Notch2 blockade enhances hematopoietic stem cell mobilization and homing

Abstract

Despite use of newer approaches, some patients being considered for autologous hematopoietic cell transplantation (HCT) may only mobilize limited numbers of hematopoietic progenitor cells (HPCs) into blood, precluding use of the procedure, or being placed at increased risk of complications due to slow hematopoietic reconstitution. Developing more efficacious HPC mobilization regimens and strategies may enhance the mobilization process and improve patient outcome. Although Notch signaling is not essential for homeostasis of adult hematopoietic stem cells (HSCs), Notch-ligand adhesive interaction maintains HSC quiescence and niche retention. Using Notch receptor blocking antibodies, we report that Notch2 blockade, but not Notch1 blockade, sensitizes hematopoietic stem cells and progenitors (HSPCs) to mobilization stimuli and leads to enhanced egress from marrow to the periphery. Notch2 blockade leads to transient myeloid progenitor expansion without affecting HSC homeostasis and self-renewal. We show that transient Notch2 blockade or Notch2-loss in mice lacking Notch2 receptor lead to decreased CXCR4 expression by HSC but increased cell cycling with CXCR4 transcription being directly regulated by the Notch transcriptional protein RBPJ. In addition, we found that Notch2-blocked or Notch2-deficient marrow HSPCs show an increased homing to the marrow, while mobilized Notch2-blocked, but not Notch2-deficient stem cells and progenitors, displayed a competitive repopulating advantage and enhanced hematopoietic reconstitution. These findings suggest that blocking Notch2 combined with the current clinical regimen may further enhance HPC mobilization and improve engraftment during HCT.

Figure 1.

Notch2 blocking antibody sensitizes hematopoietic stem cell and progenitors (HSPC) egress in response to granulocyte-colony stimulating factor (G-CSF) or/and AMD3100. Mice were given 4 doses of G-CSF in two days, or a single dose of AMD3100, or 4 doses of G-CSF followed by AMD3100 the next day, two days after a single dose (A–C) or 4 doses of Notch2-blocking or control antibody (D–F) (see details of treatment scheme in Online Supplementary Figure S1). Twenty-four hours (h) after the last dose of G-CSF, or 1 h after AMD3100, peripheral blood (PB) was analyzed for white blood cell (WBC) counts (A and D) and the presence of LSK (B and E) and LK (C and F) cells by FACS in PB (n=4–7/group). Results are pooled from 3 independent experiments and presented as mean±Standard Deviation (S.D.). Student t-test *P<0.05, **P<0.01.

Figure 2.

Notch2 deficiency causes increased hematopoietic stem cell and progenitors (HSPC) egress. (A and B) HSPCs present in the periphery (A) and in the spleen (B) of 8-week old Vav-Cre/Notch2^F/F mice and control mice (Vav-Cre/Notch2^+/+) were determined by FACS. Representative FACS profile (gated on Lin⁻c-kit⁺ cells) and bar graphs of total numbers of LSK cells and LK cells in the blood (A) and in the spleen (B) pooled from 3 similar experiments in which Vav-Cre/Notch2^F/F and control mice (n=5) were examined. (C–F) Total numbers of LSK cells (C), LK cells (D), white blood cell counts (E), and frequencies of T cells (CD4/CD/8), B cells (B220) and granulocytes (Gr-1) (F) present in the peripheral blood (PB) of recipient mice 12 weeks after receiving bone marrow (BM) transplantation from control (n=6) or Vav-Cre/Notch2^F/F mice (n=6). (G–J) Spleen-residing LSK (G) and LK (H) frequencies, as well as BM HSC subpopulations, including LT-HSC (Flt3⁻CD34⁻LSK), ST-HSC (Flt3⁻CD34⁺LSK) and MPP (Flt3⁺CD34⁺LSK) (I), and LK subsets including CMP (Lin⁻c-kit⁺Sca–1⁻CD34⁺FcγRII/III⁻), MEP (Lin⁻c-kit⁺Sca-1⁻CD34⁻FcγRII/III⁻) and GMP (Lin⁻c-kit⁺Sca-1⁻CD34⁺FcγRII/III⁺) (J) were determined by FACS in the same group of transplanted mice. Results are pooled from 2 experiments and presented as mean±Standard Deviation (S.D.). Student t-test *P<0.05, **P<0.01.

Figure 3.

Faster hematopoietic recovery from Notch2-blocked marrow progenitors after transplantation. (A and B) Platelet counts, white blood cell (WBC) counts, and hemoglobin levels on days 7, 10, 16 and 21 (A) (n=4–5/group), and at 4, 8, 10, and 13 weeks (B) after transplantation were determined (n=6/group, pooled from 2 experiments). (C–E) Bone marrow frequencies of LSK subsets (C), CMP/MEP/GMP cells (D), and CLP cells (E) were determined in the marrow of mice three months after receiving transplantation (n=6–7/group, pooled from 2 experiments). Results are presented as mean±Standard Deviation (S.D.). Student t-test *P<0.05, **P<0.01.

Figure 4.

