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Sonic Hedgehog Regulates Early Human Thymocyte Differentiation by Counteracting the IL-7-Induced Development of CD34⁺ Precursor Cells¹

Cruz Gutiérrez-Frías^2,*, Rosa Sacedón^2,, Carmen Hernández-López^*, Teresa Cejalvo, Tessa Crompton, Agustín G. Zapata^*, Alberto Varas and Angeles Vicente^3,

^* Department of Cell Biology, Faculty of Biology, and Department of Cell Biology, Faculty of Medicine, Complutense University, Madrid, Spain; and Department of Biological Sciences, Imperial College London, London, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Hedgehog (Hh) family of signaling molecules normally functions in the development of numerous tissues by regulating cellular differentiation and proliferation. Recent results have demonstrated that the different components of the Hh signaling pathway are expressed in the human thymus. In this study, we investigate the potential role of Sonic hedgehog (Shh) in human intrathymic T cell maturation. Results show that the expression of the two components of the Hh receptor, Patched and Smoothened, is mostly restricted to CD34⁺ precursor cells that are committing to the T cell lineage. Shh significantly increased the viability of CD34⁺ T cell precursors modulating bcl-2 and bax protein expression, and also inhibited their proliferation. The treatment of chimeric human-mouse fetal thymus organ cultures with Shh resulted in an arrested thymocyte differentiation and an accumulation of CD34⁺ progenitor cells. This effect was mainly attributed to the ability of Shh to counteract the IL-7-induced proliferation and differentiation of CD34⁺ cells. Shh down-regulated in the precursor cell population the expression of IL-7R as well as stromal-derived factor-1 chemokine receptor, CXCR4, and inhibited IL-7-dependent STAT5 phosphorylation. Therefore, Shh may function as a maintenance factor for intrathymic CD34⁺ precursor cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In humans, the hemopoietic progenitors that seed the postnatal thymus are CD4^–CD8^– double-negative (DN)⁴ cells that express high levels of CD34, lack CD1a, and are able to generate T cells as well as NK cells, dendritic cells (DCs), and monocytes. Later, CD34⁺CD1a⁺DN thymic progenitors lose the potential to become NK/DC/myeloid cells, commit to the T cell lineage, and gradually down-regulate CD34 and up-regulate first CD4 followed by CD8 before acquisition of CD8. After entrance into the CD4⁺CD8⁺ double-positive (DP) stage, thymocytes express low levels of CD3/TCR complex, and are the main cell targets for positive selection (1, 2). Positively selected cells then up-regulate CD3, CD69, and CD27. Finally, thymocytes, before exiting the thymus, lose CD4 or CD8, down-regulate CD1a, and switch their phenotype from CD45RO to CD45RA (3).

Hedgehog (Hh) genes encode a highly conserved family of secreted proteins whose activities include those of classic morphogens, determining cell fate and patterning during the development of many organs, but also regulating of cell survival and proliferation (4, 5, 6, 7). Hh was originally identified in Drosophila as a segment polarity gene (8), and in mammals, three proteins have been now described to compose the Hh family: Sonic (Shh), Indian (Ihh), and Desert hedgehog (Dhh), Shh being the most pleiotropic of Hh molecules (4). All Hh proteins bind to a surface receptor complex constituted by two polytopic transmembrane proteins, Patched (Ptc) and Smoothened (Smo). Ptc is the ligand binder and Smo transduces the intracellular signal. Smo activity is inhibited by Ptc in the absence of ligand, and this inhibitory effect is abrogated after Hh binding (4). The mechanism underlying Ptc inhibitory function is under extensive debate (9, 10, 11, 12, 13). The intracellular signaling cascade initiated by Smo culminates in the activation of members of the Gli family of zinc finger transcription factors, Gli 1, Gli 2 and Gli 3, which regulate the transcription of different target genes (6, 14, 15).

The involvement of Hh signaling in thymocyte differentiation has been described previously in mice (16, 17, 18). Shh is produced by thymic epithelial cells, and Hh receptors, Ptc and Smo, are mainly expressed by immature CD25⁺DN thymocytes. The addition of anti-Shh to fetal thymus organ cultures (FTOCs) accelerates the progression of DN cells into the DP stage and, by contrast, treatment with high doses of rShh arrests thymocyte differentiation at the CD25⁺DN stage (16). Likewise, the analysis of Shh^–/– embryos shows that Shh is important in the regulation of thymus cellularity as well as in the development of DN thymocytes (18).

