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. Author manuscript; available in PMC: 2024 Jul 15.
Published in final edited form as: J Thromb Haemost. 2022 Dec 22;21(3):488–498. doi: 10.1016/j.jtha.2022.10.006

Pre-existing anti-factor VIII immunity alters therapeutic platelet-targeted factor VIII engraftment following busulfan conditioning through cytotoxic CD8 T cells

Weiqing Jing 1, Christina K Baumgartner 1, Feng Xue 2, Jocelyn A Schroeder 1,2, Qizhen Shi 1,2,3,4
PMCID: PMC11249136  NIHMSID: NIHMS2006197  PMID: 36696197

Abstract

Background:

We previously demonstrated that busulfan preconditioning enabled sustained therapeutic platelet-derived factor VIII (FVIII) expression in naïve FVIIInull mice transplanted with 2bF8-transduced Sca-1+ cells. However, in mice with pre-existing inhibitors, platelet-FVIII expression was lost.

Objective:

In this study, we aimed to describe the mechanism of this platelet-FVIII loss.

Methods:

We monitored platelet-FVIII expression in FVIIInull mice that were immunized with rhFVIII to induce inhibitors and subsequently conditioned with busulfan before whole bone marrow transplantation or Sca-1+ hematopoietic stem cell transplantation (HSCT) from 2bF8 transgenic (2bF8Tg) mice. Busulfan with or without antithymocyte globulin or anti-CD8 antibody was employed before 2bF8Tg HSCT. Interferon gamma-ELISpot assay was used to assess which subset of cells was the target in platelet-FVIII loss. B-cell–deficient homozygous mutant mice were used to determine whether platelet-FVIII loss in FVIII-primed mice was mediated by antibody-dependent cellular cytotoxicity.

Results:

Platelet-FVIII expression was sustained in 2bF8Tg bone marrow transplantation but not in 2bF8Tg HSCT recipients. CD8 T-cell depletion in addition to busulfan preconditioning restored platelet-FVIII expression in 2bF8Tg-HSCT recipients. ELISpot analyses showed that FVIII-primed CD8 T cells were efficiently restimulated by 2bF8Tg-Sca-1+ cells and secreted interferon gamma, but were not stimulated by 2bF8Tg platelets/megakaryocytes, suggesting that 2bF8Tg-Sca-1+ cells are targets for FVIII-primed CD8 T cells. When 2bF8Tg-Sca-1+ cells were transplanted into FVIII-primed homozygous mutant mice preconditioned with busulfan, no FVIII expression was detected, suggesting that antibody-dependent cellular cytotoxicity was not the mechanism of platelet-FVIII loss in FVIII-primed mice.

Conclusion:

Pre-existng immunity can alter the engraftment of 2bF8Tg-Sca-1+ cells through the cytotoxic CD8 T-cell–mediated pathway. Sufficient eradication of FVIII-primed CD8 T cells is critical for the success of platelet gene therapy in hemophilia A with inhibitors.

Keywords: gene therapy, hemophilia A, inhibitors, platelets, preconditioning

1 |. INTRODUCTION

The development of anti-factor VIII (FVIII) inhibitory antibodies (inhibitors) is not only a significant problem in FVIII protein replacement therapy but also a major concern in the gene therapy of hemophilia A. We have developed a platelet-targeted FVIII gene therapy approach in which human FVIII expression is driven by the platelet-specific αIIb promoter (2bF8). We demonstrated preclinically that 2bF8 gene therapy can restore hemostasis and induce FVIII-specific immune tolerance in hemophilia A (FVIIInull) mice even with pre-existing anti-FVIII immunity when an effective preconditioning regimen is employed [1].

To advance our gene therapy protocol to the clinic for the treatment of patients with hemophilia A, we evaluated various clinically used preconditioning regimens [1–3], aiming to develop an effective clinically translatable platelet-specific FVIII gene therapy protocol using nonmyeloablative conditioning with low toxicities. Because busulfan (Bu), an alkylating agent with potent effects on primitive hematopoietic cells, is an important component of many hematopoietic stem cell transplantation (HSCT) preparative regimens in humans [4–9], we evaluated the efficacy of Bu-based conditioning regimens in our 2bF8 gene therapy [2]. We found that Bu conditioning resulted in sustained therapeutic levels of platelet-FVIII expression in FVIIInull mice that received 2bF8-transduced stem cell antigen 1–positive (Sca-1+) cells, in which recipients were not primed with FVIII before 2bF8 gene therapy and therefore had no pre-existing FVIII inhibitors, which we referred to as the “noninhibitor model” in the following. However, in the “inhibitor model,” in which recipients were preimmunized with rhFVIII to induce anti-FVIII inhibitor development, additional immune suppression, such as antithymocyte globulin (ATG), or a low dose of total body irradiation was needed to achieve therapeutic 2bF8-transduced engraftment [2].

Our further studies using platelets expressing the model antigen ovalbumin (OVA) have demonstrated that OVA-specific immune tolerance can be relatively easily achieved in the unprimed model after platelet-specific gene therapy [10]; however, there is an antagonistic dynamic process between immune responses and immune tolerance in the inhibitor model [11]. Altogether, results from our previous studies suggested that the immunologic consequences after platelet-targeted gene therapy in the immune primed setting are substantially different from those in the unprimed setting. Understanding how 2bF8-transduced cells are eliminated in the primed immune system conditioned by Bu only will allow us to determine more efficient preconditioning regimens to achieve clinically effective platelet gene therapy for both phenotypic correction and immune tolerance induction.

In the current study, we explored the mechanism of platelet-FVIII loss on Bu conditioning in the FVIII inhibitor model. More specifically, we investigated which cell type was responsible for the loss of 2bF8 genetically modified cells in FVIII-primed hemophilia A mice preconditioned with Bu only. We also explored which subset of 2bF8 genetically modified cells was eliminated in FVIII-primed FVIIInull mice after transplantation following Bu preconditioning. Because the anti-FVIII immune response is a T-cell–dependent humoral immune response [12,13], we finally examined whether the elimination of 2bF8 genetically modified cells in FVIII-primed mice conditioned with Bu was caused by antibody-dependent cell-mediated cytotoxicity (ADCC).

