29 results on '"William H. Peranteau"'
Search Results
2. Sustained Engraftment in Fanconi Anemia (FA) Mice Using in Utero Hematopoietic Cell Transplantation (IUHCT)
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Apeksha Dave, Suying Liu, John S Riley, Sourav Bose, Pallavi Menon, Peter Kurre, and William H Peranteau
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
3. Enhanced in utero allogeneic engraftment in mice after mobilizing fetal HSCs by α4β1/7 inhibition
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William H. Peranteau, Aimee G Kim, Haiying Li, John S. Riley, John F. DiPersio, Matthew M. Boelig, Matthew Holt, S Loukogeorgakis, Michael A. Conner, Alan W. Flake, Linda Eissenberg, Michael P. Rettig, and Jesse D. Vrecenak
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0301 basic medicine ,Integrins ,medicine.medical_treatment ,Immunology ,Mice, Transgenic ,Hematopoietic stem cell transplantation ,Transplantation Chimera ,Integrin alpha4beta1 ,Biochemistry ,Mice ,03 medical and health sciences ,Fetus ,Pregnancy ,medicine ,Transplantation, Homologous ,Animals ,Humans ,Hematopoietic Stem Cell Mobilization ,Mice, Inbred BALB C ,Transplantation ,business.industry ,Fetoscopy ,Plerixafor ,fungi ,Hematopoietic Stem Cell Transplantation ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Mice, Inbred C57BL ,Haematopoiesis ,030104 developmental biology ,Female ,Stem cell ,business ,medicine.drug ,Homing (hematopoietic) - Abstract
In utero hematopoietic cell transplantation (IUHCT) is a novel nonmyeloablative approach that results in donor-specific tolerance and mixed allogeneic chimerism. Clinical application is limited by low levels of donor cell engraftment. Competition from endogenous hematopoietic stem cells (HSCs) for limited "space" in fetal hematopoietic organs remains a significant barrier to successful IUHCT. AMD3100, a CXCR4 inhibitor, and firategrast, an α4β1 and α4β7 integrin inhibitor (α4β1/7), have been shown to disrupt HSC retention in the postnatal hematopoietic niche. We hypothesized that maternal administration of AMD3100 and/or firategrast prior to IUHCT would mobilize endogenous HSCs from the fetal liver (FL) and result in preferential FL homing of donor HSCs and enhanced long-term engraftment following IUHCT in an allogeneic mouse model. We demonstrate that (1) both agents cross the placenta with rapidly detectable fetal serum concentrations following maternal administration; (2) firategrast treatment alone or with AMD3100 mobilizes endogenous HSCs from the FL and results in increased FL homing of donor HSCs following IUHCT; and (3) enhanced donor HSC homing following firategrast treatment translates into increased long-term multilineage donor cell engraftment. This approach highlights the potential of mobilization strategies to overcome barriers to successful engraftment and increase the clinical promise of IUHCT.
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- 2016
4. Donor cell engineering with GSK3 inhibitor-loaded nanoparticles enhances engraftment after in utero transplantation
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Aimee G Kim, José Luiz Martins, Jesse D. Vrecenak, Antonio Fernandes Moron, S Loukogeorgakis, Camila G. Fachin, Haiying Li, Darrell J. Irvine, Alan W. Flake, Izhtak Nissim, Ilana Nissim, William H. Peranteau, Li Tang, Andre I. B. S. Dias, Paolo De Coppi, Nicholas J. Ahn, and John D. Stratigis
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medicine.medical_treatment ,Immunology ,Hematopoietic stem cell transplantation ,Biochemistry ,In utero transplantation ,Glycogen Synthase Kinase 3 ,Mice ,GSK-3 ,medicine ,Animals ,Enzyme Inhibitors ,Cell Engineering ,Progenitor ,Mice, Inbred BALB C ,Chemistry ,fungi ,Graft Survival ,Hematopoietic Stem Cell Transplantation ,Cell Biology ,Hematology ,Hematopoietic Stem Cells ,Transplantation ,Haematopoiesis ,Autocrine Communication ,In utero ,Cancer research ,Nanoparticles ,Female ,Stem cell - Abstract
Host cell competition is a major barrier to engraftment after in utero hematopoietic cell transplantation (IUHCT). Here we describe a cell-engineering strategy using glycogen synthase kinase-3 (GSK3) inhibitor-loaded nanoparticles conjugated to the surface of donor hematopoietic cells to enhance their proliferation kinetics and ability to compete against their fetal host equivalents. With this approach, we achieved remarkable levels of stable, long-term hematopoietic engraftment for up to 24 weeks post-IUHCT. We also show that the salutary effects of the nanoparticle-released GSK3 inhibitor are specific to donor progenitor/stem cells and achieved by a pseudoautocrine mechanism. These results establish that IUHCT of hematopoietic cells decorated with GSK3 inhibitor-loaded nanoparticles can produce therapeutic levels of long-term engraftment and could therefore allow single-step prenatal treatment of congenital hematological disorders.
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- 2019
5. Correction of murine hemoglobinopathies by prenatal tolerance induction and postnatal nonmyeloablative allogeneic BM transplants
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Alan W. Flake, Toshio Asakura, Satoshi Hayashi, Aziz M. Merchant, Osheiza Abdulmalik, Qiukan Chen, and William H. Peranteau
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medicine.medical_treatment ,Thalassemia ,Immunology ,Population ,Anemia, Sickle Cell ,Hematopoietic stem cell transplantation ,Biochemistry ,Immune tolerance ,Mice ,Immune Tolerance ,medicine ,Animals ,Transplantation, Homologous ,education ,Bone Marrow Transplantation ,Fetal Therapies ,education.field_of_study ,business.industry ,beta-Thalassemia ,fungi ,Hematopoietic Stem Cell Transplantation ,Beta thalassemia ,Cell Biology ,Hematology ,Allografts ,medicine.disease ,Transplantation ,Disease Models, Animal ,Tolerance induction ,medicine.anatomical_structure ,Bone marrow ,business - Abstract
Sickle cell disease (SCD) and thalassemias (Thal) are common congenital disorders, which can be diagnosed early in gestation and result in significant morbidity and mortality. Hematopoietic stem cell transplantation, the only curative therapy for SCD and Thal, is limited by the absence of matched donors and treatment-related toxicities. In utero hematopoietic stem cell transplantation (IUHCT) is a novel nonmyeloablative transplant approach that takes advantage of the immunologic immaturity and normal developmental properties of the fetus to achieve mixed allogeneic chimerism and donor-specific tolerance (DST). We hypothesized that a combined strategy of IUHCT to induce DST, followed by postnatal nonmyeloablative same donor "booster" bone marrow (BM) transplants in murine models of SCD and Thal would result in high levels of allogeneic engraftment and donor hemoglobin (Hb) expression with subsequent phenotypic correction of SCD and Thal. Our results show that: (1) IUHCT is associated with DST and low levels of allogeneic engraftment in the murine SCD and Thal models; (2) low-level chimerism following IUHCT can be enhanced to high-level chimerism and near complete Hb replacement with normal donor Hb with this postnatal "boosting" strategy; and (3) high-level chimerism following IUHCT and postnatal "boosting" results in phenotypic correction in the murine Thal and SCD models. This study supports the potential of IUHCT, combined with a postnatal nonmyelablative "boosting" strategy, to cure Thal and SCD without the toxic conditioning currently required for postnatal transplant regimens while expanding the eligible transplant patient population due to the lack of a restricted donor pool.
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- 2015
6. Stable long-term mixed chimerism achieved in a canine model of allogeneic in utero hematopoietic cell transplantation
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Jesse D. Vrecenak, Erik G. Pearson, Mark P. Johnson, William H. Peranteau, Matthew T. Santore, Alan W. Flake, Haiying Li, Carlyn A. Todorow, Tricia R. Bhatti, and Antoneta Radu
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medicine.medical_treatment ,Immunology ,Intraperitoneal injection ,Graft vs Host Disease ,Biochemistry ,Intracardiac injection ,Injections ,Dogs ,Fetal Heart ,Pregnancy ,medicine ,Animals ,Kidney transplantation ,Fetal Therapies ,Transplantation Chimera ,business.industry ,Hematopoietic Stem Cell Transplantation ,Cell Biology ,Hematology ,medicine.disease ,Allografts ,Kidney Transplantation ,Tissue Donors ,Transplantation ,Haematopoiesis ,Graft-versus-host disease ,medicine.anatomical_structure ,Microscopy, Fluorescence ,In utero ,Models, Animal ,Female ,Transplantation Tolerance ,Bone marrow ,business ,Injections, Intraperitoneal - Abstract
Evidence supporting the efficacy of in utero hematopoietic cell transplantation (IUHCT) in a valid large animal model is needed prior to clinical application. The objective of this study was to establish clinically relevant levels of hematopoietic chimerism in a canine model of maternal-to-fetal IUHCT. We first assessed immune and hematopoietic ontogeny relevant to IUHCT in the canine model and identified 40 days' gestation (term 63 days) as a time point at the initiation of thymic selection, and prior to bone marrow hematopoiesis, that might be optimal for IUHCT. We next determined that intravascular administration of donor cells via intracardiac injection was far more efficient and resulted in much higher levels of donor cell engraftment than intraperitoneal injection. By applying these findings, we achieved stable long-term multilineage engraftment in 21 of 24 surviving recipients with an average level of initial chimerism of 11.7% (range 3% to 39%) without conditioning or evidence of graft-versus-host disease. Donor cell chimerism remained stable for up to 2 years and was associated with donor-specific tolerance for renal transplantation. The levels of donor cell chimerism achieved in this study would be therapeutic for many hematopoietic disorders and are supportive of a clinical trial of IUHCT.
