Identification of the human eosinophil lineage-committed progenitor: revision of phenotypic definition of the human common myeloid progenitor

To establish effective therapeutic strategies for eosinophil-related disorders, it is critical to understand the developmental pathway of human eosinophils. In mouse hematopoiesis, eosinophils originate from the eosinophil lineage-committed progenitor (EoP) that has been purified downstream of the granulocyte/macrophage progenitor (GMP). We show that the EoP is also isolatable in human adult bone marrow. The previously defined human common myeloid progenitor (hCMP) population (Manz, M.G., T. Miyamoto, K. Akashi, and I.L. Weissman. 2002. Proc. Natl. Acad. Sci. USA. 99:11872–11877) was composed of the interleukin 5 receptor α chain+ (IL-5Rα+) and IL-5Rα− fractions, and the former was the hEoP. The IL-5Rα+CD34+CD38+IL-3Rα+CD45RA− hEoPs gave rise exclusively to pure eosinophil colonies but never differentiated into basophils or neutrophils. The IL-5Rα− hCMP generated the hEoP together with the hGMP or the human megakaryocyte/erythrocyte progenitor (hMEP), whereas hGMPs or hMEPs never differentiated into eosinophils. Importantly, the number of hEoPs increased up to 20% of the conventional hCMP population in the bone marrow of patients with eosinophilia, suggesting that the hEoP stage is involved in eosinophil differentiation and expansion in vivo. Accordingly, the phenotypic definition of hCMP should be revised to exclude the hEoP; an “IL-5Rα–negative” criterion should be added to define more homogenous hCMP. The newly identified hEoP is a powerful tool in studying pathogenesis of eosinophilia and could be a therapeutic target for a variety of eosinophil-related disorders.

TG101348, An Orally Bioavailable JAK2-Selective Inhibitor, Potently Inhibits KITD816V and FIP1L1-PDGFRA in Vitro

