Donor Bone Marrow Cells Research Articles

Differentiating Functional Roles of Gene Expression from Immune and Non-immune Cells in Mouse Colitis by Bone Marrow Transplantation

To understand the role of a gene in the development of colitis, we compared the responses of wild-type mice and gene-of-interest deficient knockout mice to colitis. If the gene-of-interest is expressed in both bone marrow derived cells and non-bone marrow derived cells of the host; however, it is possible to differentiate the role of a gene of interest in bone marrow derived cells and non- bone marrow derived cells by bone marrow transplantation technique. To change the bone marrow derived cell genotype of mice, the original bone marrow of recipient mice were destroyed by irradiation and then replaced by new donor bone marrow of different genotype. When wild-type mice donor bone marrow was transplanted to knockout mice, we could generate knockout mice with wild-type gene expression in bone marrow derived cells. Alternatively, when knockout mice donor bone marrow was transplanted to wild-type recipient mice, wild-type mice without gene-of-interest expressing from bone marrow derived cells were produced. However, bone marrow transplantation may not be 100% complete. Therefore, we utilized cluster of differentiation (CD) molecules (CD45.1 and CD45.2) as markers of donor and recipient cells to track the proportion of donor bone marrow derived cells in recipient mice and success of bone marrow transplantation. Wild-type mice with CD45.1 genotype and knockout mice with CD45.2 genotype were used. After irradiation of recipient mice, the donor bone marrow cells of different genotypes were infused into the recipient mice. When the new bone marrow regenerated to take over its immunity, the mice were challenged by chemical agent (dextran sodium sulfate, DSS 5%) to induce colitis. Here we also showed the method to induce colitis in mice and evaluate the role of the gene of interest expressed from bone-marrow derived cells. If the gene-of-interest from the bone derived cells plays an important role in the development of the disease (such as colitis), the phenotype of the recipient mice with bone marrow transplantation can be significantly altered. At the end of colitis experiments, the bone marrow derived cells in blood and bone marrow were labeled with antibodies against CD45.1 and CD45.2 and their quantitative ratio of existence could be used to evaluate the success of bone marrow transplantation by flow cytometry. Successful bone marrow transplantation should show a vast majority of donor genotype (in term of CD molecule marker) over recipient genotype in both the bone marrow and blood of recipient mice.

X-ray irradiation selectively kills thymocytes of different stages and impairs the maturation of donor – derived CD4 +CD8 + thymocytes in recipient thymus

The aim of the present study was to determine whether the sensitivity of thymocytes to X-ray radiation depends on their proliferative states and whether radiation impairs the maturation of donor-derived thymocytes in recipient thymus. We assigned 8-week-old C57BL/6J mice into three treatment groups: 1) untreated; 2) X-ray radiation; 3) X-ray radiation plus bone marrow transplantation with donor bone marrow cells from transgenic mice expressing enhanced green fluorescent protein (GFP) on a universal promoter. After 4 weeks, the size of the thymus, the number and proliferation of thymocytes and ratios of different stage thymocytes were analyzed by immunohisto-chemistry and flow cytometry. The results showed that: 1) CD4+CD8+ thymocytes were more sensitive to X-ray radiation-induced cell death than other thymocytes; 2) the proliferative capacity of CD4+CD8+ thymocytes was higher than that of other thymocytes; 3) the size of the thymus, the number of thymocytes and ratios of thymo-cytes of different stages in irradiated mice recovered to the normal level of untreated mice by bone marrow trans-plantation; 4) the ratio of GFP-positive CD4+CD8+ thymocytes increased significantly, whereas the ratio of GFP-positive CD4+ or CD8+ thymocytes decreased significantly. These results indicate that the degree of sensitivity of thymocytes to X-ray radiation depends on their proliferative states and radiation impairs the maturation of donor-derived CD4+CD8+ thymocytes in recipient thymus.

SPARC promotes the development of erythroid progenitors

Secreted protein acidic and rich in cysteine (SPARC) is a well-recognized regulator that affects cell proliferation, differentiation, and survival. Here, we investigated the impact of SPARC on erythropoiesis. Erythropoiesis in bone marrow (BM) from SPARC(-/-) mice was analyzed by flow cytometry and colony-forming assays. The mechanisms that affected erythropoiesis were investigated by BM transplantation experiments. CD34(+) cells from umbilical cord blood were isolated by MiniMACS, and the effect of SPARC on human erythropoiesis was assessed by colony-formation assay and erythroid differentiation culture. The hemoglobin level in peripheral blood (PB) was lower in SPARC(-/-) mice. Neither red blood cell count in PB nor the percentage of Ter119(+) erythrocytes in BM showed significant difference between SPARC(-/-) and WT mice. However, the ability of SPARC(-/-) bone marrow cells (BMCs) to form erythroid burst-forming units (BFU-E), as well as spleen colony-forming units (CFU-S8), was significantly decreased. The addition of exogenous SPARC could promote the formation of BFU-E from SPARC(-/-) BMCs in vitro. And the impaired ability of SPARC(-/-) BMCs to form BFU-E could be restored by transplanted into WT BM microenvironment, whereas the BFU-E formation of WT BMCs was impaired after transplanted into SPARC(-/-) recipients. Furthermore, exogenous SPARC promoted umbilical cord blood CD34(+) cells to form erythroid colonies and the hemoglobinization of erythroid, but did not affect the expression levels of glycophorin A and CD71. In conclusion, our results indicate that SPARC promotes the development of erythroid progenitors, but does not affect terminal erythroid differentiation.