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- 2014
7. Enhanced Allogeneic Engraftment Achieved Via Prenatal Tolerance Induction and Postnatal Anti-CD45 Immunotoxin Conditioned Hematopoietic Cell Transplantation
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Lauren E. McClain, Ali Y. Mejaddam, Haiying Li, John D. Stratigis, William H. Peranteau, Philip W. Zoltick, Patrick E. McGovern, John W. Urwin, Agnieszka Czechowicz, and Alan W. Flake
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0301 basic medicine ,education.field_of_study ,business.industry ,Immunology ,Population ,Cell Biology ,Hematology ,Total body irradiation ,medicine.disease ,Biochemistry ,Immune tolerance ,Transplantation ,03 medical and health sciences ,Tolerance induction ,surgical procedures, operative ,030104 developmental biology ,Graft-versus-host disease ,Medicine ,Stem cell ,business ,education ,Busulfan ,medicine.drug - Abstract
INTRODUCTION: In utero hematopoietic cell transplantation (IUHCT) is a non-myeloablative, non-immunosuppressive transplant approach that takes advantage of the immunologic immaturity of the fetus to engraft donor cells across immune barriers and induce donor specific immune tolerance (DST). Though it has the potential to treat many congenital hematologic disorders, IUHCT is limited by levels of engraftment below those anticipated to be therapeutic for most target diseases, including sickle cell anemia. Thus, a likely initial clinical application of IUHCT is to induce DST to allow for non-myeloablative, non-immunosuppressive postnatal "booster" transplants to increase engraftment. We have previously validated this approach in murine sickle cell, beta-thalassemia, and non-diseased models using low dose total body irradiation and busulfan prior to postnatal bone marrow transplantation (BMT). Although encouraging, less toxic approaches that specifically target the hematopoietic system would make IUHCT + postnatal BMT more applicable. CD45 is a membrane glycoprotein expressed by all leukocytes and hematopoietic stem cells. Recent studies have demonstrated enhanced engraftment in a congenic postnatal transplant mouse model following pre-transplant conditioning with an anti-CD45-saporin (SAP) immunotoxin (Palchaudhuri et al., Nat Biotech 2016; 34:738-45). We hypothesized that mixed allogeneic chimerism achieved by IUHCT can be enhanced after birth by conditioning with an anti-CD45-SAP immunotoxin followed by a same-donor allogeneic BMT. METHODS: 5x106 BM mononuclear cells from C57Bl/6 mice (H2Kb, CD45.2+) were injected intravenously into gestational day 14 (E14) Balb/c fetuses (H2Kd, CD45.2+). Donor cell peripheral blood (PB) chimerism was confirmed at 4 weeks of age, at which time chimeric mice were injected via tail vein with PBS (control) or CD45.2-SAP. Six days following treatment, chimeric animals were intravenously injected with 30x106 B6Pep3b (H2Kb, CD45.1+) BM cells. Additional control groups consisted of recipients of IUHCT alone without a postnatal BMT and naïve 5 week-old Balb/c mice which did not undergo IUHCT and only received CD45.2-SAP + postnatal BMT. Following postnatal BMT, PB was assessed bi-weekly by flow cytometry for total donor chimerism and the contribution of prenatal and postnatal donor cells to engraftment. Mice were weighed before each bleeding and evaluated for signs of GVHD. Statistical comparison was performed using the Student's t-test for 2 samples at each time point assuming unequal variances. RESULTS: There was no significant difference in initial donor cell chimerism levels at 4 weeks of age between treatment (IUHCT+CD45.2-SAP+BMT, 12.4±2.2%, n=5) and control (IUHCT alone, 9.3±2.7%, n=18; IUHCT+BMT, 10.0±2.6%, n=7) groups prior to therapy. In contrast to naïve non-chimeric Balb/c mice undergoing a postnatal BMT (n=10) which demonstrated no donor cell engraftment, recipients of IUHCT and a postnatal BMT (both PBS and CD45.2-SAP treated groups) demonstrated postnatal donor cell engraftment, supporting DST achieved by IUHCT. While postnatal BMT after IUHCT failed to demonstrate a significant enhancement of engraftment compared to IUHCT alone (4.4±1.3% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p = 0.7), low-level allogeneic chimerism following IUHCT was significantly enhanced to high-level chimerism following conditioning with CD45.2-SAP+BMT (55.7±9.8% vs. 7.1±4.2% donor cell engraftment at 22 weeks, p=0.0001) (Fig. 1). The majority of donor cell chimerism resulted from the postnatal donor population in recipients of CD45.2-SAP, supporting a mechanism of enhanced engraftment via a competitive advantage following recipient CD45.2-SAP treatment (Fig. 2). Chimerism was stable and multilineage at 22 weeks indicating enhanced stem cell/early progenitor engraftment. All mice demonstrated appropriate weight gain without GVHD. CONCLUSIONS: Prenatal tolerance induction achieved by IUHCT followed by postnatal CD45-SAP conditioning and same-donor BMT results in enhanced stable multilineage allogeneic engraftment at therapeutic levels for many target diseases. This work supports a potentially less toxic approach for the strategy of prenatal tolerance induction to facilitate non-myeloablative, non-immunosuppressive postnatal BMT and has the potential to significantly expand the applicability of IUHCT. Disclosures No relevant conflicts of interest to declare.
- Published
- 2016
8. Characterizing and Augmenting Peripheral Tolerance in in Utero Hematopoietic Cell Transplantation
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William H. Peranteau, Grace Lee, Haiying Li, John S. Riley, Alan W. Flake, and Lauren E. McClain
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Regulatory T cell ,business.industry ,Immunology ,Peripheral tolerance ,FOXP3 ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Donor lymphocyte infusion ,medicine.anatomical_structure ,Graft-versus-host disease ,Central tolerance induction ,medicine ,IL-2 receptor ,Central tolerance ,business - Abstract
Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism without myeloablation/immunosuppression. It has the potential to treat a number of congenital hematologic disorders, including as Sickle cell disease. Central tolerance, through which donor-reactive host T cells and host-reactive donor T cells are deleted, is instrumental to the induction of donor specific tolerance (DST) and the prevention of graft-versus-host-disease (GVHD). Central deletion, however, is incomplete, and donor-reactive host T cells remain following allogeneic IUHCT. Furthermore, central deletion alone cannot explain the absence of GVHD either following IUHCT using non-T cell depleted bone marrow (BM) or in chimeric animals that undergo postnatal donor lymphocyte infusion (DLI) with subsequent conversion to >90% allogeneic donor cell engraftment (Hayashi et al., Blood 2002;100:804-12). Studies in both mice and humans have demonstrated the importance of regulatory T cells, including traditional CD25+FoxP3+ Tregs and CD49b+LAG-3+ Type 1 regulatory T (Tr1) cells, in maintaining tolerance following postnatal allogeneic transplantation. We hypothesized that peripheral regulatory T cells are increased in chimeric animals following IUHCT and that they contribute to the establishment of DST and prevention of GVHD. Methods: 10x106 BM cells from 6-8 week old C57Bl/6 (B6) FoxP3GFP mice (H2Kb, CD45.2+) were injected intravenously (IV) into gestational day 14 Balb/c FoxP3GFP fetal recipients (H2Kd, CD45.2+). Mice were sacrificed at 2, 4, 8, and 24 weeks of age. Splenocytes were analyzed for the expression of CD4, H2Kb, H2Kd, CD49b, LAG-3, CD25, and FoxP3 to determine the percentage of traditional Tregs and Tr1 cells within the CD4+ population as well as for the expression of intracellular IL-10 - a key mediator of regulatory T cell function. A GVHD model was developed in which 10x106 B6 BM cells were injected IV into day of life 0 (P0) naive Balb/c mice. The ability of IUHCT-induced regulatory T cells to suppress GVHD was assessed in two groups: 1) 10x106 B6 BM cells injected into P0 mice that had undergone IUHCT and 2) 10x106 B6 BM cells co-injected with 5x106 CD4+ splenocytes harvested from 2 week old chimeric mice into P0 naive Balb/c mice. To evaluate the role of regulatory T cells following DLI in chimeric mice, 30x106 B6CD45.1 (H2Kb, CD45.1+) splenocytes were injected IV into 4 week old chimeric recipients. Mice were sacrificed at 48 hours and 2 weeks post-DLI, and regulatory T cells were measured as a percentage of CD45.2+CD4+ cells in the spleen. Results: IUHCT was associated with a significant increase in Tr1 cells, predominantly of donor origin, compared to naive controls (Figure 1). There was a corresponding increase in intracellular IL-10 expression in donor CD4+ splenocytes in chimeric animals compared to naive B6 FoxP3GFP controls at 2 weeks of age (MFI 7.07 vs. 3.26, p=0.0017). Elevated levels of regulatory T cells following IUHCT also had a functional effect in the GVHD model (Figure 2). Mice that had undergone IUHCT (group 1) demonstrated a trend toward improved survival compared to naive mice when injected with B6 BM at P0. Naive mice injected at P0 with B6 BM plus CD4+ splenocytes from chimeric mice (group 2) demonstrated significant improvement in survival compared to naive recipients of B6 BM alone (p=0.049). Although we detected no increase in traditional Tregs after IUHCT alone, we noted a 3-fold increase in traditional Tregs of host origin in chimeric animals at 48 hours post-DLI (25.5% vs. 8.3%, p Conclusion: The induction of tolerance following IUHCT is important for maintaining successful long-term engraftment and preventing GVHD. We demonstrate an increase in nontraditional regulatory T cells following IUHCT which can minimize GVHD as well as an increase in traditional Tregs following DLI in an established model of complete allogeneic engraftment. These studies suggest an important role for regulatory T cell populations in the induction of tolerance following IUHCT. They are significant in light of a potential obstacle to the clinical translation of IUHCT: the window of central tolerance induction in humans occurs at a gestational age that will pose significant technical challenges. Augmentation of regulatory T cell-dependent peripheral tolerance is one approach by which IUHCT could be delayed to a technically feasible point later in gestation. Disclosures No relevant conflicts of interest to declare.
- Published
- 2016
9. Evidence for an immune barrier after in utero hematopoietic-cell transplantation
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Alan W. Flake, Masayuki Endo, William H. Peranteau, and Obinna O. Adibe
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Graft Rejection ,medicine.medical_treatment ,Immunology ,Congenic ,Mice, Inbred Strains ,Hematopoietic stem cell transplantation ,Biology ,Biochemistry ,In utero transplantation ,Mice ,Immune system ,Pregnancy ,Transplantation Immunology ,medicine ,Animals ,Transplantation, Homologous ,Fetal Therapies ,Transplantation ,Fetus ,Graft Survival ,Hematopoietic Stem Cell Transplantation ,Immunity ,Cell Biology ,Hematology ,Survival Rate ,Transplantation, Isogeneic ,medicine.anatomical_structure ,In utero ,Female ,Bone marrow - Abstract
The competence of the immune system of the developing fetus to act as a barrier to in utero hematopoietic-cell transplantation (IUHCT) has been a source of debate. Until now, comparisons of allogeneic and congenic engraftment have been inconclusive due to methodologic limitations resulting in minimal and inefficient engraftment. In this study, E14 fetal mice received transplants of either allogeneic or congenic bone marrow using a new intravascular technique that allows definitive administration of much higher doses of donor cells. Our results demonstrate that 100% of surviving recipients demonstrate engraftment at 1 week of age, but that 70% of allogeneic recipients lose engraftment by 1 month of age, and 80% ultimately fail to sustain long-term chimerism. In contrast, all congenic recipients maintain stable, long-term, multilineage chimerism. These results strongly support an immune barrier to allogeneic engraftment after IUHCT.
