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Blood, Vol. 95 No. 9 (May 1), 2000:
pp. 2806-2812
HEMATOPOIESIS
Generation of T cells from adult human hematopoietic stem cells
and progenitors in a fetal thymic organ culture system: stimulation by
tumor necrosis factor-
Steven F. A. Weekx,
Hans W. Snoeck,
Fritz Offner,
Magda De Smedt,
Dirk R. Van Bockstaele,
Griet Nijs,
Marc Lenjou,
Adriaan Moulijn,
Inez Rodrigus,
Zwi N. Berneman, and
Jean Plum
From the Laboratory of Experimental Hematology and the Department of
Cardiac Surgery, University of Antwerp, Antwerp University Hospital,
Antwerp, Belgium; the Departments of Hematology and Clinical Chemistry,
Microbiology and Immunology, University of Ghent, Ghent University
Hospital, Ghent, Belgium; and the Institute for Gene Therapy and
Molecular Medicine, Mount Sinai School of Medicine, New York, NY.
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Abstract |
To investigate the T-lymphopoietic capacity of human adult bone
marrow (ABM) hematopoietic progenitor cells, CD34+Lin ,
CD34+CD38+, and CD34++CD38 cells were cultured in a severe
combined immunodeficient (SCID) mouse fetal thymic organ culture
(FTOC). Direct seeding of these progenitors resulted in a moderate to
severe cell loss, particularly for the CD34++CD38 cell fraction,
and T cells could only be generated from the CD34+Lin fraction.
Preincubation for 36 hours with interleukin-3 (IL-3) and stem cell
factor (SCF) led to an improved cell survival and proliferation,
although T-cell development was seen only in the CD34+Lin
fraction. Addition of tumor necrosis factor (TNF)- to
IL-3 + SCF-supplemented preincubation medium resulted in optimal
cell survival, cell proliferation. and T-cell generation of all 3 cell
fractions. The TNF- effect resulted in an up-regulation of CD127
(ie, the IL-7 receptor -chain) in a small subset of the CD34+
cells. No evidence could be generated to support the possibility that
TNF- inhibits a cell population that suppresses T-cell
differentiation. A quantitatively different T-cell generation
potency was still seen between the 3 subpopulations: CD34+Lin
(100% success rate) > CD34+CD38+ (66%) > CD34++CD38 (25%). These data contrast with our previous findings using fetal liver and cord blood progenitors, which readily differentiate into
T-lymphocytes in FTOC, even without prestimulation with cytokines. Our
results demonstrate that adult CD34++CD38 cells, known to contain hematopoietic stem cells, can differentiate into T-lymphocytes and that a significant difference exists in T-lymphopoietic activity of
stem cells derived from ontogenetically different sources.
(Blood. 2000;95:2806-2812)
© 2000 by The American Society of Hematology.
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Introduction |
Human CD34+ cells contain at least part of the
hematologic progenitor and stem cells. Multipotent hematopoietic stem
cells are characterized by their putative ability for self-renewal and differentiation into both the myeloid (ie, granulocytic, monocytic, erythrocytic, and thrombocytic) and the lymphoid (ie, T cell, B cell,
and natural killer [NK] cell) lineages. Hematopoietic progenitors, on
the other hand, are already committed to 1 or several of these
lineages. For a long time, lack of appropriate assays allowing in vitro
T-cell development has hampered progress in understanding
T-lymphopoiesis. Recently, however, functional analysis of T-cell
development has become possible after the introduction of techniques in
which the thymic microenvironment is mimicked, such as the in vitro
fetal thymic organ culture (FTOC) system.1-4 We have
recently described a method in which human fetal liver (FL) precursor
cells are transferred to severe combined immunodeficient (SCID) murine
fetal thymic lobes, following which the lobes are cultured in a
classical FTOC as described by Jenkinson.4,5 This in vitro
method, combined with the possibilities of detailed phenotypic
analysis, provides a unique tool to study early thymopoiesis and the
critical factors involved in this process.6
In spite of intensive research on T-lymphopoiesis using these
techniques, the question whether stem cells differentiate into committed T-cell progenitors before or after entry into the thymus remains unresolved. Although Rodewald et al7 showed that
murine T-cell commitment can occur before entry into the thymus, recent data suggest that, in humans, CD34+ progenitors derived from cord blood
(CB) and fetal bone marrow (FBM) do not undergo such a commitment before they migrate to the thymus.8
As shown by us and by others,4,8-10 human CD34+ FL and CB
cells readily generate T cells in FTOC. Stem cells derived from ABM
seem to be a more difficult source from which to generate T cells in
vitro. Galy et al2,3 studied several subsets of ABM CD34+
cells and found that a phenotypically defined subset of
CD34+LinCD45RA+ progenitor cells, which were
CD38+CD10+HLA-DR+Thy-1c-kit, were able to produce
T cells, though they were devoid of myeloid, erythroid, and
megakaryocytic potential. Because at least part of the hematopoietic
stem cells are contained within the ABM CD34++CD38 cell
fraction,11-13 we compared the T-lymphopoietic capacity of 3 subpopulations of human ABM CD34+ cells. We first evaluated survival,
proliferation, and T-cell generation of a CD34+ cell population devoid
of the lymphocytic lineage-specific cell surface markers CD2, CD7, and
CD19 (CD34+Lin). Next, we investigated CD34++CD38 and
CD34+CD38+ cells, the latter being less primitive cells containing
differentiated progenitors. We furthermore studied the effects of a
36-hour preincubation of these cells with IL-3 + SCF ± TNF- .
