Induction of Donor-Type Chimerism and Transplantation Tolerance Across Major Histocompatibility Barriers in Sublethally Irradiated Mice by Sca-1+Lin− Bone Marrow Progenitor Cells: Synergism With Non-Alloreactive (Host × Donor)F1 T Cells

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3212-3221 ◽  
Author(s):  
Esther Bachar-Lustig ◽  
Hong Wei Li ◽  
Hilit Gur ◽  
Rita Krauthgamer ◽  
Hadar Marcus ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Transplantation Tolerance ◽  
Major Histocompatibility ◽  
Hematopoietic Stem ◽  
Donor Type ◽  
Total Body ◽  
Veto Cells

Induction of transplantation tolerance by means of bone marrow (BM) transplantation could become a reality if it was possible to achieve engraftment of hematopoietic stem cells under nonlethal preparatory cytoreduction of the recipient. To that end, BM facilitating cells, veto cells, or other tolerance-inducing cells, have been extensively studied. In the present study, we show that BM cells within the Sca-1+Lin− cell fraction, previously shown to be enriched for early hematopoietic progenitors, are capable of reducing specifically antidonor CTL-p frequency in vitro and in vivo, and of inducing split chimerism in sublethally 7-Gy–irradiated recipient mice across major histocompatibility complex barriers. The immune tolerance induced by the Sca-1+Lin−cells was also associated with specific tolerance toward donor-type skin grafts. The minimal number of cells required to overcome the host immunity remaining after 7 Gy total body irradiation is very large and, therefore, it may be very difficult to harvest sufficient cells for patients. This challenge was further addressed in our study by demonstrating that non-alloreactive (host × donor)F1 T cells, previously shown to enhance T-cell–depleted BM allografts in lethally irradiated mice, synergize with Sca-1+Lin− cells in their capacity to overcome the major transplantation barrier presented by the sublethal mouse model.

Induction of Donor-Type Chimerism and Transplantation Tolerance Across Major Histocompatibility Barriers in Sublethally Irradiated Mice by Sca-1+Lin− Bone Marrow Progenitor Cells: Synergism With Non-Alloreactive (Host × Donor)F1 T Cells

Blood ◽  
1999 ◽  
Vol 94 (9) ◽  
pp. 3212-3221 ◽  
Author(s):  
Esther Bachar-Lustig ◽  
Hong Wei Li ◽  
Hilit Gur ◽  
Rita Krauthgamer ◽  
Hadar Marcus ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Transplantation Tolerance ◽  
Major Histocompatibility ◽  
Hematopoietic Stem ◽  
Donor Type ◽  
Total Body ◽  
Veto Cells

Abstract Induction of transplantation tolerance by means of bone marrow (BM) transplantation could become a reality if it was possible to achieve engraftment of hematopoietic stem cells under nonlethal preparatory cytoreduction of the recipient. To that end, BM facilitating cells, veto cells, or other tolerance-inducing cells, have been extensively studied. In the present study, we show that BM cells within the Sca-1+Lin− cell fraction, previously shown to be enriched for early hematopoietic progenitors, are capable of reducing specifically antidonor CTL-p frequency in vitro and in vivo, and of inducing split chimerism in sublethally 7-Gy–irradiated recipient mice across major histocompatibility complex barriers. The immune tolerance induced by the Sca-1+Lin−cells was also associated with specific tolerance toward donor-type skin grafts. The minimal number of cells required to overcome the host immunity remaining after 7 Gy total body irradiation is very large and, therefore, it may be very difficult to harvest sufficient cells for patients. This challenge was further addressed in our study by demonstrating that non-alloreactive (host × donor)F1 T cells, previously shown to enhance T-cell–depleted BM allografts in lethally irradiated mice, synergize with Sca-1+Lin− cells in their capacity to overcome the major transplantation barrier presented by the sublethal mouse model.

