Donor Bone Marrow Cells Research Articles

Immune tolerance across a full MHC barrier in a swine hind limb transplantation model using a combined co-stimulatory blockade and donor bone marrow cell infusion

Immune tolerance across a full MHC barrier in a swine hind limb transplantation model using a combined co-stimulatory blockade and donor bone marrow cell infusion

Reduced Efficacy of Mesenchymal Stromal Cells in Preventing Graft-Versus-Host Disease in an in Vivo Model of Haploidentical Bone Marrow Transplant with Leukemia

Mesenchymal stromal cell (MSC) immunosuppressive properties have been applied to treat graft-versus-host disease (GVHD) in allogeneic hematopoietic stem cell transplants (HSCTs). We have previously demonstrated that MSC infusions early after haplo-HSCT prevent GVHD in a haploidentical-HSCT mouse model. Now, we investigated the impact that MSCs' immunosuppressive properties have on the graft-versus-leukemia (GVL) effect. First, to mimic a chronic myeloid leukemia (CML) relapse after a haploidentical HSCT, lethally irradiated mice were coinfused with haploidentical donor bone marrow cells plus syngenic hematopoietic progenitors transduced with a retroviral vector encoding both the BCR/ABL oncogene and the ΔNGFR marker gene. As expected, a CML-like myeloproliferative syndrome developed in all the recipient animals. The addition of haploidentical splenocytes to the transplanted graft prevented CML development by a GVL effect, and all transplanted recipients died of GVHD. This GVL mouse model allowed us to investigate the impact of MSCs infused to prevent GVHD on days 0, 7, and 14 after HSCT, on the GVL effect, expecting an increase in leukemic relapse. Strikingly, a high mortality of the recipients was observed, caused by GVHD, and only few leukemic cells were detected in the recipient animals. In contrast, GVHD prevention by MSCs in the absence of BCR/ABL leukemic cells resulted in a significant survival of the recipients. In vitro data pointed to an inability of MSCs to control strong CTLs responses against BCR/ABL. Our results show that, although an evident increase in leukemic relapses induced by MSCs could not be detected, they showed a reduced efficacy in preventing GVHD that precluded us to draw clear conclusions on MSCs' impact over GVL effect.

Recovery From Overt Type 1 Diabetes Ensues When Immune Tolerance and β-Cell Formation Are Coupled With Regeneration of Endothelial Cells in the Pancreatic Islets

Immune modulation of pancreatic inflammation induces recovery from type 1 diabetes (T1D), but remission was not durable, perhaps because of an inability to sustain the formation and function of new pancreatic β-cells. We have previously shown that Ig-GAD2, carrying GAD 206–220 peptide, induced in hyperglycemic mice immune modulation that was able to control pancreatic inflammation, stimulate β-cell regeneration, and prevent T1D progression. Herein, we show that the same Ig-GAD2 regimen given to mice with overt T1D was unable to reverse the course of disease despite eradication of Th1 and Th17 cells from the pancreas. However, the regimen was able to sustain recovery from T1D when Ig-GAD2 was accompanied with transfer of bone marrow (BM) cells from healthy donors. Interestingly, alongside immune modulation, there was concomitant formation of new β-cells and endothelial cells (ECs) in the pancreas. The new β-cells were of host origin while the donor BM cells gave rise to the ECs. Moreover, transfer of purified BM endothelial progenitors instead of whole BM cells sustained both β-cell and EC formation and reversal of diabetes. Thus, overcoming T1D requires both immune modulation and repair of the islet vascular niche to preserve newly formed β-cells.

Establishment of the tracing bone marrow reconstituted nude mice model

Objective To establish the green fluorescent protein (GFP) + bone marrow reconstituted nude mice model.Methods Different doses (4,6,8,10 Gy) X-ray were used to irradiate the nude mice.The hemogram was detected separately 1,3,5,7,14 and 21 days after irradiation in order to confirm the proper irradiation dose for bone marrow damage.One-week after irradiation with such dose,allogeneic transplantation with GFP + bone marrow cells (BMCs) (2 × 107 cells/mice) were executed through the caudal vein.The proportion of GFP + donor cells in peripheral blood was tested.White blood cells (WBCs) of peripheral blood and BMCs were compared before and after transplantation.Results After irradiation with 8 Gy,the number of peripheral blood WBCs was (0.1375 ±0.0510) × 109/L,and BMCs were [(31.73 ±2.56)％] of pre-irradiation nude mice.At 28th day after BMCs transplantion,the number of peripheral blood WBCs was (12.46 ± 0.68) × 109/L and recovered to the level pre-irradiation (12.89 ± 1.04) × 109/L (P ＞ 0.05).The proportion of donor GFP + cells was (63.0 ± 0.6) ％ in peripheral blood.Conclusion Eight Gy is the suitable dose for establishment of the bone marrow damage reconstituted nude mice model.Tail vein injection of GFP + BMCs effectively indicated the reconstitution of bone marrow function,which mainly depends on the donor BMCs. Key words: Irradiation; Bone marrow; Green fluorescent protein mice

Five-year clinical effects of donor bone marrow cells infusions in kidney allograft recipients

Augmentation of microchimerism in solid organ transplant recipients by donor bone marrow cells (DBMC) infusion may promote immune hyporesponsiveness and consequently improve long-term allograft survival. Between March 2005 and July 2007, outcomes for 20 living unrelated donor (LURD) primary kidney recipients with concurrent DBMC infusion (an average of 2.19 ± 1.13 x 10⁹ donor cells consisting of 2.66 ± 1.70 x 10⁷ CD34⁺ cells) were prospectively compared with 20 non-infused control allograft recipients given similar conventional immunosuppressive regimens. With five years of clinical follow up, a total of 11 cases experienced rejection episodes (3 DBMI patients vs. 8 controls, p = 0.15). One DBMC-infused patient experienced chronic rejection vs. two episodes (1 biopsy-confirmed) in the control patients. Actuarial and death-censored 5-y graft survival was significantly higher in infused patients compared with controls (p = 0.01 and p = 0.03, respectively). Long-term graft survival was significantly associated with pre-transplant anti-HLA antibodies (p = 0.01), slightly with peripheral microchimerism (p = 0.09) and CD4⁺CD25⁺FoxP3⁺ T cells (p = 0.09). Immunosuppressant dosing was lower in infused patients than controls, particularly for mycophenolate mofetil (p = 0.001). The current findings as well as our previous reports on these patients indicates clinical improvement in long-term graft survival of renal transplant patients resulting from low-dose DBMC infusion given without induction therapy.

