Peripheral Blood Lineages Research Articles

Haemophagocytic lymphohistiocytosis: a case series from Mumbai

A retrospective review of ten patients (8 girls, 2 boys) admitted over a 9-month period with haemophagocytic lymphohistiocytosis (HLH) is presented. Presenting features included fever and hepatosplenomegaly (10), bleeding manifestations (7), lymphadenopathy (4), skin rash (4), shock (4), jaundice (3), CNS disorder (3), renal failure (2) and arthritis (2). Three infants had familial HLH (FHL) while the other seven patients had acquired (secondary) HLH. Two patients with FHL had very low perforin levels (0 and 0.05%). There was secondary HLH owing to systemic onset juvenile idiopathic arthritis in two patients, and one each had anaplastic large cell lymphoma, measles with pneumonia, disseminated tuberculosis, dengue hemorrhagic fever and lymphoproliferative disorder. Cytopenia affecting two or three lineages in peripheral blood was present in all while haemophagocytosis in bone marrow was documented in nine patients .Other important laboratory parameters were raised ferritin (9), raised LDH (9), hypertriglyceridaemia (7) and hypofibrinogenaemia (5). The patients were treated according to the HLH2004 protocol. Diagnosis of HLH should be considered early in patients presenting with unremitting fever, hepatosplenomegaly and cytopenias as without appropriate treatment HLH is usually fatal.

RhoA GTPase Is Dispensable for Hematopoietic Stem Cell Maintenance but Essential for Multipotent Progenitor and Lower Hierarchical Hematopoietic Differentiation.

AbstractAbstract 2618Rho family small GTPases are critical regulators of multiple functions of blood cells, including adhesion, migration, proliferation, survival and gene expression. Elucidation of their roles in hematopoiesis could be fundamental for understanding the mechanisms of various blood diseases and improving therapeutic outcomes for hematopoietic abnormalities. The Rho GTPases Rac1, Rac2 and Cdc42 have been found essential in maintaining hematopoietic stem cell (HSC) niche residency and regulating myelopoiesis and lymphopoiesis in previous mouse gene targeting studies. Using a dominant-negative mutant overexpression approach, an earlier study showed that suppression of RhoA activity enhanced HSC proliferation and engraftment potential; however, the bone fide role of RhoA in blood development remains unknown given limitations of such a mutant overexpression approach in specificity, dosage effect, and physiological relevance. Here, we stringently define the function of RhoA in HSC maintenance and hematopoiesis using an interferon inducible RhoA conditional knockout mouse model (Mx-cre+; RhoAloxp/loxp). Systematic deletion of RhoA caused lethality of the mice 7 days post polyIC induction due to hematopoietic failure that was accompanied by drastically decreased bone marrow (BM) cellularity (to ∼1/3 of wild type controls), a loss of splenocytes, and a significant reduction of cell counts of most cell lineages in peripheral blood, suggesting RhoA is required for multiple blood cell lineage differentiation and production. Syngenic transplant experiments yielded similar results, demonstrating that these effects are intrinsic to the hematopoietic compartment. The observed cytopenia resulting from RhoA loss was associated with the exhaustion of BM phenotypic HSPC (Lin−Sca1+c-kit+, LSK) and the hierarchical progenitor cells (Lin−c-kit+, LK) in number and frequency, a complete loss of colony forming activities, and a total engraftment failure. In addition, BrdU chase labeling and Annexin V/7-AAD staining revealed that RhoA deletion caused a transient increase of proliferation (1.7 fold increase in S phase) and reduction of survival (16.8 fold reduction in Annexin V− 7-AAD−) of remaining LSK in the BM. These results indicate that RhoA plays an indispensible, cell autonomous role in HSPC maintenance and hematopoiesis. In a competitive transplantation model where Mx-cre+;RhoAloxp/loxp or Mx-cre−;RhoAloxp/loxp CD45.2+ BM cells and WT CD45.1+ competitor cells were transplanted at 1:1 ratio into syngenic CD45.1+ recipients prior to polyIC induction, RhoA deletion also caused a complete extinction of donor derived (CD45.2+) Mac1+Gr1+ myeloid cells, B220+ B cells, as well as CD3+ T cells, in the peripheral. Interestingly, distinct from these more differentiated lineages, BM CD45.2+ LSK population was only marginally affected (88.9 % of pre-polyIC induction level) while the CD45.2+ LK cells and later hierarchical lineages were rapidly eliminated after RhoA deletion in this competitive transplant model. This was associated with increased apoptosis in CD45.2+ RhoA−/− LK, but not LSK, cells, suggesting a specific requirement of RhoA in the myeloid progenitor cell survival. Further, the CD45.2+ RhoA−/− LSK and LSKCD150+ cell populations, not differentiated donor-derived progenitors, from the primary competitive transplant BM, were able to be maintained in secondary transplant recipients 5 months post-transplantation, indicating that RhoA serves as a key regulator at an early progenitor differentiation step. Interestingly, RhoA deletion did not affect lin− cell p-MLC and p-cofilin contents and in vitro expansion in response to SCF stimulation, suggesting that RhoA is not required for actomyosin signaling nor SCF induced proliferation. Taken together, our results implicate RhoA as a unique and essential regulator of multipotent progenitor differentiation and survival that controls multi-lineage hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Suicide Gene (HSVtk) Ablation of Hematopoietic Stem Cells and Their Progeny In the Rhesus Macaque Model: An Approach to Deletion of Dangerous Clones.