CD4+CD25+FOXP3+ Regulatory T Cells In Allogeneic Hematopoietic Cell Transplantation

CD4+CD25+FOXP3+ regulatory T cells (Treg) require activation through the T cell receptor for function. CD4+CD25+FOXP3+ regulatory T cells are believed to be key players of the immune tolerance network and control the induction and effector phase of the immune system. Although these cells require antigen-specific activation, they are generally able to suppress bystander T cell responses once activated. This raises the possibility that antigen-specific Treg may be useful therapeutically by localizing generalized suppressive activity to tissues expressing select target antigens. Treg can exert a potent suppressive effect on immune effector cells reactive to host antigens and prevent graft versus host disease (GVHD) in allogeneic bone marrow transplantation (BMT). Here, we observed that co-transfer of CD4+CD25+FOXP3+ T cells derived from donor type along with the donor bone marrow cells could control GVHD-like reactions by suppressing effectors cells of host responding to the donor hematopoietic compartment, and resulted in prevention of autoimmunity and rejection. We further demonstrate that CD4+CD25+FOXP3+ regulatory T cells can control immune-based morbidity after allogeneic BMT by suppressing the development of granulocytes cells and increasing the level of B cell expression.Keywords : Bone Marrow Transplantation, Regulatory T cells, CD4+CD25+FOXP3+

Macrophage Inflammatory Protien-2/CXCR2 Blockade Attenuates Acute Graft-versus-Host Disease while Preserving Graft-versus-Leukemia Activity (126.16)

Abstract Graft-versus-host disease (GVHD) is the principal complication after allogeneic bone marrow transplantation (BMT). Strategies that effectively separate the beneficial GVL effects from GVHD toxicity are necessary for enhanced survival after BMT. GVHD mice were induced by infusion of both donor BM cells and spleen cells into BU-CY conditioned recipient female BALB/c mice. Tumor-bearing GVHD mice were also made adding A20 cells. A total of 31 cytokines and chemokines were measured in the liver, intestine, and skin in GVHD mice by Luminex Multiplex assay. Four cytokines, G-CSF, KC, MIP-2, and IL-23, were commonly unregulated in target organs at the initial GVHD stage and were reduced by neutralizing MIP2/CXCR2 axis ligation. CXCR2 blockade on donor T cells and systemic administration of anti-MIP-2 neutralizing antibodies to BMT recipients caused a significant decrease in both systemic GVHD and target organ histopathology. GVL responses (BCL2 expression or B220+/CD19+ cells in BM and PB) after MIP-2/CXCR2 blockade were largely preserved and mortality was significantly delayed compared to control allogeneic BMT recipients. These data suggest that blocking the MIP-2/CXCR2 axis represents a novel strategy to separate the toxicity of GVHD from the beneficial GVL effects after allogeneic BMT.

289 In Situ Imaging of Neonatal Tolerance Induction: Donor Bone Marrow and Spleen Cells Home Differently and Induce Tolerance by Distinct/Overlapping Mechanisms

289 In Situ Imaging of Neonatal Tolerance Induction: Donor Bone Marrow and Spleen Cells Home Differently and Induce Tolerance by Distinct/Overlapping Mechanisms

Role of bone marrow cells in the development of pancreatic fibrosis in a rat model of pancreatitis induced by a choline-deficient/ethionine-supplemented diet

Bone marrow cell (BMC)-derived myofibroblast-like cells have been reported in various organs, including the pancreas. However, the contribution of these cells to pancreatic fibrosis has not been fully discussed. The present study examined the possible involvement of pancreatic stellate cells (PSCs) originating from BMCs in the development of pancreatic fibrosis in a clinically relevant rat model of acute pancreatitis induced by a choline-deficient/ethionine-supplemented (CDE) diet. BMCs from female transgenic mice ubiquitously expressing green fluorescent protein (GFP) were transplanted into lethally irradiated male rats. Once chimerism was established, acute pancreatitis was induced by a CDE diet. Chronological changes in the number of PSCs originating from the donor BMCs were examined using double immunofluorescence for GFP and markers for PSCs, such as desmin and alpha smooth muscle actin (αSMA), 1, 3 and 8weeks after the initiation of CDE feeding. We also used immunohistochemical staining to evaluate whether the PSCs from the BMCs produce growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF) β1. The percentage of BMC-derived activated PSCs increased significantly, peaking after 1week of CDE treatment (accounting for 23.3±0.9% of the total population of activated PSCs) and then decreasing. These cells produced both PDGF and TGFβ1 during the early stage of pancreatic fibrosis. Our results suggest that PSCs originating from BMCs contribute mainly to the early stage of pancreatic injury, at least in part, by producing growth factors in a rat CDE diet-induced pancreatitis model.