Abstract 1375: Benefits of adding an exercise intervention to conventional immunosuppression in chronic graft versus host disease: insights from a murine model.

Abstract Purpose.- Graft-versus-host-disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT), and it is associated with high morbimortality and decreased patients’ physical capacity. We determined the effects of an exercise training program performed after allo-HSCT on survival, clinical evolution and physical capacity, using a murine model of chronic GVHD treated with cyclosporine A. Methods.- BALB/C female mice (aged 8 weeks) were randomly assigned to an exercise (n=11) or control group (n=12). Allo-HSCT was performed with donor bone marrow cells and splenocytes from B10.D2 female mice. Mice of the exercise group performed a moderate-intensity treadmill running program, and both groups received Cyclosporine A (15 mg/kg/day) during ∼11wks after transplant. We analyzed survival and total clinical severity score (which is the sum of five individual scores). We measured mice basal heart rate as an indicator of physical fitness status before and after allo-HSCT. We collected blood and serum samples for immune cell reconstitution at days +21 and +82, and for cytokine analysis (IL2, IL4, IL6, IL10, IL17a, IFNγ and TNFα) at days -10 and +82. On sacrifice day, we removed target tissues (liver, intestine, skin) and spleen, for analyzing the GVHD damage and the immune cell reconstitution respectively, and skeletal muscles evaluating the muscle adaptations. Results.- Exercise training resulted in an increased survival (p=0.011) and lesser deterioration of physical capacity after ∼11 weeks (p=0.03 for the between-group comparison) in the exercise group. Regarding disease clinical course/severity, the exercise intervention induced a significant group x interaction effect for total severity score (p=0.002), reflecting a better disease evolution in the exercise group. We found a beneficial effect in three individual scores: weight (a reliable indicator of the severity of the disease, p=0.02), spontaneous activity (p=0.001) and posture (p=0.002). Exercise training reduced blood IL4 and TNFα (p=0.025 and p=0.031, respectively) while increasing circulating B220 (p=0.008) and CD4 lymphocytes (p=0.043). We found no between-group difference in citrate synthase activity (p=0.322), and a trend towards higher muscle anabolism (as determined by the ratio of phospho-p70 S6 kinase / p70 S6 kinase) in the exercise compared with the control group (p=0.083). Conclusion.- A moderate-intensity exercise program mimicking the widely accepted public health recommendations for physical activity in humans (≥ 30min/day) was well tolerated and had significant positive effects on clinical and biological indicators of chronic GVHD, and resulted in better survival. Citation Format: Carmen Fiuza-Luces, Luisa Soares-Miranda, África González-Murillo, Jesús Martínez Palacio, Isabel Colmenero, Fernando Casco, Gustavo Melén, María Morán, Manuel Ramírez, Alejandro Lucía. Benefits of adding an exercise intervention to conventional immunosuppression in chronic graft versus host disease: insights from a murine model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1375. doi:10.1158/1538-7445.AM2013-1375

Cord blood transplantation for the treatment of acute leukemia

Objective This review discussed the available data on treatment outcomes of cord blood transplantation (CBT) for acute leukemia. Data sources The data cited in this review were obtained from articles listed in Medline and Pubmed. Study selection We reviewed the articles of clinical results from various registries and institutions, as well as our experiences with CBT in children, adolescents and adults. Results This research has clearly shown that cord blood (CB) has several unique characteristics resulting in distinct advantage and disadvantages when compared to transplantation with unrelated donor bone marrow or peripheral blood stem cells. The field of CBT has advanced from investigating its safety and feasibility to addressing more specific issues such as accelerating engraftment, extending access, and examining outcomes in specific subgroups of patients. Many approaches have been investigated in the attempt to improve engraftment and survival. Variable factors have been identified, such as factors related to donor choice (human leukocyte antigen (HLA) compatibility, cell dose, and others) and transplantation (conditioning and graft-versus-host disease prophylaxis regimen). Data support that CB should be considered a reasonable option in those that do not have HLA matched sibling donor and for those in whom the time to transplant is critical. Conclusions CB is a reasonable alternative to unrelated donor bone marrow or peripheral blood progenitor cells for transplantation. Recently developed strategies aimed at improving hematopoietic recovery and reducing early transplantation-related mortality could further improve treatment outcomes of CBT for patients with acute leukemia.

Establishment of an acute graft-versus-host disease animal model in EL4 T-cell leukemia/lymphoma mice