AbstractAbstract 3759For genetic blood diseases, such as primary immunodeficiencies, gene therapy targeted to hematopoietic stem cells (HSCs) is a feasible and now proven effective therapeutic option for patients who lack a histocompatible HSC. However, the risk of adverse outcomes resulting from insertional oncogenesis is a major concern. We are investigating whether inclusion of the herpes simplex virus thymidine kinase (HSVtk) gene into integrating vectors into rhesus macaque HSCs confers ganciclovir (GCV) sensitivity allowing ablation of vector-containing cells from the blood and other hematopoietic compartments, as an approach to increasing safety of gene therapy procedures. HSVtk suicide genes have been studied in detail in transduced mature T cells, but never in stem and progenitor cells. We infused autologous CD34+ cells transduced ex vivo with gammaretrovirus vectors encoding the HSVtk as suicide gene along with marker genes into 4 rhesus macaques, following myeloablative irradiation. In the first animal, a vector consisting of the MND backbone driving the sr39 high affinity tk mutant, and IRES and a truncated NGFR marker gene was used. A stable marking level of 5% NGFR+ circulating cells was observed for 6 months following transplantation, confirmed by q-PCR. The drug GCV was infused at 5 mg/Kg BID for 21 days. This animal had complete elimination of vector-containing cells in all peripheral blood lineages as assessed by flow cytometry and qPCR, and remains negative now 4 months after GCV discontinuation. Three additional animals were transplanted with autologous CD34+ cells transduced with a vector containing a standard HSVtk gene and GFP as a marker. These animals had lower stable marking levels of approximately 1% at 4 months post-transplant, and after 21 days of GCV, had a clear decrease in the level of GFP+ cells, but not complete ablation, likely due to lower drug-sensitivity of the tk protein expressed by this vector. Cells with a lower level of GFP expression were not eliminated, supporting this hypothesis. Additional animals receiving cells transduced with the sr39 tk retroviral vector and with a lentiviral vector containing a codon-optimized HSVtk are in progress. These data suggest that inclusion of a suicide gene in integrating vectors may be an effective way to address genotoxicity concerns, should clonal outgrowth occur, and increase safety of HSC-targeted gene therapy. Disclosures:No relevant conflicts of interest to declare.

Flow Cytometric Identification of CD93 Expression on Naive T Lymphocytes (CD4+CD45RA+ Cells) in Human Neonatal Umbilical Cord Blood

Human CD93 has a molecular weight of about 100kDa and is selectively expressed by myeloid cell lineages in peripheral blood (PB) mononuclear cells. Although CD93 was initially identified as a receptor for complement component 1, subcomponent q phagocytosis (C1qRp) involved in the C1q-mediated enhancement of the phagocytosis of various antigens, several recent studies have reported that CD93 is not a receptor for the C1q-mediated enhancement of phagocytosis. The expression patterns of CD93 have been previously investigated in PB mononuclear cells (lymphocytes, monocytes, and granulocytes) from adult PB and neonatal umbilical cord blood (UCB), and the expression of CD93 was not found on lymphocytes from either normal adult PB or neonatal UCB. However, the detection of CD93 expression in neonatal UCB using CD93 monoclonal antibodies (mAbs) that recognize different antigenic epitopes remains poorly understood. In this study, we examined the expression of CD93 on lymphocytes, monocytes, and granulocytes from neonatal UCB using four different types of CD93 mAb detection probes, mNI-11, R139, R3, and X-2, using flow cytometric and western blot analyses. We found that CD93, as defined using all four mAbs, was expressed on monocytes and granulocytes in PB mononuclear cells from adult PB and neonatal UCB. On the other hand, we observed for the first time that the expression of CD93 on lymphocytes in neonatal UCB can only be detected using the mNI-11 mAb, established in our laboratory, and not with commercially available CD93 mAbs (R139, R3, and X-2). However, CD93 expression on lymphocytes from normal adults was not detected using any of the four CD93 mAbs. Two-color flow cytometric analyses showed that the CD93 recognized by mNI-11 mAb was expressed on CD3(+) T lymphocytes (mainly CD4(+) helper T lymphocytes), but not on CD19(+) B lymphocytes or on CD8(+) suppressor/cytotoxic T lymphocytes from neonatal UCB. In addition, CD93 was expressed on CD45RA(+) (naive antigen) lymphocytes from neonatal UCB, but not on CD45RO(+) (memory antigen) lymphocytes from neonatal UCB or on CD45RA(+) and CD45RO(+) lymphocytes from normal adult PB. Three-color flow cytometric analysis showed that CD93 was co-expressed on naive T lymphocytes (CD4(+)CD45RA(+)) from neonatal UCB. In a western blot analysis, the CD93 mAb (mNI-11) immunoprecipitated at a molecular weight of 98kDa, identified as a CD93 molecule, in the CD4(+)CD45RA(+) cells from neonatal UCB but not from adult PB, similar to the results in the human monocyte-like cell line U937 (human CD93-positive cells). Taken together, these results provide the first direct evidence of a novel/naive cell population (CD4(+)CD45RA(+)CD93(+)) in neonatal UCB that may have an important role in cell biology, transplantation, and immature/mature immune responses.

