FURTHER-STUDIES OF VETO ACTIVITY IN RHESUS-MONKEY BONE-MARROW IN RELATION TO ALLOGRAFT TOLERANCE AND CHIMERISM

Infusing the DR(-/dim) fraction of bone marrow cells (BMC) from an all ogeneic kidney donor into rabbit antithymocyte globulin-treated transp lant recipients delivers a tolerogenic signal, leading to functional a llograft tolerance in rhesus monkeys without additional drug therapy. Our updated results in an expanded series show a median 131-day graft survival of recipients given DR(-/dim) donor BMC with a 23% 1-year sur vival (P<0.00001 vs. rabbit antithymocyte globulin controls). Removing DR(bright) cells from donor BMC appeared to have a significant effect (P<0.05). We have further investigated the tolerogenic mechanism with in the experimental framework of the veto hypothesis in this preclinic al model. In limiting dilution assays, we demonstrated the donor speci ficity of clonal inactivation of CTL precursors (CTLp) after in vitro or in vivo exposure to DR(-/dim) donor BMC, confirming specific tolera nce. Additionally, in vitro studies confirmed the allogeneic specifici ty of CTLp inactivation in 3-cell. MLR assays; minimal bystander effec ts were seen on normal CTLp responses to third party stimulator cells, while CTLp responses to the BMC donor's cells were abrogated in the s ame cultures. BMC mediating the veto effect were found to be resistant to L-leucyl-L-leucine methyl ester (Leu-leu-OMe), which excluded BMC- mediated cytotoxicity by NK or lymphokine-activated killer cells, CTL, or activated macrophages. In contrast, veto activity was abolished if the BMC were pretreated with either high dose UV-B light irradiation, mitomycin, or gamma-irradiation, indicating that BMC contained a UV-B -sensitive precursor of the veto effector, and that a proliferative st ep separated the two. Irradiation of DR(-/dim) donor BMC or administra tion of cyclophosphamide after infusion of nonirradiated BMC prevented the tolerogenic effect. Only recipients given nonirradiated DR(-/dim) donor BMC demonstrated PBL chimerism, which associated with functiona l deletion of antidonor CTLp and duration of graft survival. The Leu-l eu-OMe resistance and the other properties of the allogeneic monkey CD 3(-) CD2(+) CD8(+) BMC subpopulation that exhibits tolerance-promoting activity in vitro and in vivo lead us to postulate that a donor BMC-d erived precursor population, possibly a dendritic cell population, may induce allogeneic unresponsiveness in this model.