Objective To establish an acute graft-versus-host disease (aGVHD) model in EL4 T-cell leukemia/lymphoma mice, for providing experimental model to prevent aGVHD while maintaining graft-versus-leukemia (GVL) effect during allogeneic bone marrow transplantation (Allo-BMT). Methods Male BALB/c(H-2Kd)mice were used as donors and female C57BL/6(H-2Kb)mice were used as recipients.C57BL/6 hosts were given 9.5 Gy lethal total body irradiation at 4 hours prior to transplantation.Mice were randomly assigned into 3 groups and each group contained 17 recipients: The recipients in radiation group were injected with 0.2 mL RPMI 1640 as control.The recipients were injected with donor bone marrow cells (5×106/mouse) and splenocyte for aGVHD(5×106/mouse). The recipients in EL4 T-cell leukemia/lymphoma aGVHD group were injected with donor bone marrow cells (5×106/mouse)and plus 500 EL4 cells(5×106/mouse) .General state, life span and histopathology of the recipient mice and detected chimera were observed. Results The results showed that the mean survival time in radiation group was (10.10±0.43) days, in aGVHD group was(28.12±5.01)days, in EL4 T-cell leukemia/lymphoma aGVHD group was(31.05±5.48) days.The mean survival time in aGVHD group and EL4 T-cell leukemia/lymphoma aGVHD group were significantly longer than that in radiation group(all P 0.05). Histopathological analysis in several target organs(skin, liver and small intestine) confirmed the presence of sever GVHD in aGVHD group and EL4 T-cell leukemia/lymphoma aGVHD group.Pathological examination showed disorganization of normal tissues and leukemic cell infiltration in EL4 T-cell leukemia/lymphoma aGVHD group. Conclusion The EL4 T-cell leukemia/lymphoma aGVHD mice model is reliable to the experimental research of aGVHD. Key words: Acute graft-versus-host disease; Allogeneic bone marrow transplantation; Mouse; Animal model

Genetically Determined Severity of Anti-Myeloperoxidase Glomerulonephritis

Myeloperoxidase (MPO) is a target antigen for antineutrophil cytoplasmic autoantibodies (ANCA). There is evidence that MPO-ANCA cause necrotizing and crescentic glomerulonephritis (NCGN) and vasculitis. NCGN severity varies among patients with ANCA disease, and genetic factors influence disease severity. The role of genetics in MPO-ANCA NCGN severity was investigated using 13 inbred mouse strains, F1 and F2 hybrids, bone marrow chimeras, and neutrophil function assays. Mouse strains include founders of the Collaborative Cross. Intravenous injection of anti-MPO IgG induced glomerular crescents in >60% of glomeruli in 129S6/SvEv and CAST/EiJ mice, but <1% in A/J, DBA/1J, DBA/2J, NOD/LtJ, and PWK/PhJ mice. C57BL6J, 129S1/SvImJ, LP/J, WSB/EiJ, NZO/HILtJ, and C3H mice had intermediate severity. High-density genotypes at 542,190 single nucleotide polymorphisms were used to identify candidate loci for disease severity by identifying genomic regions that are different between 129S6/SvEv and 129S1/SvImJ mice, which are genetically similar but phenotypically distinct. C57BL/6 × 129S6 F2 mice were genotyped at 76 SNPs to capture quantitative trait loci for disease severity. The absence of a dominant quantitative trait locus suggests that differences in severity are the result of multiple gene interactions. Invivo studies using bone marrow chimeric mice and invitro studies of neutrophil activation by anti-MPO IgG indicated that severity of NCGN is mediated by genetically determined differences in the function of neutrophils.

Upper-Extremity Transplantation Using a Cell-Based Protocol to Minimize Immunosuppression

To minimize maintenance immunosuppression in upper-extremity transplantation to favor the risk-benefit balance of this procedure. Despite favorable outcomes, broad clinical application of reconstructive transplantation is limited by the risks and side effects of multidrug immunosuppression. We present our experience with upper-extremity transplantation under a novel, donor bone marrow (BM) cell-based treatment protocol ("Pittsburgh protocol"). Between March 2009 and September 2010, 5 patients received a bilateral hand (n = 2), a bilateral hand/forearm (n = 1), or a unilateral (n = 2) hand transplant. Patients were treated with alemtuzumab and methylprednisolone for induction, followed by tacrolimus monotherapy. On day 14, patients received an infusion of donor BM cells isolated from 9 vertebral bodies. Comprehensive follow-up included functional evaluation, imaging, and immunomonitoring. All patients are maintained on tacrolimus monotherapy with trough levels ranging between 4 and 12 ng/mL. Skin rejections were infrequent and reversible. Patients demonstrated sustained improvements in motor function and sensory return correlating with time after transplantation and level of amputation. Side effects included transient increase in serum creatinine, hyperglycemia managed with oral hypoglycemics, minor wound infection, and hyperuricemia but no infections. Immunomonitoring revealed transient moderate levels of donor-specific antibodies, adequate immunocompetence, and no peripheral blood chimerism. Imaging demonstrated patent vessels with only mild luminal narrowing/occlusion in 1 case. Protocol skin biopsies showed absent or minimal perivascular cellular infiltrates. Our data suggest that this BM cell-based treatment protocol is safe, is well tolerated, and allows upper-extremity transplantation using low-dose tacrolimus monotherapy.

Placenta-Derived Mesenchymal Stem Cells Have an Immunomodulatory Effect That Can Control Acute Graft-Versus-Host Disease in Mice

Background and Aims: Immunomodulatory properties of mesenchymal stem cells (MSCs) have been applied to reduce the incidence of graft-versus-host disease (GVHD) after hematopoietic stem cell transplantation (HSCT). Among the various sources of MSCs that have immunomodulatory effects in vitro, only placenta-derived MSCs (PD-MSCs) have not been evaluated in an in vivo model of GVHD. In this study, we investigated the immunomodulatory properties of PD-MSCs in vitro and evaluated their clinical potential for controlling GVHD in an animal model. Methods: A GVHD animal model was established by transplanting C57BL/6 donor bone marrow cells and spleen cells into lethally irradiated BALB/c recipient mice. To control GVHD, human PD-MSCs were transplanted into recipient mice (5 × 10⁵ or 1 × 10⁶ cells). Results: PD-MSCs suppressed mitogen-stimulated T cell proliferation in vitro in a dose-dependent manner. Moreover, PD-MSCs inhibited cytokine secretion (interleukin-12, tumor necrosis factor-α and interferon-γ) of activated T cells. In vivo, the survival rate in the PD-MSC group (transplanted with 1 × 10⁶ cells) was higher than that in the control group and histological scores were low in the PD-MSC group. Conclusion: We present the first evidence that human PD-MSCs can efficiently control GVHD in an HSCT in vivo model.

