scholarly journals Robust, But Transient Expression of Adeno-Associated Virus-Transduced Genes During Human T Lymphopoiesis

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4854-4864 ◽  
Author(s):  
Jason P. Gardner ◽  
Haihong Zhu ◽  
Peter C. Colosi ◽  
Gary J. Kurtzman ◽  
David T. Scadden
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Marker Gene ◽  
T Cell Differentiation ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
The Impact ◽  
T Lymphopoiesis

Abstract Recombinant adeno-associated viruses (rAAV) have been proposed to be gene transfer vehicles for hematopoietic stem cells with advantages over other virus-based systems due to their high titers and relative lack of dependence on cell cycle for target cell integration. We evaluated rAAV vector containing a LacZ reporter gene under the control of a cytomegalovirus (CMV) promoter in the context of primary human CD34+CD2− progenitor cells induced to undergo T-cell differentiation using an in vitro T-lymphopoiesis system. Target cells from either adult bone marrow or umbilical cord blood were efficiently transduced, and 71% to 79% CD2+ cells expressed a LacZ marker gene mRNA and produced LacZ-encoded protein after exposure to rAAV-CMV-LacZ. The impact of transgene expression on the differentiation of T cells was assessed by sequential quantitation of immunophenotypic subsets of virus-exposed cells and no alteration was noted compared with control. The durability of transgene expression was assessed and found to decay by day 35 with kinetics dependent on the multiplicity of infection. In addition, vector DNA was absent from CD4 or CD8 subselected CD3+ cells by DNA-polymerase chain reaction. These data suggest that rAAV vectors may result in robust transgene expression in primitive cells undergoing T-cell lineage commitment without toxicity or alteration in the pattern of T-cell differentiation. However, expression is transient and integration of the transgene unlikely. Recombinant AAV vectors are potentially valuable gene transfer tools for the genetic manipulation of events during T-cell ontogony but their potential in gene therapy strategies for diseases such as acquired immunodeficiency syndrome is limited.

scholarly journals Robust, But Transient Expression of Adeno-Associated Virus-Transduced Genes During Human T Lymphopoiesis

Blood ◽  
1997 ◽  
Vol 90 (12) ◽  
pp. 4854-4864
Author(s):  
Jason P. Gardner ◽  
Haihong Zhu ◽  
Peter C. Colosi ◽  
Gary J. Kurtzman ◽  
David T. Scadden
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Marker Gene ◽  
T Cell Differentiation ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
The Impact ◽  
T Lymphopoiesis

Recombinant adeno-associated viruses (rAAV) have been proposed to be gene transfer vehicles for hematopoietic stem cells with advantages over other virus-based systems due to their high titers and relative lack of dependence on cell cycle for target cell integration. We evaluated rAAV vector containing a LacZ reporter gene under the control of a cytomegalovirus (CMV) promoter in the context of primary human CD34+CD2− progenitor cells induced to undergo T-cell differentiation using an in vitro T-lymphopoiesis system. Target cells from either adult bone marrow or umbilical cord blood were efficiently transduced, and 71% to 79% CD2+ cells expressed a LacZ marker gene mRNA and produced LacZ-encoded protein after exposure to rAAV-CMV-LacZ. The impact of transgene expression on the differentiation of T cells was assessed by sequential quantitation of immunophenotypic subsets of virus-exposed cells and no alteration was noted compared with control. The durability of transgene expression was assessed and found to decay by day 35 with kinetics dependent on the multiplicity of infection. In addition, vector DNA was absent from CD4 or CD8 subselected CD3+ cells by DNA-polymerase chain reaction. These data suggest that rAAV vectors may result in robust transgene expression in primitive cells undergoing T-cell lineage commitment without toxicity or alteration in the pattern of T-cell differentiation. However, expression is transient and integration of the transgene unlikely. Recombinant AAV vectors are potentially valuable gene transfer tools for the genetic manipulation of events during T-cell ontogony but their potential in gene therapy strategies for diseases such as acquired immunodeficiency syndrome is limited.

scholarly journals Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7

2020 ◽  
Author(s):  
Emilie Coppin ◽  
Bala Sai Sundarasetty ◽  
Susann Rahmig ◽  
Jonas Blume ◽  
Nikita A. Verheyden ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Regulatory Elements ◽  
T Cell Differentiation ◽  
Human Immune System ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Human T Cell ◽  
Human T Cells

AbstractHumanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T cell differentiation remains inefficient. We generated mice expressing human interleukin (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1

2017 ◽  
Vol 14 (5) ◽  
pp. 531-538 ◽  
Author(s):  
Shreya Shukla ◽  
Matthew A Langley ◽  
Jastaranpreet Singh ◽  
John M Edgar ◽  
Mahmood Mohtashami ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
T Cell Differentiation ◽  
Hematopoietic Stem

scholarly journals In vitro T lymphopoiesis of human and rhesus CD34+ progenitor cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4040-4048 ◽  
Author(s):  
M Rosenzweig ◽  
DF Marks ◽  
H Zhu ◽  
D Hempel ◽  
KG Mansfield ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
T Lymphocytes ◽  
Progenitor Cells ◽  
T Cell Differentiation ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Differentiation of hematopoietic progenitor cells into T lymphocytes generally occurs in the unique environment of the thymus, a feature that has hindered efforts to model this process in the laboratory. We now report that thymic stromal cultures from rhesus macaques can support T-cell differentiation of human or rhesus CD34+ progenitor cells. Culture of rhesus or human CD34+ bone marrow-derived cells depleted of CD34+ lymphocytes on rhesus thymic stromal monolayers yielded CD3+CD4+CD8+, CD3+CD4+CD8-, and CD3+CD4-CD8+ cells after 10 to 14 days. In addition to classical T lymphocytes, a discrete population of CD3+CD8loCD16+CD56+ cells was detected after 14 days in cultures inoculated with rhesus CD34+ cells. CD3+ T cells arising from these cultures were not derived from contaminating T cells present in the CD34+ cells used to inoculate thymic stromal monolayers or from the thymic monolayers, as shown by labeling of cells with the lipophilic membrane dye PKH26. Expression of the recombinase activation gene RAG- 2, which is selectively expressed in developing lymphocytes, was detectable in thymic cultures inoculated with CD34+ cells but not in CD34+ cells before thymic culture or in thymic stromal monolayers alone. Reverse transcriptase-polymerase chain reaction analysis of T cells derived from thymic stromal cultures of rhesus and human CD34+ cells showed a polyclonal T-cell receptor repertoire. T-cell progeny derived from rhesus CD34+ cells cultured on thymic stroma supported vigorous simian immunodeficiency virus replication in the absence of exogenous mitogenic stimuli. Rhesus thymic stromal cultures provide a convenient means to analyze T-cell differentiation in vitro and may be useful as a model of hematopoietic stem cell therapy for diseases of T cells, including acquired immunodeficiency syndrome.

Induction of T-cell development from human cord blood hematopoietic stem cells by Delta-like 1 in vitro

Blood ◽  
2005 ◽  
Vol 105 (4) ◽  
pp. 1431-1439 ◽  
Author(s):  
Ross N. La Motte-Mohs ◽  
Elaine Herer ◽  
Juan Carlos Zúñiga-Pflücker
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cord Blood ◽  
T Cell Development ◽  
T Cell Differentiation ◽  
Human Cord Blood ◽  
Hematopoietic Stem

AbstractThe Notch signaling pathway plays a key role at several stages of T-lymphocyte differentiation. However, it remained unclear whether signals induced by the Notch ligand Delta-like 1 could support full T-cell differentiation from a defined source of human hematopoietic stem cells (HSCs) in vitro. Here, we show that human cord blood–derived HSCs cultured on Delta-like 1–expressing OP9 stromal cells undergo efficient T-cell lineage commitment and sustained T-cell differentiation. A normal stage-specific program of T-cell development was observed, including the generation of CD4 and CD8 αβ–T-cell receptor (TCR)–bearing cells. Induction of T-cell differentiation was dependent on the expression of Delta-like 1 by the OP9 cells. Stimulation of the in vitro–differentiated T cells by TCR engagement induced the expression of T-cell activation markers and costimulatory receptors. These results establish an efficient in vitro coculture system for the generation of T cells from human HSCs, providing a new avenue for the study of early T-cell differentiation and function.

