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.

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-2^b) mice were strongly reduced following stimulation with DC from F₁ (C57BL/6xBalb/c; H-2^bxd) 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-2^d restricted Hemagglutinin (HA)-peptide specific T cells titrated into the system persisted among retained T cells.

In addition, adoptive transfer experiments of H-2^b 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-2^bxd) DC preloaded with CD178-CD28 into lethally irradiated F₁ mice reconstituted with 1 x 10⁷ TCD H-2^b 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-2^b 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.