It has indeed been shown that IL-3 + SCF can release hematopoietic
stem cells from their G0 cell-cycle status.14 Moreover, incubation of ABM CD34++CD38 cells with IL-3 + SCF with TNF- resulted in a potent stimulation of myeloid proliferation and differentiation, significantly and markedly more than the combination of IL-3 + SCF without TNF-.15 TNF- has
been shown by Zúñiga-Pflücker et al16 to
be related to the induction of crucial events that lead to T-lineage
commitment and differentiation in mice. Therefore, we investigated the
effects of TNF- on the generation of T-lymphocytes from
IL-3 + SCF-stimulated human ABM.
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Materials and methods |
Adult bone marrow cells
Bone marrow samples were aspirated by sternal puncture from
hematologically normal patients undergoing cardiac surgery and were
obtained from healthy bone marrow donors who gave marrow for related
family recipients. Cells were collected into sterile collection tubes
containing complete medium (Iscove's modified Dulbecco's medium
[IMDM] / 10% fetal calf serum [FCS]; Gibco, Paisley, UK) and
heparin (100 U/mL; Novo Nordisk, Bagsvaerd, Denmark) and were isolated
by density-gradient centrifugation over lymphocyte separation medium
(ICN Biomedicals, Costa Mesa, CA). Cells were washed and resuspended in
1.8 mL 90% FCS / 10% dimethyl sulfoxide solution and frozen in
liquid N2. Samples were obtained after informed consent
according to the guidelines of the Medical Ethics Committees of the
University Hospitals of Ghent and Antwerp.
Cytokines and monoclonal antibodies
Supernatant of the 43A1 hybridoma (IgG3, kindly donated
by Dr H. J. Bühring, University of Tübingen, Germany) was
used as a source of anti-CD34 antibodies.17 Fluorescein
isothiocyanate (FITC)-conjugated rabbit antimouse (RAM) immunoglobulins
(F[ab']2 fragments) were purchased from Dako
(Glostrup, Denmark). Phycoerythrin (PE)-conjugated anti-CD38 and
anti-CD34 antibodies and FITC-conjugated anti-CD2, anti-CD7, and
anti-CD19, as well as isotype-specific control antibodies, were
purchased from Becton Dickinson (Erembodegem, Belgium), and mouse
gamma-globulins were purchased from Jackson ImmunoResearch Laboratories
(West Baltimore Pike, PA). Recombinant human (rhu) stem cell factor
(SCF) (specific activity, more than 1 × 105 U/mg)
and rhuTNF- (specific activity, more than
1 × 108 U/mg) were obtained from Boehringer
Mannheim GmbH (Penzberg, Germany), and rhu interleukin-3 (IL-3)
(biological activity, 14 × 103 U/mL) was a kind
gift of Dr S. C. Clark (Genetics Institute, Cambridge, MA).
Animals
C.B.-17 scid/scid (SCID) mice, originally purchased from
Iffa Credo (l'Abresle, France), were bred in our own pathogen-free breeding facility. To obtain timed pregnancies, female and male mice
were mated overnight and the day of the plug was considered day 0. Fetal thymic lobes were dissected from embryos at day 14 to 15 of
gestation. Mice were treated and used in agreement with the
institutional guidelines.