Notch2 blockade induces enhanced reconstitution of the stem cells and myeloid progenitors from mobilized hematopoietic stem cell and progenitors (HSPC). (A) Scheme of competitive reconstitution by mobilized circulating HSPCs in which mononuclear cells from 200 mL blood collected from control antibody or Notch2 antibody-treated mice (Ly5.2) were mixed with 0.4×10⁶ competitor marrow cells (Ly5.1), and transplanted into lethally irradiated recipient mice (Ly5.1). (B) The percentage of donor Ly5.2⁺ cell chimerism in the peripheral blood (PB) mononuclear cells of recipient mice (n=5–7/group) at various time points after transplantation. (C–E) The percentage of peripheral B cells, T cells and granulocytes (C), bone marrow megakaryocyte-erythroid progenitors (MEPs) and granulocyte-macrophage progenitors (GMPs) (D), and bone marrow total LSK and LT-hematopoietic stem cells (LT-HSCs) (E) derived from Ly5.2⁺ mobilized HSPCs in recipient mice at various time points after transplantation. Results are presented as mean±Standard Deviation (S.D.). Student t-test *P<0.05, **P<0.01. wk: weeks.

Figure 5.

Notch2 loss enhances hematopoietic stem cell and progenitors (HSPC) homing and leads to altered localizations relative to the endosteum. Isolated bone marrow LK cells (0.2×10⁶) from Vav-Cre/Notch2^F/F mice and control mice were labeled with CFSE and transferred into lethally-irradiated wild-type (WT) mice. Twenty-four hours later, 2-photon imaging was performed to locate CFSE⁺ cells in the calvarium. The endosteum is highlighted by the blue second harmonic signal, while the vessel was labeled by TRITC-Dextran dye. (A) The shortest 3D distances between the LK cells and the blood vessel or the endosteum (μm) were compared for control and Notch2-deficient cells. Wilcoxon rank sum test was performed. Data shown were pooled from 3 mice (3 experiments) in each group. Cell numbers counted in the entire calvarium of each recipient were 30, 63, and 57 (total n=150) derived from Vav-Cre/Notch2^F/F mice, and 9, 26, and 22 (total n=57) derived from control (ctrl) mice, in experiments 1, 2 and 3, respectively. (B) Representative 2D images show the locations of control versus Notch2-deficient LK cells relative to the blood vessel. Bar size=100 μm.

Figure 6.

Notch2 signaling blockade increases hematopoietic stem cell and progenitor (HSPC) cell cycling. (A) Cell-cycling status of the marrow LSK cells and CD150⁺CD48⁻LSKs hematopoietic stem cells (HSCs) was determined by the proliferation marker Ki67 in conjunction with the DNA-specific dye 7-AAD in mice receiving 4 doses of control or Notch2 blocking antibody. One representative FACS profile of LSK cells, and the relative proportion of cells in G₀, G₁ and S-G₂/M phase of the cell cycle in LSK and HSC (bottom). Results are pooled from 2 experiments, and are presented as average±Standard Deviation (S.D.) (n=5/each group). (B) 1.5×10⁵ LK cells were co-cultured with confluent OP9-DLL4 or OP9-JAG1 cells in the presence of control (ctrl) or Notch2 blocking antibody (400 ng/mL) for four days before cell-cycling analysis on gated LK cells. One representative FACS profile of 3 similar experiments. Student t-test *P<0.05.

Figure 7.

Notch2 signaling regulates hematopoietic stem cell and progenitor (HSPC) CXCR4 expression. (A and B) Cell surface expression of CXCR4 in mean fluorescence intensity (MFI) was assessed by FACS analysis of bone marrow LSK cells from mice receiving 4 doses of control (ctrl) or Notch1/2 blocking antibody (n=5/each group) (A), or from Vav-Cre/Notch2^F/F mice and control mice (Vav-Cre/Notch2^+/+) (n=4/each group) (B). (C) CXCR4 promoter region has 3 potential RBP-J binding sites. (D) CHIP assay of wild-type LK bone marrow cells. Immunoprecipitation was conducted with control antibody, or anti-RBPJ followed by PCR of the CXCR4 promoter. Results shown are mean±Standard Deviation (S.D.) of 3 biological replicates. Student t-test *P<0.05, ***P<0.001. (E) CXCR4-Luc report construct with 2.0 kb CXCR4 promoter sequence containing motif 4.1 and motif 6.1 was transfected into 293T cells expressing RBPJ (RBPJ KD), Notch2 (N2 KD), or control siRNA (ctrl KD). (F) The dependence of the CXCR4-Luc reporter activity on motif 4.1 and 6.1 was assessed by transfecting the CXCR4 reporter construct with single motif 6.1 deleted (Del6.1) or both motif 4.1 and 6.1 deleted (Ddel4.1/6.1) into 293T cells that expressed control siRNA or Notch2 siRNA. (G) CXCR4-Luc reporter activity was determined by transfecting the wild-type CXCR4-Luc reporter construct (WT) or the construct with both 4.1- and 6.1-motif deleted (DD) into 293T cells expressing ICN1-expression plasmid (ICN1 OE), ICN2-expression plasmid (ICN2 OE), or control plasmid (empty vector; EV). (H) Bone marrow WT LK cells were isolated and co-cultured with OP9-DLL4 cells for 96 hours in the presence of Notch1, Notch2, or control antibody (400 ng/mL). CXCR4 expression was assessed by FACS. (E-H) Mean±S.D of 3 biological replicates. Student t-test *P<0.05, **P<0.01, ***P<0.001.