# Patent

## Title
Derivation of unlimited quantities of neutrophils or monocyte/dendritic cells

## Abstract

A method to generate unlimited numbers of macrophage/dendritic cells or neutrophils from mice, using conditional Hox oncoproteins is disclosed. The invention further includes the establishment of a system to investigate immune responses to microorganisms or diseases involving chronic inflammation.

## Background
RELATED APPLICATION
This application is a 35 USC §371 National Stage application of PCT Application No. PCT/US2005/044673 filed Dec. 9, 2005; and claims the benefit under 35 USC §119(e) to U.S. Application Ser. No. 60/635,264 filed Dec. 9, 2004, now abandoned. The disclosure of each of the prior applications is considered part of and is incorporated by reference in the disclosure of this application.


STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under Grant No. CA56876 awarded by The National Institute of Health (NIH NCI). The government may has certain rights in the invention.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to immunology and more specifically to immune responses and diseases involving chronic inflammation.
2. Background Information
Molecular pathways of normal hematopoietic cell differentiation, as well as the mechanisms by which oncogenes disrupt this process, remain poorly understood. In normal hematopoietic progenitor cells, a program of specific gene expression orchestrates commitment and differentiation of mature cells to multiple different lineages. In acute leukemias, however, oncoproteins interfere with this genetic program, resulting in the unregulated proliferation of cells that no longer retain the capacity to differentiate normally. In acute myeloid leukemias (AMLs) many known myeloid oncoproteins can block the differentiation of normal progenitors cultured in vitro in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) or interleukin-3 (IL-3). However, neither the genetic events that underlie normal hematopoietic cell differentiation nor the mechanism through which leukemic oncoproteins interfere with the execution of the program of lineage differentiation are well understood.
Macrophages and neutrophils orchestrate the inflammatory response, communicating with each other and with T and B cells to induce cell activation and cell proliferation, to recruit more inflammatory cells, to kill the invader, to protect the surrounding tissue, to induce longer-term protective immunity, and to down regulate the response once the microorganism has been eliminated. These same processes can become chronically activated, leading to a variety of human diseases, such as autoimmune disease, multiple sclerosis, liver cirrhosis, arthritis, atherosclerosis, vascular disease, and even cancer. Academic and industrial concerns have large research programs devoted to understanding processes of inflammation that arises from various insults. Determining how microorganisms evade the immune system (immune evasion) can lead to the development of microbial-specific inhibitors. Determining how innate immune cells (macrophage/dendritic cells and neutrophils) mount an immune attack to different microbes can lead to the development of drugs that promote specific responses. Characterizing how specific microbes, such as HIV, live within macrophage/dendritic cells and respond to drugs within that context can identify new therapeutic avenues. And finally, inhibiting inflammatory responses that cause the devastating non-microbial human diseases (listed above) comprises a vast potential to relieve human suffering and generate highly profitable drugs.
Research within this field is expensive and time-consuming. Because macrophage/dendritic cells and neutrophils are non-mitotic, they need to be derived from large numbers of mice when laboratories are knocking out a single gene to look at the effect. If the knockout is embryonic lethal, day 13 or 14 mice can still be used as a sources of these cells, but the labor, time, and costs increase if cells are derived from such embryos. Once a knockout mouse has been derived for a specific protein, the goal of characterizing the specific domains of the protein that are important for its function are difficult because one can not restore production of the protein, or specific mutants of the protein, within mature inflammatory cells.
SUMMARY OF THE INVENTION
The present invention relates to the discovery that estrogen receptor (ER)-fusion oncoproteins produce neutrophil-committed progenitors. Accordingly, the invention includes a method of using conditional forms of the HOX protein to immortalize monocyte/dendritic progenitor cells,
In one embodiment, a method of modulating differentiation and immortalization of progenitor cells is provided including isolating progenitor cells from a subject, contacting the isolated cells with a fusion protein comprising a HOX oncoprotein and an estrogen receptor binding domain (ERBD), culturing the contacted cells with a combination of one or more multilineage cytokines, a myeloid-specific cytokine, and an estrogen agonist, whereupon culturing, the progenitor cells become immortalized and exhibit commitment to neutrophil and/or macrophage/dendritic lineage or exhibit multi-lineage differentiation potential.
In one aspect, the HOX oncoprotein is Hoxb8, Hoxa9, or Hoxa7. In a further related aspect, the fusion protein comprises an in-frame nucleic acid sequence encoding the general structure: Xn-ERBD-HOX, where ERBD is an estrogen receptor binding domain, Xn is a nucleic acid residue and n is an integer from 0-24, and HOX is Hoxb8, Hoxa9, or Hoxa7.
In one aspect, the Xn-ERBD-HOX comprises Hoxb8, and includes a sequence as set forth in SEQ ID NO: 3.
In another aspect, the Xn-ERBD-HOX comprises Hoxa9, and includes a sequence as set forth in SEQ ID NO: 6.
In another aspect, the Xn-ERBD-HOX comprises Hoxa7, and includes a sequence as set forth in SEQ ID NO: 9.
In one aspect, the estrogen agonist is β-estradiol.
In another aspect, the method includes removing the estrogen agonist upon expression of genes indicative of a neutrophil or macrophage/dendritic cell progenitor phenotype, thereby committing the cells to differentiate into mature neutrophils and/or macrophages/dendritic cells.
In one aspect, the multilineage cytokines are selected from stem cell factor, IL-6, IL-3, GM-CSF and combinations thereof or any cytokine that functions to sustain the proliferation of myeloid progenitors. In another aspect, the myeloid specific cytokine is IL-5 or G-CSF.
In one aspect, the method includes isolating cells from bone marrow or fetal liver cells. Moreover, the cells are obtained from wild-type or genetically-altered organisms.
In one embodiment, an immortalized, isolated neutrophil, macrophage/dendritic, biphenotypic neutrophil/macrophage/dendritic, and/or multipotent neutrophil/macrophage/dendritic/eosinophil/mast cell is provided, which is obtained from a bone marrow or fetal liver cell cultured in the presence of a combination of one or more multilineage and myeloid cytokines and infected with a vector comprising a fusion protein having a HOX oncoprotein and an estrogen receptor binding domain, where the infected cell becomes immortalized in the presence of an estrogen agonist and exhibits differentiation potential to the neutrophil, macrophage/dendritic, biphenotypic neutrophil/macrophage/dendritic, and/or multipotent neutrophil/macrophage/dendritic/eosinophil/mast lineage.
In another embodiment, an isolated neutrophil, macrophage/dendritic, biphenotypic neutrophil/macrophage/dendritic, and/or multipotent neutrophil/macrophage/dendritic/eosinophil/mast cell is provided which is obtained from a bone marrow or fetal liver cell infected with a vector comprising a fusion protein having a HOX oncoprotein and an estrogen receptor binding domain and cultured in the presence of an estrogen agonist and a combination of one or more multilineage and myeloid cytokines, wherein the cultured cell differentiates into a mature neutrophil, macrophage/dendritic, biphenotypic neutrophil/macrophage/dendritic, and/or multipotent neutrophil/macrophage/dendritic/eosinophil/mast cell after removal of the estrogen agonist.
In a related aspect, the cells are obtained from a wild-type or genetically-altered organism.
In one embodiment, a method of evaluating the effect of a genetically-altered gene on a neutrophil or a macrophage/dendritic cell is provided, including knocking-out or transferring a gene of interest to a subject animal, obtaining a sample of bone marrow or fetal liver cells from the subject animal and a wild-type animal, infecting the cells from each animal with a vector comprising a fusion protein having a HOX oncoprotein and an estrogen receptor binding domain and culturing the cells in the presence of an estrogen agonist and a combination of one or more multilineage and myeloid cytokines, removing estrogen from the cultured cells upon expression of genes indicative of a neutrophil or macrophage/dendritic cell progenitor phenotype, culturing the cells in the absence of the agonist and assaying the cells for one or more inflammatory responses associated with neutrophil or macrophage/dendritic cells, where differences in one or more inflammatory responses between the cells from the knock-out/transgenic subject animal and wild-type animal correlate with the altered gene of interest.
In one aspect, the method includes culturing the cells in the presence of an agent of interest and evaluating the agent for one or more responses associated with neutrophil or macrophage/dendritic cells. In another aspect, the method includes culturing the cells with a microbial pathogen.
In another aspect, the method includes transplanting the cultured cells into model animals presenting one or more inflammatory disease symptoms or a microbial infection and determining one or more differences in behavior between the cultured cells and endogenous wild-type neutrophils or macrophages, where determined differences are indicative of the function of the knockout gene in the inflammatory process.
In one aspect, the behavior is associated with the ability of cells to mediate steps in the disease pathology related to inflammation, where the behavior includes, migration to inflammatory sites, microbial phagocytosis, cytokine release, recruitment of phagocytes, presentation of antigen to lymphoid cells, recruitment of lymphoid cells, or a combination thereof.
In one aspect, the disease symptoms are associated with autoimmune diseases, multiple sclerosis, liver cirrhosis, arthritis, or atherosclerosis.
In one embodiment, a method of identifying processes in immune cells undergoing differentiation or maturation is provided including obtaining a sample of bone marrow or fetal liver cells from a genetically altered or a wild-type control subject, infecting the cells from the subject with a vector comprising a fusion protein having a HOX oncoprotein and an estrogen receptor binding domain and culturing the cells in the presence of an estrogen agonist and a combination of one or more multilineage and myeloid cytokines, removing samples of cells from the culture at various time points, and assaying the cells for changes in morphology, physiology, and/or gene expression, where the changes observed at different time-points correlate with different processes associated with neutrophil and/or macrophage/dendritic cell differentiation or maturation.
In a related aspect, the method includes removing the estrogen agonist upon expression of one or more gene markers associated with an immune cell exhibiting a neutrophil and/or macrophage/dendritic cell progenitor phenotype and culturing the cells in the absence of the agonist.
In one embodiment, an isolated nucleic acid encoding an amino acid sequence as set forth in SEQ ID NO: 16 or SEQ ID NO: 17 is provided. In a related aspect, the nucleic acid comprises SEQ ID NO: 3 or SEQ ID NO: 6.
In another embodiment, an expression vector is provided including an operably linked nucleic acid encoding a fusion protein comprising an in-frame nucleic acid sequence encoding the general structure: Xn-ERBD-HOX. In a related aspect, a host cell is provided including the vector encoding a fusion protein of the general structure: Xn-ERBD-HOX.
In one embodiment, a method of treating an inflammatory disorder is provided including administering to a subject in need thereof a pharmaceutical composition comprising the isolated cells of the present invention and a pharmaceutically acceptable carrier.
Exemplary methods and compositions according to this invention are described in greater detail below.



BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-D show that Hoxb8-ER and Hoxa9-ER function conditionally at the biochemical and cellular levels. Panel A: Estrogen-binding domain (ERBD) of the estrogen receptor fused to Hoxa9 or Hoxb8. Epitope tags are indicated at left. HD represents the homeodomain. Panel B: Estrogen-responsive transcriptional function of Hoxa9-ER, measured as coactivation through TGAT-TTAT motifs in conjunction with E2a-Pbx1 in 293T cells. Hoxb8-ER yielded an estrogen-induced concentration curve that was superimposable on Hoxa9-ER. Panel C: Proliferation of GM-CSF-dependent progenitors immortalized by Hoxa9-ER or Hoxb8-ER. Retroviral infection was performed at day 0, followed by a 4-day selection in G418. Inserted panel represents a Western blot using anti-Hoxa9 (left) and anti-Flag antibodies (right) on G418-selected progenitors immortalized by Hoxa9-ER or Hoxb8-ER, respectively. Immortalization kinetics and progenitor doubling times were somewhat faster for under SCF culture conditions. Panel D: Proliferation of GM-CSF-dependent progenitors immortalized by Hoxa9-ER or Hoxb8-ER following estrogen withdrawal. Proliferation of SCF-dependent progenitors continued for 4 to 5 days, while that of GM-CSF-dependent progenitors was rapidly terminated by day 2.
FIGS. 2A and 2B show that Hoxb8-ER SCF progenitors execute neutrophil differentiation while Hoxb8-ER GM-CSF progenitors execute macrophage differentiation. Panel A: Morphologic changes of Hoxb8-ER SCF neutrophil progenitors (left) and Hoxb8-ER GM-CSF macrophage progenitors (right) following estrogen withdrawal. Immortalized progenitors were washed twice in 10 mL of PBS, and then cultured in Myeloid Cell Medium without added β-estradiol. Cells were collected after 6 days and subject to Wright-Giemsa staining, NBT reduction assay (for neutrophils), NSE assay (for macrophages) after cytocentrifuge preparation of cells. Panel B: FACS analysis of expression of neutrophil or macrophage-specific surface markers by Hoxb8-ER progenitors before and after differentiation. Expression of Gr-1 (neutrophil differentiation antigen), F4/80 (macrophage differentiation antigen) and Mac-1 (general myeloid differentiation antigen), in Hoxb8-ER SCF neutrophil progenitors (left) or Hoxb8-ER GM-CSF macrophage progenitors (right) cultured in the presence of estradiol (blue tracing) or following the removal of estradiol for 6 days (red tracing).
FIGS. 3A-3D demonstrate lineage-specific gene up-regulation in Hoxb8-ER SCF neutrophil progenitors and Hoxb8-ER GM-CSF macrophage progenitors. Panel A: Affymetrix gene arrays were used to quantitate the expression levels of 128 myeloid genes in a Hoxb8-ER SCF neutrophil cell line (top panel) and a Hoxb8-ER GM-CSF macrophage cell line (lower panel) in the presence of estradiol (blue) and at 6 days of differentiation following estradiol withdrawal (red). X-axis was plotted with three different subsets of genes: neutrophi-restricted (left section), macrophage-restricted (middle section) and general myeloid differentiation genes (right section). The names of the genes plotted here are underscored in Table 4. The Y-axis plots the relative abundance of RNA, with blue designating basal levels in undifferentiated progenitors and red designating levels following 6 days of differentiation. Panels B-D: Verification of changes observed on Affymetrix arrays, using immunoblotting for the macrophage scavenger receptor (SCR-A) and the transcription factors Rel-B and c-Jun three and six days following removal of estradiol from Hoxb8-ER GM-CSF macrophage progenitors (panel B), FACS analysis for CD11c in Hoxb8-ER GM-CSF progenitors in the presence of estradiol and 4 days after withdrawal of estradiol (panel C), and by Northern blotting (panel D).
FIGS. 4A-4D demonstrate that a concerted program of phagocytic differentiation follows inactivation of Hoxb8-ER. Panels A and C: Down-regulation kinetics of c-Myb, c-Myc, Hmgb3, Pontin 52 and Reptin 52 (blue tracing) mirrors down-regulation of the cell cycle genes CycB1, Shmt2, Mcm2, Nola-1, Nol5, NOP56, and Nolc1 (described in Table 1) in both Hoxb8-ER SCF neutrophil progenitors (panel A) and Hoxb8-ER GM-CSF macrophage progenitors (panel C). In each case, up-regulation of the transcription factors ATF3, Jun-B, e-Fos, JunD1, and BTG2 (purple tracings) follows cell cycle arrest. Panels B and D: Down-regulation of the promyelocytic genes Ctsg, Prt3, Cystatin F, MPO, Plac8, and Ms4a3 (brown tracings) mirrors c-Myb downregulation, while up-regulation of the terminal differentiation genes Fpr1, Fpr-rs2, Dectin-1, Mrc1, Fgr, VacATP, MKP1, Vti1b, and Sirpbeta1 (green tracings) coincides with up-regulation of the leucine zipper transcription factors (purple tracings). The X-axis designates days post estrogen withdrawal, and the Y-axis designates relative expression level of mRNA, as measured by Affymetrix arrays.
FIG. 5 demonstrates that Hoxb8-ER SCF progenitors behave as GMP, retaining an ability to differentiate into eosinophils and macrophages. Hoxb8-ER SCF progenitors were permitted to differentiate in SCF medium supplemented with the lineage-specific cytokines GCSF, IL5, MCSF, and GM-CSF, as indicated above the photographs. Cells were photographed 6 days after differentiation was initiated by removal of estradiol. In the column demarcated “SCF+IL5”, a developing eosinophil is magnified at lower left. Distribution of mature cell types is indicated below each column, as measured at the end of the 6 day differentiation.
FIG. 6 shows that inflammatory signaling pathways are preserved in macrophages derived from Hoxb8-ER GM-CSF progenitors. LPS induces rapid activation of NfkB, IkB, bZIP, and STAT transcription factors in Hoxb8-ER-derived macrophages, as well as strong activation of the mediators of inflammation. Basal levels of mRNA are represented by the first bar, and LPS-induced levels (2 hour treatment) by the second bar of each set. mRNA levels are indicated on left and gene designations at the sides or beneath each panel.
FIGS. 7A-7C demonstrate that reexpression of TRAF3 restores the signaling defect in CpG-induced transactivation of IFN and IL-10 genes in Traf3−/− macrophages produced by HoxB8-ER. Panel A: Wildtype (wt) and Traf3−/− HoxB8-ER-immortalized macrophage progenitor cells (cultured in GM-CSF) were transduced with MSCV-Puro retroviral vectors containing HA-tagged TRAF3 or a control empty vector. Transduced cells were selected with puromycin (2 μg/ml) and TRAF3 expression was measured by intracellular staining with antibodies to the HA-tag. Shown are Traf3−/− cells transduced with the control vector (co) and Traf3−/− cells transduced with the HA-TRAF3 expression construct (HA-TRAF3). For staining, cells were fixed with 2% formaldehyde/PBS and permeabilized with 0.5% saponin. A FITC-labeled antibody was used as secondary antibody to the anti-HA antibody. Cells were analyzed by flow cytometry and are presented as fluorescence intensity vs. cell number. Panel B: Wt and Traf3−/− cells, each transduced with either the control vector or the HA-TRAF3 expression vector, were differentiated for 6 days by removal of estradiol. Lysates of these cells were analyzed by immuno-blotting with antibodies to HA and p38. Panel C: Differentiated Hoxb8-ER macrophages were stimulated with CpG-DNA and analyzed for IFN and IL-10 production by bioassay and ELISA, respectively. nd=not detectable.
FIG. 8 shows a SKY karyotype analysis of Hoxb8-ER macrophage progenitors. The karyotype of 20 cells was analyzed and the frequency of cells plotted as a function of their chromosome number. 17 cells exhibit the normal 40, XX karyotype and 3 cells exhibited abnormal karyotypes, listed adjacent to their histogram bars. A typical analysis is inset at left.