We have recently reported that the three mammalian Hh proteins, Shh, Ihh, and Dhh; their specific receptors, Ptc and Smo; and other Hh-binding proteins with modulating functions, Hip and Gas-1; as well as the Gli family of transcription factors are also expressed in the human thymus, exhibiting a complex pattern of expression (19). In the current study, we show that the Hh signaling pathway is active during early human thymocyte development, and that Shh may function in the maintenance of the intrathymic progenitor cell population, promoting their survival and inhibiting their IL-7-mediated proliferation and differentiation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

C.B17 SCID mice were purchased from Harlan (Barcelona, Spain) and maintained in our own pathogen-free breeding facilities. Fetal mice were obtained by timed mating; the day of finding a vaginal plug was designated day 0 of pregnancy.

Isolation of human thymocyte subsets

Human thymus samples were obtained from children aged 1 mo to 3 years, undergoing corrective cardiovascular surgery. Thymus tissues were obtained and used according to the guidelines of the Medical Ethics Commission of the Hospitals La Zarzuela and Madrid-Montepríncipe (Madrid, Spain). Informed consent was provided according to the Helsinki Declaration. To isolate thymic CD34⁺ cells, thymuses were dissected free of surrounding connective tissue, and then gently disrupted with a Potter homogenizer until completely disaggregated. Cell suspensions were enriched for immature thymocytes by using the SRBC rosetting technique as described previously (20). Recovered cells were then depleted of mature T, NK, B, myeloid, and dendritic cells by treatment with saturating concentrations of anti-CD3, anti-CD56, anti-CD19, anti-CD14, and anti-CD11c (all from BD Biosciences, Palo Alto, CA) bound to sheep anti-mouse Ig-coated magnetic beads (Dynal, Oslo, Norway). CD34⁺ cells were then purified by magnetic sorting using VarioMACS (Miltenyi Biotec, Bergisch Gladbach, Germany) in conjunction with CD34 Multisort kit (Miltenyi Biotec) following the manufacturer’s instructions. The purity of the recovered CD34⁺ cells was 98%.

Abs and flow cytometry

Directly labeled mAbs against the following Ags were used: CD1a (HI149-FITC); CD4 (SK3-FITC, -PE, or -PerCP); CD5 (UCHT2-FITC); CD7 (M-T701-FITC); CD8 (SK1-PerCP); CD33 (HIM3-4-FITC); CD34 (8G12-PerCP/Cy5); CD38 (HIT2-FITC); CD44 (G44-26-FITC); CD45 (HI30-PE, or -allophycocyanin); CD45RA (HI100-FITC); CD56 (B159-PE); CD117/c-kit (YB5.B8-PE, or -CyChrome); CD184/CXCR4 (12G5-PE, or -allophycocyanin); HLA-DR (G46-6-FITC, or -PE); Lineage mixture 1-FITC (including CD3, CD14, CD16, CD19, CD20, CD56) from BD Biosciences; and CD127 (R34.34-PE), TCR (IMMU-510-PE) from Coulter Immunotech (Beckman Coulter, Marseille, France). Purified anti-Ptc (G-19) and anti-Smo (N-19) Abs were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), and conjugated to FITC and PE by Biogenesis (Poole, U.K.). Two-, three-, and four-color immunofluorescence stainings were performed by incubating the cells in PBS containing 1% FCS and 0.1% NaN₃ in the presence of saturating amounts of FITC-, PE-, PerCP-, CyChrome-, PerCP/Cy5-, and allophycocyanin-conjugated Abs for 30 min at 4°C. All specific mAbs against human Ags were checked for negative staining on SCID thymocytes. Isotype-matched irrelevant mAbs were used as negative controls to define background fluorescence. To analyze Ptc expression, an intracellular staining was performed because anti-Ptc (G-19) Ab recognizes an intracellular domain at the N terminus of Ptc. For the intracellular detection of Ptc, as well as bcl-2 and bax Ags, cells were treated with a FACS permeabilizing solution according to the manufacturer’s instructions (BD Biosciences), and stained with anti-Ptc-FITC Ab (Santa Cruz Biotechnology), anti-human bcl-2-PE mAb (BD Pharmingen, San Diego, CA), or anti-human bax Abs (BD Pharmingen), followed by FITC-conjugated donkey anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) for 30 min. For the intracellular staining of phosphorylated STAT5, cells were treated with Cytofix/Cytoperm solution (BD Biosciences) for 20 min at 4°C, washed with Perm/Wash buffer (BD Biosciences), and stained with Alexa Fluor 488-conjugated anti-human phospho-STAT5 (Tyr⁶⁹⁴) (BD Biosciences) diluted in Perm/Wash buffer, following the manufacturer’s instructions. The cell cycle analysis was performed by incubating cells with 5 µg/ml Hoechst 33342 (Molecular Probes, Leiden, The Netherlands) in RPMI 1640 (Invitrogen Life Technologies, Grand Island, NY) plus 10% FCS (Harlan-Sera Lab, Leicestershire, U.K.) for 30 min at 37°C. Analyses were conducted in a FACSCalibur flow cytometer (BD Biosciences) and a three-laser BD LSR flow cytometer (BD Biosciences) from the Centro de Microscopía y Citometría (Complutense University, Madrid, Spain).

