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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4090-4097
 c Gene Transfer in the Presence of Stem Cell Factor, FLT-3L,
Interleukin-7 (IL-7), IL-1 , and IL-15 Cytokines Restores T-Cell
Differentiation From c( ) X-Linked Severe Combined
Immunodeficiency Hematopoietic Progenitor Cells in Murine Fetal
Thymic Organ Cultures
By
S. Hacein-Bey,
G. De Saint Basile,
J. Lemerle,
A. Fischer, and
M. Cavazzana-Calvo
From the Institut National de la santé et de la Recherche
Médicale U429 et Centre de Transfusion Sanguine, Hôpital
Necker-Enfants Malades, Paris Cedex, France.
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ABSTRACT |
X-linked severe combined immunodeficiency (SCID-Xl) is a rare human
inherited disorder in which early T and natural killer (NK) lymphocyte development is blocked. The genetic disorder results from mutations in the common  c chain that participates in several cytokine receptors including the interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15 receptors. We have shown in a previous report that c
gene transfer into SCID-Xl bone marrow (BM) cells restores efficient NK
cell differentiation. In this study, we have focused on the
introduction of the c gene into SCID-Xl hematopoietic stem cells
with the goal of obtaining differentiation into mature T cells. For
this purpose, we used the in vitro hybrid fetal thymic organ culture
(FTOC) system in which a combination of cytokines consisting of stem
cell factor (SCF), Flt-3L, IL-7, IL-1 , and IL-15 is added
concomitantly. In this culture system, CD34+ marrow cells
from two SCID-Xl patients were able to mature into double positive
CD4+ CD8+ cells and to a lesser degree into
CD4+ TCR + single positive cells after
retroviral-mediated c gene transfer. In addition, examination of the
output cell population at the TCR DJ1 locus exhibited multiple
rearrangements. These results indicate that restoration of the
c/JAK/STAT signaling pathway during the early developmental stages
of thymocytes can correct the T-cell differentiation block in SCID-Xl
hematopoietic progenitor cells and therefore establishes a basis for
further clinical c gene transfer studies.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
THYMIC PROGENITORS acquire a number of
cell surface molecules during their highly regulated maturation
program. The earliest human precursor cells that seed the thymus from
the fetal liver and bone marrow (BM) are CD34high CD38( )
and retain multipotential capacity.1-3 The loss of myeloid differentiation potential is heralded by upregulation of the CD45RA marker. This population has been shown to contain T, natural killer (NK), and dendritic cell precursor activity.1,4-6 The next step in maturation is the commitment to bipotential T/NK cells that
express CD7, CD2, and cytoplasmic CD3. At this point, the , , and
 loci are still in a germline configuration. Acquisition of CD1a
marks the irreversible committment to the T-cell lineage. As these
pre-T cells upregulate CD4 to become immature single positive cells
(ISP) phenotypically characterized as CD34() CD38++
CD45RO+ CD7+ CD2+ CD5+
CD1+ CD4+, they gradually switch from CD45RA to
CD45RO expression.4 After the progression to the
CD4+ CD8+ double positive (DP) stage,
thymocytes that express CD3/TCR at low levels undergo positive
selection by interaction with the major histocompatibility complex
(MHC), and it is during this process that TCR+ DP
cells develop into mature CD4+ or CD8+ single
positive cells (SP).7,8 Furthermore, this positive selection signal induces the expression of CD69, Bcl-2, and
CD27.9-15 Finally, CD1a is downregulated, and CD45RA is
expressed immediately before these cells emigrate out of the thymus.
In addition to TCR/CD4/CD8-MHC class II/I interactions, this process
involves cell-cell interactions between numerous different surface
molecules on developing thymocytes and thymic cortical epithelial
cells,16 as well as soluble factors. Thymocytes have indeed
been shown to be responsive to cytokines in vitro.17 A
variety of cytokines is produced by the thymic microenvironment including interleukin-1 (IL-1), IL-3, IL-6, IL-7, IL-12, IL-15, granulocyte-macrophage colony-stimulating factor (GM-CSF), M-CSF, G-CSF, and stem cell factor (SCF).18,19 The role of IL-7 in T-cell development has been best defined in murine models.