Hemophagocytic Lymphohistiocytosis Secondary to Ehrlichia chaffeensis Infection: A Case Report

Hemophagocytic lymphohistiocytosis (HLH) is a rare but potentially life-threatening condition that is characterized by fever, splenomegaly, and cytopenia in 2 or more peripheral blood lineages, hypertriglyceridemia, hypofibrinogenemia, and hemophagocytosis. HLH may be primary or may be triggered by numerous etiologies, including infections. Identification of underlying etiology of HLH is important as proper treatment can completely resolve the disease process. We present a patient whose clinical presentation fulfilled the diagnostic criteria for HLH but whose illness was caused by infection with Ehrlichia chaffeensis, emphasizing the need to explore all possible etiologies during evaluation of patients presenting with illnesses consistent with HLH.

Combined effect of IL‐17 and blockade of nitric oxide biosynthesis on haematopoiesis in mice

The study was undertaken to extend our investigation concerning both the in vivo activity of interleukin (IL)-17 and the specific role of nitric oxide (NO) in IL-17-induced effects in the process of haematopoiesis. CBA mice were simultaneously treated with IL-17 and/or nitric oxide synthase (NOS) inhibitor, l-NAME, for 5 days and changes within various haematopoietic cell lineages in bone marrow, spleen and peripheral blood were analysed. Findings showed that administration of both IL-17 and l-NAME stimulated increase in net haematopoiesis in normal mice. IL-17-enhanced myelopoiesis was characterized by stimulation of both femoral and splenic haematopoietic progenitor cells and morphologically recognizable granulocytes. Additionally, IL-17 induced alterations in the frequency of erythroid progenitor cells in both bone marrow and spleen, accompanied with their mobilization to the peripheral blood. As a consequence of these changes in the erythroid cell compartments, significant reticulocytosis was observed, which evidenced that in IL-17-treated mice effective erythropoiesis occurred. Exposure of mice to NOS inhibitor also increased the number of both granulocyte-macrophage and erythroid progenitors in bone marrow and spleens, and these alterations were followed by the mobilization of erythroid progenitors and elevated content of reticulocytes in peripheral blood. The specific role of NO in IL-17-induced haematopoiesis was demonstrated only in the IL-17-reducing effect on bone marrow late stage erythroid progenitors, CFU-E. The results demonstrated the involvement of both IL-17 and NO in the regulation of haematopoietic cell activity in various haematopoietic compartments. They further suggest that IL-17 effects are differentially mediated depending on the haematopoietic microenvironments.

Vascularized Bone Grafts within Composite Tissue Allotransplants Can Autocreate Tolerance through Mixed Chimerism with Partial Myeloablative Conditioning: An Experimental Study in Rats

Certain composite tissue allotransplants contain vascularized bone grafts (e.g., hand/forearm composite tissue allotransplants). The authors investigated the role of the vascularized bone graft within the hind-limb osteomyocutaneous flap in inducing tolerance. Brown Norway and Lewis rats were used as composite tissue allotransplant donors and recipients, respectively. Experimental groups were as follows: group I, syngeneic controls (Lewis to Lewis); group II, allogeneic controls (both received no pretransplantation total body irradiation); and groups III, IV, and V, which received 5 mg of antilymphocyte globulin administered intraperitoneally (on days -1 and 10), 1 mg/kg of tacrolimus administered intraperitoneally (on days 0 to 10), and total body irradiation (600, 400, and 200 cGy, respectively, 1 day before composite tissue allotransplantation). Each Lewis rat in groups II, III, IV, and V received a composite tissue allotransplant on day 0 in the form of a Brown Norway hind-limb osteomyocutaneous flap. Different donor hematopoietic cell lineages in recipients' peripheral blood were assessed by flow cytometry on posttransplantation days 15, 30, 60, 90, 120, and 150. Secondary Brown Norway skin grafts in recipients with composite tissue allotransplant acceptance were performed at 150 days after transplantation. Allotransplanted hind-limb osteomyocutaneous flaps produced chimerism and donor multilineage hematopoietic cells in groups III, IV, and V; composite tissue allotransplant acceptance rates in these groups were 37.5, 16.7, and 0 percent, respectively. Graft-versus-host disease occurred in 62.5 percent of group III recipients. Recipients with acute graft-versus-host disease produced a higher percentage of donor T cells compared with composite tissue allotransplant-accepting recipients (p < 0.05). Secondary Brown Norway skin graft acceptance in composite tissue allotransplant-accepting recipients confirmed durable tolerance. Vascularized bone grafts contained within composite tissue allotransplants can autocreate chimerism and tolerance in rats with partial myeloablative tacrolimus-based conditioning.

