Progression of T cell lineage restriction in the earliest subpopulation of murine adult thymus visualized by the expression of lck proximal promoter activity
Chiori Shimizu,
Hiroshi Kawamoto2,
Masakatsu Yamashita,
Motoko Kimura,
Eisuke Kondou,
Yoshikatsu Kaneko,
Seiji Okada1,
Takeshi Tokuhisa1,
Minesuke Yokoyama3,
Masaru Taniguchi,
Yoshimoto Katsura2 and
Toshinori Nakayama
CREST (Core Research for Evolution Science and Technology) Project, Japan Science and Technology Corporation (JST), and Department of Molecular Immunology, and
1 Department of Developmental Genetics, Graduate School of Medicine, Chiba University, 1-8-1 Inohana Chuo-ku, Chiba 260-8670, Japan
2 Department of Immunology, Institute for Frontier Medical Science, Kyoto University, Kyoto 606-8507, Japan
3 Reproductive Engineering Section, Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194-8511, Japan
Correspondence to:
T. Nakayama, Department of Molecular Immunology, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
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Abstract
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The proximal promoter of lck directs gene expression exclusively in T cells. To investigate the developmental regulation of the lck proximal promoter activity and its relationship to T cell lineage commitment, a green fluorescence protein (GFP) transgenic (Tg) mouse in which the GFP expression is under the control of the proximal promoter of lck was created. In the adult GFP-Tg mice, >90% of CD4+CD8+ and CD4+CD8 thymocytes, and the majority of CD4CD8+ and CD4CD8 [double-negative (DN)] thymocytes were highly positive for GFP. Slightly lower but substantial levels of expression of GFP was also observed in mature splenic T cells. No GFP+ cells was detected in non-T lineage subsets, including mature and immature B cells, CD5+ B cells, and NK cells, indicating a preserved tissue specificity of the promoter. The earliest GFP+ cells detected were found in the CD44+CD25 DN thymocyte subpopulation. The developmental potential of GFP and GFP+ cells in the CD44+CD25 DN fraction was examined using in vitro culture systems. The generation of substantial numbers of
ß and 
T cells as well as NK cells was demonstrated from both GFP and GFP+ cells. However, no development of B cells or dendritic cells was detected from GFP+ CD44+CD25 DN thymocytes. These results suggest that the progenitors expressing lck proximal promoter activity in the CD44+CD25 DN thymocyte subset have lost most of the progenitor potential for the B and dendritic cell lineage. Thus, progression of T cell lineage restriction in the earliest thymic population can be visualized by lck proximal promoter activity, suggesting a potential role of Lck in the T cell lineage commitment.
Keywords: green fluorescence protein, T cell lineage commitment, transgenic
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Introduction
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The lck gene encodes the lymphocyte specific tyrosine kinase p56lck (Lck) (1). The expression of the lck gene is regulated by two distinct promoter elements, i.e. a proximal and a distal promoter of lck (24). The lck proximal promoter is most active in immature thymocytes, whereas the activity of the distal promoter is low in immature thymocytes and has been reported to be increased in mature thymocytes (57). Reynolds et al. detected lck proximal promoter activity in a very early thymocyte subpopulation, such as HSA+CD4CD8 double-negative (DN) cells (5). However, a fine analysis to determine which subpopulation of the developing T cells is the first to express the T lymphocyte-specific lck proximal promoter has not been reported. In addition, whether the DN thymocytes expressing the lck proximal promoter are already restricted to the T cell lineage has not been clarified.
Perlmutter and colleagues created a transgenic (Tg) cassette containing the lck proximal promoter element that directs the transgene expression exclusively in T cells (8,9). Subsequently, a number of Tg mice have been established using this cassette to address the role of a selected target molecule in the development of T cells in the thymus. For example, Tg lines bearing dominant-negative mutants of signal transduction molecules have contributed significantly to our understanding of the molecular basis of thymocyte development (1012). Nevertheless, a precise determination of the expression levels of the transgene in each subpopulation of developing T cells and mature T cells has not been elucidated.