Cell sorting
Viably frozen CB and ABM cells were thawed at 37°C, incubated
for 10 minutes in a solution containing 100 µL 0.3 mol/L
MgSO4, 100 µL DNase (specific activity, approximately
2000 U/mg; Boehringer Mannheim GmbH), 100 µL heparin (5000 U/mL), and
1.5 mL IMDM / 10% FCS, washed in IMDM and resuspended in
IMDM / 10% FCS. After an incubation of another 20 minutes, the cells
were washed again, resuspended at 107 cells/mL, incubated
with 43A1 supernatant in a 1/10 dilution for 20 minutes at 4°C,
washed twice in IMDM / 10% FCS, and incubated with RAM-FITC (1/50
dilution) for 20 minutes at 4°C. After washing twice in
IMDM / 10% FCS, the cells were incubated with a 10-fold excess of
mouse gamma-globulins for 10 minutes to avoid nonspecific staining and
were incubated with anti-CD38PE for 20 minutes at 4°C. After
washing twice in IMDM / 10%FCS, the cells were sorted on a
FACStarPlus Cell Sorter (Becton Dickinson) equipped with an
air-cooled argon ion laser (ILT model 5500A; Ion Laser Technology, Salt
Lake City, UT), tuned to 488 nm at 40-mW power. Cells with a low to
medium forward scatter and a low side scatter, a highly positive green (CD34) fluorescence, and an orange (CD38) fluorescence signal lower
than the mean fluorescence of cells labeled with an irrelevant isotype-matched control antibody plus 2 SD were retained as
CD34+CD38 cells; cells with an orange fluorescence above this
threshold were retained as CD34+CD38+ cells. For the CD34+Lin
cell fraction, thawed and washed cells were incubated with
anti-CD34-PE and with FITC-conjugated anti-CD2, anti-CD7, and
anti-CD19 for 20 minutes at 4°C and then were washed and sorted as
described for the CD34+CD38 cells. Purities were always greater
than 95%. Sorting regions are depicted in Figure
1.
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| Fig 1.
Sorting criteria for CD34++CD38 , CD34+CD38+,
and CD34+Lin cells.
(A) Side (SSC) versus forward (FSC) scatter for ABM mononuclear cells.
The rectangular region (R1) defines the gated population used for
sorting. (B) Fluorescent intensities of CD34FITC and CD38PE
for all cells within the gated region R1. R2 defines the gated
population used for sorting the CD34+CD38+ cells, and R3 defines the
gated population used for sorting the CD34++CD38 cells. (C)
Fluorescent intensities of CD34PE and CD2/CD7/CD19FITC for all
cells within the gated region R1. R4 defines the gated population used
for sorting the CD34+Lin cells. Dot plots represent the sorting
criteria for a single ABM experiment, but similar plots were obtained
for the other ABM experiments.
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Phenotypic analysis
Phenotypic analysis at day 0 and after 36 hours was performed on a
FACScan Cell Sorter (Becton Dickinson) equipped with an air-cooled
argon ion laser (Spectra-Physics Lasers, Mountain View, CA) using the following antibodies: PE-conjugated anti-CD127
(Immunotech, Marseille, France); anti-CD7 (PharMingen, San Diego, CA);
anti-CD38 (Becton Dickinson); anti-CD2 (Becton Dickinson); anti-CD4
(Becton Dickinson); FITC-conjugatedanti-CD34 (Becton Dickinson);
phycoerythrin-cyanin-(PC-)5-conjugatedanti-CD13 (Immunotech);
anti-CD19 (Immunotech); anti-CD33 (Immunotech); and tricolor
(TC)-conjugatedanti-CD14 (Caltag, Burlingame, CA). In most staining
series, the appropriate isotype control antibodies were included and
were found to be negative.
FTOC technique
Preincubation assays were started by performing liquid cultures in
24-well flat-bottomed plates in IMDM with 10% FCS and 100 ng/mL
SCF + 20 U/mL IL-3 with or without 1 ng/mL TNF-. Cells were
transferred to fetal murine lobes by the hanging drop method for 48 hours, followed by culture of the lobes in FTOC as described previously.4 After 28 days of FTOC, the total cell number
in each disrupted thymic lobe was assessed, together with the human cell fraction, which was measured by flow cytometry after staining with
monoclonal antibodies directed against human CD45 and mouse CD45. Dead
cells were gated out by propidium iodide exclusion.
Statistics
Statistical comparisons were validated using the nonparametric
Wilcoxon signed rank test (paired samples) and the nonparametric Mann-Whitney U test (unpaired samples). Results are expressed as mean ± SEM.
