scholarly journals Ganciclovir treatment of herpes simplex thymidine kinase-transduced primary T lymphocytes: an approach for specific in vivo donor T-cell depletion after bone marrow transplantation?

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1333-1341 ◽  
Author(s):  
P Tiberghien ◽  
CW Reynolds ◽  
J Keller ◽  
S Spence ◽  
M Deschaseaux ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Herpes Simplex ◽  
Marrow Transplantation ◽  
Ex Vivo ◽  
Retroviral Vector ◽  
Hematopoietic Stem

Abstract Allogeneic bone marrow transplantation (BMT) is associated with a severe complication--graft-versus-host disease (GVHD). Although effectively preventing GVHD, ex vivo T-lymphocyte marrow depletion unfortunately increases graft rejection and reduces the graft-versus- leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T cells before their infusion with hematopoietic stem cells could allow for selective in vivo depletion of these T cells with ganciclovir (GCV) if subsequent GVHD was to occur. Thus, one could preserve the beneficial effects of the T cells on engraftment and tumor control in patients not experiencing severe GVHD. To obtain T cells specifically depleted by GCV, we transduced primary T cells with a retroviral vector containing the HS-tk and neomycin resistance (NeoR) genes. Gene transfer was performed by coculturing PHA +/- CD3- or alloantigen-stimulated purified T cells on an irradiated retroviral vector producer cell line or by incubating the T cells in supernatant from the producer. Subsequent culture in G418 for 1 week allowed for the selection of transduced cells. GCV treatment of interleukin-2-responding transduced and selected cells resulted in greater than 80% growth inhibition, whereas GCV treatment of control cells had no effect. Similarly, the allogeneic reactivity of HS-tk-transduced cells was specifically inhibited by GCV. Combining transduced and nontransduced T cells did not show a bystander effect, thus implying that all of the cells inhibited by GCV were indeed transduced. Lastly, studies involving the transduction of the HUT-78 (T-lymphoma) cell line suggest that stable expression of HS-tk can be maintained over 3 months in vitro in the absence of G418. In summary, we have established the feasibility of generating HS-tk-transduced T cells for subsequent in vivo transfer with hematopoietic stem cells and, if GVHD occurs, specific in vivo GCV- induced T-cell depletion in allogeneic BMT recipients.

scholarly journals Ganciclovir treatment of herpes simplex thymidine kinase-transduced primary T lymphocytes: an approach for specific in vivo donor T-cell depletion after bone marrow transplantation?

Blood ◽  
1994 ◽  
Vol 84 (4) ◽  
pp. 1333-1341 ◽  
Author(s):  
P Tiberghien ◽  
CW Reynolds ◽  
J Keller ◽  
S Spence ◽  
M Deschaseaux ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Herpes Simplex ◽  
Marrow Transplantation ◽  
Ex Vivo ◽  
Retroviral Vector ◽  
Hematopoietic Stem

Allogeneic bone marrow transplantation (BMT) is associated with a severe complication--graft-versus-host disease (GVHD). Although effectively preventing GVHD, ex vivo T-lymphocyte marrow depletion unfortunately increases graft rejection and reduces the graft-versus- leukemia (GVL) effect. The ex vivo transfer of the herpes simplex thymidine kinase (HS-tk) suicide gene into T cells before their infusion with hematopoietic stem cells could allow for selective in vivo depletion of these T cells with ganciclovir (GCV) if subsequent GVHD was to occur. Thus, one could preserve the beneficial effects of the T cells on engraftment and tumor control in patients not experiencing severe GVHD. To obtain T cells specifically depleted by GCV, we transduced primary T cells with a retroviral vector containing the HS-tk and neomycin resistance (NeoR) genes. Gene transfer was performed by coculturing PHA +/- CD3- or alloantigen-stimulated purified T cells on an irradiated retroviral vector producer cell line or by incubating the T cells in supernatant from the producer. Subsequent culture in G418 for 1 week allowed for the selection of transduced cells. GCV treatment of interleukin-2-responding transduced and selected cells resulted in greater than 80% growth inhibition, whereas GCV treatment of control cells had no effect. Similarly, the allogeneic reactivity of HS-tk-transduced cells was specifically inhibited by GCV. Combining transduced and nontransduced T cells did not show a bystander effect, thus implying that all of the cells inhibited by GCV were indeed transduced. Lastly, studies involving the transduction of the HUT-78 (T-lymphoma) cell line suggest that stable expression of HS-tk can be maintained over 3 months in vitro in the absence of G418. In summary, we have established the feasibility of generating HS-tk-transduced T cells for subsequent in vivo transfer with hematopoietic stem cells and, if GVHD occurs, specific in vivo GCV- induced T-cell depletion in allogeneic BMT recipients.

