Donor Bone Marrow Cells Research Articles

B-Cell-Dependent Memory T Cells Impede Nonmyeloablative Mixed Chimerism Induction in Presensitized Mice

Presensitization to HLA antigens limits the success of organ transplantation. The achievement of donor-specific tolerance via mixed chimerism could improve outcomes of transplantation in presensitized patients. In presensitized B-cell-deficient μMT B6 mice, we developed nonmyeloablative bone marrow transplantation (BMT) regimens that successfully tolerized presensitized T cells, achieving long-term (LT) multilineage chimerism and tolerance to donor-type skin. To apply these regimens in wild-type (WT) animals while avoiding antibody-mediated destruction of donor bone marrow cells, presensitized WT B6 mice were rested >2 years to allow alloantibody clearance. However, chimerism and tolerance were not reliably achieved in LT presensitized WT B6 mice in which alloantibody had declined to minimal or undetectable levels before BMT. Strong antidonor memory T-cell responses were detected in LT presensitized WT B6 mice after rejection of donor bone marrow (BM) occurred, whereas levels of alloantibody remained consistently low. In contrast, presensitized μMT B6 mice had diminished memory T-cell responses compared to WT B6 mice. These data implicate T-cell memory, but not alloantibody, in rejection of donor BM in LT presensitized WT mice.

Vascularized Bone Marrow Transplantation Model in Rats as an Alternative to Conventional Cellular Bone Marrow Transplantation: Preliminary Results

The aim of the study was to follow the development of microchimerism after allogeneic vascularized bone marrow transplantation (VBMT) versus conventional bone marrow transplantation (BMT). In one group, a VBMT model consisted of donor Brown Norway rat hind limb heterotopic transplanted on recipient Lewis rats. An intravenous infusion of donor bone marrow cells in suspension equivalent to that grafted in the vascularized femur limb was administered intravenously to recipient rats in the second group. Cellular microchimerism was investigated in recipients of VBMT versus BMT. Donor-derived cells could be detected in VBMT recipients at 30 and 60 days but not in recipients of intravenous suspension of BMC. VBMT provides a theoretical alternative to conventional cellular bone marrow transplantation by addressing crucial clinical problems such as failure of engraftment or graft-versus-host disease.

Biologics and Donor Bone Marrow Cells for Targeted Immunomodulation in Vascularized Composite Allotransplantation: A Translational Trial in Swine

Introduction Bone marrow (BM) infusion following organ transplantation is a prerequisite for potential donor-antigen-specific tolerance induction. We developed a preclinical swine model to determine the optimal dose of BM cells to achieve microchimerism. Furthermore, induction therapy was optimized by augmenting the BM infusion with biologics in the form of costimulatory blockade: cytotoxic T-lymphocyte antigen 4 immunoglobulin (CTLA4-Ig). Materials and Methods Yucatan miniature swine ( n = 12) underwent total body and thymic irradiation for cytodepletion. Animal groups received 15, 30, or 60 million cells per kilogram of whole unmodified BM. The optimal dose of BM cell infusion (BMT) was then applied to subsequent experiments evaluating the addition of CTLA4lg. Group 1 (control) received no treatment. Group 2 received FK506 only; group 3 received irradiation, BMT, and FK506; group 4 received FK506 and CTLA4-lg. Results Microchimerism was established in all animals after BM cell infusion; at postoperative day 9, it was significantly increased for 60 million cells per kilogram ( P = .0001). Transplanted animals in group 1 rejected the allograft 5 to 8 days after transplantation. Group 2 rejected the allograft (skin and muscle) 30 to 32 days after transplantation (2 days after cessation of immunosuppression). Group 3 rejected the skin portion of the allograft at 50, 52, and 53 days posttransplant. Remaining allograft components (muscle, bone, nerve, vessel) survived indefinitely. Group 4 animals demonstrated significantly prolonged muscle survival beyond 150 days posttransplant; the skin component survived past 150 days in two of three animals. Skin and muscle histology in all long-term surviving animals were normal. Conclusions BM cell infusion with 60 million cells per kilogram results in stable levels of microchimerism. The addition of costimulatory blockade (CTLA4lg) prolonged allograft skin survival and overall graft survival. Such targeted immunomodulatory protocols might facilitate immune tolerance and eliminate the need for multidrug immunosuppression to maintain graft survival after vascularized composite allotransplantation.

Replacement of Liver Parenchyma in Analbuminemic Rats with Allogenic Hepatocytes is Facilitated by Intrabone Marrow-Bone Marrow Transplantation

Although hepatocyte transplantation (HCTx) is expected to become a useful therapy for human liver diseases, allogenic hepatocytes still tend to be rejected within a short period due to host immunosurveillance. In the present study, we investigated the effect of prior bone marrow transplantation (BMTx) for the engraftment of allogenic hepatocytes using the analbuminemic rat transplantation model. The hepatocytes of Lewis (LEW) rats were not accepted in the liver of retrorsine (RS)/partial hepatectomy (PH)-treated analbuminemic F344 (F344-alb) rats, which express the disparate major histocompatibility complex (MHC) against that of LEW rats. Prior BMTx with the LEW bone marrow cells (BMCs) after sublethal irradiation achieved acceptance and repopulation of LEW hepatocytes in the liver of the RS/PH-treated F344-alb rats, associated with elevation of serum albumin. The replacement of hepatic parenchyma with albumin positive (Alb(+)) donor hepatocytes and elevation of serum albumin levels were dependent on the bone marrow reconstitution by donor BMCs, which was more efficiently achieved by intrabone marrow (IBM)-BMTx than by intravenous (IV)-BMTx. Our results demonstrate that efficient bone marrow reconstitution by IBM-BMTx enables the replacement of the hepatic parenchyma with allogenic hepatocytes in RS/PH-treated analbuminemic rats without immunosuppressants.

