Ammonium Ions Derived from Spontaneous Decomposition of L-Glutamine Inhibit Lymphoid Differentiation from Hematopoietic Stem/Progenitor Cells.

Abstract The ability to study lymphocyte differentiation in culture has been greatly advanced by the availability of the OP9 bone marrow stromal cell line, which was derived from an op/op mouse and thus lacks M-CSF. As a result, the normal default myeloid differentiation from bone marrow-derived stem and progenitor cells does not occur, and lymphocyte differentiation is favored. Introduction of the Notch ligand Delta-like 1 into OP9 cells results in promotion of T cell development and parallel suppression of B cell development. While the OP9-DL1 model of T cell development works quite well when fetal liver-derived progenitors are cultured, the success of T cell development from adult bone marrow-derived progenitors has been more difficult to reproduce. We have undertaken a systematic analysis of variables that can prevent efficient T cell development in OP9-DL1 cultures, and have found that one limiting factor that impacts the efficiency of differentiation of both T and B cell lineages is the accumulation of ammonium ions as a result of the spontaneous decomposition of l-glutamine. L-glutamine, which is present at 2 to 4 mM in standard tissue culture media, is unstable and will spontaneously degrade to form ammonium ions and pyroglutamic acid at a rate of 1%/day at 4°C and at a 10-fold higher rate at 37°C. To evaluate the effects of the two major products of l-glutamine decomposition on lymphoid differentiation, we added each product to differentiation cultures at 3 mM in the presence of a stable source of l-glutamine (l-alanyl-l-glutamine). Cultures were established in 1 ml containing 4×104 stromal cells (OP9 for B cell differentiation, OP9-DL1 for T cell differentiation), 1×103 bone marrow-derived lymphoid progenitors enriched by phenotype (c-kit+LinnegSca-1+Thy-1.1neg), and 5 ng/ml Flt3L plus 5 ng/ml IL-7. Every 3 to 4 days, cultures were harvested and passaged onto fresh stromal cell monolayers; lymphoid cells were counted and evaluated for surface antigen expression at each passage. While addition of pyroglutamic acid had no inhibitory effect on lymphocyte growth or differentiation, addition of ammonium chloride slowed growth and prevented differentiation of both T and B lymphocytes. Growth of the stromal cell monolayers was not affected by ammonium chloride at the concentrations utilized in these studies. We conclude that freshly-prepared culture medium, preferably containing a stabilized form of l-glutamine, is a critical aspect contributing to the success of lymphocyte differentiation cultures established from adult bone marrow cells. We also found that decreasing IL-7 concentrations to 1 ng/ml resulted in more rapid differentiation of T cells and a more balanced representation of CD4 and CD8 single positive cells. Our studies help define optimal conditions for differentiation of bone marrow-derived lymphoid progenitor cells into T and B lineages in vitro, and provide evidence that hematopoietic differentiation displays variable degrees of sensitivity to ammonium ions derived from decomposition of l-glutamine. These results will help define optimal conditions for expansion and differentiation of hematopoietic stem and progenitor cells in vitro.

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LRF Is Indispensable for Hematopoietic Stem Cell Function Via Blocking Notch1-Mediated T Cell-Instructive Signals in the Bone Marrow Niche.

Blood ◽

10.1182/blood.v114.22.81.81 ◽

2009 ◽

Vol 114 (22) ◽

pp. 81-81 ◽

Cited By ~ 1

Author(s):