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Results |
Phenotypic analysis and effects of preincubation on cell number and
flow cytometric scatter profile
Different cell populations from the mononuclear ABM cell fraction
were isolated by fluorescence-activated cell sorting. Combinations of
CD34 with other cell surface markers (CD38, CD2, CD7, and CD19) were
used. Three CD34-positive subpopulations were thus isolated: CD34++CD38 cells, CD34+CD38+ cells, and CD34+Lin cells
(CD2CD7CD19). The CD38 fraction was
CD34-positive with high density (CD34++CD38) because the average
CD34 fluorescence was 82.4% ± 10.4% higher than that for the
CD34+CD38+ fraction (P < .001; Figure 1). Phenotypic analysis showed that 1.7% ± 0.3% of the ABM mononuclear cells were CD34+. Within this CD34+ population, 6.2% ± 1.8% were
CD38, 93.8% ± 1.8% were CD38+, and 91.1% ± 1.2%
were Lin. Sorting regions for each of the 3 cell fractions are
depicted in Figure 1.
Incubation of these cell fractions for 36 hours in the presence of
IL-3 + SCF with or without TNF- did not result in significant cell
proliferation, though the forward and side scatter profile strikingly
shifted in all 3 fractions, especially after incubation with TNF-
(Figure 2). Moreover, microscopic
evaluation of the cell fractions incubated with TNF- always revealed
the presence of numerous clusters containing 25 or fewer cells, whereas
in the absence of TNF- only dispersed cells were seen (not shown).
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| Fig 2.
Scatter profile of sorted ABM CD34+ cells.
SSC versus FSC at day 0 (A) and after a 36-hour preincubation with
IL-3 + SCF + TNF- (B). The SSC histogram (C) and the FSC
histogram (D) of the cells gated in R1. Numbers between parentheses,
mean channel number. Dot plots represent the scatter profiles for a
single ABM CD34+ experiment, but similar plots were obtained for the
other ABM experiments when using CD34++CD38, CD34+CD38+, and
CD34+Lin cells.
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FTOC supports proliferation of human ABM CD34+ subpopulations
We first investigated whether FTOC supports survival and potential
proliferation of the 3 different CD34+ subpopulations derived from
human ABM. We seeded the CD34+ subpopulations, with or without a
36-hour preincubation in IL-3 + SCF or IL-3 + SCF + TNF-, into single thymic lobes of day 14 to 15 fetal SCID mice (Table
1). After 28 days of FTOC, we assessed the
total cell number and the human cell fraction in each disrupted thymic
lobe. Using these parameters, the total human cell number per 1000 input cells was calculated. As shown in Table
2, seeding of cells without preincubation resulted in a moderate to severe loss particularly for the
CD34++CD38 cell fraction. However, taking into account the fact
that only 20% (ie, 200 cells/1000 input cells) of the seeded cells
actually enter the thymic lobe during the hanging drop time period of
48 hours,4 our results clearly show that the FTOC assay at
least sustains the survival of human ABM cells, and it may even promote proliferation (up to 3-fold) provided the cells have been preincubated with IL-3 + SCF with or without TNF- (Table 2). This is in strong contrast with our previous findings whereby we showed that after 4 weeks of FTOC, FL and CB CD34+Lin cells without cytokine
prestimulation showed a 60-fold and a 20-fold cell expansion,
respectively.4,9
CD34+ subpopulations from ABM differentiate into T cells in
FTOC
Human T-lymphopoiesis passes through different phenotypic maturation
steps before giving rise to mature CD4+ and CD8+ T cells. As we have
shown previously for FL and CB stem cells, the following stages can be
demonstrated using FTOC: part of the
CD34++CD38CD4CD8CD1 cells develop rapidly
into dendritic cells that are CD4+HLADR+cytoplasmic(cy)- CD3CD7, and CD1/+. The T-cell
differentiation proceeds through a CD4+cyCD3+CD7+CD1+HLA-DR
intermediate stage, followed by a CD4+CD8+membrane(m)CD3+CD1+CD5+ "double-positive" (DP)
stage before achieving the end maturation stage, resulting in a
CD4+CD8CD3+CD1 and CD4CD8+CD3+CD1
phenotype.4,9,18 Because of the low number of immature
progenitor cells at the start of the FTOC (resulting from the low
frequency of the CD34+ subpopulations in ABM) and because the
proliferative potential of these cells was limited, we examined the
presence of human CD4+CD8+ DP T cells, which were mCD3+ in the FTOC at
1 time point (day 28). The presence of these human CD4+CD8+mCD3+ cells
indicates an active T-lymphopoiesis starting from the seeded stem
cells.4,6,19 In 1 experiment, however, we were able to
analyze the kinetics of the T-cell development from ABM CD34+Lin
cells and found the same differentiation markers as mentioned above
(Figure 3).