- Published
- 2006
10. Mobilization of Host Hematopoietic Cells Increases Engraftment after in Utero Hematopoietic Cell Transplantation (IUHCT)
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Linda Eissenberg, Alan W. Flake, Aimee G Kim, Jesse D. Vrecenak, John F. DiPersio, and William H. Peranteau
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medicine.medical_treatment ,Plerixafor ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Biology ,Biochemistry ,CXCR4 ,Andrology ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Bone marrow ,Stem cell ,Homing (hematopoietic) ,medicine.drug - Abstract
Background: IUHCT is a non-myeloablative non-immunosuppressive transplant approach that allows for donor cell engraftment across allogeneic barriers. It has the potential to treat a large number of congenital hematologic, genetic, and immunologic disorders. A significant barrier to the clinical application of IUHCT is the low level of donor cell engraftment secondary to competition for limited space in the developing hematopoietic niche. AMD3100, an inhibitor of the chemokine receptor CXCR4, and Firategrast, a small molecule inhibitor of the α4β1 integrin, have been shown to disrupt the retention of hematopoietic stem cells (HSCs) in postnatal bone marrow (BM) transplant models. We hypothesized that administration of these agents to a pregnant dam prior to IUHCT would mobilize endogenous fetal HSCs from the fetal liver (FL) hematopoietic niche resulting in preferential homing of donor HSCs and enhanced long-term donor cell engraftment. Methods: AMD3100 (5 mg/kg) and Firategrast (100 mg/kg) were maternally administered alone or in combination to time-dated Balb/c dams at either embryonic day 17 (E17) (pharmacokinetic studies) or E14 (endogenous HSC mobilization, donor cell homing, and long-term engraftment studies). Maternal and fetal sera were analyzed by mass spectrometry at 15, 45, 90, and 180 minutes following AMD3100 or Firategrast administration to determine serum drug concentrations. Based on these pharmacokinetic studies, FLs were harvested 60 minutes following AMD3100 and/or Firategrast administration and assessed by flow cytometry for lineage- sca-1+ c-kit+ (LSK) cells to determine endogenous HSC mobilization. Short-term homing and long-term engraftment were subsequently assessed in Balb/c fetal mice (H2Kd) intravenously injected with 10x106 B6 GFP (H2Kb) BM cells following maternal administration of AMD3100, Firategrast, or AMD3100+Firategrast (Combination). For homing analysis, the proportion of donor LSK within the donor lymphocyte compartment of the FL was measured 24 hours after IUHCT. Long-term engraftment of donor cells was assessed monthly by flow cytometry of peripheral blood (PB) through 6 months of age. Multilineage PB engraftment was assessed at 9 months of age. Statistical analyses were performed using one-way or two-way ANOVA with Bonferroni post-hoc tests. Results: Maternal administration of either agent resulted in transplancental passage with peak fetal serum concentrations occurring between 45 and 90 minutes for AMD3100, and between 15 and 90 minutes for Firategrast. Treatment with Firategrast or Combination (AMD3100+Firategrast) resulted in significant mobilization of endogenous HSCs from the FL, with Firategrast eliciting the greatest mobilization vs. control (no treatment) (2.8x103 vs. 9.6x103 HSCs/FL, p Conclusion: Maternal administration of antagonists of the CXCR4/SDF-1a and a4b1/VCAM-1 pathways results in mobilization of endogenous fetal HSCs from the FL hematopoietic niche and preferential homing of donor HSCs to the FL when administered prior to IUHCT. The combination of agents targeting both the CXCR4/SDF-1a and a4b1/VCAM-1 pathways in the current study did not yield a synergistic effect on mobilization and engraftment. This work demonstrates the potential of mobilizing endogenous fetal HSCs as a strategy to overcome barriers to successful engraftment in utero bringing IUHCT closer to clinical application. Figure 1. Mobilization of HSCs from fetal liver 1 hour after treatment Figure 1. Mobilization of HSCs from fetal liver 1 hour after treatment Figure 2. Treatment enhances long-term allogeneic engraftment through 6 months Figure 2. Treatment enhances long-term allogeneic engraftment through 6 months Disclosures No relevant conflicts of interest to declare.
- Published
- 2015
11. Mechanisms of B Cell Tolerance after in Utero Hematopoietic Cell Transplantation
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Alan W. Flake, Grace Lee, Lauren E. McClain, William H. Peranteau, Aimee G Kim, Eline T. Luning Prak, and Patricia Y. Tsao
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B cell tolerance induction ,Immunology ,Naive B cell ,Receptor editing ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Molecular biology ,Haematopoiesis ,Immune system ,medicine.anatomical_structure ,Antigen ,medicine ,biology.protein ,Antibody ,B cell - Abstract
Background: In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive transplant approach that results in donor cell engraftment across immune barriers. Although a significant amount of work has investigated the fate of T cells following IUHCT, little attention has been paid to B cell tolerance and the fate of donor derived host reactive or host derived donor reactive B cells following IUHCT. B cell tolerance is broadly believed to occur by a combination of 3 mechanisms: deletion, receptor editing, and functional inactivation (anergy). In the current study we attempt to elucidate the mechanism(s) by which B cell tolerance occurs following IUHCT. Methods: 10x106 donor bone marrow (BM) cells were injected intravenously via the vitelline vein into gestational day 14 murine fetuses. IUHCT was performed in the congenic (C57Bl/6-GFP [H2Kb ] into C57Bl/6 [H2Kb ]) and allogeneic (C57Bl/6-GFP into Balb/c [H2Kd ]) strain combinations. Naive Balb/c and C57Bl/6 mice served as controls. Mice were sacrificed at day of life 3 (P3), 1 month and 4 months of age at which time their BM, spleen, and serum were harvested. To assess B cell deletion, flow cytometry was used to determine the absolute # and % of host and donor immature and pre B cells in the BM. Additionally, apoptosis of host and donor BM derived B cells was determined by annexin staining. Central receptor editing was evaluated using RT qPCR to measure the amount of Vκ-RS rearrangments in BM pre-B cells. Peripheral receptor editing was studied by calculating the % of λ light chains in mature splenocytes identified by flow cytometry. Finally, functional inactivation of donor reactive host B cells was assessed by measuring anti-H2Kb serum antibodies (ab) of allogeneic chimeras, naive, and immunized mice at 1 month of age. Results: The absolute number of BM immature B cells was decreased in allogeneic recipients of IUHCT compared to noninjected Balb/c controls at 1 month of age (fig 1). This effect was lost by 4 months of age. The decrease in B cells resulted primarily from a decrease in immature donor as opposed to host B cells compared to controls (% immature donor B cells in allogeneic recipients vs. controls: 16.2% vs. 39.9%; p Light chain receptor editing involves rearrangements within the κ and λ gene loci and may occur in BM pre-B cells or mature B cells in the spleen. We found no difference in the Vκ-RS rearrangements of pre B cells in allogeneic chimeras and controls at 1 month. In contrast, the quantity of total λ+ mature splenic B cells was increased in allogeneic chimeras at P3 (10.8 vs. 8.4%; p=0.02) and resulted from an increased host λ+ % compared to controls (10.8 vs. 8.4% p=0.03) suggesting peripheral receptor editing of host cells (fig.2). The λ+ % increase in allogeneic chimeras was lost by 1 month. Autoreactive B cells that escape deletion and receptor editing can be functionally inactivated. Neither allogeneic nor naive mice developed ab to H2Kb splenocytes, however, Balb/c mice immunized to H2kb antigen showed high ablevels (MFI fold change: allo-0.89 naive-1.37 imm-2.77; p Conclusion: B cell tolerance after IUHCT is achieved by distinct mechanisms for host and donor cell populations. Donor derived host reactive B cells undergo deletion and apoptosis while receptor editing and functional inactivation are the primary mechanisms of B cell tolerance of host derived donor reactive B cells. We hope use this and future studies of antigen specific B cell tolerance to harness the immunologic potential of IUHCT for many hematopoietic and immunologic congenital diseases. Disclosures No relevant conflicts of interest to declare.
- Published
- 2015
12. In Utero Hematopoietic Cell Transplantation Induces Peripheral Tolerance By Upregulating Two Types of Nontraditional Regulatory T Cells
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Grace Lee, Lauren E. McClain, William H. Peranteau, Haiying Li, John S. Riley, and Alan W. Flake
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T cell ,Immunology ,Peripheral tolerance ,FOXP3 ,Cell Biology ,Hematology ,Biology ,Biochemistry ,CD49b ,Tolerance induction ,medicine.anatomical_structure ,Immune system ,medicine ,IL-2 receptor ,Bone marrow - Abstract
Background: In utero hematopoietic cell transplantation (IUHCT) results in long-term, multilineage chimerism across immune barriers without myeloablation/immunosuppression. It has the potential to induce donor specific tolerance (DST) for postnatal nonmyeloablative cellular transplants to treat target diseases such as Sickle cell disease. Partial deletion of donor reactive host T cells and host reactive donor T cells is responsible for tolerance and the absence of GVHD following IUHCT. The remaining nondeleted donor and host specific T cells are thought to undergo anergy. The mechanisms by which anergy and peripheral tolerance are achieved following IUHCT are not completely understood. Studies in both mice and humans have demonstrated the importance of regulatory T cells in maintaining tolerance following postnatal allogeneic transplantation. Subsets of regulatory T cells include traditional regulatory T cells (Tregs; CD4+ CD25+ FoxP3+), Type 1 regulatory T cells (Tr1 cells; CD4+ CD49b+ CD223+), and LAP+ regulatory T cells (LAP+ cells; CD4+ CD25- LAP+). These T regulatory populations inhibit T cell activation and proliferation through, among other mechanisms, the secretion of immunosuppressive cytokines including IL-10. During pregnancy, there is upregulation of circulating IL-10 levels in maternal and fetal blood, and maternal antigen specific fetal Tregs are induced in peripheral lymphoid tissue. We propose that IUHCT takes advantage of these tolerogenic features of the fetus to promote the peripheral induction of regulatory T cells capable of suppressing reactive T cell clones that escape thymic deletion. Methods: 10x106 bone marrow cells from 6-8 week old C57Bl/6 (B6) FoxP3GFP (H2Kb) mice were injected intravenously into allogeneic gestational day 14 Balb/c FoxP3GFP fetal recipients (H2Kd). Uninjected B6 FoxP3GFP and Balb/c FoxP3GFP mice served as controls. Mice were sacrificed at 2 and 4 weeks of age. Peripheral blood donor chimerism was determined by flow cytometry at the time of sacrifice. Splenocytes were analyzed for expression of CD4, H2Kb, H2Kd, CD49b, CD223, CD25, and LAP to determine the percentage of donor and host Tregs, Tr1 cells, and LAP+ cells within the CD4+ population. CD4+ splenocytes were also assessed for intracellular IL-10 expression. Statistically significant differences between chimeric and uninjected control animals were determined using a two-tailed student's t-test. Results: Chimeric animals demonstrated a 4- and 5-fold increase in donor Tr1 cells compared to uninjected B6 FoxP3GFP controls at 2 (2.4% vs. 0.6%, p=0.03) and 4 weeks of age (2.4% vs. 0.5%, p=0.007). There was no significant difference in host Tr1 cell levels of chimeric mice compared to uninjected Balb/c FoxP3GFP controls at either 2 (0.73% vs. 0.61%, p=0.4) or 4 weeks of age (1.0% vs. 0.53%, p=0.09). Similar to Tr1 cells, LAP+ cells of donor origin were increased compared to B6 FoxP3GFP controls at 2 (2.2% vs. 0.8%, p=0.008) and 4 weeks of age (3.2% vs. 0.6%, p=0.03). Additionally, host LAP+ cells were increased compared to Balb/c FoxP3GFP controls at 2 (2.6% vs. 1.5%, p=0.0004) and 4 weeks of age (3.0% vs. 1.1%, p=0.0005). The increase in Tr1 and LAP+ cells was associated with a significant decrease in traditional Tregs in both the host and donor population at 2 and 4 weeks of age (Figure 1). Elevated levels of Tr1 and LAP+ cells did not correlate with the absolute levels of donor cell engraftment but was dependent on achieving a threshold level of chimerism known to be associated with DST. Furthermore, the MFI of intracellular IL-10 among donor CD4+ splenocytes was significantly increased compared to those from B6 FoxP3 controls at 2 weeks of age (Figure 2), demonstrating immunosuppressive activity consistent with elevated Tr1 and LAP+ levels. Conclusion: The induction of donor and host tolerance following IUHCT is important for maintaining successful long-term engraftment and preventing GVHD. DST also allows for postnatal nonmyeloablative cellular transplants to increase engraftment to therapeutically relevant levels. We demonstrate a potentially important role that nontraditional Tr1 and LAP+ regulatory T cells play in maintaining donor cell engraftment and peripheral tolerance in the setting of IUHCT. Future investigations into the ability of these cells to augment tolerance induction after inefficient IUHCT have the potential to expand the clinical promise of IUHCT. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.
