Combining Mobilizing Agents with Busulfan to Reduce Chemotherapy-Based Conditioning for Hematopoietic Stem Cell Transplantation

Abstract

In the context of hematopoietic stem cell (HSC) transplantation, conditioning with myelo- and immune-ablative agents is used to eradicate the patient's diseased cells, generate space in the marrow and suppress immune reactions prior to the infusion of donor HSCs. While conditioning is required for effective and long-lasting HSC engraftment, currently used regimens are also associated with short and long-term side effects on extramedullary tissues and even mortality. Particularly in patients with severe combined immunodeficiency (SCID), who are generally less than 1-year old at the time of transplantation and often suffer from existing comorbidities. There is a pressing need for development of alternative, less toxic conditioning regimens. Hence, we here aimed to improve efficacy of currently used myeloablative protocols by combining busulfan with stem-cell niche-directed therapeutic agents (G-CSF or plerixafor) that are approved for clinical use in stem cell mobilization. T, B and myeloid cell recovery was analyzed in humanized NSG mice after different conditioning regimens. Increasing levels of human leukocyte chimerism were observed in a busulfan dose-dependent manner, showing comparable immune recovery as with total body irradiation in CD34-transplanted NSG mice. Notably, a better T cell reconstitution compared to TBI was observed after busulfan conditioning not only in NSG mice but also in SCID mouse models. Direct effects of reducing the stem cell compartment in the bone marrow were observed after G-CSF and plerixafor administration, as well as in combination with low doses of busulfan. Unfortunately, these direct effects on the stem population in the bone marrow were not reflected in increased human chimerism or immune recovery after CD34 transplantation in NSG mice. These results indicate moderate potential of reduced conditioning regimens for clinical use relevant for all allogeneic transplants.

Keywords: G-CSF; HSC transplantation; busulfan; conditioning; immune reconstitution; plerixafor.

Figure 1

Busulfan conditioning as an alternative to TBI in immunodeficient mice: Rag2^−/− mice transplanted with wild-type BALB/c HSPCs were preconditioned by total body irradiation (TBI, 8.09 Gy) or busulfan (50 mg/kg). Immune reconstitution was analyzed up to 20 weeks after transplantation. (A) Mice were weighed weekly during the first month after transplantation. Change of weight normalized to the starting weight before conditioning is depicted in the graph for TBI (2 mice) and busulfan (3 mice) treated mice. (Unpaired t-test; * p < 0.05). (B) Survival analysis of the TBI-conditioned (16 mice from historical data) and busulfan-conditioned mice after transplantation (4 mice). (C) Cell distribution (myeloid, B and T cell) in peripheral blood (PB) over time of mice preconditioned with TBI (2 mice) or busulfan (4 mice). (Two-way ANOVA; ** p < 0.01). (D) Proportion of the different T-cell developmental subsets in the thymus after TBI or busulfan conditioning. (E) Cell distribution (myeloid, B and T cells) in PB 20 weeks after transplantation. (Two-way ANOVA; * p < 0.05).

Figure 2

Modeling busulfan conditioning in NSG mice, determining a suitable dose. NSG mice were preconditioned with increasing doses of busulfan (control, 5 mg/kg, 12.5 mg/kg, 25 mg/kg and 50 mg/kg) and transplanted with 100,000 human CD34 cells (5 mice/group). (A) Human chimerism (% hCD45 cells) achieved in PB, spleen, bone marrow (BM) and thymus 20 weeks after transplantation. Human chimerism achieved by TBI represented by dashed line. (Two-way ANOVA; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). (B) Total number of human hematopoietic stem cells (HCS) in NSG BM 20 weeks after transplantation. (C) Cell lineage distribution (myeloid, B and T cells) in PB 20 weeks after transplantation of the different conditioned groups. (Two-way ANOVA; * p < 0.05, ** p < 0.01). (D) Proportion of B cell developmental stages in BM in the different busulfan-treated mice. (E) Total B cell counts in spleen and in PB 20 weeks after transplantation. (One-way ANOVA; ** p < 0.01, *** p < 0.001, **** p < 0.0001). (F) Proportional T-cell developmental stages in the thymus in the different busulfan-treated mice. (G) Total T cell counts in spleen and in PB 20 weeks after transplantation. (One-way ANOVA; **** p < 0.0001).