Hspa9 Haploinsufficiency Induces a Cell-Intrinsic Defect in Mouse B-Cell Progenitors

AbstractAbstract 1714Patients with myelodysplastic syndromes (MDS) have a clonal hematopoietic stem cell disorder that results in dysplastic hematopoietic cells in their bone marrow as well as peripheral blood cytopenias. In addition to the commonly described erythroid and myeloid differentiation defects associated with MDS, a reduction in bone marrow B-cell progenitors exists in patients. The genetic events contributing to the reduction in B-cell progenitors remain poorly understood.The most common cytogentic abnormality identified in patients with MDS, occurring in approximately 35% of patients, is heterozygous interstitial deletion or loss of the long arm of chromosome 5 (5q). The interstitial deletions on chromosome 5 are single copy losses, and no biallelic disruptions of genes in deleted regions have been identified, implicating haploinsufficiency as the underlying genetic mechanism. We, and others, have shown that the levels of HSPA9 mRNA expression are reduced ∼50% in patients with del(5q) when compared to MDS patients without del(5q), consistent with a haploinsufficient phenotype. To model haploinsufficiency, we used shRNA to achieve ∼50% knockdown of Hspa9 in a murine bone marrow transplant model. This model showed a significant reduction in mature B-cells in the bone marrow, spleen, and peripheral blood of recipient mice, implicating HSPA9 haploinsufficiency may contribute to the B-cell alterations observed in MDS patients with del(5q).To further evaluate HSPA9 haploinsufficiency in vivo, we created a mouse model with a heterozygous deletion of Hspa9 (Hspa9+/−) and confirmed a 50% reduction in Hspa9 protein levels in bone marrow and spleen of these mice by Western blot. Hspa9+/− mice are born at normal Mendelian frequencies (N>100), however, breeding heterozygous mice suggests Hspa9−/− mice are embryonic lethal (24 Hspa9+/+:38 Hspa9+/−:0 Hspa9−/−). No significant differences in mature lineage markers, complete blood counts, and hematopoietic organ cellularity, have been identified up to 12 months of age. However, as early as 2 months of age, the numbers of bone marrow CFU-preB colonies as assessed by methylcellulose assay, are significantly reduced in Hspa9+/− mice compared to Hspa9+/+ littermates (14 vs 48 colonies/100,000 bone marrow cells plated, respectively, N=10 mice/genotype, p<0.0001). We performed noncompetitive bone marrow transplants of Hspa9+/− or Hspa9+/+ donor cells into Hspa9+/+ recipient mice and confirmed that the reduction of B-cell progenitors is a hematopoietic cell intrinsic phenotype (N=7–9 mice/genotype, p=0.002). We also confirmed that the Hspa9+/− bone marrow microenvironment did not contribute to the phenotype as transplantation of Hspa9+/+ donor bone marrow cells into Hspa9+/− recipients did not alter the number of CFU-preB colonies (N=5).Total frequencies of common lymphoid progenitors and B-cell precursors (Hardy fractions A, B/C, D, E and F) as assessed by flow cytometry are no different in Hspa9+/− and Hspa9+/+ mice. Therefore, we hypothesize that early Hspa9+/− B-cells may have an intrinsic signaling defect which can be compensated for in vivo. Early B-cell maturation is dependent on intracellular signaling mediated through cell surface receptors in response to environmental cytokines. Consistent with our hypothesis, we showed that Hspa9+/− CFU-preB in vitro colony formation is partially rescued by increasing concentrations of IL7 while Hspa9+/+ colony numbers remain unchanged (fold change in colony formation from 10ng/mL to 50ng/mL IL7 was 1.80 for Hspa9+/− vs. 0.80 for Hspa9+/+, p=0.03, N=6 mice/genotype). Supplementation of the media with another cytokine that contributes to early B-cell maturation, Flt3 ligand, does not alter Hspa9+/− or Hspa9+/+ CFU-preB colony formation, further implicating altered IL7 signaling. We are currently investigating the downstream responses to IL7 stimulation in B-cell progenitors from Hspa9+/− mice.Collectively, these data implicate loss of HSPA9 as a contributing factor in the reduction of B-cell progenitors observed in patients with del(5q) associated MDS. Disclosures:No relevant conflicts of interest to declare.

Influence of Different Genetic Alterations On the Engraftment Capacity and Serial Transplantation Efficiency of AML Patient-Derived Bone Marrow and Peripheral Blood Cells in NSG Mice