It is well established that most T cells develop in the thymus, whereas a long-standing debate on whether the progenitor cells migrating in the thymus are already committed to the T cell lineage persists (1316). The most immature thymocyte subpopulation seen in the CD3CD4CD8 triple-negative (TN) subset was first identified by unique surface phenotype signatures, such as CD4low or a c-kit+ Thy-1low, and it was shown to give rise to T, B, dendritic and NK lineage cells but not myeloid lineage cells (17,18). From these kinds of results, the recent migrants in the thymus were thought to retain multi-lineage potential, although there appeared to be a restriction to the lymphoid lineage to a certain extent. Using CD44 and CD25 molecules as differentiation markers, CD44+CD25 cells were established to be the most immature subset in DN thymocytes (14). Consistent with previous results, CD44+CD25 cells were shown to possess multi-lineage potential, whereas the next CD44+CD25+ subset had clearly lost B cell progenitor potential (14,19). Thus, T cell lineage commitment appeared to occur during the sequence of events in development from CD44+CD25 cells into CD44+CD25+ cells.
In the fetus, however, it was shown by a clonal assay that the progenitors in the earliest CD44+CD25 population were restricted to the T cell lineage, in that they are unable to generate myeloid and B cells (20). Moreover, such a T cell lineage-restricted progenitor population was also detected in prethymic hematopoietic organs, such as the fetal liver and aortagonadmesonephros (AGM) region and fetal blood (2123), although in human no evidence has been obtained for prethymic T cell lineage restriction (24). It was further shown that prethymic T cell progenitors exist in murine fetal blood and fetal liver retain NK potential (25,26). Subsequently, the earliest T cell progenitor in the fetal thymus was shown to be exclusively bipotent for the T and NK cell lineage, although the NK lineage potential was marginal once it transits into the CD44+CD25+ subset (27). The bipotent T/NK cell progenitors were observed in both mouse (2830) and human systems (31). These results suggest that T/NK bipotent progenitor cells that migrate in the fetal thymus undergo commitment processes to the T cell lineage at the CD44+CD25+ subset stage.
The nature of the recent migrants in the adult thymus, on the other hand, has not been well elucidated. Transcription of CD3
, a T cell-specific molecule, was reported to occur in the adult CD44+CD25 DN thymocyte subpopulation (32,33). Recently, the CD3
protein was reported to be expressed only in the late CD44CD25+ DN thymocyte subpopulation (34). However, a detailed analysis of the T cell progenitor cells in the thymus and in the prethymic organ, bone marrow, has not been performed. Moreover, the precise developmental stage at which T cell lineage commitment occurs in the adult thymus remains to be clarified.
Here, we generated green fluorescence protein (GFP)-Tg mice under the control of the proximal promoter of lck, to better delineate the events in the commitment of cells to the T lineage. Significant numbers of GFP-expressing cells were observed in the CD44+CD25 DN thymocyte subpopulation. From GFP+ CD44+CD25 DN thymocytes, the development of substantial numbers of T lineage cells, small numbers of NK cells and no detectable level of B lineage cells or dendritic cells was observed. We show that in the earliest CD44+CD25 DN subpopulation, the cells that express lck proximal promoter activity are restricted to the T/NK cell lineage.
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Methods
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Mice
C57BL/6 (B6) mice were purchased from SLC (Tokyo, Japan). B6 Ly5.1 mice were maintained in the animal facility of the Institute for Frontier Medical Science, Kyoto University. All mice used in this study were maintained under specific pathogen-free conditions.
Establishment of GFP transgenic mice
A pEGFP-c1 gene was purchased from Clontech (Palo Alto, CA). A BamHI site was added to the 5' and 3' end of the EGFP gene by a PCR method. Then, a 613 bp of pEGFP insert was ligated with lck proximal promoter by using BamHI sites (8) and the resulting 4.2 kb construct was microinjected into fertilized B6 eggs. The Tg (plck-GFP-Tg) mouse line analyzed in this report has ~20 copies of the transgene.