scholarly journals Cathepsin K maintains the number of lymphocytes in vivo

2020 ◽  
Author(s):  
Renate Hausinger ◽  
Marianne Hackl ◽  
Ana Jardon-Alvarez ◽  
Miriam Kehr ◽  
Sandra Romero Marquez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cathepsin K ◽  
Hematopoietic Stem ◽  
Cell Numbers ◽  
Cell Compartments ◽  
Lymphocyte Homeostasis

AbstractIn this study, we investigated the influence of the loss of Cathepsin K (Ctsk) gene on the hematopoietic system in vitro and in vivo. We found that cultures with Lineage- SCA1+ KIT+ (LSK) cells on Ctsk deficient stromal cells display reduced colony formation and proliferation, with increased differentiation, giving rise to repopulating cells with reduced ability to repopulate the donor LSK and T cell compartments in the bone marrow. Subsequent in vivo experiments showed impairment of lymphocyte numbers, but, gross effects on early hematopoiesis or myelopoiesis were not found. Most consistently in in vivo experimental settings, we found a significant reduction of (donor) T cell numbers in the bone marrow. Lymphocyte deregulation is also found in transplantation experiments, which revealed that Ctsk is required for optimal regeneration not only of T cells, but also of B cells. Interestingly, cell non-autonomous Ctsk regulates both B- and T cell numbers, but T cell numbers in the bone marrow require an additional autonomous Ctsk-dependent process. Thus, we show that Ctsk is required for the maintenance of hematopoietic stem cells in vitro, but in vivo, Ctsk deficiency most strongly affects lymphocyte homeostasis, particularly of T cells in the bone marrow.

Adoptive Transfer of In Vitro Generated T Cell Precursors Enhances Donor T Cell Reconstitution and Graft-Versus-Tumor Activity in Allogeneic Hematopoietic Stem Cell Transplantation Recipients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 63-63 ◽  
Author(s):  
Johannes L. Zakrzewski ◽  
Adam A. Kochman ◽  
Sidney X. Lu ◽  
Theis H. Terwey ◽  
Theo D. Kim ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
T Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Hematopoietic Stem ◽  
T Cell Reconstitution

Abstract Allogeneic hematopoietic stem cell transplantation (HSCT) is associated with a varying period of immunoincompetence that particularly affects he T cell lineage resulting in significant morbidity and mortality from opportunistic infections. Recent studies have shown that murine T cells and their precursors can be generated from hematopoietic stem cells (HSC) in vitro using a OP9-DL1 coculture system consisting of OP9 bone marrow stromal cells expressing the Notch 1 ligand Delta-like 1 and growth factors (interleukin 7 and fms-like tyrosine kinase-3 ligand). In this study we determined the effects of adoptively transferred in vitro generated T cell precursors on T cell reconstitution after allogeneic HSCT. We selected HSC (Lin- Sca-1hi c-kithi) from bone marrow (BM) of C57BL/6 mice and cultured these cells on a monolayer of OP9-DL1 cells in the presence of growth factors. These HSC expanded 2,000–5,000-fold within 3–4 weeks and consisted of >95% CD4-CD8-double negative (DN) T cell precursors after 16–28 days of culture. We infused these cells (8x106) with T cell depleted (TCD) BM (5x106) or purified HSC into allogeneic recipients using minor antigen mismatched and MHC class I/II mismatched transplant models. Control mice received TCD BM or purified HSC only. Progeny of OP9-DL1 derived T cell precursors were found in thymus and spleen increasing thymic cellularity and significantly improving thymic and splenic donor T cell chimerism. This effect was even more pronounced when purified HSC instead of whole BM were used as allograft. T cell receptor repertoire and proliferative response to foreign antigen (determined by third party MLR) of in vivo differentiated OP9-DL1 derived mature T cells were intact. Administration of in vitro generated T cell precursors did not induce graft-versus-host disease (GVHD) but mediated significant graft-versus-tumor (GVT) activity (determined by in vivo bioluminescence imaging) resulting in a subsequent significant survival benefit. This advantage was associated with better cytokine responses (IL-2, INF-g, TNF-a) in T cells originating from OP9-DL1 derived T cell precursors compared to BM donor derived T cells. We conclude that the adoptive transfer of OP9-DL1 derived T cell precursors significantly enhances post-transplant T cell reconstitution and GVT activity in the absence GVHD.