InVitro Culture of Human Cord Blood Hematopoietic Stem/Progenitor Cells Interfered with Their Distribution to Other Bone Marrow Compartments after Intra-Bone Marrow Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4868-4868
Author(s):  
Kohshi Ohishi ◽  
Kentaro Yamamura ◽  
Masahiro Masuya ◽  
Naoyuki Katayama
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Marrow Transplantation ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Left Tibia

Abstract Intra-bone marrow transplantation (IBMT) is a novel strategy for transplantation of hematopoietic stem cells because it can transfer various types of cells to bone marrow regardless of their homing capacity. However, reconstitution process of these cells after IBMT remains to be fully elucidated. Here, we investigated whether in vitro culture of cord blood hematopoietic stem/progenitor cells affects their reconstitution in bone marrow after IBMT. Freshly isolated AC133+ cells (5x104 cells/mouse) or all cells derived from AC133+ cells cultured with growth factors (stem cell factor, flt-3 ligand, and thrombopoietin) for 5 days were injected into the bone marrow of the left tibia in irradiated NOD/SCID mice. In the bone marrow of the injected left tibia, the engraftment levels of human CD45+ cells at 6 weeks after transplantation was not considerably different between transplantation of noncultured and cytokine-cultured cells (54±28% vs. 69±13%). However, the migration of transplanted cells to the bone marrow of other noninjected bones was extremely lower for cytokine-treated cells compared with noncultured cells (2±2% vs. 36±10%). Similar findings were observed for engraftment of CD34+ cells. To enhance the migration of cytokine-cultured cells after IBMT, we similarly transplanted cultured AC133+ cells into the bone marrow of the left tibia, assessed the engraftment in the injected and noninjected tibiae at 7 days after transplantation, and then subcutaneously administered G-CSF (250 μg/kg/d) for 5 days. Administration of G-CSF stimulated the migration of cytokine-cultured cells to the bone marrow of previously-aspirated right tibia but failed to induce their migration to intact bone marrow of femur. These data indicate that ex vivo manipulation of hematopoietic progenitor/stem cells adversely influences their migration properties to other bone marrow compartments after IBMT. Our data raise caution for future clinical applications of the IBMT method using ex vivo-manipulated hematopoietic stem cells.

Elimination of clonogenic malignant human T cells using monoclonal antibodies in combination with 2'-deoxycoformycin.

1987 ◽  
Vol 5 (12) ◽  
pp. 1900-1911 ◽  
Author(s):  
C L Schwartz ◽  
C P Minniti ◽  
P Harwood ◽  
S Na ◽  
M L Banquerigo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Monoclonal Antibodies ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Ex Vivo ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Bone Marrow ◽  
Hematopoietic Stem ◽  
T Lymphoblasts

