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IMMUNOBIOLOGY
From the Department of Cell Biology, Faculty of
Biology, and Department of Cell Biology, Faculty of Medicine,
Complutense University of Madrid, Madrid, Spain.
The present study investigated the potential role of stromal
cell-derived factor 1 (SDF-1) in human intrathymic T-cell
differentiation. Results show that SDF-1 is produced by human thymic
epithelial cells from the subcapsular and medullary areas, and its
receptor, CXCR4, is up-regulated on CD34+ precursor cells
committed to the T-cell lineage. Chimeric human-mouse fetal thymus
organ culture (FTOC) seeded with purified CD34+
thymic progenitors and treated with neutralizing antibodies against SDF-1 or CXCR4 showed a significant reduction of the number of human
thymocytes and an arrested thymocyte differentiation in the transition
between CD34+ precursor cells and CD4+ immature
thymocytes. SDF-1-treated FTOC showed an increase of human thymocyte
numbers, mainly affecting the most immature subpopulations. Moreover,
these results suggest that CXCR4/SDF-1 signaling is not critical for
the CD34+ cell precursor recruitment to the thymus. On the
other hand, SDF-1 significantly increased the viability of
CD34+ T-cell precursors modulating the expression of
BCL-2 and BAX genes, and stimulated
the proliferation of CD34+ thymic precursor cells,
particularly in synergy with interleukin 7 (IL-7), but not with other
cytokines, such as stem cell factor or flt3-ligand.
Accordingly, only IL-7 was able to up-regulate CXCR4 expression on
CD34+ thymic progenitors. In addition, deprivation of SDF-1
partially inhibited human thymocyte expansion induced by IL-7 in
human-mouse FTOC. This study indicates that SDF-1/CXCR4 signaling is
required for the survival, expansion, and subsequent differentiation of human early thymocytes and identifies a new mechanism by which IL-7
mediates its effects on human thymopoiesis.
(Blood. 2002;99:546-554) When T cells derived from fetal liver or bone
marrow hematopoietic precursors enter the thymus, they pass through a
series of discrete stages defined by the differential expression of a variety of phenotypic markers. The most primitive hematopoietic progenitors in the human thymus express high levels of CD34 and CD45RA
and low levels of CD38; they lack CD2, CD5, CD1, CD4, CD8, or
CD3.1,2 On further differentiation, these cells acquire CD2, CD5, and CD1. The CD34+CD1+ cells
gradually become CD34 Stromal cell-derived factor 1 (SDF-1), a member of the chemokine CXC
subfamily, was first identified as a pre-B-cell growth factor8 and was later demonstrated to be involved in
migration and also survival, proliferation, and activation of different cell types.9-14 CXCR4, the receptor for SDF-1, is a G
protein- coupled 7-transmembrane receptor that also functions as a
coreceptor for the entry of T-tropic strains of human immunodeficiency
virus 1 into CD4+ cells.15-17 The widespread
distribution of SDF-1, its constitutive expression pattern, and its
highly conserved nucleotide and amino acid sequences18,19
suggest that it could play a key biologic role. In correlation with
this biologic relevance, targeted gene knockout mice of either
SDF-1 or its receptor CXCR4 die in utero and show severe abnormalities
in cardiogenesis, as well as vascular, cerebellar, and hematopoietic
development.20-23 Mice deficient in CXCR4 and SDF-1 have
fewer myeloid progenitors in fetal liver and bone marrow than normal
mice and display severe defects in the generation of B cells but not in
T cells.20,22-24 Although these gene-disruption
experiments suggest that the SDF-1/CXCR4 complex is not required for
the generation of normal cell populations in the mouse thymus, other
results indicate, however, a role for this chemokine also in T-cell
differentiation: (1) SDF-1 messenger RNA (mRNA) is abundantly
expressed in fetal and adult thymus25-28; (2) CXCR4 is
expressed on different subpopulations of human and mouse
thymocytes25,28,29; (3) SDF-1 induces calcium flux
primarily in the most immature human thymocytes28; (4)
SDF-1 is a chemoattractant for thymocytes30,31; (5)
transplantation of fetal liver-derived hematopoietic cells from CXCR4
knockout mice into lethally irradiated syngenic mice results in reduced
numbers of donor-derived T cells in the thymus32; and
(6) mice reconstituted with bone marrow-hematopoietic progenitor cells
transduced with SDF-1-intrakine gene display reduced numbers of total
thymocytes and an arrest in thymocyte maturation.33
To demonstrate the possible role of SDF-1/CXCR4 signaling in human
thymocyte development, we report the production of SDF-1 protein by
thymic epithelial cells, as well as the expression of CXCR4 in the more
mature subsets of CD34+ thymocytes. Studies using a hybrid
human/severe combined immunodeficiency (SCID) mouse fetal thymus organ
culture (FTOC) system demonstrate a key role for SDF-1 in the
proliferation, survival, and subsequent differentiation of human
thymocytes. Furthermore, we demonstrate that SDF-1 partially mediates
the effects of interleukin 7 (IL-7) on human thymopoiesis.