The profound and early block observed in T and NK cell differentiation
in patients with SCID-Xl,20 caused by mutations in the c
cytokine receptor gene,21 constitutes indirect evidence for
the requirement of c-binding cytokines, such as IL-7, during early
thymocyte developmental stages. Nevertheless, the lack of signaling via
the c/Jak/STAT pathway could also be attributed to an inability to
respond to other cytokines, which also use the c chain such as IL-2,
IL-4, IL-9, and IL-15.22 In a previous report, we have
shown that c gene transfer into SCID-Xl BM cells restores efficient
NK cell differentiation in the presence of SCF and IL-15.23
Function of the vector encoded c chain was also demonstrated by
restoration of high-affinity IL-2R expression and normal JAK3
activation in SCID-Xl B cell lines after c gene transfer.24
To investigate the potential for T-cell development after c gene
transfer to CD34+ SCID-Xl BM cells, we have used the fetal
murine thymic organ culture (FTOC) system as developed by Jenkinson et
al.25 The murine FTOC environment can to some extent
support human T lymphopoiesis from fetal sources or the postnatal
thymus26-31 without the addition of mitogens or cytokines,
yet we and others3 have found that the T-cell developmental
potential of precursor cells isolated from cord blood (CB) or postnatal
BM cells is very low and variable. While the CD34+CD38()
population from human fetal BM contains both stromal and hematopoietic
progenitors, the frequency of stromal progenitors decreases
proportionally with increasing fetal gestational age.32 It
is thus possible that the poor developmental potential in the FTOC
system using CB or postnatal BM cells is related to the lack of stromal
progenitors that have the potential to develop into cells capable of
producing the human cytokines necessary for survival, growth, and
subsequent T-cell development of the input cells. We have therefore
defined FTOC culture conditions able to promote proliferation and
T-cell differentiation of postnatal CD34+ CB cells. Using
these defined conditions, we have shown that c transduced
CD34+ SCID-Xl BM cells can give rise to T lymphocyte
differentiation in vitro with full phenotypic maturation. This
experimental system, combining retroviral gene transfer and FTOC,
constitutes a preclinical step towards the treatment of SCID-Xl
patients with c gene transfer.
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MATERIALS AND METHODS |
Cell samples and CD34+ cells purification.
All samples were obtained after parental informed consent. Human
umbilical CB samples were collected immediately after delivery. SCID-Xl
BM samples were obtained from two patients (S1 and S2) at the ages of 6 and 7 months, respectively, while under general anesthesia for central
line insertion before undergoing BM transplantation.
The mutation detected in patient S1 in exon 3 (G355T) led to a glycine
 valine substitution. In patient S2, a complex rearrangement resulting in a 2-bp deletion at position 836 and a 13-bp insertion at
position 847 with a premature stop codon in exon 7 (amino acid 293).
c chain expression at the surface of both patients' B lymphocytes was undetectable. In the peripheral blood of patient S1, the lymphocyte count was 612/µL, CD3+ 2%, CD19+ 73%, and
CD56+ 1%. In patient S2, less than 1% CD3+
cells were detected.
The mononuclear cell fraction from BM and CB samples was obtained by
density gradient centrifugation (density 1.077 g/mL; Nycomed Pharma,
Oslo, Norway) and for BM was cryopreserved before further processing.
The mononuclear cell fraction was then enriched for primitive
progenitors by immunoselection using the Ceprate LC34 cell separation
system (Cell Pro, Bothell, WA). Enriched CD34+ cells were
then incubated with phycoerythrin (R-PE) labeled anti-CD34 (PE-HPCA2;
Becton Dickinson, San Jose, CA) and separated by cell sorting on a
FACS-Star Plus cell sorter (Becton Dickinson). The purity of sorted
cells, as assessed by analyzing after sorting, was greater than 99%.
Retroviral vector and packaging cell line.