Regulatory T-cell subset analysis and profile of interleukin (IL)-10, IL-17 and interferon-gamma cytokine-producing cells in kidney allograft recipients with donor cells infusion

This pilot study aimed to assess whether the perioperative infusion of donor bone marrow cells (DBMC) in renal allograft recipients can affect the appearance of peripheral regulatory T-cell subsets and the profile of cytokine-producing cells [interferon-gamma (IFN-γ), interleukin (IL)-17 and IL-10] 2 years after transplantation. Fresh blood samples were collected from 14 kidney recipients who received infusion and from 13 kidney recipients without infusion who served as controls at the end of the second post-transplantation year. Initially the percentages of CD4(+)CD25(+)FoxP3(+) T cells and CD3(+)CD8(+)CD28(-) T cells were quantified using flowcytometry. Thereafter, the frequencies of IL-10-, IL-17- and IFN-γ-producing cells were determined separately using the ELISPOT technique with peptides corresponding to mismatched donor HLA-DR molecules and phytohemagglutinin (PHA). The mean numbers of IFN-γ- and IL-17-producing cells in response to PHA were lower in infused patients than in controls (P = 0.02 and P = 0.18, respectively); however, an increased frequency of IL-10-producing cells was observed compared to controls (P = 0.07). Furthermore, the ratio of IL-10/IFN-γ-producing cells was significantly higher in the DBMC-infused group versus controls (P = 0.01). There was a negative correlation between the percentage of CD3(+)CD8(+)CD28(-)T cells and IL-17-producing cells in the infused group (r = -0.539, P = 0.04). The mean levels and the frequency of microchimerism within the first post-transplantation year were also significantly higher in infused patients than in controls (P = 0.007 and P = 0.001, respectively). Our findings suggest that DBMC infusion could partially stimulate the regulatory mechanisms against alloimmune responses in kidney allograft recipients

First Human Face Transplantation

The first human facial allotransplantation, a 38-year-old woman, was performed on November 27, 2005. The aesthetic aspect and functional recovery and the risk-to-benefit ratio are evaluated 5 years later. The facial transplantation included nose, chin, part of cheeks, and lips. The immunosuppressive protocol included tacrolimus, mycophenolate mofetil, prednisone, and antithymocyte globulins. In addition, donor bone marrow cells were infused on days 4 and 11 after transplantation. The aesthetic aspect is satisfying. The patient has normal protective and discriminative sensibility. She showed a rapid motion recovery, which has remained stable for 3 years posttransplantation. She can smile, chew, swallow, and blow normally whereas pouting and kissing is still difficult. Phonation recovery was impressive therefore the patient can talk normally. Two episodes of acute rejection developed during the first year. Donor-specific anti-human leukocyte antigen antibodies were never detected. Five-year mucosal biopsy showed a slight perivascular inflammatory infiltrate while skin biopsy was normal. The main side effect of the immunosuppressive treatment was a progressive decrease in renal function, which improved after switching from tacrolimus to sirolimus. Moreover, she developed arterial hypertension, an increase in lipid levels, and in situ cervix carcinoma treated by conization. Since 2008, she showed mild cholangitis possibly caused by sirolimus. In September 2010, bilateral pneumopathy occurred and was successfully treated with antibiotics. Despite some long-term complications, which are similar to those reported after solid organ transplantation, the patient is satisfied of her new face and has normal social interaction.

Bone Marrow-Derived Cells from Male Donors Do Not Contribute to the Endometrial Side Population of the Recipient

Accumulated evidence demonstrates the existence of bone marrow-derived cells origin in the endometria of women undergoing bone marrow transplantation (BMT). In these reports, cells of a bone marrow (BM) origin are able to differentiate into endometrial cells, although their contribution to endometrial regeneration is not yet clear. We have previously demonstrated the functional relevance of side population (SP) cells as the endogenous source of somatic stem cells (SSC) in the human endometrium. The present work aims to understand the presence and contribution of bone marrow-derived cells to the endometrium and the endometrial SP population of women who received BMT from male donors. Five female recipients with spontaneous or induced menstruations were selected and their endometrium was examined for the contribution of XY donor-derived cells using fluorescent in situ hybridization (FISH), telomapping and SP method investigation. We confirm the presence of XY donor-derived cells in the recipient endometrium ranging from 1.7% to 2.62%. We also identify 0.45–0.85% of the donor-derived cells in the epithelial compartment displaying CD9 marker, and 1.0–1.83% of the Vimentin-positive XY donor-derived cells in the stromal compartment. Although the percentage of endometrial SP cells decreased, possibly being due to chemotherapy applied to these patients, they were not formed by XY donor-derived cells, donor BM cells were not associated with the stem cell (SC) niches assessed by telomapping technique, and engraftment percentages were very low with no correlation between time from transplant and engraftment efficiency, suggesting random terminal differentiation. In conclusion, XY donor-derived cells of a BM origin may be considered a limited exogenous source of transdifferentiated endometrial cells rather than a cyclic source of BM donor-derived stem cells.