scholarly journals Anti-Human CD117 CAR T-Cells Efficiently Eliminate Hematopoietic Stem and CD117-Positive AML Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4063-4063 ◽  
Author(s):  
Renier Myburgh ◽  
Jonathan Kiefer ◽  
Norman F Russkamp ◽  
Alexander Simonis ◽  
Surema Pfister ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Cell Lines ◽  
Surface Expression ◽  
Hematopoietic Stem ◽  
Car T Cells ◽  
Car T

Abstract Introduction: Acute Myeloid Leukemia (AML) is a clonal disease of the hematopoietic system that originates from immature hematopoietic stem and progenitor cells (HSPC). Because some AML-initiating cells are comparatively resistant to conventional cytotoxic agents, disease relapses are common with current treatment approaches. As an alternative, immunological eradication of leukemic cells by adoptively transferred chimeric-antigen receptor T-cells (CAR T-cells) might be considerably more efficient. To date, however, the search for AML-specific surface antigens has remained largely elusive. To circumvent this problem, we propose to target the stem cell antigen c-Kit (CD117) that is expressed by physiological HSPC as wells as by leukemic blasts in >90% of AML patients. For translation into a clinical setting, CAR T cell treatment must then be followed by depletion of CAR T-cells as well subsequent healthy/allogeneic HSC transplantation. Methods: A lentiviral vector was generated which incorporates the CAR (scFv linked to intracellular CD3ζ and 4-1BB signaling domains via stalk and transmembrane regions derived from CD8), followed by a T2A ribosomal skip sequence and RQR8 as selection marker and depletion gene (surface expression of CD34 and CD20 epitopes). The scFv was extracted from a previously published bivalent anti-CD117 antibody (clone 79D) that was derived from an artificial human phage library (Reshetnyak et al., PNAS, 2013). 79D exhibits high binding affinity to an epitope in the membrane-proximal domain of human CD117. Human CD117 was cloned in human CD117 negative HL-60 AML cells and cell lines with stable expression of CD117 at various levels were derived from these. Results: T-cells were isolated from healthy donors or AML patients in complete remission and both healthy donor and AML pateint derived T-cells exhibited sustained growth after activation with recombinant human IL-2 and CD3/CD28 beads. Lentiviral transduction yielded consistently high transduction rates, ranging from 55 - 75% as determined by staining for RQR8 and the scFv. In co-culture assays, CAR T-cells eliminated more than 90% of CD117high leukemia cell lines within 24 hours at effector-to target ratios (E:T) of 4:1 and 1:1 and more than 50% at E:T of 1:4. CAR-mediated cytotoxicity correlated with levels of CD117 surface expression as the elimination of CD117low target cells was less efficient compared to CD117high and CD117intermediate cells. In long-term cytotoxicity assays (45d), only CD117low cells were able to escape CAR-mediated killing. In the setting of primary cells, anti-CD117 CAR T-cells effectively depleted >90% of lin-CD117+CD34+CD38+ and >70% of lin-CD117+CD34+CD38- cells from healthy bone marrow in vitro within 48 hours. Similarly, >70% of patient derived leukemic blasts were eliminated by autologous anti-CD117 CAR T-cells within 48 hours (1:1 ratio of CAR T cells:blasts). In a long-term assay, no outgrowth of leukemic blasts was observed in the presence of autologous CAR T-cells over 3 weeks. To determine effectivity of CAR T-cells in vivo, humanized mice (NSG & MTRG-SKI) were engrafted with umbilical cord blood derived CD34+ cells. A single injection of 2x106 anti-CD117 CAR T-cells resulted in >90% depletion of CD117+ cells in the bone marrow within 6 days. Finally, humanized mice transplanted with bone marrow from AML patients expressing CD117 were treated with patient-derived autologous CAR T-cells. At 6 weeks after injection of CAR T-cells, >98% of hu-CD45 CD117+ cells were depleted in the bone marrow while control human T-cell treated mice showed full-blown CD117 positive AML. Conclusions: We provide proof of concept for the generation of highly-potent CAR T-cells re-directed against CD117 from healthy human donors and AML patients. Anti-CD117 CAR T-cells exhibit high cytotoxic activity against CD117+ cell lines as well as primary healthy HSPC and patient AML cells in vitro and in vivo in murine xenograft models. Strategies for the complete elimination of CAR T-cells (immunologic or small molecule based) are required before translation of this approach to the clinical setting. Disclosures Neri: Philochem AG: Equity Ownership.