Human c chain cDNA extending from the initiation codon ATG
(nucleotides numbered 1 to 1114) was generated by reverse
transcription-polymerase chain reaction (RT-PCR) from mRNA of control
B-cell lines. Forward primer: 5 -GCAAGCGACATGTTGAAGCC-3, and reverse
primer: 5-GAGGATCCGGGTTCAGGTTTCAG-3 contain an AFL III and
BamHI site, respectively, allowing for c chain insertion in
the retroviral vector. Correct c sequence was assessed by direct
sequencing of the entire PCR amplified fragment. The human c chain
cDNA was then inserted into the Nco I and BamHI sites
of the MFG B2 c Mo-long terminal repeat (LTR) vector.33 MFG (B2) uses the Moloney murine leukemia virus
(MO-MLV) LTRs for transcription of the viral genome and contains the B2 mutation corresponding to a single G to A transition at position +160
of the MO-MLV sequence.34 This plasmid, named MFG
(B2)-c, does not contain a selectable marker. Retroviral producer
cell lines were then generated by cotransfecting the MFG (B2)-c
plasmid with the plasmid pSV2-neo into the amphotropic packaging cell line  CRIP, as previously described.35 Two
days later, the transduced cells were diluted 10 times and placed under
G418 (Geneticin; GIBCO/BRL, Gaithersburg, MD) selection at 0.8 to 1 mg/mL active concentration until individual resistant colonies formed.
Viral titering was performed by infecting 5 × 105 NIH3T3
cells with 0.5 mL of a 24-hour supernatant from the virus-producing clones in the presence of 8 µg/mL of Polybrene (Sigma Chemical Co, St
Louis, MO). Thirty clones were screened for high-titer virus production
by Southern blot analysis of the target cells. Southern blot analysis
confirmed the presence of an unrearranged proviral genome from these
producer clones and was used to determine the number of proviral copies
integrated in the target population. The clone that produced the
highest transfer efficiency of virus to NIH3T3 cells (0.5 copy of
provirus per cell) was used in the subsequent transduction experiments.
Transduction of SCID-Xl mononuclear bone marrow cells (SCID-Xl BMC).
Purified CD34+ SCID-Xl BMC were prestimulated by culturing
in the presence of SCF (100 ng/mL, kindly provided by
Amgen, Thousand Oaks, CA), Flt3 ligand (Flt-3L; 100 ng/mL,
kindly provided by Immunex, Seattle, WA), and IL-3 (20 ng/mL, kindly provided by Sandoz, Basel, Switzerland) at
37°C, 5% CO2 for 24 hours. The cells were then
transduced on human recombinant fibronectin-coated wells (fragment
CH296; Takara, Ohtsu, Japan) by culturing in vector conditioned media
in the presence of the same cytokines and protamine sulfate (4 µg/mL)
on 3 consecutive days. Viral supernatant was replaced every 24 hours
during the 3-day transduction period.
Hybrid human/mouse FTOC.
Murine thymic lobes dissected from day 14 embryos (C57/Bl6) were
treated with 1.35 mmol/L 2deoxyguanosine (Sigma) for 5 to 6 days to
remove endogenous thymocytes and hematopoietic cells before
reconstitution. The thymic lobes were then washed extensively and
cocultured in hanging drops in wells of a Terasaki plate with 1 to 3 × 104 fluorescence-activated cell sorter (FACS) Sorted
CD34+ CB cells or 5 × 104 CD34+
transduced SCID-Xl BM cells. Cells were allowed to seed the lobes during a 48-hour coculture. Thymi were then transferred to nucleopore filters (Costar, Cambridge, MA), which were layered over gelfoam sponge
(Upjohn, Kalamazoo, MI) in 6-well plates (Costar). Culture medium
consisted of RPMI (GIBCO/BRL) supplemented with 5% fetal calf serum
(FCS) (Stem Cell Technologies, Vancouver, Canada) and 7%
human AB serum. The following cytokines were used from the start of the
hanging-drop culture throughout the FTOC: SCF 100 ng/mL (Amgen), Flt-3L
100 ng/mL (Immunex), IL-7 20 ng/mL (Genzyme), IL-1 2 ng/mL
(Genzyme), IL-15 20 ng/mL (Immunex), as indicated. Cultures
were incubated at 37°C in 5% CO2 for 4 to 5 weeks.
Afterwards, the thymic lobes were mechanically dispersed into a single
cell suspension. Cells were enumerated and assessed for viability by trypan blue exclusion.
Liquid cultures in the presence of cytokines.