2'Deoxycoformycin (dCF) specifically inhibits adenosine deaminase (ADA) and causes selective cytotoxicity of normal and malignant T cells. In clinical trials, dCF caused rapid lysis of malignant T lymphoblasts. Although dCF has been associated with dose-limiting nonhematopoietic toxicities, myelosuppression has not been observed. Since dCF is relatively nontoxic to hematopoietic stem cells, we tested dCF for utility in the ex vivo purging of malignant T lymphoblasts from remission leukemic bone marrow for autologous bone marrow transplantation. We found that T lymphoblast cell lines were sensitive to dCF (plus deoxyadenosine [dAdo]) under conditions that did not ablate human hematopoietic colony-forming cells. Moreover, combined pharmacologic (dCF plus dAdo) and immunologic (anti-T cell monoclonal antibodies [McAb] plus complement) purging resulted in additive reduction in clonogenic T lymphoblasts. These results provide the basis for a clinical trial of bone marrow transplantation using combined pharmacologic/immunologic purging of T lymphoblasts from patients' harvested autologous marrow.

scholarly journals Origin of human mast cells: development from transplanted hematopoietic stem cells after allogeneic bone marrow transplantation

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2954-2959 ◽  
Author(s):  
M Fodinger ◽  
G Fritsch ◽  
K Winkler ◽  
W Emminger ◽  
G Mitterbauer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Mast Cells ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Hematopoietic Stem ◽  
Donor Genotype

Although mast cells are hematopoietic cells, little is known about the origin of their precursors in vivo. In this study, the origin (donor v recipient genotype) of human mast cells (MCs) was analyzed in a patient who underwent allogeneic bone marrow transplantation (BMT). The patient presented with secondary acute myeloid leukemia (French-American- British classification, M2) arising from refractory anemia with excess of blast cells and bone marrow (BM) mastocytosis. Transplantation was performed in chemotherapy-induced complete remission. On days 88, 126, 198, and 494 after BMT, mast cells were enriched to homogeneity from bone marrow mononuclear cells (BM MNCs) by cell sorting for CD117+/CD34- cells. Purified mast cell populations were CD117(c-kit)+ (> 95%), CD34- (< 1%), CD3- (< 1%), CD14- (< 1%), and virtually free of contaminating cells as assessed by Giemsa staining. The genotype of MCs was analyzed after amplification by polymerase chain reaction (PCR) of a variable number tandem repeat (VNTR) region within intron 40 of the von Willebrand factor (vWF) gene. Unexpectedly, on days 88 and 126 after BMT, sorted MCs displayed recipient genotype as shown by vWF.VNTR-PCR. However, on days 198 and 494, PCR analysis showed a switch to donor genotype in isolated mast cells. Peripheral blood (PB) and BM MNC as well as highly enriched (sorted) CD3+ T cells (PB, BM), CD4+ helper T cells (PB), CD8+ T cells (PB), CD19+ B cells (PB), CD14+ monocytes (PB, BM), and CD34+ precursor cells (BM) showed donor genotype throughout the observation period. Together, these results provide evidence that human MCs developed in vivo from transplanted hematopoietic stem cells. Engraftment and in vivo differentiation of MCs from early hematopoietic progenitor cells may be a prolonged process.

scholarly journals Origin of human mast cells: development from transplanted hematopoietic stem cells after allogeneic bone marrow transplantation

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2954-2959 ◽  
Author(s):  
M Fodinger ◽  
G Fritsch ◽  
K Winkler ◽  
W Emminger ◽  
G Mitterbauer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Mast Cells ◽  
Allogeneic Bone Marrow Transplantation ◽  
Allogeneic Bone ◽  
Hematopoietic Stem ◽  
Donor Genotype

