A ROLE FOR TRANSFORMING GROWTH-FACTOR-BETA IN THE VETO MECHANISM IN TRANSPLANT TOLERANCE

We have studied the veto cell-mediated induction of transplant toleran ce by allogeneic donor bone marrow cells and have achieved kidney allo graft tolerance in a preclinical rhesus monkey model. Here we extend t hese studies to investigate the veto mechanism of CTLp suppression and the role of CD8 and TGF-beta in these events. Infusion of DR-/dim don or BMC into RATG-treated rhesus monkeys induced functional deletion of donor-specific CTLp and prolongation of kidney allograft survival, wh ereas depletion of the CD8+ subset from BMC ablated these effects. A r ole of CD8 in the veto effect was further implicated by rhesus MLR-ind uced CML experiments in which pretreatment of normal responder cells w ith MAb to MHC class I, the natural ligand of CD8, blocked the suppres sive activity of allogeneic BMC. In addition, pretreatment of the BMC with anti-CDS MAbs blocked strong veto activity significantly, suggest ing that CD8 functions as an accessory or adhesion ligand. In contrast , anti-CD8 treatment significantly enhanced weak BMC-mediated veto act ivity, suggesting that CD8 might additionally serve as a signal transd ucer to increase veto activity, perhaps by the induction of cytokine r elease. The cytokine TGF-beta was studied because it has immunosuppres sive properties that are shared by veto cells. Human TGF-beta, like BM C veto cells, inhibited MLR-induced CML in a dose-dependent manner, an d anti-TGF-beta Ig relieved the BMC-mediated veto suppressive effect. Active TGF-beta was detected only in the supernatants of CML cultures containing BMC. Pretreatment of BMC with L-leucyl-leucine methyl ester (Leu-leu-OMe), which eliminates cytotoxic precursor and effector lymp hocytes and monocytes, did not affect levels of active TGF-beta. In pr evious studies, the veto effect of BMC was also shown to be Leu-leu-OM e-resistent. Finally, treatment of isolated DR-/dim BMC cultures with anti-CD8 elicited TGF-beta secretion, whereas anti-CD2 or anti-CD3 had no effect. When isolated after stimulation with anti-CD8, only the CD 8+ subset of DR-dim BMC produced detectable levels of active TGF-beta. In summary, these studies demonstrate that CD8 functions as an immuno regulatory molecule in veto effects by freshly isolated rhesus BMC and suggest that CD8-ligand interactions may induce low-level secretion o f TGF-beta to mediate or facilitate the veto mechanism of CTLp inactiv ation in a paracrine manner.