Low-dose busulphan conditioning and neonatal stem cell transplantation preserves vision and restores hematopoiesis in severe murine osteopetrosis

Infantile malignant osteopetrosis is a fatal disease caused by lack of functional osteoclasts. In most of patients, TCIRG1, encoding a subunit of a proton pump essential for bone resorption, is mutated. Osteopetrosis leads to bone marrow failure and blindness due to optic nerve compression. Oc/oc mice have a deletion in Tcirg1 and die around 3 to 4 weeks, but can be rescued by neonatal stem cell transplantation (SCT) after irradiation conditioning. However, as irradiation of neonatal mice results in retinal degeneration, we wanted to investigate whether conditioning with busulphan prior to SCT can lead to preservation of vision and reversal of osteopetrosis in the oc/oc mouse model. Pregnant dams were conditioned with busulphan and their litters transplanted with 1 x 10(6) normal lineage-depleted bone marrow cells intravenously or intraperitoneally. Mice were followed in terms of survival and engraftment level, as well as with peripheral blood lineage analysis, bone and eye histopathology and a visual-tracking drum test to assess vision. Busulphan at 15 mg/kg was toxic to oc/oc mice. However, six of seven oc/oc mice conditioned with busulphan 7.5 mg/kg survived past the normal lifespan with 10% engraftment, correction of the skeletal phenotype, and normalization of peripheral blood lineages. Busulphan, in contrast to irradiation, did not have adverse effects on the retina as determined by histopathology, and 8 weeks after transplantation control and oc/oc mice retained their vision. Low-dose busulphan conditioning and neonatal SCT leads to prolonged survival of oc/oc mice, reverses osteopetrosis and prevents blindness even at low engraftment levels.

Clonal Hematopoiesis in Philadelphia Chromosome-Negative Bone Marrow Cells of Chronic Myeloid Leukemia Patients Receiving Tyrosine Kinase Inhibitors

Patients with chronic myeloid leukemia (CML) are experiencing prolonged survival due to successful therapy with tyrosine kinase inhibitors. However, some CML patients who have achieved longstanding remissions with these agents harbor clonal cytogenetic abnormalities in their Philadelphia chromosome negative (Ph-) bone marrow cells. Because CML patients in remission often have peripheral blood count abnormalities, including cytopenias, we investigated whether these patients may have developed myelodysplastic syndrome (MDS) within the Ph- cell population. Bone marrow samples from 26 CML patients who had achieved a major cytogenetic remission (MCyR) with tyrosine kinase inhibitor therapy between 2 and 15 years after diagnosis were evaluated; 6 patients had advanced disease prior to their last therapy, 20 were in chronic phase. At the time of evaluation, 2 of the patients were receiving imatinib, 23 dasatinib, and 1 PHA739358. At least one peripheral blood lineage was abnormal in 21 patients, of whom 7 had pancytopenia. Routine metaphase cytogenetics (MC) revealed a persistent clonal chromosomal abnormality in 10% of the Ph- metaphases in 5 patients (+8 in 2, −7 in 2, and 20q- in 1). We hypothesized that clonal hematopoiesis might exist in additional patients and applied single nucleotide array (SNP-A) based karyotyping and X-linked human androgen receptor (HUMARA) clonality assay to further delineate the nature of the hematopoietic defect in these patients. HUMARA was performed on bone marrow samples and germ-line DNA from peripheral blood T lymphocytes of the female patients. Clonality, as assessed by skewing of X-chromosome inactivation in bone marrow cells compared to germline control cells, could not be demonstrated in the12 female patients. SNP-A karyptyping using 250K Affymetrix SNP array confirmed the known cytogenetic abnormalities. Several microdeletions were found, but comparison with purified T lymphocytes demonstrated that these “lesions” represented germ line-encoded copy number variants. However, SNP-A karyotyping revealed the presence of uniparental disomy (UPD) involving chromosome 17(p12-pter) in bone marrow, but not germ line cells, from one male patient with normal karyotype by routine MC. In the context of secondary AML, del17p or UPD17 have been observed always in the presence of del7/q and 5q and were associated with poor prognosis. However, in our patient UPD17 occurred as a sole defect. Because in our studies in AML, UPD of chromosome 17p was found in association with p53 mutations, genomic sequencing of this gene was performed. A 5 bp deletion destroying the splice acceptor region of exon 6 was identified in bone marrow cells from this patient. Alternative splicing leading to loss of exon 6 was predicted to result in a frame shift and premature introduction of a stop codon. These methods revealed clonal hematopoiesis in the Ph- bone marrow cells of 6/26 patients with longstanding CML in remission from tyrosine kinase inhibitors and persistent peripheral blood abnormalities. The approaches used here probably underestimate the frequency of this condition, as oligoclonal populations may be present in numbers below the limit of assay sensitivity. The Ph- clonal bone marrow populations have cytogenetic and molecular features in common with MDS. After a median follow up of two years, one patient with monosomy 7 developed acute myeloid leukemia, but longer follow up will be required to determine the natural history of the Ph- clonal disorders.

