Negative selection by apoptosis enriches progenitors in naïve and expanded human umbilical cord blood grafts.

The influence of TNF-α and Fas-ligand (FasL) on viability and function was evaluated in fresh- and expanded-umbilical cord blood (UCB) cells. CD34(+) progenitors and T cells display outstanding survival, whereas ~30% and >50% B lymphocytes and myeloid cells undergo spontaneous apoptosis within 24 and 48 h, respectively. Although the impact of exposure to toxic doses of FasL and TNF-α was undetectable in measurements of apoptosis; removal of dead cells after 2 days of incubation with the ligands revealed a twofold increase in frequency of colony-forming cells (CFU). The sensitivity of progenitors to apoptosis was also unaffected by Fas cross-linking following TNF-induced upregulation of the receptor, increasing CFU frequency without impairing SCID repopulating cell (SRC) activity. Most significant enrichment in CD34(+) progenitors and corresponding increase in CFU frequency were observed when FasL was applied during the final week of ex vivo expansion under the influence of nicotinamide, without impairing SRC activity. These data emphasize differential sensitivities of UCB progenitors and lineage-positive cells to apoptotic signaling mediated by the Fas and TNF receptors, which might be useful in improving the efficiency of ex vivo expansion and improving UCB cell engraftment.

Apoptotic signaling through Fas and TNF receptors ameliorates GVHD in mobilized peripheral blood grafts.

Mobilized peripheral blood (mPB) is a prevalent source of hematopoietic progenitors for transplantation; however, allogeneic and haploidentical transplants are often accompanied by severe GVHD. Following the observation that murine GVHD is ameliorated by pretransplant donor cell exposure to Fas-ligand (FasL) without host-specific sensitization, we assessed the susceptibility of mPB cells to spontaneous and receptor-induced apoptosis as a possible approach to GVHD prophylaxis. Short incubation for 4 h resulted in spontaneous apoptosis of 50% of the T and B lymphocytes and 60% myeloid cells. Although expression of Fas and TNF-R1 was proportionate to fractional apoptosis, cell death was dominated by spontaneous apoptosis. Functional assays revealed that the death receptors modulated mPB graft composition as compared with incubation in medium, without detectable quantitative variations. Removal of dead cells increased the frequency of mPB myeloid progenitors (P<0.001 vs medium), and recipients of mPB exposed to death ligands displayed reduced GVHD (P<0.01 vs medium) and improved survival following lipopolysacharide stimulation. mPB grafts exposed to the apoptotic challenge retained SCID reconstituting potential and graft versus tumor activity. These data emphasize that short-term exposure of mPB grafts to an apoptotic challenge is effective in reduction of GVHD effector activity.

Regulatory functions of TRAIL in hematopoietic progenitors: human umbilical cord blood and murine bone marrow transplantation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling pathway has selective toxicity to malignant cells. The TRAIL receptors DR4 and DR5 are expressed at low levels in human umbilical cord blood cells (3-15%) and are upregulated by incubation with the cognate ligand, triggering apoptosis in 70-80% of receptor-positive cells (P<0.001). Apoptosis is not induced in hematopoietic progenitors, as determined from sustained severe combined immunodeficiency reconstituting potential and clonogenic activity. Furthermore, elimination of dead cells after incubation with TRAIL for 72 h results in a threefold enrichment in myeloid progenitors. Exposure to TRAIL in semisolid cultures showed synergistic activity of DR4 and granulocyte/macrophage colony-stimulating factor in recruiting lineage-negative (lin(-)) and CD34(+) progenitors and in promoting the formation of large colonies. In murine bone marrow, approximately 30% of lin(-) cells express TRAIL-R2 (the only murine receptor), and the receptor is upregulated after transplantation in cycling and differentiating donor cells that home to the host marrow. However, this receptor is almost ubiquitously expressed in the most primitive (lin(-)SCA-1(+)c-kit(+)) progenitors, and stimulates the clonogenic activity of lin(-) cells (P<0.001), suggesting a tropic function after transplantation. It is concluded that TRAIL does not trigger apoptosis in hematopoietic progenitors, and upregulation of its cognate receptors under stress conditions mediates tropic signaling that supports recovery from hypoplasia.