scholarly journals Severe Combined Immunodeficiency Mice Engrafted With Human T Cells, B Cells, and Myeloid Cells After Transplantation With Human Fetal Bone Marrow or Liver Cells and Implanted With Human Fetal Thymus: A Model for Studying Human Gene Therapy

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1800-1810 ◽  
Author(s):  
Sergey Yurasov ◽  
Tobias R. Kollmann ◽  
Ana Kim ◽  
Christina A. Raker ◽  
Moshe Hachamovitch ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
Severe Combined Immunodeficiency ◽  
Precursor Cells ◽  
Scid Mice ◽  
Combined Immunodeficiency ◽  
Human T Cells

To develop an in vivo model wherein human hematopoiesis occurs, we transplanted severe combined immunodeficiency (SCID) mice with either human fetal bone marrow (HFBM) or human fetal liver (HFL). After transplantation of SCID mice with cultured HFBM (BM-SCID-hu mice) or HFL cells (Liv-SCID-hu mice), significant engraftment of the mouse bone marrow (BM) and population of the peripheral blood with human leukocytes was detected. Human colony-forming unit–granulocyte macrophage and burst forming unit-erythroid were detected in the BM of the BM-SCID-hu and Liv-SCID-hu mice up to 8 months after transplantation. When the HFBM or HFL cells were transduced with a retroviral vector before transplantation, integrated retroviral sequences were detected in human precursor cells present in the SCID mouse BM and in leukocytes circulating in the peripheral blood (PB) up to 7 months after transplantation. The PB of the BM-SCID-hu mice also became populated with human T cells after implantation with human thymic tissue, which provided a human microenvironment wherein human pre-T cells from the BM could mature. When the HFBM was retrovirally transduced before transplantation, integrated retrovirus was detected in sorted CD4+CD8+ double positive and CD4+ single positive cells from the thymic implant and CD4+ cells from the PB. Taken together, these data indicated that the BM of our BM-SCID-hu and Liv-SCID-hu mice became engrafted with retrovirally transduced human hematopoietic precursors that undergo the normal human hematopoietic program and populate the mouse PB with human cells containing integrated retroviral sequences. In addition to being a model for studying in vivo human hematopoiesis, these mice should also prove to be a useful model for investigating in vivo gene therapy using human stem/precursor cells.

scholarly journals Severe Combined Immunodeficiency Mice Engrafted With Human T Cells, B Cells, and Myeloid Cells After Transplantation With Human Fetal Bone Marrow or Liver Cells and Implanted With Human Fetal Thymus: A Model for Studying Human Gene Therapy

Blood ◽  
1997 ◽  
Vol 89 (5) ◽  
pp. 1800-1810 ◽  
Author(s):  
Sergey Yurasov ◽  
Tobias R. Kollmann ◽  
Ana Kim ◽  
Christina A. Raker ◽  
Moshe Hachamovitch ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Peripheral Blood ◽  
Severe Combined Immunodeficiency ◽  
Precursor Cells ◽  
Scid Mice ◽  
Combined Immunodeficiency ◽  
Human T Cells

Abstract To develop an in vivo model wherein human hematopoiesis occurs, we transplanted severe combined immunodeficiency (SCID) mice with either human fetal bone marrow (HFBM) or human fetal liver (HFL). After transplantation of SCID mice with cultured HFBM (BM-SCID-hu mice) or HFL cells (Liv-SCID-hu mice), significant engraftment of the mouse bone marrow (BM) and population of the peripheral blood with human leukocytes was detected. Human colony-forming unit–granulocyte macrophage and burst forming unit-erythroid were detected in the BM of the BM-SCID-hu and Liv-SCID-hu mice up to 8 months after transplantation. When the HFBM or HFL cells were transduced with a retroviral vector before transplantation, integrated retroviral sequences were detected in human precursor cells present in the SCID mouse BM and in leukocytes circulating in the peripheral blood (PB) up to 7 months after transplantation. The PB of the BM-SCID-hu mice also became populated with human T cells after implantation with human thymic tissue, which provided a human microenvironment wherein human pre-T cells from the BM could mature. When the HFBM was retrovirally transduced before transplantation, integrated retrovirus was detected in sorted CD4+CD8+ double positive and CD4+ single positive cells from the thymic implant and CD4+ cells from the PB. Taken together, these data indicated that the BM of our BM-SCID-hu and Liv-SCID-hu mice became engrafted with retrovirally transduced human hematopoietic precursors that undergo the normal human hematopoietic program and populate the mouse PB with human cells containing integrated retroviral sequences. In addition to being a model for studying in vivo human hematopoiesis, these mice should also prove to be a useful model for investigating in vivo gene therapy using human stem/precursor cells.