DETAILED DESCRIPTION OF THE INVENTION
Before the present compositions, methods, and computational methodologies are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “a nucleic acid” includes one or more nucleic acids, and/or compositions of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, as it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. All publications mentioned herein are incorporated herein by reference in their entirety.
As used herein “immortalized,” including grammatical variations thereof, means a cell population changed from having a finite life span to one possessing an infinite life span.
As used herein “differentiation,” including grammatical variations thereof, means a process whereby relatively unspecialized cells acquire specialized structure and/or functional features that characterize the cells, tissues, or organs of the mature organism or some other relatively stable phase of the organism's life history.
As used herein “progenitors,” including grammatical variations thereof, are the more differentiated progeny of stem cells that give rise to distinct subsets of mature blood cells and lack the capacity for self-renewal possessed by true stem cells.
As used herein “oncoprotein,” including grammatical variations thereof, means any protein associated with the causation of cancer.
As used herein “multi-lineage differentiation potential,” including grammatical variations thereof, means a progenitor cell having the capability of development into a neutrophil, macrophage/dendritic, biphenotypic neutrophil/macrophage/dendritic, and/or eosinophil/mast cell.
In the context of destroying pathogens, complexed receptor systems are used to migrate toward invaders, recognize invaders, respond to invaders, and activate the maturation and division of T and B cells that also respond to the same foreign organism. Migration toward the site of inflammation is mediated by chemokine receptors (e.g., CCR2 and CCR5) in response to ligands such as MIP1 alpha and MIP1 beta, which are secreted by activated macrophages at the site of the inflammatory response. Recognition of pathogens involved receptors that bind bacterial products (e.g., Toll pattern-recognition receptors TLR2 and TLR4 or FPR and FPRL2 receptors that bind bacterial peptides such as fMLP), lectins that bind sugars on the surface of the pathogen (e.g. Dectin-1, DCIR, DCAR, DCL-1, MCL, MGL2, MRC1), and receptors that recognize the Fc region of antibodies that are bound to the pathogen (IgG and IgE Fc receptors, more than 5 genes encode these proteins). The response of a macrophage/dendritic cell or neutrophil to a pathogen is controlled by a complex system of receptors that transmit “activating” or “inhibiting” signals and that also tell the cell whether there is evidence of “self” MHC class I antigens (e.g. PIR-A and PIR-B receptors). Macrophage/dendritic cells and neutrophils then engage in a measured inflammatory response, which includes phagocytosis/killing of the microbe using NAPH oxidase, proteinases such as cathepsin and metalloproteinases, and enzymes that break down lipid (lipoprotein lipase) and bacterial cell walls (lysozyme), which are stored in phagocytic vesicles of the mature macrophage/dendritic cell or neutrophil. To prevent broad tissue damage, macrophage/dendritic cells and neutrophils also secrete specific inhibitors of their own intracellular proteases, such as cystatins and serpins to prevent broad tissue damage). Activation of monocyte/dendritic cells and neutrophils results in the induction of a broader inflammatory gene response that is designed to recruit and activated more cells and prepare the surrounding tissue for ensuing inflammatory response. This response produces factors that activate the maturation and proliferation of killer T cells (e.g., TNF9, ICOS-L, IL12, IL23), factors that recruit more inflammatory cells (e.g. MCP1, MCP3, MIP1alpha, RANTES), and factors that elicit a broad general response within both hematopoietic and non-hematopoietic cells (e.g., IL1alpha, IL1beta, IL6, TNFalpha, and IFN-beta). Dendritic cells are intimately involved in the activation of T cells responsive to foreign antigens. Dendritic cells uniquely express MHC Class II antigens, as well as CD11c and CD86. Each of these markers is upregulated in Hoxb8-immortalized macrophage/dendritic cells (Table 3). Dendritic cells present foreign antigens on their MHC Class II complexes to T cells, and the T cell clones containing T cell receptors that recognize the same antigen are activated by interacting with both the MHC Class II/antigen complex and the co-stimulatory signals derived from the dendritic cells.
By using genomic or other profiling tools, the genetic program of progenitor differentiation can be defined by comparing gene expression in immature progenitors growing in the presence of estrogen vs. cells maturing in the absence of estrogen. The cell system can then be used to identify the mechanisms that control stage-specific gene expression by determining the transcriptional mechanisms required to activate stage-specific gene expression.
By allowing immature proliferating progenitors immortalized by Hoxb8-ER or by Hoxa9-ER to mature in the absence of estrogen, the desired number of macrophage/dendritic cells or neutrophils can be obtained. Any protein component can then be purified by classical mechanisms. Large numbers of mature macrophage/dendritic cells or neutrophils can be derived for large-scale screening efforts. Homogeneity from batch to batch is ensured by the clonal nature of the cells used for the assay.
As disclosed herein, progenitors committed to the macrophage/dendritic lineage or neutrophil lineage can be immortalized by Hoxb8-ER or Hoxa9-ER, respectively. The effect of the target gene elimination can then be observed in mature macrophage/dendritic cells or in neutrophils following their differentiation after withdrawal of estrogen. One can then go on to determine the specific domain requirements of the knockout protein by re-expressing a panel of mutants of the knockout gene. Factors that interact with functionally-important domains can then be defined by interaction with the wild-type protein, but not with specific mutants that disrupt function in the knockout macrophage/dendritic cells or neutrophils. For example, human atherosclerosis involves the accumulation of macrophages into vascular lesions. A mouse model for atherosclerosis exists. One could ask the question, is the CCR2 chemokine receptor important for homing of macrophages to atherosclerotic lesions in vivo. If this was essential, then CCR2 inhibitors might be therapeutic agents in atherosclerosis. Wild-type and CCR2 knockout macrophage/dendritic progenitors would be immortalized by Hoxb8-ER. Mature cells would be generated and infused into mice in which atherosclerotic lesions were present. By measuring the difference in homing of wild-type vs. CCR2 knockout macrophage/dendritic cells to the lesion, one can determine the role of CCR2 in macrophage accumulation in the lesion.
Herein is described a rapid and convenient method to produce unlimited macrophages or neutrophils from mice surviving past embryonic d13, a method that overcomes the significant time, cost and animal mortality involved in using mice as a source of mature phagocytes. The functional properties of macrophages derived by maturation of Hoxb8-ER GM-CSF progenitors are at least as good as those produced by cell lines, such as TPA-induce macrophage differentiation of HL60 cells, IL6-induced macrophage differentiation of M1 AML cells, or unstimulated macrophages represented by the RAW1 cell line. These models of macrophage differentiation are also accompanied by up-regulation of Egr-1, Egr-2, ATF3, c-fos, cjun, Rel-B and down-regulation of c-Myb, c-Myc transcription [Krishnaraju, K., B. Hoffman, and D. A. Liebermann, Blood, 1998. 92 (6): p. 1957-66; Liebermann, D. A. and B. Hoffman, Stem Cells, 1994. 12 (4): p. 352-69; Kharbanda, S., et al., J Clin Invest, 1991. 88 (2): p. 571-7;]. The functional maturation of neutrophils derived from Hoxb8-ER SCF progenitors is stronger than that produced by G-CSF-induced differentiation of either 32D progenitors or Hoxa9 progenitors. 32D progenitors fail to up-regulate secondary granule genes such as lactoferrin, and Hoxa9 progenitors fail to down-regulate e-Myb and the promyelocytic genes, neutrophil elastase and MPO, and fail to up-regulate the secondary granule gene lactoferrin [Calvo, K. R., et al., Mol Cell Biol, 2000. 20 (9): p. 3274-85]. The incomplete transcriptional modeling of these inducible cell lines is likely due to the persistent oncoprotein activity during differentiation induction, which contrasts the complete inactivation in oncoprotein-ER fusions. ATRA-induced differentiation of GM-CSF-dependent EPRO promyelocytes [Gaines, P., J. Chi, and N. Berliner, J Leukoc Biol, 2005. 77 (5): p. 669-79] yields results comparable to those observed in with Hoxb8-ER, and interestingly also inactivates the intrinsic oncoprotein (a dominant-negative retinoic acid receptor a), using supra-physiologic levels of ATRA.
Conditional oncoproteins described to date have not evidenced reproducible derivation of lineage-specific progenitors that execute normal differentiation. Avian v-Myb-ER immortalizes primary chicken monocyte progenitors that differentiated, unexpectedly, into multinucleated giant cells similar bone-marrow-derived osteoclasts [Engelke, U., D. M. Wang, and J. S. Lipsick, J Virol, 1997. 71 (5): p. 3760-6]. Their expansion in the presence of estrogen is also limited to 107. Mll-Enl-ER (tamoxifen-regulated) immortalizes a biphenotypic progenitor that requires 14 days to exit the cell cycle following removal of tamoxifen and to differentiate into neutrophils and monocytes [Zeisig, B. B., et al., Mol Cell Biol, 2004. 24 (2): p. 617-28]. Terminal differentiation of progenitors immortalized by E2a-ER-Pbx1 is variable (5 to 12 days), and while derivation of neutrophil-committed progenitors is common using the Δ1 E2a-Pbx1 mutant, derivation of macrophage-committed progenitors is rare [Sykes, D. B. and M. P. Kamps, Blood, 2001. 98 (8): p. 2308-18], an observation somewhat akin to the behavior of Hoxa9-ER in the present disclosure, which yielded mostly biphenotypic progenitors, a lower number of neutrophil-committed progenitors, and rare monocyte-committed progenitors. Therefore, while neutrophils and macrophages produced by inactivation of conditional oncoproteins appear more normal than those produced by many inducible systems, oncoprotein-specific functions dictate the expansion potential, the differentiation stage, and the differentiation potential of the immortalized progenitor. When matched with appropriate cytokine conditions, Hoxb8-ER simply has a useful property of generating progenitors that execute differentiation to pure populations of neutrophils or macrophages, a technique that can be used to produce unlimited supply of genetically-defined macrophages or neutrophils for bioassays, gene expression analysis, conventional protein purification protocols, and other methods used to investigate the differentiation, signaling, and effector functions of phagocytes.
One application of the Hoxb8-ER systems is understanding how Hox oncoproteins block differentiation in myeloid leukemia. Hoxb8 was the prototypic Hox oncoprotein in leukemia, first discovered due to its transcription activation by an integrated provirus in WEHI-3B leukemia cells [Perkins, A., et al., Proc Natl Acad Sci USA, 1990. 87 (21): p. 8398-402]. Based on genomic analysis, it is clear that Hoxb8 controls a switch between progenitor proliferation, characterized by expression of c-Myb, c-Myc, Pontin, Reptin, Gfi-1, and HMGB3, and cell cycle arrest/terminal differentiation, characterized by expression of ATF3, JunB, c-fos, RelB, c-Jun, JunD1, Egr-1, Egr2. From a mechanistic standpoint, c-Myb, c-Myc, Pontin, Reptin, Gfi-1, and HMGB3 are likely to be essential targets that sustain expansion of Hox-immortalized progenitors. C-Myb, the cellular homologue of the Avian Myeloblastosis virus oncogene v-Myb, is a transcriptional activator required for self-renewal of short-term reconstituting hematopoietic stem cells, is transcriptionally activated by proviral integrations in both myeloid and lymphoid leukemias [Lipsick, J. S. and D. M. Wang, Oncogene, 1999. 18 (19): p. 3047-55], and inhibits IL-6 induced macrophage differentiation of M1 AML progenitors. Removal of c-Myb down-regulates the cell cycle and induces red cell differentiation in erythroleukemia progenitors [Chen, J., C. S. Kremer, and T. P. Bender, Oncogene, 2002. 21 (12): p. 1859-69, Lyon, J. J. and R. J. Watson, Differentiation, 1995. 59 (3): p. 171-8] and induces granulocyte/macrophage differentiation in myeloid progenitors (FDCP-mix A4, [White, J. R. and K. Weston, Oncogene, 2000. 19 (9): p. 1196-205]). c-Myc overexpression is one of the most common oncogenic events in human malignancy, and its expression is required for Myb-mediated transformation, [Kumar, A., C. M. Lee, and E. P. Reddy, J Biol Chem, 2003. 278 (13): p. 11480-8]. Pontin and Reptin are DNA helicases involved in chromatin remodeling and transcriptional activation [Kurokawa, Y., et al., DNA Seq, 1999. 10 (1): p. 37-42], and expression of Pontin is required for transformation by c-Myc [Wood, M. A., S. B. McMahon, and M. D. Cole, Mol Cell, 2000. 5 (2): p. 321-30] and by beta-catenin [Feng, Y., N. Lee, and E. R. Fearon, Cancer Res, 2003. 63 (24): p. 8726-34]. Gfi-1 and Hmgb3 are also implicated in stem cell expansion. Gfi-1 is a transcriptional repressor that promotes T cell proliferation [Zhu, J., et al., Immunity, 2002. 16 (5): p. 733-44], is activated in murine T cell lymphoma [Scheijen, B., et al., J Virol, 1997. 71 (1): p. 9-16], and is normally expressed in hematopoietic stem cells (HSC), common lymphoid progenitors (CLP), and CFU-GM [Hock, H., et al., Immunity, 2003. 18 (1): p. 109-20]. Hmgb3 is related to the High Mobility Group (HMG) family of non-histone, chromatin-binding proteins that facilitate nucleosome remodeling, and its expression is also high in Lin−, c-kit+, Sca-1+, IL-7Ralpha− long-term repopulating HSC, in Ter119+erythroid progenitors, CLP, and common myeloid progenitors (CMP [Nemeth, M. J., et al., Blood, 2003. 102 (4): p. 1298-306]), and it is essential for the expansion of CLPs and CMPs from HSCs [Nemeth, M. J., et al., Blood, 2005. 105 (2): p. 627-34]. Determining how Hox oncoproteins maintain transcription of c-Myb, c-Myc, Pontin, Reptin, Gfi-1, and Hmgb3 is one application of this cell system that may explain how Hox proteins immortalize myeloid progenitors and contribute to AML.
A second application of the Hoxb8-ER system is to study transactivation mechanisms in terminal differentiated phagocytes, such as those controlling activation of the secondary granule gene Lactoferrin, the pattern recognition gene Formyl peptide receptor, or the antimicrobial gene Cathelin. This field has been encumbered by the unavailability of appropriate model systems [Gaines, P., J. Chi, and N. Berliner, J Leukoc Biol, 2005. 77 (5): p. 669-79]. Understanding such transcriptional mechanisms will also provide insight into the oncogeneic mechanism of differentiation-arrest and myeloid leukemogenesis. New genes controlling phagocyte differentiation or function may also be identified. One candidate is MKP1 (MAP kinase phosphatase 1), which was up-regulated 30-fold coincident with neutrophil or monocyte differentiation. MKP1 is a duel specificity phosphatase that negatively regulates the cell cycle by dephosphorylating and inactivating MAP kinases, and is activated by p53 during G1 arrest in response to DNA-damage [Li, M., et al., J Biol Chem, 2003. 278 (42): p. 41059-68]. Transactivation of MKP1 represents a mechanism by which Hox down-regulation could induce terminal differentiation by dephosphorylating multiple cellular targets.
Although not to be bound by theory, the accuracy of the Hoxb8-ER model predicts a broader conceptual application-cognate differentiation models of other myeloid lineages, lymphoid lineages, or even of epithelial stem cells that control ductal formation in the breast or microvillar development in the colon should be able to be derived by expressing conditional oncogenes in their tissue-specific stem cells cultured in the presence of cytokines that support the their expansion/differentiation programs. In considering this hypothesis, it would be important to use oncogenes found specifically in cancers of the cell type for which the model is being generated, and that have been proven to alter differentiation of its corresponding stem cell. Such models of tissue differentiation would be useful both in understanding how oncogenes enforce the stem cell phenotype in cancer, as well as in understanding differentiated cell functions.
In one embodiment, a method of evaluating the effect of a genetically-altered gene on a neutrophil or a macrophage/dendritic cell is provided, including knocking-out or transferring a gene of interest to a subject animal, obtaining a sample of bone marrow or fetal liver cells from the subject animal and a wild-type animal, infecting the cells from each animal with a vector comprising a fusion protein having a HOX oncoprotein and an estrogen receptor binding domain and culturing the cells in the presence of an estrogen agonist and a combination of one or more multilineage and myeloid cytokines, removing estrogen from the cultured cells upon expression of genes indicative of a neutrophil or macrophage/dendritic cell progenitor phenotype, culturing the cells in the absence of the agonist and assaying the cells for one or more inflammatory responses associated with neutrophil or macrophage/dendritic cells, where differences in one or more inflammatory responses between the cells from the knock-out/transgenic subject animal and wild-type animal correlate with the altered gene of interest.