Background: Systemic mastocytosis (SM) and hypereosinophilic syndrome (HES) are JAK2V617F-negative orphan diseases without effective long-term therapeutic options. Both of these diseases share clinical and laboratory features with FIP1L1-PDGFRA-positive chronic eosinophilic leukemia (CEL). SM is associated with KITD816V or other KIT mutations. HES is currently not molecularly characterized. The exquisite sensitivity of FIP1L1-PDGFRA to inhibition by imatinib mesylate (IM) underlies the efficacy of this agent in treating FIP1L1-PDGFRA-positive CEL. In contrast, KITD816V is resistant to inhibition by IM, and is only moderately sensitive to dasatinib (cellular IC50=micromolar concentrations), which may explain the relatively disappointing clinical responses in SM with these agents. TG101348 is an orally bioavailable JAK2-selective inhibitor that is currently being tested in a Phase 1 clinical trial for the treatment of myelofibrosis. The aim of the current study was to evaluate TG101348 (relative to the tyrosine kinase inhibitors IM, dasatinib, and sorafenib) for its ability to: inhibit growth of leukemic cell lines that carry KITD816V and FIP1L1-PDGFRA; and inhibit the in vitro growth of eosinophil colonies derived from progenitor cells from HES patients. Methods: The following cell lines were used for in vitro experiments: HMC-1, a mast cell leukemia line (KITD816V-positive); EOL-1, a CEL derived line (FIP1L1-PDFRA-positive), Ba/F3 T674I, a Ba/F3 line that express the IM-resistant FIP1L1-PDFRAT674I mutation, and HEL, a human erythroleukemia line (JAK2V617F-positive). Cell proliferation assays were performed in triplicate using the XTT assay (Leukemia. 2007 21:1658). Drug concentrations in these experiments ranged from 2.9 × 10−12 M to 10−4 M. JAK Inhibitor I (Calbiochem) is a non-selective JAK inhibitor tool compound. Eosinophil colonies were obtained by plating PBMCs from healthy controls or HES patients in methylcellulose, in the presence of IL-3, IL-5, and GM-CSF. Effects of IM and TG101348 on eosinophil colony growth were studied in parallel experiments. Results: Consistent with prior observations, the HMC-1 cell line was resistant to inhibition by IM (IC50>10 mM), but was moderately sensitive to dasatinib (Table). In contrast, TG101348 inhibited HMC-1 growth at nanomolar concentrations, similar to its effect on HEL cells that harbor the JAK2V617F mutation. TG101348 inhibited HMC-1 growth 53-fold and 4-fold more potently than IM and dasatinib, respectively (Figure). Sorafenib, a multikinase inhibitor that reportedly has activity against KIT kinase (enzyme IC50=68 nM), had only a limited effect on HMC-1 growth (IC50=4000 nM). In contrast to HMC-1, EOL-1 growth was inhibited by IM, dasatinib, sorafenib, and TG101348 at picomolar concentrations (Table). The T674I mutation (in the context of FIP1L1-PDGFRA) corresponds to the T315I mutation in BCR-ABL – it occurs infrequently in CEL and is usually found in the context of transformation to acute leukemia. Growth of Ba/F3 cells harboring FIP1L1-PDFGRA-T674I has been shown to be inhibited by sorafenib (confirmed in this report; Table) and nilotinib, at nanomolar concentrations. TG101348 is less potent at inhibiting growth of these cells, with cellular IC50 of ~2 mM – pharmacokinetic data from the ongoing Phase I study of TG101348 shows that this concentration is achievable in plasma with once daily dosing. Drug Cellular IC50 HMC-1 (nM) EOL-1 (pM) Ba/F3 T674I (nM) HEL (nM) Imatinib 18800 4 10700 - Dasatinib 1300 <1 30900 - Sorafenib 4000 2 16 - Jak Inhibitor I 926 30 4300 ? TG101348 355 <1 1950 300 Figure Figure Conclusions: The JAK2-selective inhibitor TG101348 is a potent inhibitor of KITD816V and FIP1L1-PDGFRA, warranting clinical trials using this drug in SM and FIP1L1- PDGFRA-positive CEL. Data regarding TG101348 effects on signaling intermediates in leukemic cell lines and on eosinophil colony growth will be presented at the meeting – data from the latter experiments may indicate a therapeutic role for TG10134 in the treatment of patients with HES.

Differential Re-Programming of Myeloid Stem Cell Development by Activator Versus Repressor Isoforms of Human CCAAT/Enhancer Binding Protein Epsilon (C/EBPε).

