Ex Vivo Fas Ligand Treatment Selectively Eliminates T Cells That Mediate GVHD.

The addition of recombinant human soluble Fas ligand (sFasL) to a mixed lymphocyte reaction (MLR) resulted in selective reduction of the responder T cells during their activation against irradiated allogeneic stimulator cells, while sparing other T cells with anti-tumor reactivity, in studies using mouse models (Georgantas et al, Transplantation, In Press). To move our work toward reduction of clinical graft versus host disease (GVHD), we have extended these studies using a human model system. Peripheral blood mononuclear cell (PBMC) responders from a given individual (1^st party) were stimulated in vitro with irradiated PBMC stimulators from a second person (2^nd party), in the presence of sFasL. In control MLR cultures without sFasL, Fas expression began to increase on alloantigen-activated responder T cells by day 2. In 2–3 day MLRs done with carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled 1^st party responder cells, there were greatly reduced numbers of CD25⁺CFSE^high, CD25⁺CFSE^low, and CD25⁻CFSE^low activated responder cells in the sFasL-treated cultures. In addition, the numbers of CD4^highCD25⁺CD38⁺HLADR⁺ activated T cells that otherwise appeared in response to alloantigen were severely reduced in cultures containing sFasL. Secondary MLRs, performed on cells harvested on day 2–3 of sFasL treatment in the first MLR, showed a marked depletion of anti-2^nd party alloreactivity, but preservation of immune stimulation in response to cytomegalovirus and 3^rd party antigens. These data indicate that ex vivo sFasL/MLR-mediated depletion of alloreacting anti-donor T cells was efficient and selective. The efficacy of this method is currently being tested in vivo using a xenogeneic GVHD human-immunodeficient mouse model.