Sung-UK Lee ◽

Manami Maeda ◽

Nagisa Sakurai ◽

Freddy Radtke ◽

Takahiro Maeda

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

Target Gene ◽

T Cell Development ◽

Cell Development ◽

B Cell Development ◽

Self Renewal ◽

Hematopoietic Stem ◽

Hematopoietic System

Abstract Abstract 81 Hematopoietic stem cells (HSC) have the ability to self-renew and give rise to all hematopoietic lineage cells. Understanding signals that regulate the balance between self-renewal and differentiation of HSCs is an important issue in stem cell biology as well as regenerative medicine. Notch signals are critical regulators of the lymphoid lineage fate, but their role in adult HSC function is currently under debate. We explored the role of the LRF (Leukemia/Lymphoma Related Factor), a Notch repressor (also known as Zbtb7a, pokemon, OCZF and FBI-1) in HSC function, as it plays key roles in embryonic development, oncogenesis, and hematopoiesis. Conditional inactivation of the LRF gene in mouse HSCs (LRFF/FMx1-Cre mice) led to the development of CD4/CD8 DP (double positive) T-cells at the expense of B-cell development in the bone marrow (BM) in a Notch-dependent manner. Absolute numbers of the most primitive HSCs (LT-HSCs), defined as CD150+CD48−Flt3−Vcam-1+IL7Rα−LSK (Lin−Sca1+c-Kit+), were significantly reduced, while lymphoid-biased multi-potential progenitors (LMPPs: CD150−CD48+Flt3+Vcam-1+/−IL7Rα−LSK) and common lymphoid progenitors (CLPs: Lin−CD150−CD48+Flt3+Vcam-1−IL7Rα+) were barely detectable in LRFF/FMx1-Cre mice one month after pIpC injection. Enhanced T cell development and concomitant loss of B cell development was also seen in LRF−/− fetal liver (FL). Lin−IL7Rα+c-Kit+PIR+ (Paired Immunoglobulin-like receptors) T cell precursors were significantly increased in LRF−/− FL, indicating that Notch-mediated aberrant lymphoid fate determination also takes place during fetal hematopoiesis. To address which Notch gene(s) are targeted by LRF, we studied the HSC/progenitor population of conditional LRF knockout (LRFF/FMx1-Cre) as well as LRF/Notch1 double conditional knockout mice (LRFF/FNotch1F/FMx1-Cre). In the absence of Notch1, normal B cell development was restored in LRFF/FMx1-Cre mice. Reduction of LT-HSCs in LRFF/FMx1-Cre resulted from high Notch1 activity, as loss of Notch1 rescued LT-HSC numbers, suggesting that LRF functions to maintain HSCs and normal lymphoid fate by blocking Notch1. HSCs in active cell cycle are sensitive to 5-fluoro-uracil (5-FU) treatment, which causes remaining dormant HSCs to be recruited into the cell cycle to rapidly produce new cells and to quickly re-establish the hematopoietic system. To examine the self-renewal capacity of LRF deficient LT-HSC, LRFF/FMx1-Cre mice were treated with 5-FU after pIpC injection and the recovery of LT-HSC numbers examined. While control LT-HSC numbers recovered to pretreatment levels 3 wk after 5-FU treatment, levels in LRFF/FMx1-Cre mice remained low, accompanied by DP T cell development in the BM. Furthermore, after 5-FU treatment, LT-HSC numbers of LRFF/FNotch1F/FMx1-Cre were compatible to those of control and LRFF/FMx1-Cre mice, indicating that lack of self-renewal capacity in LRF deficient LT-HSCs was due to excessive differentiation toward T cells caused by Notch1. In support of this idea, when mice were given 5-FU weekly as a challenge to assess their HSC function in vivo, the survival percentage in LRFF/FMx1-Cre mice was much lower than in controls (0% versus 50% in 1 month, P <0.0001) and that of LRFF/FNotch1F/FMx1-Cre mice was compatible to controls. Serial bone marrow transplant experiments further demonstrated functional defects of LRF deficient HSCs, as they failed to reconstitute the hematopoietic system in secondary recipients. Microarray analysis and subsequent Gene Set Enrichment Analysis demonstrated upregulation of genes that were enriched in progenitor compartments. Since LRF can act as a transcriptional repressor, mRNA levels of Notch receptors and Notch ligands were examined using the same data set. A Notch target gene Hes1, but not Notch1 itself, was upregulated, and increased levels of Hes1 was also confirmed by real-time q-PCR in FACS-sorted LT-HSCs, as well as in 10.5 d.p.c whole embryos. These data suggest that LRF does not transcriptionally regulate Notch1, as LRF loss led to Notch1 target gene activation at the LT-HSC level without affecting Notch1 mRNA. Our genetic studies clearly indicate that LRF is indispensable for the maintenance of the HSC pool by repressing T cell-instructive signals mediated by Notch1 in the BM niche. Our findings shed new light on the regulatory mechanisms underlying the balance between HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.

