Continuous in vivo infusion of interferon-gamma (IFN-γ) enhances engraftment of syngeneic wild-type cells in Fanca^–/– and Fancg^–/– mice

Fanconi anemia (FA) is a heterogeneous genetic disorder characterized by a progressive bone marrow aplasia, chromosomal instability, and the acquisition of malignancies. The progressive bone marrow failure in FA and the late developing myeloid malignancies account for 90% of the mortality in FA.¹  Currently, the only cure for the hematopoietic manifestations of FA is HLA-identical allogeneic bone marrow transplantation, a therapy available to only about 30% of patients.²  Twelve FA complementation types (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ, FANCL, and FANCM) have been identified and the cDNAs of 11 FA genes have been sequenced.^3-10  The identification of these genes raises the potential of using gene transfer technology to express the functional cDNA into autologous stem cells. The bone marrow hypoplasia commonly observed in FA patients¹  and the reduced repopulating ability of stem cells in mice containing a disruption of the murine homologue of an FA gene^11,12  led to the hypothesis that autologous, genetically-corrected stem cells would have an engraftment and proliferation advantage. However, ex vivo culture is known to reduce stem cell engraftment in both normal murine and human cells,^13-15  and in an initial phase 1 clinical trial in FA patients where myelopreparation was not routinely performed¹⁶  the detection of transduced cells was low in all patients. Furthermore, the most efficient engraftment of transduced cells in the clinical FA trial was observed in a single patient who received nonlethal ionizing radiation. Collectively, these data provide rationale for considering nonmyeloablative therapy prior to transplantation of autologous genetically-corrected stem cells.

FA cells exhibit increased chromosomal breaks in response to DNA-damaging agents in vitro.^3,17  Thus there is a theoretical potential of FA patients having an increased incidence for secondary malignancies following myelopreparation with genotoxic agents. Due to the low frequency of FA in the general population¹⁸  and the high incidence of malignant predisposition of FA patients, it is difficult to fully ascertain the risk associated with the genotoxic myelopreparation regimen in patients. However, several studies suggest an increased risk for hematopoietic and nonhematopoietic secondary malignancies^19-22  after hematopoietic stem cell transplantation in FA patients. For these reasons, a nongenotoxic myelopreparative regimen would be ideal in selecting genetically-corrected stem cells in vivo.

Interferon-gamma (IFN-γ) is a nongenotoxic immunoregulatory lymphokine with a high therapeutic index²³  that has been used to treat a number of malignancies.^24-26  Rathbun et al provided the first evidence that murine (Fancc^–/–) and human (FANCC) hematopoietic cells were hypersensitive to IFN-γ via an increase in apoptosis.²⁷  These results were subsequently confirmed in an independent Fancc^–/– murine model in vitro and in vivo.^28,29  Previous work indicates that a mechanism for the hypersensitivity of FANCC-deficient cells to IFN-γ is mediated by FANCC physically interacting with heat shock protein 70 to inhibit PKR activation and apoptosis.³⁰  Subsequent studies showed that FANCA and FANCG also participate in that complex.³¹  FANCA progenitors were found to be hypersensitive to IFN-γ via an increase in apoptosis in that same study.³¹  However, one in vitro study in a murine model of FANCA (Fanca^–/–) was unable to detect an increase in the sensitivity of Fanca^–/– myeloid progenitors to IFN-γ compared with wild-type (WT) controls.³²  Whether the differing results of the human FANCA and murine (Fanca^–/–) studies reflect differences in in vitro protocols or, alternatively, species-specific differences remains unclear. Studies to determine whether Fanca^–/– or Fancg^–/– progenitors are hypersensitive to IFN-γ in vivo have not been attempted. Since FANCA, FANCG, and FANCC patients collectively account for 90% of all FA patients,¹  determination of the hypersensitivity of hematopoietic progenitors from these complementation groups to IFN-γ could have broad relevance to the potential of IFN-γ in nongenotoxic myelopreparation. Here we tested the hypothesis that IFN-γ can be used as a single myelopreparative agent in Fanca^–/– and Fancg^–/– mice prior to transplantation of isogenic repopulating stem cells. Our results demonstrate that both primitive and mature Fanca^–/– and Fancg^–/– myeloid progenitors are hypersensitive to IFN-γ treatment in vitro and in vivo compared with syngeneic WT progenitors, similar to Fancc^–/– mice. In addition, continuous subcutaneous infusion of IFN-γ facilitated the engraftment of consistently high levels of exogenous, isogeneic WT hematopoietic stem/progenitor cells. These data support the hypothesis that IFN-γ may be useful as a nongenotoxic, myelopreparative regimen in multiple FA complementation groups.