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- 2015
13. The Intravenous Route Optimizes Engraftment and Survival in the Murine Model of in Utero Hematopoietic Cell Transplantation (IUHCT)
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Aimee G Kim, William H. Peranteau, Matthew M. Boelig, Haiying Li, and Alan W. Flake
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medicine.diagnostic_test ,business.industry ,Immunology ,Spleen ,Cell Biology ,Hematology ,Biochemistry ,Peripheral blood mononuclear cell ,Flow cytometry ,Andrology ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Bone marrow ,business ,Fetal Spleen ,Homing (hematopoietic) - Abstract
Introduction IUHCT has the potential to treat many congenital hematologic and immune disorders. It is a nonmyeloablative, nonimmunosuppressivetransplant approach that allows engraftment across immune barriers. Obtaining levels of engraftment necessary to treat target diseases remains a significant impediment to the clinical application of IUHCT. One barrier to consistent high-level engraftment is competition from host HSCs for limited niches at the time of transplant. The fetal liver (FL) is the hematopoietic organ in the mouse, large animals, and humans at the proposed time of IUHCT. Techniques that optimize donor cell delivery to the FL may enhance engraftment following IUHCT. In this respect, we sought to identify the route of injection that results in the least degree of mortality while promoting the highest level of engraftment. We compared the intravascular (IV), intraperitoneal (IP), and intrahepatic (IH) delivery of donor hematopoietic cells in a congenic murine model of IUHCT. We hypothesized that the IV route would promote direct homing to the FL resulting in increased levels of postnatal peripheral blood (PB) chimerism, and allow for larger cell doses without deleterious effects on fetal survival. Methods Five million C57Bl/6-GFP bone marrow mononuclear cells were suspended in a fixed volume of PBS (5 uL) and injected into embryonic day 14 (E14) C57Bl/6 fetuses via the IV, IP, or IH route. Flow cytometry and fluorescence stereomicroscopy (FSM) were used to assess donor cell homing to and engraftment of the FL at 4, 24 and 72 hours and the fetal spleen (FS) at 72 hours post IUHCT. Long-term postnatal PB engraftment was assessed monthly by flow cytometry. Fetal survival to E18 following increasing volumes of PBS injectate at E14 was compared between the different routes of injection. Statistical analysis was performed using ANOVA with Bonferronicorrection and FisherÕs Exact Test. Results The IV route led to significantly higher engraftment of the FL at 24 and 72 hours and the FS at 72 hours post IUHCT (Figure 1). FSM revealed important route-dependent differences in the patterns and kinetics of donor cell homing. IV injected donor cells robustly populated the FL in a homogenous manner within 4 hours of IUHCT. Most IP injected cells remained localized to the peritoneal cavity for over 24 hours post IUHCT with only modest engraftment of the FL. IH injected cells remained within the liver in a focal distribution with notable leaking into the perihepatic space beyond 24 hours post IUHCT. The increased efficiency with which donor cells homed to and engrafted the FL following IV IUHCT translated into superior and stable long-term donor cell engraftment (Figure 2). Fetal mice also demonstrated increased survival following higher injectate volumes administered via the IV route (Figure 3). Tolerance to larger volumes permitted higher donor cell doses to be administered at a given concentration (1x106 cells/mL), which translated into higher long-term donor cell engraftment (6 month chimerism, IV 5x106 donor cells: 4.8 +/- 0.5% vs. IV 20x106 cells: 13.2 +/- 1.0%; p < 0.05). Conclusions In a congenic murine model of IUHCT, the IV route of injection results in increased levels of stable long-term donor cell engraftment compared to the IH and IP routes. Mechanistically, this is related to the increased efficiency of donor cell homing to the site of the hematopoietic niche at the time of the transplant. Furthermore, the IV route allows for larger volumes of injectate, and thus potentially higher cell doses, with reduced fetal loss. These observations have important implications in the translation of IUHCT technique to future clinical trials. Disclosures No relevant conflicts of interest to declare.
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- 2015
14. The Use of Eicosanoids to Enhance Donor Cell Engraftment after in Utero Hematopoietic Cell Transplantation (IUHCT)
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Aimee G Kim, Matthew M. Boelig, Pulin Li, Michael A. Conner, S Loukogeorgakis, Leonard I. Zon, Alan W. Flake, William H. Peranteau, Haiying Li, and Jacqueline Tsai
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business.industry ,medicine.medical_treatment ,Immunology ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Biochemistry ,Immune tolerance ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Bone marrow ,Stem cell ,business ,Ex vivo ,Homing (hematopoietic) - Abstract
Introduction In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative, nonimmunosuppressive allogeneic transplant approach that has the potential to treat a number of congenital disorders, including hemoglobinopathies and immunodeficiencies. Donor cell engraftment at levels high enough to induce donor specific immune tolerance or to treat a target disease has been elusive and remains a major limitation to the clinical application of IUHCT. One of the most significant barriers to high levels of donor cell engraftment is competition with endogenous fetal hematopoietic stem cells (HSCs) for limited hematopoietic niches following transplant. 16,16-dimethyl-prostaglandin E2 (PGE2) and the epoxyeicosatrienoic acids (EETs) 11,12-EET and 14,15-EET have been shown to enhance donor HSC homing, survival, and cell cycling in postnatal murine and zebrafish models of HSC transplantation. We hypothesized that a single ex vivo treatment of donor cells with these eicosanoids would improve donor cell homing and survival following IUHCT,resulting in higher levels of postnatal donor engraftment. Methods Ten million bone marrow (BM) mononuclear cells from C57Bl/6-GFP mice (H2Kb) were injected intravenously into embryonic day (E14) Balb/c fetuses (H2Kd) via the vitelline vein. Donor cells were treated with vehicle, PGE2, 11,12-EET, or 14,15-EET immediately prior to IUHCT. Early homing to and engraftment of the fetal liver (FL) and spleen (FS) of PGE2 and vehicle treated BM cells at 4, 24 and 72 hours after IUHCT was assessed by flow cytometry. Donor cell survival and apoptosis was also assessed in the FL 96 hours post-IUHCT in these two treatment groups by flow cytometric analysis of intracellular expression of survivin (anti-apoptotic) and anti-caspase 3 (pro-apoptotic). Long-term peripheral blood donor cell engraftment was assessed monthly up to 6 months of age and multilineage engraftment (donor T cells, B cells, granulocytes, and macrophages) was determined at 6 months of age in recipients of all donor cell treatment groups. Statistical analysis was performed using ANOVA with BonferroniÕs multiple comparison test or Kruskal-Wallis with DunnÕs multiple comparison test for normal and non-normal data, respectively. Data reported as mean +/- SEM. Results PGE2 pre-treatment produced a significant increase in FL and FS engraftment at 72 hours post-IUHCT compared to vehicle treated donor cells (FL: 37.5 +/- 3.1% vs 21.6 +/- 1.3%; FS: 52.2 +/- 3.7% vs 39.2 +/- 1.8%; p < 0.05). There was no significant increase in donor cell engraftment in the FL at earlier time points associated with PGE2 treatment. PGE2 treatment was also associated with increased survival of donor cells compared to vehicle treated cells as indicated by increased donor cell expression of survivin (19.8±5.6% vs. 4.6±1.3%; p Conclusions The ex vivo treatment of donor cells with eicosanoids including PGE2, 11,12-EET, and 14,15 EET prior to IUHCT results in enhanced long-term multilineage allogeneic donor cell engraftment. Detailed studies of early homing to fetal hematopoietic organs and survival at 96 hours following IUHCT of PGE2 treated donor cells suggest the mechanism of enhanced engraftment is mainly do to a pro-survival effect of PGE2 and related eicosanoids. This work represents a novel application of these eicosanoids in an allogeneic model of IUHCT and highlights their potential to assist in future clinical applications of IUHCT. Disclosures Zon: FATE Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Scholar Rock: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.
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- 2015
15. CD26 inhibition enhances allogeneic donor-cell homing and engraftment after in utero hematopoietic-cell transplantation
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Obinna O. Adibe, Aziz M. Merchant, Alan W. Flake, William H. Peranteau, Philip W. Zoltick, and Masayuki Endo
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medicine.medical_treatment ,Dipeptidyl Peptidase 4 ,Immunology ,Hematopoietic stem cell transplantation ,Transplantation Chimera ,Biology ,Biochemistry ,Immune tolerance ,Mice ,Fetus ,Pregnancy ,medicine ,Immune Tolerance ,Animals ,Transplantation, Homologous ,Immunosuppression Therapy ,Dipeptidyl-Peptidase IV Inhibitors ,Mice, Inbred BALB C ,Transplantation ,fungi ,Graft Survival ,Uterus ,Hematopoietic Stem Cell Transplantation ,Cell Biology ,Hematology ,Haematopoiesis ,medicine.anatomical_structure ,Liver ,Female ,Bone marrow ,Stem cell ,Oligopeptides ,Homing (hematopoietic) - Abstract
In utero hematopoietic-cell transplantation (IUHCT) can induce donor-specific tolerance to facilitate postnatal transplantation. Induction of tolerance requires a threshold level of mixed hematopoietic chimerism. CD26 is a peptidase whose inhibition increases homing and engraftment of hematopoietic cells in postnatal transplantation. We hypothesized that CD26 inhibition would increase donor-cell homing to the fetal liver (FL) and improve allogeneic engraftment following IUHCT. To evaluate this hypothesis, B6GFP bone marrow (BM) or enriched hematopoietic stem cells (HSCs) were transplanted into allogeneic fetal mice with or without CD26 inhibition. Recipients were analyzed for FL homing and peripheral-blood chimerism from 4 to 28 weeks of life. We found that CD26 inhibition of donor cells results in (1) increased homing of allogeneic BM and HSCs to the FL, (2) an increased number of injected animals with evidence of postnatal engraftment, (3) increased donor chimerism levels following IUHCT, and (4) a competitive engraftment advantage over noninhibited congenic donor cells. This study supports CD26 inhibition as a potential method to increase the level of FL homing and engraftment following IUHCT. The resulting increased donor chimerism suggests that CD26 inhibition may in the future be used as a method of increasing donor-specific tolerance following IUHCT.