Animals
Histology and immunofluorescence
Isolation of human thymocyte subsets Human thymus samples were obtained from children, aged 1 month to 3 years, undergoing corrective cardiovascular surgery. Thymus tissues were obtained and used following the guidelines of the Medical Ethics Commission of the University Hospital Gregorio Marañón (Madrid, Spain). Informed consent was provided according to the Declaration of Helsinki. Thymuses were dissected free of surrounding connective tissue and then gently disrupted with a Potter homogenizer until completely disaggregated. Cell suspensions were enriched in immature thymocytes by using the sheep red blood cell rosetting technique as described previously.34 Recovered cells were then depleted of mature T, natural killer (NK), B, myeloid, and dendritic cells by treatment with saturating concentrations of anti-CD3 (SK7) (Becton Dickinson, San Jose, CA), anti-CD56 (B159), anti-CD19 (B43), anti-CD14 (M5E2), and anti-CD11c (B-ly6) (all from Pharmingen, San Diego, CA) bound to sheep anti-mouse Ig-coated magnetic beads (Dynal, Oslo, Norway). CD34+ cells or CD4+ immature thymocytes were then purified by magnetic sorting using VarioMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) in conjunction with either CD34 or CD4 Multisort kit (Miltenyi Biotec) following the manufacturer's instructions. CD4+CD8+ thymocytes were purified from the whole population using CD4 Multisort Kit and CD8-Microbeads (Miltenyi Biotec). The purity of the enriched CD34+, CD4+, or CD4+CD8+ cells was always more than 98%.Antibodies and flow cytometry Directly labeled monoclonal antibodies (mAbs) against the following antigens were used: CD4 (SK3-FITC, -phycoerythrin [PE], or -peridinin chlorophyll protein [PerCP]); CD8 (SK1-FITC, -PE, or -PerCP); CD45 (HI30-FITC); HLA-DR (G46-6-FITC); CD117(c-kit 4B5.B8-PE); CD45RA (HI100-FITC); CD7 (M-T701-FITC); CD38 (HIT2-FITC); CXCR4 (12G5-FITC or -PE); CD33 (WM53-PE); CD5 (UCHT2-FITC); CD1a (HI149-FITC); CD34 (581-FITC, -PE, or -Cychrome); CD3 (SK7-FITC, -PE, or -PerCP) from Becton Dickinson; CD127 (R34.34-PE) from Coulter Immunotech (Beckman Coulter, Marseille, France). Three-color immunofluorescence staining was performed by incubating the cells in phosphate-buffered saline (PBS) containing 1% fetal calf serum (FCS) and 0.1% NaN3 in the presence of saturating amounts of FITC-, PE-, PerCP-, and Cychrome-conjugated mAbs for 30 minutes at 4°C. All specific mAbs against human antigens were checked for negative staining on SCID thymocytes. Isotype-matched irrelevant mAbs were used as negative controls to define background fluorescence. For intracellular detection of Bcl-2 and bax antigens, cells were treated with a FACS permeabilizing solution according to the manufacturer's instructions (Becton Dickinson), and stained with antihuman bcl-2-PE mAb (Pharmingen) or antihuman bax antibodies (Pharmingen) followed by FITC-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch Laboratories) for 30 minutes at room temperature. The percentage of stained cells was evaluated by comparison with the isotype control. Analysis was conducted in a FACScan flow cytometer (Becton Dickinson) from the Servicio Común de Investigación, Faculty of Biology, Complutense University of Madrid. Debris, and dead cells