Impaired Function of Hematopoietic Stem Cells with Abrogated Hypoxia Induced Expression of Vascular Endothelial Growth Factor

Hypoxia has been suggested to be a characteristic of the bone marrow (BM) microenvironment where hematopoietic stem cells (HSC) reside. The major molecular response to hypoxia is the activation of hypoxia inducible factors (HIF). These transcription factors activate gene expression by binding to hypoxia response elements (HRE) in the promoter region of target genes, such as vascular endothelial growth factor (VEGF). It has been demonstrated that VEGF is indispensable for HSC survival in the adult and that VEGF mediated HSC survival is cell intrinsic. We are therefore investigating the role of hypoxia-regulated VEGF expression in HSC survival. For our studies we use the transgenic mouse model VEGFδ/δ. In this model, the HRE in the VEGF promoter is mutated which prevents HIF binding. Thus, hypoxia-induced VEGF transcriptional activation is abrogated while basal VEGF expression remains unaffected. Steady state hematopoiesis was not affected by the mutation since distribution between the B-cell, T-cell and myeloid lineages in both peripheral blood and BM of mice homozygous for the mutation was comparable to wild type animals. Furthermore, we could not detect any differences in the frequency of lineage-, Sca1+, c-kit+, CD34- cells in the BM between VEGFδ/δ and wild type mice. However, when assessing HSC functional properties in transplantation assays, BM from mice homozygous for the mutation was demonstrated to have dramatically reduced HSC function. Only around 60 % of lethally irradiated recipients that were transplanted with VEGFδ/δ BM could be rescued while recipients receiving wild type BM survived as expected. In addition, during early hematopoietic reconstitution, a skewing towards myeloid differentiation at the expense of B-cell differentiation could be observed in peripheral blood of recipients reconstituted with VEGFδ/δ BM. Moreover, in a competitive repopulation assay, VEGFδ/δ BM was shown to have a severe competitive disadvantage compared to wild type BM. Our findings indicate an important role for hypoxia related VEGF expression in reconstituting HSCs and suggest that low oxygen levels might be an important characteristic of the HSC niche.

Genetic influence on immune phenotype revealed strain-specific variations in peripheral blood lineages

Inbred mouse strains are routinely used as genetically defined animal models for studying a wide assortment of biological and pathological processes, including immune system function. However, no studies have presented large-scale data on the immune cell populations among the inbred strains in physiological conditions. Here we present a systematic, quantitative analysis of peripheral blood cell phenotypes of 30 mouse strains assessed by flow cytometry. This cohort of mice represents a wide range of genetic origins and includes most of the strains used in genetic, physiological, and immunological studies. We evaluated the relative percentages of peripheral blood leukocyte subtypes (lymphocytes, granulocytes, and monocytes) and lymphocyte subpopulations (CD4+ T, CD8+ T, B220+ B, and natural killer cells) of mature (6-mo-old) mice. Our comprehensive study demonstrated: 1) marked differences in the relative proportions of blood cell populations among the strains at this age, 2) considerable variation of each immune trait with more than twofold difference between strains with the highest and the lowest trait values, and 3) haplotype analysis revealed a strong correlation between eosinophil percentage and a single region on chromosome 14 containing two candidate genes. The strain differences described here provide important information for researchers applying immunophenotyping of peripheral blood in immunological and genetic studies. The data from this study are available as part of the Mouse Phenome Database at http://www.jax.org/phenome.

Heterozygous Kit Mutants with Little or No Apparent Anemia Exhibit Large Defects in Overall Hematopoietic Stem Cell Function

The evolutionarily conserved Kit receptor is vital for function of hematopoietic stem cells (HSC). Kit(W-41) (W-41) and Kit(W-42) (W-42) are single residue changes in the KIT intracellular phosphotransferase domain, while Kit(W-v) (W-v) is a single residue change in the ATP binding domain. This study tests how each mutation affects HSC function. Cells in mutant and C57BL/6J(+/+) blood and marrow were compared. Overall HSC function was measured by competitive repopulation. Functions of specific progenitor populations were tested with stage-specific competitive repopulation and standard colony-forming unit assays. Bone marrow cells from these Kit mutants are severely defective at reconstituting peripheral blood lineages and bone marrow of irradiated recipients, when compared to +/+ control marrow. These defects increased with time. Marrow from W-41/+ and W-v/+ functions similarly but better than marrow from W-41/W-41 and W-42/+, to repopulate the erythroid and lymphoid lineages. Long-term (LT) and short-term (ST) HSC from W-v/+, W-41/W-41, and W-42/+ are more defective at reconstituting bone marrow than LT- and ST-HSC from W-41/+ and +/+. Common myeloid progenitor (CMP) cells from W-42/+ and W-41/W-41 are more defective at producing spleen colonies than CMP from W-v/+ and W-41/+. Heterozygous Kit mutants with little or no apparent anemia exhibit surprisingly large defects in overall HSC function. Multiplying the fractional defects in LT-HSC, ST-HSC, and CMP can account for overall effects of W-v/+, but does not completely account for the defects observed with W-41/+, W-42/+, and W-41/W-41.