ADA-deficient SCID is associated with a specific microenvironment and bone phenotype characterized by RANKL/OPG imbalance and osteoblast insufficiency

Blood ◽  
2009 ◽  
Vol 114 (15) ◽  
pp. 3216-3226 ◽  
Author(s):  
Aisha V. Sauer ◽  
Emanuela Mrak ◽  
Raisa Jofra Hernandez ◽  
Elena Zacchi ◽  
Francesco Cavani ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Severe Combined Immunodeficiency ◽  
Intrinsic Defect ◽  
Combined Immunodeficiency ◽  
Hematopoietic Stem ◽  
Enzyme Replacement ◽  
Bone Phenotype

Abstract Adenosine deaminase (ADA) deficiency is a disorder of the purine metabolism leading to combined immunodeficiency and systemic alterations, including skeletal abnormalities. We report that ADA deficiency in mice causes a specific bone phenotype characterized by alterations of structural properties and impaired mechanical competence. These alterations are the combined result of an imbalanced receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin axis, causing decreased osteoclastogenesis and an intrinsic defect of osteoblast function with subsequent low bone formation. In vitro, osteoblasts lacking ADA displayed an altered transcriptional profile and growth reduction. Furthermore, the bone marrow microenvironment of ADA-deficient mice showed a reduced capacity to support in vitro and in vivo hematopoiesis. Treatment of ADA-deficient neonatal mice with enzyme replacement therapy, bone marrow transplantation, or gene therapy resulted in full recovery of the altered bone parameters. Remarkably, untreated ADA–severe combined immunodeficiency patients showed a similar imbalance in RANKL/osteoprotegerin levels alongside severe growth retardation. Gene therapy with ADA-transduced hematopoietic stem cells increased serum RANKL levels and children's growth. Our results indicate that the ADA metabolism represents a crucial modulatory factor of bone cell activities and remodeling. The trials were registered at www.clinicaltrials.gov as #NCT00598481 and #NCT00599781.

Highly Reproducible Engraftment of Chronic Lymphocytic Leukemia (B-CLL) Cells in NOD/SCID Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 52-52 ◽  
Author(s):  
Peter Ebeling ◽  
Jan Duerig ◽  
Florian Grabellus ◽  
Ulrich Duehrsen ◽  
Siegfried Seeber ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cell Clone ◽  
Cell Types ◽  
Scid Mice ◽  
Lymphocytic Leukemia ◽  
In Vivo Model ◽  
Acute Leukemias ◽  
Human T Cells

