CYCLOSPORINE A SUPPRESSES THE TOLEROGENIC EFFECT OF DONOR BONE MARROW CELLS BY INHIBITING TGF-β1-MEDIATED APOPTOSIS

Abstract

1027 Introduction. Donor bone marrow cell (BMC) infusion can induce prolonged tolerance of allografts in various species, including rhesus monkeys. The tolerogenic effect has been ascribed to a veto mechanism, which associates with functional deletion of donor-specific CTLp. We have postulated that a key CTLp- BMC interaction in this event involves engagement of the BMC CD8α molecule via its natural ligand, MHC Iα3, expressed on the reactive CTLp. Following activation via CD8α, BMC secrete paracrine TGF-β1, which mediates deletion of the reactive CTLp via apoptosis. Earlier murine studies demonstrated that Cyclosporine A (CsA) abolishes veto activity by BMC through an effect on the reactive CTLp rather than on the veto population. To further elucidate the potential mechanism(s) by which CsA inhibits the tolerogenic effect of donor-BMC, we examined the influence of CsA on veto activity of rhesus BMC in vitro and also asked whether CsA impairs TGF-β1 induced apoptosis in activated T cells. Methods. Replicate MLR-induced CTL cultures were set up with and without BMC from the stimulator, in the presence or absence of CsA. Results were calculated as % suppression of CTL by BMC. Additionally, rhesus PHA-activated T cells were treated with and without 2 ug/ml CsA and cocultured with transduced COS cells expressing simian TGF-β1 for 24 h. Apoptosis in the harvested rhesus T cells was assessed by propidium iodide/annexin V FITC staining and flow cytometry. Results. In the absence of CsA, BMC suppressed CTL by 60% while 0.04, 0.5 and 2 ug/ml of CsA decreased the suppression of CTL by BMC to 36%, 20% and 4%, respectively. Although CsA (2 ug/ml) increased TGF-β1 expression, it reduced TGF-β1-induced apoptosis of activated rhesus T cells by 56.6% ± 6.3. CsA had no significant effect on activated rhesus T cell TGF-β1 receptor density, implying that the partial abrogation of TGF-β1-induced apoptosis is not mediated by decreased numbers of TGF-β1 receptors on activated rhesus T cells. Conclusions. We conclude that CsA may inhibit the tolerogenic effect of donor-BMC by suppressing TGF-β1-mediated apoptosis. Suppression of TGF-β1-mediated apoptosis by CsA may involve alteration of post-receptor signaling pathways in donor-reactive CTLp.