MP-06.18 Augmentation of Chimerism by Donor Bone Marrow Cells Infusion in Kidney Allograft Recipients

MP-06.18 Augmentation of Chimerism by Donor Bone Marrow Cells Infusion in Kidney Allograft Recipients

Microchimerism in promoting graft acceptance in clinical transplantation

Infusions of bone marrow-derived cells together with 'space making' continue to be tested in clinical organ transplant tolerance protocols. These trials are based on the hypothesis that this might produce initial multilineage chimerism. There is some evidence that this in turn induces regulatory cells that control alloimmunity. Although a wealth of knowledge is available from animal models, this review deals with what we know or can speculate about donor bone marrow cells and chimerism in human organ transplantation. Calcineurin inhibitors are employed in most of these protocols to blunt the initial immune response. One protocol also has a stepwise regulatory cell generating treatment with sirolimus before total withdrawal. A number of donor chimeric lineages including stem cells, dendritic cells, myeloid precursors, and various lymphoid subpopulations have been described. Currently, it is recognized that the nature of cells that make up the chimerism could influence graft rejection versus acceptance. Tolerogenic donor chimeric cells may also generate regulatory subsets, thus controlling alloimmunity on two fronts. It might be speculated that prolonged and sustained regulation or possible anergy induced by chimerism may eventually lead to clonal deletion, thereby bringing about classical immunologic tolerance.

Immunosupressive properties of human placenta-derived mesenchymal stem cells on control of acute graft-versus-host disease in mice.

6547 Background: Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (HSCT) is still associated with significant morbidity and mortality. High-dose corticosteroids are standard in initial treatment of patients with acute GVHD achieving response rates of 30-70%. However, no standard therapy exists once a patient has developed acute GVHD refractory to corticosteroids. In this respect, the immunomodulatory properties of mesenchymal stem cells (MSC) have been applied to reduce the incidence the GVHD after allogeneic HSCT. Among the various sauces of MSC which have immunomodulatory effect in vitro, only human placenta MSC (hP-MSC) has not elucidated in the GVHD in vivo model. In this study, we characterize the phenotype of hP-MSC, investigate their immunomodualotory properties in vitro and evaluate the clinical possibilities of hP-MSC for the control of GVHD in MHC mismatch mice model. Methods: A GVHD mouse model was established by transplanting C57BL/6 donor bone marrow (BM) cells and spleen cells into lethally irradiated BALB/c recipient mice. To control the GVHD, hP-MSC was transplanted to recipient mice (5 x 105 or 1 x 106). Results: We purified and expanded the hP-MSC characterized by flow cytometry (positive for CD105, CD73, CD44, CD56 and HLA-ABC but negative for CD3, CD14, CD31, CD45, CD11a, CD34 and CD117). These cells also exhibited mesenchymal differentiation potential, as assessed by culturing in adipogenic, osteogenic, or chondrogenic medium. In vitro experiment, the hP-MSC suppressed mitogen-stimulated T cell proliferation in a dose-dependent manner. Moreover, hP-MSC inhibited cytokine secretion (IL-12, TNF-α, and INF-γ) of T-cells in response to mitogen. In vivo experiment, survival rate in hP-MSC (1x106) group was higher than in the control group, and histological scores were low in hP-MSC (1x106) group. Conclusions: We conclude that hP-MSC can efficiently control the GVHD associated with allogeneic HSCT in mice model.

A reduction of recipient regulatory T cells by cyclophosphamide contributes to an anti‐tumor effect of nonmyeloablative allogeneic stem cell transplantation in mice

We have recently established a unique model system of nonmyeloablative allogeneic stem cell transplantation (SCT) for treatment of murine solid tumors, based on cyclophosphamide-induced tolerance. An injection of allogeneic donor spleen cells and bone marrow cells (BMC) followed by cyclophosphamide treatment induced a stable mixed chimerism with long lasting tolerance to the allografts. A donor lymphocyte infusion (DLI) in the cyclophosphamide-induced tolerant mice exerted strong anti-tumor effects on an MBT-2 murine bladder tumor, MBT-2 via their graft versus tumor (GVT) activity. In the present study, we determined whether a cyclophosphamide-induced reduction of naturally occurring regulatory T cells (Tregs) was associated with the anti-tumor activity in our nonmyeloablative SCT system. The number of recipient CD4+ CD25+ Foxp3+ Tregs significantly decreased 3 days after an intraperitoneal injection of cyclophosphamide in C3H/HeN mice that had been injected with spleen cells and BMC of donor AKR/J mice, compared with the number of CD4+ CD25+ Foxp3- T cells. An adoptive transfer of CD4+ CD25+ T cells from naïve C3H/He x AKR/J F1 mice into recipient mice 1 day after DLI significantly suppressed the expansion and IFN-γ production of host-reactive donor CD4+T cells and hampered the MBT-2 anti-tumor activity when compared with the transfer of CD4+ CD25- T cells. These results indicated that cyclophosphamide-induced reduction of recipient Tregs is associated with retardation of tumor progression via the expansion of host-reactive donor T cells and IFN-γ production after DLI in our nonmyeloablative SCT system.