PU.1 Is a Critical Downstream Target of AML1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 358-358 ◽  
Author(s):  
Gang Huang ◽  
Pu Zhang ◽  
Steffen Koschmieder ◽  
Joseph D. Growney ◽  
D. Gary Gilliland ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
Fetal Liver ◽  
Proximal Region ◽  
Specific Gene ◽  
Hematopoietic Stem

Abstract PU.1 is expressed in hematopoietic stem cells (HSC), progenitors and differentiating blood cells except terminally differentiated T cells, erythrocytes and megakaryocytes. PU.1 is required for commitment of HSC to multiple lineages. PU.1 −/− embryos die perinatally and fail to generate myeloid and B cells. We previously reported that a DNase I hypersensitive site located 14 kb upstream of the PU.1 transcription start site (−14 DHS) confers myelomonocytic specific gene expression. Targeted deletion this DHS fragment in mice results in a decrease in PU.1 expression in bone marrow to 20% of wild type levels, subsequently leading to a profound decrease in macrophages and B cells. Within the DHS fragment is a “core” consisting of a distal (296bp) and a proximal (253bp) region, which are highly conserved among different species. The PU.1 promoter by itself cannot direct gene expression in vivo. However, −14 DHS confers to the promoter the ability to direct expression of a reporter gene in granulocytes, monocytes, and B-cells of transgenic mice. The proximal region can itself direct high-level gene expression. The proximal region contains 3 AML1 sites. These results, along with data indicating that PU.1 expression is selectively absent from Aml1 −/− embryos (Okada, et al, Oncogene. 1998), suggested that AML1 is likely to be upstream of PU.1. Electro-mobility gel shift assays and chromatin immunoprecipitation assays confirmed that AML1 binds to all 3 AML1 sites both in vitro and in vivo. Mutation of the 3 AML1 sites dramatically reduced the DHS activity of conferring gene expression. We used real time PCR to quantitatively measure PU.1 expression in both embryonic and adult hematopoiesis. We found that PU.1 expression was completely lost in the 9.5 dpc yolk sac, 10.5 dpc AGM and fetal liver of Aml1−/− embryos, suggesting that AML1 is required for PU.1 expression during embryonic hematopoiesis. To evaluate the effects of AML1 loss in the adult hematopoiesis, we employed a conditional Aml1 knockout allele in which LoxP flanked Aml1 (Aml1F/F) was excised by Mx1 promoter driven Cre expression following injection of pIpC. These mice show that Aml1 is not required for maturation of myeloid lineages in adult mice. However, these mice develop a mild myeloproliferative phenotype characterized by increasing in bone marrow and peripheral blood (PB) neutrophils, a 5 fold increasing in HSC, and 2–3 fold increasing myeloid progenitors. Spleen and liver contain infiltration by myeloid cells. These mice also display a dramatic decrease (~80%) in PB platelets and bone marrow megakaryocytes. Furthermore, there are significant blocks in lymphoid development, including reduced numbers of pre-B, pro-B and mature B cells, as well a block in T cell maturation at the DN2 (CD4−;CD8−;CD44+;CD25+) stage. We observed a 70% reduction of PU.1 expression in sorted HSC, progenitors, Gr1+/Mac1+ and B-cells from these mice relative to control mice. In contrast, upregulation of 3–5 fold expression in Ter119+, CD41+, and T cells in these mice compared to controls. Our data shows that PU.1 is a critical target gene of AML1, and AML1 regulates PU.1 in both positive and negative way. We are currently testing the ability of restoration of PU.1 expression to rescue specific defects in Aml1F/F; Tg (Mx1-cre) mice, as well as investigating the role of decreased PU.1 expression in human AML in which the function of AML1 is disrupted.