Abstract In contrast to normal hematopoiesis and acute leukemias, research in CLL still is hampered by the lack of a reliable in vivo model for primary B-CLL. We here report highly reproducible engraftment of B-CLL cells, when 1x10^8 MNC derived from the peripheral blood of CLL patients were transplanted i.v and i.p. into NOD/SCID mice. So far, 14 different CLL samples were investigated in 41 mice. At weeks 4, 8 or 12 mice were sacrificed and bone marrow (BM), spleen, and peritoneal fluid (PF) were analyzed by FACS for human CD19/CD5/CD23/CD45 (B-CLL) cells and CD45/CD3/CD5 (T) cells. Additionally, HE- and immunostaining was performed on spleen sections. Analysis at week 4 revealed engraftment in NOD/SCID mice for 13/14 samples (spleen: 13/14, BM: 4/14, PF: 12/14). B-CLL cells were observed predominantly in the spleen (8.9±2.4% or 9.1±4.4x10^5 cells) and PF (19.0±4.4% or 3.4±1.8x10^5 cells) with much lower engraftment in BM (0.6±0.3% or 0.1±0.1x10^5 cells). Detection of B-CLL cells in peripheral blood could be obtained in 3/14 experiments. Also substantial engraftment of human T-cells was observed in 13/14 experiments (spleen: 13/14, BM: 8/14, PF: 11/14). T-cells engraftment was highest in the spleen (23.8±9.8% or 28.7±13.1x10^5 cells) and somewhat lower in PF (16.4±8.2% or 3.0±1.6x10^5 cells) and BM (7.3±3.8% or 2.9±1.1x10^5 cells). Subpopulation analysis revealed a CD4+ phenotype in 65, 59 and 72 % of T-cells within spleen, PF and BM, respectively. Noteworthy, immunohistological analysis of HE stained spleen sections of engrafted animals revealed a pseudofollicular infiltration with human CD45LCA+ cells along splenic arterioles. Within these pseudofollicles human B-CLL but also CD3+ T-cells were detected. Contribution of B-CLL and T-cells to individual follicles was highly variable ranging from 5–95% for both cell types. When engraftment was analysed separately for the i.p and the i.v. route, engraftment of transplanted cells in PF seemed to be depended on the i.p. route whereas splenic engraftment was obtained following i.v. as well as i.p. injection. Sustained B-CLL engraftment was seen after 8 weeks (spleen: 3.1±1.4% or 7.3±3.1x10^5 total cells; PF: 57.6±23.3% or 1.0±0.5x10^5 cells; n=3 mice) and 12 weeks (spleen: 1.4±1.3% or 0.3±0.3x10^5 cells; PF: 10.2±7.3% or 0.5±0.5x10^5 cells; n=2 mice). Thus, we have shown efficient engraftment of human B-CLL cells in the spleen and PF of NOD/SCID mice. This in vivo model should significantly help to understand B-CLL biology and to test novel therapeutic approaches. The observed pseudofolicular pattern of splenic infiltration supports the theory of T-cells creating a “microenvironment” sustaining the growth of the leukemic B cell clone.

Different Pattern of CD154 and CD40 Expression On B-CLL and T Lymphocytes In Peripheral Blood, Bone Marrow and Lymph Node Microenvironment In B-Cell Chronic Lymphocytic Leukemia (B-CLL)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3607-3607
Author(s):  
Ozren Jaksic ◽  
Branimir Gizdic ◽  
Tajana Stoos Veic ◽  
Vlatka Pandzic Jaksic ◽  
Rajko Kusec ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Lymph Node ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Significant Difference ◽  
Cd40 Expression ◽  
Cell Chronic Lymphocytic Leukemia