were excluded from the analysis by forward (FSC) and side light scatter (SSC), and analysis was performed on at least 10 000 events. The data were analyzed using PC-lysis research software (Becton Dickinson).Hybrid human-mouse fetal thymic organ culture Thymic lobes removed from 15-day-old embryos of SCID mice were cocultured for 2 days in hanging drop in Terasaki wells with purified human CD34+ cells, CD4+ immature thymocytes, or CD4+CD8+ thymocytes (1-2 × 104 cells/lobe), transferred to 0.8-µm polycarbonate filters (Millipore Iberica, Madrid, Spain) that rested on stainless steel screen pieces attached to the central well of organ tissue culture dishes (Becton Dickinson), and cultured for the indicated number of days. Culture medium consisted of RPMI-1640 supplemented with 7% human AB serum and 3% FCS. Where indicated, cultures were supplemented with recombinant human SDF-1 (rhSDF-1) at a concentration of 10 ng/mL;
neutralizing mouse anti-human CXCR4 antibodies at a concentration of
10 µg/mL; neutralizing goat anti-human SDF-1 antibodies at a
concentration of 10 µg/mL; neutralizing mouse anti-human/mouse IL-7
antibodies at a concentration of 20 µg/mL (all from R & D Systems);
recombinant human IL-7 (rhIL-7) at a concentration of 1000 U/mL (ampule
code 90/530; National Institute for Biological Standards and Control,
Hertfordshire, United Kingdom) throughout the culture period, or only
during part of the experimental procedure. The doses of rhSDF-1,
rhIL-7, and the blocking antibodies were shown to be optimal on the
basis of previous titrations in FTOC (data not shown). Purified mouse
IgG2a (Pharmingen) was used as control. Medium was replaced every week.
To analyze differentiation of human cells, mouse thymuses were
dispersed into single-cell suspensions and stained with mAbs specific
for human cell surface antigens. Flow cytometric analysis was then
performed on electronically gated CD45+ human cells.
Culture of thymic CD34+ precursors Purified thymic CD34+ precursors (3-5 × 104) were cultured in 96-well flat-bottom culture plates in 0.2 mL AIMV serum-free lymphocyte medium (Life Technologies, Eragny, France) in the presence of rhSDF-1 (10 ng/mL),
rhIL-7 (1000 U/mL), recombinant human stem cell factor (rhSCF; 5 ng/mL;
Peprotech, London, United Kingdom) or rhflt3-ligand (10 ng/mL;
PeproTechec). After 2 days in culture at 37°C in a 5%
CO2- in air incubator, cells were harvested and processed
for proliferative assays, viability analysis, and bcl-2 and bax stainings.
Proliferation assay Cultures were pulsed for the last 12 hours with 10 µM 5-bromo-2'-deoxyuridine (BrdU). A specific kit from Boehringer Mannheim (Mannheim, Germany; BrdU Labeling and Detection kit III) was used to measure BrdU incorporation into newly synthesized DNA. Briefly, the labeling medium was removed, and cells were dried (2 hours at 60°C), fixed in ethanol in HCl (0.5 M) for 30 minutes at 20°C, treated with nucleases (30 minutes at 37°C), and then incubated with
peroxidase-conjugated Fab fragments of mouse anti-BrdU antibodies (30 minutes at 37°C). The peroxidase reaction was developed with ABTS substrate (Boehringer Mannheim), and the sample absorbance was measured using an enzyme-linked immunoabsorbent assay reader at 405 nm with a reference wavelength at 492 nm.