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IL-7 Effects on Thymocyte Progenitors.

Blood ◽

10.1182/blood.v106.11.3318.3318 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3318-3318

Author(s):

Nahed El Kassar ◽

Baishakhi Choudhury ◽

Francis Flomerfelt ◽

Philip J. Lucas ◽

Veena Kapoor ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cell ◽

Notch Signaling ◽

Stromal Cells ◽

T Cell Development ◽

Fold Increase ◽

Cell Development ◽

B Cell Development ◽

Over Expression

Abstract IL-7 is a non-redundant cytokine in T cell development. We studied the role of IL-7 in early T-cell development using a model of transgenic (Tg) mice with the murine IL-7 gene under control of the lck proximal promoter. At high IL-7 over-expression (x39 fold increase at day 1 in total thymic tissue), we observed a disruption of TCRαβ development along with increased B cell development in the thymus (7- to 13-fold increase) (El Kassar, Blood, 2004). In order to further explore abnormal T and B cell thymic development in these mice, we first confirmed that they both arise in parallel and were non-cell autonomous, by in vivo injection of neutralizing anti-IL-7 MAb and mixed bone marrow chimera experiments. Using a six color flow cytometry analysis, we found a dramatic decrease of the early thymocyte progenitors (ETPs, lin−CD44+CD25−c-kithiIL-7R−/lo) in the adult Tg mice (x4.7 fold decrease). Lin−CD44+CD25−c-kit+ thymocytes were sorted and cultured on OP9 and OP9 delta-like1 (OP9-DL1) stromal cells (kindly provided by Pr Zuniga Pflucker). At day 14, we observed an important decrease of T cell development (54% vs. 1% of DP cells) and an increase of NK cells (x5 fold increase) in the Tg-derived DN1 cell culture. DN2 (Lin−CD44+CD25−c-kit+) Tg thymocytes showed the same, but less dramatic abnormalities. While DN1 progenitors developed effectively into B220+CD19+ cells on OP9 stromal cells, no B cell development was observed on OP-DL stromal cells from DN1-Tg derived progenitors or by addition of increasingly high doses of IL-7 (x10, x40, x160) to normal B6-derived DN1 progenitors. Instead, a block of T-cell development was observed with increased IL-7. We hypothesized a down regulation of Notch signaling by IL-7 over-expression and analyzed by FACS Notch expression in the DN thymocytes. By staining the intra-cellular part of Notch cleaved after Notch 1/Notch ligand activation, Tg-derived DN2 cells showed decreased Notch signaling. More importantly, HES expression was decreased in the DN2, DN3 and DN4 fractions by semi-quantitative PCR. Sorted Pro/Pre B cells from Tg thymi showed TCR Dβ1-Jβ1 rearrangement indicating their T specific origin, in opposition to Pro/Pre B cells sorted from the bone marrow of the same mice. We suggest that more than one immature progenitor seeds the thymus from the bone marrow. While ETPs had T and NK proliferative capacity, another thymic progenitor with B potential may be responsible for thymic B cell development in normal and IL-7 Tg mice. Finally, IL-7 over-expression may induce a decreased Notch signaling in thymic progenitors, inducing a switch of T vs. B lineage development.