Abstract Abstract 3607 Background: In B-cell chronic lymphocytic leukemia (B-CLL) there is a well documented intraclonal and interclonal variability of B-CLL cells in different lymphoid compartments with respect to the expression of a number of surface and intracellular molecules (for example CD38 and ZAP-70). This variability in part may reflect a number of interactions of malignant B-CLL clone with supporting microenviroment including cells (T-cells, nurse-like cells, etc.), cytokines, chemokines and stroma. One of the key interactions of B-CLL clone is with T-cells, through CD154/CD40 system. It is important pathway modulating survival, drug resistance and immunity. It is known that CD154 is transiently expressed on CD4+ T cells, as well as that CD154 can be coexpressed on B-CLL cells with CD40 in a subpopulation of B-CLL patients. Its expression on B-CLL cells can be induced by gene therapy and lenalidomide, being in part responsible for their therapeutic effects. Aim of this study was to determine the level of expression of CD154 and CD40 in vivo on B-CLL cells and T lymphocytes and to evaluate intra and interclonal differences due to different microenvironment, i.e. peripheral blood, bone marrow and lymph nodes. Methods: peripheral blood, (PB), bone marrow (BM) and lymph node (LN) samples were taken by conventional techniques (venepuncture and fine needle aspiration) on the same day. The expression level of CD154 and CD40 molecules on CD19+CD5+ B-CLL cells and CD19-CD5+ T cells was analyzed by flow cytometry. Results were expressed as mean fluorescence intensity (MFI) and analyzed by paired tests. Results: samples taken from 21 typical B-CLL patients with median age of 72 years were analyzed. There were 9 males and 12 females. Mean beta-2 microglobuin was 4.3mg/l, mean Total Tumor Mass size was 8.9 and mean Tumor Distribution pattern was 0.75. There were 2, 14 and 5 patients in Rai stage 0, I+II and III+IV, respectively. There were 6 previously treated patients (but off therapy 3 months before sampling). The expression level of CD154 was absent/low on T-cells and in 14/21 patients on B-CLL cells. However in 7/21 patients B-CLL cells had higher CD154 expression (“CD154 positive” patients). There was no detectible difference in CD154 expression on T cells between compartments, while on B-CLL cells there was highest expression in lymph nodes and lowest in peripheral blood (p<0.01). CD40 expression on B-CLL cells was significantly higher than CD154, i.e. all cases were positive, and there was no significant difference between lymphoid compartments. There was no significant difference between CD154 positive and negative patients in measured disease parameters. Conclusions: our results show that CD154 expression on T-cells is absent/low and not significantly different between lymphoid compartments regardless of different microenvironment milieu. CD40 expression on B-CLL cells is high and comparable through compartments. In subset of patients there is CD154 positivity on B-CLL cells and shows strong association with lymphoid compartments possibly indicating microenviroment influence on CD154/CD40 system in B-CLL in vivo. These results warrant further studies to indentify the role of CD154 expression on B-CLL cells in pathologic process and its regulation and may eventually uncover novel or modulate existing innovative therapeutic approaches (like gene therapy or immunomodulatory agents like lenalidomide). Disclosures: No relevant conflicts of interest to declare.

Generation of primary antigen-specific human cytotoxic T lymphocytes in human/mouse radiation chimera

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 721-730 ◽  
Author(s):  
H Segall ◽  
I Lubin ◽  
H Marcus ◽  
A Canaan ◽  
Y Reisner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Human Lymphocytes ◽  
Scid Mice ◽  
Human Antibody ◽  
Adoptive Therapy ◽  
Activation Markers ◽  
Human T Cell ◽  
Human T Cells

Severe combined immunodeficient (SCID) mice are increasingly used as hosts for the adoptive transfer of human lymphocytes. Human antibody responses can be obtained in these xenogeneic chimeras, but information about the functionality of the human T cells in SCID mice is limited and controversial. Studies using human peripheral blood lymphocytes (PBL) injected intraperitoneally (IP) into SCID mice (hu-PBL-SCID mice) have shown that human T cells from these chimeras are anergic and have a defective signaling via the T-cell receptor. In addition, their antigenic repertoire is limited to xenoreactive clones. In the present study, we tested the functionality of human T cell in a recently described chimeric model. In this system, BALB/c mice are conditioned by irradiation and then transplanted with SCID bone marrow, followed by IP injection of human PBL. Our experiments demonstrated that human T cells, recovered from these hu-PBL-BALB mice within 1 month posttransplant, proliferated and expressed activation markers upon stimulation with anti-CD3 monoclonal antibody. A vigorous antiallogeneic human cytotoxic T-lymphocyte (CTL) response could be generated in these mice by immunizing them with irradiated allogeneic cells. Moreover, anti-human immunodeficiency virus type 1 (HIV-1) Net- specific human CTLs could be generated in vivo from naive lymphocytes by immunization of mouse-human chimeras with a recombinant vaccinia-nef virus. This model may be used to evaluate potential immunomodulatory drugs or cytokines, and could provide a relevant model for testing HIV vaccines, for production of antiviral T-cell clones for adoptive therapy, and for studying human T-cell responses in vivo.