Culture of thymic CD34⁺ precursors

Purified thymic CD34⁺ precursors (1 x 10⁵) were cultured in 96-well flat-bottom culture plates in 0.2 ml of AIMV serum-free lymphocyte medium (Invitrogen Life Technologies) in the presence of recombinant human (rh)IL-7 (1000 U/ml; National Institute for Biological Standards and Control, Hertfordshire, U.K.), rhSCF (5 ng/ml; PeproTech, London, U.K.), rhflt3 ligand (10 ng/ml; PeproTech), or modified human Shh (kindly provided by Curis (Cambridge, MA)). Shh protein was octylated for high activity. In vitro modification of N-Shh (aa 25–199) with a lipophilic group on the N-terminal cysteine significantly increases the specific activity (30-fold) of Escherichia coli-derived N-Shh as measured by activation of Hh signal transduction in cultured cells (21). Octyl N-Shh is a hydrophobically modified version of the Shh signaling protein generated by coupling N-octyl-malemide to the N-terminal cysteine of bacterially derived N-Shh. The neutralizing anti-Shh mAb 5E1 (25 µg/ml) (22) used in some experiments was obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences (Iowa City, CA). Cyclopamine (5 µM), an inhibitor of the Hh signaling pathway, was a kind gift from Dr. W. Gaffield (Western Regional Research Center, Albany, CA).

After 48 h in culture at 37°C in a 5% CO₂-in-air incubator, CD34⁺ cells were harvested and processed for proliferative assays, viability analysis, and flow cytometry stainings.

Proliferation assays

Cultures were pulsed for the last 12 h with 10 µM 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 h at 60°C), fixed in ethanol in HCl (0.5 M) for 30 min at –20°C, treated with nucleases (30 min at 37°C), and then incubated with peroxidase-conjugated Fab of mouse anti-BrdU Abs (30 min at 37°C). The peroxidase reaction was developed with ABTS substrate, and the sample absorbance was measured using an ELISA reader at 405 nm with a reference wavelength at 492 nm.

Apoptosis assays

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 (Centro de Microscopía y Citometría, Complutense University, Madrid, Spain) and gated according to forward scatter, side scatter, and their ability to exclude propidium iodide. Apoptotic cells were considered to be those that were annexin V positive and propidium iodide negative.

Hybrid human-mouse fetal thymic organ cultures

Thymic lobes removed from 15-day-old embryos of SCID mice were first cocultured for 2 days in hanging drop in Terasaki wells with purified human CD34⁺ cells (1–2 x 10⁴ cells/lobe), and afterward, cultured on Millipore filters (8-µm pore size) (Millipore, Madrid, Spain) in RPMI 1640 supplemented with 7% human AB serum and 3% FCS for the indicated number of days. Cultures were supplemented with human Shh at a concentration of 100 ng/ml and/or rhIL-7 at a concentration of 1000 U/ml throughout the culture period. 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 Ags. Flow cytometric analysis was then performed on electronically gated CD45⁺ human cells.