An IL-7 splicing-defect lymphopenia mouse model revealed by genome-wide mutagenesis

Homeostasis of the hematopoietic system is tightly regulated by an array of cytokines that control proliferation, differentiation and apoptosis of various cell lineages. To identify genes that are essential for hematopoietic homeostasis, we screened C57BL/6 mice that had been genome-wide mutagenized by N-ethyl-N-nitrosourea (ENU) to produce altered blood cell composition. We identified a mutant mouse line with a drastic reduction in the number of T and B cell lineages in lymphatic tissues and peripheral blood, as well as severe atrophy of the thymus and lymph nodes. Genotyping with a genome-wide single nucleotide polymorphism (SNP) marker set mapped the mutant phenotype to chromosome 3A and subsequent direct DNA sequencing revealed a G-to-A point mutation in the splicing donor site of the third exon of the candidate gene for IL-7, a lymphocyte survival cytokine. Such mutation resulted in skipping of exon 3 and production of an internally truncated IL-7 (DeltaE3-IL7). Furthermore, using recombinant proteins produced in a baculoviral system, we demonstrated that DeltaE3-IL7 had no detectable anti-apoptotic activity even at a dose that was 30 times more than that required for a wild-type protein to manifest a full activity in a naïve T cell survival assay. Our data suggest that this mutant mouse line provides an alternative animal model for the study of severe combined immunodeficiency (SCID) syndrome in humans.

WP1066 Inhibits Growth of Human Cells Carrying the JAK2 V617F Mutation.

Myeloproliferative disorders (MPDs) are characterized by proliferation of one or more myeloid cell lineages in bone marrow and peripheral blood, with relatively preserved differentiation. Recent discovery of a dominant gain-of-function mutation in the Janus kinase 2 (JAK2) gene in patients with MPDs, involving the substitution of valine for phenylalanine at position 617 of the JAK2 protein (JAK2 V617F), represents the first acquired somatic mutation in hematopoietic stem cells described in these disorders. This discovery has opened new avenues for the development of targeted therapies for MPDs. WP1066 is a small molecule, a member of a novel class of anticancer agents whose development was based upon the backbone of AG490, a tyrphostin with activity against JAK2 V617F-expressing cell lines but limited in vivo activity. We investigated the inhibitory activity of the WP1066 against the JAK2 V617F-mutant expressing erythroid leukemia HEL cell line and peripheral blood mononuclear cells from patients with polycythemia vera (PV). WP1066 significantly inhibited the phosphorylation of JAK2 and downstream signal transduction proteins STAT3, STAT5, and ERK1/2 in a dose- and time-dependent manner. It induced a time- and dose-dependent antiproliferative and pro-apoptotic effects (activation of caspase 3, release of cytochrome c, and cleavage of PARP) in the JAK2 V617F-bearing HEL cell line in the low micromolar range. Pretreatment of cells with pan-caspase inhibitor Z-VAD abolished WP1066-induced apoptosis. The expression of apoptosis related proteins bcl-2, bax, and XIAP, however, was not changed. More important, WP1066 was effective in inhibiting cell growth in clonogenic assays of mononuclear cells harboring the JAK2 V617F mutation obtained from peripheral blood of patients with PV. We conclude that WP1066 is active both in vitro and ex vivo against cells carrying the JAK2 V617F mutation and represents a solid candidate for the treatment of JAK2 V617V-expressing MPDs.

Quantitative Monitoring of Lineage-Specific Chimerism after Dual Hematopoietic Stem Cell Transplantation (Umbilical Cord Blood with Co-Infusion of Third Party Donor CD34+ Cells).

Background: Allogeneic umbilical cord blood (UCB) stem cell transplantation (SCT) shows some advantages (HLA matching requirements or availability) respect to SCT using other sources of matched unrelated donor (MUD) stem cells. However, it is correlated with slower engraftment, increasing risk of infections and early mortality. It has been recently shown that co-infusion of third party donor (TPD) CD34+ cells (dual SCT) is useful to speed up engraftment.Objective: To evaluate the usefulness of lineage-specific chimerism quantification in the management of this transplant setting.Patients and methods: 8 dual SCT (Tables 1, 2) in 7 patients (1 CML-BC, 2 AML-M2, 1 AML-M4, 1 ALL-Ph+, 1 biphen. ALL, 1 NHL). Chimerism was analyzed by STR-PCR (AmpFlSTR SGM Plus, Applied Biosystems; sensitivity 1%) and quantitative real-time PCR (qrt-PCR) of null alleles and insertion/deletion polymorphisms (Light Cycler, Roche; sensitivity 0,01%). Peripheral blood (PB) and leukocyte lineages (T cells, CD3+, and myeloid cells, CD15+), isolated by positive selection using automated immunomagnetic technology (AutoMACS, Miltenyi Biotec), were analyzed weekly. Bone marrow (BM) was analyzed at days +30, +100, +180 and +365).Results: 7/8 cases showed initially a high proportion of TPD cells in PB which were progressively replaced by UCB cells. UCB complete chimerism (UCB-CC, absence of recipient or TPD cells even in qrt-PCR assays) was acquired in a median of 22.5 days (range 18–39). In one patient, fully HLA-mismatched with the TPD, no TPD cells were observed after dual SCT. 4/8 cases showed recipient cells in PB after dual SCT during a median period of 12 days (range 4–18 days). In 3/8 cases, recipient cells were found after CC had been acquired, which allowed early diagnosis of 1 graft rejection and 2 relapses. T cells (CD3+) are mainly of UCB origin early after dual SCT and reach UCB-CC a median of 7 days (range 0–21) before PB. However, myeloid cells (CD15+) derive primarily from the TPD and reach CC together with PB. TPD cellularity favoured early engraftment (before UCB-CC took place) in 4 cases. In this context, only one important infectious complication (hepatosplenic tuberculosis) was observed, which resolved with the appropriate treatment.Conclusions: Lineage-specific chimerism quantification allowed a close monitoring of the dynamics of engraftment of cells of both donors which is of key importance in this SCT setting. Moreover, lineage-specific chimerism analysis was useful to diagnose one graft rejection and two relapses (the patient with NHL showed a ganglionar relapse in CC). [Display omitted] [Display omitted]

