Abstract
Abstract
AMD3100 is a bicyclam compound that mobilizes hematopoietic progenitor cells by inhibiting stromal cell derived factor-1 binding to its cognate receptor CXCR4 present on CD34+ cells. In healthy donors, large numbers of CD34+ cells are released into the circulation following a single injection of AMD3100, making this an attractive mobilization agent for allogeneic hematopoietic cell transplantation. To determine the suitability of AMD3100 mobilized cells for allogeneic transplantation, we investigated the phenotypic and functional properties of T-cells mobilized with AMD3100 alone or in combination with G-CSF.
Methods: Sixteen healthy donors underwent a 15–25 liter apheresis 6 hours following mobilization with a single subcutaneous injection of AMD3100 (240mcg/kg; n=8) or after 5 daily doses of G-CSF and a single dose of AMD3100 (240 mcg/kg; n=8). All donors mobilized with AMD3100 alone had undergone a prior G-CSF mobilization > 60 days earlier, with the G-CSF mobilized apheresis products serving as controls for each donor. Serum cytokine levels were measured in all donors by Luminex bead array assays immediately prior to apheresis. The Th1 (IFN-g) to Th2 (IL-4) ratio of CD4+ T-cells at baseline and following AMD3100 mobilization was measured by ELISPOT 18 hours following nonspecific mitogen stimulation with PMA/ionomycin. Allo-reactivity against 3rd party MHC mismatched cells was measured by standard mixed lymphocyte reaction (MLR) using H3+ thymidine and a CFSE dilution assay gated on T-lymphocytes.
Results: Apheresis products mobilized with AMD3100 alone had a similar CD3+ T-cell content and a lower CD34+ cell content compared to G-CSF mobilized products (215 vs 257 x 106 CD3+ cells/kg, p=0.135; 1.46 vs 5.58 x 106 CD 34+ cells/kg; p=0.0044) respectively. In contrast, apheresis products mobilized with AMD3100 had a higher CD19+ B-cell content compared to G-CSF mobilized products (79.6 vs 24.1 x 106 CD19+ cells/kg; p=0.014). A synergistic effect on mobilization of CD34+ progenitors, CD19+ B cells, CD3+ T-cells and CD14+ monocytes occurred when AMD3100 was combined with G-CSF. The following differences were also observed: 1) CD62L (L-selectin) expression on CD4+ and CD8+ T-cells decreased significantly from baseline following G-CSF mobilization. In contrast, no change in CD62L expression on T-cells occurred with AMD3100 mobilization 2) Serum levels of IL-4 were significantly higher in donors following G-CSF mobilization compared to AMD3100 3) Mobilization with AMD3100 did not induce T-cells to undergo a Th2-type cytokine polarization 4) Compared to baseline, T cells mobilized with AMD3100 had a decrease in allo-reactivity to 3rd party PBMC (measured by MLR and CFSE dilution assay) similar to the decrease observed with G-CSF mobilized T-cells.
Conclusion: Although allo-reactivity against 3rd party PBMC was similar, distinct phenotypic and functional differences were observed in T-cells mobilized with AMD3100 compared to G-CSF. Whether the differences observed in T-cells mobilized with AMD3100 will impact the incidence and severity of acute and chronic GVHD compared to G-CSF mobilized peripheral blood cell transplants is currently being evaluated in an animal model of MHC matched transplantation.