Human thymus reaggregation cultures

To isolate human thymic epithelial cells, thymic fragments were cultured floating on Millipore filters in RPMI 1640 supplemented with 5% FCS and 1.35 mM 2-deoxyguanosine (Sigma-Aldrich, Madrid, Spain). After 7 days, thymic fragments were trypsinized (0.25% trypsin in 0.02% EDTA) (Sigma-Aldrich) to form a single-cell suspension. Residual thymocytes, NK, B, myeloid, and dendritic cells were depleted as described above, adding anti-CD34 to the mixture of purified mAbs. Isolated thymic epithelial cells were mixed with freshly isolated CD34⁺ cells at a ratio of 1:1 and spun into a pellet. After removal of the supernatant, the cell pellet was dispersed into a slurry by careful mixing and then drawn into a fine capillary pipette. An aliquot of the slurry was expelled as a discrete standing drop on the surface of a filter that floated in the same culture medium used for the hybrid FTOC. The cultures were maintained for 5 days and supplemented with human Shh (100 ng/ml), anti-Shh mAb 5E1 (25 µg/ml), or cyclopamine (5 µM).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD34⁺ thymic progenitors express Hh receptors

In the human thymus, Shh is produced by thymic epithelial cells located in the subcapsular and medullary areas (19). The components of the Hh receptor complex, Ptc and Smo, as well as the three Gli transcription factors—Gli-1, Gli-2, and Gli-3—involved downstream of Hh signaling, have been demonstrated by RT-PCR to be expressed on CD34⁺ thymic precursor cells (19). This suggests that Hh signaling pathway operates in the earliest steps of human T cell development.

To better define the phenotype of the thymic progenitor cells that express Hh receptors, we conducted a flow cytometry analysis. As shown in Fig. 1A, 70–90% of thymic CD34⁺ cells were positive for the Ptc receptor, whereas only 20–30% of this thymic cell subpopulation expressed Smo. The analysis of the expression of several differentiation-specific Ags indicated that the vast majority of Smo⁺CD34⁺ cells expressed CD38, CD7, CD45RA, and the stromal cell-derived factor (SDF)-1 chemokine receptor, CXCR4, and 50–60 and 70–80% expressed the T-lineage markers CD1a and CD5, respectively. A reduced percentage of CD34⁺Smo⁺ precursors stained positively with Abs directed against c-kit, CD33, CD44, and HLA-DR Ags (Fig. 1B). In contrast, Smo^–CD34⁺ thymic precursors expressed higher levels of CD38, CD7, and CD45RA, and an increased proportion of this cell subset expressed c-kit, HLA-DR, CD33, and CD44. The expression of CD5 and CD1a Ags was lower in Smo^–CD34⁺ cells in comparison to Smo⁺CD34⁺ thymocyte precursors (Fig. 1B). These results indicate that the expression of Smo on CD34⁺ cells is mainly associated with those thymic progenitors that are in progression toward T cell lineage commitment. Supporting this, most CD34^bright cells, which correspond to the earliest non-T cell lineage-committed precursors in the human thymus, were found in the Smo-negative cell fraction (Fig. 1C).

View larger version (47K): [in this window] [in a new window]   FIGURE 1. Expression of Hh receptors on CD34+ thymic progenitor cells. A, CD34+ cells were isolated as described in Materials and Methods and stained with anti-Ptc-FITC or anti-Smo-PE Abs. The solid histograms represent the expression of Ptc and Smo on CD34+ precursors, and open histograms indicate background staining. The percentages of positive cells are shown in each histogram. B, Purified CD34+ cells were double-stained with anti-Smo-PE Abs and a range of mAbs against differentiation-specific Ags. Solid histograms represent the expression of the indicated cell surface Ags on Smo+CD34+ and Smo–CD34+ thymic precursors. The percentages of positive cells are shown in each histogram. Open histograms represent background fluorescence using isotype-matched irrelevant mAbs. Similar staining patterns were obtained in four different experiments. C, Correlated expression of CD34 and Smo Ags on isolated thymic precursor cells.