One hundred thousand sorted CD34+ CB cells were plated in
flat-bottomed 96-well plates in the same complete RPMI medium
supplemented with the same combination of cytokines used in T-cell
differentiation assay (FTOC). After 30 days, growing cells were stained
and analyzed by FACS.
Flow cytometry analysis.
Phenotypic analysis of human cells recovered from the hybrid mouse FTOC
was performed using the following monoclonal antibodies (MoAbs):
anti-CD34 (HPCA-2), anti-CD7 (Leu 9), anti-CD2 (Leu5b), anti-CD5
(Leu1), anti-CD4 (Leu 3a), anti-CD8 (Leu 2a), anti-CD3 (Leu
4), anti-CD56 (Leu 19), anti-CD16 (Leu 11b), anti-CD14 (Leu M3),
anti-CD45RO, anti-CD45RA, (mouse antihuman PE or fluorescein isothiocyanate [FITC]-conjugated) all from Becton Dickinson,
anti-TCR- and anti-TCR- from Immunotech (Marseille,
France), rat antihuman c (unconjugated) from Pharmigen (San Diego,
CA) and anti-CD1a (unconjugated) from Becton Dickinson. Control isotype
Ig FITC was obtained from Pharmigen. For conjugated antibodies, cells were stained for 30 minutes on ice and after two washings were subjected to flow cytometric analysis. For c staining, cells were
first incubated with unconjugated rat anti-human c for 30 minutes,
washed twice followed by staining with biotin-conjugated mouse antirat
Ig (Jackson, Westgrove, PA). The second antibody was detected with
PE-conjugated avidin (Caltag, San Francisco, CA). For CD1a staining,
cells were first incubated with unconjugated mouse anti-CD1a and
detected with FITC-conjugated rat antimouse Ig (Jackson, Baltimore,
MD). Cells were suspended in phosphate-buffered saline
(PBS) and subjected to flow cytometric analysis using a FACScan (Becton
Dickinson). Between 5,000 and 20,000 events were collected per sample
(depending on the availability of cells). For analysis, a live gate was
set on forward and side scatter. Human cell origin was verified by
staining for human CD45 expression.
Provirus integration study in thymocytes derived from
CD34+ c transduced cells.
To test for vector integration in the progeny of SCID-Xl
CD34+ c transduced cells, thymocytes were recovered and
their DNA isolated. To specifically amplify c proviral DNA, two
primers were used, one within the retroviral backbone and one within
c gene sequence, respectively: PM-R 5-GACCACTGATATCCTGTCTTCAAC-3 and c-F 5-CCAGCCTACCAACCTCACT-3. DNA was amplified in a 50-µL PCR reaction mixture using 30 cycles at an annealing temperature of
60°C. A 20-µL portion of the amplified product was separated on a
1% agarose gel and analyzed by ethidium bromide staining.
TCR gene rearrangement analysis.
Genomic DNA was prepared from human thymocytes recovered from organ
cultures and PCR for DJ amplification was conducted using the two
primers TBF1 (upstream to the D1 segment) 5-TGGGAGGGGCTGTTTTTGTA-3 and TBR1 (downstream to the J1-6 element)
5-TCCAGGTAAGAAGGGGTGAC-3, and the hybridization probe was TBR3
5-CTGACCTCCGTTCTTACACT-3. PCR was performed with 30 cycles of 1 minute, 94°C; 2 minutes, 61°C; and 10 minutes, 72°C. The
amplified product was separated on a 1% agarose gel, transferred to a
nylon membrane, and probed with a radiolabeled TBR3 oligonucleotide.
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RESULTS |
Effect of different cytokine combinations on T-cell development from
CD34+ CB cells.
To study the effects of different cytokine combinations on T-cell
development from CD34+ cells derived from infants, we used
CB CD34+ cells as an appropriate control for SCID-Xl BM
CD34+ cells given the lack of availability of age-matched
marrow cells.36 We compared several combinations of
cytokines to establish the experimental conditions necessary for
optimal cell proliferation and T-cell differentiation from
CD34+ CB cells in the hybrid human/mouse FTOC.
