Transgenic Bone Marrow Research Articles

Rituximab Mediated Depletion of B Cells Following Transplant of Human CD20 Transgenic Mouse Bone Marrow Results in Prolonged B Cell Depletion and Augmented Anti-Tumor Immune Response.

We previously demonstrated rejection of murine tumors due to enhanced anti-tumor cytolytic and Th1 cytokine responses in mice lacking B cells (BCDM) compared to immunocompetent mice. We wished to compare immune and anti-tumor responses in BCDM to mice functionally depleted of B cells using an anti-human CD20 antibody (Rituximab). We evaluated Rituximab effects on the outgrowth of human CD20 transgenic mouse (huCD20 Tg) B cells following the transplant of huCD20Tg CD45.2+ bone marrow (BM) into sublethally irradiated congenic CD45.1+ mice. CD45.1 mice were irradiated with 9.5Gy at day -1, followed by infusion of 3x106 huCD20Tg BM cells on day 0, and by treatment with or without Rituximab at 400ug/mouse given once weekly i.v. for 4 weeks. Without Rituximab treatment, B cell percentages in the peripheral blood (PB) were approximately 46.4%±1.3; spleen, 77.8%±1.5; and peripheral lymph nodes (pLNs), 43%±1.5 at day 47. With Rituximab treatment, CD19+ cells in the PB, spleen and pLNs were 1.8%±0.4, 4.3%±1.3, and 0.8%±0.2 respectively at 47days post transplant. Higher percentages of CD3+ T cells in PB, spleen and pLNs (66.1%±0.5, 51.7%±1.1 and 43.0%±3.8) were noted in the Rituximab treated group compared to controls (19%±0.9, 10.3%±1.0 and 32.3%±1.7 respectively). The percentage of remaining B cells in PB declined progressively from 5.6% at day 13 to 1.8% at day 47 post transplant indicating that Rituximab depletion of huCD20+ transgenic B cells was progressive, durable and very effective. In contrast, CD3+ cells in PB increased from 11.2% (day 13) to 66.1 % (day 47) following Rituximab treatment. Interestingly reconstitution with endogenous murine B cells in huCD20 transplanted mice was minimal. The huCD20 Tg donor-derived T cells in the Rituximab treated group maintained a CD44low naive phenotype.The growth of MC38 tumors implanted 15 days following transplant was significantly slowed in the Rituximab treated group. Increased IFN-γ production and higher expression of the cytolytic marker CD107a by T cells was seen in the Rituximab treated group compared to controls. The combination of myeloablative irradiation, huCD20 transgenic bone marrow transfer and Rituximab depletion, was able to generate mice devoid of CD20+ B cells. Rituximab treated huCD20+ reconstituted B cell depleted mice have substantially higher percentages of T cells than control non-Rituximab treated mice, show sustained depression of B cell numbers and manifest an increased anti-tumor Th1 response. The huCD20 transplant model will be useful in studying effects of post-transplant B cell depletion on immune reconstitution and in the design of future clinical transplant and immunization strategies.

CREB Transgenic Mice Develop Myeloproliferative Disease/Myelodysplastic Syndrome after a Prolonged Latency.

The cyclic AMP Response Element Binding Protein, CREB, is a basic leucine zipper transcription factor that induces genes that regulate cell proliferation and survival. CREB is a downstream target of GM-CSF signaling pathways in myeloid cells, resulting in transactivation of critical target genes. We previously demonstrated that both CREB protein and mRNA levels are increased in the bone marrow cells of patients with AML at diagnosis. In contrast, CREB is expressed at low levels in normal bone marrow cells. To examine the expression of CREB in specific bone marrow and leukemia stem cell populations, we performed quantitative real-time PCR. AML blast cells were sorted and were shown to have increased CREB mRNA expression in CD34+CD33+ and CD34-CD33+ population compared to normal bone marrow progenitor cells. To understand the role of CREB in normal hematopoiesis and leukemogenesis, we created transgenic mice in which the myeloid specific hMRP8 promoter controls CREB expression. Within months, these mice developed increased monocytes and neutrophils in the peripheral blood. The increased cells in the peripheral blood and bone marrow were Gr1+Mac1+. We did not observe increases in other cell lineages using FACS analysis and the markers, B220, CD3, and Ter119. Bone marrow cells from CREB transgenic mice displayed increased colony size, greater numbers of colonies, and immortalization in colony replating assays with methylcellulose containing IL-3, IL-6, and Stem Cell Factor, compared to age-matched littermate controls. Bone marrow from CREB transgenic mice grew in the absence of cytokines, demonstrating factor-independent growth. Increased numbers of CFU-M was observed with CREB transgenic mouse bone marrow in colony assays with methylcellulose containing M-CSF. Although the mice did not develop acute leukemia, 7 out of 8 CREB transgenic mice compared to 0 out of 13 control mice developed enlarged spleens and myeloproliferative disease (MPD) after 12 months of age. Histology of the spleens showed destruction of the normal architecture with aberrant myeloid cells, suggestive of a myeloproliferative disease/myelodysplastic syndrome. Both the indolent course and phenotype of our mice were similar to Chronic Myelomonocytic Leukemia (CMML) observed in humans. Interestingly, myeloid cells from patients with CML in chronic phase also express higher levels of CREB. Studies are underway to characterize CREB expression in peripheral blood or bone marrow cells from patients with MPD/MDS, including CML, CMML, and transient myeloproliferative disease of Down Syndrome. We are also analyzing the spleens from CREB transgenic mice that develop MPD/MDS. Our results demonstrate that overexpression of CREB is sufficient to induce myeloid cell transformation to a preleukemic state in vivo. Therefore, the CREB transgenic mouse provides a useful model to test novel therapies to treat MPD/MDS.