Figure 3

Effect of busulfan and mobilizing agents on BM HSCs. (A) Total BM cell numbers, 24 h after busulfan conditioning (low and high dose) compared to the control group (without busulfan). (One-way ANOVA, * p < 0.05). (B) Total spleen cell numbers and cell viability after busulfan conditioning (24 h after). (One-way ANOVA; * p < 0.05). (C) Frequency of HSPCs (LSK; Lin-Sca1 + cKit +) cells in BM 24 h after busulfan conditioning, normalized to control mice. (One-way ANOVA; * p < 0.05). (D) Frequency of long-term HSCs (Lin-Sca1+cKit+CD150+CD48-), hematopoietic progenitor cells (HPC; Lin-Sca1+cKit+CD48+) and multipotent progenitor cells (MPP; Lin-Sca1+cKit+CD150-CD48-). (Two-way ANOVA; * p < 0.05). (E) Representative FACS plots of PB HSPCs after mobilizing agents’ injection. G-CSF was measured 1 day after the last injection and plerixafor 1 h after injection. Quantification of HSPCs in PB is depicted in the graph. (One-way ANOVA; ** p < 0.01, **** p < 0.0001). (F) Mice were conditioned with busulfan (low and high dose), G-CSF or the combination G-CSF+low-dose busulfan and analyzed 24 h after the last injection (3 mice/group). Upper graph: Total BM cell count after conditioning. (One-way ANOVA; ** p < 0.01). Middle graph: Frequency of HSPC (LSK; Lin-Sca1+cKit+) cells in BM 24 h after conditioning, normalized to control mice. (One-way ANOVA; * p < 0.05, ** p < 0.01). Lower graph: Frequency of long-term HSCs, HPC and MPP cells. (Two-way ANOVA; ** p < 0.01). (G) Mice were conditioned with busulfan (low and high dose), plerixafor or the combination plerixafor+low-dose busulfan and analyzed after the last busulfan injection or 1 h after plerixafor administration (3 mice/group). Upper graph: Total BM cell count after conditioning. (One-way ANOVA; ** p < 0.01). Middle graph: Frequency of HSPC (LSK; Lin-Sca1+cKit+) cells in BM 24 h after conditioning, normalized to control mice. (One-way ANOVA; * p < 0.05). Lower graph: Frequency of long-term HSC, HPC and MPP cells. (Two-way ANOVA; ** p < 0.01).

Figure 4

Long-term immune recovery after reduced busulfan conditioning. NSG mice (5 mice/group) were preconditioned with different conditioning regimens (low-dose busulfan, high busulfan, G-CSF, G-CSF+low-dose busulfan, plerixafor and plerixafor+low-dose busulfan) and transplanted with 100,000 CD34 enriched cells from cord blood. (A) Achieved human chimerism (% hCD45 cells) in PB, spleen, bone marrow (BM) and thymus 20 weeks after transplantation. (Two-way ANOVA; * p < 0.05, *** p < 0.001, **** p < 0.0001). (B) Total number of human hematopoietic stem cells (HCS) in NSG BM 20 weeks after transplantation. (C) Proportion of B-cell developmental stages in BM in the different conditioned regimen groups. (D) Total B cell counts in spleen 20 weeks after transplantation. (One-way ANOVA; * p < 0.05; ** p < 0.01). (E,F) Proportional T-cell developmental stages (early and late) in the thymus in the different conditioning regimen groups. (G) Total T-cell numbers (CD4+ and CD8+ cells) in spleen 20 weeks after transplantation. (Two-way ANOVA; ** p < 0.01). (H) Total naïve T-cell numbers (CD4+ and CD8+ cells) in spleen 20 weeks after transplantation. (Two-way ANOVA; * p < 0.05).