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- 2006
16. Differential Development of Donor Bone Marrow-Derived Thymocytes after Allogeneic in Utero Hematopoietic Cell Transplantation in the Murine Model
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Mathew M Boelig, William H. Peranteau, Jesse D. Vrecenak, Michael A. Conner, Yan Li, Haying Li, Huimin Jia, Aimee G Kim, Alan W. Flake, and Aliza Olive
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Immunology ,Cell Biology ,Hematology ,MHC restriction ,Biology ,Major histocompatibility complex ,Biochemistry ,Transplantation ,Andrology ,Thymocyte ,Haematopoiesis ,medicine.anatomical_structure ,Immune system ,medicine ,biology.protein ,Bone marrow ,CD8 - Abstract
Introduction In Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been determined. In this study, we utilized the murine model of allogeneic IUHCT to analyze donor and host thymocyte development in the context of normal immune ontogeny. Methods Bone marrow (BM) cells (10x106) from C57/BL6 (B6, H2kb) mice were injected intravenously into Balb/c (H2kd) fetuses at embryonic day 14 (E14). E14 B6 fetuses injected with GFP B6 BM were used as congeneic controls. At indicated postnatal ages the thymocytes were delineated by multi-color flow cytometry. Cell apoptosis and proliferation were determined by Annexin V staining and in vivo BrdU incorporation, respectively. T cell alloreactivity was assessed by in vitro and in vivo MLR. Results Our findings demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocyte processing and from thymocyte development in normal control mice. While the phenotypic development of host thymocytes remained comparable with that of normal control Balb/c mice, the four major subsets of donor thymocytes showed altered distribution, with significantly higher proportions of single positive (SP) cells, and a dramatically lower proportion of CD4+CD8+ double positive (DP) cells, compared to their host-derived counterparts and B6 controls. The extent of the alteration is directly related to both BM chimerism levels and age. Higher levels of chimerism and/or older age are associated with more profound alterations in donor thymocyte distribution. Moreover, DP cells of donor origin show higher apoptosis and lower proliferation than those of the host. Donor TCR gamma/delta cells in DN cells which do not require positive selection based on MHC recognition are relatively increased compared to the host. Moreover, compared with the naive mice and congeneic chimeric mice, the donor BM-derived thymocytes in the allogeneic chimeric mice show increased proportion of DN3 and decreased proportion of DN4, but increased TCRβ+ proportions in both DN3 and DN4 cells, indicating that donor BM-derived thymocyte development is impeded during DN to DP transition, resulting from a MHC-restriction associated mechanism. In addition, in allogeneic chimeric mice, both host and donor BM-derived T cells are tolerant to allogeneic antigens in in vivo and in vitro MLR. Conclusion Our data suggests that in an allogeneic IUHCT system the immune reconstitution of the donor bone marrow-derived thymocytes differs from that of the host cells and that of normal mice. The data supports a mechanism of impaired MHC based positive selection of donor cells by the predominantly host thymic stroma resulting in lack of progression of a higher proportion of donor cells from the DN to DP stage of thymocyte development. Taken together, although donor BM-derived T cells undergo differential thymic development, permanent host-donor two-way tolerance could be achieved in the allogeneic IUHCT mouse model. These findings add to our understanding of the requirements for tolerance induction after IUHCT and have important clinical implications in choosing an optimal donor for IUHCT. Disclosures No relevant conflicts of interest to declare.
- Published
- 2014
17. Hematopoietic Engraftment of Amniotic Fluid Stem Cells Following in Utero Transplantation
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Haiying Li, Panicos Shangaris, Stavros P. Loukogeorgakis, Aimee G Kim, Martina Piccoli, Anna L. David, William H. Peranteau, Alan W. Flake, Enrica Bertin, Michela Pozzobon, Chiara Franzin, and Paolo De Coppi
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Pathology ,medicine.medical_specialty ,education.field_of_study ,Myeloid ,Amniotic fluid ,Immunology ,Population ,CD34 ,Cell Biology ,Hematology ,Biology ,Biochemistry ,In utero transplantation ,Andrology ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Stem cell ,education - Abstract
Introduction Amniotic fluid stem cells (AFSC) are an appealing cell source for prenatal treatment of congenital blood disorders. They can be obtained safely during amniocentesis, are autologous to the fetus, and have shown hematopoietic potential when transplanted postnatally. Ex-vivo expansion to obtain adequate cell numbers is required for the clinical use of AFSC for in utero transplantation (IUT). The aim of this study was to assess long-term hematopoietic engraftment of freshly-isolated and cultured AFSC post IUT in mice, and compare it to that achieved following IUT of adult, bone marrow-derived hematopoietic stem cells (BM-HSC). Methods Lineage-depleted/c-Kit+ AFSC were isolated at embryonic day 13 (E13) from C57BL/6TgN(act-EGFP)OsbY01 (GFP) dams. AFSC were cultured for 7 days on mitotically-inactivated mouse embryonic fibroblasts with mouse embryonic stem cell media, and were sorted by flow cytometry (GFP+/c-Kit+) prior to IUT. BM-HSC (lineage-depleted/c-Kit+/Sca-1+) were isolated from 6-week old GFP mice. 104 AFSC (fresh or cultured) or BM-HSC (fresh) were injected intravenously into E14 C57BL/6J fetuses representing a congenic (autologous-like) IUT. Donor cell engraftment was assessed in the peripheral blood (PB) at 4 and 12 weeks of age by flow cytometry (% GFP+ cells within CD45+ population). Lineage characterization of engrafted cells was performed at both time points using antibodies against lymphoid (CD3, B220), and myeloid (CD11b, Gr-1) cell surface markers. Results are expressed as mean±SEM, and statistical analysis was performed using 1- or 2-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children’s Hospital of Philadelphia. Results Freshly-isolated AFSC expressed pluripotency (Oct-4, c-Myc, Klf-4) and hematopoietic markers (Sca-1+: 34.1±8.3%, CD34+: 10.6±3.1%, CD45+: 97.9±2.7%). Cultured AFSC had a doubling time of 3.2±0.2 days, and 80.7±1.6% of cells maintained c-Kit expression after 7 days of culture. c-Kit+ cultured AFSC expressed pluripotency and hematopoietic markers at similar levels as fresh AFSC. Array analysis of hematopoietic genes demonstrated significant under-expression of hematopoiesis-related transcription factors and regulators (including Gata1, Gata2, and Lmo2) in fresh and cultured AFSC compared to BM-HSC. Fetal survival rate was comparable following IUT with fresh AFSC (14/23; 60.9%), cultured AFSC (13/21; 61.9%) and BM-HSC (16/26; 61.5%) (p=0.9). Successful hematopoietic engraftment was seen at 4 weeks of age in pups that underwent fresh and cultured AFSC IUT, with comparable PB levels between groups (fresh AFSC: 21.9±1.0% vs. cultured AFSC: 20.4±1.4%; p=0.9). In contrast, IUT of BM-HSC resulted in hematopoietic micro-chimerism (engraftment: 0.04±0.02%; p Conclusions Congenic (autologous-like) IUT of AFSC results in successful multi-lineage hematopoietic engraftment in immune-competent mice, which is superior to that achieved with BM-HSC. AFSC can be expanded in vitro without differentiation, and the hematopoietic potential of cultured cells post IUT is similar to that achieved with freshly-isolated AFSC. Combined with gene therapy, IUT of autologous AFSC could be a promising approach for prenatal treatment of congenital blood disorders. Disclosures No relevant conflicts of interest to declare.
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- 2014
18. Cell Engineering with Glycogen Synthase Kinase-3 Beta Inhibitor-Loaded Synthetic Nanoparticles Enhances Hematopoietic Engraftment of Bone Marrow Mononuclear Cells Following in Utero Transplantation
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Haiying Li, William H. Peranteau, Li Tang, Izhtak Nissim, Ilana Nissim, Darrell J. Irvine, Michael A. Conner, Aimee G Kim, Alan W. Flake, Jesse D. Vrecenak, Stavros P. Loukogeorgakis, and Paolo De Coppi
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education.field_of_study ,Cluster of differentiation ,medicine.medical_treatment ,Immunology ,Population ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,Pharmacology ,Biology ,Biochemistry ,In utero transplantation ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,medicine ,Bone marrow ,Stem cell ,education - Abstract
Introduction In utero hematopoietic cell transplantation (IUHCT) has tremendous potential for prenatal treatment of congenital hematological disorders, but clinical application has been limited by low engraftment levels. Host cell competition is one of the most formidable barriers to successful engraftment post IUHCT, as fetal stem cells have a proliferative advantage over their postnatal equivalents. Glycogen synthase kinase-3 beta (GSK3β) inhibition has been shown to augment expansion kinetics of bone marrow-derived hematopoietic stem cells (HSC), resulting in enhanced repopulating capacity in vivo. We have developed a strategy that allows targeted, sustained delivery of a GSK3β inhibitor to donor cells by conjugating biodegradable nanocarriers to the cell surface. The aim of the present study was to determine whether donor cell engineering with GSK3β inhibitor-loaded nanoparticles enhances hematopoietic engraftment following IUHCT. Methods Bone marrow mononuclear cells (MNC) were isolated from 6-week old C57BL/6TgN(act-EGFP)OsbY01 mice. GSK3β inhibitor (CHIR99021) loaded multilamelar lipid vehicles (MLV) were synthesized from simple liposomes with the addition of divalent cations to induce liposome fusion. The presence of thiol-reactive maleimide groups in MLV lipid bilayers allowed stable conjugation to the cell membrane of donor MNC. IUHCT was performed in Balb/c mice at E14 and 107 MNC were injected intravenously into each fetus. Donor cell engraftment was assessed in peripheral blood at 4 and 12 weeks of age by flow cytometry (% GFP+ cells within CD45+ population). Comparisons were made between animals that received untreated MNC (control), MNC conjugated with inhibitor-loaded MLV (CHIR99021-MLV), and MNC co-injected with a bolus dose of the inhibitor equivalent to that loaded in MLV (CHIR99021-bolus). Lineage characterization of donor cells was performed using antibodies against lymphoid (CD3, B220), and myeloid (CD11b, Gr-1) cell surface markers. Results are expressed as mean±SEM, and statistical analysis was performed using 1- or 2-way ANOVA with Bonferroni post-hoc tests. Experimental protocols were approved by the Institutional Animal Care and Use Committee at The Children’s Hospital of Philadelphia. Results MLV had a diameter of 489±8nm, and contained 21.7±2.1μg CHIR99021 per 1010 nanoparticles. Inhibitor release was gradual, with 92.7±0.2% of the encapsulated mass released within 7 days. 64±9 nanoparticles were conjugated on each donor cell, resulting in inhibitor dose of 1.3*10-7μg per cell (1.3μg or 4mg/kg per fetus). MLV conjugation did not affect the ability of MNC to migrate to fetal hematopoietic organs post IUHCT. Fetal survival was comparable in control (20/27; 74.1%) and CHIR99021-MLV (10/12; 83.3%) groups, but was reduced in the CHIR99021-bolus group (7/15; 46.7%; p Conclusion Cell engineering with GSK3β inhibitor-loaded synthetic nanoparticles enhances hematopoietic engraftment of BM-MNC following IUHCT. Prolonged retention of the biodegradable nanocarriers on donor cell surfaces enables sustained CHIR99021 release, and allows pseudo-autocrine bioactivity. Conjugation of drug-loaded particles directly to donor cells allows targeted augmentation of HSC function, and could markedly increase the therapeutic potential of IUHCT. Disclosures No relevant conflicts of interest to declare.
