CR. Allograft biopsies of all six recipients with positive ADA later developed C4d in their renal allograft biopsies. In contrast, five recipients without ADA have never developed C4d or CR. In Group A, there was no significant difference found between tolerant recipients and rejecters in the level and duration of mixed chimerism or postoperative MLR and CML. In the ELISPOT assay, where posttransplant peripheral blood mononuclear cells (PBMC) were activated via either direct (donor APC) or indirect allorecognition pathway (sonicated donor cells), tolerant recipients showed specific inhibition of donor-reactive, gamma-interferon (gIFN) production in the indirect pathway (6.5 ±6 spots per 106 PBMC). In contrast, the recipients with CR showed vigorous gIFN production (132 ±17). In the direct pathway, however, there was no significant difference detected in gIFN production between tolerant recipients and rejecters (31.5 ±15 vs 82 ±25). Conclusions: Long-term stable renal allograft survival was achieved in 50% of recipients treated with a chimerism inducing nonmyeloablative regimen with perioperative CD154 blockade. Posttransplant ADA by flow cytometry, positive C4d in renal biopsies and positive gIFN production in the indirect pathway appear to correlate with the development of chronic rejection.