Human C/EBPε is expressed as four isoforms (32, 30, 27, 14kD) through alternative promoter usage, RNA splicing, and translational start sites. We are studying the C/EBPε isoforms in vivo in cord blood (CB) CD34+ progenitors to define their roles in granulocyte differentiation and hematopoietic lineage specification. The C/EBPε32/30 isoforms function as transcriptional activators. We reported that C/EBPε27 interacts with and is a repressor of GATA-1 transactivation of eosinophil-specific genes (Du J et al. JBC2002; 277:43481). C/EBPε14, which contains DNA binding and bZIP domains but lacks a transactivation domain, may function as a dominant negative repressor of C/EBPε32,30 or other C/EBPs through heterodimerization or competition for C/EBP binding sites. We assessed mRNA expression of the C/EBPε isoforms during eosinophil differentiation of CB CD34+ progenitors to determine their temporal patterns of expression. CD34+ progenitors (>95% pure) induced to differentiate into eosinophils by SCF, IL-3, and IL-5 showed differentiation to >90% eosinophils by 17–19 days. Semi-quantitative RT-PCR using C/EBPε isoform-specific primers showed that CD34+ progenitors initially express only the repressor C/EBPε14 isoform, with induction of all isoforms by day 3 to different peak levels of expression by days 9–11 during the promyelocyte to myelocyte transition. These results define temporal changes in the expression ratios of the C/EBPε activator vs. repressor isoforms during eosinophil development that may differentially regulate gene transcription in this process. We subcloned the C/EBPε isoform cDNAs into an MSCV-based bicistronic retroviral vector (pGCDNsam IRES-EGFP) and ectopic expression was induced in CB CD34+ progenitors by retroviral transduction for 72hrs. The CD34+/GFP+ cells were sorted by FACS and plated in Collagen Cult™ media containing SCF, IL-3 and a lineage-specific cytokine (i.e. EPO, G-CSF, or IL-5), or in suspension culture containing SCF, IL-3 and IL-5 to drive eosinophil differentiation. Total and differential colony and cell counts were performed after 15–17 days based on colony morphology, histochemical and enzyme staining of the cells. The activator C/EBPε32 isoform significantly altered myeloid development, favoring eosinophil over neutrophil or erythroid development. Even in cultures containing EPO, cells transduced with C/EBPε32 failed to develop into erythroid colonies (BFU-E). C/EBPε27, a potent repressor of GATA-1 in vitro, inhibited erythroid colony growth by ~50%, and doubled the numbers of granulocyte-macrophage colonies compared to C/EBPε14 or empty vector control. C/EBPε32 strongly induced eosinophil colony formation at the expense of neutrophil other myeloid lineages regardless of the cytokines used, inducing ~90% eosinophil colonies. C/EBPε27 reduced eosinophil colonies by >50%. Likewise, >90% of cells transduced with C/EBPε32 and grown in suspension culture were eosinophils, whereas C/EBPε27 and C/EBPε14 inhibited eosinophil differentiation by ~50% and >98%, respectively. Thus, the C/EBPε isoforms: (1) are differentially expressed during eosinophil development, (2) have the capacity to reprogram stem cells to different myeloid lineages consistent with their predicted activator vs. repressor activities and interactions with hematopoietic transcription factors such as GATA-1 or other C/EBPs, and (3) may be useful in their ability to reprogram myeloid terminal differentiation for the development of novel approaches to treat myeloid or other leukemias.

The Ordered Expression of Transcription Factors Directs Hierarchical Lineage Specification of Eosinophils, Basophils and Mast Cells.

Here we show that eosinophil progenitors (EoPs) and basophil/mast cell progenitors (BMCPs) are prospectively isolatable in normal hematopoiesis, and that their lineage decisions are regulated principally by GATA-2 and C/EBPα. These progenitors were isolated downstream of granulocyte/monocyte progenitors (GMPs), and BMCPs further generated monopotent basophil progenitors (BaPs) and mast cell progenitors (MCPs). Gene expression analysis showed that neither GATA-1 nor GATA-2 was expressed in GMPs, whereas both of them were upregulated in EoPs, BMCPs, BaPs and MCPs. Importantly, C/EBPα was expressed in EoPs and BaPs as well as GMPs, but was downregulated in BMCPs and MCPs. We have reported that GATA-1 is critical primarily for megakaryocyte/erythrocyte commitment or conversion of stem and progenitor cells. We therefore focused on GATA-2 and C/EBPα functions in this study. Since both EoPs and BaPs co-expressed GATA-2 and C/EBPα while GMPs expressed only C/EBPα, we first transduced GATA-2 into GMPs via a GFP-tagged retrovirus. Strikingly, all GATA-2+ GMPs gave rise to pure eosinophil colonies but not basophil colonies, indicating that enforced GATA-2 can instruct GMPs to become EoPs. Next, since BMCPs only expressed GATA-2 but not C/EBPα, we maintained the expression of C/EBPα in GMPs by retroviral transduction. Interestingly, the sustained expression of C/EBPα blocked basophil/mast cell differentiation from GMPs, indicating that C/EBPα downregulation is required for GMPs to choose the basophil/mast cell fate. As a reciprocal experiment, we conditionally disrupted C/EBPα gene at the level of GMPs by retrovirally transducing Cre gene into GMPs purified from mice in which C/EBPα gene is flanked by loxP sequences (floxed: F). The frequency of mast cell read-out from C/EBPα-disrupted GMPs was 5-fold higher than that from C/EBPα F/F (Cre−) GMPs. C/EBPα-disrupted GMPs, however, did not give rise to BaPs. Furthermore, MCPs transduced with C/EBPα were converted into BaPs. Thus, C/EBPα is required to be reactivated during transition from BMCPs to BaPs. We further tested their interplay in specification of these lineages by using common lymphoid progenitors (CLPs), which do not express GATA-2 or C/EBPα. We enforced the expression of each transcription factor in CLPs in different orders by using the two-step retroviral transduction system. Interestingly, C/EBPα transduction reprogrammed CLPs into GM lineages, and subsequently-transduced GATA-2 instructed C/EBPα + CLPs to select the eosinophil fate. Next, we switched the order of transduction. Strikingly, GATA-2 transduction converted CLPs into BMCPs, and subsequently-transduced C/EBPα specified GATA-2+ CLPs to become BaPs. Thus, at the branchpoint for EoPs and BMCPs, GATA-2 upregulation instructed EoP development if C/EBPα was present, whereas it instructed BMCP development if C/EBPα was absent. After the BMCP stage, C/EBPα had to remain suppressed for MCP development, whereas BaPs developed by C/EBPα reactivation. These data collectively suggest that the order of expression of GATA-2 and C/EBPα is critical for their interplay to selectively activate developmental programs for the eosinophil, the basophil and the mast cell lineages.