In one aspect, the method includes culturing the cells in the presence of an agent of interest and evaluating the agent for one or more responses associated with neutrophil or macrophage/dendritic cells. In another aspect, the method includes culturing the cells with a microbial pathogen.
In another aspect, the method includes transplanting the cultured cells into model animals presenting one or more inflammatory disease symptoms or a microbial infection and determining one or more differences in behavior between the between the cultured cells and endogenous wild-type neutrophils or macrophages, where determined differences are indicative of the function of the knockout gene in the inflammatory process.
In one aspect, the behavior is associated with the ability of cells to mediate steps in the disease pathology related to inflammation, where the behavior includes, migration to inflammatory sites, microbial phagocytosis, cytokine release, recruitment of phagocytes, presentation of antigen to lymphoid cells, recruitment of lymphoid cells, or a combination thereof.
In one aspect, the disease symptoms are associated with autoimmune diseases, multiple sclerosis, liver cirrhosis, arthritis, or atherosclerosis.
Hox genes are developmental regulators whose persistent expression has been found to underlie myeloid leukemia, a disease in which the progenitors of macrophages, dendritic cells, and neutrophils are blocked in their differentiation, and can continue to divide as progenitor cells. Here in described is the use of the ability of Hox proteins to block differentiation to control cell differentiation and immortalize specific types of progenitor cells. Using a conditional form of HOX, a means of generating unlimited numbers of immature progenitors that can differentiate into mature, normal macrophage/dendritic cells when directed to do so is disclosed.
In one embodiment, a method of modulating differentiation and immortalization of progenitor cells is provided including isolating progenitor cells from a subject, contacting the isolated cells with a fusion protein comprising a HOX oncoprotein and an estrogen receptor binding domain (ERBD), culturing the contacted cells with a combination of one or more multilineage cytokines, a myeloid-specific cytokine, and an estrogen agonist, whereupon culturing, the progenitor cells become immortalized and exhibit commitment to neutrophil and/or macrophage/dendritic lineage or exhibit multi-lineage differentiation potential.
In a related aspect, the HOX oncoprotein is Hoxb8, Hoxa9, or Hoxa7. In a further related aspect, the fusion protein comprises an in-frame nucleic acid sequence encoding the general structure: Xn-ERBD-HOX, where ERBD is an estrogen receptor binding domain, Xn is a nucleic acid residue and n is an integer generally between 0-24, where n is sufficient to encode an immunologic tag for the purposes of identifying the fusion protein using anti-epitope antibodies, and HOX is Hoxb8, Hoxa9, or Hoxa7.
In one aspect, the Xn-ERBD-HOX comprises Hoxb8, and includes the sequence as set forth in SEQ ID NO: 3. In another aspect, the Xn-ERBD-HOX comprises Hoxa9, and includes the sequence as set forth in SEQ ID NO: 6. In another aspect, the Xn-ERBD-HOX comprises Hoxa7, and includes the sequence as set forth in SEQ ID NO: 9.
These cell lines can be made from any normal mouse or any genetic mutant of a mouse that survives birth, using bone marrow as the source of cells subjected to the cell line derivation procedure. These cell lines can be made from any normal mouse or any genetic mutant of a mouse that survives to day 13, using fetal liver progenitors as the source of cells for the immortalization procedure. The mature macrophage/dendritic cells or neutrophils are functionally normal by all criteria. They express a vast complement of receptors involved in immune function, and produce a wide spectrum of pro-inflammatory cytokines in response to activation by pathogens. In a related aspect, such animals may be transgenic, knockin, or knockout animals. In one embodiment, the knockin animal is a mouse. In another embodiment, the animal is a knockout mouse. One embodiment comprises a disruption in an endogenous alleles encoding a gene of interest thought to be associated with inflammation or an immune response.
Transgenic/knockin/knockout animals may be mice, rats and rabbits, or mammals such as pigs, goats, sheep, and monkeys. Other standard animals used in the act for transgenic knockin or knockout models an be used in the present invention. For example, such a transgenic, knock-out or knockin animal can be used as a control, when identifying and testing drugs that can be useful treating an inflammatory disorder. Thus the transgenic, knockin, and knockout animals of the present invention can be used in drug screens and the like. Cells from the transgenic, knockin and knockout mice are also part of the present invention.
Transgenic vectors, including viral vectors, or cosmid clones (or phage clones) corresponding to the wild type locus of candidate gene, can be constructed using the isolated gene of interest. Cosmids may be introduced into transgenic mice using published procedures (Jaenisch, Science, 240:1468-1474 (1988)).
Gene expression is disrupted, according to the invention, when no functional protein is expressed. One standard method to evaluate the phenotypic effect of a gene product is to employ knock-out technology to delete a gene as described in U.S. Pat. No. 5,464,764, Issued Nov. 7, 1995; and U.S. Pat. No. 5,777,195, Issued Jul. 7, 1998 (both of which are hereby incorporated by reference herein in their entireties).
Monocyte/dendritic progenitor cell lines immortalized by HOX fusion proteins are disclosed using CCR2 knockout cells, demonstrating the prediction that progenitors from mice harboring genetic mutations will also be susceptible to immortalization by the disclosed methods. Further, the present invention demonstrates that day 13 fetal liver cells can be immortalized by HOX containing fusion oncoproteins, permitting the derivation of either macrophage/dendritic or neutrophil progenitor cells lines, respectively, from knockout mice having embryonic lethal phenotypes produce by ablation of genes controlling the immune system. In one case, progenitors from a mouse in which the DAP12 gene was removed were immortalized. DAP12 signals downstream of Toll receptors, which bind bacterial components.
The function of the HOX oncoprotein is made conditional by fusing it to the estrogen-binding domain of the estrogen receptor (e.g, SEQ ID NO: 10). The Hoxb8-ER cDNA is inserted into a retroviral vector, and the Hoxb8-ER retrovirus are used to infect mouse marrow progenitors expanded from marrow by culturing in one or more multi-lineage cytokines (e.g., but not limited to, stem cell factor, IL-6, and IL-3). In one aspect, a viral vector that comprises a nucleic acid encoding a fusion protein is provided. For example, but not limited to, such fusion proteins may comprise the amino acid sequence as set forth in SEQ ID NO: 16 or SEQ ID NO: 17.
In one aspect, the viral vector is a herpes simplex viral vector, an adenoviral vector, or an adeno-associated viral vector (AAV). In another aspect, the viral vector is a retroviral vector, for example but not limited to, an HIV retroviral vector, a VL 30 vector, a MSCV retroviral vector, or a Harvey Murine Sarcoma Vector. In a related aspect, a progenitor cell is transduced by being co-cultured with a retroviral producer cell line. In another aspect, transducing a progenitor cell with Xn-ERBD-HOX is performed with a DNA vector (i.e., a naked DNA) that comprises a nucleic acid encoding the fusion protein.
Infected/transfected progenitors can then be cultured in the presence of tissue culture medium containing an estrogen agonist (to keep the fusion protein active) and a myeloid specific cytokine (e.g., GM-CSF, G-CSF and FI.T-3), which maintains proliferation of progenitors committed to the neutrophil or macrophage/dendritic lineage. In one aspect, the agonist may be β-estradiol, raloxifene, tamoxifen, toremifene, and clomiphene. Such agonists may be present at about 0.1 to about 0.5, about 0.5 to about 1.0, or about 1 to about 5 micromolar.
Subsequent to infection, populations of immortalized progenitors emerge (FIG. 1A, pictures grow in the presence of estrogen are designated with a “+”) that express numerous genes indicative of their macrophage/dendritic cell progenitor phenotype (Table 1).
These progenitors proliferate indefinitely. Following oncoprotein inactivation, they stop dividing and mature into cells having the typical morphology of macrophage/dendritic cells (FIG. 1A, lanes designated “−” estrogen for 1, 4, or 6 days). The progenitors stop cell division using a well-orchestrated process that involves downregulation of genes involved in cell cycle control, nucleotide biosynthesis, DNA replication, and RNA maturation (FIG. 2, Table 2). In one embodiment, a method of identifying processes in immune cells undergoing differentiation or maturation is provided including obtaining a sample of bone marrow or fetal liver cells from a genetically altered or a wild-type control subject, infecting the cells from the subject with a vector comprising a fusion protein having a HOX oncoprotein and an estrogen receptor binding domain and culturing the cells in the presence of an estrogen agonist and a combination of one or more multilineage and myeloid cytokines, removing samples of cells from the culture at various time points, and assaying the cells for changes in morphology, physiology, and/or gene expression, where the changes observed at different time-points correlate with different processes associated with neutrophil and/or macrophage/dendritic cell differentiation or maturation.
In a related aspect, the method includes removing the estrogen agonist upon expression of one or more gene markers associated with an immune cell exhibiting a neutrophil and/or macrophage/dendritic cell progenitor phenotype and culturing the cells in the absence of the agonist.
Coincident with ceasing proliferation, progenitors immortalized by fusion proteins of the present invention differentiate into mature macrophage/dendritic cells, down-regulating genes expressed specifically during the middle stages of myeloid differentiation (Table 2) and up-regulating a large number of cell surface receptors characteristic of macrophage/dendritic cells (FIG. 3, Table 3). Note, for example, specific expression of the MHC class II genes, of CD83, and of CD11c, all of which are markers for the dendritic cell, a specialized antigen-presenting cell derived from the monocyte lineage.
The progenitors immortalized by the method of the present invention mature visually into cells that look identical to normal macrophage/dendritic cells (FIG. 1). In one embodiment, an immortalized, isolated neutrophil, macrophage/dendritic, biphenotypic neutrophil/macrophage/dendritic, and/or multipotent neutrophil/macrophage/dendritic/eosinophil/mast cell is provided, which is obtained from a bone marrow or fetal liver cell cultured in the presence of a combination of one or more multilineage and myeloid cytokines and infected with a vector comprising a fusion protein having a HOX oncoprotein and an estrogen receptor binding domain, wherein the infected cell becomes immortalized in the presence of an estrogen agonist and exhibits differentiation potential to the neutrophil, macrophage/dendritic, biphenotypic neutrophil/macrophage/dendritic, and/or multipotent neutrophil/macrophage/dendritic/eosinophil/mast lineage.
In another embodiment, an isolated neutrophil, macrophage/dendritic, biphenotypic neutrophil/macrophage/dendritic, and/or multipotent neutrophil/macrophage/dendritic/eosinophil/mast cell is provided which is obtained from a bone marrow or fetal liver cell infected with a vector comprising a fusion protein having a HOX oncoprotein and an estrogen receptor binding domain and cultured in the presence of an estrogen agonist and a combination of one or more multilineage and myeloid cytokines, where the cultured cell differentiates into a mature neutrophil, macrophage/dendritic, biphenotypic neutrophil/macrophage/dendritic, and/or multipotent neutrophil/macrophage/dendritic/eosinophil/mast cell after removal of the estrogen agonist.
Following oncoprotein inactivation the mature macrophage/dendritic cells can be activated by pro-inflammatory stimuli such as bacterial lipopolysaccharide (LPS). When activated, these mature macrophage/dendritic cells secrete the normal broad array of pro-inflammatory cytokines (FIG. 4, Table 4).
Using a different HOX oncoproteins (e.g., but not limited to, Hoxa9 and Hoxa7) fusion proteins were found to immortalize progenitors that are committed to principally to neutrophil differentiation. Half of the clones exhibited principally, but not exclusively, neutrophilic differentiation. Approximately one-third of clones exhibit exclusive or >95% differentiation to neutrophils. For example, for Hoxa9, the differentiation outcomes of analysis of 23 clones is listed in Table 5, and the morphology of representative clones committed to the neutrophil, macrophage, or bi-phenotypic (both neutrophils and macrophages) is represented in FIG. 5.
These progenitors can be expanded indefinitely and differentiate into mature neutrophils following removal of estrogen (i.e., inactivation of HOX fusion protein), based on morphologic criteria. They exhibit normal morphologic differentiation, suggesting they have executed a normal genetic program of normal neutrophils. They die within 5 days of maturation, the same lifespan as normal neutrophils have in vivo. For example, using Affymetrix, for example, genomic arrays show that Hoxa9-ER progenitors down-regulate their cell-cycle within 48 hrs of estrogen withdrawal (FIG. 6), evidencing the same genomic fingerprint evidenced by progenitors immortalized by Hoxb8-ER. For example, genomic analysis over a shorter time-frame, examining changes after 24, 48, and 72 hours following estrogen withdrawal. It is evident that the process of downregulating cell division and executing the program of differentiation gene expression occurs rapidly, being significantly underway within 24 hours following estrogen withdrawal.
The Hoxb8-ER and Hoxa9-ER cell technology of the present invention forms a rapid and convenient means to study the role of the immune system in microbial pathogenesis or in inflammatory human diseases. The system is extremely cost-effective when considering the comparable costs involved in housing mice and deriving mature monocyte/dendritic cells or neutrophils from adult mice. The system eliminates the needless death of mice, and its use should be encouraged in all contexts of studying innate immunity and the role of macrophage/dendritic cells and neutrophils to the stimulation of the acquired immune system (B and T cells). Applying these systems to marrow from genetically modified (transgenic or gene knockout) mice provides a convenient system to delineate biochemical signaling pathways by reconstituting pathways with normal or mutant versions of the knockout cDNA, introduced into progenitors prior to their differentiation, and functionally assessed after maturation.
The invention also provides a system for the testing of various pharmacological compounds and therapeutic reagents for the treatment of disease. The ability of a therapeutic agent to promote differentiation induced by a variety of single oncoproteins in a defined background could only be studied using the system of the instant invention. Expression of a heterologous oncogenes in a cell line of the invention allows for the direct comparison of the efficacy of a single compound on cells with identical genetic backgrounds other than the single oncogene. Alternatively, a series of compounds can be tested on cells containing a defined genetic alteration to determine if they are capable of inducing differentiation.
In a related aspect, a method of treating an inflammatory disorder is provided including administering to a subject in need thereof a pharmaceutical composition comprising the isolated cells of the present invention and a pharmaceutically acceptable carrier.
The following examples are intended to illustrate but not limit the invention.
Example 1
Methods
Construction of Tagged, Estrogen Receptor Fusions of Hoxb8 and Hoxa9.
A murine stem cell provirus (Mscv) expressing Hoxb8-ER or Hoxa9-ER was generated by inserting estrogen-binding domain (ERBD) of estrogen receptor (ER) at an N-terminal MluI restriction site engineered into epitope-tagged murine Hoxb8 or Hoxa9 proteins. Tagged Hoxb8 was generated by PCR using the 5′ primers (FLAG tag: g gaa ttc gcc acc ATG GAC TAC AAG GAC GAC GAT GAC AAA GGA ACG CGT GGA AGC TCT TAT TTC GTC AAC TCA C (SE ID NO:11); HA tag: g gaa ttc gcc acc ATG GGA TAC CCA TAC GAT GTT CCG GAT TAC GCT ACG CGT GGA AGC TCT TAT TTC GTC AAC TCA C (SEQ ID NO: 12)) and the common 3′ primer: ccg ctc gag tta CTA CTT CTT GTC ACC CTT CTG CG (SEQ ID NO: 13). Underlined sequences complement the 5′ sense strand sequences for amino acid positions 2 to 7 and the 3′ antisense strand sequences encoding the last 7 amino acids. Sequences encoding the Flag (DYKDDDDKG) (SEQ ID NO: 14) or HA (YPYDVPDYA) (SEQ ID NO: 15) tags are in italics. There is a unique EcoRI site (bold lower case) followed by canonical Kozac sequence (gcc ace) preceding the initiating ATG (first capitalized codon), and a unique XhoI site (bold lower case) after the stop codon (bold upper case). Following sequences encoding the tag (italics), there is a unique in-frame MluI site in each 5′ PCR primer (bold upper case) as well as an additional GGA codon encoding glycine which could facilitate flexibility between the tag and Hox protein domains. This is an important consideration, because the N-terminal domain of Hox proteins is critical for their immortalizing function. In preparation for cloning, the PCR product was digested by EcoRI and XhoI, and ligated into the pMscvNeo proviral vector (Clontech). To generate ER fusions of Hoxb8, in-frame sequences encoding the estrogen-binding domain of the human estrogen receptor (residues 282 to 595; ERBD, see, e.g., SEQ ID NO: 10) containing a Gly400Val mutation were produced by PCR using primers containing in-frame MluI sequences at their 5′ ends followed by digestion with MluI and ligation into the MluI site of each tagged Hoxb8 construct. The Gly400Val mutant ER was used because this point mutation renders the receptor insensitive to the low levels of estrogen found in fetal bovine serum (FBS) as well as to the estrogenic effects of other compounds, such as phenol red.