2 |. MATERIALS AND METHODS

2.1 |. Antibodies and reagents

The anti-mouse CD41 monoclonal antibody (MoAb) directly conjugated with phycoerythrin was purchased from Santa Cruz Biotech. The anti-mouse CD42b MoAb conjugated with DyLight-649 was purchased from Emfret Analytics. Mouse BD Fc Block was purchased from BD Pharmingen. Anti-CD3 MoAb (Clone 145–2C11) was purchased from eBioscience/Invitrogen/Thermo Fisher Scientific. Anti-CD8 MoAb (Clone 2.43) was purchased from Bioxcell. The EasySep Mouse SCA1 Positive Selection Kit and EasySep Mouse CD8+ T-Cell Isolation Kit were purchased from StemCell Technologies Inc. Recombinant human B-domain-deleted FVIII (rhFVIII, Xyntha) was purchased from Pfizer. Recombinant human full-length FVIII (rhfFVIII, Kogenate FS) was purchased from Bayer Pharma. Bu was purchased from American Regent Inc. Anti-(murine)-thymocyte globulin (ATG) was purchased from Fitzgerald. 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate was purchased from MP Biomedicals. The Chromogenix Coatest SP4 Factor VIII:C kit was purchased from Diapharma. The Mouse IFN-γ ELISpot kit was purchased from R&D Systems Inc.

2.2 |. Mice

FVIII knockout (FVIIInull) mice used in this study were in a 129/Sv × C57BL/6 mixed genetic background with a targeted disruption of exon 17 of the FVIII gene [14], a kind gift from Dr. Haig Kazazian at the University of Pennsylvania School of Medicine, and the colony was maintained in our facility. 2bF8 transgenic (2bF8Tg) mice were generated by the Transgenic Core Facility of the Medical College of Wisconsin and Blood Research Institute using either embryonic stem (ES) cell-mediated or lentivirus-mediated transgenesis as reported previously [15–18]. 2bF8Tg mice used in most of the studies were in a 129/Sv × C57BL/6 mixed genetic background unless otherwise specified. B-cell deficient homozygous mutant (μMT) and C57BL/6J wild-type (WT) mice were purchased from the Jackson Laboratory. Both male and female mice were used in our studies. Isoflurane or ketamine was used for anesthesia. All animals were kept in pathogen-free micro-isolator cages at the animal facilities operated by the Medical College of Wisconsin. Animal studies were performed according to a protocol approved by the Institutional Animal Care and Use Committee of the Medical College of Wisconsin.

2.3 |. Transplantation of 2bF8 genetically modified bone marrow cells into FVIIInull mice with pre-existing anti-FVIII immunity

To induce anti-FVIII immune responses, FVIIInull mice were immunized with rhFVIII by intravenous injection at 50 U/kg weekly for 4 to 5 weeks. For μMT and WT mice, which express normal levels of mouse FVIII, they were immunized with rhfFVIII at a dose of 200 U/kg weekly for 8 weeks. One week after the last immunization, plasma samples were collected from mice and inhibitor titers were determined by a modified Bethesda assay as described in our previous reports [1,15]. Animals were preconditioned with either Bu at 25 mg/kg at days −2 and −1 via intravenous (i.v.) administration, i.v. injection of Bu+ATG at a dose of 10 mg/kg, or intraperitoneal cavity injection of anti-CD8 antibody at 250 μg/mouse on day −2 before receiving whole bone marrow transplantation (BMT) or Sca-1+ HSCT from 2bF8Tg mice.

Bone marrow (BM) cells were collected from 2bF8Tg mice following procedures described in our previous reports [1,15]. Sca-1+ cells were isolated from the 2bF8Tg BM using the EasySep Mouse SCA1 Positive Selection Kit according to the protocol provided by the manufacturer. The percentage of Sca-1+ cells in whole BM and the purity of isolated Sca-1+ cells were analyzed by flow cytometry. Then, 1.2 to 1.4 × 106 Sca-1+ cells or the corresponding whole BM cells containing an equal amount of Sca-1+ cells based on the percentage determined by flow cytometry analysis were transplanted into each animal preconditioned with either Bu or Bu+anti-CD8 antibody. After at least 3 weeks of BM reconstitution, blood samples were collected by retro-orbital bleeds as previously reported [1]. Plasmas were isolated for a chromogenic-based Bethesda assay to determine anti-FVIII inhibitor titers, as previously reported [15]. Platelets were isolated and lysed in 0.5% CHAPS for the platelet lysate FVIII activity (FVIII:C) assay to determine platelet-FVIII expression levels following the protocol described in our previous report [2]. The bleeding phenotype was assessed in some recipients by a 6-hour tail bleeding test according to the procedures described in our previous reports [19,20].

2.4 |. Determination of the target cell subset that was 2bF8 genetically modified in the FVIII-primed FVIIInull model

The enzyme-linked immunosorbent spot (ELISpot)–based interferon gamma (IFN-γ) production assay was used to assess which subset of 2bF8 engineered cells was targeted in the inhibitor model. Three subsets of cells—platelets, Sca-1+ cells, and megakaryocytes (MKs)—were isolated from either 2bF8Tg or naïve FVIIInull mice. Because some T lymphocytes express Sca-1 antigen [21], mice were pretreated with anti-CD3 antibody at a dose of 200 μg per mouse to deplete T cells 2 days before cell isolation. For platelet isolation, blood samples were collected from 2bF8Tg mice via the vena cava and platelets were isolated following a previously described protocol [1]. For 2bF8Tg-Sca-1 cell isolation, the EasySep Mouse SCA1 Positive Selection Kit was used according to the protocol provided by the manufacturer. For MK isolation, BM was collected and stained with anti-CD41 and CD42 antibodies. CD41+CD42+ cells were sorted using a BD FACSAria cell sorter. FVIII-primed CD8+ effector T cells were isolated from splenocytes of rhFVIII-immunized FVIIInull mice using the EasySep Mouse CD8+ T-Cell Isolation Kit following the protocol provided by the manufacturer. Serial dilutions of CD8+ T cells were incubated with each subset of 2bF8Tg cells on an anti-mouse IFN-γ antibody-coated polyvinylidene difluoride 96-well microplate, and cocultured for 24 hours. IFN-γ produced by CD8 T cells was detected using the Mouse IFN Gamma ELISpot kit following the protocol provided by the manufacturer. Cells from naïve FVIIInull mice were used as controls in parallel. The IFN-γ secreting spots were determined by CTL-ImmunoSpot S6 Macro Analyzer.