CD8+/TCR− Facilitating Cells Induce Generation of Regulatory T cells in Vivo: Chimeric Regulatory T Cells are Antigen-Specific and Enhance Engraftment of Allogeneic HSC in Non-Obese Diabetic (NOD) Mice.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2318-2318
Author(s):  
Yiming Huang ◽  
Larry D Bozulic ◽  
Thomas Miller ◽  
Hong Xu ◽  
Lala-Rukh Hussain ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
T Cells ◽  
Regulatory T Cells ◽  
Nod Mice ◽  
Strong Suppression ◽  
Hematopoietic Stem ◽  
Cell Based Therapy

Abstract CD8+/TCR− graft facilitating cells (FC) are a novel tolerogenic cell population in bone marrow that potently enhance engraftment of hematopoietic stem cells (HSC) in allogeneic and syngeneic recipients. The CD11c+/CD11b−/B220+ plasmacytoid precursor dendritic cell (p-preDC) subpopulation of FC (p-preDC FC) comprises over 60% of FC total and plays a critical and nonredundant role in facilitation. FC prevent graft-versus-host disease and remain tolerogenic after in vivo infusion. Regulatory T cells (Treg) are immunomodulatory cells that maintain tolerance in vivo. They can be generated in vitro via co-culture with p-preDC. There is great interest regarding the use of Treg as a cell-based therapy to induce graft/host tolerance in vivo. However, a major challenge to the clinical use of Treg has been to obtain sufficient numbers of cells for in vivo use and maintain their tolerogenic properties in vivo after in vitro expansion. Here, we evaluated whether FC function by inducing the production of Tregin vivo and examined the function of these chimeric Tregin vivo and in vitro. HSC (c-Kit+Sca-1+Lin−; KSL) were sorted from donor B6 and NOD mice. 10,000 B6 HSC and 1,000 NOD HSC were transplanted by tail-vein injection into recipient NOD mice conditioned with 950 cGy of total body irradiation (TBI). Spleen, thymus, and bone marrow were harvested from recipient NOD mice 5 weeks after transplantation. CD4+CD25+Foxp3+ Treg were analyzed by flow cytometry. FC induced the generation of both donor and recipient CD4+CD25+Foxp3+ Tregin vivo; the majority of Treg were recipient-derived (89% to 97%). To test the function of Treg from HSC + FC chimeras (chimeric Treg), CD8− CD4+CD25+ Treg were sorted from the spleen of chimeras 5 weeks after transplantation. 50,000 chimeric Treg plus 10,000 B6 HSC were transplanted into NOD recipients conditioned with 950 cGy TBI. Recipients of 50,000 Treg from naïve B6 spleens (B6 Treg) + HSC or HSC alone served as controls. Five of 26 recipients of HSC alone engrafted and survived up to 100 days. Only 2 of 5 recipients of HSC plus 50,000 B6 Treg engrafted and none of the recipients exhibited durable engraftment beyond 100 days. In striking contrast, 100% (4 of 4) recipients of HSC + 50,000 chimeric Treg engrafted durably, with survival ≥ 100 days. Chimeric Treg function was confirmed in vitro by MLR suppressor assays, as evidenced by strong suppression of T cell proliferation. Sorted chimeric Treg demonstrated an 87.2% suppression of cell proliferation when plated in a 1:1 ratio with naïve NOD responder cells and B6 stimulator cells. Moreover, when plated at a 1:4 and 1:8 ratio with naïve NOD responders, Treg suppressive function titrated to 62.7% and 43.3%, respectively. In contrast, sorted Treg from naïve B6 animals showed 75.8%, 35.4, and 29.4% suppression when plated in ratios of 1:1, 1:4, and 1:8, respectively. Taken together, these data suggest that FC induce the production of antigen-specific Tregin vivo and chimeric Treg are superior to naïve Treg in suppressing the proliferation of effector T cells and potently enhance engraftment of allogeneic HSC.