Bioimaging for the monitoring of thein vivodistribution of infused mesenchymal stem cells in a mouse model of the graft-versus-host reaction

Cell therapy using MSCs (mesenchymal stem cells) might be effective treatment for refractory GVHD (graft-versus-host disease). However, the fate and distribution of MSCs after transplantation remains unclear. In this study, an animal model was developed to monitor the dynamic distribution of MSCs in mice with GVHD. A GVHD mouse model was established by transplanting C57BL/6 donor bone marrow cells and C57BL/6 EGFP (enhanced green fluorescent protein) splenocytes into lethally irradiated BALB/c nude recipient mice. Donor MSCs were obtained from MHC-identical C57BL/6 RFP (red fluorescent protein) mice and infused into the recipient mice on the same transplantation day. In vivo movement of the donor splenocytes (EGFP) and MSCs (RFP) were evaluated by measuring the biofluorescence (IVIS-Xenogen system). Donor splenocytes and MSCs reached the lungs first, and then the gastrointestinal tract, lymph nodes and skin, in that order; the transit time and localization site of these cells were very similar. In the recipient mouse with GVHD, the number of detectable cells declined with time, as assessed by biofluorescence imaging and confirmed by RT (real-time)-PCR. This bioimaging system might be useful for preclinical testing and the design of therapeutic strategies for monitoring the dynamic distribution of MSCs with GVHD.

Serum Levels of Interleukin (IL)-10, IL-17, Transforming Growth Factor (TGF)-β1, and Interferon-γ Cytokines and Expression Levels of IL-10 and TGF-β1 Genes in Renal Allograft Recipients After Donor Bone Marrow Cell Infusion

Background Cytokine storm generated by an alloimmune response after transplantation can lead to either graft survival or rejection. The aim of this study was to evaluate the serum levels of interleukin (IL)-10, IL-17, transforming growth factor (TGF)-β1, and interferon (IFN)-γ and expression levels of IL-10 and TGF-β1 in renal allograft recipients with or without donor bone marrow cell infusion (DBMI). Methods We retrospectively followed 28 living unrelated kidney recipients, including 14 with and 14 without DBMI infusion for 2 years. Also, 14 healthy subjects were included as a normal control group. PBMC gene expression analysis for mRNA levels of IL-10 and TGF-β1 cytokines relative to β-actin as a reference gene was performed using quantitative fluorescence real-time polymerase chain reaction at the end of 2 years posttransplantation. Also, serum levels of IL-10, TGF-β1, IFN-γ, and IL-17 in the 3 groups were measured by enzyme-linked immunosorbent assay at the same time. Results Both patient groups showed increased gene expression and serum content of IL-10 compared with normal controls. The expression levels were only significant between control patients and normal subjects ( P = .02). Serum levels of IFN-γ and IL-17 were higher in untreated patients compared with normal controls ( P = .03 and P = .07, respectively). DBMI patients showed significantly lower levels of serum TGF-β1 and IL-17 compared with normal subjects ( P = .05 and P = .06, respectively). Also, infused patients showed a positive correlation between circulating levels of IL-17 and IL-10 ( r = 0.692; P = .006), and an inverse correlation between serum creatinine and TGF-β1 levels ( r = −0.580; P = .03). Conclusion The decreased levels of inflammatory cytokines besides IL-10 with increased TGF-β1 levels and better allograft function with improved clinical outcomes were observed among infused patients, possibly indicating immunomodulatory effects of this approach in kidney allograft patients.

Dendritic cell depletion and repopulation in the lung after irradiation and bone marrow transplantation in mice

Macrophages and dendritic cells are essential for innate and adaptive immunity, respectively and have been reported to exhibit variable radiosensitivities in response to irradiation in various bone marrow transplantation (BMT) protocols. In order to address this controversy, we analyzed the magnitude of depletion and repopulation of both “conventional“ CD11bpos DC and CD103pos DC subsets as well as different pulmonary macrophage subsets in the lung tissue in response to irradiation and BMT in a mouse model. In our study, CD45.2pos donor bone marrow cells were transplanted into irradiated CD45.1pos recipient mice, in order to examine the depletion of recipient-type and the repopulation of donor-type DC and macrophage subsets. We observed an apoptosis- and necrosis-mediated >90% depletion of the recipient CD103pos DC subset and only a 50–60% depletion of recipient CD11bpos DC from lung parenchymal tissue at day 3 and 5, whereas recipient alveolar and lung macrophages were much less radiosensitive, showing ˜50% depletion by days 14–21 post-treatment. Complete repopulation of lung tissue with donor DC subsets was observed by days 10 and 28 for CD11bpos DC and CD103pos DC, whereas alveolar and lung macrophages were repopulated by 6 and 10 weeks post-treatment. Furthermore, infection of mice with S. pneumoniae highly accelerated the turnover of lung DC as well as lung macrophage subsets. Our data illustrate the vulnerability of lung CD103pos DC and CD11bpos DC to irradiation and indicate an accelerated turnover of lung DC subsets relative to pulmonary macrophages. Our findings may have important implications for the development of adjuvant immune-stimulatory protocols which could reduce the risk of opportunistic infections in patients undergoing BMT.