CD34+ CB cells were selected by an immunoaffinity column,
further purified by FACS, and cultured together with mouse thymic lobes
(10,000 to 30,000 cells per lobe) in the presence of SCF + Flt-3L + IL-7, SCF + Flt-3L + IL-7 + IL-1, or SCF + Flt-3L + IL-7 + IL-1 + IL-15. After an incubation period of 4 to 5 weeks, recovered
cells were enumerated and stained with antihuman CD45 to determine the
total number of human cells. The cell expansion rate in FTOC was very
low in the presence of SCF + Flt-3L + IL-7, with a less than twofold
increase in the cell number per thymic lobe (Table
1). CB CD34+ cells were shown
to develop into double positive (DP), as well as
CD4+TCR+ mature T cells. However, the
vast majority of the recovered cells had an immature (ISP) phenotype.
CD56+ NK cells were also detected. The addition of IL-1
(2 ng/mL) considerably enhanced cell proliferation (Table 1), but did
not result in differentiation beyond the DP
CD4+CD8+ stage (Table 1). The recovered
population expressed CD4, CD7, CD2, CD5, CD1a with or without
CD8low (DP or ISP, respectively, Table 1). Leclercq et
al19 have recently shown that the addition of a low
concentration of IL-15 to FTOC colonized by murine fetal thymocytes
increased cell proliferation. To address whether IL-15 could play a
role in proliferation and/or differentiation of human
CD34+ CB cells in mouse thymic lobes, T-cell
differentiation was assessed in FTOC in the presence of this cytokine
(20 ng/mL). Although some variability between experiments was observed,
the thymic cell recovery was high (fivefold to sixfold increase) in
this culture condition. Cells acquired surface expression of CD7, CD2, CD5, CD1a, and CD4. Although the ISP subset was predominant (Table 1),
the next stages of DP and CD4+ SP mature T cells could also
be detected. A typical experiment is depicted in Fig
1, showing that 15% of cells were
CD4+ SP TCR + cells. Fifteen percent were
CD4+CD8+CD3low DP and 15%
CD4+ ISP after 5 weeks of culture (not shown). All of these
cell subsets coexpressed CD7, CD2, CD5, and CD1a. In addition, in this
experiment, 2% of the TCR + cells were
CD8+. However, in most experiments no CD8+ SP
mature cells could be detected. As also shown in Fig 1,
CD56+ and CD16+ CD14() NK cells were
detectable. The CD4+ CD3high SP thymocyte
subset expressed some features of cells that have responded to positive
selection signals, as they were CD27 (Fig 1) and CD69 positive (not
shown). However, these cells were still expressing CD1a as found in all
other experiments. This finding is consistent with data published by
Res et al31 indicating that another species-specific signal
is required for the downregulation of CD1a in the further maturation of
the CD3high TCR + cell subset. To exclude
possible contamination of CD34+ sorted cells by mature T
cells, an aliquot of sorted CD34+ CB cells used in the FTOC
system was placed in liquid culture supplemented with the same cytokine
combination. Three major subsets of cells emerged under these culture
conditions: two populations bearing NK-cell markers CD56 and CD16, and
a third population expressing the monocytic antigen CD14 (data not
shown). This liquid culture failed to promote T
lymphopoiesis, indicating that no expansion of contaminating
T cells took place. Together, these data conclusively
demonstrate that the addition of SCF, Flt-3L, IL-7, IL-1, and IL-15
resulted in human T-cell differentiation from human CB
CD34+ cell in murine FTOC. These culture conditions were
therefore chosen for assessing c gene transfer into
CD34+ c() cells.
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Table 1.
Effect of Different Cytokine Combinations on Cell Yield
and T-Cell Immunophenotype Acquisition by CD34+ Cells
in Fetal Thymic Organ-Cultured Lobes
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| Fig 1.
CD34+ cord blood cells in FTOC acquire a
T-cell immunophenotype in the continuous presence of SCF, Flt-3L, IL-7,
IL-1, and IL-15. CD34+ cells were sorted and cultured
in fetal thymic lobes with the cytokines for 35 days. FTOC were then
harvested, and T-cell differentiation was assessed by flow cytometric
analysis using the indicated antibodies.
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Retrovirus-mediated c gene transfer into
c() CD34+ marrow cells from
two SCID-Xl patients.
