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Department of Clinical Chemistry, Microbiology, and Immunology, University of Ghent, Ghent University Hospital, Ghent, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells, whereas the other T cell subpopulations were
largely unaffected. Interestingly, when injected in vivo in
SCID-nonobese diabetic mice, the transduced cells generated ectopically
human CD4+CD8+ TCR-
cells in the bone
marrow, cells that are normally only present in the thymus, and lacked
B cell differentiation potential. Our results show unequivocally that,
in human, Notch signaling inhibits the monocyte and B cell fate,
promotes the T cell fate, and alters the normal T cell differentiation
pathway compatible with a pretumoral state. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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gene in a subset of human T cell leukemias that gave a first
clue as to the important role of Notch in T cell regulation
(6). These Notch translocations result in the
expression of truncated Notch1 polypeptides that lack most of the
extracellular domain and constitutively activate the Notch pathway. In
addition, induced overexpression of constitutively active
Notch1
(ICN)3 in mouse bone
marrow (BM) stem cells causes T cell leukemia, suggesting a
causative role for Notch1 in T cell oncogenesis (7). A
role for Notch signaling in promoting the commitment of lymphoid
progenitor cells to the T cell lineage has been recently proposed and
reviewed (8, 9, 10). Critical findings in this respect were a
severe block in the further differentiation of the most immature
CD4-CD8- thymocytes after
inducible deletion of Notch1 in murine BM stem cells,
whereas the development of other hematopoietic populations was
apparently undisturbed (5). To elucidate the importance of Notch1 activation on the differentiation of human hematopoietic cells, we transduced cord blood (CB) CD34+ progenitor cells with ICN and evaluated their differentiation toward different hematopoietic lineages by the following approaches. First, in coculture experiments with the murine BM MS-5 cell line (11), differentiation toward B lymphocyte, monocyte, and NK lymphocyte lineages was evaluated by the determination of the expression of CD19, CD14, and CD56, respectively. The determination of the coexpression of CD7 and cyCD3 allowed the evaluation of differentiation toward T/NK precursor cells. Second, the introduction of transduced human CD34+ progenitor cells in SCID-nonobese diabetic (NOD) mouse FD14 thymi followed by fetal thymus organ culture (FTOC) allowed us to estimate the impact of Notch1 on human T lymphocyte differentiation in vitro. Finally, the transduced human CD34+ progenitor cells were i.v. injected in SCID-NOD mice to estimate the impact of Notch1 on human lymphopoiesis in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNA encoding a constitutively active form of Notch consisting of the intracellular domain (ICN) (base pairs 5308–7665; amino acids 1770–2555) (12) was subcloned into the multicloning site of the retroviral vector MSCV-EGFP (kindly provided by N. Carlesso, Massachusetts General Hospital, Charlestown, MA) (13). The puromycin amino transferase gene was cloned in the pBlue(II)KS vector (Stratagene, La Jolla, CA) into the KpnI/ClaI restriction sites. Propagated plasmids were purified with resin columns (Qiagen, Hilden, Germany). The Phoenix-A cell line, which was derived from 293T cells (kindly provided by Dr. P. Achacoso and Dr. G. P. Nolan, Stanford University School of Medicine, Stanford, CA) (14), was cotransfected with the pBlue(II)KS-puro plasmid and either the MSCV-ICN-EGFP or the MSCV-EGFP plasmid (control) using calcium phosphate precipitation (Life Technologies, Paisley, U.K.). After three rounds of selection with IMDM containing puromycine (2 µg/ml), cells were cultured for 48 h in IMDM. Cell culture supernatant from confluent cultures in 175-cm2 tissue culture flasks (Falcon; BD Labware, Franklin Lakes, NJ) was collected 24 h after refreshment of the medium. Pooled supernatants were spun (350 x g for 10 min at room temperature) and aliquots were stored at -70°C until use.
Cells and sorting of cells
Jurkat cells (American Type Culture Collection, Manassas, VA) were cultured in complete IMDM as described previously (15). Child thymi and CB samples were obtained and used following the guidelines of the Medical Ethical Commission of the University Hospital of Ghent.