The Fanca^+/– and Fancg^+/– lines used in these studies have been previously described.^33,34 

Fanca^+/– mice (C57Bl/6 × SV129), Fancg^+/– mice, and Fancc^+/– mice were backcrossed into a C57Bl/6J strain (CD45.2⁺) and then bred to generate Fanca^–/– or Fancg^–/– and WT mice. Congenic C57Bl/6J (CD45.2⁺) and B6.SJL-PtrcaPep3b/BoyJ (B6.BoyJ) mice (CD45.1⁺) were purchased from Jackson Laboratories (Bar Harbor, ME) and maintained in our animal facility.¹² 

All experimental mice were housed in the Indiana University School of Medicine Laboratory Animal Resource Center and examined regularly by one of the investigators. Murine IFN-γ was purchased from Peprotech (Rocky Hill, NJ), reconstituted as recommended by the manufacturer, and placed within micro-osmotic pumps to administer a defined volume (0.5 μL/h for 7 days; DURECT, Cupertino, CA). Micropumps containing either IFN-γ or phosphate-buffered saline (PBS) were implanted subcutaneously into the backs of WT, Fancc^–/–, Fanca^–/–, and Fancg^–/– mice and removed following a 7-day infusion. IFN-γ was administered at a dosage of 0 or 400 μg/kg per day. These studies were reviewed and approved by the Indiana University School of Medicine Institutional Animal Care and Use Committee.

Complete peripheral blood counts were obtained using an automated cell counter (Sysmex, Kobe, Japan) 7 days after treatment. The accuracy of abnormal blood counts was verified by direct examination of blood smears. Renal and hepatic functions of the mice were also examined. Spleen weight and bone marrow (BM) cellularity were determined. The hematopoietic tissues were collected for clonogenic assays and histologic analysis.

Bone marrow cells were flushed from the tibiae and femurs using Iscoves modified Dulbecco media (IMDM; Gibco-BRL, Gaithersburg, MD) containing 5% fetal calf serum (FCS; Hyclone Laboratories, Logan, UT). Low-density mononuclear cells (LDMNCs) were prepared by centrifugation on ficoll-hypaque (density, 1.119; Sigma, St Louis, MO).²⁸ 

Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin 3 (IL-3), and stem cell factor (SCF) were obtained from Peprotech; recombinant murine IL-1 (mIL-1) and macrophage CSF (M-CSF) were purchased from R&D Research Laboratories (Minneapolis, MN). LDMNCs were placed into culture in triplicate 35-mm plates (Becton Dickinson, Franklin Lakes, NJ) at a final concentration of 2 × 10⁴ LDMNCs or 5 × 10⁵ spleen LDMNCs per plate. To culture low proliferating potential colony-forming cells (LPP-CFCs) and high proliferating potential colony-forming cells (HPP-CFCs), LDMNCs and recombinant growth factors were added to (0.66%-1.0%) agar, and the solution was thoroughly mixed before plating as previously described.³⁵  Growth factors used for culture of HPP-CFCs and LPP-CFCs included SCF, IL-1, M-CSF, GM-CSF, and IL-3.³⁵  To evaluate growth-inhibitory effects of IFN-γ on progenitor cells, LDMNCs were plated in the absence or presence of IFN-γ (0.3-1 ng/mL) in clonogenic assays as described elsewhere.²⁸ 