To generate conditional Hoxa9 proteins, codons encoding amino acids 4 to 5 of Hoxa9 were mutated into an MluI site in pGEM3zf-EE-Hoxa9 [Calvo, K. R., et al., Mol Cell Biol, 2000. 20 (9): p. 3274-85], and the same fragment encoding the mutant human ERBD described above was ligated into the Hoxa9 MluI site. The EE-ER-Hoxa9 coding sequence was excised by EcoRI and inserted into pMscvPuro (Clontech). All plasmids were sequenced over their cloning junctions to verify integrity.
Retrovirus preparation. Helper-free retrovirus was collected as culture supernatant following calcium phosphate cotransfection (Invitrogen, Carlsbad, Calif.) of 293T cells with MSCV tag-ER-Hox retroviral constructs and an ecotropic packaging construct.
Producing Retrovirus by CaPO4 Transfection of 293T Cells.
Helper-free retrovirus is produced in 293T cells by CaPO4 co-transfection of the retroviral construct with an ecotropic or amphotropic packaging construct (CellGenesys), using Invitrogen's CaPO4 Transfection Kit (#44-0052).
At day 0, 2×106 293T were seeded cells into a 10 cm dish with 10 ml DMEM (High glucose)+10% FBS+penicillin/streptomycin/glutamine. At day 1, the media was removed and replaced with 10 ml of fresh, pre-warmed media. Cells should be at  ̃60-70% confluence. 10 μg of retroviral construct+10 μg of packaging construct as per protocol was used to transfect the cells, and the cells were incubated overnight.
At day 2, media was removed and replaced with 6 ml of fresh, pre-warmed media. At day 3 6 ml of virus was harvested to a 15 ml conical tube. The tube was centrifuged briefly to pellet all cell debris. 1-2 ml aliquots were frozen in 2 ml freezing tubes and stored at −80° C.
Alternatively, the virus supernatant can be filtered and used immediately, where another 6 ml of fresh, pre-warmed media was added to transfected cells.
Depending on the size of the insert, viral titers between 105-106/ml are routinely obtained.
Spin Infection Protocol—for Infection in 12-Well Plates.
A non-TC treated plate was coated with Fibronectin (Falcon, 12-well #351153 or 6-well #351146) supplied as a 1 mg/ml solution from Sigma (F-0895). The fibronectin was diluted 1:100 in PBS to a final 10 μg/ml solution, 1 ml of the solution was aliquoted into each well of a 12-well non-tissue treated plate (or 2 ml per well in a 6-well plate), and the plates were incubated at 37° for 1-4 hrs or at 4° overnight.
Cells were counted and resuspend at 105-106/ml in “Progenitor Outgrowth Medium” (OptiMem 10% FBS 1% PSG, 10 ng/ml stem cell factor or 1% culture supernatant from an SCF-producing cell line 30 uM beta mercapto ethanol (1 ul neat into 500 mls medium) 1 uM estradiol). 1 μl of Lipofectamine was added per ml of cells. The fibronectin was aspirated and 250 μl ( ̃25,000 to 250,000 cells) were aliquoted into each well. 1-2 ml of virus were added to each well of the 12-well plate. The final Lipofectamine concentration was 1× (1:1000).
Spinoculation.
The plate(s) were wrapped in Saran Wrap with an equivalent balance plate, the plates were then spun in plate carriers at 1500 g for 60-90 minutes at 22°-32° in a Gernot Walter's Beckman JS5.2 rotor at 2800 rpm (r=20 cm,  ̃1300 g). The Lipofectamine/Polybrene was diluted with 3 ml of fresh “Progenitor Outgrowth Media” and the cells were incubated at 37° C.
Progenitor Outgrowth Medium.
Virus was stored at −80° C. after filtration through a 0.45 μM membrane. Virus titers ranged from 105 to 106, as calculated by the outgrowth of G418-resistant clones on NIH3T3 fibroblasts.
Cytokine Pre-Stimulation of the Cells.
For a good retroviral infection, the cells must be actively cycling. Therefore, the cells from either negatively-selected progenitors, some of which may be quiescent in marrow, should be transferred to a cytokine-rich media for 2 days. Stem Cell Media (IMDM (Iscove's)+15% FBS+1% pen/strep/glutamine, 10 ng/ml murine IL-3 (5 ng/ml) 20 ng/ml murine IL-6 25 ng/ml murine SCF (up to 100 ng/ml)) is very effective, however, other media is equally effective, especially those that include G-CSF, Flt3-ligand, and the like.
Derivation of Neutrophil Progenitors.
Derivation of neutrophil progenitors is dependent upon the cytokine conditions used during pre-expansion of target stem cells and during selection of immortalized progenitors following infection with Hoxb8-ER retrovirus. Bone marrow was harvested from the femur and tibia of female Balb/c mice, as described. Lineage-negative progenitors obtained by negative selection using an antibody cocktail reactive against MacI, B220, and Thy1.2 followed by removal of lin+ cells on a magnetic column (Stemcell Technologies, Vancouver, BC, Canada). Progenitors were pre-stimulated for 48 hours in Iscoves modified Dulbecco medium (IMDM) containing 15% FBS, 1% PSE, 50 ng/mL stem cell factor (SCF), 25 ng/mL IL-3, and 25 ng/mL IL-6 (also in Methods Supplement 4). 25,000 marrow progenitors were infected with 1 mL ER-Hoxb8 retrovirus by spinoculation (2,500 g, 2 hours, 22° C.) in the presence of lipofectamine (1:1000, Gibco BRL), as described. Infected progenitors were cultured in OptiMem medium containing 10% FBS, 1% PSE, 10 ng/ml SCF, 30 uM beta mercaptoethanol (1 ul into 500 mls medium), and 1 uM β-estradiol (Sigma). An infection efficiency of 10% was approximated based on comparison of the initial rates of progenitor outgrowth in the presence or absence of G418 selection. Immortalized myeloid progenitors were enriched by the serial passage of non-adherent cells every 3 days into new 12-well tissue culture plates. Immortalized progenitors predominated cultures infected by Hox-ER retroviruses by day 14, while control cultures evidenced reduce proliferation and stopped dividing by day 21. ER-Hox proteins continued to divide with a generation time of 18-20 hours. Recombinant SCF was replace by a 1% culture supernatant (approximately 10 ng/ml) from an SCF-producing cell line immediately following infection with Hoxb8-ER retrovirus. SCF-dependent Hoxb8-ER progenitors have proliferated over 9 months, maintaining a normal 40, XX karyotype in 19 of 20 chromosome spreads in an analysis done after 8 months (analysis for Hoxb8-ER macrophage progenitors is illustrated in FIG. 1).
Derivation of Macrophage Progenitors.
Harvesting Marrow.
Female Balb/c mice were sacrificed (generally 8-12 weeks) and intact femurs and tibia were removed into sterile dishes of PBS on ice. The ends of the bones were cut off and 10 ml syringes (filled with RPMI 10%) and 25 G needles were used to shoot the marrow into 50 ml conical tubes. The tubes were topped off up at 50 ml with PBS and the cells pelleted. The cells were resuspended in 10 ml ACK red blood cell lysis buffer (150 mM NH4Cl 10 mM KHCO3 0.1 mM Na2EDTA Adjust to pH 7.2-7.4 with 1N HCl Filter sterilize and store at 4° C.), followed by incubation for 5 min at RT. The tube was topped off at 50 ml with PBS and the cells are pelleted. The cells are finally resuspended in 4 ml of PBS.
Harvesting Fetal Liver Cells.
Pregnant mice were sacrificed, and subsequently, embryos were removed (can be used as early as day 11). Using a plunger from a 5 ml syringe, the cells are dispersed through a 70μ cell strainer (Falcon #352350) and pelleted. Pelleted cells are resuspended in 10 ml of ACK red blood cell lysis buffer and subsequently incubated 5 min at RT. 50 ml centrifuge tubes are Topped off at 50 ml with PBS and the cells are pelleted. Pelleted cells are rinsed 1× in PBS, and resuspended in 4 ml of PBS.
Using 5-Flurouracil Prior to Isolation of Marrow and Progenitors.
Mice can be injected with 5-Flurouracil (5-FU) 3-5 days prior to harvesting the bone marrow. Injections are done at 100-150 mg/kg I.P. The 5-FU reduces the total cellularity of the marrow with an increased % of progenitors. The advantage of the 5-FU is that the marrow from more mice can be processed on the same Ficoll gradient and on the same StemCell Technology column (using less reagent).
Bone marrow was isolated from the femurs of mice following ammonium sulfate lysis of red cells and centrifugation onto a cushion of Ficoll-Paque (Pharmacia, Piscataway, N.J.) as described. 100,000 Ficoll-purified mononuclear cells were subjected to spinoculation with one ml of Hoxb8-ER or Hoxa9-ER retrovirus. Infected progenitors were cultured in “Myeloid Cell Medium” (RPMI 1640 with 10% FBS, 1% Pen-Strep-Glut (PSQ, Gibco BRL, Rockville, Md.), 1% GM-CSF-conditioned media from B16 melanoma expressing the GM-CSF cDNA (approximately 10 ng/mL GM-CSF) and 1 uM β-estradiol (Sigma)). β-estradiol was kept as 1,000× (1 mM) or 10,000× (10 mM) stocks in 100% ethanol and stored at −20° C. An infection efficiency of 10% was approximated based on comparison of rates of progenitor outgrowth in the presence or absence of G418, which selects for expression of the neomycine phosphotransferase gene encoded by the MSCV retroviral vector.
Purifying early progenitors using Ficoll-Hypaque centrifugation for derivation of macrophage-committed progenitors immortalized by Hoxb8-ER or biphenotypic, neutrophil, or macrophage progenitors immortalized by Hoxa9-ER.
In a 15 ml conical tube, 3 ml of room-temperature Ficoll-Paque (Pharmacia, Piscataway, N.J.) was added and 4 ml of total marrow cells in PBS was gently layered on top. The cells were spun for 30 minutes at 1500 rpm at 20° in a Sorvall 6000B rotor (450 g). The cells were harvested from the interface and all supernatant within  ̃0.5 ml of the pellet were collected. The collected supernatant was diluted to 50 ml in Myeloid Medium (RPMI 1640 \10% FBS, 1% Pen-Strep-Glut (PSQ, Gibco BRL, Rockville, Md.), 20 ng/ml GMCSF or 1% culture supernatant from a GM-CSF-producing cell line and 1 uM β-estradiol (Sigma)). The cells were subsequently pelleted and counted.
Immortalized myeloid progenitors were selected by removal of non-adherent progenitor cells every 3 days to a new well in a 6-well culture plate. This protocol was continued over 3 weeks, at which time cultures contained immortalized macrophage progenitors (detailed protocol in Methods Supplement 7). Selection for G418 resistance permitted derivation of immortalized progenitors in a shorter time-frame (10 to 14 days). Eleven lines were derived from Black/6 and Balb-c mice using this protocol and each differentiated quantitatively into macrophages. Differentiation to macrophages is achieved by removal of estrogen with continued culture in the same medium. Macrophage chromosome analysis using chromosome painting demonstrated that greater than 85% of progenitors exhibited a normal karyotype (FIG. 8). Cells were maintained in a 37° C. humidified incubator with 5% CO2. Progenitors can be store in liquid nitrogen.
Cell Culture.
Cell lines 293T, Nalm6, 3T3 and cytokine-producing cell lines (for SCF and GM-SCF) were cultured and maintained as previously described [Calvo et al., (2000), Sykes, D. B. and M. P. Kamps, Mol Cell Biol, 2004. 24 (3): p. 1256-69].
Luciferase Reporter Assay.
Estradiol-responsiveness was evaluated by the ability of Hox-ER and activated form of E2a-Pbx1 (or Vp16-Pbx1) to induce cooperative activation of a luciferase reporter gene driven by tandem repeats of TGAT-TTAT motifs in Nalm6 (for Hoxa9-ER) or 293T (for Hoxb8-ER) cells cultured in medium supplemented with a wide range of concentration of β-estradiol (10-11M to 10-5 M), as previously described [Calvo et al., (2000), Sykes and Kamps (2001)]. Transcription activation was calculated as the value of relative light units for firefly luciferase versus that for a control renilla luciferase construct that was cotransfected in all samples.
Spectral Karyotyping (SKY) Analysis.
SKY analysis of myeloid progenitors were performed as previously described [Yang, A. H., et al., J Neurosci, 2003. 23 (32): p. 10454-62].
Western Blot and Northern Blot.
Antibodies α-Hoxa9, α-Flag, α-HA, α-RelB, α-c-Jun, α-p65, α-p38, and a-SRC-A were used for western blot as previously described [Calvo et al., (2000), Sykes and Kamps (2001), Wang, G. G., M. P. Pasillas, and M. P. Kamps, Blood, 2005, Park, J. M., et al., Immunity, 2005. 23 (3): p. 319-29]. Northern blot was performed as described previously [Sykes, D. B. and M. P. Kamps, Leuk Lymphoma, 2003. 44 (7): p. 1131-9].
Affymetrix Array Analysis.
Gene expression profiling analysis was performed and analyzed using affymetrix mouse total genome array as previously described [Wang et al., (2005)].
Flow Cytometric Analysis (FACS), Wright-Giemsa Staining, Nitroblue Tetrazolium Reduction Assay (NBT) & Nonspecific Esterase Assay (NSE).
Phenotypic characterization of myeloid progenitors and differentiated cells analyzed by FACS, Wright-Giemsa staining, and use of the NBT and NSE assays was performed as described previously [Sykes and Kamps (2001)].
ER fusions of Hoxb8 and Hoxa9 exhibit estrogen-regulated transcriptional function. The estrogen-binding domain of the estrogen receptor (ER) was fused to the N-terminus of Hoxb8 and Hoxa9, and the fusion cDNA's expressed in the retroviral vector MSCV (FIG. 1A). N-terminal epitope tags were added to facilitate subsequent identification. While Hoxa9 nor Hoxb8 exhibit independent transcriptional activation through their TAAT or TTAT DNA recognition motifs, both heterodimerize with Pbx on the composite TGAT-TTAT Pbx-Hox motif, and their binding to DNA can be measured indirectly through their ability to cooperate with a transcriptionally activated forms of Pbx (oncoprotein E2a-Pbx1) to activate transcription of a reporter driven by TGAT-TTAT elements. Using this assay, ER-Hoxa9 and ER-Hoxb8 co-activated transcription 10- and 3-fold, respectively, in response to β-estradiol. Half-maximal activation occurred at 10 nM β-estradiol (FIG. 1B). Therefore, Hoxb8-ER and Hoxa9-ER exhibit estradiol-regulated access to DNA.
Hoxb8-ER immortalizes neutrophil-committed or monocyte-committed progenitors, which can be selected based on their cytokine-dependent properties. In the presence of 1 uM estradiol, infection of primary marrow cultured in interleukin 3 (IL3), stem cell factor (SCF), or granulocyte-macrophage colony-stimulating factor (GM-CSF) with retrovirus expressing Hoxb8-ER or Hoxa9-ER resulted in production of immortalized factor-dependent progenitors (FIG. 1C; ER fusion proteins identified by Western blotting in insert), while infection in the presence of granulocyte colony stimulating factor (G-CSF) or macrophage colony stimulating factor (M-CSF) did not. The lineage-commitment of immortalized progenitors was tested by observing cell fate after withdrawal of estradiol. SCF-dependent Hoxb8-ER progenitors differentiated into 99% neutrophils (1% mast cells) while GM-CSF-dependent Hoxb8-ER progenitors differentiated to macrophages at levels >99% (FIG. 2A). By contrast, Hoxa9-ER progenitors exhibited principally biphenotypic neutrophil/macrophage differentiation regardless of the cytokine that was used during their derivation. After cloning, 20% of clones executed neutrophil-restricted differentiation and rare clones (1 in 30) executed macrophage-restricted differentiation. Because the use of Hoxb8-ER obviated the need for cloning, characterization of the Hoxb8-ER-immortalized progenitors was pursued.
Neutrophil or macrophage progenitors immortalized by Hoxb8-ER were next characterized for surface antigens and enzyme activities characteristic of neutrophils or macrophages. Neutrophils produced by differentiation of SCF-dependent Hoxb8-ER progenitors up-regulated NADPH oxidase (FIG. 2A), the neutrophil surface antigen Gr-1 and the myeloid integrin Mac-1, and down-regulated the macrophage marker F4/80 (FIG. 2B). By contrast, macrophages produced by differentiation of GM-CSF-dependent Hoxb8-ER macrophage progenitors exhibited activation of macrophage nonspecific esterase (FIG. 2A), up-regulation of F4/80 and Mac1, and down-regulation of Gr-1 (FIG. 2B).
Hoxb8-ER SCF neutrophil progenitors have proliferated continuously for 8 months with a cell division time of approximately 21 hours and Hoxb8-ER GM-CSF have a generation time of approximately 23 hours. Throughout this time-frame, progenitors exhibited stable karyotypes and differentiation responses. 19 of 20 metaphase spreads from neutrophil progenitors cultivated for 8 months yielded a normal karyotype (40, XX) (FIG. 8). Therefore, Hoxb8-ER progenitors do not become aneuploidy as a requirement for immortalization, they do not become aneuploidy at a significant rate over long durations of passage, and there is no selection for karyotypically abnormal cells. Progenitors also retained stable differentiation phenotype. Hoxb8-ER SCF progenitors have differentiated quantitatively into neutrophils for 8 months. In one testing (8 months) one of two lines yielded 5% adherent monocytes following differentiation.
Transcription profiling defines the differentiation program and the mature characteristics of neutrophil and macrophage progenitors immortalized by Hoxb8-ER. Affymetrix genome arrays (430 2.0 Array; probe sets for 39,000 transcripts including over 34,000 for characterized mouse genes) were interrogated with RNA from SCF Hoxb8-ER progenitors undergoing neutrophil differentiation and from GM-CSF Hoxb8-ER progenitors undergoing macrophage differentiation (Summary in Table 1; Down-regulated genes in Table 2, up-regulated genes in Table 3, LPS-induced genes in Table 4).







