We have postulated that a small number of donor-derived leukocytes and the recipient immune system mutually interact and reciprocally attenuate both host-vs-graft (HVG) and graft-vs-host (GVH) reactions.1 Multiple cell and organ transplant models confirm the coexistence of donor cell traffiking and graft acceptance.2 However, the outcome of GVH and HVG reactions after drug discontinuation remains unpredictable. In this study, we hypothesize that both the graft's antigenicity and migratory cell load playa determinant role in the development of chimerism, graft-vs-host disease (GVHD), and rejection during the final drug-free stage. MATERIALS AND METHODSBN recipients underwent different cell, organ, or composite tissue allografts from LEW donors. Immunosuppression involved short (28 days) or prolonged (100 days) treatment with tacrolimus (1 mg/kg/d IM for 14 days, then weekly). Cell inoculi (spleen, bone marrow) and organ allografts (liver, heart, small bowel) were studied and compared to composite tissue allografts (CTA; rat hind limb) which conversely offer both a strong antigenic barrier as well as a continuous source of viable migratory donor leukocytes (ie, vascularized bone marrow). 3 RESULTS AND DISCUSSIONFollowing short-term immunosuppression, tolerogenic allografts (bone marrow, liver) reached long-term survival and tissue-detectable microchimerism (Table 1). Only heart allografts, with high antigenicity and low migratory cell load, underwent moderate to severe chronic rejection and disappearance of mixed tissue microchimerism by flow cytometric techniques. Those allografts with a heavy migratory cell load such as the bone marrow, spleen, and small bowel presented significant expansion of tissue and peripheral chimerism and concomitantly, the greatest incidence of GVHD. Bone marrow inoculi displayed low GVHD susceptibility. CTAs, which are highly antigenic and contain the greatest viable migratory cell load, underwent delayed rejection after short-term immunosuppressive coverage. However, when exposed to prolonged immunosuppression, the chimeric expansion was allowed to stabilize throughout tacrolimus coverage. After its discontinuation, transient, nonlethal GVH reaction ensued in 9 of 13 animals. Their recovery was then consistently followed by slow reduction of cytometrically detectable chimeric leukocytes from peripheral blood, repopulation of the donor bone marrow with recipient elements, and allograft chronic rejection. Of notice, allografted

Allogeneic chimerism and tolerance have been successfully achieved by infusing allogeneic bone marrow cells (BMC) into irradiated or neonatal recipients; however, engraftment of xenogeneic BMC using the same procedure has been difficult to achieve. One of the reasons for this difficulty was the lack of a species-specific hematopoietic microenvironment, such as donor stromal cells and species-specific growth factors. In order to overcome this problem, we applied bone fragments obtained from neonates (NBF) for induction of xenogeneic chimerism and subsequent xenograft acceptance. Results were compared to those of BMC. MATERIALS AND METHODSInbred Lewis rats and outbled Syrian hamsters were used as recipients and donors, respectively. NBF were obtained from the tibias and femurs of hamster neonates (within 48 hours of the birth) and 15 to 25 pieces were implanted subcutaneously or under the kidney capsule of rat recipients. BMC (250 × 10 6 ) prepared from the tibias and femurs of 6 to 10-week-old hamsters were IV infused into Lewis rat recipients. All recipients received 4 Gy whole body irradiation (WBI, day 0), cyclophosphamide (CP) 15 mg/kg/d for 5 days (day −3 to 1), and oral form tacrolimus (TAC) 1 mg/kg/d for 30 days (day 0 to 30). On day 30, they were challenged with hamster heart transplantation (HTx). TAC (1 mg/kg/d) was given for 10 days after HTx, then xenografts were followed without any treatment. For comparison, BMC were IV infused into lethally irradiated (9.5 Gy) rat recipients, and recipients that survived for more than 30 days were challenged with hamster HTx.