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| Fig 3.
Kinetic analysis of the development of ABM CD34+Lin
cells in hybrid FTOC.
CD34+Lin ABM cells were sorted and cultured together with mouse
thymic lobes. After various incubation times (as indicated), the lobes
were minced and a flow cytometric analysis was performed after staining
the cells with monoclonal antibodies directed against human antigens as
indicated in the axis of the dot plot. CyCD3, cytoplasmic CD3; mCD3,
membrane CD3.
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As shown in Table 3, starting from
unstimulated CD34+ cells, only the CD34+Lin cells generated
CD4+CD8+ DP T cells in 3 of 7 experiments analyzed at day 28, yielding
7.5% ± 4.0% DP cells. No DP T cells were generated in the
CD34++CD38 and the CD34+CD38+ subfractions, except for 1 CD34+CD38+ experiment, in which traces (less than 0.1%) of DP cells
were found. Preincubation of the CD34+Lin cells during 36 hours
in the presence of IL-3 + SCF or IL-3 + SCF + TNF- did not
result in a significant rise in DP T-cell generation. Starting from the
CD34++CD38 and the CD34+CD38+ cell fractions, only traces of DP
T cells were generated after preincubation with IL-3 + SCF. Addition
of TNF- to this preincubation cocktail resulted in a significantly
higher output of DP T cells for CD34+CD38+ 5.9% ± 2.3%
(P = .008) and CD34++CD38 6.0% ± 4.1% (P = .03) cells. In terms of absolute DP T-cell
generation/1000 input cells, Table 4
clearly shows that we were able to generate T cells starting from all 3 different CD34+ subpopulations, with an optimal output after
preincubation with IL-3 + SCF + TNF-.
Preincubation with TNF- stimulates ABM stem cells to generate
CD4+CD8+ T cells
To make a qualitative assessment of the effects of TNF- on the
generation of human T cells, we analyzed the pattern of T-cell development in the FTOC, which resulted in a classification with 3 distinctive growth patterns. In pattern A, only murine CD45+ cells and
no or few (less than 0.1%) human CD45+ cells were found after 28 days
of FTOC. Pattern B was defined by the presence of human CD45+ cells
that could develop into CD4+CD8CD3 single positive cells
but of which less than 0.5% reached the CD4+CD8+ stage. In pattern C,
a clearly distinguishable population of human double-positive CD4+CD8+
T cells could be observed. As summarized in Table
5, only part (43%) of the CD34+Lin
samples could generate T cells without prior preincubation, whereas
none of the CD34++CD38 or of the CD34+CD38+ fractions developed
beyond the CD4 single-positive stage, not even after a 36-hour
preincubation with SCF + IL-3. It was only after adding TNF- to
IL-3 + SCF that 25% of the experiments using CD34++CD38 cells,
66% of those using CD34+CD38+ cells, and 100% of those using
CD34+Lin samples generated CD4+CD8+ T cells (Table 5).
To understand the mode of action of TNF-, we first performed
extensive phenotypic analyses on CD34+Lin cells preincubated in
the presence and absence of TNF-. We performed these experiments on
CD34+Lin cells for two reasons. First, to analyze a maximum number of markers on each bone marrow sample, an adequate cell number
is required, making it almost impossible to use the low-frequency CD34++CD38 cell population for this purpose. Second, as shown in
Table 5, preincubation of CD34+Lin cells in the presence of
TNF- resulted in a C pattern in 100% of the experiments, which was
in contrast to the outcome of the growth patterns of the CD34+CD38+ and
the CD34++CD38 cell fractions, making the CD34+Lin
fraction the most interesting population to use for phenotypic
analysis. The following markers were analyzed: CD34, CD33, CD13,
CD38, CD2, CD4, CD7, CD14, and CD127 (ie, IL-7R). Preincubation
of the cells resulted in a down-regulation of CD34, an
up-regulation of CD13 and CD33, a slight up-regulation of CD38, and no
effect on CD19 and CD7 expression. There was also an up-regulation of
CD14 and of CD4, but all the CD4+ cells were also positive for CD14.