Reagents
For phenotypic analyses, the following mAb were used; allophycocyanin (APC)-conjugated anti-CD4 (RM4-5APC; PharMingen, San Diego, CA), anti-CD8APC (CT-CD8
APC, Caltag, San Francisco, CA), anti-TCRßAPC (H57-597APC; PharMingen), anti-CD3
APC (145-2C11APC; PharMingen), phycoerythrin (PE)-conjugated anti-CD4 (RM4-5PE; PharMingen/GK1.5PE; Caltag), anti-CD44PE (IM7PE; PharMingen), anti-NK1.1PE (PK136PE; PharMingen), anti-TCR
PE (GL3PE; PharMingen), biotin-conjugated anti-TCRß (H57-597biotin; PharMingen), anti-CD25biotin (7D4biotin; PharMingen), Cy5-conjugated anti-CD8
(53-6.72Cy5; prepared in our laboratory). Red613-conjugated avidin (Life Technologies, Rockville, MD) and Red670-conjugated avidin (Life Technologies) were also used for visualization of biotinylated mAb.
Immunofluorescent staining and flow cytometry (FCM) analysis
Freshly prepared thymocytes were suspended in PBS supplemented with 2% FCS and 0.1% sodium azide. In general, 106 cells were incubated on ice for 30 min with appropriate staining reagents as described (35). For direct staining, cells were first incubated with 2.4G2 anti-Fc
R mAb to prevent non-specific binding of mAb via FcR interactions. FCM analysis was performed on a FACSCalibur (Becton Dickinson Immunocytometry Systems, San Jose, CA), and fluorescence data were collected as a list mode on 40,000 viable cells as determined by light scatter parameters and propidium iodide (PI) exclusion. Where indicated, 400,000 events were collected. CellQuest software was used for collecting and analyzing data.
Confocal laser microscopic analysis
Thymus and spleen of plck-GFP-Tg mice were fixed with 4% paraformaldehyde in PBS overnight. After replacing the paraformaldehyde with 20% sucrose, the fixed organ was washed with PBS twice and embedded in OCT compound (TissuTec; Sakura Finetechnical, Tokyo, Japan) and frozen with dry ice. Sections (10 µm thick) were prepared by a Cryostat (CM1800; Leica, Nossloch, Germany), dried immediately and permeabilized with a blocking solution containing 20% goat serum, 2% BSA, 0.05% Tween 20 and 100 µg/ml 2.4G2 for 60 min at room temperature for immunostaining. Then, the sections were stained with anti-Thy1.2PE (x100 dilution; PharMingen) and anti-B220Cy5 (x100 dilution; PharMingen) in blocking solution for 60 min at room temperature. After washing twice in TBS, the coverslips were mounted on glass slides using phenylenediamine medium. Slides were examined on a confocal laser scanning microscope (Carl Zeiss; LSM 510) using x10 and x20 Plan Apo objectives. GFP fluorescence was visualized with 488 nm argon laser excitation and a 515540 nm band pass emission filter. PE fluorescence and Cy5 fluorescence were simultaneously viewed using 543 nm He/Ne excitation and a 560615 nm band pass emission filter, and 633 nm He/Ne excitation and a 670810 nm band pass emission filter respectively.
Proliferation assay
Splenic T cells were purified by panning with plastic dishes coated with goat anti-mouse Ig, and then stimulated with immobilized anti-TCR mAb (H57-597, 3 µg/ml), anti-CD3
(145-2C11, 10 µg/ml) or phorbol myristate acetate (3 ng/ml) and ionomycin (250 nM) for 40 h. [3H]Thymidine (0.5 µCi/well) was added to the stimulation culture for the last 16 h and the incorporated radioactivity was measured using a ß-plate (36).
Sorting of progenitor cells
Thymocytes from 4-week-old Tg mice were treated with mAb specific for CD8 (3.155) and CD3 (145-2C11), and rabbit complement for 1 h at 37°C. Two cycles of the treatment were performed. The cells were then stained with anti-CD25PE, anti-CD44APC, and a mixture of biotin-conjugated mAb specific for CD3 (145-2C11; PharMingen), CD4 (CT-CD4; Caltag), CD8 (CT-CD8
; PharMingen), NK1.1 (PK136; PharMingen), TER119 (established by Dr T. Kina, University of Kyoto, Kyoto, Japan), Mac-1 (M1/70; Caltag) and B220 (6B2; Caltag). Both GFP+ and GFP cells in the CD4CD8CD3Lineage marker (Lin)CD44+CD25 subpopulation were sorted by a FACS Vantage (Becton Dickinson) as described (27).