Combined Sustained Delivery of Basic Fibroblast Growth Factor and Administration of Granulocyte Colony-Stimulating Factor:Synergistic Effect on Angiogenesis in Mouse Ischemic Limbs

To investigate whether the efficacy of a single angiogenic therapy (sustained delivery of basic fibroblast growth factor [bFGF] or administration of granulocyte colony-stimulating factor [G-CSF]) can be enhanced further by combining the therapies. One day after surgical induction of hind-limb ischemia, groups of 6 mice were randomized to receive either no treatment, sustained delivery (SD) of bFGF, endothelial progenitor cell (EPC) mobilization with G-CSF administration, or a combination of bFGF SD + G-CSF administration. G-CSF administration increased significantly (p < 0.05) the number of EPC lineages (CD34 + /AC133 + cells) in both peripheral blood and bone marrow compared to no G-CSF administration. The bFGF SD and G-CSF administration individually increased the capillary and arteriole densities significantly versus no treatment (capillary density: 659 +/- 48/mm2 and 385 +/- 59/mm2, respectively, versus 280 +/- 28/mm2; p < 0.05; arteriole density: 34 +/- 9/mm2 and 41 +/- 6/mm2, respectively, versus 15 +/- 2/mm2; p<0.05). Importantly, bFGF SD + G-CSF further increased the capillary and arteriole densities compared to either strategy alone (capillary density: 786 +/- 40/mm2 versus 659 +/- 48/mm2 and 385 +/- 59/mm2, respectively, p < 0.05; arteriole density: 55 +/- 10/mm2 versus 34 +/- 9/mm2 and 41 +/- 6/mm2, respectively, p < 0.05). This study demonstrates that the combined therapy of sustained bFGF delivery and G-CSF administration potentiates the angiogenic efficacy of either single therapy in mouse hind-limb ischemia models.

Correction of Murine Hemophilia A by Hematopoietic Stem Cell Gene Therapy.

A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally-delivered FVIII transgene offers the possibility to permanently correct the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based oncoretroviral vector encoding a secretion-enhanced B domain-deleted human FVIII transgene linked to a downstream EGFP reporter gene into immunocompetent hemophilia A mice (FVIII exon 16 knockout mice on a C57BL/6 background) that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy) or a nonmyelablative preparative regimen involving busulfan (two intraperitoneal doses of 10 mg/kg). Both groups of irradiated mice were transplanted with 2 × 106 sorted EGFP+ bone marrow cells. At 26 weeks, 48 ± 24% (n = 10) and 18 ± 11% (n = 12) EGFP+ nucleated peripheral blood cells were detected in mice conditioned with 800 and 550 cGy irradiation, respectively. The busulfan-conditioned mice (n = 4) were transplanted with 15–20 × 106 transduced unsorted bone marrow cells. One mouse died at 4 weeks posttransplant due to an unknown cause. The reconstitution kinetics of the remaining three mice was very similar to the group of mice conditioned with 550 cGy irradiation (18 ± 7% of their nucleated peripheral blood cells were EGFP+ at 26 weeks posttransplant). Broad transcriptional activity of the vector was observed in cells belonging to both the myeloid and lymphoid lineages in peripheral blood, and in donor-derived cells residing within the bone marrow, spleen and thymus. Importantly, therapeutic levels of FVIII (42%, 18% and 11% of normal, respectively, by COATEST assay) were detected in the plasma of all recipients 22–26 weeks posttransplant. When the mice were subsequently challenged with high doses of recombinant human FVIII (up to eight intravenous injections of 5–10 IU of recombinant full-length human FVIII at weekly intervals) to investigate the durability of tolerance induction, only minimal levels of inhibitor antibodies were detected in a subset of the corrected animals (0.8 ± 0.6, 0.7 ± 0.5 and 3 ± 4 Bethesda units per ml) in contrast to the robust anti-FVIII inhibitor response seen following immunization of naive hemophilia A mice (98 ± 48 Bethesda units per ml; n = 11). Suppression of the immune response to human FVIII was specific, as gene-treated mice mounted a normal humoral immune reaction to an unrelated antigen, tetanus toxoid. While we make no claims as to the nature of the hyporesponsive states elicited in each case, the results obtained in the small cohort of busulfan-conditioned animals are particularly exciting since the experimental protocol more closely approximates a clinically-acceptable situation, both in terms of a mild conditioning regimen as well as the lack of a preselection step for transduced bone marrow cells. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.