Apoptosis assay Cells were washed twice with PBS containing 1% FCS and then stained with annexin V-FITC (Boehringer Mannheim) according to the supplier's instructions. Cells were analyzed on a FACScan and gated according to FSC, SSC, and their ability to exclude propidium iodide. Apoptotic cells were considered as those positive for annexin V and negative for propidium iodide.
Expression of SDF-1 on human thymus The expression of SDF-1 was assayed by immunofluorescence on frozen sections of human thymus (Figure 1A). SDF-1 protein was detected in
epithelial cells as demonstrated by double staining with TE-4 mAbs,
which recognize subcapsular and medullary epithelial cells (Figure
1B,C). SDF-1+ epithelial cells appeared as small cell
clusters in the subcapsular region (Figure 1B), and scattered SDF
1-expressing epithelial cells were also found in the medullary
compartment (Figure 1C). No reactivity for SDF-1 was found in
cortical epithelial cells as evidenced by double staining with TE-3
mAbs (Figure 1D,E). Furthermore, no coexpression of SDF-1 protein and
fibroblast-associated type I collagen was found in the human thymus
(Figure 1F,G).
Phenotypic characterization of CD34+CXCR4  4+8 intermediate
cell population and more in CD4+CD8+ DP
thymocytes (Figure 2A). The analysis of the expression of differentiation-specific antigens indicated that the vast majority of
CD34+CXCR4 cells expressed CD45RA, c-kit,
CD33, CD7, and CD38; about 40% to 50% of this subpopulation was
positive also for HLA-DR and CD127 antigens, but was mostly negative
for CD5 and CD1 antigens (Figure 2B). Progenitor cells exhibiting this
surface phenotype represent primitive tripotential T/dendritic
cell/NK precursors in the postnatal human
thymus.35 In contrast, a reduced percentage of
CD34+CXCR4+ cells positively reacted with
c-kit, CD45RA, CD33, and HLA-DR antigens, and about 60% of this
subpopulation expressed the T- lineage markers, CD5 and CD1 (Figure
2B). These results indicate that the
CD34+CXCR4 subpopulation contains more
primitive cells than the CD34+CXCR4+ cell
subset. Supporting this, most CD34bright cells, which
contain the earliest T-cell precursors in the human thymus,4,36 were found to be in the CXCR4
fraction (data not shown).
SDF-1 deprivation drastically reduces the yield of human thymocytes and arrests their differentiation in chimeric human-mouse FTOC To establish the role of SDF/CXCR4 signaling in human T-cell development, we investigated the effects of the addition of neutralizing antibodies against human CXCR4 or SDF-1 proteins to chimeric human-mouse FTOC. Addition of anti-CXCR4 (10 µg/mL) or anti-SDF-1 (10 µg/mL) antibodies resulted in a dramatic decrease in the cell recovery from the thymus seeded with CD34+ thymocyte precursors. By 10 to 18 days of FTOC, a 60% to 80% reduction in human cellularity was observed. The reduction in cell number was always more pronounced in the anti-CXCR4-treated cultures (Table 1).
We next examined the thymocyte subsets affected by the treatments.
After 18 days of culture, the frequency of CD34+ thymic
precursors was increased 2 to 3 times (control, 5.4% ± 3%;
anti-CXCR4, 11.5% ± 4.8%; anti-SDF-1
Given that both anti-CXCR4 and anti-SDF-1 antibodies arrested
human thymocyte development, we tested the effects of rhSDF-1
Because blocking anti-SDF-1 antibodies and rhSDF-1
These results indicated a role for the SDF-1 signal in the early steps
of human T-cell differentiation. To further characterize the thymocyte
subpopulations that are the main targets for this chemokine, we next
established chimeric human-mouse FTOC seeded with either
CD34+ or immature CD4+ thymocytes. As shown in
Figure 6, although both types of cultures were affected by the treatment with rhSDF-1
Therefore, the current data show that SDF-1 plays a pivotal role in the expansion and differentiation of early human T-cell precursors at a stage between the transition of CD34+ cells toward CD4+ cells. SDF-1 does not play a functional role in thymic colonization Previous reports have described a role for SDF-1 in the migration of CD34+ progenitor cells.12 To determine whether SDF-1 has a role in thymus colonization, we examined the effects of anti-CXCR4 antibodies or rhSDF-1 on the ability of human
thymic CD34+ precursors to repopulate alymphoid
fetal thymus lobes. As shown in Table 2,
exposure of CD34+ T-cell precursors to inhibiting
anti-CXCR4 antibodies or rhSDF-1 only in the hanging drop did not
significantly modify the recolonization of recipient thymus lobes by
T-cell precursors. The addition of anti-CXCR4 antibodies after hanging
drop resulted in a drastic reduction of human cell number, comparable
to the inhibition that was found after continuous treatment during the
hanging drop period and the FTOC afterward (Table 2). Similar increases
in the human thymocyte yields were observed when rhSDF-1 was present
during either the FTOC period or the whole culture period.