To determine the functional relevance of Hh signaling in T cell precursors, experiments were performed in which CD34⁺ thymocytes were cultured for 48 h in serum-free medium with different doses of Shh, and cellular viability was evaluated. Cultures supplemented with cytokines such as IL-7, stem cell factor (SCF), or flt3 ligand, were conducted in parallel to compare the effects on CD34 cell survival. The addition of different doses of Shh, ranging from 0.05 to 500 ng/ml, to thymocyte precursor cultures always increased the viability of CD34⁺ cells (Fig. 2). The survival effect of Shh was maximal for concentrations from 5 to 100 ng/ml, and followed a bell-shaped curve (Fig. 2A).

View larger version (40K): [in this window] [in a new window]   FIGURE 2. Shh enhances the survival of human thymocyte precursors. A, Determination of CD34+ thymic cell viability after culture for 48 h in serum-free medium alone or supplemented with either different doses of Shh or cytokines such as IL-7, SCF, or flt3 ligand (number of input cells, 105). Percentage of viable cells was determined by staining with annexin V and propidium iodide. Viable cells were defined as annexin V negative and propidium iodide negative. Data represent the mean ± SD of three to four independent experiments, including three cultures per point. Asterisks refer to the statistical significance differences between control and the different treatments. *, p 0.05; **, p 0.01. B, Annexin V, bcl-2, and bax expression (open histograms) measured by flow cytometry in CD34+ thymocytes harvested from control, Shh (100 ng/ml)-, and Shh (100 ng/ml) plus 5E1 (25 µg/ml)-treated cellular suspensions. Background staining is shown by isotype-matched control Abs (solid histograms). Data are representative results from three independent experiments. Mean fluorescence intensity (MFI) is shown for bcl-2 staining.

Shh inhibits the proliferation of human thymocyte precursors

We next assessed the proliferative response of thymic CD34⁺ progenitors to Shh. As shown in Fig. 3, the addition of Shh to CD34⁺ cell cultures induced a dose-dependent inhibition of proliferation. In the presence of the highest doses (100–500 ng/ml), Shh treatment inhibited the proliferative response by 40–50% (Fig. 3). This inhibitory effect was abolished in the presence of anti-Shh mAb or cyclopamine (Fig. 3).

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Shh inhibits the proliferation of human thymocyte precursors. Determination of CD34+ thymic cell proliferation after culture for 48 h in serum-free medium alone or supplemented with different doses of Shh. Proliferation in the presence of both Shh and anti-Shh 5E1 mAb or cyclopamine is also shown. Cells were pulsed for 12 h with BrdU. A specific kit was used to measure BrdU incorporation into newly synthesized DNA. Full details are given in Materials and Methods. Results are the mean of four independent experiments, each with three cultures per point. Asterisks refer to the statistical significance differences between control and the different treatments. *, p 0.05.

Because Shh affected the survival and proliferation of thymic precursors, we tested the effect of Shh on CD34⁺ cell differentiation in organ cultures. We initially used thymus reaggregation cultures, in which isolated human thymic CD34⁺ precursors were reaggregated with human thymic epithelial cells in the presence or absence of Shh. After 5 days in culture, the production of DP thymocytes was totally blocked in the Shh-treated cultures. The proportion of immature single-positive CD4⁺ (ISPCD4) thymocytes was also reduced, and concomitantly, DN cells accumulated in these cultures. By contrast, thymocyte differentiation was promoted in those reaggregation cultures grown in the presence of Hh signaling inhibitors (Fig. 4A).