Effects of intracoronary delivery of allogenic bone marrow-derived stem cells expressing heme oxygenase-1 on myocardial reperfusion injury

Heme oxygenase-1 (HO-1) decreases apoptosis, inflammation and oxidative stress. The aim of the study was to investigate the effects of intracoronary infusion of allogenic bone marrow cells (BMC) overexpressing HO-1 in the porcine model of myocardial infarction (MI). MI was produced by balloon occlusion of a coronary artery. BMC were transduced with adenoviruses encoding for HO-1 (HO-1 BMC) or GFP (GFP-BMC) genes. Prior to reperfusion animals received HO-1 BMC, control BMC (unmodified or GFP-BMC) or placebo. Left ventricular (LV) ejection fraction (EF), shortening fraction (SF), end-systolic and end-diastolic diameters (EDD, ESD) were assessed by echocardiography before, 30 minutes (min) and 14 days after reperfusion. BMC significantly improved LVEF and SF early (30 min) after reperfusion as well as after 14 days. Early after reperfusion HO-1 BMC were significantly more effective than control BMC, but after 14 days, there were no differences. There were no effect of cells on LV remodelling and diastolic function. Both HO-1 BMC and control BMC significantly reduced the infarct size vs. placebo (17.2 ± 2.7 and 18.8 ± 2.5, respectively, vs. 27.5 ± 5.1, p= 0.02) in histomorphometry. HO-1-positive donor BMC were detected in the infarct border area in pigs receiving HO-1-cells. No significant differences in expression of inflammatory genes (SDF-1, TNF-α, IL-6, miR21, miR29a and miR133a) in the myocardium were found. In conclusion, intracoronary delivery of allogeneic BMC immediately prior to reperfusion improved the LVEF and reduced the infarct size. HO-1 BMC were not superior to control cells after 14 days, however, produced faster recovery of LVEF. Transplanted cells survived in the peri-infarct zone.

Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting

Donor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs. We performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4-T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler. The majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2 × 10(10) total cells, 6.2 ± 2.2 × 10(8) of which were CD45(+) CD34(+). Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34(+) CD90(+) CD117(dim) hematopoietic stem cells (15.5 ± 7.5% of the CD34 (+) cells) and CD45(-) CD73(+) CD105(+) mesenchymal stromal cells (0.04 ± 0.04% of the total cells). This procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.

Aortic Tissue as a Niche for Hematopoiesis

The stem cell theory of hematopoiesis predicts that all mature blood cell lineages circulating in the bloodstream or resident in tissues arise via a hierarchy of hematopoietic progenitor cell (HPC) intermediates that are ultimately derived from hematopoietic stem cells (HSC). In the adult human, bone marrow–resident HSC and HPC generate billions of circulating blood cells daily, ranging from the short-lived neutrophils and platelets to long-lived B and T lymphocytes and monocyte macrophages. Recent interest in the roles of macrophages as modulators of angiogenesis1 and as participants in atheroma formation2 highlight the need to better understand the mechanisms of recruitment and retention of macrophages and their progenitor precursors within tissues. In this issue of Circulation , Psaltis and colleagues3 identify clonogenic HPC activity in aortic adventitia (that is enriched in HPC compared with peripheral blood or muscle tissue), which appears skewed toward colony-forming unit-macrophage (CFU-M) differentiation. In addition, stem cell antigen-1–expressing cells in the aortic adventitia coexpressed a variety of HSC antigens, and adoptive transfer of these cells into irradiated recipient mice led to low but sustained multilineage reconstitution, a property of HSC. Although bone marrow (BM)–derived donor hematopoietic cells reconstituted the aorta of lethally irradiated animals, the majority of aorta-derived CFU-M appeared to be constitutive or long term residents of the vessel wall (with sustained but only partial replacement from donor BM cells), suggesting that either the aortic adventitial microenvironment imparts specific differentiation properties to marrow-derived precursors or that a local origin for HSC/HPC exists in the adult vasculature. Is there any evidence to suggest that HSC/HPC may originate and/or migrate through the aortic adventitia? Article see p 592 Hematopoiesis occurs in a site- and tissue-specific pattern that varies throughout mammalian development. The first blood cells (including macrophages) arise in the extraembryonic yolk sac, and the yolk …

Bone marrow−derived cells do not engraft into skeletal muscle microvasculature but promote angiogenesis after acute injury

The skeletal muscle is supported by a vast network of microvessels with the capacity to regenerate in response to injury. However, the dynamics of microvascular repair and the origin of reconstituted endothelial cells in the skeletal muscle are poorly understood. A growing body of literature exists to indicate bone marrow (BM)-derived cells engraft into regenerating vascular endothelium and muscle macrovasculature. Therefore, we investigated the extent of BM contribution to skeletal muscle microvasculature after acute injury. Because reporters and markers commonly used to trace donor BM cells are not endothelial specific but are also expressed by leukocytes, we generated novel BM chimeras utilizing Tie2-green fluorescent protein BM cells transplanted into CD31 and Caveolin-1 knockout recipients. In turn, we surveyed BM vascular contribution, not just by the presence of green fluorescent protein, but also CD31 and Caveolin-1, respectively. After stable BM reconstitution, chimera limb muscles were cardiotoxin (CTX) injured and examined 21 days post-injury for the presence of green fluorescent protein, CD31, and Caveolin-1. Acute muscle injury by CTX is characterized by initial microvasculature death followed by rapid endothelial regeneration within 14 days post-damage. Histological analysis of injured and uninjured contralateral limb muscles revealed a complete absence of BM engraftment in the muscle vasculature of wild-type and CD31/Caveolin-1 knockout chimeras. In contrast, F4/80(+) cells isolated from CTX-injured muscle, expressed endothelial-related markers and promoted angiogenesis invitro. Therefore, despite the absence of BM engraftment to regenerated skeletal muscle microvasculature, macrophages recruited after injury promote angiogenesis and, in turn, vascular regeneration.

