Peripheral Blood Stem Cell Mobilization with Single Agent Plerixafor Induces FoxP3 Expression in Donor T Cells and Enables Durable Long Term Hematopoietic Engraftment Across Mismatched Major Histocompatibility Complex Barriers

Abstract 1891

Plerixafor is a novel stem cell mobilizing agent that functions by blocking the interaction between the CXCR4 receptor and stromal-derived factor 1a. Several studies have suggested that compared to granulocyte colony stimulating factor (G-CSF), plerixafor mobilizes peripheral blood stem cells (PBSC) with a greater capacity for bone marrow homing and repopulation. Prior studies have shown that plerixafor-mobilized PBSC (P-PBSC) can induce long term donor engraftment in major histocompatibility complex matched hematopoietic cell transplantation (HCT). In the current study we compare the cellular composition, engraftment kinetics and efficacy of P-PBSC and G-CSF PBSC (G-PBSC) in high-dose and nonmyeloablative (NMA) dog leukocyte antigen (DLA) haploidentical HCT. Fourteen dogs were transplanted with P-PBSC (collected by apheresis 4–6 hours after administration of 4 mg/kg) after conditioning with 920 cGy total body irradiation (TBI) (n=4) or 200 cGy TBI (n=10) (data from 2 dogs previously published, Blood 2010; 115:918). Cellular P-PBSC composition was compared to 11 G-PBSC products (G-CSF 5 μg/kg day −5 to −1, and 10 μg/kg on day 0 two hours before apheresis) and transplant outcome in the P-PBSC group was compared to 6 dogs treated with G-PBSC after 200 cGy TBI (Cytotherapy, 2011, in press). Post-transplant immunosuppression after P-PBSC or G-PBSC consisted of cyclosporine and MMF. Two days of apheresis (day 0 and 1) were planned for 12 donors, whereof 8 accomplished both procedures, while 4 only had 1 due to low platelets (<50,000/μl) after the first apheresis. Sufficient numbers of mononuclear cells (MNC) (>5×10⁸ /kg) were obtained during the first day of apheresis in all 14 dogs (mean 14.9×10⁸ /kg). Although number of infused cells tended to be higher for P-PBSC after 1 or 2 apheresis (n=14) compared to G-PBSC (n=11) for most cell subsets, statistical significance was only observed for CD3⁺ cells (mean number of cells, plerixafor vs. G-CSF: MNC, 16.7 vs. 10.2 x10⁸/kg, p=0.06; CD34⁺, 8.5 vs. 5.6 x10⁶/kg, p=0.43; CD3⁺, 5.6 vs. 3.4×10⁸/kg, p=0.05; CD4⁺, 3.2 vs. 3.8 x10⁸/kg, p=0.71; CD8⁺, 0.8 vs. 0.5 x10⁸/kg, p=0.09; CD14⁺, 4.2 vs. 3.8 x10⁸/kg, p=0.51). No skewing of the CD4:CD8 ratio was observed (CD4:CD8 ratio, 4.9 vs. 7.5, p=0.36), but quantitative PCR for FoxP3 showed a 3.8 fold increase in expression in donor peripheral blood CD3⁺ cells 4–6 hours after administration of plerixafor (n=12; pre-plerixafor, mean 2489 FoxP3 copies/10⁵ G3PDH; post-plerixafor, mean 9657 FoxP3 copies/10⁵ G3PDH; p<0.01). All dogs had initial engraftment, but 3 of 10 dogs (30%) conditioned with 200 cGy TBI rejected after 42—161 days, as compared to 4 of 6 of the G-PBSC treated dogs (67%, p=0.15). With a median follow up of 59 (range, 6–284) days for the remaining 7 dogs conditioned with 200 cGy TBI, final granulocyte and MNC donor chimerisms ranged between 61–85% and 36–80%. In the 920 cGy TBI group, 3 of 4 dogs had final granulocyte and MNC donor chimerisms of 100%, while the dog that was euthanized on day 6 had 80% in both compartments. Median granulocyte, lymphocyte and platelet nadirs in the 920 cGy TBI group (3 dogs evaluable) were 0 (0–53), 39 (26–98) and 4500 (1500–7500) cells/μl and were observed between days 5–7, 3–16 and 9–22. In the P-PBSC 200 cGy TBI group, median granulocyte, lymphocyte and platelet counts were 689 (8–1320), 154 (0–521) and 5750 (1500–15000) cells/μl between days 8–20, 4–28 and 11–22. All dogs that rejected had autologous recovery and were euthanized in good condition. None of the dogs receiving P-PBSC developed acute skin graft versus host disease (GVHD) and one dog developed chronic GVHD of the skin on day 270, 9 weeks after discontinuation of immunosuppression. Compared to the G-PBSC 200 cGy TBI conditioned dogs, there were no differences in engraftment kinetics, nadir depth or time to nadir and overall survival.

The current study shows that PBSC mobilized with plerixafor as a single agent have the ability to establish long-term engraftment across DLA-haploidentical barriers after high dose or NMA conditioning with transplantation outcomes, blood cell and engraftment kinetics at least equivalent to G-PBSC. The higher T-cell content of the P-PBSC graft with increased expression of the T regulatory marker, FoxP3, low rejection and GVHD rates, suggest a graft with a more favorable phenotype that combined with the convenience of 1–2 injections of plerixafor over 6 of G-CSF, could be a future alternative to G-PBSC.