Hematopoietic stem cell and marrow stromal cell for spinal cord injury in mice

We compared the effects of hematopoietic stem cell and marrow stromal cell transplantation for spinal cord injury in mice. From green fluorescent protein transgenic mouse bone marrow, lineage-negative, c-kit- and Sca-1-positive cells were sorted as hematopoietic stem cells and plastic-adherent cells were cultured as marrow stromal cells. One week after injury, hematopoietic stem cells or marrow stromal cells were injected into the lesioned site. Functional recovery was assessed and immunohistochemistry was performed. In the hematopoietic stem cell group, a portion of green fluorescent protein-positive cells expressed glial marker. In the marrow stem cell group, a number of green fluorescent protein and fibronectin-double positive cells were observed. No significant difference was observed in the recovery between both groups. Both hematopoietic stem cells and marrow stromal cells have the potential to restore the injured spinal cord and to promote functional recovery.

Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: An investigation using green fluorescent protein transgenic bone marrow chimeric mice

Activated microglia and hematogenous macrophages are known to be involved in infarct development after cerebral ischemia. Traditionally, hematogenic macrophages are thought to be the primary cells to remove the ischemic cell debris. However, phagocytosis is a well known property also of activated microglia. Due to a lack of discriminating cellular markers, the cellular origin of phagocytes and the temporal course of phagocytosis by these two cell types are largely unknown. In this study, we used green fluorescent protein (GFP) transgenic bone marrow chimeric mice and semithin serial sections after methyl methacrylate embedding of the brains to dissect in detail the proportion of identified activated resident microglial cells and infiltrating hematogenous macrophages in phagocytosing neuronal cell debris after 30 min of transient focal cerebral ischemia. Already at day one after reperfusion, we found a rapid decrease of neurons in the ischemic tissue reaching minimum numbers at day seven. Resident GFP-negative microglial cells rapidly became activated at day one and started to phagocytose neuronal material. By contrast, hematogenous macrophages incorporating neuronal cell debris were observed in the ischemic area not earlier than on day four. Quantitative analysis showed maximum numbers of phagocytes of local origin within 2 days and of blood-borne macrophages on day four. The majority of phagocytes in the infarct area were derived from local microglia, preceding and predominating over phagocytes of hematogenous origin. This recruitment reveals a remarkable predominance of local defense mechanisms for tissue clearance over immune cells arriving from the blood after ischemic damage.

Macrophage-specific expression of group IIA sPLA2 results in accelerated atherogenesis by increasing oxidative stress