Fancc^–/–, Fanca^–/–, Fancg^–/–, and WT mice (CD45.2⁺) were treated with 400 μg/kg per day IFN-γ or PBS for 7 days as described in “Treatment and monitoring” and then received transplants of 1 × 10⁷ BM nucleated B6.BoyJ cells (CD45.1⁺). The percentage of CD45.1⁺ cells in the peripheral blood was analyzed at 1, 4, and 6 months after transplantation as described previously.^11,36  To evaluate multilineage reconstitution of donor CD45.1⁺ cells, peripheral blood of IFN-γ–treated or untreated mice was costained with phycoerythrin (PE)–conjugated lineage marker antibodies (Gr1, Mac1, B220, and CD3; BD PharMingen, San Diego, CA) and fluorescein isothiocyanate (FITC)–conjugated CD45.1 antibody (BD PharMingen) and analyzed by fluorescence cytometry 6 months following transplantation. Six months following transplantation, secondary transplantation studies were conducted using BM cells from IFN-γ–treated or untreated recipients as donor cells.

Computations were carried out using the Graph Pad Prism software package (San Diego, CA). Statistical comparisons among all groups were compared using analysis of variance and Student t test. A P value less than .05 was considered statistically significant.

To compare the sensitivity of Fanca^–/– and Fancg^–/– myeloid progenitors to Fancc^–/– and WT myeloid progenitors, clonogenic assays for the growth of CFU-GMs were established in a range of IFN-γ concentrations. The mean ± the standard error of the mean of 4 independent experiments is shown in Figure 1. Consistent with previous studies,^27,28 Fancc^–/– myeloid progenitors were hypersensitive to IFN-γ compared with WT control cells. Fanca^–/– and Fancg^–/– progenitors exhibited a sensitivity to IFN-γ that was comparable with Fancc^–/– myeloid progenitors.

To determine whether Fanca^–/– and Fancg^–/– myeloid progenitors are hypersensitive to IFN-γ in vivo comparable with concentrations used clinically,^23,24  osmotic micropumps containing IFN-γ were implanted subcutaneously into the backs of age-matched Fanca^–/–, Fancg^–/–, Fancc^–/–, and WT mice to deliver 400 μg/kg per day IFN-γ or PBS alone for 7 days. The peripheral white blood cell (WBC) counts of all genotypes were similar following the administration of PBS (Figure 2A). Administration of IFN-γ had no significant reduction in the peripheral WBC count of WT mice following a 7-day infusion of 400 μg/kg per day IFN-γ. Similarly, IFN-γ treatment had no effect on the hematocrit or platelet counts in any genotype compared with mice treated with vehicle control (data not shown). However, the peripheral WBC counts of Fanca^–/– and Fancg^–/– mice had a significant reduction in peripheral WBC counts, similar to that observed in Fancc^–/– mice. A similar decrease in bone marrow cellularity was observed in all complementation groups of FA-deficient mice (Figure 2B).

In order to determine the effect of IFN-γ treatment on primitive and mature hematopoietic progenitors in vivo, high proliferative potential colony-forming cells (HPP-CFC) and low proliferative potential colony-forming cells (LPP-CFC) were cultured from bone marrow of the respective genotypes. Even in the absence of IFN-γ treatment, Fanca^–/– and Fancg^–/– mice, similar to Fancc^–/– mice, had a reduction in the number of HPP-CFCs per femur compared with WT mice (Figure 3A). Of importance, following administration of IFN-γ, a dramatic reduction in the number of HPP-CFCs and LPP-CFCs was observed in all 3 FA complementation groups compared with equivalently-treated WT mice (Figure 3B). Similar reductions were observed in burst-forming unit–erythroid (BFU-E) and multipotent myeloid progenitors (CFU-GEMM) (data not shown). Collectively, these data indicate that in vivo IFN-γ treatment preferentially reduces the number of primitive and mature myeloid progenitors in all 3 genotypes of FA-deficient mice.

To determine whether the myelopreparative protocol with IFN-γ is sufficient to allow engraftment and proliferation of isogenic WT repopulating cells, 10⁷ WT CD45.1⁺ BM nucleated cells were transplanted into Fanca^–/–, Fancg^–/–, Fancc^–/–, and WT animals pretreated with IFN-γ or PBS. Figure 4A shows the chimerism of individual recipients in 1 of 2 independent experiments with similar results. The percentage of CD45.1⁺ donor cells in peripheral blood was examined using fluorescence cytometry 4 months following transplantation. Isogenic cells failed to engraft and contribute to hematopoiesis in any of the 3 respective FA or WT experimental groups following treatment with vehicle only (Figure 4A). IFN-γ was not sufficient to allow engraftment of exogenous, isogenic BM cells into WT recipients (Figure 4A). However, both Fanca^–/– and Fancg^–/– recipients treated with IFN-γ prior to transplantation had a significant donor chimerism in peripheral blood 4 months following transplantation comparable with the donor chimerism of Fancc^–/– mice. Representative FACS analyses showing multilineage reconstitution in Fanca^–/– and Fancg^–/– mice (Figure 4B-C) are shown.