AbstractAbstract 3511 Introduction:In order to allow a better understanding of acute myeloid leukemia (AML) and develop more promising therapeutic strategies the establishment of functional and reproducible in vivo models is widely pursued. Of available model systems, xenografts in immunodeficient mice reproduce the clinical situation best. Here, we performed extensive analysis of AML engraftment in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice comparing tail vein versus intratibial injection and growth behavior of AML patient-derived bone marrow versus peripheral blood cells. Furthermore, tumor growth characteristics in the murine host were correlated with the disease stage and the molecular risk factor profile of the individual donors. Methods:Bone marrow and peripheral blood cells from 17 AML patients were injected intratibially into NSG mice (n=4–8/patient, 82 mice in total). As controls, 14 mice received bone marrow from three different donors and 5 mice were mock-injected. Tumor growth was monitored via a) determination of overall survival, b) fluorescence-based in vivo imaging (IVI, Kodak FX, Alexa750 labeled anti-human CD45 or CD33 and c) confirmation of IVI data by histological and immunohistochemical examination of bone marrow and spleen. When highly positive IVI signals and/or the overall condition of individual mice indicated enlarged tumor burden, the respective animals were sacrificed and the human AML cells transferred to another animal. In parallel the engraftment pattern of AML cells 2–4 weeks posttransplant was correlated with clinical disease activity, application route and origin of the particular tumor cells. Results:Patients included in the present study represent multiple different French-American-British (FAB) subtypes, various karyotypes and molecular features in terms of the mutational status of NPM1 and FLT3. All patient-derived specimens were capable of recapitulating the disease in NSG mice at 4–6 weeks after transplantation. Over a period of 13 months 12 out of 17 xenografts could be passaged once and 9 at least twice. Up to six passages were performed for an individual AML xenograft. In contrast, engraftment of healthy donor bone marrow cells could be determined merely until day 56 after implantation. The human bone marrow cells of the healthy donors did not engraft in serial passages. Mean survival time of AML bearing animals ranged between 21 and 82 days for a respective xenograft. No differences could be determined between engraftment capacities of peripheral blood or bone marrow cells of one patient. Neither karyotype, FAB classification nor leucocyte count or the percentage of monomorphic blasts in the bone marrow seemed to have an impact on engraftment capacity in the murine organism. However, mice bearing AML xenografts with mutations in FLT3 as well as in NPM1 showed particular short overall survival times and high tumor cell engraftment determined by IVI. This phenomenon became more obvious along the different passages. The intratibial approach proved to be superior in comparison to the intravenous application as cells of an individual patient engrafted faster when injected directly into the bone marrow microenvironment. Determination or tumor load via IVI permits to closely monitor not only the growth behavior but also the homing characteristics of the human cells over time. The positive IVI signals in bone marrow and spleen could be confirmed by histological examination as well as by immunohistochemistry specific for human CD45 and CD33. Conclusions:Our xenografts show a close resemblance to the AML-disease regarding the level of dissemination and organ involvement. Collection of whole-body IVI data proved to be a time- and animal-saving analysis that allows to closely monitor AML growth. As AML is characterized by an increasing number of molecular subtypes with completely different therapeutic options it seems to be extremely worthwhile to develop patient derived xenograft models representing as many AML subtypes as possible. Our results suggest that this model reflects the heterogeneity and important clinical characteristics of the disease, and thus may serve as a tool for preclinical drug testing and investigation of the pathophysiology of AML. Disclosures:No relevant conflicts of interest to declare.

Anti-CD45 Radioimmunotherapy Facilitates Donor Engraftment and Prolongs Survival in the Absence of TBI Prior to Haploidentical Bone Marrow Transplantation in a Disseminated Murine Leukemia Model

AbstractAbstract 4101 Background:Despite the curative promise of hematopoietic cell transplantation (HCT), many patients with hematologic malignancies relapse and others may not proceed to HCT due to the unavailability of a matched donor. Toxicities remain high with HCT, due in part to the administration of non-specific therapies such as total body irradiation (TBI) as part of preparative regimens. We aim to overcome these limitations by replacing TBI with anti-CD45 radioimmunotherapy (RIT) for haploidentical HCT to deliver radiation directly to leukemic cells while sparing normal organs and minimizing non-specific toxicities. Methods:We established an initial TBI HCT regimen in B6SJLF1/J mice (H-2Db haplotype) conditioned with fludarabine (FLU, days -6 to -2), followed by TBI (250, 500, 750 cGy; day -1). The mice then received 15 million donor (CB6F1/J, H-2Dd) BM cells (day 0), followed by cyclophosphamide (CY) for graft-versus-host disease (GvHD) prophylaxis (day +2). Subsequent RIT HCT studies involved B6SJLF1/J mice conditioned with and without fludarabine (FLU) and escalating doses (200–400 μCi) of 90Y-anti-CD45 Ab (30F11) RIT without TBI, followed by infusion of haploidentical BM cells from CB6F1/J mice and a single dose of cyclophosphamide (CY) 2 days after HCT. Chimerism studies were performed using flow cytometric analysis to assay for engraftment of donor CD8+ cells. Therapeutic studies were performed in B6SJLF1/J mice given 105 syngeneic leukemia cells via tail vein (day -5), followed by 200 or 400 μCi 90Y-30F11 (day -3), and 1.5 × 107 BM donor cells (day 0) and two doses of CY (days -2 and +2) without FLU. Results:Using this model we have demonstrated that mixed chimerism was established in mice transplanted with TBI or escalating doses (200–400 μCi) of 90Y-30F11 RIT followed by injection of haploidentical BM donor rescue cells. TBI-based HCT showed that chimerism as determined by flow cytometric analysis for donor CD8+ cells was TBI dose-dependent; mice receiving ≥500 cGy were fully chimeric 4 weeks post-HCT, and persisted ≥12 months. RIT-based HCT also revealed mice with mixed chimerism, with up to 89% of donor CD8+ cells 1 month after HCT. Elimination of FLU from the conditioning regimen did not significantly decrease chimerism, as mice transplanted without FLU showed up to 70% donor CD8+ cells 1 month after HCT. Subsequent RIT experiments in B6SJLF1/J mice harboring AML were treated with escalating doses of 90Y-30F11 prior to HCT without FLU. Mice treated with anti-CD45 RIT using 200 μCi and 400 μCi of 90Y-30F11 had a median overall survival (OS) of 73 (p<0.001) and 107.5 (p=0.0015) days, respectively, compared to untreated leukemic control mice which had a median OS of 34 days after HCT (p values by Log rank (Mantel-Cox) testing)(Figure). Two mice in the 400 μCi-90Y-30F11 group were euthanized on day 3 for excessive weight loss, without gross histology abnormality in kidneys or liver. Conclusion:These studies suggest that anti-CD45 RIT in the absence of TBI and FLU prior to haploidentical HCT can lead to establishment of mixed chimerism. Moreover, this anti-CD45 RIT in combination with haploidentical HCT can lead to improvement in survival for mice with AML. These results suggest that clinical studies with anti-CD45 RIT in lieu of TBI and FLU in a haploidentical HCT regimen should be considered for further investigation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Preconditioning with AMD3100 Mobilization Promotes Homing and Long-Term Engraftment of Hematopoietic Stem Cells During Syngeneic Transplantation in the Murine Model