CD34+ CB cells were purified by positive
selection with CD34 MACS beads (Miltenyi Biotec, Bergisch Gladbach,
Germany) and stained with CD34-allophycocyanin, CD8-FITC, CD4-FITC,
CD3-FITC, CD19-FITC, and CD7-FITC (all mAb from BD Immunocytometry
Systems, Mountain View, CA). Subsequently,
CD34+Lin- cells were
sorted for by flow cytometry (FACSVantage; BD Immunocytometry Systems).
CD34+ thymocytes were purified by positive
selection with MACS beads (Miltenyi Biotec) and sorted for
CD1-CD4-CD8-CD3-CD34+
progenitor cells by flow cytometry. Purity of the cells was always at
least 98%.
Retroviral transduction
Sorted CD34+Lin- CB cells were resuspended in complete IMDM supplemented with 100 ng/ml recombinant human c-kit ligand (stem cell factor (SCF)), 100 ng/ml flt3/flk-2 ligand (FL), and 20 ng/ml thrombopoietin (TPO) (all cytokines from R&D Systems, Abingdon, U.K.) and cultured in 96-well round-bottom plates (Falcon) for 48 h. Sorted CD34+ thymocytes were cultured in complete IMDM supplemented with 10 ng/ml SCF and 10 ng/ml IL-7 for 24 h and then cells were put in RetroNectin (Takara Biomedicals, Otsu Shiga, Japan)-coated 96-well flat-bottom plates (Falcon) and cultured with the same volume (100 µl) of retroviral supernatants, supplemented with cytokines (to keep the final cytokine concentrations unchanged). After 24 h, cells were harvested to determine transduction efficiency by flow cytometric analysis of enhanced green fluorescent protein (EGFP) expression and were used in subsequent assays. ICN overexpression was also determined by immunoblotting and flow cytometric analysis of intracellular ICN labeled with a mouse mAb against the cdc10-NCR region of mNotch1 (a kind gift from L. A. Milner, Fred Hutchinson Cancer Research Institute, Seattle, WA) (16, 17), revealed with second-step PE-labeled anti-mouse Ig Ab.
Immunoblotting
Cell lysates were run on 4–12% Bis-Tris polyacrylamide gels (Nupage; Invitrogen, Carlsbad, CA) in 2-(N-morpolino) ethane sulfonic acid buffer in reducing conditions and proteins were blotted on polyvinylidene fluoride membranes (Invitrogen). Blots were stained with a mAb against ICN (L. A. Milner) and anti-mouse Ig alkaline phosphatase conjugate (Santa Cruz Biotechnology, Santa Cruz, CA).
Assessment of human T cell potential in FTOC
Isolation of murine embryonic thymic lobes, incubation with human cells using the hanging drop procedure, and organotypic cultures were performed as described previously (18). NOD-LtSz-scid/scid (NOD-SCID) mice, originally purchased from The Jackson Laboratory (Bar Harbor, ME), were obtained from our own specific pathogen-free breeding facility. NOD-SCID mice were treated according the guidelines of the Laboratory Animal Ethical Commission of the University Hospital of Ghent.
After 3–4 wk of FTOC, cells were harvested. After blocking with
anti-mouse FcRII/III (clone 2.4.G2; a kind gift of Dr. J.
Unkeless, Mount Sinai School of Medicine, New York, NY) to avoid
aspecific staining of the murine cells, cells were stained with
anti-mouse CD45-CyChrome (BD PharMingen, San Diego, CA) in
combination with one or more of the following anti-human Abs:
CD4-allophycocyanin, TCR--PE, CD3-allophycocyanin,
CD34-allophycocyanin (all from BD Immunocytometry Systems), CD8-PE,
and TCR--PE (Coulter, Miami, FL). Human viable cells were gated
by exclusion of propidium iodide and anti-mouse
CD45-positive cells, and were examined for the expression of the Ags by
fluorescence analysis performed on a FACSCalibur using CellQuest Pro
software (BD Immunocytometry Systems).