2.5 |. Statistical analysis

Data are presented as mean ± SD. Statistical comparisons of 2’ experimental groups were evaluated by a 2-tail Student s t-test. Fisher exact test was used to compare the incidence of animals with platelet-FVIII expression levels >1 mU/108 platelets. The 1-way analysis of variance test was used to determine whether there were statistically significant differences between the means of 3 groups, and the Tukey test was used for multiple pairwise comparisons using GraphPad Prism 7 Software (GraphPad Software). A p value of <.05 was considered statistically significant.

3 |. RESULTS

3.1 |. Platelet-FVIII transgenic whole BMT supported FVIII expression in the inhibitor model on busulfan conditioning only

Our previous study showed that platelet-FVIII expression was abolished in the inhibitor model after 2bF8 lentiviral gene delivery to Sca-1+ cells under Bu preconditioning alone [2]. Therefore, we first tested if transplantation of the whole BM from 2bF8-expressing mice would also lead to platelet-FVIII expression loss in FVIIInull mice that were preimmunized with rhFVIII and had developed anti-FVIII inhibitors (inhibitor model) upon Bu preconditioning. As shown in Figure 1A, 2bF8 transgenic mice with a low platelet-FVIII expression level of 0.76 mU/108 platelets (2bF8-ESTg+/−), which were generated via ES cell –mediated transgenesis [15], were used as donors. Whole BM cells collected from donors were transplanted into FVIII-primed FVIIInull mice preconditioned with either Bu or Bu+ATG. After BM reconstitution, similar expression levels of platelet-FVIII were achieved in the Bu only and Bu+ATG conditioning groups, and their platelet-FVIII expression levels were sustained during the study period (Figure 1B–D). The inhibitor titers declined with time after BMT in both the Bu and Bu+ATG groups (Figure 1E–F). However, there was a negative correlation between the anti-FVIII inhibitor titers and platelet-FVIII expression levels in FVIII-primed recipients (Figure 1G).

FIGURE 1.

FIGURE 1

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Platelet-targeted FVIII (2bF8) transgenic whole bone marrow transplantation does not mimic platelet-FVIII expression loss on busulfan (Bu) conditioning in the inhibitor model. To test if whole bone marrow transplantation mimics platelet-FVIII expression loss on Bu conditioning in hemophilia A mice with pre-existing immunity, whole bone marrow cells from low-expressor (~0.76 mU/108 platelets) 2bF8-ESTg+/− transgenic mice were transplanted into FVIIInull mice preimmunized with recombinant human B-domain deleted FVIII (rhFVIII), and preconditioned with either Bu alone or Bu+ATG. Blood samples were collected for plasma or platelet isolation. Platelet-FVIII expression was determined by a chromogenic assay on platelet lysates. Anti-FVIII inhibitor titers were determined by a modified Bethesda assay. (A) A schematic diagram of the experimental design. (B) Platelet-FVIII expression in recipients under Bu conditioning alone. (C) Platelet-FVIII expression in recipients under Bu+ATG conditioning. (D) Comparison of the platelet-FVIII expression levels between the Bu group and the Bu+ATG group at 6 months after BMT. (E) Anti-FVIII inhibitor titers in the Bu group. (F) Anti-FVIII inhibitor titers in the BU+ATG group. (G) The correlation between platelet-FVIII expression and inhibitor titers at 4 weeks post-BMT. These results demonstrate that 2bF8 transgenic whole bone marrow engraftment was viable and platelet-FVIII expression was sustained on whole bone marrow transplantation in FVIII-sensitized hemophilia A mice under Bu preconditioning alone. ATG, antithymocyte globulin; BMT, bone marrow transplantation.

To investigate if the platelet-FVIII expression level impacts 2bF8-engineered BM engraftment, we used donor BM from 2bF8-LV18Tg+/− mice that were generated by lentivirus-mediated oocyte transduction transgenesis to express FVIII with a level of 6 mU/108 platelets [16], an 8-fold higher platelet-FVIII expression compared with 2bF8-ESTg+/− mice (Figure 2A). After whole BMT, platelet-FVIII expression levels in FVIII-primed recipients of the Bu conditioning only and Bu+ATG groups were comparable and sustained during the study period (Figure 2B–D). At 8 weeks after 2bF8-LV18Tg+/− BMT, all recipients (n = 10) survived the 6-hour tail bleeding test, which was significantly different compared with the FVIIInull controls, in which 7 of 12 mice died within 6 hours (Figure 2E). The percentage of remaining hemoglobin levels in the Bu group after the tail bleeding test was comparable with that in the Bu+ATG group, which was significantly higher than that in the FVIIInull group (Figure 2F). These data suggest that a higher platelet-FVIII expression does not impact the engraftment of 2bF8Tg BMT in FVIII-sensitized FVIIInull mice under Bu conditioning only and Bu+ATG.

FIGURE 2.

FIGURE 2

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Platelet-FVIII expression level does not impact 2bF8 genetically engineered engraftment in the inhibitor model under busulfan (Bu) conditioning. Whole bone marrow cells from 2bF8-LV18Tg+/− transgenic mice with an intermediate level of platelet-FVIII expression (6 mU/108 platelets) were transplanted into FVIIInull mice preimmunized with rhFVIII to induce anti-FVIII inhibitor development and preconditioned with either Bu alone or Bu+ATG. Blood samples were collected for plasma or platelet isolation. Platelet-FVIII expression was determined by a chromogenic assay on platelet lysates. Anti-FVIII inhibitor titers were determined by a modified Bethesda assay. (A) A schematic diagram of the experimental design. (B) Platelet-FVIII expression in recipients under Bu conditioning alone. (C) Platelet-FVIII expression in recipients under Bu+ATG conditioning. (D) Comparison of the platelet-FVIII expression levels between the Bu and Bu+ATG groups at 5 months after BMT. (E) The percentage of animals that survived the 6-hour tail bleeding test. Two months after transplantation, a 6-hour tail bleeding test was performed. The tail tip was transected by a scalpel using a 1.6-mm diameter template. Fifty microliters of blood was collected before and after the test, and hemoglobin (Hb) levels were measured. The Hb level in each animal before the test was defined as 100%. (F) The percentage of remaining hemoglobin in recipients after a 6-hour tail bleeding test. These results demonstrated that on whole bone marrow transplantation, higher level of platelet-FVIII expression did not abolish 2bF8-engineered therapeutic engraftment in FVIII-sensitized hemophilia A mice under Bu preconditioning alone. ATG, antithymocyte globulin; BMT, bone marrow transplantation; Bu, busulfan; Hb, hemoglobin; HSCT, hematopoietic stem cell transplantation.