Abstract 1886 IntroductionTransplantation efficacy can be limited by inadequate hematopoietic stem cell (HSC) number, homing, engraftment, or self-renewal. Traditional conditioning prior to HSC transplantation by myeloablative chemotherapy with or without radiation provides therapeutic benefit but also results in immunosuppression and tissue damage that contribute to increased morbidity and mortality, therefore, development of safe myeloablative strategies is a clinical priority. AMD3100 has been shown to be a potent mobilizer of HSCs in multiple animal species and humans and acts by specifically blocking the CXCR4/CXCL12 (SDF-1α) axis that is critical for HSC homing and retention in the niche. Therefore, we hypothesized that rapid mobilization of host HSC out of the bone marrow would result in increased availability of niches for subsequently infused donor HSCs. MethodsC57/BL6 mice were injected subcutaneously with 5 mg/kg AMD3100 and at indicated time points, 40×106 C57/BL6 GFP+ donor bone marrow mononuclear cells were intravenously delivered. The control group received injection of PBS. For additive treatment of the donor cells with PGE2 and Diprotin A, B6 GFP+ HSCs were treated with 1μM PGE2 or 5 mM Diprotin A and then delivered into the recipient mice 60 min post-AMD3100 administration. For combinatorial treatment with both drugs, the cells were treated with PGE2 for 2 hours at 4 °C and then treated with Diprotin A at RT for 15 min prior to transplantation. For homing studies, twelve hours after transplantation, the mononuclear cells from peripheral blood and bone marrow were harvested for flow cytometry and CFU-C assay. Fold increases were based on comparison with control mice that received PBS injection and DMEM-treated donor cells. For long-term engraftment studies, the assays were performed four months after transplantation. ResultsThe results demonstrated that a single administration of AMD3100 resulted in significantly increased availability of bone marrow niches by mobilizing the host HSCs into the periphery in a time-dependent manner. The maximal effect of up to 90% vacancy of the niche was observed at 1 hour after AMD3100 administration. Infusion of syngeneic donor bone marrow cells between 45 and 60 min post-AMD3100 administration resulted in maximum homing and engraftment efficiency. Donor CXCR4+CD49d+ cells preferentially homed to the host bone marrow niche. Pretreatment of donor HSCs with PGE2 or Diprotin A (CD26 blockade) further enhanced the homing efficiency following AMD3100 preconditioning. Moreover, AMD3100 preconditioning with or without PGE2 or Diprotin A also resulted in long-term engraftment of donor HSCs. The occupancy of the marrow niche by HSCs was restored to the full capacity in the long term, indicating treatment with these drugs did not result in toxicity. PGE2 and Diprotin A pre-treatment, each of which have been demonstrated to selectively improve donor cell homing and engraftment, further enhances the positive effects of AMD3100 conditioning alone. ConclusionOur data support the efficacy of AMD3100 mobilization, alone or in combination with donor cell pre-treatment strategies, as a safe-and-effective preconditioning method for hematopoietic stem cell transplantation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

STAT5 Promotes Hematopoietic Stem Cell Maintenance by Increasing Quiescence-Associated Gene Expression and Suppressing Lymphoid Commitment

AbstractAbstract 851Signal transducer and activator of transcription 5 (STAT5) is a critical regulator of lympho-myeloid hematopoiesis through activation downstream of early-acting cytokines, their receptors (e.g. c-Mpl), and janus kinases (JAKs). We have previously reported that hematopoietic stem cells (HSC) in pI:pC treated Mx1-Cre conditional STAT5 knockout mice, like c-Mpl deficient HSC, are persistently more actively cycling and become apoptotic, reducing long-term HSC number. Since down-regulation of c-Mpl is associated with HSC heterogeneity defined by differential CD150/Flk2 expression and skewing toward lymphoid-primed multipotential progenitors (LMPP), we set to investigate whether STAT5-deficient cycling KLS cells are similarly lymphoid-primed. First, we generated novel Vav1-Cre/+STAT5abfl/fl hematopoietic-specific conditional knockout mice with 98% deletion efficiency in sorted KLS cells as determined by real-time PCR (N=3 mice). The majority of Vav1-Cre/+STAT5abfl/fl mice were viable with significantly reduced white blood cell counts (1.84±1.01 x103/μl, N=17 vs 8.46±2.26 x103/μl, N=27), red blood cell counts (7.53±0.78 x106/μl vs 10.19±0.80 x106/μl) and hematocrits (33.2±4.3% vs 47.1±3.2%) relative to wild-type. Competitive repopulation was 16-fold reduced CD45.2 donor chimerism with Vav-Cre/+STAT5abfl/fl (3.3±0.7%; N=5) compared to wild-type (55±2%; N=5) in a 1:1 mix with wild-type Boy J bone marrow cells. In non-ablative bone marrow transplantation, injection of a single dose of 5 million donor bone marrow cells gave rise to high levels of engraftment (84±8%, N=5) in the peripheral blood of congenic adult Vav-Cre/+STAT5abfl/fl recipients compared to no detectable engraftment controls. The KLS fraction was analyzed using CD34 and FLK2 markers. Despite severe reduction in the long-term repopulating HSC CD34-FLK2-KLS (N=3, p<0.001) and short-term repopulating HSC CD34+FLK2-KLS (p=0.03) fractions, the CD34+FLK2+KLS fraction enriched for LMPP was relatively maintained. We obtained similar results using CD150/CD48 in both Vav-Cre/+STAT5abfl/fl and STAT5ab null/null fetal liver transplanted mice. Using Affymetrix mouse gene chips to compare global gene expression between CD45.2+ KLS cells from STAT5ab null/null vs. wild-type fetal liver transplanted mice, several HSC-related genes were among those decreased whereas the majority of increased genes were associated with B-lineage commitment and immunoglobulin production. Furthermore, single cell gene expression analysis on the Fluidigm BioMark 48.48 chip (20 individual cells each genotype) was consistent with the microarray profiling. Half of wild-type KLS cells expressed high levels of HSC transcripts such as c-Mpl and Gfi1b and low levels of lymphoid transcripts whereas half were the opposite. In contrast, all STAT5 deficient KLS cells were lymphoid-primed and deficient in HSC transcripts. Due to the STAT5-dependent block at the pre-pro-B-cell stage, we are unable to functionally readout the donor B-lymphoid cells in the peripheral blood of transplanted mice. Since the combination of bcl-2 and myc induces aggressive B-cell acute lymphoblastic leukemia, we set out to use both bcl-2 and myc transgenic mouse models to test whether lymphoid-primed KLS cells lacking STAT5 would promote B cell development through a non-canonical pathway. Transgenic myc driven from an immunoglobulin H chain enhancer (Eμ-Myc) combined with H2K-bcl-2 induced faster B-ALL in STAT5ab null/null vs. wild type control fetal liver transplanted mice. The latency was significantly reduced from a median survival of 91 days in the wild type control to a median survival of 44 days in lethally-irradiated recipients (P<0.001; N range from 8–14 mice/group). Similar results were obtained in sub-lethally irradiated recipients (P=0.007; N range 10–20 mice/group). Loss of STAT5 promoted the accumulation of B-lineage committed progenitor cells in combination with pre-pro-B blockage. We also found a recently described Sca1+c-Kit-Lin- alternative lymphoid progenitor population to be expanded only in the STAT5ab null/null genotype expressing myc and bcl-2. Whether this population possesses the leukemia-initiating potential is still under investigation. Overall, we have found that STAT5 plays an unexpected role in hematopoietic stem cell differentiation by limiting early lymphoid lineage commitment. Disclosures:No relevant conflicts of interest to declare.

