Mobilizing Agents G-CSF, Cyclophosphamide or AMD3100 (Plerixafor) Have Distinct Effects on Osteoblasts, Hematopoietic Stem Cell Niches, and B-Lymphopoiesis,

Abstract 4005

The CXCR4 antagonist AMD3100 is progressively replacing cyclophosphamide as adjuvant to G-CSF to mobilize hematopoietic stem cells (HSC) for autologous transplants in patients who failed prior mobilization with G-CSF alone. We and others have recently demonstrated that G-CSF-induced mobilization and the associated response of HSC niches and bone formation depend on bone marrow (BM) macrophages^1–3. Moreover medullar B lymphopoiesis is dependent on bone-forming osteoblasts in vivo. We therefore compared the effects of these three mobilizing agents (6 day course of G-CSF, versus a single injection of cyclophosphamide, versus 6 day course of AMD3100) on endosteal osteoblasts, bone formation, BM macrophages, expression of HSC-supportive cytokines and B lymphopoiesis in the mouse.

G-CSF administration significantly reduced the number of endosteal osteoblasts and niche-supporting macrophages. G-CSF also inhibited expression of chemokines and cytokines such as CXCL12, Kit-ligand, angiopoietin-1 and IL-7 in the endosteal region resulting in a profound inhibition of medullar B lymphopoiesis with suppression of pre-pro-B, pro-B, pre-B cells and mature sIgM+ B cells as long as G-CSF was administered and HSC mobilization persisted.

Cyclophosphamide also suppressed osteoblasts, niche-supportive macrophages and inhibited endosteal expression of CXCL12 and angiopoietin-1 but did not inhibit KIT-ligand expression. Of note, the down-regulation of CXL12 was more pronounced in response to cyclophosphamide than G-CSF. This could explain the higher mobilizing effect of cyclophosphamide. In contrast to G-CSF, medullar B lymphopoiesis rebounded in synchrony with endosteal IL-7 expression during cyclophosphamide-induced mobilization despite persistent suppression of osteoblasts and bone formation. Despite a marked rebound in the number of immature B cell progenitors in cyclophosphamide-mobilized BM, B cell progenitors could not mature into sIgM+ B cells which remained very depressed in BM, spleen and blood even 10 days after a single cyclophosphamide injection.

Finally, while AMD3100 was effective at mobilizing HSC, it did not suppress osteoblasts, cytokine expression at the endosteum or B lymphopoiesis.

In conclusion, although G-CSF, cyclophosphamide and AMD3100 efficiently mobilize HSC into the blood, their effects on HSC niches, bone formation and B lymphopoiesis are distinct. Furthermore the contrasted effects of G-CSF, cyclophosphamide and AMD3100 on medullar B lymphopoiesis indicates that grafts mobilized with these three agents may have different immune properties.

(1) Winkler IG, Sims NA, Pettit AR, et al. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood. 2010;116(23):4815–4828. (2) Christopher MJ, Rao M, Liu F, Woloszynek JR, Link DC. Expression of the G-CSF receptor in monocytic cells is sufficient to mediate hematopoietic progenitor mobilization by G-CSF in mice. J Exp Med 2011;208(2):251–260. (3) Chow A, Lucas D, Hidalgo A, et al. Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. J Exp Med 2011;208(2):261–271.