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

Abstract

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.

Antigen-Specific Priming is Dispensable in Depletion of Apoptosis-Sensitive T Cells for GvHD Prophylaxis

“…Even megadoses of 10 7 progenitors/kg (2) can be safely administered as T cell-replete grafts following exposure to the apoptotic challenge, provided that the progenitor:T cell ratio is below 1:20. Evidently, the duration of graft preparation is determined by the differential sensitivities of T cells from various origins: UCB-derived T cells are relatively resistant to 48 h of exposure to death ligands, whereas 40–50% of T cells in BM and MPB are depleted within 18–32 and 4–8 h, respectively (30, 32, 34, 38). The duration of these cultures is shorter than the critical period of 48 h associated with significant decline in efficiency of engraftment (39, 40).…”

“…At the second level, functional depletion by apoptosis affects all immune cells within the graft including professional antigen-presenting cells (APC) and disrupts the activation cascades at multiple levels (22). For example, B lymphocytes and myeloid cells endowed with antigen-presenting capacity are generally more sensitive to apoptosis than unstimulated T cells in hematopoietic grafts derived from UCB, BM, and MPB (30, 32–35, 38). At the third level, the apoptotic challenge particularly but incompletely removes T cells expressing activation markers such as CD25 and CD69 (29, 30), which impact experimental (17, 18) and clinical GvHD (44).…”

“…It will be interesting to determine whether fractional depletion of unstimulated donor immune cells further protects from GvHD by polarizing the sensitivity to apoptosis using IL-2 to preserve regulatory T cells (21, 36, 37). In view of the potential of UCB-derived T cells to elicit potent GvH reactions (12) and insensitivity of these naïve cells to apoptosis under unstimulated conditions (38), AICD may be achieved on a shorter time scale using various antigen-non-specific stimuli (67–69). It remains to be determined whether the apoptosis-mediated approach to GvHD restrains chronic disease, which is less characterized and largely unresponsive to immunosuppressive therapy.…”