Dysregulation of T-Cell Development in Adrenal Glucocorticoid-Deprived Rats

2009 ◽  
Vol 234 (9) ◽  
pp. 1067-1074 ◽  
Author(s):  
Zorica Stojić-Vukanić ◽  
Aleksandra Rauški ◽  
Duško Kosec ◽  
Katarina Radojević ◽  
Ivan Pilipović ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
T Cell Differentiation ◽  
Male Rats ◽  
Double Positive ◽  
Cell Repertoire ◽  
Single Positive ◽  
The Impact

A number of different experimental approaches have been used to elucidate the impact of basal levels of adrenal gland-derived glucocorticoids (GCs) on T cell development, and thereby T cell-mediated immune responses. However, the relevance of the adrenal GCs to T cell development is still far from clear. This study was undertaken to explore the relevance of basal levels of GCs to T cell differentiation/maturation. Eight days post-adrenalectomy in adult male rats the thymocyte yield, apoptotic and proliferative rate and the relationship amongst major thymocyte subsets, as defined by TCRαβ/CD4/CD8 expression, were examined using flow cytometry. Adrenal GC deprivation decreased thymocyte apoptosis and altered the kinetics of T cell differentiation/maturation. In the adrenalectomized rats there was increased thymic hypercellularity and an over-representation of the CD4+CD8+ double positive (DP) TCRαβlow cells entering selection, as well as increased numbers of their DP TCRαβ− immediate precursors. These changes were accompanied with under-representation of the postselected DP TCRαβhigh and the most mature CD4−CD8+ and, particularly, CD4+CD8− single positive (SP) TCRαβhigh cells. This data suggests that withdrawal of adrenal GCs produces alterations in the thymocyte selection processes, possibly affecting the diversity of functional T cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of CD4−CD8− double negative TCRαβhigh cells. In addition, it indicates that GCs influence the post-selection maturation of thymocytes and plays a regulatory role in controlling the ratio of mature CD4+CD8−/CD4−CD8+ SP TCRαβhigh cells.

scholarly journals Homeostasis-Stimulated Proliferation Drives Naive T Cells to Differentiate Directly into Memory T Cells

2000 ◽  
Vol 192 (4) ◽  
pp. 549-556 ◽  
Author(s):  
Bryan K. Cho ◽  
Varada P. Rao ◽  
Qing Ge ◽  
Herman N. Eisen ◽  
Jianzhu Chen
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
T Cell ◽  
Cd8 T Cells ◽  
Interleukin 2 ◽  
T Cell Differentiation ◽  
Target Cells ◽  
Central Feature ◽  
Memory Cd8 T Cells ◽  
Memory T Cell

The developmental requirements for immunological memory, a central feature of adaptive immune responses, is largely obscure. We show that as naive CD8 T cells undergo homeostasis-driven proliferation in lymphopenic mice in the absence of overt antigenic stimulation, they progressively acquire phenotypic and functional characteristics of antigen-induced memory CD8 T cells. Thus, the homeostasis-induced memory CD8 T cells express typical memory cell markers, lyse target cells directly in vitro and in vivo, respond to lower doses of antigen than naive cells, and secrete interferon γ faster upon restimulation. Like antigen-induced memory T cell differentiation, the homeostasis-driven process requires T cell proliferation and, initially, the presence of appropriate restricting major histocompatibility complexes, but it differs by occurring without effector cell formation and without requiring interleukin 2 or costimulation via CD28. These findings define repetitive cell division plus T cell receptor ligation as the basic requirements for naive to memory T cell differentiation.

scholarly journals Intrathymic transplantation of bone marrow–derived progenitors provides long-term thymopoiesis

Blood ◽  
2010 ◽  
Vol 115 (10) ◽  
pp. 1913-1920 ◽  
Author(s):  
Rita Vicente ◽  
Oumeya Adjali ◽  
Chantal Jacquet ◽  
Valérie S. Zimmermann ◽  
Naomi Taylor
Keyword(s):  
Bone Marrow ◽  
Cell Differentiation ◽  
T Cell ◽  
Large Population ◽  
Severe Combined Immunodeficiency ◽  
T Cell Differentiation ◽  
Hematopoietic Stem ◽  
Insufficient Entry ◽  
Deficient Mice