Assessment of lymphoid and myeloid differentiation
After transduction of CD34+Lin- purified CB cells, 1000 cells were incubated in 24-well plates precoated with confluent murine marrow-derived MS-5 cells (11) (kindly provided by L. Coulombel, Institut Gustave Roussy, Villejuif, France) in IMDM supplemented with 5% human serum and 5% FCS. For assessment of CD34 maintenance and myeloid differentiation, the following mix of six human recombinant cytokines was used: 50 ng/ml SCF, 50 ng/ml FL, 20 ng/ml IL-7, 10 ng/ml IL-15, 5 ng/ml IL-2, and 10 ng/ml TPO. For assessment of B lymphoid differentiation, 50 ng/ml SCF and 20 ng IL-7 was used. For the assessment of NK cells, 50 ng/ml SCF, 5 ng/ml IL-2, and 10 ng/ml IL-15 were used (all reagents from R&D Systems).
After 2 wk of culture cells were collected and counted under the microscope, and their phenotype was assessed by flow cytometry after labeling with the following mAbs: CD34-allophycocyanin, CD19-PE, CD14-PE, CD56-allophycocyanin, HLA-DR-allophycocyanin, CD7-PE, CD4-PE or allophycocyanin, CD3-PE or allophycocyanin. For intracellular staining, cells were fixed and permeabilized using Fix and Perm (Caltag Laboratories, San Francisco, CA) according to the guidelines of the manufacturer.
NOD-SCID repopulation assay
NOD-SCID mice aged 8–10 wk were given a sublethal dose of
whole-body irradiation (350 cGy, 12–15 cGy/min) with a cobalt
radiation source and injected i.p. with 200 µg of TM-1, a rat mAb
functionally blocking the mouse IL-2R chain (19)
(kindly provided by Dr. T. Tanaka, Tokyo Metropolitan Institute of
Medical Science, Tokyo, Japan).
Within 24–48 h after irradiation, the mice were injected i.v. with 75,000–100,000 human transduced CD34+ CB cells. Eight to 10 wk after injection, mice were killed and peripheral blood, thymus, spleen, and femora were used for analysis. Cell suspensions from these organs were put on a 70-µm cell strainer (Falcon). RBCs were lysed with hypotonic lysing buffer. Cells were counted, cell viability was checked with eosin (>85%), and, after blocking the FcR of mouse cells, cells were labeled with mAbs and analyzed by flow cytometry.
Statistical analysis
A paired Student t test was used and differences were considered to be significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fig. 1
A shows a diagram
of the complete Notch protein, of the active intracellular part of the
Notch protein that is formed after protein processing upon
ligation of the receptor, and of the constructs that were used in the
study. The efficiency of MSCV-ICN-EGFP or control MSCV-EGFP retroviral
transduction was assessed by Western blot (Fig. 1B) and flow
cytometric analysis (Fig. 1C). Independent experiments
showed a mean infection efficiency of 20 and 27% with MSCV-ICN-EGFP or
control MSCV-EGFP retroviruses, respectively, for Jurkat cells.
Infection was more variable in CD34+ cells,
ranging from 0.7 to 12.7% of cells (average 5.3 ± 3.2,
n = 8) expressing EGFP for MSCV-ICN-EGFP and ranging
from 2.7 to 24.1% (average 14.8 ± 6.6) for control
MSCV-EGFP retrovirus, after transduction during 24 h.
After another 24 h, the proportion of cells stably expressing EGFP
increased to 10.6 ± 6.2 and 23.2 ± 9.9% for the cells
transduced with MSCV-ICN-EGFP and control MSCV-EGFP retrovirus,
respectively.
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B), whereas control Jurkat
cells and control vector-transduced CB progenitor cells were negative.
In a representative experiment, wherein the proportion of
ICN-transduced cells increased to 30% in FTOC after 30 days of
culture, cells were stained intracellularly with anti-Notch mAb. It
is clear that Notch could be detected in ICN-transduced CB cells (Fig. 1C, upper panel), whereas control
vector-transduced cells were negative (Fig. 1C, lower
panel). Moreover, there was a good correlation between the
intensity of ICN staining and EGFP fluorescence in the transduced CB
cells, showing that EGFP intensity is a parameter for intensity of
Notch production (Fig. 1C, upper panel). Isotype
control Abs did not stain transduced or untransduced cells
aspecifically (data not shown). Constitutively active Notch does not support CD34 expansion and inhibits myeloid differentiation
It has been shown previously that murine BM-derived MS-5 cells
support B cell differentiation from human CD34+
CB cells (20). Depending on the mix of cytokines added to
the culture, either the growth of stem cells with a CD34 phenotype is
supported or the differentiation toward myelocytes or lymphocytes is
promoted. We used a combination of following cytokines: SCF plus FL
plus IL-2 plus IL-7 plus IL-15 plus TPO. These conditions allow the
growth of stem cells and the differentiation toward monocytes. In a
series of four to six separate experiments these conditions triggered
active proliferation, and after 2 wk of culture the number of the human
cells increased 
1000-fold for the nontransduced or
control-transduced CD34+ CB cells, whereas the
number of ICN-transduced cells increased 2650-fold (Table I). However, ICN-transduced
CD34+ CB cells had a significant lower frequency
of CD34+ cells (10-fold) as compared with
nontransduced cells in the same culture, or as compared with control
vector-transduced progenitor cells (Table II).