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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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GSK3β Inhibition Promotes T Cell Reconstitution and Preserves Naive T Cell Phenotype in Bone Marrow Reconstituted Mice

Blood ◽

10.1182/blood.v120.21.1890.1890 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1890-1890

Author(s):

Shen Sylvie ◽

Ning Xu ◽

Guy Klamer ◽

Tracey O'Brien ◽

Alla Dolnikov

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

B Cell ◽

Progenitor Cells ◽

Cell Development ◽

Memory T Cell ◽

T Cell Reconstitution ◽

Human T Cells

Abstract Abstract 1890 Stem cell transplantation has become a widely used procedure in the treatment of haematological and non-haematological clinical disorders. Unfortunately, cure is often hampered by relapse of the underlying disease, graft-versus-host disease (GVHD), or severe opportunistic infections. Slow T-cell reconstitution is regarded as primarily responsible for infections, GVHD, and relapse, therefore, enhancing immune reconstitution is important. Glycogen synthase kinase-3β (GSK3β) was recently identified as an important regulator of T cell function acting through the Wingless (Wnt) pathway. The effect of in vivo administration of GSK3β inhibitor 6-Bromoindirubin 3'-oxime (BIO) was examined in a humanised mouse model. Mice transplanted with highly purified cord blood CD34+ stem cells demonstrated efficient multilineage reconstitution including myeloid, B and T cell lymphoid compartments. The presence of human CD4 and CD8 single positive human T cells was abundant in peripheral blood (PB). De novo generated T cells exhibited low CD31 expression in the naïve CD4+ T cells suggesting prolonged post-thymic proliferative history of these cells. This is not completely surprising considering that graft recipient mice are characterized by impaired thymopoiesis following irradiation. Human T cells at various stages of differentiation including late effector T cells were recorded by detecting the expression of CD62L, CD45RA and CD45RO. Late memory T cell skewing was observed in PB and spleen of graft recipient mice. Activation of human T cells expressing CD25 was registered in the spleen, however, the recipients of the graft did not exhibit any signs of GVHD suggesting normal positive and negative selection occurring in the thymus during human T cell development in this mouse model. Human T cells isolated from the spleen of transplanted mice exhibited strong proliferative responses to mitogenic and allogeneic stimulation, however, they did not demonstrated any CTL activity tested following vaccination with human leukaemia U937 cells. In vivo administration of GSK3β inhibitor promoted T cell reconstitution in mice transplanted with human CD34+haematopoietic progenitor cells. Per cell output of T cells from CD34+ and CD34+CD38- primitive bone marrow (BM) progenitor cells was higher in BIO-treated mice while CD19+ B cell output was reduced suggesting T-cell developmental skewing in expense of B cell development. In vitro analysis of CD34+ progenitor cells co-cultured with bone marrow stroma MS5 cells has demonstrated inhibited B-cell development following BIO-treatment. CD31 expression in naïve CD4+ T cells was not up-regulated by BIO suggesting that GSK3β inhibition does not act to increase thymic output of T cells. GSK3β inhibition also increased naïve/memory T cell ratio in reconstituted mice. A similar effect was observed in mice transplanted with mature cord blood (CB)-derived T cells. delayed naive to memory T cell transition is likely related to decreased T cell activation and proliferation demonstrated ex vivo. BIO reduced IFNγ and TNFα production in human T cells. BIO increased naïve T cell production in mitogenically stimulated T cells and in mixed lymphocyte cultures. GSK3β inhibition preserved naïve T cell gene expression profile and suppressed the expression of genes activated during effector T cell differentiation. BIO actrivated β-catenin sigbnaling and up-regulated IL7Rα expression. IL7 signalling prevents activated T cell death following effector differentiation suggesting that the mechanism triggered by BIO may act through the inhibition of activated T cell death. In addition, BIO down-regulated negative regulator of IL7Rα SOCS1 as well as CTLA4 and PDCD1 both up-regulated during effector differentiation. Thus clinically GSK3β inhibition acting to prevent late memory T cell skewing and preserving a subset of naïve T cells may increase T cell diversity and improve T cell responses in the recipients of CB transplant particularly in adult patients with impaired thymic function. Disclosures: No relevant conflicts of interest to declare.