Pro- and Antiapoptotic Proteins Expression on Bone Marrow and Peripheral Blood CD34+Cells in Acute Leukemia (AL) Patients

Abstract 4996It was shown that drug resistance, poor-risk cytogenetics and poor prognosis in AL is associated with high level of Bcl-2 expression and low Bax/Bcl-2 ratio (<0,3). Fas-antigen (CD95) as a protein triggering the extrinsic apoptotic pathway is differently expressed on hematopoietic precursors. More immature CD34+/CD38- AML blast cells have lower expression of Fas/Fas-L and lower Fas-induced apoptosis than CD34+/CD38+cells. CD34+/CD38− leukemia precursors also have a reduced sensitivity to daunorubicin in vitro and increased expression of multidrug resistance genes (mrp/lrp). CD34+ leukemia cells have not yet been properly characterized regarding the expression of angiotensin converting enzyme (ACE) which regulatory influence on hematopoiesis is now beeing extensively investigated. ACE expression on blast cells is high, but it’s still unknown how CD34+ACE+ leukemia cells behave after chemotherapy. Recent publications indicate that CD34+ACE+ hematopoietic precursors transplanted into NOD/SCID mice contribute 10-fold higher numbers of multilineage blood cells than their CD34+ACE- counterparts.We have studied the dynamics of Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in peripheral blood (PB) and bone marrow (BM) in AL pts during treatment. PB and BM samples were collected before and on +36 day after chemotherapy. The antigens were detected by flow cytometry using monoclonal antibodies. We calculated 10 000 cells in each sample.19 pts were included in the study: 10 - AML and 9 - ALL. The control group comprised 8 healthy donors. At time of diagnosis there were 40±5,7% of CD34+ cells in BM and 26±4,9% - in PB. There was no significant difference between AML and ALL. CD34+ cells in BM and PB of healthy donors constituted 1,6% and 0,27%, respectively. After induction therapy (+36 day) CD34+ cells decreased in BM to 6,1%±3,3 (p=0,0001), in PB to 3,7%± 2,7 (p=0,0008) in all pts. The data on antigens expression on CD34+ cells of BM and PB are presented in table 1CD34+/Bcl-2+CD34+/Bax+CD34+/CD95+CD34+/ACE+BMPBBMPBBMPBBMPBAML pts n=100 day38±11,6*41±1424,4±7,929,2±7,6*16,4±8,523,2±7,821,7±9,520,8±8,7*36 day13,5±3,4**23,7±5**46,2±11,550,3±1119,9±5,536,4±1034±6,635±9,2**ALL pts n=90 day36±1133,7±1246,2±9,437,4±3,7*3,4±1,1*7,1±2,5*41±10,933,2±9,7*36 day18,4±5,826±8,938±11,840,5±1026,2±9,1**40,9±9,2**34±1062,8±10**Donors n=811,7±1,626,1±5,922,8±467,8±6,713,4±3,247,7±11,628±5,368,2±10,2*− p<0.05 compare with donors**− p<0.05 compare with day 0CD34/Bcl-2 expression in BM in AML pts is significantly higher (p=0,04) at the diagnosis comparing with donors. CD34/Bcl-2 expression in PB in AML pts and in BM and PB in ALL pts is higher too, but not significantly. This expression level decreased substantially in BM and PB in AML pts on +36 day comparing with day 0 (p<0,05). We did not found significant changes in ALL pts. CD34/Bax expression in PB is significantly lower (p=0,003) both in AML and ALL pts in comparison with donors. In AML, not in ALL, chemotherapy caused augmentation of Bax expression in CD34+ BM and PB cells on +36 day.BM and PB CD34+ cells in donors had different expression characteristics of Bcl-2 and Bax, demonstrating much higher level of pro- and antiapoptotic markers in PB cells. On the contrast CD34+ leukemia cells in BM and PB had similar characteristics regarding CD34/Bcl-2 and CD34/Bax expression. This fact demonstrates the heterogeneity of donor CD34+cells in BM and PB and points that leukemia CD34+cells in BM and PB are rather similar.CD95 expression on CD34+ BM and PB before treatment is significantly lower (p=0,01, p=0,008) in ALL pts in comparison with donors, and this expression level increased after chemotherapy (p<0,05). CD34/CD95 expression in AML pts is similar with donors, and we didn’t find changes after treatment.CD34/ACE coexpression in BM cells of leukemia pts and donors did not differ much at any time of evaluation. But CD34/ACE expression in PB cells of AML and ALL pts was much lower (p<0,05) than in donors and substantially increased on the day 36.So, our data demonstrate that Bcl-2, Bax, CD95 and ACE expression on CD34+ cells in AL pts and donors significantly differs. The chemotherapy provokes critical changes in CD34/CD95 expression in BM and PB in ALL pts, CD34/Bcl-2 expression in AML pts and ÑÂ34/ACE expression in PB in all AL pts. Disclosures:No relevant conflicts of interest to declare.

