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

Abstract

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 (Po0.001 vs medium), and recipients of mPB exposed to death ligands displayed reduced GVHD (Po0.01 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.

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

“…UCB cells are safely maintained in liquid suspensions for 24-48 h, whereas in mobilized-peripheral blood cells similar rates of apoptosis are observed after 4-12 h of incubation. 27 The relatively activated state of mobilized-peripheral blood cells is also recognized in the shorter period required to activate progenitor function by receptor-mediated trophic signals 22 and the remarkable difference in viability of T cells in culture. 27 It is difficult to predict the potential benefit of application of the current approach to overcome the particular limitations of UCB cells, and whether it can reduce the 10-15 day delay in UCB cell engraftment as compared with bone marrow and mobilizedperipheral blood cells.…”

“…[7][8][9][10]16 We have recently demonstrated that exposure of donor inoculum to an apoptotic challenge in the absence of host-specific sensitization reduces GvHD severity in mice grafted with haploidentical T cells 26 and human mobilized-peripheral blood xenografts. 27 In view of the preserved-engraftment facilitating and graft versus tumor activities of T cells pretreated with death ligands, it remains to be determined whether UCB T cells have reduced-GvH reactivity following functional depletion. T cells in UCB were largely insensitive to receptor-mediated apoptosis, as immature cells [40][41][42] with reduced-cytotoxic activity in response to cytokine stimulation in vitro 43 that become sensitive to negative regulation only after activation.…”

“…19,20,22 Resistance of human UCB progenitors to receptor-mediated apoptosis, consistently observed in mobilized-peripheral blood cells 22,27 and murinebone marrow, [17][18][19]21 evolves as an inherent characteristic of hematopoietic precursors. 20 Furthermore, we demonstrate by direct measurements of viability and functional hematopoieticreconstitution assays that TNF-induced Fas expression in 40% of the CD34 + progenitors does not sensitize to apoptosis induced by receptor cross-linking, similar to observations in murine-bone marrow.…”

“…CD34 + cells were identified using antigen-presenting cells-labeled antibodies (clone 8G12, Pharmingen, San Diego, CA, USA) and lineages were quantified using phycoerythrin-labeled mAb: anti-human CD3 (clone OKT3, Pharmingen), anti-human CD19 (clone HD37, IQ Products, Groningen, Netherlands) and anti-human CD33 (clone WM53, IQ Products). 27 Expression of the receptors was determined using mAb against human Fas (clone DX2, Miltenyi Biotec), TNF-R1 and TNF-R2 (clones 16803 and 22235, respectively, R&D Systems, Abingdon, UK). 22 Cell death and apoptosis were determined in cells incubated with 5 μg/mL 7-aminoactinomycin-D (7-AAD, Sigma, St Louis, MO, USA) and Annexin-V (IQ Products), respectively.…”

“…17,18 Insensitivity of hematopoietic progenitors to apoptosis can be used to improve the outcome of transplants, conferring immune privilege to the graft 24,25 and fostering progenitor activity. [19][20][21][22] A pretransplant apoptotic challenge with Fas-ligand (FasL) and TNF-α reduces significantly the incidence and severity of GvHD in haploidentical-and xenogeneic-murine transplants, 26,27 and improves early myeloid reconstitution. 20,22 In this study we assessed possible implementation of apoptotic signaling in several aspects of UCB cell transplantation.…”