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T and B cell development in pituitary deficient insulin-like growth factor-II transgenic dwarf mice

Journal of Endocrinology ◽

10.1677/joe.0.1550165 ◽

1997 ◽

Vol 155 (1) ◽

pp. 165-170 ◽

Cited By ~ 7

Author(s):

R Kooijman ◽

SC van Buul-Offers ◽

LE Scholtens ◽

RG Reijnen-Gresnigt ◽

BJ Zegers

Keyword(s):

Bone Marrow ◽

T Cell ◽

B Cells ◽

B Cell ◽

Bone Marrow Cells ◽

T Cell Development ◽

Cell Development ◽

B Cell Development ◽

Igf I ◽

Dwarf Mice

Treatment of mice with IGF-I stimulates T and B cell development. We showed that overexpression of IGF-II in transgenic FVB/N mice only stimulated T cell development. In the present study, we further addressed the in vivo effects of IGF-II in the absence of IGF-I to get more insight into the potential abilities of IGF-II to influence T and B cell development. To this end, we studied lymphocyte development in IGF-II transgenic Snell dwarf mice that are prolactin, GH and thyroid-stimulating hormone deficient and as a consequence show low serum IGF-I levels. We showed that T cell development was stimulated to the same extent as in IGF-II transgenic FVB/N mice. Furthermore, IGF-II increased the number of nucleated bone marrow cells and the number of immature B cells without having an effect on the number of mature B cells in spleen and bone marrow. Our data show that IGF-II has preferential effects on T cell development compared with B development, and that these preferential effects also occur in the absence of measurable IGF-I levels.

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Physioxia enhances T-cell development ex vivo from human hematopoietic stem and progenitor cells

Stem Cells ◽

10.1002/stem.3259 ◽

2020 ◽

Author(s):

Dong-Yeop Shin ◽

Xinxin Huang ◽

Chang-Hyun Gil ◽

Arafat Aljoufi ◽

James Ropa ◽

...

Keyword(s):

T Cell ◽

Progenitor Cells ◽

Ex Vivo ◽

T Cell Development ◽

Cell Development ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

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Inflammatory Cytokines Regulate T-Cell Development from Blood Progenitor Cells in a Stage and Dose-Specifc Manner

10.1101/2021.01.18.427186 ◽

2021 ◽

Author(s):

John M. Edgar ◽

Peter W. Zandstra

Keyword(s):

T Cells ◽

T Cell ◽

Progenitor Cells ◽

T Cell Development ◽

Cell Development ◽

Cell Maturation ◽

Hematopoietic Stem ◽

Lineage Differentiation ◽

T Cell Maturation

ABSTRACTT-cell development from hematopoietic stem and progenitor cells (HSPCs) is tightly regulated through Notch pathway activation by the Notch ligands Delta-like (DL) 1 and 4 and Jagged-2. Other molecules, such as stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L) and interleukin (IL)-7, play a supportive role in regulating the survival, differentiation, and proliferation of developing progenitor (pro)T-cells. Numerous other signaling molecules are known to instruct T-lineage development in vivo, but little work has been done to optimize their use for T-cell production in vitro. Using a defined T-lineage differentiation assay consisting of plates coated with the Notch ligand DL4 and adhesion molecule VCAM-1, we performed a cytokine screen that identified IL-3 and tumor necrosis factor α (TNFα) as enhancers of proT-cell differentiation and expansion. Mechanistically, we found that TNFα induced T-lineage differentiation through the positive regulation of T-lineage genes GATA3, TCF7, and BCL11b. TNFα also synergized with IL-3 to induce proliferation by upregulating the expression of the IL-3 receptor on CD34+ HSPCs, yielding 753.2 (532.4-1026.9; 5-95 percentile)-fold expansion of total cells after 14 days compared to 8.9 (4.3-21.5)-fold expansion in conditions without IL-3 and TNFα. We then optimized cytokine concentrations for T-cell maturation. Focusing on T-cell maturation, we used quantitative models to optimize dynamically changing cytokine requirements and used these to construct a three-stage assay for generating CD3+CD4+CD8+ and CD3+CD4−CD8+ T-cells. Our work provides new insight into T-cell development and a robust in vitro assay for generating T-cells to enable clinical therapies for treating cancer and immune disorders.