Interleukin-3 in vivo: kinetic of response of target cells

Human recombinant interleukin-3 (IL-3; Sandoz AG, Basel, Switzerland) was administered for 7 days to patients with neoplastic disease and normal hematopoiesis. The purpose of the study was to assess IL-3 toxicity, to identify target cells, to define their kinetics of response at different dose levels, and to determine if IL-3 in vivo increased the sensitivity of bone marrow (BM) progenitors to the action of other hematopoietic growth factors. A total of 21 patients entered the study; the dosage ranged from 0.25 to 10 micrograms/kg/d. The effect on peripheral blood cells during treatment showed no significant changes in the number of platelets, erythrocytes, neutrophils, or lymphocytes (and their subsets). A mild monocytosis and basophilia occurred. Eosinopenia, present in the first hours of treatment, was followed by a dose-and time-dependent eosinophilia. IL-3 treatment affected BM cell proliferation by increasing the percentage of BM progenitors engaged in the S-phase of the cell cycle. The effect was dose dependent, with the various progenitors showing different degrees of sensitivity. The most sensitive progenitors were the megakaryocyte progenitors (colony-forming unit-megakaryocyte), then the erythroid progenitors (burst-forming unit-erythroid), and finally the granulo- monocyte progenitors (colony-forming unit-granulocyte-macrophage) whose proliferative activity was stimulated at the higher doses of IL-3. Only a slight increase in the proliferative activity of myeloblasts, promyelocytes, and myelocytes was observed, whereas the activity of erythroblasts was unchanged. The priming effect was such that BM progenitors, purified from patients treated with IL-3, produced more colonies in vitro in the presence of granulocyte colony-stimulating factor (G-CSF; granulocyte colonies), IL-5 (eosinophil colonies), and granulocyte-macrophage CSF (GM-CSF; predominantly eosinophil colonies). These data indicate that even in vivo IL-3 acts essentially as a primer for the action of other cytokines. Therefore, optimum stimulus of myelopoiesis will require either endogenous or exogenous late-acting cytokines such as G-CSF, erythropoietin, GM-CSF, and IL-6 for achieving fully mature cells in peripheral blood. If exogenous cytokines are used with IL-3, it is likely that G-CSF will yield more neutrophils, whereas GM-CSF may enhance eosinophils, monocytes, and neutrophils. Attention to the clinical relevance of each cell type will be necessary and should determine the selection of the combination of cytokines.