We have previously shown that cocultivation of bone marrow mononuclear
cells23 or SCID-Xl EBV-transformed B-cell
lines24 on the packaging cell line CRIP MFG (B2)c
allowed efficient c gene transfer. To improve transduction
efficiency under clinically applicable conditions, we used a strategy
combining a standard supernatant transduction with the use of
fibronectin fragment-coated culture wells.37-39 It has been
shown previously that this fibronectin fragment colocalizes retroviral
particles and target cells, optimizing gene transfer
efficiency.39 Previously cryopreserved SCID X-l marrow
mononuclear were thawed and cultured for 18 hours before CD34+ cell selection and sorting. The
CD34+c() sorted cells were prestimulated by culture
for 24 hours in the presence of SCF, Flt3-L, and IL-3, followed by
culture in MFG (B2)c-conditioned supernatant for 3 cycles of 24 hours each on fibronectin-coated wells as described in Materials and Methods. As a control, CD34+ c() cells were
mock-transduced by culture under the same conditions without viral
supernatant addition. As shown in Fig 2,
under these experimental conditions, 35% to 70% of CD34+
cells expressed c on their surface immediately after the three cycles of infection.
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| Fig 2.
c Chain expression on CD34+ cells
immediately after c gene transfer. Immunofluorescence staining of
surface c chain on CD34+ S1 and S2 BM cells after c
transduction (left panel) or on uninfected CD34+ S1 and
S2 cells (right panel).
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Development of CD34+ c()
cells into T cells after c gene transfer and culture in
fetal thymic lobes.
Fifty thousand CD34+c transduced or mock-transduced
cells (from the two patients) were used to reconstitute
deoxyguanosine-treated fetal thymic lobes. The organ cultures were
maintained 4 to 5 weeks in the continuous presence of SCF + Flt3-L + IL-7 + IL-1 + IL-15. Thymocytes were then recovered and assayed for
c and other cell surface markers expression. As shown in a
representative experiment (Fig 3), 15%
(patient S1) and 30% (patient S2) of recovered cells expressed c at
their surface (Fig 3A and B). These cells were all positive for CD45
staining showing their human origin. Conversely all CD45+
cells were c(+). c(+) cells acquired the capacity for CD4 CD8 DP
development in the thymic microenvironment. The latter subset represented 30% of c(+) cells for S1 and 20% for S2. These data are consistent with those obtained from CD34+ CB cells (Fig
3D). In addition to the DP subset, SP CD4+
CD3high TCR+ cells were also detected.
Figure 3A shows that this population represented 6% of the human
thymocytes. In some experiments, development of CD56+ NK
cells was also observed (data not shown). In contrast, no cell
differentiation could be achieved from CD34+c()
mock-transduced cells. Only a small population of c() CD4low CD8() CD3() TCR() was detected (Fig
3C). It was not possible to further characterize this population
because the harvest sample size was very low (<50,000 cells). c
Gene integration into thymocytes was analyzed by direct PCR. As shown
in Fig 4, cells derived from S1- and
S2-c-transduced CD34+ cells contain the integrated c
gene after 5 weeks of organ culture. We could not detect the c gene
containing provirus in mock-transduced cells.
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| Fig 3.
c gene transfer restores T-cell differentiation of
CD34+ SCID-Xl patients' cells in FTOC.
CD34+ c transduced S1 (A), S2 (B) cells as well as
untransduced CD34+ S1 cells (C) were cultured with the
preestablished combination of cytokines (SCF, Flt-3L, IL-7, IL-1,
IL-15) into fetal thymic lobes for 35 days. FTOC were harvested and
cells were stained for expression of CD4, CD8, CD3, and TCR (dot
plots) or for c expression (histograms). T-cell differentiation
obtained from CD34+ normal CB cells is indicated on panel
(D) as a control.
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| Fig 4.
Presence of c provirus gene in cells derived from
transduced S1 and S2 CD34+ BM cells. Thymic lobes were
seeded with transduced (c+) or untransduced (c) S1 and S2
CD34+ BM cells. After 35 days of organ culture, genomic
DNA was isolated from the thymocytes and analyzed by PCR using primers
to amplify the c gene. DNA from the retroviral producer cell line
served as the positive control.
