scholarly journals Induced Pluripotent Stem Cells Provide an Unlimited T-cell Source for CAR-T cell Development and A Potential Source for Off-the-shelf Products

2021 ◽  
Author(s):  
Muhammad Sadeqi Nezhad
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Pluripotent Stem Cells ◽  
T Cell Development ◽  
Cell Development ◽  
Car T Cell ◽  
Car T ◽  
Cell Source

CAR-T cell therapy has been increasingly conducted for cancer patients in clinical settings. Progress in this therapeutic approach is hampered by the lack of a solid manufacturing process, T lymphocytes, and tumor-specific antigens. T-cell source used in CAR-T cell therapy is predominantly derived from the patient’s own T lymphocytes, which makes this approach impracticable to patients with progressive diseases and T leukemia. Autologous CAR-T cell generation is time-consuming due to lack of readily available T lymphocytes and is not applicable for third-party patients. Pluripotent stem cells, such as human induced pluripotent stem cells (hiPSCs), could provide an unlimited T-cell source for CAR-T cell development. iPSC-derived T cells would be a promising infinite T-cell source and are phenotypically defined, expandable and functional as physiological T cells. iPSC-derived T cells provide a feasible T-cell source for the development of off-the-shelf T cells and CAR-T cells. The combination of iPSC and CAR technologies provides an extraordinary opportunity to oncology and greatly facilitates cell-based therapy for cancer patients. T-iPSCs in combination with CAR is in early stage of development and the pre-clinical and clinical studies concerning the combination of these novel technologies are not sufficient. This article critically reviews the progress in iPSC-derived T cell development, and it considers the opportunity to convert iPSC-derived T cells into off-the-shelf T cells for universal CAR-T cell treatment.

scholarly journals Induced Pluripotent Stem Cells Provide an Unlimited T-cell Source for CAR-T cell Development and A Potential Source for Off-the-shelf Products

2021 ◽  
Author(s):  
Muhammad Sadeqi Nezhad
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Pluripotent Stem Cells ◽  
T Cell Development ◽  
Cell Development ◽  
Car T Cell ◽  
Car T ◽  
Cell Source

CAR-T cell therapy has been increasingly conducted for cancer patients in clinical settings. Progress in this therapeutic approach is hampered by the lack of a solid manufacturing process, T lymphocytes, and tumor-specific antigens. T-cell source used in CAR-T cell therapy is predominantly derived from the patient’s own T lymphocytes, which makes this approach impracticable to patients with progressive diseases and T leukemia. Autologous CAR-T cell generation is time-consuming due to lack of readily available T lymphocytes and is not applicable for third-party patients. Pluripotent stem cells, such as human induced pluripotent stem cells (hiPSCs), could provide an unlimited T-cell source for CAR-T cell development. iPSC-derived T cells would be a promising infinite T-cell source and are phenotypically defined, expandable and functional as physiological T cells. iPSC-derived T cells provide a feasible T-cell source for the development of off-the-shelf T cells and CAR-T cells. The combination of iPSC and CAR-Technologies provides an extraordinary opportunity to oncology and greatly facilitates cell-based therapy for cancer patients. T-iPSCs in combination with CAR is in early stage of development and the pre-clinical and clinical studies concerning the combination of these novel technologies are not sufficient. This article critically reviews the progress in iPSC-derived T cell development and provides a roadmap for development of CAR iPSC-derived T cells and off-the-shelf T-iPSCs. Keywords: CAR-T cell; iPSC; T cell; iPSC-derived T cell; tumor cell; therapeutic; off-the-shelf

scholarly journals Induced Pluripotent Stem Cells Provide an Unlimited T-cell Source for CAR-T cell Development and A Potential Source for Off-the-shelf Products

2021 ◽  
Author(s):  
Muhammad Sadeqi Nezhad
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Pluripotent Stem Cells ◽  
T Cell Development ◽  
Cell Development ◽  
Car T Cell ◽  
Car T ◽  
Cell Source