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5-fold) as compared with nontransduced cells or compared with
progenitor cells, whether they were transduced with the control vector
or not (Table II and Fig. 2A).
Taken together, it is clear that not only the frequencies but also the
absolute numbers of CD34+ and
CD14+ cells were decreased in the ICN-transduced
cells.
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Interestingly, when CD34+ cells were
cocultured on MS-5 stromal cells with SCF and IL-7, known to promote B
lymphocyte differentiation, ICN-transduced cells almost did not
generate any CD19+ B cells. The
nontransduced cells in the same culture had a significant number of
CD19+ cells (Table II and Fig. 2B).
This number was comparable to the number of CD19+
B lymphocytes in both fractions, nontransduced or transduced,
observed in cultures where progenitor cells were transduced with the
control vector.
Both control vector nontransduced and transduced
CD34+ cells yielded a very low number of cells
(<2%) with a CD7+cyCD3+
phenotype. In contrast, the frequency of
CD7+cyCD3+ cells was
significantly more abundant in ICN-transduced cells (>80%) (Table II
and Fig. 2B). This phenotype is analogous to the phenotype
of early T/NK precursor cells that arise when
CD34+ progenitor cells are cultured in FTOC
(21). These culture conditions resulted in a much higher
increase (1390-fold) in the number of ICN-transduced
(EGFP+) cells as compared with the
control-transduced cells and the nontransduced cells from both control
or ICN-transduced cells, which increased from 176- to 202-fold (Table I). Taken together, it is clear that the 30-fold lower frequency of
CD19+ B cells and the 80-fold higher frequency of
early T/NK precursors also reflect changes in the absolute numbers of
those cells, and that the increase in cell number is due to the
expansion of T/NK precursors in the ICN-transduced cell population.
To further demonstrate that the CD7+ cells that
were obtained from the ICN-transduced CB cells after culture on MS-5
cells with IL-7 and SCF were a bipotent T/NK population we did the
following experiment. First, CD7+ cells were
generated by culture of ICN-transduced CB cells on MS-5 cells with SCF
and IL-7 for 2 wk, which we will refer to as primary culture. Then the
EGFP+CD7+CD56-
cells obtained in the primary culture were sorted by flow cytometry. As
shown in Fig. 3 we obtained a population
that was >99% pure. Intracellular staining for cyCD3 on those sorted
cells showed that >90% were positive (data not shown). This
EGFP+CD7+CD56-
population was put in a secondary coculture on MS-5 cells with SCF,
IL-2, and IL-15. After 1 wk we were able to obtain human cells that
were 85% CD56+, indicating the NK potential of
the
EGFP+CD7+CD56-
population (Fig. 3). In addition,
EGFP+CD7+CD56-
cells from the primary culture were seeded in D15 fetal thymus by
hanging drop, and after 16 days of FTOC we obtained human cells that
were >75% CD3+; almost all the
CD3+ cells were TCR-+
cells (Fig. 3). This shows the T cell potential of the
EGFP+CD7+CD56-
population.
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Conversely, in the presence of SCF/IL-2/IL-15,
CD56+ NK cells were significantly more abundant
in ICN-transduced cells as compared with the moderate amounts in
nontransduced cells. A comparable lower number of
CD56+ NK cells was obtained in both populations,
transduced or nontransduced, when the control vector was used (Table II
and Fig. 2C). The increase in absolute number of cells in
these culture conditions was 120-fold for all cell fractions,
control or ICN-transduced cells and nontransduced cells (Table I).