Cytokine-supplemented high oxygen submersion (HOS) culture
HOS culture was performed as described (37,38). In brief, single dGuo-treated Ly5.1 B6 15 day post-coitum (d.p.c.) fetal thymus lobes were placed into wells of a 96-well V-bottom plate, to which a suspension of sorted progenitor cells (100 cells) was added. The plates were sealed in plastic bags and the air inside was replaced by a gas mixture (70% O2, 25% N2 and 5% CO2). The bags were incubated at 37°C. The medium was changed every 45 days. After cultivation for 21 days, cells in the well were harvested and then subjected to FCM analysis.
Cultures for the investigation of B cell and dendritic cell potential
For B cell generation, sorted progenitor cells (100 cells) were cultured on a stromal cell line (TSt-4) for 14 days in a well of a six-well plate (Costar) (23). For dendritic cell generation, 100 cells were cultured in a well of a 96-well flat-bottom plate. Culture medium was supplemented with 10 ng/ml murine stem cell factor (SCF; Genzyme, Cambridge, MA), 10 ng/ml murine IL-7 (a gift from Dr T. Sudo, Basic Research Laboratory, Toray, Kanagawa, Japan), 10 ng/ml of murine granulocyte macrophage colony stimulating factor (GM-CSF; Life Technologies, Rockville, MD), 10 ng/ml IL-3 (Genzyme), 10 ng/ml tumor necrosis factor (TNF)-
(Genzyme) and 10 ng/ml IL-1
(Genzyme). Cells were harvested on day 10.
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Results
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GFP expression in the developing thymocytes
To determine whether the expression of the GFP would have any effect on thymocyte development, we began the study by examining CD4/CD8 profiles of plck-GFP-Tg mice. Thymocytes from 6-week-old heterozygous (Tg+/) and homozygous (Tg+/+) plck GFP-Tg mice were stained with anti-CD4PE and anti-CD8Cy5 (Fig. 1
, left panels). The cellularity and the CD4/CD8 profiles of both heterozygous and homozygous plck Tg thymocytes were very similar to those of littermate (LM) controls, indicating that thymocyte development is normal in the presence of the expressed GFP. Concurrently, GFP profiles of electronically gated CD8 single-positive (SP), CD4+CD8+ double-positive (DP), CD4 SP and CD4CD8DN thymocytes were generated. Representative profiles are presented in Fig. 1
(A, right panels). Greater than 90% of CD4 SP and DP thymocytes were highly positive for GFP, and also the majority of CD8 SP and DN cells were GFP+. As expected, the expression levels of GFP in homozygous Tg mice are significantly higher than the levels expressed in heterozygous Tg mice. The expression levels of GFP in DN thymocytes were significantly lower than the levels of the other three subpopulations. Similar GFP expression profiles were observed in developing thymocytes obtained from the 16.5 d.p.c. fetus, 17.5 d.p.c. fetus and day 2 newborn plck-Tg animals (data not shown).


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Fig. 1. GFP expression in the developing thymocytes of plck-GFP-Tg mice. (A) Thymocytes from plck-GFP-Tg heterozygous (Tg+/) and homozygous (Tg+/+) and LM mice were stained with anti-CD4PE and anti-CD8Cy5. GFP expression profiles on the electronically gated CD4+CD8+ (DP), CD4+CD8 (4SP), CD4CD8+ (8SP) and CD4CD8 (DN) thymocytes are shown with background fluorescence obtained from LM thymocytes (shaded area). Cell yield is indicated as the boxed numbers and the percentages of cells in each CD4/CD8 subpopulation are shown in the respective quadrants. Percent positive cells and mean channel number of the GFP-expressing population (the gate is indicated in the histograms) are summarized in the lower right panel. (B) Thymocytes from plck-GFP-Tg heterozygous (Tg+/) and homozygous (Tg+/+) and LM mice were stained with anti-CD4APC, anti-CD8Cy5, anti-CD44PE and anti-CD25biotin, followed by avidinRed613. A total of 400,000 events was collected and CD44/CD25 profiles of CD4CD8 DN thymocytes are shown. GFP expression profiles of the electronically gated four CD44/CD25 subset from heterozygous (solid line) and homozygous (bold line) mice are shown. Percent positive cells and mean channel number of the GFP-expressing population (the gate is indicated in the histograms) are summarized in the lower right panel.