CAR-T cell therapy has been increasingly conducted for cancer patients in clinical settings. Progress in this therapeutic approach is hampered by the lack of a solid manufacturing process, T lymphocytes, and tumor-specific antigens. T-cell source used in CAR-T cell therapy is predominantly derived from the patient’s own T lymphocytes, which makes this approach impracticable to patients with progressive diseases and T leukemia. Autologous CAR-T cell generation is time-consuming due to lack of readily available T lymphocytes and is not applicable for third-party patients. Pluripotent stem cells, such as human induced pluripotent stem cells (hiPSCs), could provide an unlimited T-cell source for CAR-T cell development. iPSC-derived T cells would be a promising infinite T-cell source and are phenotypically defined, expandable and functional as physiological T cells. iPSC-derived T cells provide a feasible T-cell source for the development of off-the-shelf T cells and CAR-T cells. The combination of iPSC and CAR-Technologies provides an extraordinary opportunity to oncology and greatly facilitates cell-based therapy for cancer patients. T-iPSCs in combination with CAR is in early stage of development and the pre-clinical and clinical studies concerning the combination of these novel technologies are not sufficient. This article critically reviews the progress in iPSC-derived T cell development and provides a roadmap for development of CAR iPSC-derived T cells and off-the-shelf T-iPSCs. Keywords: CAR-T cell; iPSC; T cell; iPSC-derived T cell; tumor cell; therapeutic; off-the-shelf

scholarly journals NOTCH and CAR Signaling Control T Cell Lineage Commitment from Pluripotent Stem Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Sjoukje van der Stegen ◽  
Pieter Lindenbergh ◽  
Roseanna Petrovic ◽  
Benjamin Whitlock ◽  
Raedun Clarke ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Research Funding ◽  
Cell Lineage ◽  
Lineage Commitment ◽  
Publicly Traded ◽  
Car T Cell ◽  
Car T ◽  
Single Positive

Chimeric Antigen Receptor (CAR) T cells are a new treatment paradigm for relapsed/refractory hematopoietic malignancies. However, their autologous nature imposes manufacturing constraints that can delay CAR T cell availability and increase their cost. We previously established proof of principle that αβ T cell-derived induced pluripotent stem cells (TiPSCs) can provide a self-renewing source for in vitro CAR T cell production (Themeli, Nat Biotechnol, 2013). The use of cloned TiPSC further enhances the feasibility of verifying genome integrity of the genetically engineered stem cells and should in principle yield highly homogenous cell products. Using αβ T cell-derived TiPSCs transduced with a well-defined CD19-specific CAR (1928z; Park, NEJM, 2018), we previously demonstrated that TiPSCs can be differentiated into CAR T cells. These T cells retained their endogenous T cell receptor (TCR) and also displayed characteristics of innate lymphoid cells. We have now examined how the timing of CAR expression as well as the CAR signaling strength influence T cell lineage commitment, enabling better control towards αβ T cell lineage commitment. αβ T cell lineage development depends in part on a precisely orchestrated interactions between NOTCH and (pre)TCR signaling, the timing and strength of which are crucial for αβ lineage commitment. Because TiPSCs harbor rearranged TCRα and TCRβ genes, mature TCR expression occurs earlier than if it required VDJ recombination, skewing differentiation towards acquiring innate features including CD4-CD8- double-negative or CD8αα single-positive phenotypes. We show that providing strong NOTCH stimulation counteracts the effects of early antigen receptor expression, facilitating CD4+CD8αβ+ double positive (DP) formation. We hypothesized that CAR signaling in the absence of ligand binding (tonic signaling) may mimic a TCR signal, the strength and timing of which could re-direct lineage commitment. We therefore investigated CARs providing different levels of signaling strength and the impact of delaying the onset of CAR expression. Tonic CAR signaling was measured in peripheral blood T cells expressing 1928z or 1928z-1XX, a construct in which the second and third ITAM in the CD3ζ domain have been mutated to be non-functional (Feucht, Nat Med, 2019), following either retroviral transduction (SFG vector) orTRAC-targeted cDNA integration, placing CAR expression under the transcriptional control of the TCRα promoter (Eyquem, Nature, 2017). CAR signaling in the absence of antigen exposure, measured by phosphorylation of ITAM3, ERK1/2 and ZAP70, was reduced by bothTRAC-targeting and reduction of functional ITAMs, with additive effects when combined inTRAC-1928z-1XX. Three of these engineering strategies (virally expressed 1928z,TRAC-1928z andTRAC-1928z-1XX) were evaluated in the context of TiPSC-derived T cell differentiation. Virally expressed 1928z (resulting in constitutive CAR expression throughout differentiation) resulted in the predominant generation of innate-like CD8αα T cells, associated with the absence of early T cell lineage markers such as CD5, CD2 and CD1a. Delayed expression of 1928z throughTRACtargeting resulted in increased CD5, CD2 and CD1a, but did not yield any more CD4+CD8αβ+ DP cells. In TiPSC expressingTRAClocus-encoded 1928z-1XX, a greater DP population emerged, from which CD8αβ single-positive T cells could be induced. Phenotypic analyses of clonal TRAC-1928z-1XX TiPSC lines further establish the interplay between CAR and NOTCH1 in determining αβ lineage commitment. Together these data show that early TCR and CAR expression skew T cell lineage commitment towards an innate-like T cell fate, which can be overcome by controlling the strength and timing of NOTCH, TCR and CAR signaling. These studies pave the way for the predetermined generation of a variety of CAR T cell types endowed with different functional attributes. Disclosures Whitlock: Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Clarke:Fate Therapeutics Inc.:Current Employment, Current equity holder in publicly-traded company.Valamehr:Fate Therapeutics, Inc:Current Employment, Current equity holder in publicly-traded company.Riviere:Juno Therapeutics:Other: Ownership interest, Research Funding;Takeda:Research Funding;Fate Therapeutics Inc.:Consultancy, Other: Ownership interest , Research Funding;FloDesign Sonics:Consultancy, Other: Ownership interest;Atara:Research Funding.Sadelain:Atara:Patents & Royalties, Research Funding;Fate Therapeutics:Patents & Royalties, Research Funding;Mnemo:Patents & Royalties;Takeda:Patents & Royalties, Research Funding;Minerva:Other: Biotechnologies , Patents & Royalties.