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The Ataxia Telangiectasia Nude Mouse with No Risk of Thymoma: A Model to Investigate Tumour Development of B Cell and Myeloid Origin Associated with ATM Loss

Blood ◽

10.1182/blood.v120.21.1320.1320 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1320-1320

Author(s):

Tatjana Stankovic ◽

Phil Byrd ◽

Tegan Francis ◽

Teresa Marafioti ◽

Zbigniew Rudzki ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

Mouse Model ◽

B Cell ◽

Ataxia Telangiectasia ◽

Cell Lymphoma ◽

T Cell Development ◽

Cell Development ◽

Double Knockout ◽

Tumour Development

Abstract Abstract 1320 Constitutional inactivation of Ataxia Telangiectasia Mutated (ATM), a principal double strand breaks (DSB) response gene leads to ataxia telangiectasia syndrome characterised by a high risk of development of a wide range of leukaemias and lymphomas. Unfortunately, the existing Atm null mouse model provides the opportunity to study only a single tumour type as affected animals die of thymic T cell lymphoma by age of 3 months. In order to produce an Atm−/− mouse model that recapitulates the human disease, and exhibits a wider tumour range, we bred Atm−/−nu−/− double knockout mice. This led to the absence of thymomas due to defective T cell development caused by the loss of Foxn1 alleles in nude mice. We generated 29 Atm−/−nu−/− double knockout animals exhibiting a severe reduction in T cell compartment. In comparison with single Atm−/− mice or Atm−/−nu+/−displayed a greater than doubling of the median survival time (212 days vs 90 days), delayed onset and a decreased frequency of tumour development. Importantly, these animals also showed a change in tumour phenotype. Of 29 mice, 12 developed lymphomas between 2 and 10 months of age. Importantly, in contrast to the exclusively observed T-cell lymphomas in Atm−/− animals, Atm−/−nu−/−lymphoma appeared to be of B-cell origin. Closer morphological evaluation of tumour nodular proliferation was suggestive of germinal centre derived cells. We performed immunocytochemistry and found tumour cells to be positive for B cell markers B220 and Pax5, GC markers Bcl-6 and AI, weakly positive for IRF4 and negative for T cell marker CD3. Absence of cyclin D1 staining argued against a mantle cell lymphoma phenotype, whereas absence of staining for additional GC markers LMO2, HGAL, GCET1, CD10 and post GC marker Blimp-1 suggested that clonal proliferation has originated from GC cells that had not completed the GC reaction. In addition, the B cell tumour phenotype was also confirmed by FACS analysis that revealed positivity for surface marker B220 and variable positivity for IgM. Interestingly, we observed that tumour nodular formations were surrounded by rare cells that exhibited helper cell phenotype. This was consistent with incomplete abolition of T cell development in Atm−/−nu−/− animals, that might play a role in B lymphoma development. Tumours of non-haematological origin were rarely seen – one Atm−/−nu−/−mouse without a lymphoma developed an aggressive spindle cell sarcoma, topographically associated with the surface of the brain, most likely of meningeal origin. The most intriguing observation however, was the finding of an increased extramedullary haematopoiesis in the Atm−/−nu−/− mice. We have observed the presence of megakaryocytic proliferation as well as their extensive clustering, both in the bone marrow and spleen reminiscent of human myeloproliferative disease (MPD) in 10/22 analysed Atm−/−nu−/− animals. These changes were occasionally accompanied by small foci of haematopoiesis within the liver. The increased number of megakaryocytes in Atm−/−nu−/− animals was not accompanied by bone marrow fibrosis, bone remodelling, increase in blasts or other abnormalities, frequently seen in MPD. Finally, as another indicator of increased myelopoiesis Atm−/−nu−/− mice showed elevated proportions of myeloid progenitors in the spleen. Interestingly, this expansion in the myeloid cell progenitors was not accompanied by an increase in proliferative potential in vitro indicating that this myeloproliferation could be a result of an altered in vivo microenvironment in the Atm−/−nu−/−mice. In conclusion, we have produced an Atm null animal model that recapitulates a further part of the spectrum of tumours observed in humans lacking ATM function and additionally, points to a novel role of ATM in myeloproliferation. This model facilitates elucidation of mechanisms involved in tumorigenesis at different stages of haematopoietic differentiation and in the longer term may assist development of targeted treatments for these aggressive, currently incurable malignancies. Disclosures: No relevant conflicts of interest to declare.