These results indicate, therefore, that SDF-1/CXCR4 signaling is not involved in the migration of human T-cell precursors into the thymus. SDF-1 enhances the proliferation and the survival of human thymocyte precursors To determine whether the results obtained with chimeric human-mouse FTOC could be reflecting a role of SDF-1 in promoting the expansion or survival of human T-cell precursors, experiments were performed in which CD34+ thymocytes were cultured for 48 hours in serum-free medium with rhSDF-1 alone or in combination with
various cytokines, and cellular proliferation and viability were
evaluated. The addition of rhSDF-1 to thymocyte precursor cultures
always increased the viability of CD34+ cells. The
percentage of annexin-positive propidium iodide-negative apoptotic cells in the absence of the chemokine was
29% ± 6% (n = 4), decreasing to
12% ± 4% (n = 4) in cultures supplemented with rhSDF-1 (Figure 7A). To elucidate
possible mechanisms of the inhibitory effects of SDF-1 on serum
depletion-induced apoptosis in T-cell progenitors, we examined the
expression of bcl-2 and bax proteins on CD34+ thymocyte
suspensions cultured in the presence or absence of rhSDF-1. As
previously described, the ratio of bcl-2/bax can determine survival or
death on triggering of apoptosis.37 As shown in Figure 7A,
rhSDF-1 significantly induced an increase of the bcl-2/bax ratio in
CD34+ thymocytes, due to both an up-regulation of bcl-2
expression (4-5 times in mean fluorescence intensity and 1.5-2 times in
percentage of positive cells) and down-regulation of bax expression
(2.5-3 times in percentage of positive cells). On the other hand, the ability of SDF-1 alone to promote CD34+ thymic precursor
survival did not increase in the presence of SCF or flt3-ligand, and
the viability of IL-7-treated cultures was not improved by the
addition of rhSDF-1 (Figure 7B).
We next determined the direct proliferative response of
CD34+ thymocyte precursors to SDF-1. Figure
8A shows that rhSDF-1
It has been previously shown that SDF-1 stimulates the expansion of
peripheral blood CD34+ cells in synergy with thrombopoietin
(TPO), SCF, and flt3-ligand.11 However, our
results show that neither SCF nor flt3-ligand alone or in combination
with rhSDF-1 These data indicate that the SDF-1-mediated expansion of early human
thymic precursors involve both survival and proliferative signals.