Abstract Although mast cells are hematopoietic cells, little is known about the origin of their precursors in vivo. In this study, the origin (donor v recipient genotype) of human mast cells (MCs) was analyzed in a patient who underwent allogeneic bone marrow transplantation (BMT). The patient presented with secondary acute myeloid leukemia (French-American- British classification, M2) arising from refractory anemia with excess of blast cells and bone marrow (BM) mastocytosis. Transplantation was performed in chemotherapy-induced complete remission. On days 88, 126, 198, and 494 after BMT, mast cells were enriched to homogeneity from bone marrow mononuclear cells (BM MNCs) by cell sorting for CD117+/CD34- cells. Purified mast cell populations were CD117(c-kit)+ (> 95%), CD34- (< 1%), CD3- (< 1%), CD14- (< 1%), and virtually free of contaminating cells as assessed by Giemsa staining. The genotype of MCs was analyzed after amplification by polymerase chain reaction (PCR) of a variable number tandem repeat (VNTR) region within intron 40 of the von Willebrand factor (vWF) gene. Unexpectedly, on days 88 and 126 after BMT, sorted MCs displayed recipient genotype as shown by vWF.VNTR-PCR. However, on days 198 and 494, PCR analysis showed a switch to donor genotype in isolated mast cells. Peripheral blood (PB) and BM MNC as well as highly enriched (sorted) CD3+ T cells (PB, BM), CD4+ helper T cells (PB), CD8+ T cells (PB), CD19+ B cells (PB), CD14+ monocytes (PB, BM), and CD34+ precursor cells (BM) showed donor genotype throughout the observation period. Together, these results provide evidence that human MCs developed in vivo from transplanted hematopoietic stem cells. Engraftment and in vivo differentiation of MCs from early hematopoietic progenitor cells may be a prolonged process.

scholarly journals Outcome of alloanergized haploidentical bone marrow transplantation after ex vivo costimulatory blockade: results of 2 phase 1 studies

Blood ◽  
2008 ◽  
Vol 112 (6) ◽  
pp. 2232-2241 ◽  
Author(s):  
Jeff K. Davies ◽  
John G. Gribben ◽  
Lisa L. Brennan ◽  
Dongin Yuk ◽  
Lee M. Nadler ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
T Cell ◽  
Marrow Transplantation ◽  
Viral Infections ◽  
Ex Vivo ◽  
Chronic Gvhd ◽  
Costimulatory Blockade ◽  
Donor T Cells

AbstractWe report the outcomes of 24 patients with high-risk hematologic malignancies or bone marrow failure (BMF) who received haploidentical bone marrow transplantation (BMT) after ex vivo induction of alloantigen-specific anergy in donor T cells by allostimulation in the presence of costimulatory blockade. Ninety-five percent of evaluable patients engrafted and achieved full donor chimerism. Despite receiving a median T-cell dose of 29 ×106/kg, only 5 of 21 evaluable patients developed grade C (n = 4) or D (n = 1) acute graft-versus-host disease (GVHD), with only one attributable death. Twelve patients died from treatment-related mortality (TRM). Patients reconstituted T-cell subsets and immunoglobulin levels rapidly with evidence of in vivo expansion of pathogen-specific T cells in the early posttransplantation period. Five patients reactivated cytomegalovirus (CMV), only one of whom required extended antiviral treatment. No deaths were attributable to CMV or other viral infections. Only 1 of 12 evaluable patients developed chronic GVHD. Eight patients survive disease-free with normal performance scores (median follow-up, 7 years). Thus, despite significant early TRM, ex vivo alloanergization can support administration of large numbers of haploidentical donor T cells, resulting in rapid immune reconstitution with very few viral infections. Surviving patients have excellent performance status and a low rate of chronic GVHD.

scholarly journals Identification and expansion of highly suppressive CD8+FoxP3+ regulatory T cells after experimental allogeneic bone marrow transplantation

Blood ◽  
2012 ◽  
Vol 119 (24) ◽  
pp. 5898-5908 ◽  
Author(s):  
Renee J. Robb ◽  
Katie E. Lineburg ◽  
Rachel D. Kuns ◽  
Yana A. Wilson ◽  
Neil C. Raffelt ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
Regulatory T Cells ◽  
Marrow Transplantation ◽  
Allogeneic Bone Marrow Transplantation ◽  
Class I ◽  
Allogeneic Bone ◽  
Mesenteric Lymph Nodes

Abstract FoxP3+ confers suppressive properties and is confined to regulatory T cells (Treg) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4+ Treg are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8+ population of FoxP3+ Treg that convert from CD8+ conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8+ Treg undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-β. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4+FoxP3+ population and is more potent in exerting class I–restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8+FoxP3+ Treg are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8+FoxP3+ Treg thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I–restricted T-cell responses after bone marrow transplantation.

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