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Thymus-autonomous T cell development in the absence of progenitor import

Journal of Experimental Medicine ◽

10.1084/jem.20120846 ◽

2012 ◽

Vol 209 (8) ◽

pp. 1409-1417 ◽

Cited By ~ 74

Author(s):

Vera C. Martins ◽

Eliana Ruggiero ◽

Susan M. Schlenner ◽

Vikas Madan ◽

Manfred Schmidt ◽

...

Keyword(s):

Bone Marrow ◽

T Cell ◽

T Cell Development ◽

Cell Development ◽

Self Renewal ◽

Hematopoietic Stem ◽

Short Life Span ◽

Receptor Repertoire ◽

T Cell Progenitors ◽

Transplantation Experiments

Thymus function is thought to depend on a steady supply of T cell progenitors from the bone marrow. The notion that the thymus lacks progenitors with self-renewal capacity is based on thymus transplantation experiments in which host-derived thymocytes replaced thymus-resident cells within 4 wk. Thymus grafting into T cell–deficient mice resulted in a wave of T cell export from the thymus, followed by colonization of the thymus by host-derived progenitors, and cessation of T cell development. Compound Rag2−/−γc−/−KitW/Wv mutants lack competitive hematopoietic stem cells (HSCs) and are devoid of T cell progenitors. In this study, using this strain as recipients for wild-type thymus grafts, we noticed thymus-autonomous T cell development lasting several months. However, we found no evidence for export of donor HSCs from thymus to bone marrow. A diverse T cell antigen receptor repertoire in progenitor-deprived thymus grafts implied that many thymocytes were capable of self-renewal. Although the process was most efficient in Rag2−/−γc−/−KitW/Wv hosts, γc-mediated signals alone played a key role in the competition between thymus-resident and bone marrow–derived progenitors. Hence, the turnover of each generation of thymocytes is not only based on short life span but is also driven via expulsion of resident thymocytes by fresh progenitors entering the thymus.

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Notch Repression by LRF Is Necessary for the Maintenance of Adult Hematopoietic Stem Cell Pool.

Blood ◽

10.1182/blood.v116.21.2633.2633 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2633-2633

Author(s):