Insights into the ontogeny and activation of T cells

1994 ◽  
Vol 40 (11) ◽  
pp. 2128-2131 ◽  
Author(s):  
T W Mak
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cell Surface ◽  
Immune Responses ◽  
Tyrosine Phosphatase ◽  
T Cell Development ◽  
Cell Development ◽  
Mutant Mice ◽  
Target Cells

Abstract T lymphocytes recognize antigen peptides and major histocompatibility complex products through their T-cell antigen receptors (TcR), consisting of alpha and beta chains. The interaction between T cells and their target cells or antigen-presenting cells is also assisted by a series of other cell-surface polypeptides, most notably CD4 and CD8, which are selectively expressed on mature helper/inducer and killer/suppressor T cells, respectively. Upon engagement of their ligands, a series of signals is transduced intracytoplasmically via some of these molecules and their associated proteins. Perhaps the most important enzyme in this signal transduction process is the lymphocyte-specific tyrosine kinase lck. Another important component is the cell-surface tyrosine phosphatase CD45. This molecule is alternatively spliced and the different isoforms are expressed on the various hematopoietic and lymphopoietic cells. Signaling through the TcR-CD4 D8-lck-CD45 complex is thought to be insufficient to activate T lymphocytes. A costimulatory signal is believed to be essential, and many investigators have suggested that CD28, a ligand for B7/BB1, is such a signal. Immune responses are also controlled by a number of cytokines and soluble factors. Signaling through the tumor necrosis factor receptor p55 is required for clearance of intracellular pathogens. Transcriptional factors involved in controlling interferon production are also important in T-cell development and immune responses. In an attempt to gain a better understanding of the roles of these molecules in T-lymphocyte functions and ontogeny, we generated a series of mutant mice with disruptions in the genes coding for these molecules. We are analyzing the mutant mice to evaluate the importance of these genes in T-cell development.

Bcr-Abl Impairs T Cell Development From Murine Induced Pluripotent Stem Cells and Hematopoietic Stem Cells; A Partial Explanation for the Concept That Ph+ Clone Never Involves T Cell Lineage in CML,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3748-3748
Author(s):  
Bidisha Chanda ◽  
Kiyoko Izawa ◽  
Ratanakanit Harnprasopwat ◽  
Keisuke Takahashi ◽  
Seiichiro Kobayashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
T Cell Development ◽  
Cell Lineage ◽  
Cell Development ◽  
Hematopoietic Stem