Advanced Donor Age Impairs Bone Marrow Cell Therapeutic Efficacy for Cardiac Disease

Therapeutic results of clinical autologous bone marrow cell (BMC) therapy trials for cardiac disease have been modest compared to results of BMC implantation into rodent hearts post-myocardial infarction (MI). In clinical trials, autologous BMCs are typically harvested from older patients who have recently suffered an MI. In contrast, experimental studies in rodent models typically utilize donor BMCs isolated from young, healthy, inbred mice that are not the recipients. Using unfractionated BMCs from donor mice at ages of young, middle-aged, and old, we discovered that recipient left ventricular function post-MI was significantly improved by young donor BMC implantation but was only preserved by middle-aged donor BMCs. Notably, old donor BMCs did not slow the decline in recipient post-MI cardiac function, suggesting BMC impairment by advanced donor age. Furthermore, we also show here that BMCs that are therapeutically impaired by donor age can be further impaired by concurrent donor MI. In conclusion, our findings suggest that therapeutic impairment of BMCs by advanced age is one of the important factors that can limit the success of clinical autologous BMC-based therapy.

Bone marrow cells participate in tumor vessel formation that supports the growth of Ewing’s sarcoma in the lung

An MHC-mismatch bone marrow (BM) transplant Ewing's sarcoma mouse model was used to investigate whether BM cells participate in the vessel formation that support Ewing's sarcoma lung metastasis. BM cells from H-2K(b/d) donor mice were transplanted into sublethally irradiated H-2K(d) recipient mice. Donor BM cells were identified using the H-2K(b) marker. Engraftment was confirmed by identifying the H-2K(b) IL-1β-type specific polymorphism. After engraftment highly lung metastatic TC71-PM4 cells were injected intravenously. Mice were sacrificed 10 weeks after tumor cell injection. Hematoxylin-and-eosin staining was performed to identify lung metastatic foci. These tumors were then evaluated using immunohistochemical analysis. H-2K(b)-positive cells were found in lung metastases but not in normal lung, liver or spleen tissues. Injection of CM-Dil-labeled BM cells into tumor bearing and control mice showed that nonspecific organ migration occurred at 24 h, but that these cells were absent 1 week later in control mice. These data suggest that the migration of the H-2K(b) BM cells to lung nodules was specific because these cells were observed 14 weeks after transplantation. Co-localization of H-2K(b) and CD31 or VE-Cadherin demonstrated that some endothelial cells were BM-derived. Co-localization of H-2K(b) and Desmin, smooth muscle actin (α-SMA) or PDGFR-β indicated that a fraction of pericytes was also BM-derived. These results suggest that BM cells participate in the vascular formation that supports the growth of Ewing's sarcoma lung metastases. BM cells migrated to the metastatic tumor and differentiated into endothelial cells and pericytes. These data indicated that targeting this process may have therapeutic potential.

Dendritic Cell Depletion and Repopulation in the Lung after Irradiation and Bone Marrow Transplantation in Mice

Dendritic cells (DCs) are essential for innate and adaptive immunity, but are purported to exhibit variable radiosensitivity in response to irradiation in various bone marrow transplantation (BMT) protocols. To address this controversy, we analyzed the magnitude of depletion and repopulation of both lung CD11b(pos) DC and CD103(pos) DC subsets in response to irradiation and BMT in a murine model. In our study, CD45.2(pos) donor bone marrow cells were transplanted into irradiated CD45.1(pos) recipient mice to examine the depletion of recipient DC subsets and the repopulation of donor DC subsets. We observed an apoptosis-mediated and necrosis-mediated depletion (> 90%) of the recipient CD103(pos) DC subset, and only a 50-60% depletion of recipient CD11b(pos) DCs from lung parenchymal tissue on Days 3 and 5, whereas recipient alveolar and lung macrophages were much less radiosensitive, showing an approximately 50% depletion by Days 14-21 after treatment. A repopulation of lung tissue with donor DC subsets had occurred by Days 10 and 28 for CD11b(pos) DCs and CD103(pos) DCs, whereas alveolar and lung macrophages were repopulated by 6 and 10 weeks after treatment. Furthermore, the infection of mice with Streptococcus pneumoniae further accelerated the turnover of lung DCs and lung macrophage subsets. Our data illustrate the vulnerability of lung CD103(pos) DCs and CD11b(pos) DCs to irradiation, and indicate that an accelerated turnover of lung DC subsets occurs, relative to pulmonary and lung macrophages. Our findings may have important implications in the development of adjuvant immune-stimulatory protocols that could reduce the risk of opportunistic infections in patients undergoing BMT.

