Dissociating graft-versus-tumor (GVT) effect from acute graft-versus-host disease (GVHD) still remains a great challenge in allogeneic bone marrow transplantation (allo-BMT). Bortezomib, a proteasome inhibitor, has shown impressive efficacy as a single agent in patients with hematologic malignancies but can result in toxicity when administered late after allogeneic transplantation in murine models of GVHD. In the current study, the effects of T-cell subsets and their associated cytokines on the efficacy of bortezomib in murine allogeneic BMT were investigated. Increased levels of serum tumor necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) were observed after allo-BMT and continuous bortezomib administration. Bortezomib-induced GVHD-dependent mortality was preventable by depletion of CD4(+) but not CD8(+) T cells from the donor graft. The improved survival correlated with markedly reduced serum TNFalpha but not IFNgamma levels. Transfer of Tnf(-/-) T cells also protected recipients from bortezomib-induced GVHD-dependent toxicity. Importantly, prolonged administration of bortezomib after transplantation of purified CD8(+) T cells resulted in enhanced GVT response, which was dependent on donor CD8(+) T cell-derived IFNgamma. These results indicate that decreased toxicity and increased efficacy of bortezomib in murine allo-BMT can be achieved by removal of CD4(+) T cells from the graft or by inhibiting TNFalpha.