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The GFP expression profiles of the four subsets in DN thymocytes as defined by CD44 and CD25 expression are shown in Fig. 1
(B). The CD44/CD25 profiles of DN thymocytes were not distinguishable among heterozygous and homozygous plck-Tg mice and LM mice. A significant proportion (1920%) of CD44+CD25 DN thymocytes, which are thought to be the most immature subset, was found to be positive for GFP. Greater than 70% of the cells were GFP+ in CD44+CD25+ DN thymocytes and the majority of cells were GFP+ in the CD44CD25+ subset in which TCRß rearrangement occurs (39,40). These results are in good agreement with those reported in a previous publication, in which the activity of lck proximal promoter was examined in T antigen-coupled Tg mice (5). The expression levels of GFP in the GFP+ CD44+CD25+ DN subpopulation were significantly lower than those of other three DN subpopulations. Again, the expression levels of GFP of homozygous Tg mice are significantly higher than those of heterozygous Tg mice. However, it is worth noting that the percentages of GFP+ cells in each subpopulation were not significantly different between heterozygous and homozygous plck-Tg mice (see numbers in Fig. 1B
). These results suggest that most of the cells expressing lck proximal promoter can be visualized by GFP expression even in the heterozygous plck-Tg mice. No GFP+ cells was detected in CD3 bone marrow cells (data not shown), suggesting that the proximal promoter of lck is not active in progenitor cells in the bone marrow of young adult mice.
GFP expression in splenocytes assessed by FCM analysis
The activity of the proximal promoter of lck is thought to be very high in immature thymocytes, whereas only marginal levels are expected in peripheral mature T cells, such as splenic T cells (7). Therefore, we evaluated the GFP expression in splenocytes of plck-GFP-Tg mice. Splenocytes were stained with anti-CD4PE and anti-CD8Cy5, and GFP expression profiles of electronically gated CD4 SP T cells and CD8 SP T cells were analyzed (Fig. 2A
). Surprisingly, we observed that >80% of CD8 SP T cells and >60% of CD4 T cells were highly positive for GFP, although the expression levels were significantly lower than those of CD4 or CD8 SP thymocytes (compare mean channel numbers in Figs 1A and 2A
). Essentially no GFP+ cells was present in CD4CD8 spleen cells. A similar proportion of GFP+ cells was detected in lymph node cells and Peyer's patch cells (data not shown).

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Fig. 2. GFP expression and TCRCD3-mediated proliferative response of splenic T cells of plck-GFP-Tg mice. (A) Splenocytes from heterozygous plck-GFP-Tg mice were stained with anti-CD4PE and anti-CD8Cy5. GFP expression profiles on the electronically gated CD4+CD8 (4SP), CD4CD8+ (8SP) and CD4CD8 (DN) cells are shown along with background fluorescence profiles (shaded area). Percentages of cells in each CD4/CD8 subpopulation are shown in each quadrant and the cell yield is indicated as the boxed numbers. In the histograms, percentages of GFP+ cells are shown (top) along with a mean channel number (bottom). (B) Splenic T cells from LM (open bars) and heterozygous plck-GFP-Tg mice (hatched bars) were stimulated with immobilized anti-TCR mAb (H57-597), anti-CD3 mAb (145-2C11) or phorbol myristate acetate plus ionomycin (P + I). Proliferative responses were assessed by the incorporation of [3H]thymidine.
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To assess the T cell function of GFP-expressing splenic T cells from plck-GFP-Tg mice, purified splenic T cells were stimulated with immobilized anti-TCR mAb and anti-CD3 mAb, and [3H]thymidine uptake was measured. Equivalent responses were detected between LM control and plck-GFP-Tg T cells (Fig. 2B
). In addition, equivalent levels of IL-2 were produced in these stimulation cultures (data not shown). There was no significant difference in the expression of cell-surface marker antigens, including CD3, TCRß, CD25, CD69 and CD44 (data not shown). These results indicate that the expression of GFP did not affect the development of functional T cells.