P90RSK2 Is Essential for FLT3-ITD-, but Dispensable for BCR-ABL-Induced Myeloid Leukemia

AbstractAbstract 1716p90 ribosomal S6 kinase 2 (p90RSK2) is a serine/threonine kinase that plays an active role in diverse cellular processes, including gene expression, cell proliferation, and survival. We previously demonstrated that RSK2 signaling plays a key role in the pathogenesis and disease progression of leukemogenic FGFR3 associated hematopoietic malignancies, including FGFR3-expressing t(4;14) multiple myeloma and TEL-FGFR3-expressing t(4;12)(p16;p13) peripheral T cell lymphoma. In this study, we found that p90RSK2 is commonly activated in diverse leukemia cell lines expressing different leukemogenic tyrosine kinases, including K562 (BCR-ABL), Molm14 and Mv4;11 (FLT3-ITD), HEL (JAK2 V617F), and EOL-1(FIP1L1-PDGFR alpha). We next examined the role of RSK2 in myeloid transformation induced by BCR-ABL and FLT3-ITD due to their high frequency of occurrence in CML and AML, respectively. Interestingly, although RSK2 is activated by BCR-ABL in both stably transduced Ba/F3 cells and K562 human leukemia cells, we found that genetic deficiency of RSK2 does not affect the pathogenesis or disease progression of myeloproliferative disease induced by BCR-ABL in a murine bone marrow transplant (BMT) model using wild type or RSK2-/- donor bone marrow cells. This finding suggests that RSK2 is dispensable for BCR-ABL induced myeloproliferative disease. Moreover, targeting RSK2 by treatment with potent and highly specific RSK inhibitor fmk did not effectively induce apoptosis in K562 human leukemia cells expressing BCR-ABL, or in primary leukemia cells from BCR-ABL positive CML patients. In contrast, we found that targeting RSK2 may represent an effective therapy to treat patients with FLT3-ITD positive AML. Treatment with fmk induced significant apoptotic cell death in Molm14 and Mv4;11 human leukemia cells expressing FLT3-ITD, as well as in primary leukemia cells from FLT3-ITD positive AML patients. In consonance with these results, FLT3-ITD induced T-cell lymphoma in a BMT assay using RSK2-/- donor bone marrow cells, phenotypically distinct from the myeloproliferative disease induced by FLT3-ITD using wild type donor bone marrow cells. These results suggest that RSK2 is required for FLT3-ITD induced hematopoietic transformation, likely playing a role in pathogenesis and lineage determination. Together these findings suggest that the role of RSK2 in hematopoietic transformation may depend on different upstream oncogenic signals mediated by different leukemogenic tyrosine kinases. Our data also demonstrate that RSK2 may represent an alternative therapeutic target in the treatment of FLT3-ITD positive leukemia. Disclosures:No relevant conflicts of interest to declare.

The Scd1 Gene Functions as a Tumor Suppressor In Leukemia Stem Cells

AbstractAbstract 201We have previously shown that the arachidonate 5-lipoxygenase gene (Alox5) functions as a critical regulator of leukemia stem cells (LSCs) in BCR-ABL-induced chronic myeloid leukemia (CML) in mice. The Alox5 pathway appears to represent a major molecular network in LSCs. Taking advantage of our DNA microarray analysis for the identification of critical genes regulated by BCR-ABL in LSCs, we identified a small group of candidate genes that likely play tumor suppressor roles in these stem cells, and among them, a gene called stearoyl-CoA desaturase 1 (Scd1), an endoplasmic reticulum enzyme catalyzing the biosynthesis of monounsaturated fatty acids from saturated fatty acids, was shown to have a strong inhibitory effect on survival of LSCs in CML mice. BCR-ABL transduced bone marrow cells from Scd1-/- mice induced CML much faster than BCR-ABL transduced wild type bone marrow cells, and overexpression of Scd1 dramatically delayed CML development. Therefore, we further investigated whether Scd1 suppresses LSCs. FACS analysis showed that the percentages and total numbers of LSCs (GFP+ Lin−c-Kit+Sca-1+) and long-term (GFP+ Lin−c-Kit+Sca-1+ CD34−) or short-term (GFP+ Lin−c-Kit+Sca-1+ CD34+) LSCs in bone marrow of recipients of BCR-ABL transduced Scd1-/- donor bone marrow cells were significantly higher than those in bone marrow of recipients of BCR-ABL transduced wild type donor bone marrow cells, suggesting that Scd1 suppresses LSCs. Next we did a competitive repopulation assay to examine the function of LSCs. LSCs were sorted by FACS from bone marrow of mice with primary CML induced by transplanting BCR-ABL-transduced Scd1-/- (CD45.2) or wild type (CD45.1) bone marrow cells. The sorted CD45.2 and CD45.1 LSCs were mixed in a 1:1 ratio, followed by transplantation into lethally irradiated recipient mice to induce secondary CML. At 8 weeks after transplantation, only less than 10% of GFP+Gr-1+ cells were CD45.1 leukemia cells derived from wild type mice, whereas more than 75%-80% of GFP+Gr-1+ cells in peripheral blood of the mice were CD45.2 leukemia cells derived from Scd1-/- mice. To determine how Scd1 deficiency affects the maintenance of LSCs, we examined the cell cycle and apoptosis of LSCs. We found the percentages of apoptotic LSCs (Annexin V+ cells) were significantly decreased in bone marrow and spleens from Scd1-/- CML mice compared to the wild type group; however, we did not observe significant differences in the cell cycle status of LSCs from bone marrow and spleen, indicating that Scd1 regulates apoptosis but not cell cycle of LSCs.PPARγ agonist rosiglitazone can increase Scd1 expression, and our real time PCR data showed that rosiglitazone significantly induced Scd1 expression in bone marrow cells from CML mice. Therefore, we used PPARγ agonist rosiglitazone to treat these cells, and observed that rosiglitazone treatment dramatically decreased LSCs and that loss of Scd1 partially rescued the effect of PPARγ agonist on LSCs. Further, we investigated the molecular mechanisms that may contribute to the acceleration of CML development resulting from Scd1 deficiency. The decreased apoptosis in LSCs from Scd1-/- CML mice led us to focus on apoptosis-related genes. Real time PCR analysis showed a significant decrease of p53 in Scd1-/- immature leukemia cells as compared with that in wild type immature leukemia cells; however, loss of Scd1 resulted in dramatically and modest increased expression of Bcl-2 and Mcl-1 respectively in immature leukemia cells. We also found that the tumor suppressor gene Pten was significantly downregulated in Scd1-/- LSCs. Together, our results demonstrate a novel tumor suppressor function of Scd1 in LSCs, and provide a rationale for suppressing LSCs by enhancing Scd1 expression. Disclosures:No relevant conflicts of interest to declare.