Group IIA secretory phospholipase A2 (sPLA2) is an acute-phase protein mediating decreased plasma HDL cholesterol and increased atherosclerosis. This study investigated the impact of macrophage-specific sPLA2 overexpression on lipoprotein metabolism and atherogenesis. Macrophages from sPLA2 transgenic mice have 2.5 times increased rates of LDL oxidation (thiobarbituric acid-reactive substances formation) in vitro (59 +/- 5 vs. 24 +/- 4 nmol malondialdehyde/mg protein; P < 0.001) dependent on functional 12/15-lipoxygenase (12/15-LO). Low density lipoprotein receptor-deficient (LDLR-/-) mice were transplanted with bone marrow from either sPLA2 transgenic mice (sPLA2--> LDLR-/-; n = 19) or wild-type C57BL/6 littermates (C57 BL/6-->LDLR-/-; n = 19) and maintained for 8 weeks on chow and then for 9 weeks on a Western-type diet. Plasma sPLA2 activity and plasma lipoprotein profiles were not significantly different between sPLA2-->LDLR-/- and C57BL/6-->LDLR-/- mice. Aortic root atherosclerosis was increased by 57% in sPLA2-->LDLR-/- mice compared with C57BL/6-->LDLR-/- controls (P < 0.05). Foam cell formation in vitro and in vivo was increased significantly. Urinary, plasma, and aortic levels of the isoprostane 8,12-iso-iPF2alpha-VI and aortic levels of 12/15-LO reaction products were each significantly higher (P < 0.001) in sPLA2-->LDLR-/- compared with C57BL/6-->LDLR-/- mice, indicating significantly increased in vivo oxidative stress in sPLA2--> LDLR-/-. These data demonstrate that macrophage-specific overexpression of human sPLA2 increases atherogenesis by directly modulating foam cell formation and in vivo oxidative stress without any effect on systemic sPLA2 activity and lipoprotein metabolism.

1039. Ex Vivo Pre-Selection Increases Marking Following Transplant of Mixtures of Bone Marrow from Wild Type and Green Fluorescent Protein-Methylguanine Methyltransferase P144K Fusion Protein Transgenic Mice

Top of pageAbstract Methylguanine methyltransferase (MGMT) repairs alkylation of DNA resulting from 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) treatment. Mutant MGMT (MGMT|[ast]|) is resistant to inactivation by the irreversible inhibitor, O6-benzylguanine (BG), while retaining DNA repair activity. MGMT|[ast]| have been incorporated into retrovirus vectors for marking of hematopoietic stem cells, facilitating use of in vivo selection regimens that combine BG with BCNU or Temozolomide to enhance gene marking. Green fluorescent protein (GFP) is a convenient marker for monitoring transgene expression. We showed that GFP-MGMT|[ast]| fusion protein retains high fluorescence and MGMT|[ast]| activity when expressed by retrovirus vector transduced cells, and is a convenient model system to explore optimization of in vivo selection (Exp. Hematol. 32 (2004) 709). We engineered a GFP-MGMT|[ast]| fusion protein transgenic mouse (C57BL/6 oocyte injection; CMV enhancer/chicken ?-actin-promoter driven expression). All organs demonstrate strong GFP fluorescence with heterogeneity. With respect to blood cells, T-cells, B-cells, granulocytes, and Sca-1+ cells show 40%, 95%, 90%, and 70% green fluorescence, respectively. Transgenic mouse, but not wild-type mouse tissues retained high DNA repair enzymatic activity following 200?M BG treatment. Transgenic mouse bone marrow (BM) cells mixed with different ratios of wild-type BM cells were transplanted into myeloablated C57BL/6 mice. GFP fluorescent blood cells in transplanted mice were proportional to the ratio of transgenic to wild-type BM in the graft, where mice transplanted with 100% transgenic marrow demonstrated marking similar to that seen in transgenic mice. Transplanted mice were treated with BG/BCNU (20/6 mg/kg) two times 7 days apart. 3 weeks after selection, mice with baseline marking between 0.3% and 10% demonstrated 10-fold to 4-fold increase in marking, respectively. Because of potential in vivo toxicities, we also explored use of a single short time ex vivo selection (1 hour BG 40?M followed by addition of BCNU 80uM for 2 additional hours; cells washed before transplant) using 1:4, transgenic:wild-type mixture before transplantation (1|[times]|10exp6 BM cells). Animals transplanted with untreated mixtures showed 9|[plusmn]|2.2 % of GFP+ cells in blood 4 weeks after transplantation whereas ex vivo selected cells transplanted mice showed 68.8|[plusmn]|7.1 % (P< 0.0001). There was no significant difference in absolute white blood cell numbers. This demonstrates the feasibility of using a single short time ex vivo selective treatment with BG/BCNU to select for cells that express GFP-MGMT|[ast]|. There does not appear to be any requirement for either continued presence of the BG/BCNU or prolonged ex vivo growth of the cells to achieve selective enhancement of in vivo marking. Studies are in progress to determine whether this approach can also be used with retrovirus GFP-MGMT|[ast]| transduced BM cells. This also demonstrates the utility of GFP-MGMT|[ast]| transgenic mice to explore selection regimens.