Anti-CD19 Chimeric Antigen Receptor Controlled By The Suicide Gene HSVsr39TK In Hematopoietic Stem Cells For Immunotherapy Of B-Lineage Malignancies

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1659-1659 ◽  
Author(s):  
Sarah M. Larson ◽  
Andy Tu ◽  
Shanta Senadheera ◽  
Michelle Ho ◽  
Donald B. Kohn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
Suicide Gene ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Specific Cytotoxicity ◽  
B Lineage

Abstract Background Although significant improvements have been made, patients with relapsed or refractory B-cell malignancies continue to have unfavorable clinical outcomes. We hypothesize that transduction of hematopoietic stem cells (HSCs) with an anti-CD19 Chimeric Antigen Receptor (CAR) will produce a multi-lineage, persistent immunotherapy that can be controlled by the HSVsr39TK suicide gene. Methods First generation anti-CD19 CAR lentiviral constructs containing the HSVsr39TK suicide gene were developed to compare vectors containing the human elongation factor alpha short (EFS) or myeloproliferative sarcoma virus U3 (MNDU3) promoters for transduction efficiency, antigen-specific cytotoxicity and ganciclovir (GCV)-induced cell death in primary human T-cells. The CD28 costimulatory domain was added to the selected construct, and high titer lentiviral vectors were generated to evaluate transduction of human umbilical cord blood (UCB) HSCs for in vitro and in vivo assays. In vitro assays were performed after culture under myeloid differentiation conditions, followed by assessment of phenotype, transduction efficiency, cytotoxic function and GCV-induced cell death. In vivo assays were conducted through transplantation of gene-modified human HSCs into irradiated NSG pups, compared to humanized NSG injected with non-modified human HSCs. Once engraftment was identified, mice from each cohort were further separated into GCV treated and untreated groups. Following GCV administration, mice were harvested to evaluate the presence of human and CAR-modified cells in the bone marrow, spleen and peripheral blood. Results In human primary T cells, the MNDU3 promoter resulted in higher percentage of CAR expressing cells and mean fluorescence intensity compared to the EFS promoter. Cytotoxicity by the transduced T cells against the huCD19+Raji cell line showed similar target cell specific lysis among the constructs. Treatment with GCV effectively decreased the in vitro survival of the cells containing the HSVsr39TK gene compared to the non-transduced and control vector. The construct with MNDU3 promoter was then used with a CD28-containing second-generation anti-CD19 CAR (CCL-MND-αCD19/z/28-sr39). Once transduction efficiency and CAR function were validated in primary human T cells, this vector was used to transduce human UCB CD34+ cells. Following transduction, these cells were evaluated in vitro and in vivo. The cells used for the in vitro studies were cultured under myeloid differentiation conditions. The average number of CAR expressing cells was 45% at the clinically relevant vector copy number of 0.5-1 copies/cell. The myeloid cells transduced with the CCL-MND-αCD19/z/28-sr39 vector demonstrated CD19-specific killing and were eliminated by GCV. In vivo studies demonstrated successful engraftment of transduced HSC with CAR-expressing cells in the different hematopoietic lineages (T, NK, myeloid) detected among human cells in the bone marrow (1.2-15.4%, mean 7.6%), spleen (0.3-15.4%, mean 5.6%), and peripheral blood (0.5-30%, mean 9.2%). Mice engrafted with anti-CD19 CAR-modified HSCs exhibited decreased huCD19+ populations, compared to the mice engrafted with non-modified HSCs. Treatment with GCV resulted in significant decrease in CAR-expressing cells only in the mice transplanted with CD34+ cells transduced with the HSVsr39TK-containing vector. Discussion Here we demonstrate that HSCs can be effectively transduced with an anti-CD19 CAR linked to the HSVsr39TK suicide gene. The CAR was detected in human cells in the bone marrow, spleen and peripheral blood and resulted in decreased B-lineage populations as an index of antigen-specific cytotoxicity; the HSVsr39TK gene conferred sensitivity to ganciclovir which eliminated transduced cells. These results provide pre-clinical support for the use of a CD19 targeted CAR in HSCs for the treatment of B-cell malignancies. Disclosures: Larson: Millenium: Speakers Bureau.