Preincubation furthermore resulted in the appearance of a small
population of CD2+ cells, though no difference was seen between the
cells preincubated with or without TNF-. Interestingly, in all
samples analyzed, a small but clearly distinguishable population
of CD127+ cells became evident only after preincubation in the presence
of TNF- (P = .01) (Figure 4).
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| Fig 4.
Effect of IL-3 + SCF and IL-3 + SCF + TNF-
on the expression of CD34 and CD127.
CD34+Lin cells were analyzed at day 0 immediately after cell
sorting and after a preincubation period of 36 hours. Numbers in
quadrants refer to percentage of cells within that quadrant. This
figure is representative of a series of six independent experiments.
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In a second set of experiments, we performed 6 mixing experiments to
examine the possibility that TNF- might remove a cell population
that suppresses T-cell differentiation (Table
6). In these experiments, an equal number
of cells preincubated in IL-3 + SCF (X in Table 6) and of cells
preincubated in IL-3 + SCF + TNF- (Y in Table 6) were mixed
before they were transferred in the hanging drop. After 28 days of
FTOC, the A-B-C growth pattern of these mixed cells was compared with
that of each of the preincubation settings separately. These mixing
experiments did not alter the positive effect of TNF- on T-cell
generation capacity, indicating that an inhibitory population that can
be suppressed by TNF- is not present (Table 6).
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Table 6.
Analysis of growth pattern after mixing equal numbers of
cells preincubated in IL-3 + SCF and IL-3 + SCF + TNF-
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In a third set of experiments, we investigated the potential role of
accessory cell populations such as dendritic cells. Figure 3 shows that
at days 11 to 14, a population with a CD4+HLA-DR+ phenotype is present
in the FTOC. This population can be further delineated according to a
high forward and side scatter profile, and it is CD14
(data not shown). We have recently shown that these cells represent a
dendritic cell population that is CD7 and
cyCD3.20 No difference in dendritic cell frequency
was seen after preincubation with IL-3 + SCF with or without TNF- (17.9% ± 8.1% vs 12% ± 5%, respectively;
P = .17), though the absolute dendritic cell numbers in the
presence of TNF- were always higher (264.1 ± 138.6 vs
698.6 ± 220.9, respectively; P = .03).
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Discussion |
In this report we demonstrate that T cells can be generated from
hematopoietic stem cell- and progenitor cell-containing fractions derived from human adult bone marrow in an ex vivo xenotransplant model. In this FTOC assay, human hematopoietic progenitor cells can
differentiate to mature T cells in isolated fetal thymic lobes of SCID
mice, which have a mutated scid gene, resulting in a deficient murine thymocyte maturation21 but allowing concomitant
T-cell development of xenogeneic cells. We furthermore show that T
cells can be generated from adult bone marrow, not only from the
CD34+Lin and CD34+CD38+ fractions, which still are rather
heterogeneous populations containing differentiated progenitors, but
also from the phenotypically and functionally most primitive
hematopoietic cells, characterized by their high expression of CD34 and
the absence of CD38 expression (CD34++CD38
cells).11-13,22,23
It has previously been shown by others and by us that human
hematopoietic stem cell-containing populations derived from different ontogenetic sources such as CB, FBM, or FL can be stimulated to T-cell
differentiation in a murine SCID fetal thymic environment. Res et
al10 showed that only the CD34++CD38 FL cells had
the potential to develop into T cells, whereas their CD34+CD38+
counterparts did not have this capacity. The same group, however,
observed that both CD34++CD38 and CD34+CD38+ fractions from FBM
and CB were able to generate T cells,8 which was recently
confirmed by us.9 However, little information is available
on whether T cells can be generated from ABM stem cells and on the
possible differences herein between the CD34++CD38 and the
CD34+CD38+ cell fractions. Freedman et al24 and Rosenzweig
et al25 were able to generate T cells from human ABM cells
by using a thymic stromal culture system derived from either fetal
human or fetal rhesus macaque thymus glands. However, these authors
used high concentrations (1 × 105 cells/well) of
CD34+ cells, which are relatively heterogeneous cells containing
progenitors already committed to T-cell differentiation.3 In addition, because this CD34+ population is globally more
differentiated than CD34++CD38 cells,11-13,22,23 it
is likely that the early differentiation steps from multipotent