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- 2014
19. High-level allogeneic chimerism achieved by prenatal tolerance induction and postnatal nonmyeloablative bone marrow transplantation
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Aimen F. Shaaban, Satoshi Hayashi, William H. Peranteau, Alan W. Flake, and Michael H. Hsieh
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medicine.medical_treatment ,Immunology ,Mice, Inbred Strains ,Transplantation Chimera ,Hematopoietic stem cell transplantation ,Biology ,Biochemistry ,In utero transplantation ,Lymphocyte Depletion ,Mice ,Mice, Congenic ,Transplantation Immunology ,medicine ,Immune Tolerance ,Animals ,Transplantation, Homologous ,Bone Marrow Transplantation ,Cell Biology ,Hematology ,Total body irradiation ,Donor Lymphocytes ,Hematopoiesis ,Transplantation ,Tolerance induction ,Fetal Diseases ,Kinetics ,medicine.anatomical_structure ,Models, Animal ,Bone marrow ,Whole-Body Irradiation - Abstract
Clinical application of allogeneic bone marrow transplantation (BMT) has been limited by toxicity related to cytoreductive conditioning and immune response. In utero hematopoietic stem cell transplantation (IUHSCT) is a nonablative approach that achieves mixed chimerism and donor-specific tolerance but has been limited by minimal engraftment. We hypothesized that mixed chimerism achieved by IUHSCT could be enhanced after birth by nonmyeloablative total body irradiation (TBI) followed by same-donor BMT. To test this hypothesis, mixed chimerism was created by IUHSCT in a major histocompatibility complex-mismatched strain combination. After birth, chimeric animals received nonmyeloablative TBI followed by transplantation of donor congenic bone marrow cells. Our results show that: (1) low-level chimerism after IUHSCT can be enhanced to high-level chimerism by this strategy; (2) enhancement of chimerism is dependent on dose of TBI; (3) the mechanism of TBI enhancement is via a transient competitive advantage for nonirradiated hematopoietic stem cells; (4) engraftment observed in the tolerant, fully allogeneic IUHSC transplant recipient is equivalent to a congenic recipient; and (5) host-reactive donor lymphocytes are deleted with no evidence of graft-versus-host disease. This study supports the concept of prenatal tolerance induction to facilitate nonmyeloablative postnatal strategies for cellular therapy. If clinically applicable, such an approach could dramatically expand the application of IUHSCT.
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- 2002
20. Complete allogeneic hematopoietic chimerism achieved by a combined strategy of in utero hematopoietic stem cell transplantation and postnatal donor lymphocyte infusion
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Satoshi Hayashi, Alan W. Flake, Aimen F. Shaaban, and William H. Peranteau
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Lymphocyte Transfusion ,medicine.medical_treatment ,Immunology ,Transplantation Chimera ,Hematopoietic stem cell transplantation ,Thymus Gland ,Biology ,Biochemistry ,Donor lymphocyte infusion ,In utero transplantation ,Mice ,Pregnancy ,medicine ,Immune Tolerance ,Animals ,Transplantation, Homologous ,Mice, Inbred BALB C ,Graft Survival ,Hematopoietic Stem Cell Transplantation ,Cell Biology ,Hematology ,Tolerance induction ,Fetal Diseases ,medicine.anatomical_structure ,Animals, Newborn ,Models, Animal ,Female ,Bone marrow ,Stem cell - Abstract
In utero hematopoietic stem cell transplantation (IUHSCTx) can achieve mixed hematopoietic chimerism and donor-specific tolerance without cytoreductive conditioning or immunosuppression. The primary limitation to the clinical application of IUHSCTx has been minimal donor cell engraftment, well below therapeutic levels for most target diseases. Donor lymphocyte infusion (DLI) has been used in postnatal circumstances of mixed chimerism as targeted immunotherapy to achieve a graft-versus-hematopoietic effect and to increase levels of donor cell engraftment. In this report we demonstrate in the murine model that a combined approach of IUHSCTx followed by postnatal DLI can convert low-level, mixed hematopoietic chimerism to complete donor chimerism across full major histocompatibility complex barriers with minimal risk for graft-versus-host disease (GVHD). Time-dated embryonic day 14 (E14) to E15 Balb/c (H-2Kd, CD45.2) fetuses underwent intraperitoneal injection of 5 × 106T-cell–depleted B6 (H-2Kb, CD45.2) bone marrow cells. Chimeric recipients then received transplants at either 4 or 8 weeks of age with 1 of 3 doses (5, 15, or 30 × 106cells) of donor congenic splenocytes (B6-Ly5.2/Cr, H-2Kb, CD45.1). The response to DLI was dose dependent, with conversion to complete donor peripheral blood chimerism in 100% of animals that received high-dose (30 × 106 cells) DLI. Only 1 of 56 animals receiving this dose succumbed to GVHD. This study directly supports the potential therapeutic strategy of prenatal tolerance induction to facilitate nontoxic postnatal cellular therapy and organ transplantation, and it has broad implications for the potential treatment of prenatally diagnosed genetic disorders.
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- 2002
21. Donor T Cell Content Of Whole Bone Marrow Is Responsible For Maternal Immunization Following In Utero Hematopoietic Cell Transplantation
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Jesse D. Vrecenak, William H. Peranteau, Erik G. Pearson, and Alan W. Flake
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biology ,T cell ,Immunology ,Cell Biology ,Hematology ,Acquired immune system ,Major histocompatibility complex ,Biochemistry ,medicine.anatomical_structure ,Immune system ,Antigen ,medicine ,biology.protein ,Bone marrow ,Antigen-presenting cell ,CD8 - Abstract
Introduction In Utero Hematopoietic Cell Transplantation (IUHCT) represents a potential therapeutic approach for many genetic disorders by inducing mixed hematopoietic chimerism and associated donor specific tolerance. We have shown previously that IUHCT of adult bone marrow (BM) cells in the allogeneic murine model induces a maternal alloimmune response against donor antigen. This results in the transfer of maternal alloantibodies via breast milk triggering an adaptive immune response in the pup with loss of chimerism in 70% of recipients. In contrast to our results, other investigators using a murine model of IUHCT with fetal liver (FL) derived cells, identified no significant maternal alloresponse and implicated prenatal maternal-fetal cellular trafficking and maternal T-cells as a primary mechanism of engraftment loss. As these two different observations have profound implications for clinical IUHCT, we sought to reconcile the data by investigation of differences between the two models. One major difference is the difference in antigen presenting cell (APC) and T cell content between the BM and FL grafts. We hypothesized that depletion of the APC or T-cell content of the donor graft would reduce maternal immunization after IUHCT. Methods Donor APC, defined as macrophages (CD11b+), dendritic cells (CD11c+) and B cells (CD45R/B220+) make up a substantial portion of murine BM and contribute to the allohumoral response by indirect presentation of antigen via class II MHC. Donor T cells (CD3+) have also been implicated in the host immune response by indirect and direct activation of host immune cells. In this series of experiments, Balb/c mice underwent IUHCT at E14 with the following B6 donor cell populations: 1) BM; 2) FL; 3) APC depleted BM (containing an increased relative fraction of T cells) 4) T cell depleted BM 5) APC and T cell depleted BM with T cell add back to 2.5% (to approximate T cell concentration in WBM) 6) CD4 Depleted BM and 7) CD8 Depleted BM. Serial post-natal maternal alloantibody assays measured by Fluorescent Activated Cell Sorting (FACS) analysis were used to quantify the postnatal maternal immune response. Results We found a significant difference in the postnatal maternal humoral response in each of these groups. In the WBM and APC depleted BM groups there was a significant increase in maternal serum IgG levels reaching a 24 fold increase compared to negative controls (no serum) at 4 weeks. When we measured the maternal immune response following IUHCT in the fetal liver and T cell depleted BM groups, both of which have decreased relative CD3+ cells, we found a minimal increase (2 fold) in maternal serum IgG levels at 4 weeks. We could not identify a difference between CD4 depleted and CD8 depleted WBM. Conclusion These findings identify that donor T cells within whole bone marrow are responsible for maternal immunization following IUHCT. Modification of donor cell content may allow IUHCT from non-maternal donor sources by avoiding the maternal immune response. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
22. Donor Specific Tolerance Of Renal Allografts Following Haploidentical In Utero Hematopoietic Cell Transplantation In The Canine Model
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Alan W. Flake, Tricia R. Bhatti, Erik G. Pearson, Jesse D. Vrecenak, and William H. Peranteau
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medicine.medical_specialty ,medicine.diagnostic_test ,business.industry ,medicine.medical_treatment ,Interstitial nephritis ,Immunology ,Urology ,Immunosuppression ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry ,Organ transplantation ,Nephrectomy ,Transplantation ,Tolerance induction ,Blood chemistry ,medicine ,Renal biopsy ,business - Abstract
Introduction In utero hematopoietic cell transplantation (IUHCT) can result in allogeneic mixed hematopoietic chimerism and associated donor specific tolerance (DST). We have reported a 1-2% donor chimerism threshold for consistent DST in the murine model as defined by the ability to enhance engraftment postnatally with a same donor minimal conditioning BMT, and have confirmed this finding in the canine model of IUHCT. Recently, we have optimized the canine model and achieved higher levels of chimerism (average>10%). As prenatal tolerance induction for postnatal organ transplantation is one of the clinical goals of IUHCT, we hypothesized that the presence of donor chimerism (>2.0%) after haploidentical IUHCT in the canine model would be sufficient to allow same donor renal transplants without immunosuppression. Method Stable mixed hematopoietic macrochimerism was documented by VNTR after haploidentical IUHCT performed at 40 days gestation using maternal donor BM cells in 4 pups (chimerism levels 3-38%). One positive control canine with no detectable chimerism following IUHCT also underwent haploidentical renal transplantation. Renal transplantation was performed from the maternal donor at ages between 12 and 18 months, and the pups were serially followed by ultrasound of the graft, blood chemistry and urinalysis post transplant. At 60 days an open biopsy of the allograft was taken and at 6 months a graft nephrectomy was performed for histologic analysis. Results Following transplantation, all recipients demonstrated blood flow to the renal cortex and all laboratory values were within normal ranges. At 60 days and 6 months 3 of the 4 recipients demonstrated a graft without evidence of acute or chronic rejection. The recipient with the lowest level of chimerism (3%) demonstrated evidence of mild interstitial nephritis (Banff class 1 rejection) following transplantation at the time of renal biopsy which increased in severity to severe interstitial fibrosis and moderate tubular atrophy (Banff class 3 rejection) at the time of graft nephrectomy. The positive control canine without detectable chimerism demonstrated clinical and histologic evidence of acute rejection within one week of transplantation. Conclusion Our results support the ability of IUHCT to induce DST for haploidentical organ transplantation, in a large animal model, without the need for immunosuppression. In agreement with previous studies in the murine model there appears to be a threshold level of donor chimerism required for associated DST. Although our numbers are not adequate to establish absolute thresholds of chimerism predictive of DST for organ transplantation, it appears to be slightly higher than the 1-2% threshold established for cellular transplantation. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