A STAT5 modifier locus on murine chromosome 7 modulates engraftment of hematopoietic stem cells during steady-state hematopoiesis

AbstractHomologous disruption of expression of signal transducer and activator of transcription 5a (STAT5a) and STAT5b (STAT5ab–/–) in mice results in hematopoietic stem cells (HSCs) that can engraft irradiated hosts alone but are noncompetitive against wild-type HSCs. To explore mechanisms for this phenotype, we crossed the STAT5 mutations onto an HW80 background congenic to the original C57BL/6 that differs in a small chromosome 7 genomic locus. We previously demonstrated that C57BL/6 or HW80 background STAT5ab–/– bone marrow (BM) cells showed equal repopulating function either competitively or noncompetitively in irradiated hosts. However, one intraperitoneal injection of wild-type green fluorescent protein (GFP) transgenic BM cells into unconditioned newborn STAT5ab–/– recipients of either background was sufficient for high-level donor engraftment. Furthermore, haploinsufficiency of STAT5 (STAT5ab+/–) allowed improved engraftment over wild-type recipients, indicating a dose-dependent requirement for STAT5 activation. In reciprocal experiments, STAT5ab–/– BM was transplanted into nonirradiated W/Wv hosts. In these mice, C57BL/6 STAT5ab–/– BM cells were 10-fold more defective in long-term engraftment than control wild-type BM cells and HW80 STAT5ab–/– BM cells were 5- to 10-fold more defective than C57BL/6 STAT5ab–/– BM cells. Therefore, we conclude that STAT5 plays a critical role during steady-state HSC engraftment and a chromosome 7 modifier locus regulates this activity.

Analysis of Distinct Tartrate-resistant Acid Phosphatase Promoter Regions in Transgenic Mice

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFkappaB ligand/macrophage colony-stimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone- and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.

Macrophage-specific overexpression of group IIa sPLA2 increases atherosclerosis and enhances collagen deposition

Atherosclerosis is a chronic inflammatory disease of the vessel wall characterized by the accumulation of lipid-laden macrophages and fibrotic material. The initiation of the disease is accompanied by the accumulation of modified lipoproteins in the vessel wall. Group IIa secretory phospholipase A2 (sPLA2 IIa) is a key candidate player in the enzymatic modification of low density lipoproteins. To study the role of sPLA2 IIa in macrophages during atherogenesis, transgenic mice were generated using the human sPLA2 IIa gene and the CD11b promoter. Bone marrow transplantation to LDL receptor-deficient mice was performed to study sPLA2 IIa in atherosclerosis. After 10 weeks of high-fat diet, mice overexpressing sPLA2 IIa in macrophages showed 2.3-fold larger lesions compared with control mice. Pathological examination revealed that sPLA2 IIa-expressing mice had increased collagen in their lesions, independent of lesion size. However, smooth muscle cells or fibroblasts in the lesions were not affected. Other parameters studied, including T-cells and cell turnover, were not significantly affected by overexpression of sPLA2 IIa in macrophages. These data clearly show that macrophage sPLA2 IIa is a proatherogenic factor and suggest that the enzyme regulates collagen production in the plaque and thus fibrotic cap development.

Targeted expression of Cre recombinase in macrophages and osteoclasts in transgenic mice

To develop specific conditional gene ablation in the hematopoietic myeloid-osteoclast lineage, transgenic mice expressing Cre recombinase under the control of the CD11b promotor were generated on the C57BL/6 background. The cellular specificity of Cre activity following recombination was quantified in the Z/EG reporter transgenic mice by FACS analysis with lineage-specific markers and EGFP coexpression. A high degree of recombination, as evidenced by EGFP-positive cells, was demonstrated in macrophages and granulocytes of bone marrow and spleen by the presence of double-positive cells CD11b/EGFP and Gr1/EGFP, respectively. Interestingly, the peritoneal macrophage population showed almost complete DNA recombination at large. Most important, mature osteoclast cells derived from the double transgenic bone marrow and spleen progenitors were EGFP-positive. Hence, these CD11b-Cre mice will provide a unique tool to unravel novel gene function and activities involved during osteoclast and macrophage differentiation and maturation processes.