Targeting RANKL for Immunotherapy of Multiple Myeloma

Abstract 2905The TNF family member RANKL and its receptors RANK and osteoprotegerin are key regulators of bone remodelling, but also largely influence progression of cancers of various origins (Tan et al., Nature 2011; Sordillo et al., Cancer 2003). Recently we reported that multiple myeloma (MM) and chronic lymphocytic leukemia (CLL) cells can express RANKL. Moreover, we generated RANK-Ig fusion proteins with wild type (RANK-Fc-WT) or modified human IgG1 Fc parts displaying highly enhanced (RANK-Fc-ADCC) or abrogated (RANK-Fc-KO) affinity to the NK cell Fc receptor to induce antibody-dependent cellular cytotoxicity (ADCC) against RANKL-expressing targets (Schmiedel et al., Blood 2010 116, 21 :1252–1253). Here we extended our analyses on RANKL expression and release in B cell-derived malignancies and evaluated the effects of RANKL targeting with our fusion proteins in vivo. We found substantial expression of RANKL on plasma cells in 35 of 44 (80%) MM patients. RANKL in soluble form (sRANKL) was detectable in culture supernatants of bone marrow (BM) cells of MM patients (6/10) and MM cell lines (all 7). In contrast, RANKL release was neither observed with blood (n=10) and BM (n=4) cells of healthy donors nor with primary CLL cells (n=10) despite the fact that all CLL cells expressed RANKL on the cell surface. In line, mRNA of the splice variant for sRANKL was expressed only in BM cells of MM patients (7/10) and all 7 MM cell lines, but not in blood and BM samples of CLL patients and healthy donors, respectively (n=10 each). In light of available data that neutralization of RANKL prevented disease progression in a MM mouse model (Sordillo et al., Cancer 2003) we went on to evaluate the anti-tumor efficacy and also potential side effects of our Fc-modified RANK-Fc fusion proteins in vivo. First we took advantage of the fact that human RANK binds both human and mouse RANKL (Bossen et al., JBC 2006) to determine potential in vivo toxicity of the fusion proteins. Neither RANK-Fc-ADCC nor RANK-Fc-KO application altered cell counts or distribution in peripheral blood or BM in DBA/2N mice, and analysis by computed tomography after 10 and 30 days revealed no alterations of bone density. In a syngeneic DBA/2N tumor model, treatment with RANK-Fc-WT and RANK-Fc-ADCC significantly (both p<0.05, Mann-Whitney U test) prolonged survival of mice inoculated with RANKL-expressing P815 cells as compared to treatment with RANK-Fc-KO or isotype controls. No effects of treatment were observed in mice inoculated with RANKL-negative P815 cells confirming the target antigen restriction of the effects induced by RANK-Fc-WT and RANK-Fc-ADCC. Surprisingly, no difference between the effects of RANK-Fc-ADCC and RANK-Fc-WT was observed. Since RANK-Fc-ADCC induced by far higher ADCC against RANKL-expressing targets than RANK-Fc-WT in analyses with human NK cells in vitro we next aimed to elucidate why this was not mirrored in our mouse model. Comparative analyses with human PBMC and splenocytes of two different mouse strains (DBA/2N and C57/BL6) revealed that ADCC of NK cells was generally rather weak in mice as compared to the human system, and in line with the in vivo results no benefit of the Fc-optimization was observed with mouse NK cells. These results indicate that ADCC induction by human Fc parts is less potent in mice compared to humans raising doubt regarding the validity of mouse models to study ADCC in general and immunostimulatory effects of Fc-modifications of therapeutic proteins or antibodies in particular. However, when interpreting the results of our tumor model in light of these findings it seems likely that the therapeutic effects of RANK-Fc-ADCC in humans will exceed that observed in mice. Thus, our data provide a clear rationale for evaluation of RANK-Fc-ADCC treatment in patients with MM and potentially also other B cell-derived hematopoietic malignancies. Disclosures:No relevant conflicts of interest to declare.

MKL2 Functions in the Absence of MKL1 to Promote Megakaryocyte Maturation

Abstract 2336 Background:The transcriptional cofactors, MKL1 and MKL2, are members of the myocardin related factor family, which bind and activate the transcription factor Serum Response Factor (SRF) (Cen et al J Cell Biochem, 2004). Megakaryocytes, large, highly polyploid myeloid cells give rise to platelets, which promote blood clotting in response to tissue damage. The Krause lab has reported a significant decrease in Mk ploidy with a 50% decrease in mature platelets in Mkl1 knockout (KO) mice, indicating that MKL1 may contribute to megakaryocyte maturation (Cheng et al Blood, 2009). The phenotype of the Mk specific (PF4-Cre) Srf conditional KO (SRF PF4-cKO) further supports the importance for the MKL1/SRF pathway in Mk differentiation (Halene et al Blood, 2010); although similar to the Mkl1 KO mouse, the Srf PF4-cKO mouse has a more severe phenotype with a 90% decrease in platelet counts suggesting the presence of compensatory proteins or redundant pathways in Mkl1 KO mice. Objective:This study addresses the hypothesis that MKL2 is critical for normal megakaryocytopoiesis particularly in the absence of MKL1. Methods and Results:Using bone marrow (BM) transplantation, we determined that the Mkl1 KO phenotype (decreased megakaryocyte ploidy and decreased platelets) is cell autonomous. Lethally irradiated WT recipients of Mkl1 KO BM cells had a 50% decrease in platelet count, as well as significantly decreased ploidy indicated by the percentage of Mk with 2N (60% and 40% in the recipients of Mkl1 KO vs WT BM, respectively, p<0.0001). Transduction of Mkl1 KO donor BM cells with retrovirus encoding MKL1 cDNA rescued the ploidy phenotype. Although the Mkl1 KO mice are thrombocytopenic, their bleeding times are not significantly increased. In vitro studies show that MKL2 and MKL1 activate the same promoters in luciferase assays and have similar subcellular localization patterns, suggesting redundancy.To assess MKL2’s compensatory function in Mkl1 KO mice, we crossed Mkl2F/F mice with PF4-Cre mice to induce Mkl2 deletion in the Mk compartment (Mkl2 PF4-cKO). The Mkl2 PF4-cKO mice were mated onto the Mkl1 KO background to create double KO (DKO) megakaryocytes. The megakaryocyte ploidy and platelet counts (decreased by 90%) of DKO mice are more severe than the Mkl1 KO and comparable to the Srf PF4-Cre cKO mice In addition, bleeding times were significantly increased compared to WT and Mkl1 KO mice (p<0.0001). Electron microscopy and immunofluorescence of DKO megakaryocytes and platelets indicate abnormal cytoskeletal and membrane organization with decreased granule complexity. These morphological changes are consistent with abnormal in vitro platelet activation in response to ADP or thrombin. Conclusions:Our investigation reveals that both MKL1 and MKL2 are important for megakaryocyte differentiation, and that MKL2 mitigates the phenotype of MKL1 deficient megakaryocytes. The Mkl1 KO/Mkl2 PF4-cKO (DKO) mice phenocopy the Srf PF4-cKO mice suggesting that SRF activity in megakaryocyte differentiation is dependent on the myocardin related transcription factor family of proteins. Disclosures:No relevant conflicts of interest to declare.