Abstract Abstract 3748 Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder generally believed to originate from a hematopoietic stem cell carrying the BCR-ABL fusion gene, which generally encodes 210kD and 190kD constitutively active tyrosine kinases termed as p210 and p190, respectively. In spite of the putative stem cell origin and the competence for differentiation toward mature B cells, there is a longstanding consensus that CML never involves the T cell lineage at least in chronic phase. To gain insight into this apparent conflict, we used in vitro T cell differentiation model from murine pluripotent stem cells (PSCs) as well as hematopoietic stem cells (HSCs). C57BL/6 MEFs were reprogrammed using a polycistronic lentiviral Tet-On vector encoding human Oct4, Sox2 and Klf4, which were tandemly linked via porcine teschovirus-1 2A peptides, together with another lentiviral vector expressing rtTA driven by the EF-1a promoter. Almost all the vector sequences including the transgenes were deleted by adenovirus-mediated transduction of Crerecombinase after derivation of iPSCs, and only remnant 291-bp LTRs containing a single loxP site remained in the genome. A clone of MEF-iPSCs were retrovirally transduced with p190DccER, a ligand-controllable p190-estrogen receptor fusion protein, whose tyrosine kinase activity absolutely depends on 4-hydroxytamoxyfen (4-HT).For T cell lineage differentiation, p190DccER-MEF-iPSCs were recovered from a feeder-free culture supplemented with LIF and plated onto a subconfluent OP9-DL1 monolayer in the presence of Flt3 ligand and IL7 with or without 0.5 mM 4-HT.After 3 weeks of culture, iPSC-derived blood cells were collected and subjected to FACS analysis for their lineage confirmation. About 70% of lymphocyte-like cells from the 4-HT(-) culture expressed CD3, but only 20% of counterparts from the 4-HT(+)culture expressed CD3, suggesting impaired T cell development by Bcr-Abl. Next, c-Kit+Sca1+Lin− (KSL) bone marrow cells were prepared by FACS from 8-weeks old C57BL/6 mice treated with 5-FU. KSL cells were similarly transduced with p190DccER and were subjected to the OP9-DL1co-culture system with or without 0.5 mM 4-HT.After 2 weeks of culture, 90% of lymphocytes from the 4-HT(-)culture revealed CD3+TCRβ+ phenotype, but only 30% of those were double positive in the presence of 4-HT(+). In addition, 96% of lymphocytes from the 4-HT(-) culture progressed to the DN2 stage with c-Kit−CD44+CD25+phenotype, whereas 40% of those from the 4-HT(+) culture arrested at the DN1 stage showing c-Kit+CD44+CD25−.Since IL7 plays a central role at the stage from DN1 to DN2 of progenitor T cells, Bcr-Abl is suggested to impair T cell development possibly through interfering with the IL7 signal. The precise mechanism underlying impaired T lymphopoiesis by Bcr-Abl is under investigation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals In Vitro Generation of Human Pluripotent Stem Cell-Derived T Cells for Immunotherapy

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 691-691
Author(s):  
Amélie Montel-Hagen ◽  
Christopher S. Seet ◽  
Suwen Li ◽  
Brent Chick ◽  
Patrick Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Pluripotent Stem Cells ◽  
Research Funding ◽  
Cytokine Release ◽  
Highly Efficient ◽  
Car T ◽  
Engineered T Cells