Taken together, it is clear that not only the frequencies but also the
absolute numbers of CD56+ cells were increased in
the ICN-transduced cells.
Constitutively active Notch favors differentiation of
CD34+ cells toward TCR-+ thymocytes in
FTOC
To assess T cell differentiation in vitro, CB
CD34+ progenitor cells were transferred to
NOD-SCID FD14 thymic lobes and cultured in FTOC. In this
microenvironment, human T cells develop (15, 18). We found
a significantly higher amount of TCR- cells in ICN-transduced
thymocytes than in the nontransduced cells, which were comparable to
control vector-transduced or nontransduced cells (Table III and Fig. 4, A and B).
Accordingly, the number of CD3+ cells was also
significantly increased as compared with the cells that were
nontransduced or to the cells of the FTOC seeded with CB cells
transduced with control vector, irrespective of whether those cells
were transduced or not (Table III and Fig. 4, A and
B). The other populations, including
TCR-+ and
CD4+CD8+ double
positive (DP) cells, were not significantly different, although the
frequency of CD34+ cells was slightly diminished
in the ICN-transduced cell population (data not shown). However, the
large CB-to-CB variation in the number of TCR- and
CD4+CD8+ DP cells obtained
in the 11 FTOC performed did not allow a good estimation of the
influence of ICN on those subsets. Interestingly, in cultures where a
high number of TCR- and DP cells were generated, ICN-transduced
CD34+ cells generated a lower number of
TCR- DP cells than the nontransduced cells and the control
vector-transduced CD34 cells (data not shown).
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DP cells in FTOC. For this reason, we
looked at the influence of ICN overexpression on the fate of those
cells in FTOC. This approach confirmed the higher frequency of
TCR- cells in the ICN-transduced cells as compared with the
nontransduced cells, or to the control vector-transduced or
nontransduced cells. In addition, the frequency of TCR- cells was
significantly lower in the ICN-transduced cells as compared with the
nontransduced cells or to the control vector-transduced or
nontransduced cells (Table III and Fig. 4, C and
D). The same goes for the frequency of DP cells, as these
cells were significantly lower in the ICN-transduced cells as compared
with the nontransduced cells, or to the control vector-transduced or
nontransduced cells (Table III). The frequency of
CD3+ cells did not differ significantly. Constitutively active Notch promotes ectopic differentiation of CD34+ progenitor cells toward CD4+CD8+ DP T lymphocytes in BM in vivo
To assess T cell differentiation in vivo, ICN-transduced
CD34+ progenitor cells from CB were injected i.v.
in 8- to 10-wk-old NOD-SCID mice that were sublethally irradiated and
treated with TM-1 mAb 1 or 2 days before injection. We have shown
previously that CD34+ CB cells give rise to
myeloid cells and to B and T lymphoid cells in these mice (22, 23). When examined 2 mo after injection, in the BM of four of
five injected mice we found cells that strongly expressed EGFP, and
thus ICN. Those cells coexpressed CD4 and CD8, a phenotype normally
found only in the thymus. All these cells were
CD1+; the majority were
CD3+TCR-+. We noticed
a small population of
CD4+CD8- cells (3%) that
were partly CD1+ (and therefore most likely
represent immature CD4 precursor cells) and partly
CD1-, a phenotype compatible with mature CD4
single positive (SP) cells. Because we could not find
CD14+ cells, we conclude that monocytes were not
present in this CD4+ cell fraction. This is in
contrast with the untransduced cells, because
CD4+ cells in the EGFP-
cell fraction were
CD1-CD3-CD8-
and partly CD14+ and can therefore be considered
monocytes (data not shown).
CD19+ B lymphocytes were nearly absent within the
EGFP+ population. In addition, in the
nontransduced EGFP- cell fraction we found no DP
cells and a high number of CD19+ B lymphocytes
(Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because an activated form of Notch1 is able to inhibit G-CSF-induced granulocytic differentiation of 32D myeloid progenitors (17), it is indicated that Notch activity plays a negative role in mediating cell fate decisions in the myeloid lineage. This is compatible with the lower number (absolute and frequency) of CD14 cells that we obtained after overexpression of ICN in human CD34+ progenitor cells. The inhibition of monocytic and granulocytic differentiation reported by Carlesso et al. (13) is in line with our observations that the number of generated CD14+ cells is diminished upon ICN transduction. Preferential transduction of a cell population with lower differentiation capacity toward monocytes is unlikely, because both EGFP+ and EGFP- cell fractions after transduction with a control vector produced CD14+ cells in equal frequencies and absolute numbers. It is possible that, upon ICN transduction, not only was the differentiation of monocytes impaired, but their survival was impaired as well. Recently it has been shown that, in cell suspension cultures wherein Notch was activated in isolated human monocytes by immobilized Delta, apoptosis of monocytes was induced and differentiation into macrophages was impaired (27).