Dll4 Blockade Modifies the Bone Marrow Vascular Niche and Improves Donor Bone Marrow Cells for Bone Marrow Transplant

Abstract 1321The Delta:Notch signaling pathway regulates multiple aspects of hematopoietic cell differentiation and function and more recently Delta-like 4 (Dll4) has been shown also to regulate adult neo-vascularization (angiogenesis), namely in tumors, which led to the development of therapeutic strategies targeting Dll4 for the treatment of different cancers. Nevertheless, the current therapeutic strategies to target Dll4, which were suggested to result in non-functional vessels in the tumors, appear also to affect the vasculature of normal organs. Given the importance of the Bone marrow (BM) vascular niche in regulating hematopoiesis, we hypothesized Dll4 targeting might also affect BM function. In the present study, we describe 2 means of targeting Dll4: one using Dll4 specific neutralizing monoclonal antibodies (systemic treatment), and another using VECadCreERT2Dll4lox/lox mice, where Dll4 is specifically ablated on VE-Cadherin expressing cells. Using these 2 models, we investigated if Dll4 targeting affected the BM vascular niche and consequently affected hematopoietic recovery following irradiation and BM recovery in a transplant setting.First, we show that the genetic ablation of Dll4 on VE-Cadherin+ vessels (VECadCreERT2Dll4lox/lox mice) and also anti-Dll4 antibody treatment of WT sub-lethally irradiated mice (300rad) does affect the BM vascular niche, resulting in increased megakaryocytes (CD41+), CD31+ and VE-Cadherin+ vessels, without significant changes on CD105+ vessels. Interestingly, the systemic anti-Dll4 treatment of non-irradiated and sub-lethally irradiated WT mice increased the BM and peripheral blood myeloid (CD11b+) content and promoted hematopoietic progenitor cell (Sca1+Flk1−) mobilization, as determined by FACS analysis. The genetic model (VECadCreERT2Dll4lox/lox mice) showed no changes in hematopoietic cells percentage or mobilization.However, BM recovery in a transplant setting was improved in both models. Lethally irradiated WT mice transplanted with anti-Dll4 treated BM following sub-lethal irradiation or BM from VECadCreERT2Dll4lox/lox mice showed faster hematopoietic recovery than control mice transplanted with WT BM alone.Taken together, we show that Dll4 blockade perturbs the BM vascular niche, increasing VE-Cadherin+ and CD31+ vessel number and also megakaryocyte BM content, both in the setting of Dll4 genetic ablation (using VECadCreERT2Dll4lox/lox mice) and also using a systemic approach to target Dll4. However, these vascular changes do not, at least following sub-lethal irradiation, significantly affect the BM recovery or affect the normal distribution of the hematopoietic populations. Nevertheless, the significant effects observed in a BM transplant setting (both where donor had been exposed to the systemic anti-Dll4 blockade and where Dll4 had been specifically ablated on VE-Cadherin+ vessels) suggest the existence of cell autonomous (and BM vascular niche-autonomous) effects of Dll4 targeting which deserve further detailed analysis. These data also suggest targeting Dll4-expressing vessels in the setting of BM malignancies may have therapeutic benefit, since it appears to have limited effects on normal BM/hematopoietic function. We are currently investigating the therapeutic efficacy of Dll4 targeting using the genetic model and the antibody-based approaches, in the setting of malignant myeloma and also acute leukemias. Disclosures:No relevant conflicts of interest to declare.