Together, these results demonstrate that although the intensity of the immune response impacts platelet-FVIII expression, both Bu and Bu+ATG preconditioning prior to 2bF8Tg whole BMT led to successful 2bF8 engraftment and expression of therapeutic levels of platelet-FVIII in FVIII-primed FVIIInull mice.

3.2 |. Platelet-FVIII expression was abolished in FVIII-primed mice that received transgenic Sca-1+ hematopoietic cell transplantation on Bu conditioning

We then examined if transgenic Sca-1+ HSCT mimics platelet-FVIII expression loss upon Bu conditioning in 2bF8 lentiviral gene delivery to HSCs in the inhibitor model. Sca-1+ cells isolated from 2bF8Tg mice with a high platelet-FVIII expression level (18 mU/108 platelets) were transplanted into FVIII-primed FVIIInull mice preconditioned with Bu alone, as shown in Figure 3A. Whole BMT was used as a control in parallel. Before transplantation, the anti-FVIII inhibitor titer in the HSCT group was not significantly different compared with the BMT group (Figure 3B). However, after transplantation and 4 weeks of BM reconstitution, only 13% (n = 15) of recipients in the HSCT group had a platelet-FVIII expression level of >1 mU/108 platelets. In contrast, 80% (n = 5) of the recipients in the group that received whole BMT had a level of platelet-FVIII expression greater than 1 mU/108 platelets (Figure 3C). The level of platelet-FVIII expression in recipients transplanted with 2bF8Tg Sca-1+ cells (HSCT) was 0.55 ± 1.02 mU/108 platelets, which was significantly lower than that in animals that received whole BMT (7.19 ± 8.59 mU/108 platelets) (Figure 3D).

FIGURE 3.

FIGURE 3

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Platelet-FVIII expression loss occurs in platelet-targeted FVIII (2bF8) transgenic Sca-1+ HSCT on Bu conditioning. Sca-1+ cells were isolated from 2bF8-LV17/18Tg transgenic mice with a high level of platelet-FVIII expression (18 mU/108 platelets) and were transplanted into FVIII-primed FVIIInull mice preconditioned with Bu alone. Whole bone marrow transplantation was performed as a control in parallel. Blood samples were collected for plasma or platelet isolation. Anti-FVIII inhibitor titers were determined by a modified Bethesda assay. Platelet-FVIII expression was determined by a chromogenic assay on platelet lysates. (A) A schematic diagram of the experimental design. (B) Inhibitor titers in the inhibitor model before the transplantation. (C) The percentage of recipients with platelet-FVIII expression greater than 1 mU/108 platelets at 4 weeks after the transplantation. (D) Platelet-FVIII expression in recipients at 4 weeks after transplantation. (E) Platelet-FVIII expression in recipients at 6 months after the transplantation. These results demonstrate that 2bF8 transgenic Sca-1+ engraftment was abolished in FVIII-primed FVIIInull mice on Bu preconditioning alone. BM, bone marrow; HSCT, hematopoietic stem cell transplantation; Bu, busulfan.

In contrast, animals that were primed with rhFVIII but failed to develop detectable levels of anti-FVIII inhibitors and received Sca-1+ cells transplantation from 2bF8Tg mice under Bu conditioning alone had a FVIII expression of >4 mU/108 platelets with an average of 11.57 ± 5.45 mU/108 platelets (n = 6; Figure 3D). Platelet-FVIII expression levels were sustained long-term in the primed mice without inhibitors after 2bF8Tg-HSCT. The significant differences in platelet-FVIII expression between the BMT and HSCT groups were maintained during the entire study period of 6 months (Figure 3E). These data demonstrated that 2bF8 transgenic Sca-1+ cell transplantation mimics platelet-FVIII loss in 2bF8 lentiviral gene delivery to HSCs on Bu conditioning in the inhibitor model.

3.3 |. CD8 T-cell depletion enhanced platelet-FVIII expression in FVIII-primed recipients that received 2bF8Tg-HSCT under Bu preconditioning

We next explored if CD8 T cells owing to their cytotoxic potential are responsible for platelet-FVIII loss in the inhibitor model on Bu peconditioning. We depleted CD8 T cells in FVIII-primed mice using anti-CD8 antibodies in addition to Bu preconditioning before the transplantation of Sca-1+ cells from 2bF8Tg donors, and compared this treatment regimen with Bu preconditioning without CD8 T-cell depletion (Figure 4A). FVIII-primed FVIIInull mice with comparable FVIII inhibitor titers were randomly assigned to the 2 groups before Sca-1+ cell transplantation (Figure 4B). After 2bF8Tg-HSCT and BM reconstitution, platelet-FVIII expression levels in recipients preconditioned with Bu+anti-CD8 antibody were significantly higher than that in recipients preconditioned with Bu alone at both the early time point of 4 weeks (Figure 4C) and the later time point of 6 months (Figure 4D). Platelet-FVIII expression levels in recipients preconditioned with Bu+anti-CD8 antibody significantly increased from 2.14 ± 2.35 at week 4 to 6.46 ± 5.87 mU/108 platelets at 6 months after 2bF8Tg-HSCT. In contrast, the platelet-FVIII levels in recipients preconditioned with Bu only were 0.29 ± 0.52 at week 4 and decreased to 0.12 ± 0.33 mU/108 platelets at 6 months. These data suggest that FVIII-primed CD8 T cells in FVIIInull mice are responsible for platelet-FVIII loss on Bu conditioning in the inhibitor model.

FIGURE 4.