The ability of ICN to preferentially inhibit the B cell fate in
lymphoid differentiation conditions was evident by comparing the
proportion of cells in the
EGFP+CD19+ population. It
is strikingly evident that almost no CD19+ cells
were EGFP+ in the ICN-transduced cultures. The
percentage of CD19+ cells was 12% in the
EGFP- and <1% in the
EGFP+ fraction. These results clearly indicate
that the B cell fate is negatively influenced by ICN. The negative role
of Notch on B cell development is well documented in the murine system.
In mice, retrovirally transduced BM cells that overexpress ICN do not
develop into B cells in lethally irradiated recipients
(28). Moreover, immature T cells with a
CD4+CD8+ phenotype
accumulate in the BM of these chimeric mice. Our observations provide
the first direct evidence that expression of an active form of Notch1
inhibits human B lymphopoiesis. Here we show that, in culture
conditions that allow B cells to develop, retrovirally transduced
CD34+ progenitor cells that overexpress ICN
scarcely develop into B lymphocytes. Also, when injected i.v. in
immunodeficient mice, ICN-transduced cells do not develop into B
lymphocytes. In contrast, those transduced cells develop ectopically in
human
CD4+CD8+CD3+TCR-+
T cells in the BM. Therefore, we can conclude that also in humans these
gain-of-function experiments clearly show that Notch inhibits the B
cell fate and promotes the T cell fate.
There is ample evidence from our experiments that the T cell fate is favored in ICN-transduced cells. After 2 wk of MS-5 coculture assays, ICN-transduced cells developed in CD7+cyCD3+ cells, a phenotype that is considered to identify pre-T/NK cells (21). After 3 wk CD7+cyCD3+CD4+ cells were found, a phenotype that is considered to identify T precursor cells (21). In addition, when those CD7+ cells obtained after 2 wk of MS-5 coculture assay were purified by flow cytometry, those cells generated NK cells in secondary MS-5 coculture assays with IL-2, IL-15, and SCF after 1 wk, whereas T cells were generated in FTOC after 16 days. We can conclude that these experiments confirm that ICN favors the generation of progenitor cells with T/NK potential.
When ICN-transduced CD34+ precursor cells were
cultured in FTOC, differentiation toward
CD7+cyCD3+CD4+
cells (data not shown) and toward TCR- and TCR- cells was
seen. Whereas generation of TCR- T cells was unaffected,
a significant increase in the number of TCR- T cells was
observed. This is in contrast with studies in mice wherein reduced,
rather than increased, activity of Notch favors the T cell fate
over the T cell fate (29). In FTOC, starting with
CD34+ cells from human thymus, one obtains a
faster kinetic of T cell development and a higher yield of TCR-,
DP, CD4 SP, and CD8 SP cells, because thymic
CD34+ cells are already a further step of
differentiation toward the T cell lineage than
CD34+ cells from CB. Using ICN-transduced thymic
CD34+ cells we observed a significant increase in
the number of TCR- T cells, whereas no changes in CD4 SP and CD8
SP cells (data not shown) and even a decrease in
TCR-+ T cells and DP cells were observed.
This is in contrast with the observation that expression of an
activated form of Notch-1 in murine thymocytes under the lck promoter
leads to an increase in CD8+ SP
TCR-+ cells and a parallel decrease in
CD4+ T cells (30). These
discrepancies can be related to the dose effect of Notch or to species
differences. It is clear that complete deletion of the
Notch1 gene in mice by cre/lox recombination
leads to severe thymic atropy due to a block of the expansion and
differentiation of immature
CD4-CD8- T cell
precursors (5). In studies where a reduction in
Notch gene expression was examined, it was shown that T
cells from mice that are heterozygous for Notch1
(N+/-) adopt preferentially the TCR- and
CD4 TCR- fate as compared with the T cell fate of wild-type mice
that are homozygous for Notch1 (N+/+)
(29). The same conclusion could be drawn from studies
wherein the use of -secretase inhibitors leads to Notch deficiency
by inhibition of the formation of the intracellular domain of Notch,
which is essential for signal transduction in vivo (16).