TABLE 1







Expression of diagnostic myeloid and cell cycle genes in SCF Hoxb8-ER neutrophil differentiation and in GM-CSF


Hoxb8-ER macrophage differentiation















Neutrophil

Macrophage




Gene
Comment
Prog
Diff
Prog
Diff
Genbank










Myeloid lineage markers (unchanged)













Fcgr2b
IgG Fc gamma receptor 2 beta, low affinity
32
34
34
52
BM224327


Fcgr3
IgG Fc gamma receptor 3, low affinity
38
38
32
58
NM_010188


CCR2
Receptor for macrophage chemotactic protein
138
62
84
192
U47035



(MCP1)


Fcer1g
IgE Fc receptor 1 gamma, high affinity
32
30
44
103
NM_010185







Neutrophil markers up-regulated













IL8Rbeta
IL8 Receptor beta, neutrophil chemokine receptor
<1
94
<1
<1
NM_009909


LF
Lactoferrin
<1
138
<1
<1
NM_008522


LRG1
Leucine-rich alpha-2-glycoprotein, granulocyte
<1
50
2
1
NM_029796



marker of unknown function


NB-1
CD177, Neutrophil marker of unknown function
<1
22
1
<1
BC027283


Cnlp
Cathelin, anti-bacterial peptide
4
56
<1
<1
NM_009921


Lip2
Lipocalin 2. Neutrophil granual protein. Function
3
62
3
5
X14607



unknown.


Itgb2l
Integrin beta 2-like. Neutrophil granual protein
<1
75
<1
<1
NM_008405



released on activation. Function unknown.


MMP9
Neutrophil gelatinase, Gelatinase B
<1
104
<1
9
NM_013599


Pglyrp1
Peptidoglycan recognition protein 1. In neutrophil
4
140
8
8
NM_009402



granuals in traps. Hydrolyzes peptidoglycan.


Stefin A1
Cystein proteinase inhibitor. Aka Cathepsin, Stefin
4
214
2
11
AW146083



3, Cystatin A


Arginase 1
Arginase 1, inflammation modulation. Hydrolysis of
<1
330
<1
<1
NM_007482



L-arginine into ornithine.


Ceacam1
CEA-related cell adhesion molecule 1. Expressed on
3
41
4
6
BC016891



leukocytes, epithelia, and endothelia mediates



homophilic cell adhesion. Promotes motility.



Matrix-dependent binding to talin.



Carcinoembryonic antigen-related cell adhesion



molecule 1 (CEACAM1) is expressed on epithelia,



blood vessel endothelia, and leukocytes. A variety of



physiological functions have been assigned to



CEACAM1. It is involved in the formation of glands



and blood vessels, in immune reactions, and in the



regulation of tumor growth. As a homophilic and



heterophilic adhesion receptor, it signals through



different cellular pathways. CEACAM1 is a tumor



suppressor whose expression is known to be lost in



the great majority of early adenomas and



carcinomas. CEACAM1 and alpha(v)beta(3) integrin



are functionally interconnected with respect to the



invasive growth of melanomas. Can bind SHP1 and



SHP2 and suppress activation in T cells.


Ceacam10
CEA-related cell adhesion molecule 10. Highly
<1
160
<1
1
NM_007675



related to Ceacam1. Expressed in placenta, colon,



marrow.


Olfactomedian
Secreted neutrophil glycoprotein. Aka pDP4.
<1
112
8
6
AV290148


4
Function unknown.







Monocyte markers up-regulated













ICSBP/IRF-8
Interferon Consensus Sequence binding protein.
4
<1
18
16
NM_008320



Macrophage commitment transcription factor


MMP12
Macrophage elastase, Matrix metalloproteinase 12.
<1
8
36
196
NM_008605


Macrosialin
CD68, Class D scavenger receptor
6
5
26
120
BC021637


Dectin-
Dendritic cell C-type lectin, Clecsf10. Marker of
<1
<1
72
360
NM_020001


2beta
inflammatory monocytes. Function unknown.


CD11c
Integrin alpha X, ItgaX, Dendritic marker
<1
5
14
117
NM_021334


Msr1
Macrophage scavenger receptor 1 (SR-A)
<1
<1
3
30
NM_031195


Msr2
Macrophage scavenger receptor 2
<1
<1
<1
32
BC016551


mMGL2
Macrophage galactose N-acetyl-galactosamine
<1
7
12
132
AW494220



specific lectin


CCL17
CCR4 ligand, T cell chemokine produced by
<1
<1
5
48
NM_011332



macrophages


CCL22
CCR4 ligand, T cell chemokine produced by
<1
<1
<1
17
BC012658



macrophages


Plet1
Unknown function, 285aa
<1
<1
4
116
BC022950







Myeloid differentiation markers up-regulated in both neutrophils and monocytes













Fpr1
Formyl peptide receptor 1
<1
86
<1
30
NM_013521


Fpr-rs2
Formyl peptide receptor-related sequence 2
1
104
4
118
NM_008039


Dectin-1
Clecsf12, beta glucan receptor, fungal recognition
4
278
20
147
NM_020008


CD300lf
CD300-like factor, Pigr3, CLIM1, Polymeric Ig
<1
260
5
35
BM230330



Receptor III


Mrc1
Mannose receptor. Binds bacterial C-terminal
<1
80
13
226
NM_008625



mannose


TLR2
Toll-like receptor 2
<1
28
10
25
NM_011905


CD14
TLR coreceptor
6
92
40
136
NM_009841


MMP8
Matrix metalloproteinase 8
6
154
74
154
NM_008611


Mac1
CD11b/Integrin alpha M/CD18
<1
24
9
48
NM_008401


Fgr
Src-family myeloid tyrosine protein kinase
<1
36
2
52
NM_010208


Lgmn
Legumain. Specific protease. Activates cathepsins B,
<1
29
1
65
NM_011175



H, L


SHPS-1
Adhesion receptor coupled to SHP-2. Cytoskeleton
6
63
17
128



reorganization. Apoptotic cell engulfment


Sirpbeta1
Receptor. Activates macrophage/MAP kinase.
<1
140
4
82
AI662854



Promotes phagocytosis. Binds DAP12


Vti1b
Vesicle transport through interaction with tSNAREs,
<1
54
<1
26
AV002218



facilitates exocytosis


MKP1/dusp1
Ptpn16. Inactivates Jun. Prevents re-entry into cell
7
109
5
114
NM_013642



cycle. Negative regulator of inflammation.







Myeloid differentiation markers down-regulated in both neutrophils and monocytes













MPO
Myeloperoxidase. In azurophilic (primary) granuals
340
4
240
<1
NM_010824


Prtn3
Proteinase 3, myeloblastin, serine proteinase, in
294
4
400
2
U97073



azurophilic (primary) granuals


Ela2
Elastase 2, Neutrophil elastase, serine proteinase
20
<1
300
<1
NM_015779


Cnn3
Calponin 3, actin-binding protein
136
3
46
<1
BB724741


Nedd4
Ubiquitination regulation. Developmentally regulated.
110
12
9
<1
NM_010890


Plac8
Placental 8, unknown function
240
4
180
1
AF263458


Ms4a3
Membrane-spanning 4-domains A3, HTm4, unknown
56
3
144
3
NM_133246



function







Common myeloid differentiation markers upregulated in SCF Hoxb8-ER neutrophil progenitors and expressed persistantly in


GM-CSF Hoxb8-ER progenitors













Lyzs
Lysozyme
6
260
200
380
AW208566


Gsn
Gelsolin, involved in podosome formation
2
30
74
80
NM_010354


CD14
TLR coreceptor
5
40
40
136
NM_009841


Lilrb4
Leukocyte Ig-like inhibitory receptor B4
<1
54
30
121
U05264


Pira1
Paired-Ig-like activating receptor A1, binds
<1
24
34
58
NM_011087



FcRgamma


Pira6
Paired-Ig-like activating receptor A6, binds
<1
14
44
86
NM_011093



FcRgamma


Pilrb1
Paired-Ig-like type II activating receptor beta
2
158
24
90
NM_133209


Gp49b1
Ig inhibitory receptor
<1
54
30
120
NM_013532


DC-HIL
Dendritic cell transmembrane protein, adhesion, binds
4
104
108
266
NM_053110



RGD/proteoglycans







Progenitor Genes correlating with high expansion potential of SCF Hoxb8 neutrophil progenitors













Sox4
HMG protein, oncoprotein
32
<1
<1
<1
NM_009238


HMG14
HMGN1, High mobility group protein 14
94
6
<1
<1
NM_008251


HMGa2
High mobility group protein a2
44
6
<1
<1
NM_010441







Transcription factors up-regulated













c-fos
FBJ osteosarcoma proto-oncogene, bZIP transcription
8
116
12
256
NM_010234



factor


c-Jun
bZIP transcription factor
2
40
2
13
BC002081


ATF3
c-jun-related bZIP transcription factor
<1
32
3
56
NM_007498


JunD1
Jun family member D, bZIP transcription factor
9
168
22
100
NM_010592


JunB
Jun family member B, bZIP transcription factor
16
80
8
54
NM_008416


CEBP beta
upregulated in myeloid differentiation
14
102
82
136
NM_009883


Btg2
B-cell translocation gene 2. Downregulates Cyclin D1
3
52
12
200
BG965405



and cell cycle. Promotes differentiation.


Mad
Max dimerization protein
4
88
7
15
BB036846







Transcription factors down-regulated













c-Myc
Myelocytomatosis proto-oncogene
88
<1
37
3
BC006728


Pontin52,
Tip 49, Helicase, binds TATA-binding protein, Myc,
30
<1
14
4
NM_019685


Tip49,
E2F, and b-catenin activation complexes


Ruvb1


Reptin52,
Tip 48, Helicase, transcription regulation
43
3
29
6
NM_011304


Tip48,


Ruvb2


c-Myb
Myelocytomatosis proto-oncogene
5
1
156
<1
NM_033597


B-Myb
Myb-like 2, regulates cell cycle via E2F-binding
8
<1
14
<1
NM_008652



protein p107, maintains ES cell stem-likeness


Rbb4
Retinoblastoma binding protein 4
62
<1
36
10
BF011461


Hmgb3/H
High mobility group member b3, Embryonic
72
4
12
2
NM_008253


mg4
expression, hemopoietic stem cells, inhibits dif.