FIGURE 4

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Elimination of CD8 T cells improves the engraftment of 2bF8-engineered Sca-1+ cell transplantation upon Bu conditioning. Sca-1+ cells were isolated from 2bF8-LV17/18Tg transgenic mice with a high level of platelet-FVIII expression (18 mU/108 platelets), and were transplanted into FVIIInull mice preimmunized with rhFVIII, followed by Bu alone or Bu+anti-CD8 antibody conditioning. Blood samples were collected for plasma or platelet isolation. Anti-FVIII inhibitor titers were determined by Bethesda assay. Platelet-FVIII expression was determined by a chromogenic assay on platelet lysates. (A) A schematic diagram of the experimental design. (B) Inhibitor titers before transplantation. (C) Platelet-FVIII expression in recipients at 4 weeks after the transplantation. (D) Platelet-FVIII expression in recipients at 6 months after the transplantation. These results demonstrate that adding an anti-CD8 antibody to Bu conditioning to deplete FVIII-primed CD8 T cells could rescue 2bF8 transgenic Sca-1+ engraftment in FVIII-primed FVIIInull mice on 2bF8-HSCT. BM, bone marrow; Bu, busulfan; HSCT, hematopoietic stem cell transplantation.

3.4 |. Sca-1+ cells from 2bF8Tg mice restimulated CD8 T cells from FVIII-primed FVIIInull mice to secrete IFN-γ

We next determined which subset of cells from 2bF8Tg mice were targeted by CD8 T-cell–mediated killing, leading to platelet-FVIII expression loss after 2bF8Tg-HSCT on Bu conditioning in the inhibitor model. As shown in Figure 5A, we cocultured rhFVIII-primed CD8 T cells with platelets, Sca-1+ cells, or MKs isolated from either 2bF8Tg with 18 mU/108 platelet-FVIII expression or FVIIInull mice, and measured IFNγ production by CD8 T cells using ELISpot assays. CD8 T cells from rhFVIII-primed FVIIInull mice were efficiently restimulated by Sca-1+ cells from 2bF8Tg mice, but not by platelets or MKs, and secreted IFNγ (Figure 5B). As controls, without 2bF8Tg-Sca-1+ cell stimulation, FVIII-primed CD8 T cells did not secrete IFNγ (Figure 5B), and similarly, as expected, Sca-1+ cells from FVIIInull mice did not activate rhFVIII-primed CD8 T cells to secrete IFNγ (Figure 5C). When IFNγ spot-forming units in CD8 T cells were calculated, there were 14.1 ± 2.1 spots/105 total CD8 T cells that secreted IFNγ when restimulated with 5 × 104 2bF8Tg-Sca-1+ cells. In contrast, there were only 1.5 ± 1.4 spots/105 CD8 T cells secreting IFNγ when stimulated with 107 platelets and 2.3 ± 0.9 spots/105 CD8 T cells secreting IFNγ when stimulated with 2 × 104 MKs (Figure 5D). Together, these results suggest that Sca-1+ cells from 2bF8Tg mice are the targets for rhFVIII-primed CD8 T-cell–mediated killing on Bu conditioning in the inhibitor model and that IFNγ production from rhFVIII-primed CD8 T cells stimulated with 2bF8Tg-Sca-1+ cells was the FVIII-specific response.

FIGURE 5.

FIGURE 5

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Determination of the 2bF8-engineered target cells attacked by FVIII-primed CD8 T cells in the inhibitor model of hemophilia A. Three subsets of cells—platelets, Sca-1+, and megakaryocytes (MKs)—were isolated from 2bF8 transgenic mice and incubated with CD8 T cells isolated from FVIIInull mice that were preimmunized with rhFVIII. Serial dilutions of FVIII-primed CD8+ T cells were incubated with each subset of 2bF8Tg cells on an anti-mouse IFN-γ antibody-coated polyvinylidene difluoride 96-well microplate and cocultured for 24 hours. IFN-γ produced by CD8 T cells was detected using the Mouse IFN Gamma ELISpot kit. Subsets of cells isolated from naïve FVIIInull mice were used as controls in parallel. (A) A schematic diagram of the experimental design. (B) Representative IFNγ ELISpot results from all subsets of cells. (C) Magnified representative IFN-γ ELISpot results from coincubation of FVIII-primed CD8 T cells with 2bF8 transgenic Sca-1+ cells. (D) IFN-γ spot-forming units per 105 FVIII-primed CD8 T cells when incubated with each 2bF8-engineered subsets of cells. These results demonstrate that 2bF8 genetically modified Sca-1+ cells could activate FVIII-primed CD8 T cells to produce IFN-γ, indicating that Sca-1+ cells are the target of CD8 T cells, leading to FVIII expression loss on Bu conditioning. Bu, busulfan; IFN, interferon.

3.5 |. Platelet-FVIII expression loss in the inhibitor model preconditioned with Bu is not mediated by ADCC

Lymphocyte-mediated cytotoxicity and ADCC are the 2 pathways by which immune cells kill target cells. Because the anti-FVIII immune response is a CD4 T-cell–dependent humoral immune response [12,13], we wanted to explore whether the elimination of 2bF8 genetically modified cells in FVIII-primed FVIIInull mice conditioned with Bu alone is caused by ADCC. To this end, we used a B-cell–deficient mouse model (μMT) [22], which is in a C57BL/6J background with no mature B cells but with normal T cells and dendritic cells [23] for this study. Due to a lack of mature B cells, μMT mice failed to produce antibodies after antigenic stimulation [24]. We transplanted 2bF8Tg-Sca-1+ cells isolated from 2bF8-LV18Tg, which is in a C57BL/6J background with 12 mU/108 platelet-FVIII expression, into rhfFVIII-primed μMT mice preconditioned with Bu only (Figure 6A). As expected, no anti-FVIII antibodies were detected in μMT mice even after rhfFVIII immunization (Figure 6B). As a control, all WT mice developed FVIII inhibitors with titers of 138 ± 61 BU/mL when they were immunized using the same rhfFVIII immunization protocol. After 2bF8Tg-Sca-1+ transplantation and BM reconstitution, there was no platelet-FVIII detected in FVIII-primed μMT recipients even though the μMT mice did not produce anti-FVIII antibodies (Figure 6C). The platelet-FVIII expression level in donor 2bF8Tg-LV17/18 mice was 12.32 ± 0.73 mU/108 platelets. The platelet-FVIII expression level in unprimed μMT mouse was similar to that in donors, as expected. Our data from this study suggest that the loss of platelet-FVIII expression in the inhibitor model is not mediated by T-cell–dependent anti-FVIII antibody production or the ADCC pathway but rather directly by CD8 T cells.

FIGURE 6.

