Role of the IL-7 Receptor in γδ T-Cell Development from Hematopoietic Stem Cells

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.

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Early human T cell development: analysis of the human thymus at the time of initial entry of hematopoietic stem cells into the fetal thymic microenvironment.

Journal of Experimental Medicine ◽

10.1084/jem.181.4.1445 ◽

1995 ◽

Vol 181 (4) ◽

pp. 1445-1458 ◽

Cited By ~ 96

Author(s):

B F Haynes ◽

C S Heinly

Keyword(s):

Stem Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

T Cell Development ◽

Fold Increase ◽

Cell Receptor ◽

Cell Development ◽

Hematopoietic Stem ◽

Human Thymus ◽

Human T Cell

To determine events that transpire during the earliest stages of human T cell development, we have studied fetal tissues before (7 wk), during (8.2 wk), and after (9.5 wk to birth) colonization of the fetal thymic rudiment with hematopoietic stem cells. Calculation of the approximate volumes of the 7- and 8.2-wk thymuses revealed a 35-fold increase in thymic volumes during this time, with 7-wk thymus height of 160 microM and volume of 0.008 mm3, and 8.2-wk thymus height of 1044 microM and volume of 0.296 mm3. Human thymocytes in the 8.2-wk thymus were CD4+ CD8 alpha+ and cytoplasmic CD3 epsilon+ cCD3 delta+ CD8 beta- and CD3 zetta-. Only 5% of 8-wk thymocytes were T cell receptor (TCR)-beta+, < 0.1% were TCR-gamma+, and none reacted with monoclonal antibodies against TCR-delta. During the first 16 wk of gestation, we observed developmentally regulated expression of CD2 and CD8 beta (appearing at 9.5 wk), CD1a,b, and c molecules (CD1b, then CD1c, then CD1a), TCR molecules (TCR-beta, then TCR-delta), CD45RA and CD45RO isoforms, CD28 (10 wk), CD3 zeta (12-13 wk), and CD6 (12,75 wk). Whereas CD2 was not expressed at the time of initiation of thymic lymphopoiesis, a second CD58 ligand, CD48, was expressed at 8.2 wk, suggesting a role for CD48 early in thymic development. Taken together, these data define sequential phenotypic and morphologic changes that occur in human thymus coincident with thymus colonization by hematopoietic stem cells and provide insight into the molecules that are involved in the earliest stages of human T cell development.

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NKL homeobox gene activities in hematopoietic stem cells, T-cell development and T-cell leukemia

PLoS ONE ◽

10.1371/journal.pone.0171164 ◽

2017 ◽

Vol 12 (2) ◽

pp. e0171164 ◽

Cited By ~ 13

Author(s):

Stefan Nagel ◽

Claudia Pommerenke ◽

Michaela Scherr ◽

Corinna Meyer ◽

Maren Kaufmann ◽

...

Keyword(s):

Stem Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

T Cell Development ◽

Homeobox Gene ◽

Cell Development ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Hematopoietic Stem

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Characterization in vitro and engraftment potential in vivo of human progenitor T cells generated from hematopoietic stem cells

Blood ◽

10.1182/blood-2008-10-187013 ◽

2009 ◽

Vol 114 (5) ◽

pp. 972-982 ◽

Cited By ~ 89

Author(s):

Génève Awong ◽

Elaine Herer ◽

Charles D. Surh ◽

John E. Dick ◽

Ross N. La Motte-Mohs ◽

...

Keyword(s):

Stem Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

T Cell Development ◽

Cell Development ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Hematopoietic Stem ◽

Human T Cell

T-cell development follows a defined set of stage-specific differentiation steps. However, molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. To address this, human umbilical cord blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T lineage in OP9-DL1 cocultures. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. Quantitative clonal analyses demonstrated that CD34+CD38− and CD34+CD38lo subsets of UCB contain a similarly high T-lineage progenitor frequency, whereas the frequency in CD34+CD38+/hi cells was 5-fold lower. Delta-like/Notch-induced signals increased the T-cell progenitor frequency of CD34+CD38−/lo cells differentiated on OP9-DL1, and 2 distinct progenitor subsets, CD34+CD45RA+CD7++CD5−CD1a− (proT1) and CD34+CD45RA+CD7++CD5+CD1a− (proT2), were identified and their thymus engrafting capacity was examined, with proT2 cells showing a 3-fold enhanced reconstituting capacity compared with the proT1 subset. Furthermore, in vitro–generated CD34+CD7++ progenitors effectively engrafted the thymus of immunodeficient mice, which was enhanced by the addition of an IL-7/IL-7 antibody complex. Taken together, the identification of T-progenitor subsets readily generated in vitro may offer important avenues to improve cellular-based immune-reconstitution approaches.

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In Utero Transplantation of Transduced Hematopoietic Stem Cells Results in Deletion of Transgene-Specific T Cells.