Previous reports have shown that IL-7 is also an essential growth and
survival factor in early human T-cell development,38 suggesting that SDF-1 and IL-7 could be functionally redundant. To test
this hypothesis, we used chimeric human/mouse FTOC and analyzed the
effects of the addition of rhSDF-1
Stromal cell-derived factor 1 has been described as an important factor in the maturation and proliferation of different cell types of the hematopoietic and immune systems. We show here that in addition to its apparent role in myelopoiesis, B differentiation, or the peripheral immune system, SDF/CXCR4 signaling is critical for early human T-cell development. In the present report, we show SDF-1 expression on human thymic subcapsular and medullary epithelial cells. In agreement, other authors have described SDF-1 mRNA expression in mouse thymic epithelium positive for major histocompatibility complex class II,26 especially in the subcapsular compartment.25 However, Zaitseva et al28 reported SDF-1 mRNA expression on collagen III+ fibroblastlike cells derived from human thymic stromal cell monolayers. The preferential expression of SDF-1 in the thymic subcapsular area
suggests a role for this chemokine in the early steps of T-cell
differentiation, because primitive progenitors in the human thymus have
been described to be located in that thymic compartment.4
We describe the expression of CXCR4 on thymic CD34+
progenitors, confirming the observations of Zaitseva et
al28 and extending them by further specifying the
expression of SDF-1 receptor on those thymic CD34+
progenitors committed to the T-cell lineage. In agreement, Suzuki et
al25 described that in adult mice the population of
CD44+CD25 To test whether SDF/CXCR4 signaling might be important for human
thymocyte development, we added anti-SDF-1 antibodies, anti-CXCR4 antibodies, or rhSDF-1 Because various studies have involved SDF-1 in the homing and
mobilization of CD34+ progenitor
cells,12,29,39 the severe reduction of thymocyte number
obtained in the absence of SDF-1/CXCR4 signaling could be reflecting an
altered migration of T-cell progenitors into the thymus. Our data
suggest that SDF-1/CXCR4 signaling is not critical for
CD34+ precursor cell recruitment to the thymus, because the
treatment with either rhSDF-1 The reduction in thymocyte numbers and the blockade of T-cell
differentiation observed in both anti-CXCR4- and anti-SDF-1-treated FTOC may be also related to an alteration in the intrathymic
trafficking of developing thymocytes toward the appropriate stromal
cell niches where they would receive growth- and maturation-promoting
signals. In this respect, a similar migratory behavior of different
thymocyte subsets to SDF-1 has been described, unlike other chemokines
such as thymus-expressed chemokine (TECK), secondary lymphoid tissue chemokine (SLC), or macrophage inflammatory protein-3 Stromal cell-derived factor 1 was first described as a growth
factor for murine pre-B cells.8 More recently, Lataillade et al11 have extended this proliferative effect of SDF-1
also to adult peripheral blood CD34+ cells. We demonstrate
that SDF-1 alone stimulates the proliferation of CD34+
thymic precursors. In the presence of IL-7, but not other cytokines, such as SCF or flt3-ligand, this effect is significantly enhanced, indicating that SDF-1 and IL-7 synergistically act in the human thymus
to promote the proliferation of early T-cell precursors. Interestingly,
we also show that IL-7, but not SCF or ftl3-ligand, up-regulates CXCR4
expression on CD34+ thymic precursor cells. In addition,
SDF-1 deprivation with anti-SDF-1 antibodies partially inhibits the
IL-7-mediated thymocyte expansion, whereas the profound reduction in
thymic cellularity induced by anti-IL-7 antibodies cannot be
counteracted by the addition of rhSDF-1 In summary, our findings provide evidence that SDF-1/CXCR4 signaling plays a key role in human thymocyte development, promoting survival and expansion of human early T-cell progenitors. Therefore, SDF-1 may be a good additional candidate to include in the list of trophic cytokines that could be used to therapeutically promote reconstitution of T-cell immunity by enhancing thymopoiesis in some of those pathologic situations associated with loss or damage to the peripheral T-cell pool.
We thank Dr Barton F. Haynes for the generous gift of mAbs, and the Pediatric Cardiosurgery Unit from the Hospital Gregorio Marañón (Madrid) for the thymus samples. The flow cytometry analysis was performed on a FACScan flow cytometer from the Servicio Común de Investigación, Facultad de Biología, Universidad Complutense de Madrid.
Submitted February 5, 2001; accepted July 2, 2001.
Supported by grant 98/0041 from the Fondo de Investigaciones Sanitarias; grants 08.3/0014/1997, 08.3/0027/1998, 08.3/0041./2000 from the Comunidad Autónoma de Madrid; and grants PB97-0032 and PM99-0060 from the Ministerio de Educación y Cultura.
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.
Reprints: Angeles Vicente, Departamento de Biología Celular, Facultad de Biología, Universidad Complutense, 28040 Madrid, Spain; e-mail: avicente{at}bio.ucm.es.
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Abstract
Introduction
.
). (B) FTOC were
cultured with anti-SDF-1
.05; **P 