Insights into the Biology of Mobilized Cells through Innovative Treatment Schedules of the CXCR4 Antagonist AMD3100.
Authors: Bonig Halvard , Papayannopoulou Thalia 
Published: April 2020

Abstract
The CXCR4 antagonist AMD3100 mobilizes hematopoietic stem/progenitor cells (HSPC), but most available data are on combination with G-CSF, rather than its use as a single modality for HSPC mobilization and transplantation. Because of low efficacy of the standard single-dose regimen, we additionally tested in mice the effect of pulsed or continuous AMD3100-infusion: Repeat injections as little as 4 hours apart for up to 6 days did not result in CFU-C accumulation or curtail the size of AMD3100-responsive HSPC pools. In contrast to the 3-fold increase in circulating CFU-C after bolus or repeat injections, after 5 days of continuous AMD3100-infusion circulating CFU-C were increased 30-fold over baseline, from 301±41/mL to 10,687±757/mL compared to 1,088±75/mL after a bolus. These numbers were sustained at least until day 8 and, of interest, were not further augmented by injection of an AMD3100-bolus, suggesting satiating doses of AMD3100-infusion. Furthermore, at least equally efficient mobilization by AMD3100-bolus or -infusion was documented in splenectomized hosts, indicating that the contribution of spleen to mobilization by AMD3100 is negligible. Long-term repopulating cells in AMD3100-infusion-mobilized blood, assessed by limiting dilution transplantation, were also increased, to 273(205–362) CRU/mL, vs. 77(56–106) CRU/mL after bolus. Of interest, the number of HSPC mobilized by AMD3100-infusion was much higher than by standard twice-daily dosing of G-CSF, and AMD3100 effectively mobilized two G-CSF resistant/refractory mouse models (G-CSFR−/−, Plasminogen−/−), while not surprisingly, mice treated with the Gi protein inhibitor Pertussis toxin were resistant to AMD3100-induced mobilization. Subsequent studies addressed the phenotype and the in vitro and in vivo functional behavior of AMD3100-mobilized HSPC. The immunophenotype of AMD3100-mobilized c-kit+ cells differed significantly from that of steady-state bone marrow (BM) c-kit+ cells. Thus expression of several integrins was lower, while CD26 expression was significantly higher than on BM c-kit+ cells. AMD3100-infusion-mobilized blood and BM CFU-C migrated equally efficiently towards SDF-1 in vitro. In vivo marrow homing of AMD3100-infusion-mobilized blood CFU-C was &gt;50% increased over BM CFU-C, similarly to what we recently showed for G-CSF-infusion-mobilized blood. These properties differ from those of in vitro AMD3100-incubated BM-HSPC, in which migration was blocked, and homing was normal or reduced. Kinetics of hematopoietic recovery after transplantation of 100 μl AMD3100- or G-CSF-infusion-mobilized blood to lethally irradiated hosts were similar except for delayed platelet recovery in AMD3100-mobilized blood recipients. Partial BrdU labeling experiments for up to 3 days before mobilization (resulting in 20–40% BrdU labeling of c-kit+ cells in BM) documented that labeled cells were consistently underrepresented (&lt;5%) among AMD3100-mobilized c-kit+ cells. These data defend the notion that only non-cycling HSPC are found in mobilized blood (&gt;80% of AMD3100-mobilized c-kit+ cells are in G0), but extend it to conclude that such a status precedes their exit from BM, rather than being a consequence of modification in the blood environment. In summary, we are presenting a novel efficacious mobilization scheme which fully supports transplantation demands in normal and G CSF-refractory donors. Furthermore, our data expand previous knowledge about size and turn-over of AMD3100-mobilizable HSPC pools.