In these studies, a high dose of -secretase inhibitors administered
to murine FTOC leads to a complete block of T cell development at an
early stage of differentiation, resulting in an increase in
CD4-CD8- double
negative thymocytes and a decrease in
CD4+CD8+ DP thymocytes. A
lower dose of -secretase inhibitors leads to changes at later steps
of differentiation, resulting in a higher number of TCR- and CD4
SP thymocytes (31). Thus, complete deficiency of Notch
leads to an early block of T cell development, whereas partial
deficiency of Notch allows further T cell development, but at the
expense of TCR- and CD8 SP development. Based on these findings
obtained in murine models, one would expect that overexpression of ICN
in human CD34+ progenitor cells should also favor
these cells to adopt the TCR- and CD8 SP cell fate. However, we
consistently found in the mixed human-mouse FTOC that human cells
adopted more frequently the TCR- fate and that the frequency of
CD8 SP cells was not increased. This contradiction could be ascribed to
the fact that overexpression ICN may be detrimental, that the mixed
human-murine FTOC is not optimal to allow the effect on TCR-
development to be seen, or that species differences are important.
Our data are remarkably in line with data recently obtained by Jaleco
et al. (32), using human CD34+
progenitor cells in a different approach. Instead of introducing the
active form of Notch in hematopoietic stem cells, these
authors transduced a ligand for Notch, Jagged-1, or
Delta-1 in the S17 murine stromal cell line. They were able
to show that Delta-1-transduced S17 stromal cells induced
CD34+ CB cells to adopt the T/NK developmental
pathway and even to allow the differentiation of a few DP
CD4+CD8+ cells, whereas the
B cell pathway was inhibited. In contrast,
Jagged-1-transduced S17 stromal cells did not have an effect
on the differentiation of CD34+ CB cells. It is
clear that our results not only confirm but also extend those
experimental data in the following ways. First, the specificity of the
ligands for the Notch receptors is largely unknown (25).
Theoretically each of the four known Notch receptors may be triggered
in the experiments of Jaleco et al. (32). Our approach is
a direct proof that activation of Notch1 alone suffices to impose the
CD34+ progenitor cells to adopt a T/NK pathway
and to inhibit their differentiation potential toward B cells and
myeloid cells. Furthermore, it is obvious that the presence of the few
DP cells with a CD4/CD8 DP phenotype that were seen in coculture
with the S17 Delta-transduced stromal cells is suggestive
for a transition toward a further step of differentiation of the T
cell lineage; nevertheless, these
CD4+CD8+ DP cell numbers
are small (32). In our hands, in vivo experiments have
shown a high number of CD4/CD8 DP TCR- cells in the BM of
reconstituted mice. These cells are phenotypically similar to
CD4+CD8+ DP cells normally
found in the thymus. Importantly, we found that there was a
dose-dependent effect: when Notch1 ICN was expressed at
lower levels as estimated by the lower expression of the reporter gene
EGFP, the CD4/CD8 DP TCR- cells were not present (data not
shown). Therefore, we assume that it is likely that the differential
effect seen between the two ligands of Notch, Delta-1 and
Jagged-1, may be related to the efficiency of those ligands to activate
the Notch receptor. In this respect, the authors were able to show that
Hes-1 up-regulation was clearly seen when the cells received signals
from Delta-1-transduced S17 stromal cells and not from
Jagged-1-transduced S17 stromal cells (32). It
remains an open question whether this reflects a physiological
difference in the triggering capacity of those two ligands or whether
the expression levels of the ligands were different.