progenitor cells to the T-lineage have been missed. On the other hand,
Galy et al26 used a SCID-human thymus assay in which
irradiated fetal thymuses were injected with human ABM cells, following
which the thymuses were inoculated during 6 weeks under the kidney
capsule of SCID mice. They found that lymphoid cells can be generated
from immature progenitors such as
CD34+LinThy-1CD45RA cells and
CD34+LinThy-1+CD45RA cells. Both these cell fractions
were also able to generate erythroid and myeloid progeny, comparable
with the lymphopoietic (this study) and the myelopoietic capacities of
CD34++CD38 cells.11,15,22,27,28
Our data further indicate that 36-hour preincubation of ABM cells with
IL-3 + SCF before seeding to FTOC improves cell survival and
proliferation of the CD34++CD38 fraction (shift from pattern A
 B). This is in strong contrast with the findings of Hirayama and
Ogawa,29 who show that the addition of IL-3 to murine
LinLy-6A/E+ immature hematopoietic progenitors in
methylcellulose cultures containing SCF + IL-11 + IL-7 for 8 days
strongly suppressed T-lymphopoiesis. This may have been the result of
an irreversible commitment of these progenitor cells to the myeloid
lineage after 8 days of culture in the presence of IL-3 + SCF. A
shorter preincubation period of 36 hours, however, may be just enough
to release stem cells from their G0 cell-cycle status
without directing them irreversibly to a specific
lineage.14 This is supported by the results of Moore and
Zlotnik,30 who found that a 3-day incubation of
murine thymic CD4lo cells
(CD44+CD25CD3CD4loc-kit+CD8cells; ie,
the first cells to seed the thymus from the bone marrow) with
SCF + IL-3 resulted in the maintenance of cell survival, whereas the
addition of IL-7 to this cocktail even induced a capacity for
repopulation in FTOC. That IL-7 is also a critical growth factor in
early human T-cell development in the FTOC system has recently been
confirmed by us.31
The addition of TNF- to SCF + IL-3 resulted in a T-cell generation
success rate (pattern C) of 100% for the CD34+Lin fraction and
in a further shift from pattern B C (66%) for the CD34+CD38+ cells
and from patterns A B and B C (25%) for the CD34++CD38 cells (Table 5). These effects of TNF-, which is produced within the
thymic microenvironment, could be related to the induction of crucial
events leading to T-lineage commitment and differentiation as shown by
Zúñiga-Pflücker et al.16 However, this
group could not conclude from their study whether TNF- acted
directly on murine T-cell progenitors or indirectly on a non-T-cell
subset that is critical for T-cell maturation. We investigated both
possibilities. First, we showed that in all CD34+Lin samples
analyzed, a small but clearly distinguishable population of CD127+
cells appeared only after preincubation with IL-3 + SCF + TNF-.
We hypothesize that TNF- induces the up-regulation of CD127 on a
small fraction of the preincubated cells, resulting in a positive
effect (be it direct or indirect) on T-cell generation in the FTOC.
This is in agreement with the 1997 findings of Kondo et
al,32 who identified a clonogenic common progenitor in
mouse bone marrow. This common lymphoid progenitor is Lin
IL-7R + Thy-1Sca-1loc-Kitlo, and it
has a lymphoid-restricted (T, B, and NK) reconstitution capacity in
vivo but completely lacks myeloid differentiation potential. To
address the role of this CD127+ cell fraction on T-cell generation in a
direct manner, it would be interesting to isolate (sort) the CD127+
cells after 36 hours of preincubation and to seed them directly into
the thymic lobes of the FTOC. Unfortunately, the low number of cells
and the potential of CD127 antibodies to block the development of FL
cells in the FTOC31,33 or of sorted CD127+CD34+ intrathymic
precursor cells in FTOC (Plum J, unpublished data) present
important obstacles to test whether CD127+ cells, generated after
incubation with TNF- , favor T-cell development. In addition, we
examined the potential role of the dendritic cells that were generated
from CD34+Lin cells in the FTOC assay. We could not show a
difference in dendritic cell frequency after preincubation with
IL-3 + SCF with or without TNF- , though the absolute dendritic
cell numbers in the presence of TNF- were always higher. Because of
the overall low absolute cell numbers generated, it was not feasible to
investigate further phenotypical and functional (eg, mixed lymphocyte
culture assays) properties of these dendritic cells. Therefore, we
cannot exclude that the dendritic cells generated in the presence or
absence of TNF- have different functional properties, resulting in
an indirect TNF- effect on T-cell generation. Finally, within the
limitations of qualitative analysis when using the FTOC system, the
evidence we gathered does not support the hypothesis that TNF-
inhibits a cell population that suppresses T-cell differentiation.