Transcriptional regulation of hematopoietic stem cell development in zebrafish

The zebrafish (Danio rerio) is a well-established vertebrate model for studying hematopoiesis. The major advantages of this system include robust experimental techniques in both genetics and embryology, which have been utilized to model many aspects of human development and disease. Although much is known about the transcription factors involved in the terminal differentiation of peripheral blood lineages, little is known about the development and maintenance of the hematopoietic stem cell (HSC). This review will focus on the current knowledge of the transcriptional regulation of the HSC in the context of the zebrafish. Future studies using new technologies in the zebrafish model will enhance our understanding of the molecular networks regulating HSC pluripotency and differentiation.

706. Retrovirus Integration Site Distribution in Long-Term Gene-Corrected Hematopoiesis of the SCID-X1 Gene Therapy Clinical Trial

An amphotropic pseudotyped Simian Immunodeficiency Virus (SIV) based lentiviral vector developed previously efficiently transduces rhesus macaque cells (Blood 102: 989a, 2003) in contrast to HIV-based lentiviral vectors, which are blocked from optimal transduction of monkey cells due to species-specific factors. Due to current interest in using lentiviral vectors for gene therapy applications, this SIV vector system can be useful as a pre-clinical model for evaluation of safety and efficiency of such gene therapy protocols. Three animals previously reconstituted with transduced G-CSF+SCF mobilized peripheral blood CD34+ cells, exhibited genetically modified cells in multiple lineages. We have now extended the period of observation and these animals still have stable marking levels of 5–20% in different peripheral blood lineages at 6 to 9 months after transplant. The recent report of insertional mutagenesis within the LMO2 proto-oncogene following hematopoietic stem cell directed retroviral gene therapy prompted us to investigate the distribution pattern of the SIV vector genome in cells of the hematopoietic system. We used a modified LAM-PCR methodology to retrieve the insertion sites in circulating granulocytes and mononuclear cells of these animals at steady state, at least 6 months after transplant. The LAM-PCR generated products were sequenced. A sequence was considered a genuine insertion site if it contained the SIV-LTR and the linker sequences, and showed at least 90% identity to the NCBI build 34 of the human genome with a unique best hit in the BLAT ranking. So far we have sequenced a total of 241 insertion sites from these three animals (Table). 79 insertions sites were found to be in repetitive elements (Line, Sine, etc.) of the genome so the exact location of these insertion sites can not be ascertained. Of the remaining 162 insertion sites, 36 (22%) are located in non-coding regions of the genome and 126 (78%) are found to be located inside coding regions of the RefSeq set of genes, with 5 genes being independently interrupted twice. Four of the genes interrupted twice occurred in two different animals (EYA3: eyes absent 3; Scap1:Src family associated phosphoprotein 1; PACS1:phosphofurin acidic cluster sorting protein 1; and ITGAL:integrin alpha L precursor) and one occurred twice in the same animal (STK38:serine/threonine kinase 38). We are currently in the process of sequencing and analyzing additional insertion sites, and comparing the pattern to the over 400 insertion sites we have generated from rhesus macaques previously transplanted with CD34+ cells transduced with MLV-derived onco-retroviral vectors. These findings advance our understanding of lentiviral vector insertional mutagenesis risk in hematopoietic stem and progenitor cells of nonhuman primates, a highly relevant pre-clinical model, and ultimately will have significant impact on the design of lentiviral gene therapy trials.

Altered phenotype and reduced function of muscle-derived hematopoietic stem cells.

Skeletal muscle-derived cells have the potential to repopulate the major peripheral blood lineages of lethally irradiated mice and thus behave like hematopoietic stem cells (HSC). We have recently shown that muscle cells with HSC activity (ms-HSC) express CD45 and Sca-1, suggesting a hematopoietic origin. Here we sought to clarify contradictions in the literature regarding the phenotype of ms-HSC and precisely define the hematopoietic origin of these cells. Skeletal muscle-derived cells fractionated based on the expression of CD45 and c-kit and efflux of Hoechst 33342 and were examined for HSC activity in vivo. WBM HSC expressing beta-galactosidase were transplanted into lethally irradiated recipients, whose ms-HSC compartment was later analyzed for beta-galactosidase activity to determine if ms-HSC were derived from WBM HSC. Muscle-derived HSC fall exclusively in the c-kit(dim)CD45(pos) compartment of the muscle side population (msSP). Furthermore, the CD45(pos) msSP compartment of skeletal muscle is derived from WBM HSC. CD45(pos)c-kit(dim) msSP are about 22-fold less potent in HSC activity than WBM HSC cells in competitive repopulation assays and express low levels of c-kit relative to WBM HSC. In our transplantation experiments, WBM HSC gave rise to ms-HSC, suggesting that WBM HSC and ms-HSC likely represent the same stem cell population in distinct environments. However, these two related populations are both functionally distinct in their ability to repopulate the peripheral blood of irradiated mice and phenotypically distinct.