View larger version (44K): [in this window] [in a new window]   FIGURE 4. Shh influences the development of human CD34+ precursors. A, Thymus reaggregation cultures were performed with human thymic CD34+ cells and thymic epithelial cells, and grown for 5 days in the presence or absence of either Shh or the Hh inhibitors, cyclopamine and anti-Shh mAb. Dot plots show CD4 vs CD8 expression. Data are representative of two independent experiments. Mean cell recoveries: control, 1.5 x 104; cyclopamine, 2.6 x 104; anti-Shh, 2.1 x 104; Shh, 0.8 x 104. B–F, Chimeric human-mouse FTOC were conducted as described in Materials and Methods and cultured for 6 days in the absence or presence of Shh. B, The scatter plot shows the human cell recovery from these chimeric FTOC. The number of human cells recovered in each experiment was divided by the mean number of human cells recovered from the control cultures, to give the relative cell number from eight individual experiments. C, Cells recovered from the hybrid FTOC were triple-labeled with anti-human CD45, anti-CD4, and anti-CD5. Dot plots show the expression of CD4 and CD5 on gated human CD45+ cells from hybrid FTOC grown in the absence or presence of Shh. D, Expression of CD34 on human cells derived from control and Shh-treated FTOC. The proportion of annexin V-positive cells as well as the percentage of cycling cells (cells in S plus G2 plus M phases of cell cycle) on gated human CD34+ cells is also shown. E, Proportion of CD1+ and CD1– in the subpopulation of CD34+ human thymocytes found in the hybrid FTOC cultured in the presence or absence of Shh. F, Absolute numbers of human TCR+ thymocytes, NK cells, and DCs from control and Shh-treated thymic lobes. NK cells were defined as CD56+ cells and DCs as CD4+HLA-DR+CD7–Lin-1–. The absolute numbers were calculated by multiplying the percentage of each cell subset by the total number of human cells. The bars represent the mean ± SD from three independent experiments.

As the above findings indicate that Shh disturbs the development of TCR thymocytes, we examined whether Shh alters the differentiation of TCR thymocytes, NK cells, and DCs using chimeric human-mouse FTOC. Fig. 4F shows that Shh also interfered with the development of TCR⁺ thymocytes, whereas the development of DCs and NK cells was unaltered in the Shh-treated FTOC.

Shh inhibits IL-7-induced proliferation and differentiation of CD34⁺ progenitor cells

The expansion and survival of human intrathymic CD34⁺ precursors is controlled by several factors, including cytokines, some of which also have prodifferentiative effects (24, 25, 26, 27, 28, 29).

To determine the mechanism by which Shh regulates human intrathymic CD34⁺ precursors, we next investigated the effect of Shh addition on the proliferation induced by different cytokines in CD34⁺ cell suspension cultures. The presence of SCF or flt3 ligand minimally stimulated the proliferation of CD34⁺ cells, in comparison with the strong proliferative response shown in the presence of IL-7 (Fig. 5A). Remarkably, the addition of Shh drastically reduced the IL-7-driven expansion of CD34⁺ progenitors (Fig. 5A).

View larger version (24K): [in this window] [in a new window]   FIGURE 5. Shh inhibits the IL-7-mediated development of human precursor cells. A, Determination of CD34+ thymic cell proliferation after culture for 48 h in serum-free medium alone or supplemented with different doses of Shh, IL-7, SCF, flt3 ligand, or a combination of Shh plus IL-7, SCF, or flt3 ligand. Cells were pulsed for 12 h with BrdU, and a specific kit was used to measure BrdU incorporation into newly synthesized DNA. Results are the mean of three to four independent experiments, each with three cultures per point. B, SCID fetal thymic lobes were seeded with CD34+ human thymic precursors and organ-cultured for 8 days in the presence of Shh, IL-7, or Shh plus IL-7. The numbers of human cells were calculated by multiplying the total number of cells per lobe by the percentage of human CD45+ cells determined by flow cytometry analysis. The scatter plot shows the human cell recovery from these chimeric FTOC. The number of human cells recovered in each experiment was divided by the mean number of human cells recovered from the control cultures, to give the relative cell number from four individual experiments. C, Cells from the cultures in B were stained with anti-human CD45, CD4, and CD8, or anti-human CD45 and CD34, and the proportions of human CD34+ progenitor cells, immature CD4+ cells, and CD4+CD8+ thymocytes were analyzed and used to calculate the absolute number of each cell subset. The number of human cells recovered in each experiment was divided by the mean number of human cells recovered from the control cultures, to give the relative cell number of each human thymocyte subpopulation from four individual experiments.

Using the chimeric FTOC system, it was also evident that Shh was able to inhibit the IL-7-dependent expansion of human thymocytes (Fig. 5B). Likewise, Shh blocked the IL-7-induced stimulation of thymocyte differentiation (Fig. 5C), while maintaining the numbers of CD34⁺ precursor cells increased over the control values (C).