Abstract Adoptive cell therapy using T cells engineered to express antigen-specific T cell receptors (TCR-T) or chimeric antigen receptors (CAR-T) offer targeted and potentially curative treatments for malignancy. Current approaches rely on the genetic modification and expansion of mature circulating T-cells. Such processes are limited to autologous T cells due to the risk of graft-versus-host (GvHD) disease from allogeneic T cells through endogenous TCR expression as well as rejection through MHC incompatibility. Furthermore, prolonged ex-vivo expansion of T cells may reduce in vivo efficacy and harvesting sufficient T cells from lymphopenic patients is challenging. Direct in vitro differentiation of engineered T cells from human pluripotent stem cells (HSPCs) may overcome these problems by providing an unlimited source of cells that can be genetically edited, permitting the suppression of endogenous TCR expression through allelic exclusion, and the de novo generation of naïve antigen-specific T cells. We have developed an in vitro Artificial Thymic Organoid (ATO) system that induces highly efficient and reproducible production of mature naïve T cells from human hematopoietic stem cells and progenitor cells (HSPC). Here, we report the preclinical development of a modified ATO system that supports highly efficient in vitro differentiation and positive selection of naive human T cells from at least 5 different lines of human pluripotent stem cells (PSC), including Embryonic stem cells (ESC) and induced Pluripotent Stem Cells (iPSC). T cell differentiation from PSC was very similar phenotypically to that from HSPC. As in normal human thymopoiesis, the first evidence for T cell commitment was expression of CD7 and CD5, followed by the CD3-CD8lo "ISP8" stage, then CD4+CD8+ "DP" stage and finally production of CD3+CD8+CD4- "CD8SP" and Cd3+CD4+CD8- "CD4SP". As is typical with both monolayer cultures and ATOs (and opposite to normal thymus), CD8SP predominated over CD4SP. Surprisingly, differentiation occurred more rapidly from PSC than with HSPC. As with HSPC-ATOs, CD8SP from PSC ATOs showed a mature naïve conventional T cell phenotype i.e. CD3+TCRab+CD4- CD45RA+CD62L+CD27+ and exhibited a diverse, thymic-like TCR repertoire, and robust TCR-dependent cytokine release and proliferation. The differentiation in ATOs of an ESC line that expresses an HLA-A*02:01-restricted αβ TCR specific for NY-ESO-1 resulted in a markedly increased cell yield with an enhanced generation of naïve CD3+TCRαβ+CD8αβ+ conventional T cells, the majority of which were antigen-specific by tetramer staining. TCR-engineered T cells produced from PSC in ATOs displayed a near complete lack of endogenous TCR Vβ expression, consistent with induction of allelic exclusion by the exogenous TCR during T cell development. The TCR engineered T cells underwent polyfunctional cytokine release, and proliferation in response to artificial APCs. Moreover, the differentiation in ATOs of an ESC line that expresses a CD19-specific 2nd generation (CD28/CD3zeta) CAR construct resulted in the production of CD5+CD7+ CD45RA+ CAR T cells. As reported previously, the ESC-derived CAR T cells did not express CD4, CD8 or CD3; however, they responded to PMA/ionomycin and underwent specific cytokine release and degranulation in response to target cells expressing CD19. PSC-derivedATOs thus present a highly efficient platform for the generation of clinically relevant mature naïve and potentially non-alloreactive TCR and CAR engineered T cells for adoptive immunotherapy. Disclosures Montel-Hagen: Kite Pharma: Research Funding. Seet: Kite Pharma: Research Funding. Crooks: Kite Pharma: Research Funding.

scholarly journals Human Delta Like 1-Expressing Human Mesenchymal Stem Cells Promote Human T Cell Development and Antigen-Specific Response in Humanized NOD/SCID/IL-2Rgnull (NSG) Mice

2021 ◽  
Author(s):  
Ki-Young Lee ◽  
Do Hee Kwon ◽  
Jae Berm Park ◽  
Joo Sang Lee ◽  
Sung Joo Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
T Cell Development ◽  
Cell Development ◽  
Specific Response ◽  
Cell Fate Decisions ◽  
Nsg Mice ◽  
Human T Cells

Abstract Human delta-like 1 (hDlk1) is known to be able to regulate cell fate decisions duringhematopoiesis. Mesenchymal stem cells (MSCs) are known to exhibit potentimmunomodulatory roles in a variety of diseases. Herein, we investigated in vivofunctions of hDlkl1-hMSCs and hDlk1+hMSCs in T cell development and T cell responseto viral infection in humanized NOD/SCID/IL-2Rγnull (NSG) mice. Co-injection ofhDlk1-hMSC with hCD34+ cord blood (CB) cells into the liver of NSG mice markedlysuppressed the development of human T cells. In contrast, co-injection of hDlk1+hMSCwith hCD34+ CB cells into the liver of NSG dramatically promoted the development ofhuman T cells. Human T cells developed in humanized NSG mice represent markedlydiverse in terms of TCR Vβ usages, functionally active, and the restriction to human MHCmolecules. Upon challenge with Epstein-Barr virus (EBV), EBV-specific hCD8+ T cellsin humanized NSG mic were effectively mounted with phenotypically activated T cellspresented as hCD45+hCD3+hCD8+hCD45RO+hHLA-DR+ T cells, suggesting thatantigen-specific T cell response was induced in the humanized NSG mice. Taken together,our data suggest that the hDlk1-expressing MSCs can effectively promote thedevelopment of human T cells and immune response to exogenous antigen in humanizedNSG mice. Thus, the humanized NSG model might have potential advantages for thedevelopment of therapeutics targeting infectious diseases in the future.