Dendritic cells derived from HOXB4-immortalized hematopoietic bone marrow cells

2011 ◽  
Vol 236 (11) ◽  
pp. 1291-1297 ◽  
Author(s):  
Abdul Mannan Baru ◽  
Jayendra Kumar Krishnaswamy ◽  
Anchana Rathinasamy ◽  
Michaela Scherr ◽  
Matthias Eder ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Dendritic Cells ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Surface Expression ◽  
Hematopoietic Stem ◽  
Hematopoietic Bone Marrow

Dendritic cells (DCs) are essential for the generation and modulation of cell-mediated adaptive immunity against infections. DC-based vaccination involves transplantation of ex vivo-generated DCs loaded with antigen in vitro, but remains limited by the number of autologous or allogeneic cells. While in vitro expansion and differentiation of hematopoietic stem cells (HSCs) into DCs seems to be the most viable alternative to overcome this problem, the complexity of HSC expansion in vitro has posed significant limitations for clinical application. We immortalized lineage-depleted murine hematopoietic bone marrow (lin−BM) cells with HOXB4, and differentiated them into CD11c+MHCII+ DCs. These cells showed the typical DC phenotype and upregulated surface expression of co-stimulatory molecules on stimulation with various toll-like receptor ligands. These DCs efficiently presented exogenous antigen to T-cells via major histocompatibility complex (MHC) I and II and viral antigen on infection. Finally, they showed migratory capacity and were able to generate antigen-specific primed T-cells in vivo. In summary, we provide evidence that HOXB4-transduced lin−BM cells can serve as a viable means of generating fully functional DCs for scientific and therapeutic applications.

scholarly journals Primitive Human Hematopoietic Cells Are Enriched in Cord Blood Compared With Adult Bone Marrow or Mobilized Peripheral Blood as Measured by the Quantitative In Vivo SCID-Repopulating Cell Assay

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3919-3924 ◽  
Author(s):  
Jean C.Y. Wang ◽  
Monica Doedens ◽  
John E. Dick
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Hematopoietic Cells ◽  
Allogeneic Transplantation ◽  
Functional Assay ◽  
Hematopoietic Stem ◽  
Mobilized Peripheral Blood

Abstract We have previously reported the development of in vivo functional assays for primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of severe combined immunodeficient (SCID) and nonobese diabetic/SCID (NOD/SCID) mice following intravenous transplantation. Accumulated data from gene marking and cell purification experiments indicate that the engrafting cells (defined as SCID-repopulating cells or SRC) are biologically distinct from and more primitive than most cells that can be assayed in vitro. Here we demonstrate through limiting dilution analysis that the NOD/SCID xenotransplant model provides a quantitative assay for SRC. Using this assay, the frequency of SRC in cord blood (CB) was found to be 1 in 9.3 × 105 cells. This was significantly higher than the frequency of 1 SRC in 3.0 × 106 adult BM cells or 1 in 6.0 × 106 mobilized peripheral blood (PB) cells from normal donors. Mice transplanted with limiting numbers of SRC were engrafted with both lymphoid and multilineage myeloid human cells. This functional assay is currently the only available method for quantitative analysis of human hematopoietic cells with repopulating capacity. Both CB and mobilized PB are increasingly being used as alternative sources of hematopoietic stem cells in allogeneic transplantation. Thus, the findings reported here will have important clinical as well as biologic implications.

Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Gisele Olinto Libanio Rodrigues ◽  
Julie Hixon ◽  
Hila Winer ◽  
Erica Matich ◽  
Caroline Andrews ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
T Cell Leukemia ◽  
Cell Leukemia ◽  
Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

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