The expression of IL-7R/CD127 was also analyzed on the population of CD34⁺ cells that accumulated in the Shh-treated FTOC to know the mechanism by which Shh modulates IL-7 activity. CD127 expression was highly down-regulated in Shh-treated CD34⁺ cells (Fig. 6A). In addition, CD34⁺ progenitors expressed CXCR4 in a lower proportion and with a lower intensity in the presence of Shh, in agreement with our previous data showing that the IL-7-dependent effects in the early steps of human thymocyte development are partially mediated through the chemokine SDF-1 (27). The expression of other cytokine receptors such as c-kit remained unaltered (Fig. 6A).

View larger version (30K): [in this window] [in a new window]   FIGURE 6. Shh down-regulates IL-7R expression and inhibits IL-7-dependent STAT5 phosphorylation. A, Expression of c-kit, CD127/IL-7R, and CXCR4 was analyzed on the CD34+ precursor cell population found in control and Shh-treated chimeric FTOC. The percentage of positive cells and the mean fluorescence intensity (MFI) are indicated in each histogram. Background fluorescence values were set by use of isotype-matched irrelevant mAbs. Results are representative of three independent experiments. B, Purified CD34+ cells were cultured for 48 h in serum-free medium alone or supplemented with Shh (100 ng/ml). For the last 12 h, IL-7 (1000 U/ml) was added to the cultures, and CD34+ cells were then assessed for intracellular STAT5 phosphorylation. Background staining is shown by isotype-matched control Abs (dashed lines). The results are representative of three separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we show that the Hh signaling pathway plays an important role in early human T cell development. Most intrathymic CD34⁺ progenitors express Ptc, the component of Hh receptor that binds Shh, and about one-third express Smo, the signal-transducing component. This low proportion of thymic precursors expressing Smo on the cell surface is probably reflecting that the expression of Smo receptor and, therefore Hh signaling in the thymus, is tightly regulated. Several authors have shown that Smo expression on the cell membrane, which is a prerequisite to activate Hh target gene transcription, occurs after a Hh signal is received (30, 31, 32, 33). In the human thymus, Shh seems to be provided to the CD34⁺ thymocytes in a paracrine fashion by the epithelial cells from the subcapsullary area (19), where thymocyte precursors are located (34).

The subpopulation of Smo-expressing CD34⁺ cells lacks the Ags CD33, HLA-DR, and c-kit, express low levels of CD44, CD7 and CD45RA, and is starting to express CD1a. This phenotype indicates that the intrathymic precursor subset where Hh signaling is active is that which is becoming committed to the T cell lineage and concomitantly losing the NK/DC/myeloid potential (1, 2). Therefore, in the process of T cell lineage commitment and differentiation to ISP CD4⁺ and CD4⁺CD8⁺ cells, intrathymic CD34⁺ precursors come through an Hh-sensitive stage in which Shh can perform its function modulating thymocyte differentiation.

Our current results show that Shh maintains the human intrathymic CD34⁺ precursor cell pool by increasing their viability and inhibiting their expansion and concomitant progression to the ISPCD4 cell stage. In the murine model, we have reported that the addition of high doses of Shh to FTOC arrested thymocyte differentiation at the DN cell stage (16), whereas the treatment with very low doses of Shh promoted thymocyte development and increased the production of DP cells (18). These latter in vitro results are in agreement with the reduced thymocyte differentiation found in Shh^–/– embryos (18). However, we have not detected that different concentrations of rShh can produce distinct outcomes using human thymocyte precursors. These differences could be attributed to a distinct capacity to respond to Shh shown by fetal and adult cells. Alternatively, Shh could differentially regulate thymocyte development in humans and mice, and supporting this, the expression of Hh proteins in the human thymus gland differs from that described in mice (16, 19).

Antiapoptotic effects of Shh have been also demonstrated during the development of the nervous system (35), tooth (36), and external genitalia (37), as well as in somite cells (38) and certain neuron populations (39). Our data demonstrate that Shh promotes CD34⁺ thymocyte survival, inducing both the down-regulation of the proapoptotic bax protein and the up-regulation of the antiapoptotic bcl-2 protein. In agreement, other authors have also shown that Shh induces the expression of bcl-2 in keratinocytes (40, 41) and peripheral T cells (42).