Engraftment Capacity of Intratibialy Injected Multiple Myeloma Patient-Derived Bone Marrow-Cells In Vivo

AbstractAbstract 3010 Introduction:In order to allow a better understanding of multiple myeloma (MM), the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation advantageously. Here, the engraftment capacity of MM patient-derived bone marrow cells implanted into NOD/SCID-IL2-receptor-gamma-chain-/- (NSG) mice was meticulously investigated. Material and Methods:Bone marrow cells from 7 MM patients were injected intratibialy into NSG mice (n=5/patient). As controls, 5 mice received healthy donor bone marrow and 5 mice were mock-injected. Tumor growth was monitored via a) daily MM-symptom acquisition, such as hind limb paresis, apathy and consistent foot dragging, b) FACS (human HLA-A,B,C; CD138; CD45; CD38) and c) fluorescence-based in vivo imaging (FI, Kodak FX, Alexa750 labeled anti-human CD138, CD38, CD45 and HLA-ABC) in bone marrow, peripheral blood, spleen and lymph node sites of the respective animals. Results:There were significant differences in engraftment capacity, persistence of human cells and expression of selected markers between bone marrow of MM patients and healthy donors: 1.) infiltration of the spleen and lymph nodes was exclusively detected in NSG-mice bearing patient-derived MM cells, whereas cells of healthy donors were - if detected - exclusively found within the murine bone marrow; 2.) mean FI-areas in the bone marrow of MM-patient-derived injected mice were significantly increased as compared to mice bearing bone marrow cells of healthy donors (p=0.006); 3.) patient-derived MM cells expressed CD138, CD38 and HLA-ABC. In contrast, bone marrow cells of healthy donors expressed exclusively CD45 and CD138. The CD138 cell population determined by FACS in patients' bone marrow cells (before NSG-injection) decreased from a median of 11.3% to 0.8% 56 days after implantation (in NSG mice), either due to preferably CD138-negative plasma cell engraftment or the CD138 loss within the murine environment as previously described.Fifty-six days after implantation, patient-derived MM cells could be detected in all animals via FACS-analysis. Follow-up analyses by FI confirmed, that bone marrow engraftment was prominent and observed in all (35/35) NSG mice, albeit also in others organs. Patient-derived MM cells within the bone marrow could be detected in parallel via FACS- and FI-analyses in 10 NSG mice and within the peripheral blood in 12 NSG mice (total of 35 mice being examined). Maximal bone marrow-, peripheral blood- and spleen-engraftment numbers in NSG mice were as high as 4%, 25% and 52%, respectively, suggesting that in peripheral blood- and spleen-sites, MM-cell engraftment could even surmount that of bone marrow-sites. Spleen and other organ involvement observed in our xenografts have been confirmed in previous murine MM-models (Murillo et al. Clin Cancer Res, 2008), postulating that similarly to spleen-colony-forming-cells in hematopoiesis, spleen and other sites serve as fertile tumor engraftment locations.Differences in engraftment capacity and expression pattern between respective patient-derived MM specimen were evident, but did not strikingly correlate with MM-patients' characteristics, such as MM-subtypes, disease stage or expression pattern of the primary material; this observation also well correlating with previous reports (e.g. (Pilarski et al. Blood, 2000). Conclusions:Murine MM-models have shown to be exceedingly challenging in their ability to induce valid and trustworthy MM-patient-derived cell engraftment; here our NSG model suggest to harbor MM-cells. Our data demonstrates that intratibially-injected NSG mice mimic the clinical MM disease with respect to the disseminated nature of the disease and the indispensable engraftment of clonogenic plasma cells into the bone marrow. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor MM growth. Disclosures:No relevant conflicts of interest to declare.

Role of the Spleen In CML: Lessons from an Inducible Transgenic Mouse Model.