23. Posttranslational Modification Of The Thrombopoietin Receptor Regulates Cytokine Signal Transduction and Hematopoietic Stem Cell Engraftment
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Aimee G Kim, Alan W. Flake, William H. Peranteau, Emily A. Partridge, Jesse D. Vrecenak, and Miroslaw Kozlowski
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chemistry.chemical_classification ,MAPK/ERK pathway ,Glycosylation ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Cell biology ,Intracellular signal transduction ,Transplantation ,chemistry.chemical_compound ,chemistry ,Cell surface receptor ,Receptor clustering ,Signal transduction ,Glycoprotein - Abstract
Background Thrombopoietin (TPO) is a key cytokine regulating hematopoietic stem cell (HSC) quiescence and self-renewal, and has been shown to augment ex vivo expansion and maintenance of HSCs in culture. Impaired TPO signaling is associated with bone marrow failure and thrombocytopenia. TPO mediates intracellular signal transduction by binding its cell surface receptor, cMpl, and transgenic mice deficient in cMpl expression have an age-progressive reduction in HSC content. An improved understanding of the mechanisms regulating HSC proliferation, self-renewal, and quiescence is essential to optimize transplantation, expansion, and other therapeutic applications of HSCs. Posttranslational modification of proteins in the Golgi results in conjugation of complex-type N-glycans on glycoproteins destined for the cell surface. A complete deficiency of glycosylation results in failure of transport of proteins to the plasma membrane, while glycosylation with complex branched carbohydrates regulates a wide variety of functions at the cell surface including receptor clustering, cross-linking and trafficking. N-glycosylation of tyrosine kinase cytokine receptors has been shown to enhance their signaling potential by sparing receptor internalization via endocytosis, thus promoting receptor retention at the cell surface (Partridge EA et al, Science 2004). cMpl is known to undergo N-linked glycosylation, with defects in posttranslational processing associated with polycythemia vera. However, little is known about the functional significance of glycosylation of cMpl on TPO signaling and HSC function. Here we show that glycosylation of cMpl regulates TPO signal transduction in an endocytosis-dependent manner, resulting in enhanced short-term engraftment in a mouse model of in utero hematopoietic cell transplantation (IUHCT). Methods 293T cells stably overexpressing the TPO receptor cMpl were treated with either the α-mannosidase II inhibitor swainsonine (1 μg/ml) to inhibit complex N-glycan branching or the hexosamine biosynthetic pathway substrate UDP-GlcNAc (12.5 – 50 mM) to increase N-glycan production. Activation of signaling proteins was measured by immunoblotting and immunofluorescence studies. Clathrin- and caveolin-dependent endocytosis was inhibited by potassium depletion and nystatin treatment, respectively. The effect of posttranslational cMpl modification on donor cell engraftment was assessed in a murine model of IUHCT. Fetal liver from gestational day 14 Balb/c recipient fetuses were assessed for donor cell chimerism 48 hours after transplantation with 10e6 B6-GFP bone marrow cells with or without UDP-GlcNAc treatment. Results Inhibition of N-linked glycosylation by swainsonine treatment resulted in reduced downstream signal transduction following TPO stimulation, with significantly decreased levels of phosphorylation of MAPK (Figure 1A). Pharmacologic disruption of endocytosis was found to ablate the effects of swainsonine treatment, with equivalent and robust activation of MAPK in swainsonine-treated and untreated cells. Upregulation of N-linked glycosylation by supplementation of the glycosylation pathway with UDP-GlcNAc treatment resulted in a dose-dependent increase in signaling following TPO stimulation, with increased levels of phosphorylation of MAPK (Figure 1B). Finally, UDP-GlcNAc treatment of donor cells resulted in significantly higher engraftment levels at 48 hours post IUHCT compared to controls (20.18 +/- 2.77 versus 30.27 +/- 1.985, p=0.0417). Conclusions These results support a role for cMpl glycosylation in modulating TPO signal transduction in an endocytosis-dependent manner, and suggest that enhanced cMpl glycosylation can optimize HSC function resulting in increased levels of engraftment following IUHCT. Ongoing studies of receptor trafficking, including internalization by clathrin-dependent and –independent endocytosis, are needed to further elucidate the functional significance of complex N-glycans on cMpl fate. These studies will ultimately improve our understanding of how TPO signaling may be manipulated to control HSC self-renewal and quiescence to optimize in vitro expansion and HSC transplantation. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
24. Differential Development Of Donor Bone Marrow-Derived Thymocytes After Allogeneic In Utero Hematopoietic Cell Transplantation In The Murine Model
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Yan Li, Jesse D. Vrecenak, Haying Li, Huimin Jia, William H. Peranteau, and Alan W. Flake
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Abstract
Introduction In Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been determined. In this study, we utilized the murine model of allogeneic IUHCT to analyze donor and host thymocyte development during postnatal ontogeny. Methods Bone marrow (BM) cells (10x106) from Foxp3GFP C57/BL6 (B6, H2kb) mice were injected intravenously into Foxp3GFP Balb/c (H2kd) fetuses at embryonic day 14 (E14). At indicated postnatal ages the thymocytes were delineated by multi-color flow cytometry. Cell apoptosis and proliferation were determined by Annexin V staining and in vivo BrdU incorporation, respectively. Results Our findings demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocyte processing and from thymocyte development in normal control mice. While the phenotypic development of host thymocytes remained comparable with that of normal control Balb/c mice, the four major subsets of donor thymocytes showed altered distribution, with significantly higher proportions of immature TCRβ-CD4+CD8- and TCRβ-CD4-CD8+ single positive (SP) cells, a slightly higher proportion of CD4-CD8- (DN) cells, and a dramatically lower proportion of CD4+CD8+ double positive (DP) cells, compared to their host-derived counterparts and B6 controls. The extent of the alteration is directly related to both BM chimerism levels and age. Higher levels of chimerism and/or older age are associated with more profound alterations in donor thymocyte distribution. Moreover, DP cells of donor origin show higher apoptosis and lower proliferation than those of the host. Donor DN cells and TCR gamma/delta cells which do not require positive selection based on MHC recognition are increased compared to the host. Donor T-regulatory cells were present at a higher frequency in the thymus and secondary lymphoid organs of chimeric animals compared to T-regulatory cells in host and normal strain controls. Conclusion Our data suggests that in an allogeneic IUHCT system the immune reconstitution of the donor bone marrow-derived thymocytes differs from that of the host cells and that of normal mice. The data supports a mechanism of impaired MHC based positive selection of donor cells by the predominantly host thymic stroma resulting in lack of progression of a higher proportion of donor cells from the DN to DP stage of thymocyte development. In contrast, generation of donor T-regulatory cells was not impaired and appeared to be increased in chimeric animals. The alloreactivity, regulatory suppression, and MHC restriction of the donor derived T cells, and the potential importance of these observations for clinical IUHCT remain to be determined. Disclosures: No relevant conflicts of interest to declare.
- Published
- 2013
25. Altered Thymocyte Development in Allogeneic in Utero Hematopoietic Cell Transplantation in the Mouse Model
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Alan W. Flake, Haiying Li, William H. Peranteau, Yan Li, Jacqueline Tsai, Miho Watanabe, and Jesse D. Vrecenak
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medicine.diagnostic_test ,T cell ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry ,Flow cytometry ,Andrology ,Transplantation ,Haematopoiesis ,Thymocyte ,medicine.anatomical_structure ,Immune system ,medicine ,Bone marrow ,CD8 - Abstract
Abstract 4668 Introduction In Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been analyzed. In this study, we utilized the murine model of allogeneic IUHCT and analyzed donor and host thymocyte development. Methods Bone marrow (BM) cells (10×106) from Foxp3GFP C57/BL6 (B6, H2kb) mice were injected intravenously into Foxp3GFP Balb/c (H2kd) fetuses at embryonic day 14 (E14). At indicated postnatal time points the thymocytes were analyzed by multi-color flow cytometry. Results The results demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocytes and from thymocyte development in normal B6 and Balb/c control mice. Though each subpopulation of the host's thymocytes showed comparable levels to the normal untransplanted Balb/c mice, the donor BM-derived thymocytes demonstrated significantly higher proportions of CD4+CD8- and CD4-CD8+ single positive cells, and a dramatically lower proportion of CD4+CD8+ double positive cells compared to their donor-derived counterparts, respectively. These discrepancies increased with each analysis time point up to 12 weeks. Immature single positive cells, including both TCRb-CD4+CD8- and TCRb-CD4-CD8+ cells, were significantly higher in the donor-derived thymocytes than the host, indicating the development of the donor BM-derived thymocytes were impeded at the DN-DP stage in this specific allogeneic IUHCT setting. In addition, we also found higher frequencies and more potent suppressive capacity of regulatory T cells in the donor-derived T cell compartment than in the host T cell compartment. In the reversed allogeneic strain combination, i.e. IUHCT of Foxp3GFP Balb/c BM MNCs into the E14 Foxp3GFP B6 recipients, thymocytes of both origins showed imbalanced intra-thymic development that differed from the normal mouse. Conclusion Our data in the current study suggests that immune reconstitution of the donor bone marrow derived thymocytes differs from that of the host cells and that of normal mice in an allogeneic IUHCT system. The mechanism of the altered thymic development in allogeneic IUHCT, and the potential importance of these observations for clinical IUHCT remains to be determined. Bone marrow (BM) cells (10×106) from Foxp3GFP C57/BL6 (B6, H2kb) mice were injected intravenously into Foxp3GFP Balb/c (H2kd) fetuses at embryonic day 14 (E14). At indicated postnatal time points the thymocytes were analyzed by multicolor flow cytometry (Fig. A), and the expression of TCR¦Â in each individual subpopulation in 4-week old mice was measured (Fig. B). Disclosures: No relevant conflicts of interest to declare.