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TCR rearrangements in control and c
transduced thymocytes.
Previous studies have shown that human thymocytes developing in murine
thymic organ cultures express a broad TCR repertoire.40 To
analyze the rearrangement status at the TCR locus, we studied DJ
rearrangement in the human T cells generated in the chimeric thymic
organ cultures. Genomic DNA was isolated from differentiated CD34+ CB control cells or CD34+c transduced
SCID-Xl marrow cells after 5 weeks of culture and amplified by PCR to
detect D1-J1 rearrangements (Fig 5).
Human thymocytes and fibroblasts were used as positive and negative controls, respectively. As expected, human thymocytes generated in the
FTOC system were characterized by the presence of distinct DJ
rearrangements. Multiple rearrangements of D1 to the
J1.1-J1.6, particularly those corresponding to short-sized
products (DJ1.6, DJ1.5, DJ1.4), were also detected in SP
mature T cells generated from the c transduced CD34+
patient cells (Fig 5).
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| Fig 5.
Fetal thymic organ cultured cells expressed multiple
DJ rearrangements. Genomic DNA was isolated from thymocytes
generated from CD34+ CB cells (lobes D35/control) or from
CD34+ c transduced BM patient cells (lobes
D35/ + S1) after 35 days of organ culture. DNA was then
amplified by PCR using TBF1 and TBR1 primers to detect D1-J1
rearrangements. PCR products were blotted and hybridized with the TBR3
probe. Total thymocytes and fibroblasts served as positive and negative
controls, respectively. (Right) Positions of the genomic fragment and
the specific rearrangements of D1 to the J1-1-J1-6 elements.
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DISCUSSION |
In this work, we have defined culture conditions required for human
T-cell differentiation from CD34+ CB cells, and have used
this system to show restoration of T-cell differentiation of
CD34+c() BM cells from SCID-Xl patients after
retroviral-mediated c gene transfer. The culture conditions required
the additional cytokines SCF, Flt-3L, IL-7, IL-1, and IL-15. The
thymic stroma produces a number of cytokines, including IL-7, which
have been shown to be necessary for early lymphoid development. This
has been well demonstrated by the generation of mice mutant for
IL-7,41 IL-7R,42 and c,43 all
of which display a dramatic decrease in thymic cellularity. The same
phenomenon was observed in mice treated with anti-IL-744
or anti-IL-7R.45 Furthermore, the use of these
antibodies in vitro in FTOC resulted in the inhibition of T-cell
development.46 IL-7 is also indispensable for human T-cell
development. Plum et al30 described an early block at the
CD34+ stage in T-cell differentiation in chimeric
human/mouse FTOC treated with anti-IL-7 or anti-IL-7R MoAbs.
Another receptor-ligand pair, cKit/SCF, has also been shown to drive
expansion of immature thymocytes in the mouse.17,47
Moreover, the recent report showing complete abrogation of thymocyte
development in mice lacking both cKit and c indicates essential and
synergistic functions of these two distinct signaling
pathways.48 As Flk-2/Flt-3 ligand (Flt-3L) has been shown
to induce proliferation/expansion of the most immature CD4low murine thymocytes,49 we also decided to
include Flt-3L in the cytokine combination. Our data indicate that
human CB-derived CD34+ cells cultured in the presence of
SCF + Flt-3L + IL-7, although being able to repopulate FTOC, poorly
proliferate and differentiate along the T-cell lineage. Previous
studies have shown that IL-1 is involved in murine
lymphopoiesis.18 Zuniga-Pflücker et al50 provided evidence that IL-1 (or TNF) promotes the transition of
the CD117+ CD25() subset to the CD25+ stage,
which is a prerequisite for further maturation to the DP stage. When
IL-1 was added in our FTOC culture system, it resulted in an
increase in human thymic cellularity, while early thymic progenitors
differentiated to the ISP/DP stages. In the presence of SCF + Flt-3L + IL-7 + IL-1 and IL-15, CD34+ cells developed mainly into
CD4+ immature single positive (ISP),
CD4+CD8+ (DP) and CD4+SP cells, as
well as CD56, CD16 NK cells. Compared with cultures in which IL-15 was
omitted, all of these subsets expanded more efficiently. The
CD56+ NK cell subset expanded particularly well in the
presence of IL-15. These results suggest that, in addition to the known
role of IL-15 in cell differentiation towards the NK pathway, IL-15 in
association with other cytokines promotes both proliferation and
differentiation of progenitor cells towards the T-cell lineage.