AbstractAbstract 3384Spleen size is one of the most important independent pre-treatment risk factors (Sokal and Hasford Scores) in patients with chronic myeloid leukemia (CML). However, the role of the spleen in the pathogenesis of CML remains poorly defined. We used an inducible transgenic mouse model of CML (ScltTA/Bcr-Abl) in which we have previously shown that the disease phenotype is transplantable using unfractionated BM cells or Lin−c-kit+Sca-1+ (LSK) cells. Here, we compared the effects of Bcr-Abl induction on cells derived from the bone marrow (BM) versus spleen and assessed primary mice as well as transplanted and serially transplanted congenic recipients by flow cytometry, clonogenic assays, Bcr-Abl expression levels, and histology. In addition, we assessed the effect of imatinib treatment on leukemic spleen cells in comparison to BM cells in transplanted mice. The results showed that splenomegaly and splenic neutrophilia occurred as early as seven days after induction of Bcr-Abl by tetracycline withdrawal, and this was followed by an increase of clonogenic growth in the spleen but not BM as well as disruption of the splenic architecture on day 14. The percentage of LSK cells was increased 4.9-fold in the spleen but only 1.5-fold in the BM on day 28 after Bcr-Abl induction. To address functional differences including the self-renewal potential between BM- and spleen-derived CML-initiating cells, we conducted comparative transplantations of induced vs. un-induced BM and spleen cells. While Bcr-Abl positive BM-transplanted recipients developed CML-like disease including neutrophilia and splenomegaly, there was no significant difference between recipients transplanted with induced vs- un-induced BM donor cells. Conversely, mice transplanted with induced spleen cells showed significantly increased percentages of granulocytic Gr1+ donor cells in BM and spleen and increased splenomegaly compared to recipients transplanted with un-induced spleen cells. Moreover, upon serial transplantation of spleen cells from these primary transplant recipients, peripheral blood analysis revealed an increased percentage of donor Gr1+ granulocytic cells in mice receiving spleen cells that had initially been induced before the first transplantation as compared to un-induced cells. Interestingly, upon serial transplantation of BM-derived cells, recipient mice developed neither an increase of granulocytic Gr1+ cells in the BM nor splenomegaly. However, percentages of immature granulocytes (Gr1low/CD11b+) were significantly increased in the spleen of mice transplanted with induced and un-induced BM cells but not in BM. Imatinib treatment in BM-transplanted recipients turned out to be less effective in spleen cells compared to BM cells. While percentages of mature donor granulocytes (Gr1high) in BM and numbers of granulocytes in PB were decreased to control levels upon 4 weeks of imatinib treatment, donor granulocytes cells were still significantly increased in the spleen. In conclusion, our data show significant differences in the dynamics and function of Bcr-Abl positive BM vs. spleen hematopoiesis. While our previous data showed that Bcr-Abl enhances differentiation of LT-HSCs in BM, our current findings suggest that leukemic, self-renewing cells are increased in the spleen thus implying that the spleen provides a reservoir for proliferating CML-initiating cells. Disclosures:Koschmieder:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Membership on an entity's Board of Directors or advisory committees.

Rational Targeting of the Signaling GTPase Cdc42 Establishes a Non-Myeloid Ablative Regimen for Hematopoietic Stem Cell Engraftment In Mouse Models

AbstractAbstract 73Hematopoietic stem cell (HSC) transplantation has become a standard of care for the treatment of many hematological disorders such as Fanconi anemia. However, current myeloid ablative or chemotherapy conditioning regimens are associated with significant morbidity and mortality. In addition, outcomes of transplantation can be limited by low infused stem cell numbers, especially in the setting of umbilical cord blood or autologous transplantation. Thus, availability of agents that could optimize entry of stem cells into bone marrow (BM) niche would be of great value in improving HSC transplant outcome. Recent studies have shown that HSC mobilization before transplantation might vacate BM niche and thus allow improved engraftment of donor HSCs (Blood 113, 4856). Previously we reported that conditional knockout of the Rho GTPase Cdc42 from BM cells led to massive egress of hematopoietic stem/progenitor cells (HSPCs) from BM to peripheral blood (PB) in mice (PNAS 104, 5091). We have also shown that a novel Cdc42 activity-specific inhibitor, CASIN, is able to transiently mimic Cdc42 knockout phenotype in mice in inducing HSPC mobilization from mouse BM by suppressing actin polymerization, adhesion, and directional migration (Blood 112, 68). In the present studies, we hypothesize Cdc42 targeting can open up BM niche to facilitate subsequent HSC engraftment. First, in a conditional Cdc42 knockout mouse transplant model (CD45.2+ MxCre;Cdc42flox/flox BM engrafted in congenic recipients) we showed that poly I:C induction followed by transplant of congenic donor BM cells resulted in significant engraftment of donor blood accompanied by a loss of Cdc42−/− cells. The donor-derived cells in both BM and PB made up of ∼70% chimerism in multiple blood lineages including Lin−Sca+c-Kit+ primitive progenitors (74.62±5.5%). The multi-lineage donor chimerism was maintained in secondary transplant, indicating effective long-term HSC engraftment. In contrast, no significant engraftment was observed in mice of MxCre;Cdc42wt/wt BM genotype after similar polyI:C induction. These experiments indicate that genetic deletion of Cdc42 in host HSCs can allow efficient donor engraftment without irradiation or myeloid ablative conditioning. Second, we found CASIN treatment of recipient mice transiently suppressed low density BM cell Cdc42 activity and mimicked the Cdc42 knockout effect in mobilizing long-term HSCs to allow congenic donor HSC engraftment. Three i.p. injections of CASIN to recipient mice prior to infusion of donor BM cells resulted in 8.66±2.3% chimerism of multi-lineage donor derived cells 4 months post-transplant, while the vehicle treated mice showed less than 1% chimerism. Third, similar CASIN preconditioning allowed the engraftment of 5.53±2.68% CD34+ human cord blood cells into immunodeficient NSG mice compared with less than 1% by vehicle. Fourth, CASIN preconditioning alone was able to achieve 13.08±2.94% and 11.17±2.86% chimerisms of WT and Fanca gene-corrected Fanca−/− donor BM cells, respectively, in Fanca−/− mouse recipients. This effect in engraftment was particularly evident when CASIN was administrated in combination with the immunosuppressant, Fludarabine, as donor chimerism reached 22±3.05% compared with 3.63±0.87% by Fludarabine alone or 11.3±1.08% by CASIN alone. Throughout these studies, no toxicity was detected in CASIN-treated recipient mice based on examinations of CASIN treated blood, BM, spleen, liver, and other organs at one or seven days. Together, our studies demonstrate that Cdc42 is a useful target for enhancing HSC engraftment in transplantation by opening BM niche. The results present a novel preclinical regimen of pharmacological targeting Cdc42 to facilitate the engraftment of murine HSCs and human cord blood HSPCs and to improve Fanconi anemia HSC transplantation therapy, clinical settings in which stem cell numbers critically limit success. Disclosures:No relevant conflicts of interest to declare.