FIGURE 6

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Platelet-FVIII expression loss in Bu preconditioning is not dependent on anti-FVIII antibody-mediated cytotoxicity. To explore if platelet-FVIII expression loss is because of antibody-mediated cytotoxicity, we used B cell–deficient μMT mice. Sca-1+ cells were isolated from 2bF8Tg mice, which were in a C57BL/6J background with a FVIII expression level of 12 mU/108 platelets and transplanted into μMT mice preimmunized with rhfFVIII. WT C57BL/6J mice were used as a control in parallel. Blood samples were collected for plasma or platelet isolation. Inhibitor titers were determined by Bethesda assay. Platelet-FVIII expression was determined by a chromogenic assay on platelet lysates. (A) Schematic diagram of the experimental design. (B) Anti-FVIII inhibitor titers in recipients before the transplantation. (C) Platelet-FVIII expression. These results demonstrate that platelet-FVIII expression loss in 2bF8-HSCT was not mediated by anti-FVIII antibody-dependent cytotoxicity. BM, bone marrow; Bu, busulfan; HSCT, hematopoietic stem cell transplantation; μMT, homozygous mutant; WT, wild-type.

4 |. DISCUSSION

The immune response to transgene or viral proteins is a fundamental problem in gene therapy [25–27]. Our previous studies have demonstrated that pre-existing anti-FVIII immunity does not negate 2bF8 genetically modified therapeutic engraftment and that immune tolerance can be established with platelet-targeted gene therapy when a sufficient preconditioning regimen is employed [1,2]. However, our studies also showed that in mice with pre-existing FVIII inhibitors (inhibitor model), 2bF8 transgene expression was abolished in FVIII-primed recipients preconditioned with Bu alone (without ATG), indicating that 2bF8-transduced engraftment was abolished [2]. In the current study, we report that platelet-FVIII expression loss in the inhibitor model under Bu conditioning resulted from the elimination of 2bF8 genetically modified Sca-1+ cells by FVIII-primed CD8 T cells.

Immune responses can be significant obstacles to gene therapy of hemophilia A in the clinical setting of pre-existing anti-FVIII immunity as it requires the primed immune system to be tolerized to the neoprotein introduced by the gene therapy. Inducing immune tolerance in the face of a primed immune system is more challenging than the prevention of an immune response in the unprimed setting. Our previous studies have demonstrated that lentiviral-mediated gene delivery to HSC platelet-targeted gene therapy can induce antigen-specific immune tolerance in both unprimed and primed models [10,11]. However, we found that transgene protein was diminished when animals had pre-existing high immune responses before gene therapy, even under an optimized preconditioning regimen, although immune tolerance was still established [11]. We hypothesize that there is an antagonistic dynamic process between immune responses to neoprotein and tolerization through clonal deletion of antigen-specific CD4 and CD8 T cells after platelet-targeted gene therapy in the immune primed model. This hypothesis is supported by the data from our previous study [11] as well as the data from the current study.

In this study, we found that platelet-FVIII expression was sustained in the inhibitor model mice that received transplantation of 2bF8Tg whole BM cells (BMT) but not in those that received 2bF8Tg Sca-1+ cell transplantation. It is well known that Bu mainly exerts myelosuppressive effects [28–30] and barely impacts lymphocytes when it is employed as a preconditioning regimen for transplantation [31]. Thus, anti-FVIII immunity was maintained in FVIII-primed mice after Bu conditioning. In the setting of 2bF8Tg BMT, it was likely that the 2 events happened simultaneously after transplantation in FVIII-primed recipients preconditioned with Bu. One was that FVIII-primed CD8 T cells were reactivated by 2bF8Tg-Sca-1+ stem/progenitor cells and eliminated the Sca-1+ cells through lymphocyte-mediated cytotoxicity. The other could be that MKs/platelets from 2bF8Tg BMT induced immune tolerance through the clonal deletion of FVIII-primed CD4 and CD8 T effector cells, as demonstrated in our previous OVA model studies [10,11]. We speculate that immune tolerance induction prevailed over the elimination of 2bF8-engineered Sca-1+ stem/progenitors by the remaining endogenous FVIII-primed CD8 T cells, leading to sustained platelet-FVIII expression in 2bF8Tg-BMT recipients.

In the setting of 2bF8-Sca-1+ transplantation under Bu conditioning, the remaining FVIII-primed cytotoxic CD8 T cells in the body, in turn, could attack and kill the cells that express FVIII peptides on MHC class I molecules. FVIII-primed CD8 T cells could be readily reactivated by 2bF8-engineered Sca-1+ stem/progenitor cells upon transplantation of 2bF8Tg-Sca-1+ cells and kill the target cells. We reason that, in this setting, FVIII-primed CD8 T cells were exposed to 2bF8Tg-Sca-1+ cells directly, almost with no opportunity to be desensitized/tolerized by 2bF8-engineered MKs/platelets, because it takes time to differentiate 2bF8-Sca-1+ stem/progenitor cells into MKs/platelets [32] after 2bF8Tg HSCT for immune tolerance induction in recipients. At the time when 2bF8-Sca-1+ stem/progenitor cells were eradicated by FVIII-primed CD8 T cells, there were no tolerizing 2bF8Tg MKs/platelets produced yet. The other potential explanation is that the killing effect of active FVIII-primed CD8+ T cells on 2bF8Tg-Sca-1+ stem/progenitor cells can overcome the immune tolerance induced by 2bF8Tg MKs/platelets in the setting of 2bF8Tg HSCT. These could explain why there was no platelet-FVIII expression detected in 2bF8Tg-HSCT recipients in the current study and why 2bF8 lentiviral gene delivery to Sca-1+ cells failed to induce platelet-FVIII expression in the inhibitor model under Bu conditioning only [2].

Although our current study suggests that 2bF8-engineered Sca1+ cells are the target of FVIII-primed CD8 T cells in 2bF8 gene therapy, we still do not know which subset(s) of Sca-1+ cells is attacked and killed. Besides being activated in MKs/platelets, the αIIb promoter is also activated in MK progenitors and pluripotent HSCs [33,34]. If FVIII was produced by MK progenitors that do not have α-granules, FVIII would be secreted and those cells might become targets of FVIII-primed CD8 T cells. In addition, it has been suggested that the αIIb promoter is also activated in the early stage of embryonic HSCs (from E11.5 embryos) but not in more mature HSCs [35]. We speculate that during the early stage of BM reconstitution, long-term engrafting HSCs may have a similar phenotype as a certain stage of embryonic HSCs, eg, the αIIb promoter gets activated because HSCs need to be rapidly expanded to reconstitute BM. Thus, FVIII may be expressed in those embryonic-like HSCs transiently in the BM of recipients after receiving 2bF8-engineered Sca-1+ cell transplantation, and 2bF8Tg-HSCs/progenitors become a target of FVIII-primed CD8 T cells. Further investigation is warranted to characterize the precise target cells of the FVIII-primed cytotoxic CD8 T cells in 2bF8 gene therapy in hemophilia A.