Fusion-protein truncation provides new insights into leukemogenesis

AML1-ETO (also known as RUNX1-ETO) is the fusion-protein transcription factor product of the 8;21 translocation. The translocation is present in up to 40% of leukemias of the French-American-British M2 subtype and is one of the most common events associated with myeloid leukemia. Clarifying the role of AML1-ETO in leukemogenesis has been difficult because the expression of the oncoprotein is not sufficient to cause disease. The challenge is reflected by the number of models that have been devised to study AML1-ETO in mice. In this issue of PNAS, Zhang and colleagues (1) advance the understanding of AML1-ETO another step with the discovery that a C-terminal truncation mutation produces penetrant leukemia. The earliest attempts to model AML1-ETO-associated leukemia in mice showed that the oncoprotein has detrimental developmental effects when expressed during embryogenesis (2, 3). The phenotype of the AML1-ETO knock-in embryos strongly resembled AML1 knockout embryos. The similarity provided compelling in vivo evidence supporting the hypothesis that AML1-ETO interferes with AML1. Although the embryonic lethal phenotype of the AML1-ETO knock-in was a temporary setback in the production of a leukemia model, the observations provided important clues to the action of AML1-ETO. Subsequent mouse models were engineered to bypass the embryonic lethality of AML1-ETO. Transgenic models, inducible systems, and bone marrow transplant strategies for expressing AML1-ETO were unable to reliably produce leukemia in mice even after 24 months (4–7). However, the failure to cause leukemia was extremely enlightening. When stem cells were transduced with AML1-ETO and transplanted into lethally irradiated recipient animals, the stem cell compartment expanded dramatically (6). Similarly, direct targeting of AML1-ETO expression to stem cells by using the SCA-1 promoter enhanced myeloid progenitor expansion (7). These experiments suggested that AML1-ETO could promote the expansion of a compartment that ultimately would acquire additional “hits” leading to disease. In …

Immune-Mediated Hepatitis Drives Bone marrow to Hepatocyte Plasticity.

Liver dysfunction is a major health burden world-wide. Future cell-based therapies for liver regeneration may benefit from the fact that bone marrow cells can fuse with or transdifferentiate into hepatocytes. All models demonstrating bone marrow to hepatocyte plasticity presented so far, however, have used highly artifical conditions of liver regeneration - applying toxins, genetic pressure models or liver resection. We have set up a model of transgenic T cell induced bystander hepatitis in bone marrow chimeras to assess the effect of hepatitis, a common liver pathology in humans, as an enhancer of bone marrow to hepatocyte plasticity events.MHC haplotype (Kb) transgenic bone marrow from 178.3 mice or control bone marrow from B10.BR (Kk) mice was transplanted into sublethally irradiated B10.BR (Kk) mice. Hepatitis was induced by repeated injections of Des Kk T cell receptor transgenic T cells against the Kb antigen. In additonal groups Retrorsine was used as an agent inhibiting endogenous hepatocyte proliferation and GCSF for mobilisation of bone marrow stem cells.Repeated injections of transgenic T cells induced subsequent waves of hepatitis in recipients of MHC haplotype transgenic bone marrow but not in control animals confirmed by serum ALT levels. Hepatocyte single cell suspensions from animals suffering from hepatitis revealed an increased expression of donor bone marrow derived antigen. This could be further enhanced by either increasing the number of circulating stem cells or by inhibiting the endogenous response of resident hepatocytes. FISH analysis showed fusion nuclei on a single cellular level.T cell receptor transgenic T cells induce bystander hepatitis in an antigen specific manner. This inflammatory response drives the plasticity of bone marrow cells to hepatocytes and their potential contribution to liver regeneration. Fusion between donor cells and resident hepatocytes is the underlying mechanism of liver regeneration in this model mimicking a common liver pathology.

Cooperative Pathways to Acute Myeloid Leukemia Include the Combining of Transcription Factor Alterations: PML-RARα Cooperates with SOX4.

One paradigm for transformation of normal myeloid cells into acute myeloid leukemia (AML) is the combining of transcription factor alteration with activation of signaling pathways, including cytokine receptor activation. To identify possible alternative pathways to leukemogenesis we crossed MRP8-PML/RARA transgenic mice with BXH-2 mice, which harbor an endogenous retrovirus that causes AML. Approximately half of the leukemias that arose in this cross showed features of acute promyelocytic leukemia (APL). We identified 22 insertions sites in 8 APL-like leukemias. Of these, 7 represented common insertion sites in the Mouse Retroviral Tagged Cancer Gene Database (RTCGD, http://rtcgd.ncifcrf.gov/mm4/index.html). We introduced into a retroviral vector cDNAs encoding 6 genes located at retroviral insertion sites identified in PML/RARA leukemias (Sox4, Meis1, Lck, Sfpi1, Nfil3, Cblb). PML/RARA transgenic bone marrow was transduced with these retroviruses and the marrow was used to reconstitute lethally irradiated recipient animals. Recipients of PML/RARα transgenic marrow transduced with a SOX4 retrovirus developed APL-like leukemias in 3 months. SOX4 is an HMG box transcription factor that exhibits sequence specific DNA binding and transactivation. It is the most common insertion site in RTCGD, and has been found to be overexpressed in small cell lung cancer and medulloblastoma, as well as other tumors. In light of other data indicating that combining transcription factor abnormalities can rapidly induce leukemia (e.g. Hoxa9 + Meis1), our finding that SOX4 + PML/RARα is potently leukemogenic supports the hypothesis that such cooperativity represents another important paradigm in myeloid leukemogenesis.