A Critical Role for Innate Immunity In Allogeneic Hematopoietic Cell Rejection Via the TLR4/TRIF Pathway

AbstractAbstract 77Adaptive immunity, especially T cells, has long been believed to be the dominant immune barrier in allogeneic transplantation. Targeting host T cells significantly reduces conditioning for bone marrow cell (BMC) engraftment. Innate immunity has been recently shown to pose a significant barrier in solid organ transplantation, but has not been addressed in bone marrow transplantation (BMT). Using T cell deficient (TCR-β/δ−/−) or T and B cell deficient (Rag−/−) mice, we found that allogeneic BMC rejection occurred early before the time required for T cell activation and was T- and B-cell independent, suggesting an effector role for innate immune cells in BMC rejection. Therefore, we hypothesized that by controlling both innate and adaptive immunity, the donor BMC would have a window of advantage to engraft. Survival of BMC in vivo was significantly improved by depleting recipient macrophages and/or NK cells, but not neutrophils. Moreover, depletion of macrophages and NK cells in combination with co-stimulatory blockade with anti-CD154 and rapamycin as a novel form of conditioning resulted in 100% allogeneic engraftment without any irradiation and T cell depletion. Donor chimerism remained stable and durable up to 6 months. Moreover, specific Vβ5½ and Vβ11 clonal deletion was detected in host CD4+ T cells in chimeras, indicating central tolerance to donor alloantigens.Whether and how the innate immune system recognizes or responds to allogeneic BMCs remains unknown. Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system. The signaling function of TLR depends on intracellular adaptors. The adaptor MyD88 transmits signals emanating from all TLR, except TLR3 while TRIF specifically mediates TLR3 and TLR4 signaling via type 1 IFN. To further determine the innate signaling pathways in allogeneic BMC rejection, B6 background (H2b) MyD88−/− and TRIF−/− mice were conditioned with anti-CD154/rapamycin plus 100 cGy total body irradiation and transplanted with 15 × 106 BALB/c (H2d) BMC. Only 33.3% of MyD88−/− recipients engrafted at 1 month, resembling outcomes for wild-type B6 mice. In contrast, 100% of TRIF−/− mice engrafted. The level of donor chimerism in TRIF−/− mice was 5.1 ± 0.6% at one month, significantly higher than in MyD88−/− and wild-type B6 controls (P < 0.005). To determine the mechanism of innate signaling in BMC rejection, we examined whether TRIF linked TLR3 or TLR4 is the key pattern recognition receptor involved in BMC recognition. To this end, TLR3−/− and TLR4−/− mice were transplanted with BALB/c BMC with same conditioning. None of the TLR3−/− mice engrafted. In contrast, engraftment was achieved in 100% of TLR4−/− mice up to 6 months follow up. Taken together, these results suggest that rejection of allogeneic BMC is uniquely dependent on the TLR4/TRIF signaling pathway. Thus, our results clearly demonstrate a previously unappreciated role for innate immunity in allogeneic BMC rejection. Our current findings are distinct from prior reports demonstrating a critical role of MyD88 in rejection of allogeneic skin grafts and lung, and may reflect unique features related to BMC. The findings of the role of innate immunity in BMC rejection would lead to revolutionary changes in our understanding and management of BMT. This would be informative in design of more specific innate immune targeted conditioning proposals in BMT to avoid the toxicity. Disclosures:Bozulic:Regenerex LLC: Employment. Ildstad:Regenerex LLC: Equity Ownership.