Finally, ICN-transduced cells developed ectopically into
CD4+CD8+CD3+TCR-+
T cells in BM when injected i.v. in immunodeficient mice. These
findings are in line with previous murine studies, wherein ICN promotes
the differentiation of cells with similar
CD4+CD8+CD3+TCR-+
cells in BM (33). These cells may be considered pretumoral
cells, because mice harboring ICN transgenes for Notch1 (7, 33, 34, 35) or Notch3 transgenes (36) develop acute
leukemia-like tumors. We were not able to find transduced cells in the
spleen, thymus, or blood. It is likely that the transduction procedure,
wherein CD34+ cells are precultured over 24
h, inhibited the homing capacity of those cells. Our study indicates
that mice injected with human progenitor cells transduced with
Notch1-ICN accumulate human T cells in the BM with an
unusual phenotype. Additional experiments are now performed to address
whether these cells eventually develop into tumoral cells in function
of length of time.
We were not able to show non-cell-autonomous modifications. We found
that ICN nontransduced cells (EGFP-) had a
remarkably similar distribution of cell subpopulations (B lymphocytes,
monocytes, NK cells, and immature progenitor cells) as compared with
control-transduced and nontransduced cells in the MS-5 coculture
experiments (see Table II). Similarly, the distribution of the
different cell subpopulations (CD3, TCR-, TCR-,
CD4+CD8+) of the ICN
nontransduced cells and the control-transduced and nontransduced cells
were comparable in the thymuses in FTOC experiments (see Table III).
This was also the case for the in vivo experiments, where, as expected,
a significant amount of B cells were produced in the ICN nontransduced
cell population (Fig. 4). This is in contrast with a recent study
reported by Kawamata et al. (37) wherein mice
reconstituted with Notch1ICN stem cells also showed a profound
suppression of the B lineage in the nontransduced compartment. It is
possible that a close cell contact within the BM microenvironment,
together with a high transduction efficiency, is required to observe an
influence on the surrounding cells.
In conclusion, our results show that, in human Notch, signaling determines the adoption of the T cell fate at the expense of the B cell fate, and that continuous stimulation of Notch drives the cells to develop ectopically into CD4+CD8+ DP cells in the BM. Therefore, a thorough knowledge of the Notch signaling pathway is of high potential relevance to modulate the T/B fate of human lymphocytes. These strategies are needed to shorten the lag time in T cell recovery after stem cell transplantation or after highly active antiretroviral therapy in HIV patients, and to address Notch-induced leukemogenesis.
|
Acknowledgments |
|---|
chain, Dr. D. Scadden and A. Carlesso
(Massachusetts General Hospital) for the MSCV-ICN-IRES-EGFP construct
and MSCV-IRES-EGFP control vector, Prof. Dr. G. Van Nooten
(Department of Heart Surgery, Ghent University Hospital) for the supply
of human thymic tissue, Caroline Collier and Greet De Smet for animal
care, and Christian De Boever for artwork. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Jean Plum, Department of Clinical Chemistry, Microbiology, and Immunology, University of Ghent, Ghent University Hospital, 4BlokA, De Pintelaan 185, B-9000 Ghent, Belgium. E-mail address: jean.plum{at}rug.ac.be
3 Abbreviations used in this paper: ICN, constitutively active form of Notch; BM, bone marrow; CB, cord blood; NOD, nonobese diabetic; EGFP, enhanced green fluorescent protein; SCF, stem cell factor; FL, flt3/flk-2 ligand; TPO, thrombopoietin; FTOC, fetal thymus organ culture; DP, double positive; SP, single positive.
Received for publication March 22, 2002. Accepted for publication July 16, 2002.
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References |
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,
hydrophobic leader; 
, EGP-like repeats; 
, LNR repeats;
right 
, ram; 
, ankyrin
repeats; Q, glutamine-rich region; P, PEST sequence. For the two
retroviral constructs, the MSCV LTR, IRES motif, and EGFP gene of the
MSCV vector are indicated, as well as ICN. B, Western
blot analysis for Notch1. Protein lysates were prepared from
nontransduced Jurkat cells (lane 2, control), Jurkat
cells infected with the ICN retroviral vector (lane 3),
ICN-transduced CD34 CB cells (lane 4), or the control
vector-transduced CB cells (lane 5) cultured for 4 wk in
FTOC. Lysates were separated on a 4–12% SDS-PAGE, transferred to
nitrocellulose, and probed with anti-ICN Ab and alkaline
phosphatase anti-mouse Ig. ICN with a molecular mass of 