Our current study suggests that FVIII-specific cytotoxic CD8 T cells could be an obstacle in platelet-targeted FVIII gene therapy in patients with hemophilia A and pre-existing anti-FVIII immunity. Elimination of FVIII-specific CD8 T cells in recipients is critical for the success of platelet-targeted FVIII gene therapy in the inhibitor model. Our study suggests that FVIII-primed CD8 T cells could be eradicated by either sufficient preconditioning before transplantation, such as depletion of CD8 T cells using anti-CD8 antibodies, or immune tolerance induction via 2bF8-engineered MKs/platelets from the transplant. In the clinic, patients’ CD34+ HSCs will be harvested for ex vivo transduction with 2bF8 lentivirus, followed by autologous transplantation. Therefore, applying effective preconditioning to eradicate FVIII-primed CD8 T cells will become critically important for platelet-FVIII gene therapy of patients with hemophilia A and inhibitors because there are no 2bF8-engineered MKs/platelets to drive immune tolerance induction at the time of reintroduction of 2bF8-transduced CD34+ HSCs.

In summary, our studies demonstrate that pre-existing anti-FVIII immunity can negatively impact therapeutic engraftment of 2bF8 genetically manipulated Sca-1+ hematopoietic stem/progenitor cells in FVIIInull mice with pre-existing anti-FVIII immunity via cytotoxic CD8 T cells. Efficient preconditioning is essential for viable engraftment in platelet-targeted FVIII gene therapy to achieve clinical efficacy of hemostasis and immune tolerization in patients with hemophilia A along with inhibitors. Sufficient pretransplant conditioning to eradicate FVIII-sensitized CD8 T cells, which may be achieved with agents, such as clinically used ATG, is key to achieving successful platelet-targeted FVIII gene therapy in HA in a setting of pre-existing anti-FVIII immunity.

Essentials.

  • Effective preconditioning is critical for the success of hematopoietic stem cell–based platelet-specific gene therapy of hemophilia A with pre-existing immunity.

  • Platelet-factorVIII (FVIII) expression was abolished in FVIII-primed mice receiving transgenic Sca-1+ cell transplantation on busulfan conditioning.

  • 2bF8-engineered Sca-1+ cells can be a target of FVIII-primed CD8 T cells in hemophilia A with pre-existing immunity.

  • The loss of platelet-FVIII expression in the inhibitor model is not mediated by T-cell–dependent anti-FVIII antibody production but rather directly by CD8 T cells.

ACKNOWLEDGMENTS

This work was supported by the National Institutes of Health grant (HL-102035; to Q.S.) and generous gifts from the Midwest Athletes Against Childhood Cancer and Bleeding Disorders (MACC) Fund and Children’s Hospital of Wisconsin Foundation (to Q.S.). We thank H. Kazazian at the University of Pennsylvania School of Medicine for the FVIIInull mice.

Funding information

National Institutes of Health grant (HL-102035; to Q.S.)

Midwest Athletes Against Childhood Cancer and Bleeding Disorders (MACC) Fund

Children’s Hospital of Wisconsin Foundation (to Q.S.).

Footnotes

DECLARATION OF COMPETING INTEREST

The authors declare no competing financial interests.