CREB Overexpression In Vivo Results in Increased Proliferation, Blast Transformation, and Earlier Engraftment of Myeloid Progenitor Cells.

CREB or cAMP responsive element binding protein is a 43-kDa-basic/leucine zipper (bZip) transcription factor that regulates gene expression through the activation of cAMP-dependent or -independent signal transduction pathways. CREB promotes growth and survival in a variety of cell types and is overexpressed in the bone marrow of greater than 60% of AML patients. To understand the role of CREB in myelopoiesis, we characterized the effects of CREB overexpression in transgenic mice. We created mice in which CREB expression was targeted to the myeloid lineage using the hMRP8 promoter. CREB transgenic mice showed evidence of monocytosis, compared to age-matched littermate controls. We performed colony assays with methylcellulose containing SCF, IL-6, and IL-3. Bone marrow cells from CREB transgenic mice formed robust colonies earlier and had increased numbers of colony forming units (CFU-GM) when compared to control mice. Cytospin analysis of these cells showed the presence of more immature myeloid cells compared to controls. At day 12, cells from colonies were 50% c-Kit positive, 83% Gr-1 positive, and 67% Mac-1 positive by FACS analysis. To assess self-renewal of progenitors from CREB transgenic mice, serial replating experiments were performed. Bone marrow cells from transgenic mice were highly successful in repopulating the methylcellulose containing SCF, IL-6, and IL-3, in contrast to the control cells, which were unable to grow after serial replating. Following tertiary replating of the CREB transgenic mouse bone marrow, we observed that the colonies (96+3.5) appeared more homogeneous with immature cells that were >99% c-Kit positive and <1% GR-1, Mac-1 positive. These results suggest that persistent expression of CREB leads to a blast-like phenotype in the absence of differentiation. To determine whether increased CREB expression confers growth factor-independence, we cultured bone marrow cells in methylcellulose that did not contain cytokines. We observed a 10-fold increase in the numbers of cells from CREB transgenic mice (two different founder lines) compared to normal bone marrow. When cultured in methylcellulose containing M-CSF, the bone marrow cells from CREB transgenic mice formed larger and significantly greater numbers of colonies. However, these cells did not grow in the presence of G-CSF or EPO alone. To determine if the myeloproliferative (monocytosis) phenotype was transplantable into wild type recipient mice we injected 4x106 bone marrow cells from CREB transgenic mice into wild type C57/BL6 recipient mice. Serial analysis of the peripheral blood counts and cell surface markers by FACS analysis showed earlier myeloid engraftment at 6 weeks following transplantation compared to normal control mice. The transgenic recipients showed increased monocytes and neutrophils in the peripheral blood with a corresponding increase in Mac-1 positive, Gr-1 positive cell populations at 8 weeks after transplantation. At the same time, the lymphocyte count was significantly lower in CREB transgenic recipient mice than controls. Our results suggest that CREB plays a critical role in the regulation of normal hematopoiesis and stem cell self-renewal.

Homodimerization of Retinoic Acid Receptor alpha through its Fusion Partners Underlies Pathogenesis of Acute Promyelocytic Leukemia.