Infused Bone Marrow Fails to Prolong Vascularized Composite Allograft Survival in Nonhuman Primates

Introduction: Clinical protocols for hand and face vascularized composite allografts (VCA) have included varying amounts donor bone marrow cell (BMC) infusion after lymphocyte depletion as an immunomodulatory strategy. We previously demonstrated that vascularized bone marrow (VBM) protects facial VCA from rejection in unconditioned nonhuman primates. To investigate the potential immunologic benefit of infused BMC, we substituted infused BMC for VBM in the transplanted facial segment. Methods: Three cynomolgus macaques underwent heterotopic facial segment transplantation with mandibular segment removed, followed by central venous infusion of BMC from the same MHC-mismatched donor on postoperative day 1. Bone marrow cells were collected from the donor lumbar vertebrae following euthanasia. Cells counts and viability were confirmed using trypan blue and automated complete blood count. Donor BMC were infused at 200 million cells per kg of recipient weight, a standard for bone marrow transplantation. CD34+ hematopoeitic stem and progenitor cells were identified using a modified ISHAGE gating strategy. Immunosuppression was maintained with tacrolimus and mycophenolate mofetil. Rejection episodes were not treated. Chimerism was measured in blood samples using flow cytometry. Results: Mean cell content of the mandibular segments set aside before transplantation was 48.2 million cells/kg of donor weight. Hematopoeitic stem and progenitor cells comprised 5.63% of vertebral bone marrow BMC and 7.60% of mandibular BMC. Animals in the previous VCA+VBM treatment group had no graft loss while on immunosuppression. Additionally, graft survival was longer vs. VCA alone (205-430 vs. 9-42 days), as was rejection-free survival (44-358 vs. 7-15 days). In the VCA + BMC group all animals developed acute rejection (Banff grade I or higher) by day 14. One progressed to grade IV rejection and graft loss by day 28, while another resolved but progressed to chronic rejection by day 50 and endpoint at day 115 for post-transplant lymphoproliferative disease with chronic rejection. Only one animal receiving BMC demonstrated transient macrochimerism days 85-98. Chimerism was similarly observed transiently in only one animal receiving VCA without either VBM or BMC. In contrast, 3 of 4 animals with VBM demonstrated macrochimerism.Figure: [Graft Survival]Conclusion: Despite larger cell counts, BMC infusion failed to prolong facial VCA survival compared to VBM. These data support that VBM is a more effective immunomodulatory strategy than infusion of donor BMC without depletional pre-conditioning. The addition of irradiation and T cell depletion therapies to VBM may further enhance the amount and durability of chimerism, and potentially allow for the development tolerance.

Systemic Immunomodulation with SA-FasL Protein-Engineered Donor Splenocytes Under Transient Cover of Rapamycin Induces Robust Tolerance to Cardiac Allografts