Interleukin-3 in vivo: kinetic of response of target cells

Human recombinant interleukin-3 (IL-3; Sandoz AG, Basel, Switzerland) was administered for 7 days to patients with neoplastic disease and normal hematopoiesis. The purpose of the study was to assess IL-3 toxicity, to identify target cells, to define their kinetics of response at different dose levels, and to determine if IL-3 in vivo increased the sensitivity of bone marrow (BM) progenitors to the action of other hematopoietic growth factors. A total of 21 patients entered the study; the dosage ranged from 0.25 to 10 micrograms/kg/d. The effect on peripheral blood cells during treatment showed no significant changes in the number of platelets, erythrocytes, neutrophils, or lymphocytes (and their subsets). A mild monocytosis and basophilia occurred. Eosinopenia, present in the first hours of treatment, was followed by a dose-and time-dependent eosinophilia. IL-3 treatment affected BM cell proliferation by increasing the percentage of BM progenitors engaged in the S-phase of the cell cycle. The effect was dose dependent, with the various progenitors showing different degrees of sensitivity. The most sensitive progenitors were the megakaryocyte progenitors (colony-forming unit-megakaryocyte), then the erythroid progenitors (burst-forming unit-erythroid), and finally the granulo- monocyte progenitors (colony-forming unit-granulocyte-macrophage) whose proliferative activity was stimulated at the higher doses of IL-3. Only a slight increase in the proliferative activity of myeloblasts, promyelocytes, and myelocytes was observed, whereas the activity of erythroblasts was unchanged. The priming effect was such that BM progenitors, purified from patients treated with IL-3, produced more colonies in vitro in the presence of granulocyte colony-stimulating factor (G-CSF; granulocyte colonies), IL-5 (eosinophil colonies), and granulocyte-macrophage CSF (GM-CSF; predominantly eosinophil colonies). These data indicate that even in vivo IL-3 acts essentially as a primer for the action of other cytokines. Therefore, optimum stimulus of myelopoiesis will require either endogenous or exogenous late-acting cytokines such as G-CSF, erythropoietin, GM-CSF, and IL-6 for achieving fully mature cells in peripheral blood. If exogenous cytokines are used with IL-3, it is likely that G-CSF will yield more neutrophils, whereas GM-CSF may enhance eosinophils, monocytes, and neutrophils. Attention to the clinical relevance of each cell type will be necessary and should determine the selection of the combination of cytokines.

Leukemia inhibitory factor/human interleukin for DA cells: a growth factor that stimulates the in vitro development of multipotential human hematopoietic progenitors

We investigated the in vitro hematopoietic stimulatory activity of leukemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) on bone marrow progenitor populations in 17 normal individuals. In serum-free cultures LIF/HILDA did not induce colony growth. In serum containing media, LIF/HILDA stimulated the growth of colony forming unit (CFU)-MIX and CFU-EO in a dose-dependent fashion and resulted in an increased CFU-MIX and burst forming unit-erythrocytes (BFU-E) colony size. Similar stimulatory effects were seen on a highly purified hematopoietic progenitor population obtained after immunomagnetic depletion of mature myeloid precursors and lymphoid cells. Addition of LIF/HILDA to cultures containing maximally stimulatory concentrations of recombinant human interleukin-3 (rhuIL3), rhuIL3 + rhuIL6, or rhu granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) in serum containing media significantly increased the number of CFU-MIX and eosinophil colonies and increased size and cluster number of CFU-MIX and BFU-E. Depletion of accessory T lymphocytes or monocytes from bone marrow progenitors did not alter the response of hematopoietic precursors to LIF/HILDA. A similar increased colony growth was seen when LIF/HILDA was added to cultures of positively selected CD34/HLA- DR+ or CD34+/HLA-DR- bone marrow hematopoietic progenitor cells stimulated with maximally stimulatory concentrations of rhuIL3 + rhuIL6. LIF/HILDA is a novel cytokine capable of stimulating growth and proliferation of multi-lineage, erythroid, and eosinophil colonies in the presence of serum. LIF/HILDA exerts its activity by direct interaction with highly purified immature bone marrow progenitor cells, has an additive effect when used with other cytokines known to stimulate primitive hematopoietic precursors, and does not require accessory cells.