Abstract The sustained differentiation of T cells in the thymus cannot be maintained by resident intrathymic (IT) precursors and requires that progenitors be replenished from the bone marrow (BM). In patients with severe combined immunodeficiency (SCID) treated by hematopoietic stem cell transplantation, late T-cell differentiation defects are thought to be due to an insufficient entry of donor BM progenitors into the thymus. Indeed, we find that the intravenous injection of BM progenitors into nonconditioned ζ-chain–associated protein kinase 70 (ZAP-70)–deficient mice with SCID supports short- but not long-term thymopoiesis. Remarkably, we now show that the IT administration of these progenitors produces a significant level of donor-derived thymopoiesis for more than 6 months after transplantation. In contrast to physiologic thymopoiesis, long-term donor thymopoiesis was not due to the continued recruitment of progenitors from the BM. Rather, IT transplantation resulted in the unique generation of a large population of early c-Kithigh donor precursors within the thymus. These ZAP-70–deficient mice that received an IT transplant had a significantly increased prothymocyte niche compared with their untreated counterparts; this phenotype was associated with the generation of a medulla. Thus, IT administration of BM progenitors results in the filling of an expanded precursor niche and may represent a strategy for enhancing T-cell differentiation in patients with SCID.

scholarly journals Treatment of Post- HSCT Immunodeficiency By Infusion of Ex Vivo- Generated T Cell Precursors from Adult and Cord Blood Hematopoietic Stem and Progenitor Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1887-1887
Author(s):  
Laura Simons ◽  
Corinne De La Chappedelaine ◽  
Christian Reimann ◽  
Elodie Elkaim ◽  
Sandrine Susini ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
T Cell ◽  
Cord Blood ◽  
Conflicts Of Interest ◽  
T Cell Differentiation ◽  
Cell Compartment ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
T Cell Progenitors

Abstract Non-HLA identical hematopoietic stem cell transplantation (HSCT) provides a corrective therapy for most life-threatening primary immunodeficiencies (PID) and some malignant hemopathies. Despite advances made, severe complications following the treatment such as the prolonged persistence of T cell immunodeficiency still limit the use of this partially incompatible HSCT. After HSCT, the reconstitution of a functional T cell compartment relies on the availability of T cell precursors to rapidly seed the thymus and differentiate into mature T cells. We have previously demonstrated that an in vitro culture system based on the use of a modified Delta-like-4 (DLL4) Notch ligand and T cell cytokines allows for the effective generation of human T cell precursors from cord blood within 7 days. Moreover, once injected into NOD/SCID/gcko mice, T cell precursors generated in this system were able to colonize the thymus and generate a diversified and functional T-cell compartment. Here, we aimed at testing the capacity of adult HSPCs in this reconstitution system. We found that, like their CB- derived counterparts, T cell precursors generated from adult HPSCs phenotypically resembled thymic CD34+CD7+ cells with high in vitro T-cell differentiation potential. Interestingly, the peak of T cell progenitors for adult HSPCs occurred around day 3, compared to day 7 in CB. At this timepoint, T cell precursors derived from adult HSPC already expressed all critical genes for T cell lineage development, as well as the major chemokine receptors implicated in thymus homing. The introduction of retronectin further improved differentiation and proliferation of T cell progenitors from both HPSC sources in our in vitro system. Comparative molecular analysis of adult- and CB- derived progenitors suggested, that differential requirements for Notch receptor/ligand interactions may explain the differences in kinetics observed during the culture of the two types of HSPC. It remains to be further evaluated, whether targeted modifications of the Notch signaling pathway can improve the outcome of this in vitro T cell differentiation system for adult HPSCs. Overall our results suggest that adult HSPCs, like their CB- derived counterparts, provide an effective source of in vitro cultured T cell progenitors harboring all the necessary requirements for the in vivo -reconstitution of a functional T cell compartment. This is particularly important in the context of future clinical applications in HSCT where adult HSPCs are more available and more frequently used than CB HSPCs. Based on our results, we propose that upon injection into a patient, DLL4- cultured T cell precursors from both HSPC sources could significantly accelerate the reconstitution of the adaptive immune system after a partially HLA-incompatible HSCT. Currently, we are translating these results into a phase I clinical trial including adult and pediatric patients transplanted for malignant hemopathies or PIDs requiring an allogeneic HSCT from a HLA-partially mismatched donors. Disclosures No relevant conflicts of interest to declare.

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