Enhancement of T Cell Development in Murine Bone Marrow Transplant Recipients by Transfer of the IL-7 or SCF Gene into Marrow-Derived Mesenchymal Stem Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3572-3572
Author(s):  
Brile Chung ◽  
Dullei Min ◽  
Mark Krampf ◽  
Won Jong Ju ◽  
Kenneth I. Weinberg
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Bone Marrow ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
T Cell ◽  
Immune Reconstitution ◽  
T Cell Development ◽  
Cell Development ◽  
Allogeneic Bmt

Abstract The ability of the thymus to generate T cells diminishes with increasing age, the use of chemotherapy, bone marrow transplantation (BMT), anti-retroviral therapy for HIV, and graft-versus-host disease (GVHD) which can lead to a major clinical problem. Therefore, developing a clinically relevant strategy for the rapid development of T lymphocytes is crucial for treating immune deficiency. Stem cell factor (SCF: also known as kit ligand) and interleukin-7 (IL-7) are stroma–derived cytokines that induce proliferation, differentiation, and survival of developing immature T cells in the thymus. Studies have shown that administration of recombinant human IL-7 following murine BMT resulted in improved thymopoiesis and immune function. However, our previous studies have shown that that IL-7 treatment post-HSCT to enhance immune reconstitution in the allogeneic setting may have adverse effects because of the dual role of IL-7 in supporting both thymopoiesis and mature T lymphocyte expansion. Therefore it raises the question of whether IL-7 treatment after allogeneic BMT will increase the frequency or severity of GVHD. The purpose of this study was to examine whether: administration of IL-7 and SCF with infusion of mature T cell depleted (TCD) BM cells can induce enhancement of donor-derived immune reconstitution more rapidly than treatment with either cytokine alone and whether IL-7 and SCF are synergistic and partially complementary signals for the proliferation, survival, and differentiation of immature T cells. To evaluate the combinatory effect of IL-7 and SCF in T cell development following BMT, we developed a gene therapy approach using retrovirally-mediated transduction of BM-derived mesenchymal stem cells (MSC) with the human IL-7 or murine SCF gene (soluble isoform). C57BL/6J (CD45.2) recipient mice were irradiated (1300 cGy) and co-transplanted with 1 × 10 6 T cell depleted (TCD) bone marrow cells from congenic donor B6.SJL mice (CD45.1) and different doses (0.1 × 10 6 or 0.3 × 10 6) of eGFP (control), IL-7, SCF, or combination of IL-7 and SCF MSC. At day 30 following BMT, we observed that transplantation of both IL-7 and SCF MSC resulted in significantly higher numbers of donor-derived thymocytes and peripheral lymphocytes than either IL-7 or SCF MSC transplantation alone. Most noticeably, the number of donor-derived immature and mature T cells recovered from the animals receiving transplantation of 0.1 × 10 6 IL-7 MSC and 0.3 × 10 6 SCF MSC was similar to that of animals receiving 0.3 × 10 6 IL-7 MSC alone, demonstrating that the reduced proliferative signals produced by 0.1 × 10 6 IL-7 MSC can be compensated by co-transplantation of 0.3 × 10 6 SCF MSC. Moreover, transplantation of IL-7 and SCF MSC significantly increased the number of donor-derived common lymphoid progenitors (CLP [Lin-, Sca-1 low, Thy1-, c-Kit low, IL-7R+]) in the BM, suggesting that transplanted CLPs are induced to differentiate or expand more rapidly in response to IL-7 and SCF and may have contributed to increased immune reconstitution. Collectively, our findings demonstrate that IL-7 and SCF gene therapy may be a therapeutically useful method to promote enhancement of T cell development in de novo. Furthermore, the experiments resulted in important knowledge about complementary signals provided between IL-7 and SCF, and suggest various doses of IL-7 and SCF therapy may enhance development of T cells with limited expansion of mature T cells responsible for causing GVHD in allogeneic BMT setting.

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