Sung-UK Lee ◽

Min Li ◽

Manami Maeda ◽

Nagisa Sakurai ◽

Yuichi Ishikawa ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Stem Cell ◽

T Cell ◽

T Cell Development ◽

Cell Development ◽

Self Renewal ◽

Hematopoietic Stem ◽

Deficient Mice

Abstract Abstract 2633 Among the different stem cells, hematopoietic stem cells (HSCs) are one of the best studied and characterized stem cells. To maintain life-long hematopoiesis in the bone marrow (BM), signals governing the balance between self-renewal and differentiation are tightly regulated in HSC compartment. Notch signals are critical regulators of the lymphoid lineage fate, but their role in adult HSC function in the BM is currently under debate. LRF (Leukemia/Lymphoma Related Factor, also known as Zbtb7a/pokemon) is a transcription factor that acts as a proto-oncogene and plays a key role in lymphoid and erythroid development. Previously we reported that the pool of LT-HSCs, CD150+CD48−Flt3−Vcam-1+/−IL7Rα−LSK (Lin−Sca-1+c-Kit+), was significantly reduced, while lymphoid-biased multi-potential progenitors (LMPPs: CD150−CD48+Flt3+Vcam-1+/−IL7Rα−LSK) and common lymphoid progenitors (CLPs: Lin−CD150−CD48+Flt3+Vcam-1−IL7Rα+) were barely detectable in LRF deficient mice. This was due to excessive differentiation of HSC into aberrant CD4/CD8 DP (double positive) T cell development in the BM caused by high Notch activity, implicating LRF role on HSC maintenance. Both gene expression profile (GSEA and DAVID analysis) and Q-PCR results indicated that LRF deficient LT-HSCs had loss of stem cell signature; but gain of T cell signature and up-regulated Notch-target gene, Hes-1, without affecting mRNA expression of Notch (1-4) or related (DLL1, DLL4, Jagged-1) genes. To determine LRF function in HSCs, we performed in vivo and in vitro experiments: 1) 5-FU (5-fluorouracil, the chemotherapy agent) treated LRF deficient mice were not able to compensate for their loss of LT-HSCs; 2) multi-lineage defects were shown in second recipient mice transplanted with 1 million of LRF deficient bone marrow cells in serial bone marrow transplantation assays, suggesting that LRF deficient LT-HSCs had defect in self-renewal and 3) LRF deficient FL-HSCs (CD150+CD48−LSK cells) were cultured on OP9 cells expressing delta-like ligand (DLL1, DLL4 and Jagged1), and enhanced T cell differentiation was only observed when they were co-cultured with delta-expressing OP9 cells. Among the Notch family, these phenotypes were Notch1-dependent. In fact, Notch1flox/floxLRFflox/floxMx1-Cre+ mice demonstrated normal LT-HSC numbers and restored B cell development, and prolonged survival over LRFflox/floxMx1-Cre+ mice in sequential 5-FU treatment in vivo. To explore which Notch-ligand(s) in BM niche is responsible for aberrant T-cell development in LRF deficient mice as well, we treated wild-type and LRFflox/floxMx1-Cre+ with anti-DLL4 antibody twice per week for 3 weeks. DLL4 blockage in LRF deficient mice rescued B cell development and prevented the development of aberrant DP T-cell development in LRF deficient mice. To further elucidate the relationship between LRF and Notch in adult HSC function, we analyzed Notch protein expression levels in HSCs and performed in-depth analysis of HSC/progenitor (HSC, LMPP and CLP) compartments in wild-type and LRF knockout (KO). Interestingly, Notch1 proteins were differentially expressed in LT-HSCs and ~50 % of them were positive for Notch1, while Notch2 was abundantly expressed in LT-HSCs. Notch1 expressing LT-HSCs were in more active cell-cycle (S phase) and absent in LRF conditional knockout mice. It is most likely that Notch1 expressing LT-HSCs were continually differentiating toward T cells in the absence of LRF, as CD4+CD8+ T cells were evident in the BM 10 months after pIpC injection. Taken together, our data strongly indicate that LRF is indispensable for hematopoietic homeostasis by preventing the lymphoid-primed HSCs from Notch/Delta-mediated T-instructive signal in the BM niche. Currently we're investigating the functional significances of Notch1 expressing LT-HSCs in detail. Our studies help us to better understand the underlying mechanism for HSC fate decision (self-renewal v.s. differentiation) in stem cell biology and its therapeutic approach in regenerative medicine. Disclosures: No relevant conflicts of interest to declare.

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