To confirm that IFN-γ myelopreparation was sufficient to allow engraftment of donor long-term repopulating stem cells, 2 million BM LDMNCs from selected primary recipients of a second cohort were transplanted into irradiated, secondary recipients and their chimerism was evaluated 6 months following transplantation (Figure 5A-B). The chimerism of recipients that received BM cells from Fanca^–/– or Fancg^–/– mice previously given the vehicle control only as a myelopreparative agent was low as expected. However, the chimerism of secondary recipients who received bone marrow cells from IFN-γ–treated Fanca^–/– (Figure 5A) and Fancg^–/– (Figure 5B) recipients consistently retained high chimerism. The bone marrow and splenic cellularity of these recipients at postmortem exam was normal comparable with previous studies in Fancc^–/– mice (data not shown). Collectively, these data indicate that in vivo administration of IFN-γ in Fanca^–/– and Fancg^–/– mice is sufficient as a myelopreparative agent to enhance the engraftment of repopulating syngeneic WT cells in Fanca^–/– and Fancg^–/– mice.

Although palliative therapy has improved the survival of patients with Fanconi anemia over the past 10 years,¹  bone marrow failure remains the leading cause of death.³⁷  Allogenic bone marrow transplantation or cord blood transplantation is available to a subset of these patients.²  However, for individuals who lack an HLA-compatible donor, non–HLA-matched transplantation is rarely used because of the particularly high morbidity of this therapy in FA patients.³⁸  Thus transplantation of genetically-corrected autologous stem cells remains an important potential therapy for this uniformly fatal disease.

In addition to the usual challenges in gene transfer protocols, FA disease itself creates an additional set of potential obstacles. Patients that are considered for gene transfer often have bone marrow hypoplasia.³⁷  In addition, emerging data in FA patients³⁹  and in Fancc^–/– mice⁴⁰  suggests that granulocyte colony-stimulating factor, the current cornerstone of stem cell mobilization, inefficiently mobilizes FA-deficient stem/progenitor cells. Further, the propensity of Fancc^–/– cells to apoptosis ex vivo¹²  and the known challenges in homing of ex vivo–cultured cells⁴¹  are additional challenges in having an adequate graft of genetically-transduced cells. These observations, together with previous clinical data showing that patients with FA have an increased risk for secondary malignancies following genotoxins,^19,20,42-44  have provided the impetus for considering IFN-γ as a myelopreparative conditioning agent in FA preclinical models as a proof of concept.

Given previous in vitro and in vivo studies demonstrating that Fancc^–/– cells are hypersensitive to IFN-γ, we evaluated whether a similar phenotype is present in other FA complementation groups. In 2 independent studies (6-7 mice per study group), we found that continuous subcutaneous infusion of IFN-γ enhances the engraftment of WT cells in Fanca^–/– and Fancg^–/– mice. The high level of engraftment was maintained in secondary recipients, indicating the engraftment of long-term repopulating stem cells. The treatment of IFN-γ was well tolerated in all the genotyped mice tested in the study. No severe hematologic sequelae were observed in the IFN-γ–treated mice in either primary or secondary recipients. The establishment of the IFN-γ BM conditioning in Fanca^–/–, Fancg^–/–, and Fancc^–/– models could have implications in using this approved drug in the clinic as a means to enhance the engraftment of exogenous genetically-corrected stem/progenitor cells.

Conflict-of-interest disclosure: the authors declare no competing financial interests.

The authors would like to acknowledge Dr Alan D'Andrea at the Dana Farber Cancer Research Institute at Harvard University for providing Fancg^–/– mice. We also thank Natascha Karlova for preparing the paper.

This work was supported by National Institutes of Health (NIH) grant P01HL053586.