770. Selective Elimination of Alloreactive GVHD-Mediating Donor T Cells by Ex Vivo sFasL Treatment

Abstract

Graft-versus-host disease (GVHD), the major cause of morbidity and mortality after allogeneic blood and marrow transplantation, is mediated mainly by donor T cells. However, when all or most T cells are removed from donor allografts in an attempt to prevent GVHD, problems with graft failure, reduced immune reconstitution, and tumor relapse have emerged. Our goal was to develop a clinically applicable strategy to selectively deplete from the donor allograft the anti-recipient T cells that mediate GVHD, without ablating the remainder of the T cells that mediate immune responses against tumor antigens and infectious agents. Previous work done in our lab, using mouse models, has shown that recombinant human soluble Fas ligand (sFasL) can efficiently delete alloreactive donor T cells during their activation in response to irradiated recipient cells in a mixed lymphocyte reaction (MLR). This ex vivo sFasL treatment abrogated GVHD while sparing donor T cells with anti-tumor reactivity (Georgantas et al, submitted). The present work was performed in a human model system, in order to extend our work toward clinical application. In our human model system, peripheral blood mononuclear cells (PBMCs: 1st party; responder cells) from a given human donor were activated in vitro in response to irradiated PBMCs from a second donor (2nd party; stimulator cells), in the presence (or absence) of sFasL. Analyses of day 1–7 MLR cultures showed that recipient alloantigen-activated responder (donor) T cells began to up-regulate Fas expression on day 2 and that indirect (non-specific) activation of the 2nd party stimulator cells was detectable by day 5. Based on these findings, the sFasL treatment was limited to 2–3 days. Secondary MLRs, performed on cells harvested after the 2–3-day sFasL treatment during the first MLR, showed a marked depletion of anti-2nd party alloreactivity, but preservation of competence to different non-alloreactive antigens, such as cytomegalovirus (CMV), and 3rd party stimulator cells. In addition, sFasL treatment efficiently deleted or prevented the development of CD25+CFSEhigh, CD25+CFSElow, and CD25−CFSElow subsets of activated responder cells.