Epithelial stem cell-mediated development of the human respiratory mucosa in SCID mice

2000 ◽  
Vol 113 (5) ◽  
pp. 767-778 ◽  
Author(s):  
A. Delplanque ◽  
C. Coraux ◽  
R. Tirouvanziam ◽  
I. Khazaal ◽  
E. Puchelle ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Sex Chromosome ◽  
Severe Combined Immunodeficiency ◽  
Scid Mice ◽  
Functional Assay ◽  
Combined Immunodeficiency ◽  
Freezing And Thawing ◽  
Normal Epithelium ◽  
Tracheobronchial Epithelium

We have developed an in vivo assay for progenitor cells of the human tracheobronchial epithelium relying on the transplantation of human prenatal respiratory tissues into severe combined immunodeficiency mice. Engrafted embryonic or fetal open tracheobronchial rudiments are rapidly closed at each end by a neoformed membrane that we named the operculum. After 2–4 weeks, differentiated human respiratory epithelium covers both the native airway matrix and the new operculum. Human epithelial cells dissociated from either emerging embryonic lung primordia or mature xenografts were seeded in host human airway grafts, of which native epithelium had been eliminated by several cycles of freezing and thawing. All grafts seeded with donor epithelial cells and implanted back into SCID mice recovered a surface mucociliary epithelium expressing expected markers and secreting mucus. Spontaneous epithelium regrowth was never observed in control unseeded, denuded grafts. In some experiments, donor epithelial cells and host denuded airway were sex-mismatched and the donor origin of newly formed epithelial structures was confirmed by sex chromosome detection. After two rounds of seeding and reimplantation, a normal epithelium was observed to line the 3rd generation operculum. These observations substantiate a functional assay for human candidate airway epithelium stem cells.

scholarly journals High frequency of normal DJH joints in B cell progenitors in severe combined immunodeficiency mice.

1993 ◽  
Vol 178 (3) ◽  
pp. 1007-1016 ◽  
Author(s):  
J L Pennycook ◽  
Y Chang ◽  
J Celler ◽  
R A Phillips ◽  
G E Wu
Keyword(s):  
Bone Marrow ◽  
B Cell ◽  
Fetal Liver ◽  
Severe Combined Immunodeficiency ◽  
Cell Activity ◽  
Scid Mice ◽  
Combined Immunodeficiency ◽  
Cell Stage ◽  
Reading Frame ◽  
B Lineage

The severe combined immunodeficiency (scid) mouse has a defective V(D)J recombinase activity that results in arrested lymphoid development at the pro-B cell stage in the B lineage. The defect is not absolute and scid mice do attempt gene rearrangement. Indeed, approximately 15% of all scid mice develop detectable levels of oligoclonal serum immunoglobulin and T cell activity. To gain more insight into the scid defect and its effect on V(D)J rearrangement, we analyzed DJH recombination in scid bone marrow. We determined that DJH structures are present in scid bone marrow and occur at a frequency only 10-100 times less than C.B-17+/+. The scid DJH repertoire is limited and resembles fetal liver DJH junctions, with few N insertions and predominant usage of reading frame 1. Moreover, 70% of the DJH structures were potentially productive, indicating that normal V(D)J recombinants should be arising continually.

