Separation of GvH- and GvL-Immune Responses in Allogeneic Stem Cell Transplantation by CD95-Mediated Depletion of Alloreactive T-Cells Using CD178 Based Proapoptotic Chimeric Molecules.

Abstract

Depletion of donor T cells (TCD) from the graft effectively ameliorates graft-vs-host (GvH) disease (GvHD) in allogeneic stem cell transplantation (SCT). TCD, however, is associated with impaired engraftment, immunosuppression, and abrogation of the beneficial graft-vs-leukemia (GvL) effect. Thus, based on previous results (Hartwig et al., Blood 2002) to eliminate alloreactive specificities but preserve oligoclonal antiviral- and GvL- immunity we explored proapoptotic chimeric molecules composed of the extracellular domains of human CD178 (FasL) and CD28 or RANK to selectively deplete GvH-reactive T cells by AICD in vitro and in vivo. As these fusion proteins are expressed as soluble Flag-tagged trimers and reconstitute bioactivity only upon binding to well defined cell-surface molecules (Samel et al., J. Biol. Chem., 2003), they can be specifically targeted onto e.g. recipient derived DC or donor T cells via the molecules CD80/86 or RANKL/TRANCE, respectively. Using an MHC-mismatched murine BMT model proliferative responses of purified CD90+ alloreactive T cells from C57BL/6 (H-2b) mice were strongly reduced following stimulation with DC from F1 (C57BL/6xBalb/c; H-2bxd) mice in vitro in the presence of CD178-CD28 or CD178-RANK to levels comparably obtained with agonistic anti-CD95 mAb (Jo2). Depletion was specific to alloantigen since H-2d restricted Hemagglutinin (HA)-peptide specific T cells titrated into the system persisted among retained T cells. In addition, adoptive transfer experiments of H-2b derived CD90+ T cells depleted of alloreactivity in vitro prior to injection into irradiated F1 recipients confirmed the in vitro results as these mice did not develop clinical signs of GvHD as compared to controls. Moreover, in current studies the CD178-fusion proteins are applied in vivo in our BMT model by injecting 2.5 to 5.0 x 106 syngeneic (H-2bxd) DC preloaded with CD178-CD28 into lethally irradiated F1 mice reconstituted with 1 x 107 TCD H-2b donor marrow cells. Alternatively, soluble CD178-fusion proteins are applied i.v. following BMT. Preliminary results obtained in first experiments suggest that host DC expressing cell surface bound CD178 fusion proteins confer protection to acute GvH-reactions in recipients challenged with purified CD90+ T cells of H-2b origin as these mice show prolonged survival compared to controls. Furthermore, studies on evaluating GvL-reactivity in this model using donor T cells from HA-primed Balb/c mice and A20 cells expressing HA as a defined tumor surrogate antigen are in progress, and their results will be discussed. Taken together, these results suggest that CD178-based proapoptotic fusion proteins devoid of systemic toxicity due to cell-surface antigen-restricted activation might provide a promising tool to separate GvH- and GvL-responses in vivo without inducing severe side effects on CD95-mediated apoptosis in sensitive tissues.