GM-CSF Signaling Abnormalities in Chronic Myelomonocytic Leukemia

Abstract 1713 Background:Chronic Myelomonocytic Leukemia (CMML) and Juvenile Myelomonocytic leukemia (JMML) are classified as MDS/MPN in the WHO classification system. Despite sharing clinical and histological features, CMML is characterized by a heterogeneous collection of molecular lesions while JMML is defined by well-established molecular aberrations clustered along the RAS pathway leading directly to GM-CSF hypersensitivity; a pathognomonic characteristic of JMML. Here we test whether a molecular signature for GM-CSF hypersensitivity in JMML, determined by the pSTAT5 activation assay, is also present in CMML and whether this signature clusters within a specific CMML subgroup. Methods:Cryopreserved bone marrow aspirates from 24 patients with newly diagnosed or relapsed CMML were obtained from the Moffitt Cancer Center Tissue Repository. Cells were thawed and rested in Stem Span H3000 with 10% FBS for 2 hours and then either starved for one hour in serum-free media, serum free group (n=12), or rested in Stem Span for an additional hour, serum group (n=12), prior to stimulation with G-CSF, IL-3, or GM-CSF for 15 minutes and then fixed and permeabilized with formaldehyde and methanol, as previously described. Samples were stained with an anti-pSTAT5(Y-694) antibody and analyzed by flow cytometry (Kotecha, Cancer Cell. 2009). Cells stained with isotype-control antibody were used to establish the threshold for basal STAT5 phosphorylation. Because STAT5 was constitutively phosphorylated in serum, and to a lesser extent in serum-free conditions, inducible cytokine activation was defined as the percentage of pSTAT5 positive cells above untreated samples in both CMML and healthy controls. A retrospective chart review was performed to obtain clinical variables including age, sex, WHO classification, Dusseldorf scoring system, MD Anderson scoring system, WBC, peripheral monocyte count, blast percentage, anemia, platelet count, splenomegaly, and metaphase cytogenetics. Results:The percentage of pSTAT5 responsive cells after G-CSF stimulation with doses up to 10 ng/ml was similar in cases and normal BM controls (p=0.14), whereas, a statistically significant increase in the percentage of inducible pSTAT5 positive cells was observed with GM-CSF 0.1 ng/ml (p=0.04), GM-CSF 1 ng/ml (p=0.02), and GM-CSF 10 ng/ml (p=0.01) in CMML BM cells compared to healthy donor BM cells, as shown in Figure 1. Using one standard deviation below the mean as a cut point, only 5 patients failed to show GM-CSF hypersensitivity in the serum (n=3) and serum-free groups (n=2), respectively. IL-3 and GM-CSF play similar roles in hematopoietic growth through the activation of JAK2/STAT5 and share a common beta-chain required for signaling. Signaling mediated by GM-CSF and IL3 converge to activate RAS and other downstream intermediates that regulate DNA synthesis, cell-cycle progression and suppression of apoptosis. The concentration of IL3 required to induce STAT5 phosphorylation was 10-fold greater than GM-CSF in CMML cells, but the percentage of cells responsive to IL3 was greater in CMML cases compared to controls at 10 ng/ml (p=0.02). Analysis of the percentage of GM-CSF hypersensitive cells and clinical parameters revealed no associations with age at onset, WHO classification, Dusseldorf scoring system, MD Anderson scoring system, blast percentage, anemia, platelet count, splenomegaly, or karyotype. The percentage of pSTAT5 positive cells with GM-CSF 0.1 ng/ml positively correlated with the total leukocyte (p=0.03) and total monocyte (p=0.02) count indicating that the JAK2/STAT5 signaling response is indicative of disease burden. [Display omitted] Conclusions:Based on the threshold for cytokine stimulation and percentage of cells that display pSTAT5 induction, CMML appears to preferentially utilize GM-CSF for survival and/or expansion. Although RAS mutations were not assessed, CMML cells were preferentially sensitive to GM-CSF in newly diagnosed cases independent of cytogenetic abnormalities suggesting that JMML and CMML share biological features of GM-CSF hypersensitivity. Disclosures:Padron:KaloBios Pharmaceuticals, Inc.: Research Funding. Bebbington:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership. Baer:KaloBios Pharmaceuticals, Inc.: Employment, Equity Ownership.

Nonobese Diabetic Natural Killer Cells: A Barrier to Allogeneic Chimerism That Can be Reduced by Rapamycin

Induction of allogeneic hematopoietic chimerism is a promising strategy to induce tolerance to donor islets for treating type 1 diabetes. Successful induction of chimerism requires overcoming host alloimmunity. In diabetes-prone nonobese diabetic (NOD) mice, this is challenging due to their general tolerance resistance. Although the adaptive alloimmunity of NOD mice is a known barrier to allogeneic chimerism, whether NOD natural killer (NK) cells are an additional barrier has not been examined. Because NOD NK cells exhibit functional defects, they may not inhibit chimerism generation. Antibody depletion of NK cells in vivo, or transplantation of F1 hybrid donor cells to eliminate the "missing-self" trigger of NK cells, was preformed to test the NK-mediated rejection of donor bone marrow cells. We also studied the capacity of rapamycin to block the NK cell response against allogeneic cells in vivo. Depleting NK cells or rendering them unresponsive to the donor greatly improved the level of chimerism obtained in NOD mice. Rapamycin significantly reduced the resistance to allogeneic chimerism mounted by NOD NK cells; however, it was much less effective than NK cell depletion by antibodies. Contrary to the view that NOD NK cells are defective, we found these cells to be a substantial barrier to allogeneic chimerism in the presence or absence of adaptive immunity. Moreover, rapamycin will need to be combined with other approaches to fully overcome the NK cell barrier.