Based on our previous results concerning the stimulatory effect of
TNF- on the myeloproliferative capacity of CD34++CD38 cells,
it is possible that TNF- promotes the proliferation of stem cells
and immature progenitor cells, making them more sensitive to
T-lymphopoietic influences.15 Another explanation would be that TNF- (in combination with SCF + IL-3) directly makes the ABM
CD34+ cells more able to enter the thymic lobes. Further
investigation is required to elucidate the exact role of TNF- in
this matter.
The results presented in this study also revealed important ontogenetic
differences in T-cell generation potential between fetal, neonatal, and
adult hematopoietic stem cells. From our previous findings, we know
that highly purified CD34++Lin cells from FL develop into T
cells in 99% of the FTOC experiments, with a 60-fold cell
expansion.4 CB progenitors show a success rate of around
85% (Plum J, unpublished data) and a 20-fold
cell expansion.9 Our recent data demonstrate
that for ABM stem cells the success rate progressed from 25%
(CD34++CD38 cells) to 66% (CD34+CD38+ cells) to
100% (CD34+Lin cells) and a 1 to 3-fold cell expansion (Tables
2 and 5). Different hypotheses can be postulated to explain these
differences in success rate and expansion capacity. They may develop
because FL cells enter the thymic lobes far more easily than ABM cells,
because FL contains more T-cell progenitors than ABM, or because there
is an important difference between the proliferative capacity of T-cell
progenitors derived from FL and those derived from ABM. Interestingly,
the observed differences in T-cell generation potential of FL, CB, and
ABM cells when using the FTOC model are comparable with the important
variations that exist in their myelopoietic capacities. Indeed,
reports28,34 have shown that, as far as myelopoiesis is
concerned, there is an at least 100-fold difference in proliferative
capacity between FL and ABM stem cells and that ABM cells have much
higher cytokine requirements than their FL counterparts. It is, of
course, still possible that ABM stem cells need an additional stimulus,
unknown thus far, to enter or to grow and develop in the thymic
environment. It is also possible that facilitating cells, which display
a positive feedback mechanism for the developing stem
cells,35 are generated from FL and CB cells but not from
ABM cells. Our data, however, suggest that when using the FTOC system,
providing an additional stimulus (eg, TNF-) besides
survival-enhancing factors (eg, IL-3 + SCF) could be of benefit to
augment the T-lymphopoietic potential of ABM CD34+ subpopulations.
Finally, although ABM CD34+ subpopulations are used in a clinical
set-up to try and purge malignant cells or to diminish the role of
graft-versus-host reactions,36,37 transplantation of purified CD34+ cells could have the disadvantage of slowing down thymus
repopulation, resulting in delayed T-cell immune responsiveness. Therefore, finding a system that would allow an accelerated graft take and T-cell repopulation, using ex vivo generated (and expanded) T cells from purified CD34+ cells, could obviate these disadvantages. Understanding the process that governs T-lymphopoiesis is
also of importance in potential applications such as gene transfer to
correct genetic disorders of the T-cell lineage and restoration of the
T-lymphocyte compartment after depletion caused by HIV infection.
In conclusion, we show for the first time T-cell differentiation
starting from CD34++CD38 human hematopoietic stem cells by using
an in vitro FTOC model. This provides additional proof that at least
some of the multipotent stem cells are contained within this cell
fraction. We also demonstrate, again for the first time in the human
system, that TNF- promotes T-cell differentiation from different
subsets of CD34+ progenitors and that TNF- induces up-regulation of
CD127. Finally, we show that a significant difference exists in the
T-cell progenitor activity of hematopoietic stem cells derived from
ontogenetically different sources.
| |
Footnotes |
Submitted December 2, 1998; accepted December 22, 1999.
Supported by grants G.0096.95, 3.0109.96, and G.0157.99 of the Fund for
Scientific Research, Flanders, Belgium; by the Geconcerteerde Onderzoeksactie of the Special Research Fund of the University of
Antwerp (GOA 21/1996); and by private charities organized for the HEBA
Foundation by Martine Julien, Gerald Dauphin, and the musical friends
of the late Luke Walter, Jr., to whose memory this article is
dedicated. S.F.A.W. is a research assistant of the Fund for Scientific
Research, Flanders, Belgium.
Reprints: Zwi N. Berneman, Laboratory of Experimental
Hematology, Antwerp University Hospital (UZA), Wilrijkstraat 10, B-2650 Edegem, Belgium.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
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Abstract
Introduction