TSC22d1
Tgfb1i4, Growth factor inducible, HLH transcription
44
2
96
9
AU016382



factor







Cell division genes down-regulated













cycB1
cyclin B1, binds cdk1/cdc2, G2/M progression
40
<1
18
3
X58708


Shmt2
Serine hydroxymethyltransferase. Nucleotide
21
<1
37
4
BM222403



biosynthesis


Rrm2
Ribonucleotide reductase m2. Nucleotide biosynthesis
46
<1
52
4
NM_009104


mcm2-
Mini chromosome maintenance deficient 2-DNA
84
2
38
<1
NM_008564


G1/S peak
replication licensing helicase


cdc6-G1/S
Cell division cycle 6 (loads MCM7), licensing factor.
40
7
12
<1
NM_011799


peak
DNA replication


CDT1
DNA replication licensing--loads mcm2-7.
27
8
60
8
AF477481



Oncoprotein in 3T3


Nola1
Nucleolar protein family A, member 1, snRNP. RNA
32
10
30
<1
NM_026578



maturation.


Nol5
Nucleolar protein 5, Nop5. RNA maturation.
44
<1
24
<1
BB729616


U50snoRNA
mU50, snoRNA. RNA maturation.
42
4
23
<1
AK012825


NOP56
Nucleolar protein 56. RNA maturation, ribosome
66
6
28
1
BM249243



Biogenesis.


Nolc1
Nucleolar and coiled body phosphoprotein 1. RNA
22
<1
24
2
BM236574



maturation, ribosome Biogenesis


SET
PP2A inhibitor. High in cancer/regenerating cells.
104
15
72
6
BF134272



Early development. translocated in leukemia
















TABLE 2







Gene down-regulation in SCF Hoxb8-ER neutrophil progenitors and in GM-CSF Hoxb8-ER macrophage


progenitors











Neut
Mac




Prog
Prog













Gene
Comment
0
6d
0
6d
Genbank










Cell cycle genes













cycB1
cyclin B1, binds cdk1/cdc2, G2/M
40
<1
18
130
X58708



progression


Cenph
Centromere protein H
14
2
8
<1
NM_021886


Wee-1
Inhibits cdk1
10
<1
12
2
BC006852


ARD1 homologue
N-acetyltransferase (maintains
20
5
14
3
NM_019870



HIF1a instability)


Cdc20
Promotes cyclin B ubiquitination and
42
20
60
12
BB041150



degredation (M phase arrest)


Cyclin B1-related
cyclin-D dependent kinase 4, c-Myc
48
6
60
17
NM_007629


cdk4
target gene
25
6
13
4
NM_009870


cycD2

46
1
8
7
NM_009829







Nucleotide


biosynthesis













Apex
Apurinicapyrimidinic endonuclease
32
6
30
3
AV263745


Shmt2
Serine hydroxymethyltransferase
21
<1
37
4
BM222403


PRAT
Phosphoribosyl pyrophosphate
33
2
8
<1
BG064988



amidotransferase


Rrm2
Ribonucleotide reductase m2
46
<1
52
4
NM_009104


Tmk
Thymidylate kinase
52
10
27
3
NM_023136


Prps1
Phosphoribosyl pyrophosphate
28
2
12
3
NM_021463



synthetase 1


Gart
Phosphoribosylglycinamide
19
6
40
10
NM_010256



formyltransferase


Impdh2
inosine 5-phosphate dehydrogenase
106
3
40
4
NM_011830



2, rate-limiting step in guanosine



biosynthesis







DNA replication













mcm2-G1/S peak
Mini chromosome maintenance
84
2
38
<1
NM_008564



deficient 2-replication licensing



helicase


Srm
Spermidine synthase
48
2
52
2
NM_009272


dUTPase
Deoxyuridine triphosphatase,
38
9
17
<1
AF091101



eliminates dUTP to prevent



incorporated into DNA


Hells
Helicase, lymphoid specific
18
10
17
<1
NM_008234


cdc6-G1/S peak
Cell division cycle 6 (loads MCM7),
40
6
12
<1
NM_011799



licensing factor


CDT1
DNA replication licensing. Loads
27
8
60
8
AF477481



mcm2-7. Oncoprotein in 3T3


mcm3-G1/S peak
Mini chromosome maintenance
24
4
19
3
BF606890



deficient 3


mcm5-G1/S peak
Mini chromosome maintenance
42
3
27
4
NM_008566



deficient 5-replication licensing



helicase


mcm6-G1/S peak
Mini chromosome maintenance
90
4
50
7
NM_008567



deficient 6-replication licensing



helicase


mcm7-G1/S peak
Mini chromosome maintenance
134
20
100
15
BB464359



deficient 7-replication licensing



helicase


Rpa2
replication protein A2
30
1
19
4
AK011530


priA
DNA polymerase primase p49
24
6
14
3
NM_008921



subunit


Smu1
DNA replication, activation of cdc2
26
3
20
12



kinase, spindle assembly,



chromosome integrity







Ribosome biogenesis, RNA maturation













Nola1
Nucleolar protein family A, member
32
10
30
<1
NM_026578



1, snRNP


Nol5
Nucleolar protein 5, Nop5
44
<1
24
<1
BB729616


U50snoRNA
mU50, snoRNA
42
4
23
<1
AK012825


NOP56
Nucleolar protein 56
66
6
28
70
BM249243


Nolc1
Nucleolar and coiled body
22
<1
24
2
BM236574



phosphoprotein 1


p38-2G4
Proliferation-associated nuclear non-
44
3
46
5
NM_011119



nucleolar factor PA2G4


Lyar
Zinc-finger nucleolar protein that
54
10
24
4
NM_025281



promotes oncogenesis


U22snRNP
U22 small nucleolar ribonuclear
50
6
40
7
BQ177137



protein


Hnrnpa1
heterogeneous nuclear
92
10
90
24
AK007802



ribonucleoprotein A1


ASF/SF2
splicing factor, arginine/serine-rich 1
32
3
30
7
NM_173374


SF3a
splicing factor 3a
30
2
10
4
BC009141







Differentiation-related or unknown functions













Ela2
Elastase 2, Neutrophil elastase,
20
<1
156
<1
NM_015779



serine proteinase


MPO
Myeloperoxidase
320
3
176
3
NM_010824


Prtn3
Proteinase 3, myeloblastin, serine
294
4
188
<1
U97073



proteinase, in azurophilic granuals


Ctsg
Cathepsin G, granual protein, serine
400
20
194
2
NM_007800



proteinase


Cystatin F
Cystein proteinase inhibitor,
82
8
82
<1
NM_009977



hematopoietic-specific


Cnn3
Calponin 3, actin-binding protein
136
3
54
<1
BB724741


Bcrp1
Responsible for Hoechst 33342
48
3
28
14



exclusion of “side population” of



stem cells


Ms4a3
Membrane-spanning 4-domains
50
4
154
1
NM_133246



subfamily A, member 3,, HTm4


Plac8
Placental 8, unknown function
240
4
180
1
AF263458


Igfbp4
Insulin-like growth factor binding
32
3
34
2
BC019836



protein 4


Slc16a1, MCT1
monocarboxylate transporter
66
5
41
4
NM_009196


Cyclophilin D
Peptidylprolyl isomerase D
48
8
28
3
BC011499


Slc19a1
Folate carrier, methotrexate
26
2
32
4
AI323572



importer


F13a1
Coagulation factor XIII, A1 subunit
112
20
170
25
NM_028784


FKBP4
FK506 binding protein 4, FKBP52
34
4
31
5
NM_010219


ICAM2
Intercellular adhesion molecule 2
26
6
17
4
NM_010494


Timm8a
Translocase of inner mitochondrial
30
7
24
7
NM_013898



membrane


Csda
Cold shock domain protein
50
10
30
9
AV216648


Icdh
Isocitrate dehydrogenase 3, subunit
31
3
34
6
AK003393



alpha


Nedd4
Ubiquitination (developmentally
38
1
9
<1
NM_010890



regulated)


Tilz1b
TSC22-related, Tgfb1i4, Growth
44
2
94
9
AU016382



factor inducible


Unknown
hematopoietic-specific
72
2
30
<1
BC035044







Transcription factors













c-Myc
Myelocytomatosis proto-oncogene
102
2
65
11
BC006728


Pontin52, Tip49, Ruvb1
Helicase, binds TATA-binding
24
<1
20
6
NM_019685



protein, Myc, E2F, and b-catenin



transactivation complexes


Reptin52, Tip48, Ruvb2
Helicase, transcription regulation
75
4
31
6
NM_011304


c-Myb
Myelocytomatosis proto-oncogene
116
28
62
<1
NM_033597


B-Myb
Myeloblastosis oncogene-like 2,
8
<1
14
<1
NM_008652



regulates cell cycle through



interaction with E2F-binding protein



p107, maintains ES cell stem-



likeness


Gfi-1
Growth factor independent--Zinc
33
8
41
<1
NM_010278



finger transcriptional repressor


Nsbp1, NBP-45, GARP45
Nucleosome binding protein 1, has
21
3
18
2
NM_016710



transactivation function, embryonic



expression


Hmgb3/Hmg4
High mobility group member b3,
70
5
48
11
NM_008253



Embryonic expression, hemopoietic



stem cells


Rbb4
Retinoblastoma binding protein 4
61
<1
36
10
BF011461


Sox4
HMG protein, oncoprotein
36
<1
<1
<1
NM_009238


HMG14
HMGN1
94
6
<1
<1
NM_008251


Unknown



















FIG. 3. Gene up-regulation in SCF Hoxb8-ER neutrophil progenitors and in GM-CSF Hoxb8-ER


macrophage progenitors















Neutrophil

Macrophage




Gene
Comment
Prog
Diff
Prog
Diff
Genbank










Early myeloidmarkers













Fcgr2b
IgG Fc gamma receptor 2 beta, low
32
34
34
52
BM224327



affinity


Fcgr3
IgG Fc gamma receptor 3, low affinity
38
38
32
58
NM_010188


CCR2
Receptor for macrophage chemotactic
138
62
84
192
U47035



protein (MCP1)


Fcer1g
IgE Fc receptor 1 gamma, high affinity
32
30
44
104
NM_010185







Neutrophil markers













IL8Rbeta
IL8 Receptor beta
<1
38
<1
<1
NM_009909


IL17R
IL17 Receptor
11
60
<1
2
NM_008359


LF
Lactoferrin/Lactotransferrin
<1
190
<1
<1
NM_008522


LRG1
LRG1, Leucine-rich alpha-2-glycoprotein
<1
74
2
1
NM_029796


NB-1
NB-1
<1
20
1
<1
BC027283


Cnlp
Cathelin, anti-bacterial peptide
<1
440
<1
<1
NM_009921


Lip2
Lipocalin 2, In granulocytic precursors
3
240
3
5
X14607



and epithelia cells


MMP9
Neutrophil gelatinase, Gelatinase B
<1
152
<1
<1
NM_013599


NG
Neutrophil gelatinase, MMP9
<1
196
2
14
NM_013599


Pglyrp
Peptidoglycan recognition protein,
4
76
8
9
NM_009402



neutrophil granual protein


Stefin A1,
Cystein proteinase (Cathepsin) inhibitor,
10
520
2
10
AW146083


Cystatin A
Stefin 3


Stefin A2-
Cystein proteinase inhibitor
4
460
1
17
BB667930


like 1


Itgb2l,
Neutophil-specific Integrin beta 2-like
<1
50
<1
<1
NM_008405


Pactolus


Stefin A3
Cystein proteinase inhibitor
<1
340
<1
10
NM_025288


Ngp,
Neutrophilic granual protein, Cathelin-
13
170
22
2
NM_008694


Bactinecin
and Cystatin-homology


Arg1
Arginase 1
<1
330
<1
<1
NM_007482


Bcl6
Zn-finger transcriptional repressor
<1
76
<1
2
NM_009744


Rnf11
Ring Finger 11, Ubiquitination specificity
8
224
4
7
BI150320


Rnf144
Ring Finger 144, Ubiquitination
16
102
<1
<1
BB125275



specificity


Pdi4
Histone H3 specific pepdidyl arginine
<1
44
<1
<1
NM_011061



deiminase, transcriptional repression


CDCRel1
GTP-binding protein
3
96
10
4
AF033350


Trim 12
Tripartite motif protein 12
1
22
<1
<1
BM244351


Ceacam1
CEA-related cell adhesion molecule 1
3
41
4
6
BC016891


Ceacam10
CEA-related cell adhesion molecule 2
<1
160
<1
1
NM_007675


Wdnm1
Proteinase inhibitor
2
173
15
11
AV006463


Olfactomedian 4
Secreted neutrophil glycoprotein, pDP4
<1
112
8
6
AV290148


Crispld2
cysteine-rich secretory protein LCCL
<1
56
<1
<1
BB558800



domain containing 2


mDia1
Diaphanous protein homolog 1, Rho
<1
44
2
4
AW554652



effector, cell motility


Olfl1
Olfactomedian-like1
<1
22
5
6
AV230978


Slc2a3
Facilitated glucose transporter
20
274
3
2
BB414515


Olfml2b
Olfactomedian-like 2b, photomedian 2
<1
19
<1
<1
BC025654


St3ga14
ST3 beta-galactoside alpha-2, 3-
2
50
<1
<1
BE954474



sialyltransferase 4, neutrophil rolling


Mcp8
Mast cell protease 8
58
164
<1
<1
NM_008572


Syne1
Nuclear organization
<1
54
<1
<1
BI734306


cyp450
Cytochrome p450 4f18, unknown
2
54
<1
<1


4f18
function


Stk17b
Serine-threonine kinase, apoptosis-
<1
32
<1
2
NM_133810



inducing


Cd73
ecto-5′-nucleotidase
<1
30
<1
<1
AV273591


Unknown
1691 aa,
<1
17
<1
<1
BG070087


Unknown
216 aa, Protein ID BAE42230


Unknown
786 aa, Denn and Madd domains.
3
52
<1
5
BB168293



Conserved to C. Elegans, Gene 1437121


Unknown
463 aa, conserved to C. elegans, Gene
2
64
<1
<1
NM_133829



1424463


Unknown
537 aa, conserved to C. elegans, PL48,
8
66
2
2
NM_178658



Up in neuts from HL60, Gene 1460555







Monocyte/Dendritic markers













F4/80
Emr1
<1
1
10
26
U66888


ICSBP/IRF-8
Interferon Consensus Sequence binding
4
<1
18
16
NM_008320



protein


MMP12
Macrophage elastase, Matrix
<1
5
56
300
NM_008605



metalloproteinase 12,


Macrosialin
CD68, Class D scavenger receptor
333
5
26
120
BC021637


Dectin-
Dendritic cell-associated C-type lectin,
<1
<1
72
360
NM_020001


2beta
Clecsf10


CD11c
Integrin alpha X, ItgaX, Dendritic
<1
5
14
117
NM_021334



marker


Msr1
Macrophage scavenger receptor 1 (SR-
<1
<1
2
30
NM_031195



A)


Msr2
Macrophage scavenger receptor 2
<1
<1
<1
32
BC016551


mMGL2
Macrophage galactose N-acetyl-
<1
7
12
132
AW494220



galactosamine specific lectin


CCL17
CCR4 ligand, T cell chemokine produced
<1
<1
5
48
NM_011332



by macrophages


CCL22
CCR4 ligand, T cell chemokine produced
<1
<1
<1
19
BC012658



by macrophages, aka MDC


MHC-H2-
Histocompatibility 2, class II, antigen E
<1
<1
2
109
NM_010381


Ealpha
alpha


MHC-H2-
Histocompatibility 2, class II antigen A,
<1
<1
5
95
BC008168


Abeta1
beta 1


MHC-H2-
Histocompatibility 2, class II antigen A,
<1
20
9
164
AV018723


Aalpha
alpha


MHC-H2-
Histocompatibility 2, class II, antigen IE
<1
<1
9
133
AF119253


Ealpha
alpha


MHC-H2-
Histocompatibility 2, class II antigen E
<1
3
4
142
NM_010382


Ebeta1
beta 1


MHC-H2-
Histocompatibility 2, class II, locus
2
3
9
69
NM_010388


DM beta2
DMb2


MHC-H2-
Histocompatibility 2, class II, locus Dma
15
2
10
94
NM_010386


DM alpha


Csf2rb1
GM-CSF receptor low-affinity Beta 1
10
14
38
134
NM_007780


DCL-1
Dendritic cell receptor, C-type lectin--
<1
<1
11
66
NM_025422



unknown function


DCAR
Dendritic cell activating immunoreceptor
<1
<1
7
54
NM_027218



(FcReceptor gamma-associated)