Blood ◽

10.1182/blood.v108.11.3266.3266 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3266-3266

Author(s):

Pablo Laje ◽

William H. Peranteau ◽

Masayuki Endo ◽

Philip W. Zoltick ◽

Alan W. Flake

Keyword(s):

Stem Cells ◽

T Cells ◽

T Cell ◽

Hematopoietic Stem Cells ◽

T Cell Receptor ◽

Peripheral Blood ◽

T Cell Development ◽

Cell Receptor ◽

In Utero ◽

Hematopoietic Stem

Abstract The developing fetal immune system provides a unique opportunity to manipulate normal immunologic development for therapeutic prenatal and anticipated postnatal interventions. In previous studies we have shown that allogeneic in utero hematopoietic cell transplantation (IUHCT) results in donor specific tolerance that can subsequently facilitate non-myeloablative postnatal cellular or organ transplants. It follows that in utero injection of transduced hematopoietic stem cells (HSC) could potentially induce tolerance to a transgene encoded protein. We hypothesized that expression of a transduced antigenic protein by HSC and their progeny would alter thymic T cell development resulting in deletion of antigen specific T-cells. To test this hypothesis, we used the mammary tumor virus (MTV) superantigen system to evaluate the effect of IUHCT of transduced HSC on T cell development. In this system, expression of different MTV oncogenes by different I-E+ strains of mice results in deletion of T cells expressing the relevant Vβ T cell receptor. Specifically, mice which are Mtv7+ delete T cells expressing the Vβ6 T-cell receptor. In this study, CD150+CD48− enriched Balb/c (I-E+ Mtv7−) HSC were transduced with an HIV-based lentivirus expressing MTV7 under an MND promoter. 1.5E+05 transduced cells were injected intravascularly via the vitelline vein into E14 Balb/c fetuses. Non-injected age matched naive Balb/c mice served as the control group. The peripheral blood (PB) and thymuses of injected fetuses and control mice were harvested at day of life (DOL) 10, 20 and 60 and analyzed by flow cytometry for T lymphocyte Vβ6 expression. Additionally, the T cell composition of the thymus was assessed at DOL10 for CD4 and CD8 single positive (SP) and CD4/CD8 double positive (DP) cells. Thymic flow cytometric analysis at DOL10 revealed that greater than 98% of the T cells were CD4CD8 DP, a stage that has not yet undergone negative selection. No significant difference was noted in the percentage of thymic Vβ6+ DP T-cells at this time point or at DOL20 and DOL60. In contrast, there was a significant decrease in the percentage of Vβ6+ peripheral blood SP cells in those mice injected with MTV7 transduced HSC relative to control mice at DOL10, DOL20 and DOL60 (p<0.05) (Fig 1). The current study supports the ability of enriched transduced HSC to induce deletion of transgene specific T cells after IUHCT. In the future, this strategy may be useful to promote tolerance for pre or postnatal cellular or gene therapy. Figure Figure

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Role of Itk in γδ T cell development and function

The FASEB Journal ◽

10.1096/fasebj.22.2_supplement.410 ◽

2008 ◽

Vol 22 (S2) ◽

pp. 410-410

Author(s):

Catherine Chih‐tzu Yin ◽

Martin Felices ◽

Yoko Kosaka ◽

Joonsoo Kang

Keyword(s):

T Cell ◽

T Cell Development ◽

Cell Development ◽

Γδ T Cell ◽

And Function

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A Hypomorphic Cbfb Allele Reveals a Critical Dosage-Sensitive Function of Core Binding Factors at the Earliest Stages of T Cell Development.

Blood ◽

10.1182/blood.v106.11.124.124 ◽

2005 ◽

Vol 106 (11) ◽

pp. 124-124

Author(s):

Ivan Maillard ◽

Laleh Talebian ◽

Zhe Li ◽

Yalin Guo ◽

Daisuke Sugiyama ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Dna Binding ◽

B Cell ◽

Bone Marrow Cells ◽

T Cell Development ◽

Cell Development ◽

B Cell Development ◽

Hematopoietic Stem

Abstract The family of core binding factors includes the DNA-binding subunits Runx1-3 and the common non-DNA binding partner CBFβ. Runx1 and CBFβ are essential for the emergence of hematopoietic stem cells during fetal development, but not for stem cell maintenance during later ontogeny. Runx1 is also required for megakaryocyte differentiation, B cell development, and for the DN2 to DN3 transition in thymocyte development. Runx2/CBFβ are critical for normal osteogenesis, and Runx3 for CD4 silencing in CD8+ T cells, but their contribution to other steps of hematopoietic development is unknown. To examine the collective role of core binding factors in hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss). CBFβ protein levels were reduced by approximately 2–3 fold in fetuses homozygous for the Cbfbrss allele (Cbfbrss/rss), and 3–4 fold in fetuses carrying one hypomorphic and one knockout allele (Cbfbrss/−). Cbfbrss/rss and Cbfbrss/− fetuses had normal erythroid and B cell development, and relatively mild abnormalities in megakaryocyte and granulocyte differentiation. In contrast, T cell development was very sensitive to an incremental reduction of CBFβ levels: mature thymocytes were decreased in Cbfbrss/rss fetuses, and virtually absent in Cbfbrss/−fetuses. We next assessed the development of Cbfbrss/rss and Cbfbrss/− fetal liver progenitors after transplantation to irradiated adult recipients, in competition with wild-type (wt) bone marrow cells. Wt, Cbfbrss/rss and Cbfbrss/− fetal progenitors replenished the erythroid, myeloid and B cell compartments equally well. The overall development of Cbfbrss/rss T cells was preserved, although CD4 expression was derepressed in double negative thymocytes. In Cbfbrss/− chimeras, mature thymocytes were entirely derived from competitor cells. Furthermore, the developmental block in Cbfbrss/− progenitors was present at the earliest stages of T cell development within the DN1 (ETP) and DN2 subsets. Our data define a critical CBFβ threshold for normal T cell development, and they situate an essential role of core binding factors during the earliest stages of T cell development. In addition, early thymopoiesis appeared more severely affected by reduced CBFβ dosage than by the lack of Runx1 (Ichikawa et al., Nat Med 2004; Growney et al., Blood 2005), suggesting that Runx2/3 may contribute to core binding factor activity in the T cell lineage.

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