The culture conditions we designed to study T-cell development of
CD34+ progenitors from postnatal origin provided us with
the tool to study restoration of human T-cell development after gene
transfer and expression of the c gene in the CD34+
selected transduced SCID-Xl marrow cells. Although efficient progenitor
cell transduction can be achieved by cocultivation with the packaging
cell line, we used a viral supernatant transduction on wells coated
with a recombinant human fibronectin fragment38,39 to both
improve transduction efficiency and develop a clinically-applicable protocol. Under these conditions, high transduction levels were reached, with between 35% and 70% of the cells expressing c. These
progenitor cells were able to differentiate along the T-lymphoid lineage in the presence of SCF, Flt-3L, IL-7, IL-1, and IL-15. Fifteen percent to 30% of harvested cells were shown to be of human
origin. All of these cells expressed c chain after a 4- to 5-week
culture period. c chain expression by SCID-Xl CD34+
marrow cells enabled them to mature into DP cells and CD4+
TRC+ SP cells. Polyclonal DJ rearrangements of the
TCR locus were shown in the cell population. The inability of
mock-transduced CD34+c() cells placed in the same
culture condition to acquire a mature T-cell phenotype confirms that,
in humans, T-cell development is strictly dependent on c chain
expression. The almost complete failure of the generation of
CD8+ SP T cells from both cord blood CD34+ and
c transduced SCID-Xl CD34+ cells could be attributed to
the inability of murine MHC class I molecules to interact efficiently
with human CD8+ T cells or to the requirement of other
signals in addition to class I MHC.31 It is possible that a
mutated form of c may have been expressed in S1, which exerted no or
only a limited influence on the function of the normal c protein. It
does not indicate, however, that other mutated products could not exert a dominant inhibitory effect.
The ability of c-transduced CD34+ cells from SCID-Xl
patients to mature into T cells (this study), as well as NK
cells,24 sets the basis for a clinical study of ex vivo
c gene transfer into CD34+ cells from SCID-Xl patients.
Despite the low efficiency of retrovirally-mediated gene transfer into
human hematopoietic stem cells (HSC), it is indeed expected that a
strong selective advantage will be conferred to the few transduced
cells potentially able to differentiate into T, NK, and possibly B
cells. The observation of a significant development of long-lasting T
cells after c gene reverse mutation into a T-cell precursor observed
in a patient51 demonstrates that a strong positive pressure
for survival, proliferation, and differentiation of c(+) precursors
does exist in vivo. In conclusion, this study shows that T-cell
differentiation to mature CD4 T cells can be achieved in murine FTOC
from CB and postnatal CD34 cells in the presence of a cytokine
combination, which compensates for the lack of human stromal cells. In
addition, this system has enabled us to provide evidence that after
c gene transduction, SCID-Xl CD34 cells can mature into T cells,
paving the way for a clinical study of c gene transfer in SCID-Xl
patients.
| |
ACKNOWLEDGMENT |
We are indebted to the patients' families for support in this study,
to Dr Adrian Thrasher and Dr Bert Gerritsen for providing sample from
one patient, to Dr Tony Troutt from Immunex, to Dr Setsuko Yoshimura
(from Takara Shuzo Co) for providing useful reagents, to C. De Coene
for excellent technical assistance to F. Selz and C. Garcia for cell
sorting, and to Dr Jane Peake for checking the manuscript.
| |
FOOTNOTES |
Submitted January 22, 1998;
accepted August 5, 1998.
Supported by grants from INSERM, Association Française contre les
Myopathies, Ministère de la Recherche et de la Technologie, Agence Française du Sang (contrat No. 9600 13 701 76) and
Assistance Publique et Hôpitaux de Paris (AP-HP).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to S. Hacein-Bey, PhD, INSERM U 429, Hôpital Necker-Enfants Malades, 149, rue de Sèvres, 75743 Paris Cedex 15, France.
| |
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Abstract
Introduction