Car4
Carbonic anhydrase 4
<1
<1
42
30
NM_007607


CD74
Macrophage migration inhibitory factor
<1
6
11
292
BC003476



receptor, Ia-gamma, CLIP, DHLAG


Capg
Gelsolin-like. Involved in receptor-
6
11
18
92
NM_007599



mediated ruffling, phagocytosis


F7
Coagulation factor VII
<1
14
166
250
NM_010172


Lpl1
Lipoprotein Lipase, Secreted, Hydrolyzes
<1
27
140
250
NM_008509



VLDL


Lrp1
LDL-related protein, macrophage
<1
1
8
30
NM_008512



marker


Ifi30
Lysosomal thiol reductase, interferon
1
<1
28
76
NM_023065



gamma inducible protein 30


NOV
Connective tissue growth factor
<1
<1
22
54
X96585


CCL9
MIP1 gamma, Scya9/Scya10
90
<1
120
140
NM_011338


EST1
456 aa, Widely expressed
<1
<1
24
44
BB559293







Myeloid differentiation markers upregulated in


both neutrophils and monocytes













Fpr1
Formyl peptide receptor 1
<1
48
<1
30
NM_013521


Fpr-rs2
Formyl peptide receptor-related
1
104
4
118
NM_008039



sequence 2


Dectin-1
Clecsf12, beta glucan receptor, fungal
4
138
20
147
NM_020008



recognition


CD300lf
CD300-like factor, Pigr3, CLIM1,
<1
260
5
35
NM_145634



Polymeric Ig Receptor III


Mrc1
Mannose receptor, C1,
<1
76
13
226
NM_008625



macrophage/dendritic, binds bacterial C-



terminal mannose


TLR2
Toll-like receptor 2
<1
30
10
25
NM_011905


P2ry6
Pyrimidinergic receptor P2Y activated by
<1
18
<1
38
BC027331



UDP


CD14
TLR coreceptor
5
40
40
136
NM_009841


Mac1
CD11b/Integrin alpha M/CD18
2
28
9
48
NM_008401


Fgr
Src-family myeloid tyrosine protein
2
70
2
52
NM_010208



kinase


MMP8
Matrix metalloproteinase 8
6
154
74
154
NM_008611


Lyzs
Lysozyme
6
260
200
380
AW208566


Gsn
Gelsolin, involved in podosome
2
30
74
80
NM_010354



formation


Lgmn
Legumain, activates cathepsins B, H, L
<1
29
1
65
NM_011175


Vacuolar
Subunit d2 isoform
<1
80
17
126
AV204216


H+ ATPase
Vesicle transport through interaction
<1
54
<1
26
AV002218


Vti1b
with tSNAREs, facilitates exocytosis


Sirpbeta1
Signal regulatory protein beta 1,
<1
140
5
82
AI662854



activation promotes phagocytosis,



induces filopodia and lamellipodia, binds



DAP12


Mcl
Macrophage C-type lectin, Clecsf8,
31
156
27
308
NM_010819



endocytic recptor


TLR1
Toll-like receptor 1
<1
6
<1
14
NM_030682


TLR4
Toll-like receptor 4
2
14
16
14
NM_021297


TLR6
Toll-like receptor 6
3
14
<1
6
NM_011604


Lilrb4
Leukocyte Ig-like inhibitory receptor B4
<1
54
30
121
U05264


Pira1
Paired-Ig-like activating receptor A1,
<1
24
34
58
NM_011087



binds FcRgamma


Pilra
Paired IG-like type II inhibitory receptor
<1
54
18
64
BB775785



alpha


Pira6
Paired-Ig-like activating receptor A6,
<1
14
44
86
NM_011093



binds FcRgamma


Pilrb1
Paired-Ig-like type II activating receptor
2
158
24
90
NM_133209



beta


Pirb5
Paired-Ig-like inhibitory receptor B5,
<1
40
14
30
U96693



dendritic/B cells


Gp49b1
Ig inhibitory receptor
<1
54
30
120
NM_013532


CCR1
CC chemokine receptor 1
<1
30
10
56
BC011092


CD157
fMLP receptor, ADP-ribosyl cyclase.
<1
48
2
20
NM_009763



Mediates adhesion, motility, and



chemotaxisis. In uropod


C3aR1
Complement component 3a receptor 1-
1
24
6
28
BC003728



high affinity


DCIR3
Dendritic cell inhibitory receptor 3--
<1
43
8
92
AK014135



unknown function


DCIR
Dendritic cell immunoreceptor-
2
92
10
94
NM_011999



inhibitory, Clecsf6


DC-HIL
Dendritic cell transmembrane protein,
4
104
108
266
NM_053110



adhesion, binds RGD/proteoglycans


Fcgrt
IgG Fc receptor, alpha chain transporter
<1
2
34
60
NM_010189


IL1R2
Interleukin 1 receptor, type II (pro-
3
40
12
42
NM_010555



inflammatory)


St2L
IL1-like receptor negative feedback of
44
212
2
32
D13695



pro-inflammatory function


IEX-1, IER-3
Immediate early response gene 3,
12
32
11
70
NM_133662



protects against apoptosis


CD200
Ig superfamily, myeloid-restricted,
5
10
4
52
NM_021325


Receptor
negative regulation of cell activation


TNFR-1
Tumor necrosis factor receptor 1, pro-
10
36
12
128
M59378



inflammatory


betaig-h3
Integrin-binding ECM protein, adhesion,
16
50
<1
25
NM_009369



TGF beta-induced 68 kDa


CD166
ALCAM (activated leukocyte cell
<1
34
5
44
AV315205



adhesion molecule)


IL1b
Interleukin 1 beta
5
186
6
68
BC011437


MIP-2
CXCL2, Scyb2, Macrophage
<1
38
<1
74
NM_009140



inflammatory protein 2, dendritic cell



inflammatory protein


CCL6
CC chemokine ligand 6, Scya6, C10,
<1
42
132
540
NM_009139



macrophage chemokine


Tubulin,
microtubules,
<1
44
5
56
BC003475


beta 2


Myadm
Myeloid dif. Marker, unknown function
<1
50
18
96
NM_016969


ADAM8
A disintegrin and metalloproteinase 8
<1
150
12
32
NM_007403


gpnmb
Osteoactivin
<1
54
66
320
NM_053110


Hck
Src-family tyrosine protein kinase
9
28
5
14
NM_010407


YM1
Glycosidase, chitinase 3-like 3, ECF-L
<1
163
<1
19
NM_009892


YM2
Secretory protein precursor
3
33
3
12
AY065557


Arg2
Arginase, type II (NO biosynthesis)
<1
72
<1
16
NM_009705


L-Selectin
Mediates roling of leukocytes on
2
36
2
10
NM_011346



endothelium


CC3
Complement component C3
5
40
10
34
K02782


Timp2
Tissue inhibitor of metalloproteinase 2
2
44
2
17
BF168458


CD9/MRP-1
Mobility related protein 1, tetraspanin
6
42
6
72
NM_007657



protein


RhoC
Migration, organization at the leading
<1
20
2
12
AI503490



edge


Puma-g
Interferon-gamma inducible gene
<1
162
5
22
NM_030701


IFITM3
Similar to interferon-induced TM protein 3
<1
66
4
19
BB193024


Zfp36
TIS11b, Zinc finger protein 36,
4
28
13
78
X14678



Brf1, ERF1, cMG1, Berg36, mRNA



stability


Rgs2
Regulator of G protein signaling 2
9
80
6
24
NM_009061


ASK1
Links TRAF6 to p38 signaling in innate
5
31
2
6
AV377656



immunity


Csf2ra
Colony-stimulating factor 2 receptor
9
108
54
78
NM_009970



alpha, GM-CSF R alpha, low-affinity


Osteopontin
Th1 immune reaction, ECM formation
6
96
202
148
NM_009263



and Ca deposition in the bone/kidney


Sema4a
Dentritic cell expression,
<1
32
20
34
BB114323



immunomodulation


ifitm3
interferon-induced transmembrane 3
<1
68
3
18
BB193024


Lyst
Lysozomal trafficking regulator
8
168
12
22
BB463428


Cybb
Cytochrome b-245, gp91phox
2
64
52
210
NM_007807


Cathepsin S

7
217
108
457
NM_021281


Gpsm3
G-protein signaling modulator 3,
4
280
18
52
NM_134116



regulates Gi alpha activation


MRP8
S100a8; pro-inflammatory, activates
6
312
140
258
NM_013650



endothelial cells, high in neutrophils


MRP14
S100a9; pro-inflammatory, activates
4
368
42
48
NM_009114



endothelial cells, high in neutrophils


SASPase
Skin aspartyl protease
<1
30
3
10
AK004007


RGS14
Regulator of G protein signaling 14
3
25
10
18
NM_016758


Slc15a3,
Histidine, dipeptide transporter
<1
37
2
35
NM_023044


PHT2


TMEM23
Sphingomyelin synthase 1, MOB, SMS1
<1
32
3
40
AV244175


MKP1,
Ptpn16, inactivates Jun kinase, prevents
7
109
5
114
NM_013642


dusp1
reentrance into the cell cycle, negative



regulator of inflammation


Samhd1
SAM and phosphohydrolase domain,
2
68
6
31
NM_018851



Unknown function


Ier5
Immediate early response 5, Unknown
2
20
2
22
NM_010500



function


Unknown
Unclassifiable transcript, BC025215
5
160
106
216
AK018202







Transcription factors













ICSBP
Interferon Consensus Sequence binding
4
<1
18
16
NM_008320



protein


Mad
Max dimerization protein
4
88
7
15
BB036846


ATF3
c-jun-related bZIP transcription factor
<1
31
3
56
NM_007498


JunB
Jun family member B, bZIP transcription
7
54
4
54
NM_008416



factor


c-fos
FBJ osteosarcoma proto-oncogene, bZIP
8
72
12
214
NM_010234



transcription factor


Btg2
B-cell translocation gene 2, anti-
2
52
12
200
BG965405



proliferative, Tis21, Pc3


ID2
Inhibitor of Helix-Loop-Helix
4
28
16
218
NM_010496



differentiation proteins


Klf2
Krupple-like factor 2, activates
4
54
7
18
NM_008452



p21WAF1/CIP1


c-Jun
bZIP transcription factor
2
40
2
13
BC002081


Egr-2
Early growth response 2, Zinc-finger
3
28
4
48
X06746



transcription factor, Krox20


Egr-1
Early growth response 1, Zinc-finger
44
120
18
135
NM_007913



transcription factor


JunD1
Jun family member D, bZIP transcription
4
52
22
100
NM_010592



factor


Ets1
E26 proto-oncogene
<1
8
7
<1
BB151715


KLF6
Krupple-like factor 6
6
76
4
20
NM_011803


CEBP beta
upregulated in myeloid differentiation
11
54
54
92
NM_009883


KLF7
Krupple-like factor 7
2
30
3
6
BE851797
















TABLE 4







Inflammatory genes induced by LPS or sBLP in GM-CSF Hoxb8-ER-derived macrophages.

















LPS
sBLP
Fold


Gene
Comment
Genbank
Basal
2 hr
2 hrs
stim










Secreted proteins/Ligands













IL1a
IL1 alpha
BC003727
4
134
26
>30


IL6
Interleukin 6 (signals through STAT3)
NM_031168
<1
55
<1
>30


IL23p19
Interleukin 23
NM_031252
<1
50
1
>30


LIF
Leukemia inhibitory factor
BB235045
1
81
20
>30


RANTES
RANTES, Scya5, CCL5
NM_013653
<1
53
<1
>30


CXCL10
IP10, Scyb10, T cell chemoattractant (produced by
NM_021274
<1
182
121
>30



Macrophages)


Serpin b2
PAI-2; plasminogen activator inhibitor 2 (uPA inhibitor)
NM_011111
2
76
19
25


TNFsf9
TNF ligand 9, binds 4-1BB receptor on T cells
NM_009404
<1
24
<1
25


IL12p40
Interleukin 12p40 (STAT4) drives T helper cell differentiation
NM_008352
<1
19
<1
19


Serpinb9g
NK21B, Inhibits granzyme b, the killing protease of CTL, NK cells
AF425083
5
94
40
19


Dermokine
Dermokine alpha/beta, secreted peptide, unknown function
BI452905
<1
18
12
18


IFN-beta
Interferon beta (signals through STAT1 and STAT2)
NM_010510
<1
16
<1
16


CCR11
A receptor for MCP-1, LPS-induced
AJ318863
7
104
54
15


Hamp
Hepcidin antimicrobial peptide, iron homeostasis, LPS/IL6
NM_032541
<1
14
<1
14



upregulated


Activin
Inhibin beta (dimer = activin) TGF-b/DPP superfamily; neg reg of
NM_008380
5
71
8
14



inflammation, promotes differentiation), signals through smad



proteins


IL1beta
IL1 beta
BC011437
18
215
86
12


TNF alpha
TNF alpha
NM_013693
30
320
145
11


MCP3
Monocyte chemotactic protein 3; MCP3, Scya7, CCL7
AF128193
<1
11
2
11


MCP1
Monocyte chemoattractant protein 1; Scya2/CCL2
AF065933
4
51
9
11


CXCL11
IFN-inducible T cell alpha chemoattractant (I-TAC), scyb11
AF136449
2
18
3
10


Serpin e1
PAI-1; plasminogen activator inhibitor 1 (tPA and uPA inhibitor)
NM_008871
11
110
23
10


MIP1 alpha
Macrophage inflammatory protein 1 alpha; MIP1alpha, Scya3,
NM_011337
26
254
114
10



CCL3


MIP2
Macrophage inflammatory protein 2 (CXCL2)
BB829808
6
54
9
9


ICOS ligand
Inducible costimulatory receptor ligand, binds B7-H2,
NM_015790
4
28
11
7



coactivates T cells







Signaling regulators













ISG15
Interferon-stimulated gene 15, covalently modifies protein
AK019325
<1
54
44
>30


Usp18
Ubiquitin specific protease 18, aka U8P43 (hydrolyzes ISG15
NM_011909
<1
46
42
>30



from protein)


Rtp4
28 kDa IFN alpha responsive protein, receptor transport protein 4
NM_023386
<1
51
48
>30


MIC-1
Macrophage inhibitory cytokine 1, GDF15, TGF beta family
NM_011819
<1
80
58
>30



member


A20/Tnfaip3
A20, removes ubiquitin from TRAF6, downregulates Toll/NF-kB
NM_009397
2
42
17
21



signaling


SOCS3/Cish3
Cytokine-inducible SH2-containing protein 3, negative regulation
NM_007707
<1
21
10
21


TDAG51
Signaling protein that promotes survival
NM_009344
4
60
24
13


PAC1/Dusp2
Dual (Y/T) specificity phosphatase PAC-1, Map kinase
L11330
1
10
10
10



phosphatase


Pellino1
Required for NF-kB activation through IL-1R
NM_023324
4
30
8
7


MKP-7/Dusp16
Dual specificity phosphatase 16 (targets Jun kinase), MKP-7,
NM_130447
3
20
4
6