In almost all cases of acute promyelocytic leukemia (APL), t(15;17)(q22;q12) fuses the promyelocytic leukemia (PML) gene with the retinoic acid receptor alpha (RARA) gene. In rare cases of APL, other genes are fused to RARA including PLZF, NPM, NuMA, and STAT5b. These chimeras are collectively referred to as X-RARA fusions. Common features of the X-RARα fusions include their ability to form dimers through the X domain proteins while retaining the ability to interact with rexinoid receptors (RXRs, the proteins with which RARα normally heterodimerizes). We previously created an artificial X-RARα: p50-RARα (a fusion of the dimerization interface of p50 NFκB with the portion of RARα found in naturally occurring X-RARα proteins). p50-RARα has effects similar to PML-RARα including (i) enhancing repression at retinoic acid response elements, due to impaired release of co-repressors at low levels of all-trans retinoic acid (ATRA), and (ii) inhibiting differentiation of myeloid cell lines. We generated transgenic mice expressing p50-RARα under the control of the MRP8 promoter. p50-RARα had a minimal effect on myelopoiesis and initiated myeloid leukemias at a rate of <5% in the first year of observation. Nevertheless, transduction of p50-RARA transgenic bone marrow with a retrovirus expressing an activated cytokine receptor (βcV449E) generated leukemias with features of APL including therapeutic responsiveness to ATRA. Complementing our work with the p50-RARA transgenic mice, retroviral co-transduction of normal bone marrow with βcV449E plus either p50-RARA or PML-RARA generated APL-like myeloid leukemias. Although retrovirally generated βc/p50-RARα and βc/PML-RARα leukemias were nearly identical, the PML fusion appeared associated with a subtle decrease in differentiation relative to the p50 fusion. In contrast to these X-RARα fusions, preliminary experiments suggest that RCRα (a homodimeric form of RARα in which the dimerization interface of RARα has been replaced by the dimerization domain of the homodimeric COUP-TF transcription factor) does not readily cooperate with βcV449E to cause leukemia. This finding lends support to the hypothesis that the decreased binding site selectivity of X-RARα fusions contributes to APL pathogenesis. Furthermore, we have generated a novel X-RARα fusion, F3-RARα, in which three copies of the F36M mutant of the Rapamycin-binding protein have been fused to RARα. This protein mimics the in vitro effects of PML-RARα on transcription & differentiation, and de-dimerization of F3-RARα by rapamycin reverses these effects. Additional experiments in vivo with F3-RARα are expected to further confirm the importance of abnormal transcription factor dimerization in leukemia pathogenesis.

Adult Bone Marrow-Derived Cell Fusion with Cardiomyocytes and Purkinje Neurons in Response to Irradiation but Not in Steady State.

Several recent studies have suggested that bone marrow (BM) cells can contribute to non-hematopoietic cell lineages through cell fusion rather than transdiffentiation. As this phenomenon has been observed in multiple organs, including the brain and heart, without prior infliction of organ-specific insults, it has been proposed that BM cells might contribute to replacement of non-hematopoietic cell lineages during steady state, and that BM transplantation might be developed as a therapeutic modality in diseases of these organs. However, as all observations of BM-derived cell fusion in vivo have been made in lethally irradiated mice reconstituted with genetically marked BM cells, we addressed to what degree cell fusion occurs normally and/or in response to whole body irradiation. To be able to distinguish between these possibilities we used c-kit deficient (w41/w41) mice, which unlike wild type mice do not require irradiation-induced myeloablation to facilitate reconstitution of transplanted BM cells. Noteworthy, no BM-derived cell fusion events were observed in the brain (purkinje neurons) or heart (cardiomyocytes) when unconditioned w41/w41 mice were reconstituted with beta actin GFP transgenic BM cells. In striking contrast, following whole body irradiation (875 rad), BM-derived cell fusion was observed in recipient cardiomyocytes and purkinje neurons of all BM transplanted mice. Thus, spontaneous adult BM-derived cell fusion does not occur in steady state but is potently facilitated by irradiation-induced injuries to the organs in which cell fusion occurs.

PERIPHERAL CD4 T CELL TOLERANCE INDUCED BY GENE THERAPY

P938 Aims: Reconstitution of lethally irradiated B10.AKM (H-2Kk) mice with syngeneic bone marrow cells infected with retroviruses carrying the allogeneic MHC class I gene H-2Kb resulted in stable and lifelong expression of Kb on bone marrow-derived cells. More importantly, these mice were specifically tolerant to H-2Kb skin grafts while still retaining the ability to reject third party skin grafts. Using CD8 transgenic mice, we discovered that alloreactive CD8 T cells underwent negative selection in the thymus, leading to the absence of these potentially alloreactive T cells in the periphery. In this study, we utilize Tg361 CD4 transgenic mice (CBA/Ca (H-2k)) expressing a TCR specific for the MHC class I gene H-2Kb to determine the fate of potentially alloreactive CD4 T cells using our gene therapy protocol. Methods: Tg361 CD4 TCR transgenic mice which express an alloreactive TCR on CD4 T cells specific for Kb were utilized to determine the mechanism by which genetic engineering of bone marrow induces donor specific CD4 T cell tolerance after bone marrow transplantation. Lethally irradiated B10.AKM mice were reconstituted with a 6:1 ratio of mock or Kb-transduced B10.AKM bone marrow to Tg361 bone marrow. Starting at four weeks after bone marrow transplantation, mice were analyzed for H-2Kb expression as well as for the development of Tg361 CD4 T cells. Results: Lethally irradiated recipients that received CD4 transgenic bone marrow in combination with autologous bone marrow transduced with virus encoding H-2Kb displayed stable and long-term expression of Kb on bone marrow-derived cells. Alloreactive CD4 transgenic T cells were readily detectable in the peripheral blood, spleen and thymus of chimeric recipients; although their levels were 2-fold lower than mock-transduced bone marrow recipients. Despite the presence of potentially alloreactive CD4 T cells in the periphery, chimeric recipients were specifically tolerant to H-2Kb expressing skin grafts. Interestingly, chimeric mice which received Kb-transduced and CD4 transgenic bone marrow expressed CD25 on 20-30% of Tg361-derived CD4 T cells, compared to only 6% of Tg361-derived CD4 T cells in mock transduced controls. Conclusions: We conclude that genetic engineering of bone marrow induces donor specific CD4 T cell tolerance through deletional and non-deletional mechanisms. We further hypothesize that expression of Kb on bone marrow derived cells induces CD4 T cells capable of inhibiting alloreactive T cells.