REFERENCES

  • [1].Shi Q, Fahs SA, Wilcox DA, Kuether EL, Morateck PA, Mareno N, Weiler H, Montgomery RR. Syngeneic transplantation of hematopoietic stem cells that are genetically modified to express factor VIII in platelets restores hemostasis to hemophilia A mice with preexisting FVIII immunity. Blood. 2008;112:2713–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [2].Kuether EL, Schroeder JA, Fahs SA, Cooley BC, Chen Y, Montgomery RR, Wilcox DA, Shi Q. Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity. J Thromb Haemost. 2012;10:1570–80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Gao C, Schroeder JA, Xue F, Jing W, Cai Y, Scheck A, Subramaniam S, Rao S, Weiler H, Czechowicz A, Shi Q. Nongenotoxic antibody-drug conjugate conditioning enables safe and effective platelet gene therapy of hemophilia A mice. Blood Adv. 2019;3:2700–11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].McPherson ME, Hutcherson D, Olson E, Haight AE, Horan J, Chiang KY. Safety and efficacy of targeted busulfan therapy in children undergoing myeloablative matched sibling donor BMT for sickle cell disease. Bone Marrow Transplant. 2011;46:27–33. [DOI] [PubMed] [Google Scholar]
  • [5].Buggia I, Locatelli F, Regazzi MB, Zecca M. Busulfan. Ann Pharmacother. 1994;28:1055–62. [DOI] [PubMed] [Google Scholar]
  • [6].Diaz MA, Vicent MG, Madero L. High-dose busulfan/melphalan as conditioning for autologous PBPC transplantation in pediatric patients with solid tumors. Bone Marrow Transplant. 1999;24:1157–9. [DOI] [PubMed] [Google Scholar]
  • [7].Ghalie R, Reynolds J, Valentino LA, Manson S, Korenblit AD, Feingold JM, Adler SS, Pruett J, Kaizer H. Busulfan-containing pretransplant regimens for the treatment of solid tumors. Bone Marrow Transplant. 1994;14:437–42. [PubMed] [Google Scholar]
  • [8].Rohaly J The use of busulfan therapy in bone marrow transplantation. A nursing overview. Cancer Nurs. 1989;12:144–52. [PubMed] [Google Scholar]
  • [9].Trame MN, Bergstrand M, Karlsson MO, Boos J, Hempel G. Population pharmacokinetics of busulfan in children: increased evidence for body surface area and allometric body weight dosing of busulfan in children. Clin Cancer Res. 2011;17:6867–77. [DOI] [PubMed] [Google Scholar]
  • [10].Luo X, Chen J, Schroeder JA, Allen KP, Baumgartner CK, Malarkannan S, Hu J, Williams CB, Shi Q. Platelet gene therapy promotes targeted peripheral tolerance by clonal deletion and induction of antigen-specific regulatory T cells. Front Immunol. 2018;9:1950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Li J, Chen J, Schroeder JA, Hu J, Williams CB, Shi Q. Platelet gene therapy induces robust immune tolerance even in a primed model via peripheral clonal deletion of antigen-specific T cells. Mol Ther Nucleic Acids. 2021;23:719–30. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Lollar P Pathogenic antibodies to coagulation factors. Part one: factor VIII and factor IX. J Thromb Haemost. 2004;2:1082–95. [DOI] [PubMed] [Google Scholar]
  • [13].Hausl C, Ahmad RU, Schwarz HP, Muchitsch EM, Turecek PL, Dorner F, Reipert BM. Preventing restimulation of memory B cells in hemophilia A: a potential new strategy for the treatment of antibody-dependent immune disorders. Blood. 2004;104:115–22. [DOI] [PubMed] [Google Scholar]
  • [14].Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD, Kazazian HH Jr. Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A. Nat Genet. 1995;10:119–21. [DOI] [PubMed] [Google Scholar]
  • [15].Shi Q, Wilcox DA, Fahs SA, Weiler H, Wells CW, Cooley BC, Desai D, Morateck PA, Gorski J, Montgomery RR. Factor VIII ectopically targeted to platelets is therapeutic in hemophilia A with high-titer inhibitory antibodies. J Clin Invest. 2006;116:1974–82. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Chen Y, Schroeder JA, Chen J, Luo X, Baumgartner CK, Montgomery RR, Hu J, Shi Q. The immunogenicity of platelet-derived FVIII in hemophilia A mice with or without preexisting anti-FVIII immunity. Blood. 2016;127:1346–54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [17].Baumgartner CK, Zhang G, Kuether EL, Weiler H, Shi Q, Montgomery RR. Comparison of platelet-derived and plasma factor VIII efficacy using a novel native whole blood thrombin generation assay. J Thromb Haemost. 2015;13:2210–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Baumgartner CK, Mattson JG, Weiler H, Shi Q, Montgomery RR. Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice. J Thromb Haemost. 2017;15:98–109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [19].Chen Y, Schroeder JA, Kuether EL, Zhang G, Shi Q. Platelet gene therapy by lentiviral gene delivery to hematopoietic stem cells restores hemostasis and induces humoral immune tolerance in FIX(-null) mice. Mol Ther. 2014;22:169–77. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Schroeder JA, Chen Y, Fang J, Wilcox DA, Shi Q. In vivo enrichment of genetically manipulated platelets corrects the murine hemophilic phenotype and induces immune tolerance even using a low multiplicity of infection. J Thromb Haemost. 2014;12:1283–93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Spangrude GJ, Aihara Y, Weissman IL, Klein J. The stem cell antigens Sca-1 and Sca-2 subdivide thymic and peripheral T lymphocytes into unique subsets. J Immunol. 1988;141:3697–707. [PubMed] [Google Scholar]
  • [22].Kitamura D, Roes J, Kuhn R, Rajewsky K. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature. 1991;350:423–6. [DOI] [PubMed] [Google Scholar]
  • [23].Linton PJ, Harbertson J, Bradley LM. A critical role for B cells in the development of memory CD4 cells. J Immunol. 2000;165:5558–65. [DOI] [PubMed] [Google Scholar]
  • [24].Melamed D, Miri E, Leider N, Nemazee D. Unexpected autoantibody production in membrane Ig-mu-deficient/lpr mice. J Immunol. 2000;165:4353–8. [DOI] [PubMed] [Google Scholar]
  • [25].Herzog RW, Dobrzynski E. Immune implications of gene therapy for hemophilia. Semin Thromb Hemost. 2004;30:215–26. [DOI] [PubMed] [Google Scholar]
  • [26].High KA. The gene therapy journey for hemophilia: are we there yet? Blood. 2012;120:4482–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Scott DW, Lozier JN. Gene therapy for haemophilia: prospects and challenges to prevent or reverse inhibitor formation. Br J Haematol. 2012;156:295–302. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Santos GW, Tutschka PJ, Brookmeyer R, Saral R, Beschorner WE, Bias WB, Braine HG, Burns WH, Elfenbein GJ, Kaizer H. Marrow transplantation for acute nonlymphocytic leukemia after treatment with busulfan and cyclophosphamide. N Engl J Med. 1983;309:1347–53. [DOI] [PubMed] [Google Scholar]
  • [29].Haddow A, Timmis GM. Myleran in chronic myeloid leukaemia; chemical constitution and biological action. Lancet. 1953;264:207–8. [DOI] [PubMed] [Google Scholar]
  • [30].Elson LA, Galton DA, Till M. The action of chlorambucil (CB. 1348) and busulphan (myleran) on the haemopoietic organs of the rat. Br J Haematol. 1958;4:355–74. [DOI] [PubMed] [Google Scholar]
  • [31].Kang EM, Hsieh MM, Metzger M, Krouse A, Donahue RE, Sadelain M, Tisdale JF. Busulfan pharmacokinetics, toxicity, and low-dose conditioning for autologous transplantation of genetically modified hematopoietic stem cells in the rhesus macaque model. Exp Hematol. 2006;34:132–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Thon JN, Italiano JE. Platelet formation. Semin Hematol. 2010;47:220–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [33].Fraser JK, Leahy MF, Berridge MV. Expression of antigens of the platelet glycoprotein IIb/IIIa complex on human hematopoietic stem cells. Blood. 1986;68:762–9. [PubMed] [Google Scholar]
  • [34].Ody C, Vaigot P, Quere P, Imhof BA, Corbel C. Glycoprotein IIb-IIIa is expressed on avian multilineage hematopoietic progenitor cells. Blood. 1999;93:2898–906. [PubMed] [Google Scholar]
  • [35].McKinney-Freeman SL, Naveiras O, Yates F, Loewer S, Philitas M, Curran M, Park PJ, Daley GQ. Surface antigen phenotypes of hematopoietic stem cells from embryos and murine embryonic stem cells. Blood. 2009;114:268–78. [DOI] [PMC free article] [PubMed] [Google Scholar]

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