LPS-activated


Pde4b
phosphodiesterase 4B, inflammatory cell activation
AF326555
4
26
10
6


Arhe
Ras homology gene, member E
NM_028810
<1
6
<1
6


TLR7
Toll-like receptor 7
NM_133211
6
30
22
5


Tnfrsf23
Tumor necrosis factor receptor super family member 23, TRAIL
NM_024290
<1
18
17
18



decoy receptor







Others













Cox-2
Cyclooxygenase 2, involved in prostaglandin synthesis
M88242
2
119
59
>30


TG2
Transglutaminase 2 (upreguated in inflammation)
BC016492
2
74
62
>30


Gbp2
Interferon gamma-induced guanylate nucleotide binding protein 2
NM_010260
<1
38
26
>30


Ifit-2/Ifi54
Interferon-induced protein with tetratricopeptide repeats 2
NM_008332
<1
98
56
>30


Ifi16/Ifi204
Interferon-induced protein 204, transcriptional coactivator,
NM_008329
<1
35
26
>30



induces differentiation


Vlperin
Interferon-induced anti-viral glycoprotein
NM_021384
<1
118
84
>30


Tyki
LPS-induced thymidylate kinase
NM_020557
<1
35
30
>30


Ifit3/Ifi49
Interferon-induced protein with tetratricopeptide repeats 3
NM_010501
<1
56
46
>30


Ifit1/Ifi56
Interferon-induced protein with tetratricopeptide repeats 1
NM_00833
<1
116
48
>30


Ifi205
Interferon-inducible gene 205, macrophage expression
AI481797
<1
48
28
>30


MIP-2
CXCL2, Scyb2, Macrophage inflammatory protein 2, dendritic
NM_009140
2
118
85
>30



cell inflammatory protein


Mpa2l, GBP1
Macrophage activation 2-like, G-protein
BM241485
2
52
32
>30


BimL
BimL--proapoptotic protein
AF032460
2
44
15
24


Trim30
Tripartite motif protein
BM241342
<1
22
21
22


Bcl2a1a
Bcl2a1a
AI326167
<1
8
4
15


Serpine 1
Plasminogen activator inhibitor type I
NM_008871
3
50
10
15


Irg1
Immune-responsive gene 1, propionate catabolism
L38281
9
104
16
12


ICAM-1
ICAM-1, intracellular adhesion molecule 1
BC008626
2
28
10
12


MIP-2, Dcip1
Dedritic cell inflammatory protein 1 (LPS induced)-neutrophil
BB829808
4
54
8
12



chemotaxis


Saa3
Serum amyloid A3 (LPS-activate)
NM_011315
5
55
6
11


Myd116
Myd116, induced by IL-6, PP1 regulatory subunit 16
NM_008654
5
46
9
10


FIG. 1
L-amino acid oxidase, interleukin-4 induced (maps to
NM_010215
3
22
3
8



autoimmune succeptibility site; lupus site)


AdoRA2b
Adenosine A2b receptor, inflammation regulation
NM_007413
6
50
17
8


Rdh11
Retinol dehydrogenase 11 (IL4-induced)
BC018261
3
22
5
7







Transcription factors/regulators of mRNA abundance













NFkB2
Nuclear factor kappa B subunit p100

<1
54
28
>30


Nfkbie
Nuclear factor kappa B inhibitor epsilon
AK011965
<1
36
24
>30


Ikbke
Inhibitor of kappaB kinase epsilon
NM_019777
<1
52
24
>30


Fra-2
fos-like antigen 2
NM_008037
<1
32
15
>30


STAT1
Signal transducer and activator of transcription 1 (IFN signaling)
BB229853
<1
51
48
>30


STAT2
Signal transducer and activator of transcription 2 (IFN signaling)
AF088862
<1
38
32
>30


Nfkbib
Nuclear factor kappa B inhibitor beta
NM_010908
2
54
29
27


Nfkbia
Nuclear factor kappa B inhibitor alpha
NM_010907
8
210
165
24


IkappaBzeta
IkappaB zeta, IkappaB MAIL, negative regulator of nuclear NFKB
NM_030612
3
58
24
17


Six1
Sine oculis-related homeobox 1 homolog, development
BB137929
1
24
9
17


IkappaBalpha
IkappaBalpha
NM_010907
16
260
210
16


I kappa BNS
I kappa BNS, Negative regulator of NF-kappa-B
AW495632
10
122
72
12


c-Rel
c-Rel, required for LPS-induced transcription of IL12p40 subunit
NM_009044
3
28
10
9


c-Maf
bZIP transcription factor. Activates LPS-induced IL-10
BC022952
3
25
12
9



transcription


Nupr1/p8
p8 transcription factor. Induced by LPS
NM_019738
6
42
10
7


JunD1
bZIP transcription factor
NM_010592
38
250
220
7


Jundp2
Jun dimerization protein 2, inhibits AP1, promotes differentiation
NM_030887
7
45
8
6


MALT1
MALT1, paracaspase involved in NF-Kappa-B activation
BB296321
8
44
28
6


RelB
Reticuloendotheliosis oncogene related B
NM_009046
9
52
40
5


NFkB1
Nuclear factor kappa B subunit p105
L28118
20
100
44
5


Egr-1
Early growth response 1
NM_007913
10
38
26
4


Egr-2
Krox20, Zinc-finger transcription factor
X06746
6
25
22
4









The expression levels of 128 myeloid genes is plotted in FIG. 3 (genes plotted are underlined in supplement tables). IL8R-beta, LF, LRG1, NB-1, Lip2 and other neutrophil markers were up-regulated selectively during differentiation of SCF Hoxb8-ER neutrophil progenitors while MMP12, Macrosialin, Dectin-2β, CD11c, Msr1, Msr2, and other macrophage-specific markers were up-regulated coincident with differentiation of GM-CSF Hoxb8-ER macrophage progenitors, and IRF-8, a marker of macrophage lineage commitment, was expressed persistently. Fpr1, Fpr-rs2, Mrc1, TLR2, MMP8, Mac1, Fgr, Lgmn, and other common myeloid differentiation markers were up-regulated during maturation of both progenitor cell types, while common promyelocytic markers, including MPO, PRT3, Ela2, and Cnn3 were strongly down-regulated. This transcriptional profile indicates that Hoxb8-ER progenitors exhibit normal neutrophil and macrophage differentiation. Macrophage-committed GM-CSF Hoxb8-ER progenitors appeared to be positioned at a later stage of myeloid differentiation because they exhibit high basal levels of a subset of late differentiation genes, such as Lyzs, Gsn, CD14, Lilrb4, Pira1, Pira6, Pilrb1, Gp49b1, and DC-HIL (see elevated blue signals in FIG. 3), which were not expressed, but strongly up-regulated during SCF Hoxb8-ER neutrophil differentiation. Changes in gene expression predicted by Affymetrix arrays were verified by a variety of analysis. Immunoblot analysis confirmed up-regulation of the macrophage scavenger receptor (SRC-A) and transcription factors Rel-B and c-Jun (FIG. 3B), FACS analysis confirmed up-regulation of CD11c (FIG. 3C), and Northern blotting confirmed down-regulation of Gfi-1, c-Myb, NOP52, neutrophil elastase, and up-regulation of c-fos (FIG. 3D).
Inactivation of Hoxb8-ER by removal of estrogen resulted in cell cycle arrest following a 4-fold expansion of GM-CSF Hoxb8-ER macrophage progenitors (FIG. 1D) and a 90-fold expansion of SCF Hoxb8-ER neutrophil progenitors. Cell cycle arrest coincided with down-regulation of c-Myb, c-Myc, and Hmgb3, as well as the helicases Pontin 52/Tip49 and Reptin 52/Tip48, which serve as coactivators for c-Myc, TBP, β-catenin, and E2F (Blue tracings in FIGS. 4A and 4C, Table 1). Cell cycle genes (e.g. CycB1, Mcm2), as well as c-Myc target genes (e.g., Nolc1, Shmt2) fell in parallel with expression of c-Myc, Pontin52/Tip49, and Reptin 52/Tip48 (Yellow tracings in FIGS. 4A and 4C). Promyelocyte-specific expression of the c-Myb targets, ELA2 (leukocyte elastase), Ctsg (Cathepsin G), Prtn3 (Proteinase 3/myeloblastin), and MPO (myeloperoxidase) fell in parallel with c-Myb (Brown tracings in FIGS. 4B and 4D). Synchronous expression of differentiation markers (e.g., Fpr1, Fpr-rs2, Dectin-1, Mrc1, Fgr, VacATP; green tracings in FIGS. 4B and 4D) paralleled that of the leucine zipper transcription factors ATF3, Jun-B, c-Fos, and JunD1. This suggests that a broad program of cell cycle gene down-regulation (Supplement Table 2) and differentiation gene up-regulation (Supplement Table 3) is being driven by inactivating c-Myc and c-Myb and up-regulating bZIP transcription factors (ATF3, JunB, c-fos, RelB, c-Jun, JunD1, Egr-1, Egr2; [Krishnaraju et al (1998), Libermann and Hoffman (1994), Kharbanda et al., (1991)]).
Neutrophil and macrophage progenitors immortalized by Hoxb8-ER exhibit normal terminal differentiation based on expression analysis. Mature macrophages evidenced up-regulation of genes involved in adhesion (CD11c, CD11b, DC-HIL, CD157, CD166), migration (CD74, CCR1, CCR5), phagocytosis (SIRPbeta1, DCAR, DCIR3), activation (DCL1, DCIR), pathogen pattern recognition (Mgl2, Mc1, Mrc1, Dectin 1, Fpr1, Fpr-r2, CD157), recognition of necrotic cell debris (Msr1, Msr2, CD36, CD68), T cell stimulation/activation (CD83, MHC2 class II complex antigens E alpha, A beta 1, A alpha, E alpha, Ebeta1, DM loci alpha and beta2, IL-18), migration (MIP-2/CXCL2, MCP-1), bacterial killing (Bactenecin, Cathelin), opsinophagocytosis (CC3, C3aR1), proteolysis/MHC class II peptide generation (LGNM, MMP9, uPA, Cathepsin H, Cathepsin B), protease inhibition (Stefin A1, Stefin A2, Stefin A3, Timp2, PAI-2, Serpinb9 g, Wdnm1), nitric oxide biosynthesis (Arg2, Pdi4), metal ion transport (Slc11a1, LF, transferrin), and receptor signaling via tyrosine kinases (Hck, Fgr). Mature neutrophils up-regulated a largely overlapping group of myeloid maturation genes, in addition to neutrophil-specific granule genes such as lactoferrin (FIG. 4, Table 1, and Table 3).
SCF Hoxb8-ER progenitors behave as Granulocyte/Macrophage Progenitors (GMP), retaining eosinophil differentiation potential. While the strong expression of promyelocytic genes, such as MPO and Prt3, established them as myeloid, the SCF-responsiveness and negligible expression of any terminal differentiation gene in Hoxb8-ER progenitors suggested they might retain the ability to execute alternative differentiation fates in response to other lineage-specific cytokines. To test this hypothesis, SCF Hoxb8-ER progenitors were allowed to differentiate in SCF medium supplemented with GCSF, IL5, MCSF, GM-CSF, or erythropoietin (FIG. 5). Inclusion of GCSF augmented chromatin condensation and increased expansion from 70- to 120-fold. Inclusion of IL5 induced eosinophilic granules in a third of maturing granulocytes (enlarged example inset). Inclusion of GM-CSF increased expansion to 830-fold, and produced 16% macrophages, while inclusion of M-CSF did not alter expansion, but induced 6% of progenitors to mature as macrophages. Erythropoietin had no impact on neutrophil-committed differentiation. Therefore, SCF Hoxb8-ER progenitors are similar to normal granulocyte-macrophage progenitors (GMP), which retain the ability to differentiate into eosinophils, as well as neutrophils and macrophages. Hoxb8-ER progenitors execute neutrophil differentiation as a default program in SCF alone.
Macrophages produced by maturation of Hoxb8-ER GM-CSF progenitors exhibit normal inflammatory responses. The genetic inflammation response of Hoxb8-ER macrophages was evaluated in response to lipopolysaccharide (LPS), an activator of toll-like receptor 4 (TLR4) and sBLP, an activator of toll-like receptor 2 (TLR2). LPS activated strong transcription of the genes encoding NF-kappa-B, STAT, Jun, and Egr, as well as over 50 additional genes involved in inflammation (Genes responding greater than 10-fold in FIG. 6 and Table 4), including those encoding coactivators of T cell migration and proliferation (TNF9, IL12, IL23), monocyte chemokines (MCP1, MCP3, MIP1a, RANTES) and pleotropic cytokines (IFN-beta, TNFa, IL6, IL1a, IL1b, LIF).
Hoxb8-ER target progenitors can be immortalized from d13 fetal liver. The most useful application of the conditional immortalization system described above is immortalization of progenitors from genetically-engineered mice—those with transgenic or knockout alleles designed to address questions pertaining to issues of immunologic and inflammatory functions. As a simple proof of concept, TLR-induced transcription of IFN and IL-10 in Traf3−/−myeloid cells was tested by restoring Traf3 expression in Traf3−/− progenitors conditionally immortalized by Hoxb8-ER, and testing CpG-induced production of IFN and IL-10 following 6 days. TRAF3 is essential for induction of type I IFNs and the anti-inflammatory cytokine IL-10, but is dispensable for induction of pro-inflammatory cytokines. TRAF3 is also recruited to the adaptor TRIF and is required for marshalling the protein kinase TBK1/NAK into TIR signaling complexes, thereby explaining its unique role in activation of the IFN response. The Traf3−/− mice exhibit a d15 embryonic lethal phenotype, so characterizing their defective responses and restoration of those responses by expression of Traf3 is particularly challenging using the small numbers of myeloid cells that can be expanded from progenitors in fetal liver.
Traf3−/− progenitors were immortalized by infection of GM-CSF-dependent progenitors from d13 fetal liver with Hoxb8-ER retrovirus. Immortalized Traf3−/− progenitors grew from the cultures following kinetics identical to those derived from wild-type mice. Traf3−/− HoxB8-ER GM-CSF progenitors were transduced with retroviral vectors containing expression cassettes for HA-tagged TRAF3 and puromycin resistance or a control vector containing the puromycin resistance cassette only. Transduced cells were selected with puromycin (2 μg/ml) and specific expression of TRAF3 was demonstrated by intracellular staining with antibodies to the HA-tag (FIG. 7a). Progenitors transduced with either the control vector or the HA-TRAF3 expression vector, were differentiated for 6 days by withdrawing estrogen. Lysates of these cells were analyzed by immunoblotting with antibodies to HA and p38, and exhibited retention of HA-TRAF3 expression in the differentiated monocytes (FIG. 7b). Differentiated HoxB8-ER monocytes were then stimulated with CpG-DNA and analyzed for IFN and IL-10 production by bioassay and ELISA, respectively. While TRAF3−/− macrophages produced no IFN or IL-10, robust expression was detected in those expressing exogenous HA-TRAF3 (FIG. 7c). Thus, conditional immortalization of knockout progenitors using Hoxb8-ER provides an effective system to study inflammatory signal transduction based on reconstitution of essential protein functions, and can be applied in the circumstance of an embryonic lethal phenotype in order to provide an abundant source of phagocytes for experiments at the cellular or biochemical levels.
Although the invention has been described with reference to the above example, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

## Claims

1. A method of differentiating and immortalizing progenitor cells comprising:
a) contacting isolated progenitor cells with a fusion protein comprising a HOX oncoprotein and an estrogen receptor binding domain (ERBD), wherein the ERBD consists of the sequence as set forth in SEQ ID NO: 10, wherein said isolated progenitor cells are progenitor cells that give rise to subsets of mature blood cells and wherein the contacting comprises infecting the progenitor cells with a

vector comprising a nucleic acid sequence which encodes the fusion protein, and wherein the ERBD is fused to the N-terminus of the HOX oncoprotein; and
b) culturing the progenitor cells of step a) with a combination of one or more multilineage cytokines, a myeloid-specific cytokine, and an estrogen agonist, whereupon culturing, the progenitor cells become immortalized and exhibit commitment to neutrophil, macrophage, and/or dendritic lineage or exhibit multi-lineage blood cell differentiation potential.

2. The method of claim 1, wherein the vector is a retroviral vector.