Dynamic reorganization of chromatin structure and selective DNA demethylation prior to stable enhancer complex formation during differentiation of primary hematopoietic cells in vitro

AbstractIn order to gain insights in the true molecular mechanisms involved in cell fate decisions, it is important to study the molecular details of gene activation where such decisions occur, which is at the level of the chromatin structure of individual genes. In the study presented here we addressed this issue and examined the dynamic development of an active chromatin structure at the chicken lysozyme locus during the differentiation of primary myeloid cells from transgenic mouse bone marrow. Using in vivo footprinting we found that stable enhancer complex assembly and high-level gene expression are late events in cell differentiation. However, even before the onset of gene expression and stable transcription factor binding, specific chromatin alterations are observed. This includes changes in DNA topology and the selective demethylation of CpG dinucleotides located in the cores of critical transcription factor binding sites, but not in flanking DNA. These results firmly support the idea that epigenetic programs guiding blood cell differentiation are engraved into the chromatin of lineage-specific genes and that such chromatin changes are implemented before cell lineage specification. (Blood. 2004;103:2950-2955)

Interleukin-4 elicits apoptosis of developing mast cells via a Stat6-dependent mitochondrial pathway

Objective The aim of this study was to assess the effects of interleukin-4 and signal transducer and activator of transcription (Stat)-6 on IL-3 + SCF-induced mast cell development. Materials and Methods Unseparated mouse bone marrow cells were cultured in IL-3 + SCF, giving rise to mast cells and monocytes/macrophages. The addition of IL-4, the use of Stat6-deficient bone marrow cells, and expression of a constitutively active Stat6 mutant were employed to assess the effects of IL-4 and Stat6 on cell viability, proliferation, and differentiation. Bax-deficient and bcl-2 transgenic bone marrow cells were used to assess the importance of the mitochondria in IL-4-mediated effects. Results IL-4 elicited apoptosis and limited the cell cycle progression of developing bone marrow cells, without affecting cell differentiation. Apoptosis required that IL-4 be present during the first 8 days of the 21-day culture period. Cell death correlated with loss of mitochondrial membrane potential. Accordingly, IL-4-mediated apoptosis was inhibited by Bax deletion or bcl-2 overexpression. Lastly, Stat6 activation was both necessary and sufficient to inhibit cell survival. Conclusion IL-4 exerts potent apoptotic effects on developing mast cells and monocyte/macrophages through mitochondrial damage and Stat6 activation.

Specific abrogation of transforming growth factor-β signaling in T cells alters atherosclerotic lesion size and composition in mice

AbstractA large body of evidence supports a role for proinflammatory mediators in atherosclerotic disease progression and instability. However, only few endogenous mechanisms have been suggested that could alter disease progression. One such mechanism is thought to be mediated by transforming growth factor β (TGF-β). Transgenic mice that express a dominant-negative TGF-β receptor type II under a T-cell-specific promoter were generated. Bone marrow transplantation from transgenic mice into irradiated low density lipoprotein receptor knock-out (LDLr KO) mice, subsequently fed an atherogenic diet, resulted in T-cell-specific blockade of TGF-β signaling in the recipient mice and increased differentiation of T cells toward both T helper 1 (Th1) and Th2 phenotypes. These mice showed a significant decrease in atherosclerotic lesion size in the aortic sinus compared with mice receiving transplants with the wild-type bone marrow. Atherosclerotic plaques of mice receiving transplants with the transgenic bone marrow showed increased T-cell infiltration and expression of major histocompatability complex (MHC) class II, along with a decrease in smooth muscle cell and collagen content, a plaque phenotype that is potentially vulnerable to rupture. These results identify for the first time an important role for specific and selective T-cell-TGF-β signaling in atherosclerosis. (Blood. 2003;102:4052-4058)