- Published
- 2012
26. In Utero Hematopoietic Cell Transplantation Using Haploidentical Parental Donors Reverses the Lethal Phenotype in Dogs with Canine Leukocyte Adhesion Deficiency
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Andrea T. Badillo, Susan W. Volk, Mark P. Johnson, William H. Peranteau, Adam Kaye, Dennis D. Hickstein, Laura M. Tuschong, Thomas R. Bauer, Yu-chen Gu, and Alan W. Flake
- Subjects
business.industry ,medicine.medical_treatment ,Immunology ,CD18 ,Cell Biology ,Hematology ,Hematopoietic stem cell transplantation ,medicine.disease ,Biochemistry ,Transplantation ,Graft-versus-host disease ,In utero ,Intensive care ,medicine ,Leukocytosis ,medicine.symptom ,business ,Leukocyte adhesion deficiency - Abstract
Juvenile dogs with the genetic disease leukocyte adhesion deficiency or CLAD, like children with leukocyte adhesion deficiency or LAD, experience recurrent life threatening bacterial infections due to the inability of leukocytes to migrate to sites of infection. Both CLAD and LAD result from defects in the leukocyte integrin CD18 molecule. We have used the CLAD model to develop new therapeutic approaches to children with LAD. Previous studies demonstrate that low levels of donor chimerism following matched littermate transplant reverses the disease phenotype in CLAD. However, most children with LAD lack a matched sibling donor. In utero hematopoietic stem cell transplantation (IUHCT) has been shown to result in low levels of allogeneic chimerism in the normal mouse model. In the current study we evaluate IUHCT in the CLAD model using a haploidentical paternal donor. A previously transplanted CLAD female was mated with a paternal CLAD carrier. IUHCT was performed at gestational day 50 by ultrasound guided intraperitoneal injection of 1.7E+08 paternal CD34+ enriched BM cells/kg estimated fetal weight reconstituted with nonenriched paternal BM to provide 2.4% CD3+ cells. Seven fetuses were injected. One pup was still born and one died on day 2 from maternal neglect. Flow cytometry for CD18 expression in PB, spleen, liver, thymus and BM from the two deceased pups confirmed the diagnosis of CLAD with donor cell engraftment (PB:1.3–3%, spleen:3.5–4%, liver:3.4–4.2%, thymus:1.7–4.4%, BM:3.3–21.3%). Histology demonstrated no evidence of GVHD. Of the 5 surviving pups, 3 are CLAD carriers (Louie, Miles, Ella) and 2 are CLAD offspring (Billie, Duke). Currently all 5 pups are alive at 5 months of age. Engraftment analysis in those in which it is possible by CD18 expression (Billie and Duke) or the presence of the Y chromosome (Billie and Ella) demonstrates donor cells in all analyzable pups at 5 months. The PB levels of CD18+ donor cells in CLAD offspring are low but stable and contribute to multiple lineages (Fig 1A). Clinically, Billie and Duke are alive and active at 5 months compared to historical controls with 4 and 6 month mortalities of 75% and 100%. Both have had mild leukocytosis (Billie:17.6–23.8K/uL, 21.4K/uL @ 5 months; Duke:23.4–39.5K/uL, 23.8K/uL @ 5 months) compared to historical CLAD controls of 50–100K/uL. Billie has had no clinical episodes consistent with the CLAD phenotype. Duke has experienced five CLAD phenotypic episodes which have resolved without the need for intensive care (Fig 1B). There was no evidence of GVHD in any injected animals. This study highlights the ability to safely achieve levels of haploidentical donor CD18+ leukocytes following IUHCT which markedly improve the lethal disease phenotype in a disease-specific large-animal model of a human genetic disease. It supports the potential therapeutic value of IUHCT for diseases, such as LAD, which can be successfully treated with low levels of hematopoietic chimerism. Figure Figure
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- 2006
27. In Utero Transplantation of Transduced Hematopoietic Stem Cells Results in Deletion of Transgene-Specific T Cells
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Pablo Laje, Masayuki Endo, William H. Peranteau, Philip W. Zoltick, and Alan W. Flake
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T cell ,Immunology ,Cell Biology ,Hematology ,T lymphocyte ,Biology ,Biochemistry ,Molecular biology ,In utero transplantation ,Transplantation ,Haematopoiesis ,medicine.anatomical_structure ,Immune system ,medicine ,Stem cell ,CD8 - Abstract
The developing fetal immune system provides a unique opportunity to manipulate normal immunologic development for therapeutic prenatal and anticipated postnatal interventions. In previous studies we have shown that allogeneic in utero hematopoietic cell transplantation (IUHCT) results in donor specific tolerance that can subsequently facilitate non-myeloablative postnatal cellular or organ transplants. It follows that in utero injection of transduced hematopoietic stem cells (HSC) could potentially induce tolerance to a transgene encoded protein. We hypothesized that expression of a transduced antigenic protein by HSC and their progeny would alter thymic T cell development resulting in deletion of antigen specific T-cells. To test this hypothesis, we used the mammary tumor virus (MTV) superantigen system to evaluate the effect of IUHCT of transduced HSC on T cell development. In this system, expression of different MTV oncogenes by different I-E+ strains of mice results in deletion of T cells expressing the relevant Vβ T cell receptor. Specifically, mice which are Mtv7+ delete T cells expressing the Vβ6 T-cell receptor. In this study, CD150+CD48− enriched Balb/c (I-E+ Mtv7−) HSC were transduced with an HIV-based lentivirus expressing MTV7 under an MND promoter. 1.5E+05 transduced cells were injected intravascularly via the vitelline vein into E14 Balb/c fetuses. Non-injected age matched naive Balb/c mice served as the control group. The peripheral blood (PB) and thymuses of injected fetuses and control mice were harvested at day of life (DOL) 10, 20 and 60 and analyzed by flow cytometry for T lymphocyte Vβ6 expression. Additionally, the T cell composition of the thymus was assessed at DOL10 for CD4 and CD8 single positive (SP) and CD4/CD8 double positive (DP) cells. Thymic flow cytometric analysis at DOL10 revealed that greater than 98% of the T cells were CD4CD8 DP, a stage that has not yet undergone negative selection. No significant difference was noted in the percentage of thymic Vβ6+ DP T-cells at this time point or at DOL20 and DOL60. In contrast, there was a significant decrease in the percentage of Vβ6+ peripheral blood SP cells in those mice injected with MTV7 transduced HSC relative to control mice at DOL10, DOL20 and DOL60 (p Figure Figure
- Published
- 2006
28. Evidence for an Adaptive Immune Barrier after in Utero Hematopoietic Cell Transplantation
- Author
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Masayuki Endo, William H. Peranteau, Obinna O. Adibe, and Alan W. Flake
- Subjects
Fetus ,business.industry ,Immunology ,Congenic ,Cell Biology ,Hematology ,Biochemistry ,In utero transplantation ,Transplantation ,Andrology ,Haematopoiesis ,medicine.anatomical_structure ,Immune system ,Medicine ,Bone marrow ,Stem cell ,business - Abstract
In utero hematopoietic cell transplantation (IUHCT) is a nonmyeloablative approach that takes advantage of normal immunologic development to achieve donor specific tolerance. Despite the many potential advantages of the fetal recipient, IUHCT across MHC barriers has been limited by low levels of engraftment and the inability to consistently achieve allochimerism. Although the immature immune system of the developing fetus has long been appreciated as a principal advantage of IUHCT, the competence of the fetal immune system to act as a barrier to IUHCT has been a source of debate. Until now, comparisons of allogeneic and congenic engraftment have been inconclusive due to methodologic limitations resulting in minimal and inefficient engraftment. In this study, a new intravascular technique that allows definitive administration of much higher doses of donor cells was employed to directly compare the incidence and levels of engraftment following in utero transplantation of either congenic or allogeneic bone marrow (BM) or enriched hematopoietic stem cells (HSCs). 20E+06 B6 GFP BM cells (H2Kb+, GFP+) or 1E+05 cKit+Sca-1+Lin- B6 GFP cells (H2Kb+, GFP+) were intravenously injected via the vitelline vein into gestational day 14 Balb/c (H2Kd+, allogeneic) or C57Bl/6 (H2Kb+, congenic) fetal mice. The peripheral blood (PB) of recipients was serially analyzed by flow cytometry for GFP+ donor cells at 1, 2, 4 and 6 months of age. A separate group of animals was harvested at 1 week of age (2 weeks after injection) to assess donor chimerism in PB and BM. Our results demonstrate that 100% of surviving recipients of whole BM demonstrate engraftment at 1 week of age, but that 70% of allogeneic recipients lose engraftment by 1 month of age, and 80% ultimately fail to sustain long-term chimerism. In contrast, all congenic recipients maintain engraftment at 6 months of age (Table 1). Chimerism levels in allogeneic recipients drop significantly after 1 month of life while those in congenic recipients remain stable. This results in a significant difference in engraftment levels in allogeneic and congenic recipients beyond 1 month of life (Fig 1). Similar results were seen when enriched HSCs were the donor cell source. 100% (2/2) of congenic recipients of enriched HSCs demonstrated stable low level PB engraftment up to 6 months of life (0.14–0.55% GFP+ donor cells). In contrast, no allogeneic recipients (0/9) of enriched HSCs were chimeric from 1 to 6 months of life. In combination, these results demonstrating a 100% efficiency of long-term engraftment in congenic recipients and loss of engraftment by 1 month of age in the majority of allogeneic recipients strongly implicate an adaptive immune barrier to allogeneic engraftment after IUHCT. Better understanding of the immune mechanisms limiting allogeneic engraftment after IUHCT is required to allow the development of successful strategies for IUHCT. | | 1 week of age (BM) | 1 week of age (PB) | 1 month of age (PB) | 6 months of age (PB) | |:---------- | ------------------ | ------------------ | ------------------- | -------------------- | | congenic | 100% (8/8) | 100% (8/8) | 100% (25/25) | 100% (25/25) | | allogeneic | 100% (8/8) | 100% (8/8) | 29% (9/31) | 19% (6/31) | Efficiency of Engraftment after IUHCT in Congenic and Allogeneic Recipients ![Figure][1] Figure [1]: pending:yes
- Published
- 2006
29. Murine Mesenchymal Stem Cells Fail To Suppress Acute Graft Versus Host Disease
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Alan W. Flake, William H. Peranteau, Andrea T. Badillo, Stephanie Filice, and Keith A. Alcorn
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education.field_of_study ,biology ,business.industry ,Immunology ,Mesenchymal stem cell ,Population ,Spleen ,Cell Biology ,Hematology ,medicine.disease ,Major histocompatibility complex ,Biochemistry ,In vitro ,medicine.anatomical_structure ,Graft-versus-host disease ,In vivo ,biology.protein ,Medicine ,Bone marrow ,business ,education - Abstract
Mesenchymal stem cells (MSCs) are multipotent cells of potential clinical interest given their capacity for in vitro expansion and intriguing immunologic properties. Studies using human and murine MSCs demonstrated their ability to suppress stimulated T cells in vitro. Consequently, much interest has been generated in the ability of MSCs to prevent graft versus host disease (GVHD). In fact, a limited number of case reports suggest a therapeutic role of human MSCs in the treatment of GVHD. Although encouraging, no systematic study has been performed to assess the ability of MSCs to suppress GVHD in vivo. In the current study we utilize a purified population of adult bone marrow derived murine MSCs previously shown to be immunosuppressive in vitro to evaluate the therapeutic potential in vivo of MSCs in an established model of murine GVHD. Methods: 8–12 week old C57Bl/6xBalb/c F1 mice were given 750cGy irradiation in a Cs135 gamma irradiator. 16–20 hours after irradiation, the mice received one of four groups of donor cells via lateral tail vein injection: 1) 10e6 C57Bl/6 (B6) bone marrow cells (BM) (n=5), 2) 10e6 B6 BM cells + 30e6 B6 spleen cells (n=12) (GVHD inoculum), 3) 10e6 B6 BM cells + 30e6 B6 spleen cells + 1e6 B6 MSCs (n=4) or 4) 10e6 B6 BM cells + 30e6 B6 spleen cells + 1.5e5 B6 MSCs (n=7). Mice were weighed and assessed for physical signs of GVHD such as ruffled fur, desquamation, diarrhea and hunching prior to receiving irradiation and on a weekly basis following irradiation and injection of the cellular inoculum. Results: In accordance with previous studies, the injection of 30e6 parental (B6) spleen cells combined with 10e6 (B6) parental BM cells into an F1 (B6xBalb/c) recipient following 750cGy irradiation resulted in a reliable model of GVHD. All mice receiving this inoculum demonstrated physical signs of GVHD including hunching and ruffled fur by three weeks post injection with the progression to desquamation and diarrhea by 5 weeks post injection. Similar to mice receiving the GVHD inoculum, mice receiving 30e6 B6 spleen cells + 10e6 B6 BM cells + either 1e6 B6 MSCs or 1.5e5 B6 MSCs demonstrated physical signs of GVHD by 3 weeks post injection. Control mice receiving only 10e6 B6 BM cells after 750cGy irradiation remained healthy and did not demonstrate any signs of GVHD. As demonstrated in figure 1, coinjection of either 1e6 B6 MSCs or 1.5e5 B6 MSCs with 30e6 B6 spleen cells + 10e6 B6 BM cells did not result in any significant change in weight loss compared to those mice receiving the GVHD inoculum. Similarly, the survival of mice receiving the GVHD inoculum was not improved by the coinjection of either 1e6 B6 MSCs or 1.5e5 B6 MSCs (25% vs 0% vs 28.57% at 6 weeks post injection). Conclusion: Previous studies have supported an in vitro immunosuppressive function of MSCs and a limited number of human studies have highlighted the potential ability of human MSCs to suppress GVHD. Despite these previous findings the current study demonstrates that the intravenous injection of MHC matched murine MSCs at the time of GVHD induction in an established murine model of GVHD does not affect the onset or severity of GVHD as measured by physical exam, weight loss and survival. Figure Figure
- Published
- 2005
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