Leukemia inhibitory factor/human interleukin for DA cells: a growth factor that stimulates the in vitro development of multipotential human hematopoietic progenitors

We investigated the in vitro hematopoietic stimulatory activity of leukemia inhibitory factor/human interleukin for DA cells (LIF/HILDA) on bone marrow progenitor populations in 17 normal individuals. In serum-free cultures LIF/HILDA did not induce colony growth. In serum containing media, LIF/HILDA stimulated the growth of colony forming unit (CFU)-MIX and CFU-EO in a dose-dependent fashion and resulted in an increased CFU-MIX and burst forming unit-erythrocytes (BFU-E) colony size. Similar stimulatory effects were seen on a highly purified hematopoietic progenitor population obtained after immunomagnetic depletion of mature myeloid precursors and lymphoid cells. Addition of LIF/HILDA to cultures containing maximally stimulatory concentrations of recombinant human interleukin-3 (rhuIL3), rhuIL3 + rhuIL6, or rhu granulocyte-macrophage colony-stimulating factor (rhu GM-CSF) in serum containing media significantly increased the number of CFU-MIX and eosinophil colonies and increased size and cluster number of CFU-MIX and BFU-E. Depletion of accessory T lymphocytes or monocytes from bone marrow progenitors did not alter the response of hematopoietic precursors to LIF/HILDA. A similar increased colony growth was seen when LIF/HILDA was added to cultures of positively selected CD34/HLA- DR+ or CD34+/HLA-DR- bone marrow hematopoietic progenitor cells stimulated with maximally stimulatory concentrations of rhuIL3 + rhuIL6. LIF/HILDA is a novel cytokine capable of stimulating growth and proliferation of multi-lineage, erythroid, and eosinophil colonies in the presence of serum. LIF/HILDA exerts its activity by direct interaction with highly purified immature bone marrow progenitor cells, has an additive effect when used with other cytokines known to stimulate primitive hematopoietic precursors, and does not require accessory cells.

In vivo administration of antibody to interleukin-5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice

Bone marrow of mice parasitized with Nippostrongylus brasiliensis showed increased numbers of eosinophils as early as 4 days after infection. By day 7, their bone marrow also contained elevated numbers of progenitors that form small eosinophil colonies (20 to 50 cells) in soft agar cultures supplemented with interleukin-5 (IL-5). However, when mice were infused with anti-IL-5 antibodies at the time of infection, the number of recognizable eosinophils present in bone marrow remained low and eventually dropped below normal levels. The antibody treatment also prevented increased generation of IL-5- responsive precursors capable of differentiating into mature eosinophils in liquid culture and inhibited the generation of progenitor cells capable of forming small eosinophil colonies or clusters in soft agar cultures. The results of these in vivo experiments directly show that IL-5 is an essential regulatory molecule required for the bone marrow-dependent phase of a parasite-induced eosinophilia.

In vivo administration of antibody to interleukin-5 inhibits increased generation of eosinophils and their progenitors in bone marrow of parasitized mice

Bone marrow of mice parasitized with Nippostrongylus brasiliensis showed increased numbers of eosinophils as early as 4 days after infection. By day 7, their bone marrow also contained elevated numbers of progenitors that form small eosinophil colonies (20 to 50 cells) in soft agar cultures supplemented with interleukin-5 (IL-5). However, when mice were infused with anti-IL-5 antibodies at the time of infection, the number of recognizable eosinophils present in bone marrow remained low and eventually dropped below normal levels. The antibody treatment also prevented increased generation of IL-5- responsive precursors capable of differentiating into mature eosinophils in liquid culture and inhibited the generation of progenitor cells capable of forming small eosinophil colonies or clusters in soft agar cultures. The results of these in vivo experiments directly show that IL-5 is an essential regulatory molecule required for the bone marrow-dependent phase of a parasite-induced eosinophilia.