Introduction: We previously demonstrated that donor splenocytes engineered to display on their surface a novel form of FasL molecule chimeric with streptavidin (SA-FasL) induced tolerance in ˜ 60% of ACI rats transplanted with allogeneic WF heart grafts. The main objective of this study is to further improve the efficacy of this tolerance protocol by using different routes of injections, a short course of rapamycin as immunosuppressant, and bone marrow cells engineered with SA-FasL as a clinically practical approach. Methods: A heterotopic cardiac allograft model using BALB/c as donors and C57BL/6 mice as recipients was used. Donor splenocytes (Spl) or bone marrow cells (BMCs) were biotinylated under physiological conditions followed by incubation with SA-FasL protein (70 ng/106 cells). After extensive washing to remove the unbound protein, the level of SA-FasL on the cell surface was assessed by flow cytometry. Five million of SA-FasL-engineered cells were injected intraperitoneally or intravenously into C57BL/6 mice on day 1, 3, 5, post cardiac transplantation performed on day 0. Graft recipients were also treated with a short course of rapamycin (0.2 mg/kg) administered daily from day 0 to 14 post-transplantation. Ventricular contractions were assessed by daily abdominal palpation and rejection was defined as cessation of heartbeat verified by autopsy and pathology. Results: Intraperitoneal treatment with donor SA-FasL-engineered splenocytes induced tolerance in all cardiac graft recipients (n=15; >100 days), as compared to SA-FasL-engineered BMCs treated group [n=3, mean survival time (MST) = 22.7±3.5 days], or streptavidin-engineered control group (n=7; MST = 20.9±2.2 days). Intravenous treatment exhibited the same efficacy in inducing tolerance with SA-FasL-engineered splenocytes (n=5; >100 days). Interestingly, only live allogeneic splenocytes, but not syngeneic or irradiated allogeneic splenocytes, induced tolerance, as the grafts from the latter two groups were rejected at a similar tempo to SA control group (n=4, MST = 28±0.6 days and n=5, MST = 26 days). Multilineage phenotypic analysis of splenocytes and BMCs showed high percentages of lymphocytes (58% T cells and 26% B cells) in splenocytes, while BMCs primarily included granulocytes (74%), suggesting that either B or T cells or the combination of both of these cell types are responsible for the tolerogenic effect of splenocytes. Conclusions: SA-FasL-engineered live allogeneic splenocytes, but not BMCs, induce robust tolerance to allogeneic cardiac grafts. Ongoing studies are aimed at identifying the cell type in the spleen that is responsible for tolerance to expedite the application of this approach to the clinic. Supported in parts by AHA 09GRNT2380136, KDR-PP09-23,and NIH T32 AI055456.

Co-Stimulatory Blockade and Donor Bone Marrow Cell Infusion for Immunomodulation and Tolerance Induction in Vascularized Composite Allografts - A Large Animal Translational Trial

Introduction: Particular Vascular Composite Allografts (VCA), such as hand or arm transplants, contain vascularized long bones that instantly and continuously produce bone marrow (BM) cells after engraftment and thus can be considered to be both a VCA and vascularized BM graft. The creation of hematopoietic chimerism through BM transplantation remains the most reliable method for inducing transplantation tolerance. However, the contribution of a vascularized bone graft in VCA towards such effects is largely unknown. Therefore, this study investigates the long-term effects of vascularized BM within VCA under costimulation blockade-based immunosuppression. Materials and methods: Complete MHC-mismatched miniature swine (n=12) underwent total body and thymic irradiation for cytodepletion. Animal groups received 15, 30 or 60 × 106 cells/kg of whole, unmodified BM. The optimal dose of BM cell infusion was then applied to subsequent experiments evaluating the addition of CTLA4Ig. MGH miniature swine (n=24) underwent heterotopic hind limb transplantation and received a short course of 30 days of Tacrolimus monotherapy. Group 1 (control) received no treatment. Group 2 received FK506 only; Group 3 received irradiation and FK506; Group 4 received irradiation, BM infusion and FK506; Group 5 received FK506 and CTLA4-lg; Group 6 received irradiation, BM infusion, FK506 and CTLA4Ig; and Group 7 received BM infusion, FK 506 and CTLA4Ig fusion protein. Results: Microchimerism was established in all animals after BM cell infusion; at postoperative day 9, it was significantly increased for 60 ×106 cells/kg (P = .0001). Transplanted animals in Group 1 rejected the allograft 5 to 8 days post-transplant. Group 2 rejected the allograft (skin and muscle) 30-32 days post- transplant. Group 3 rejected the allograft (skin and muscle) 35-37 days post-transplant. Group 4 rejected the skin portion of the allograft at 50-53 days post-transplant. The remaining allograft components (muscle, bone, nerve, and vessel) survived indefinitely. Group 5 animals demonstrated significantly prolonged muscle survival beyond 150 days post-transplant; the skin component survived past 150 days in 3 out of 5 animals. Skin and muscle histology in all long-term surviving animals was rejection-free. Two animals in Group 5 developed parasitic diarrhea and were euthanized at post-operative day 45. Both skin and muscle components were rejection free at the time of euthanasia. All animals in Group 6 had unexpected weight loss leading to sudden death (n=1) or euthanasia (n=2). Group 7 is currently in progress with rejection-free survival beyond 52 days post-transplant. Conclusion: High-dose BM cell infusion results in stable levels of micro-chimerism. The addition of co-stimulatory blockade (CTLA4Ig) enabled us to optimize induction therapy, further reduce maintenance immunosuppression, and indefinitely prolong graft survival. Such targeted immunomodulatory protocols that combine BM cell-based strategies and biologics might facilitate immune tolerance and eliminate the need for multi-drug immunosuppression to maintain graft survival after vascularized composite allotransplants.

Mesenchymal Stromal Cells Fail to Alleviate Experimental Graft‐Versus‐Host Disease in Rats Transplanted with Major Histocompatibility Complex‐Mismatched Bone Marrow

Mesenchymal stromal cells (MSC) can be used to treat graft-versus-host disease (GVHD) caused by allogeneic stem cell transplantation (allo-SCT). The effectiveness of this therapy has been variable in clinical trials and in experimental animal models. In this study, we investigated the ability of bone marrow (BM)-derived MSC to alleviate GVHD in an experimental rat model of allo-SCT using two different combinations of major histocompatibility complex (MHC) mismatch with survival as the primary endpoint. Recipient rats received total body irradiation and a transplant of T cell-depleted donor BM cells with either a full [PVG.7B → BN] or a partial MHC mismatch [PVG.1U → PVG.R23] restricted to the class II and non-classical class I sub-regions (RT1-B/D-CE/N/M). GVHD was invoked by infusion of graded doses of donor leukocytes 2 weeks after allo-SCT. Weekly doses of MSC were injected starting on the day of donor leukocyte infusion. No significant overall improvement of mortality and morbidity was observed in the two transplantation settings. Stimulation of MSC with exogenous tumor necrosis factor α and interferon (IFN)γ prior to infusion could not rescue BM-transplanted rats from lethal acute GVHD. In conclusion, repeated administrations of MSC failed to alleviate GVHD after fully or partially MHC-mismatched allo-SCT in the rat.