Our results also show that Shh blocks the proliferation of CD34⁺ intrathymic precursors, as well as their differentiation into T cells. In contrast, the differentiation of thymic DCs and NK cells was unaffected by Shh, and as discussed above, this is in agreement with the phenotype of Smo-expressing intrathymic progenitors, which indicates that this precursor cell subset is committing to the T cell lineage. An antiproliferative role for Shh has been described previously in other organs, such as prostate (43), pancreas (44), and stomach (45). Likewise, several authors have reported that Shh can function as an inhibitory signal for the differentiation of distinct cell types in prostate, pancreas, gut, and limbs (43, 46, 47, 48). We show that the mechanism by which Shh abrogates the expansion and differentiation of CD34⁺ cells is to counteract the functions of IL-7, a cytokine that has been repeatedly demonstrated to play a major role in human thymopoiesis (24, 25, 49, 50, 51). These effects are mediated by the Shh-induced down-regulation of IL-7R expression and inhibition of IL-7-dependent STAT5 phosphorylation in CD34⁺ precursor cells. In contrast, Bhardwaj et al. (52) described that Shh enhanced the proliferation induced by a cytokine mixture in human bone marrow-derived CD34⁺CD38^– progenitor cells. Although the type of cells used and the culture conditions are different, we have, however, observed that, in addition to its ability to inhibit IL-7-induced proliferation, Shh is also able to reduce the proliferation of intrathymic CD34⁺ precursors in the presence of a cytokine combination (our unpublished observations).

Taken together, our results provide evidence that the Hh signaling pathway is active during early human thymocyte development. We show that, in the T cell differentiation pathway, intrathymic CD34⁺ precursor cells are targets of the Hh signals at an early developmental stage when the signaling component of the Hh receptor complex is transiently expressed. This developmental stage overlaps with that of sensitivity to IL-7 (51), the main growth factor with prodifferentiative effects functioning in the human thymus (24, 25, 49, 50, 51). Shh provided by the subcapsular thymic epithelial cells might counteract the activity of IL-7, inducing a loss of sensitivity to this cytokine and retaining, therefore, the precursor cells in an undifferentiated state, contributing to the maintenance of the intrathymic progenitor cell population.

On the other hand, the chemokine SDF-1 is another thymic factor whose activity could be modulated by Shh. In fact, we show in this study that Shh down-regulates the expression of its receptor, CXCR4, on CD34⁺ precursor cells. Presumably, other morphogens, such as bone morphogenetic proteins, which are also able to retain thymic precursors in an undifferentiated stage (53), could function regulating the activity of cytokines or other thymic factors, in some cases in collaboration with Shh, as already described in bone marrow (52). Thus, the relationships between growth factors and morphogens in the thymus need further investigation.

	Acknowledgments

 
We thank Curis for providing human Shh protein, Dr. W. Gaffield for the gift of cyclopamine, and Dr. F. Villagrá and the Pediatric Cardiosurgery Units from Hospital La Zarzuela and Hospital Madrid-Montepríncipe for the thymus samples.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Grants PM99-0060, BMC2001-2025, and BMC2003-01901 from the Ministerio de Ciencia y Tecnología, and CAM08.3/0018.1/2001 and CAM08.1/0038.1/2003 from the Comunidad Autónoma de Madrid.

² C.G.-F. and R.S. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Angeles Vicente, Department of Cell Biology, Faculty of Medicine, Complutense University, 28040 Madrid, Spain. E-mail address: avicente{at}bio.ucm.es

⁴ Abbreviations used in this paper: DN, double negative; DP, double positive; DC, dendritic cell; Hh, Hedgehog; Shh, Sonic hedgehog; Ptc, Patched; Smo, Smoothened; FTOC, fetal thymus organ culture; rh, recombinant human; SCF, stem cell factor; SDF, stromal cell-derived factor; ISP, immature single positive.

Received for publication February 25, 2004. Accepted for publication August 17, 2004.

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