Preferential Mobilization of CD34+ Plasmacytoid Dendritic Cell Precursors by Plerixafor.

Plerixafor (Mozobil^®) is a CXCR4 antagonist that was recently approved by the Food and Drug Administration for use in combination with granulocyte-colony stimulating factor (G-CSF) to mobilize hematopoietic stem cells (HSCs) in patients with non-Hodgkin's lymphoma and multiple myeloma undergoing autologous transplantation. As part of our ongoing clinical evaluation of plerixafor, we observed that the drug efficiently mobilized a CD34^dim population of HSCs. Here, we characterize the CD34^dim population and assess its relative frequency following mobilization with G-CSF and/or plerixafor in apheresis samples and in non-mobilized cord blood samples. Methods: Aliquots of apheresis products were obtained from 16 healthy donors following treatment with a single injection of plerixafor or 5 days of G-CSF (10 μg/kg/day). In a separate study, five patients with lymphoma were mobilized sequentially with plerixafor (240 μg/kg), G-CSF (10 μg/kg/day), and plerixafor + G-CSF. These patients received an injection of plerixafor on day −5. Twenty-four hours later, patients were treated with G-CSF for 4 days. On the fifth day (day 0), patients received a dose of G-CSF and a dose of plerixafor followed four hours later by apheresis. Samples were collected after treatment with plerixafor on day −5 as well as immediately before and 4 hrs after plerixafor administration on day 0. Cord blood units were obtained from the St. Louis Cord Blood Bank. Human CD34⁺ cells were purified by positive selection with a Magnetic Affinity Cell Selection CD34 isolation kit. We used Affymetrix Human Genome U133+2 arrays to generate gene expression profiles for 24 samples of CD34⁺ cells collected following mobilization of healthy donors with either Plerixafor (n=12) or G-CSF (n=12). Results: Human CD34⁺ cells can be divided into three distinct subsets based on their cell surface expression of CD45RA and CD123 (IL-3Rα): (i.) CD34⁺CD45RA⁻CD123^+/− primitive HSCs, (ii.) CD34⁺CD45RA⁺CD123^+/− committed progenitors, and (iii.) CD34^dimCD45RA⁺CD123^hi cells. Table 1 shows the relative frequencies of each of these CD34⁺ cell subsets in apheresis products obtained from healthy donors mobilized with G-CSF or plerixafor as well as in cord blood units. Strikingly, we observed that each graft type was significantly enriched for one of the CD34⁺ cell subsets compared to the other two grafts. G-CSF mobilized grafts contained more CD45RA⁻CD123^+/− primitive HSCs, cord blood units were enriched with CD45RA⁺CD123^+/− committed progenitors and plerixafor mobilized products had significantly more CD34^dimCD45RA⁺CD123^hi cells. Table 1 also shows the CD34 cell subset distribution following sequential mobilization of lymphoma donors with plerixafor, G-CSF, and plerixafor + G-CSF (PL+G). This data agrees with the results we obtained from healthy donors and confirms that G-CSF grafts are enriched with CD34⁺CD45RA⁻CD123^+/− cells while plerixafor preferentially mobilizes the CD34^dimCD45RA⁺CD123^hi subset. Extensive FACS and functional analyses determined that the CD34^dim population represents a plasmacytoid pro-DC2 (for progenitor of pre-dendritic cell type 2) progenitor compartment as indicated by their CD45RA⁺CD123^hiBDCA−2⁺BDCA−4⁺CD36⁺CXCR4^hiCD4^dimCD25⁻CD13⁻ phenotype and inability to form CFU-GM colonies in vitro. Finally, we found that the gene signature of plerixafor-mobilized CD34⁺ cells is distinct from that of G-CSF-mobilized CD34⁺ cells. Plerixafor-mobilized CD34⁺ cells expressed significantly more of the specific transcriptional regulator of plasmacytoid DC (pDC) development, E2-2, as well as additional transcriptional factors (SpiB, IRF7, IRF8) and cell surface markers (BDCA-2, ILT7) that are specific to the pDC lineage. Conclusions: This data suggest that the CD34^dimCD45RA⁺CD123^hi cells preferentially mobilized by plerixafor are precursor pDCs. Further study is required to determine the impact these cells have on the engraftment and function of plerixafor mobilized grafts after hematopoietic stem cell transplantation.

DiPersio:Genzyme Corp.: Honoraria.