Long-Term Chimerism and B-Cell Function After Bone Marrow Transplantation in Patients With Severe Combined Immunodeficiency With B Cells: A Single-Center Study of 22 Patients

Blood ◽  
1999 ◽  
Vol 94 (8) ◽  
pp. 2923-2930 ◽  
Author(s):  
Elie Haddad ◽  
Françoise Le Deist ◽  
Pierre Aucouturier ◽  
Marina Cavazzana-Calvo ◽  
Stephane Blanche ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Bone Marrow Transplantation ◽  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Severe Combined Immunodeficiency ◽  
Combined Immunodeficiency ◽  
B Cell Function ◽  
Host Origin

We retrospectively analyzed the B-cell function and leukocyte chimerism of 22 patients with severe combined immunodeficiency with B cells (B+ SCID) who survived more than 2 years after bone marrow transplantation (BMT) to determine the possible consequences of BMT procedures, leukocyte chimerism, and SCID molecular deficit on B-cell function outcome. Circulating T cells were of donor origin in all patients. In recipients of HLA-identical BMT (n = 5), monocytes were of host origin in 5 and B cells were of host origin in 4 and of mixed origin in 1. In recipients of HLA haploidentical T-cell–depleted BMT (n = 17), B cells and monocytes were of host origin in 14 and of donor origin in 3. Engraftment of B cells was found to be associated with normal B-cell function. In contrast, 10 of 18 patients with host B cells still require Ig substitution. Conditioning regimen (ie, 8 mg/kg busulfan and 200 mg/kg cyclophosphamide) was shown neither to promote B-cell and monocyte engraftment nor to affect B-cell function. Eight patients with B cells of host origin had normal B-cell function. Evidence for functional host B cells was further provided in 3 informative cases by Ig allotype determination and by the detection, in 5 studied cases, of host CD27+ memory B cells as in age-matched controls. These results strongly suggest that, in some transplanted patients, host B cells can cooperate with donor T cells to fully mature in Ig-producing cells.

scholarly journals Interleukin-2 production and response to interleukin-2 by peripheral blood mononuclear cells from patients after bone marrow transplantation: II. Patients receiving soybean lectin-separated and T cell-depleted bone marrow

Blood ◽  
1987 ◽  
Vol 70 (5) ◽  
pp. 1595-1603 ◽  
Author(s):  
K Welte ◽  
CA Keever ◽  
J Levick ◽  
MA Bonilla ◽  
VJ Merluzzi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Interleukin 2 ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

Abstract The ability of peripheral blood mononuclear cells (PBMC) to produce and respond to interleukin-2 (IL-2) was evaluated in 50 recipients of HLA- identical bone marrow (BM) depleted of mature T cells by soybean agglutination and E rosetting (SBA-E-BM). In contrast to our previous findings in recipients of unfractionated marrow, during weeks 3 to 7 post-SBA-E-BM transplantation (BMT), PBMC from the majority of patients spontaneously released IL-2 into the culture medium. This IL-2 was not produced by Leu-11+ natural killer cells, which were found to be predominant in the circulation at this time, but by T11+, T3+, Ia antigen-bearing T cells. The IL-2 production could be enhanced by coculture with host PBMC frozen before transplant but not by stimulation with mitogenic amounts of OKT3 antibody, thus suggesting an in vivo activation of donor T cells or their precursors by host tissue. Spontaneous IL-2 production was inversely proportional to the number of circulating peripheral blood lymphocytes and ceased after 7 to 8 weeks post-SBA-E-BMT in most of the patients. In patients whose cells had ceased to produce IL-2 spontaneously or never produced this cytokine, neither coculture with host cells nor stimulation with OKT3 antibody thereafter induced IL-2 release through the first year posttransplant. Proliferative responses to exogenous IL-2 after stimulation with OKT3 antibody remained abnormal for up to 6 months post-SBA-E-BMT, unlike the responses of PBMC from recipients of conventional BM, which responded normally by 1 month post-BMT. However, the upregulation of IL- 2 receptor expression by exogenous IL-2 was found to be comparable to normal controls when tested as early as 3 weeks post-SBA-E-BMT. Therefore, the immunologic recovery of proliferative responses to IL-2 and the appearance of cells regulating in vivo activation of T cells appear to be more delayed in patients receiving T cell-depleted BMT. Similar to patients receiving conventional BMT, however, the ability to produce IL-2 after mitogenic stimulation remains depressed for up to 1 year after transplantation.

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