GFP expression of NK and NKT lineage cells
GFP expression in NK1.1+TCRß+ NKT cell population in the thymus was assessed and only a fraction (16.8%) was found to be positive for GFP (Fig. 3A
). In the spleen, as shown in Fig. 3
(B), splenic NK1.1+TCRß NK cells were completely negative for GFP. Interestingly, however, in the NK1.1+TCRß+ NKT cell subpopulation, about half (46.4%) of the cells express GFP. A similar proportion of NKT cells in the liver was positive for GFP (data not shown). These results indicate that the proximal promoter of lck is not expressed in NK lineage cells, but expressed in a certain proportion of NK1.1+TCRß+ NKT cells.

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Fig. 3. GFP expression in the NK and NKT lineage cells in thymus and spleen. (A) Thymocytes from heterozygous plck-GFP-Tg and LM mice were stained with anti-TCRßbiotin and anti-NK1.1PE, followed by avidinCy5. GFP profiles of the total population and of the indicated subpopulation are depicted. TCRß+ NK1.1+ cells are NKT cells. (B) Splenocytes were stained with anti-TCRßbiotin and anti-NK1.1PE, followed by avidinCy5. GFP profiles of the total population and those of the indicated subpopulation are depicted. NK cells are TCRß NK1.1+ cells (area #1) and NKT cells are TCRß+ NK1.1+ cells (area #2).
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GFP expression in the thymus and spleen examined by confocal microscopic analysis
Thymus and spleen sections of the plck-GFP mice were stained with anti-Thy1PE and anti-B220Cy5 mAb, and analyzed by confocal microscopy to assess the localization patterns of the GFP in situ. As can be seen in Fig. 4
(A), strong green fluorescence by GFP was detected in the cortical areas of the thymus. We also observed significant GFP fluorescence in the medulla. Most of the green fluorescence appears to be coincident with the Thy-1+ cells, as visualized by yellow in the superimposed profiles in the lower panel. In the spleen, as expected, the cells with green fluorescence were found in the T cell area, but not in the B cell areas of white pulp (Fig. 4B
). These results are consistent with those obtained by FCM analyses shown in Figs 1 and 2
. Therefore, at the tissue level the expression patterns of GFP is very consistent with what might have been predicted from the results of the analysis of isolated cells by FCM.

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Fig. 4. GFP expression in the thymus and spleen assessed by confocal microscopic analysis. Thymus (A) and spleen (B) of 4-week-old heterozygous plck-GFP mice were examined by confocal microscopic analysis with anti-Thy1PE and anti-B220Cy-5 mAb. The areas of cortex (C) and medulla (M) in the thymic section are indicated. The PE staining and Cy-5 staining are depicted by red and blue respectively. A superimposed profile of the thymus with anti-Thy1 staining and GFP profile is shown in the lower panel in (A). An another profile generated with anti-Thy-1, anti-B220 and GFP, the profile of the spleen is shown in the lower right hand corner in (B). The white bar represents 50 µm.
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Generation of
ß and 
T cells from CD44+CD25 DN thymocytes
The data obtained thus far suggest that the earliest GFP+ cells are found in CD44+CD25 DN thymocyte subset and that the proximal promoter of lck becomes active before TCRß rearrangement. Recently, the presence of progenitor cells for non-T lineage cells, such as B, dendritic and myeloid cells, was demonstrated in the CD44+CD25 subset from the fetal thymus (20). Consequently, we wished to evaluate the progenitor activity for various lineages in the GFP+ CD44+CD25 cells. Since the expression of the lck proximal promoter is T lineage specific, it might be expected that the GFP-expressing CD44+CD25 cells would not have demonstrable progenitor activities for non-T lineage cells. To begin to address this question, we performed in vitro HOS cultures, and compared the progenitor potential of GFP+ CD44+CD25 cells and GFPCD44+CD25 cells for the generation of
ß and 
T cells and NK cells.
To increase the efficiency of sorting, thymocytes from 4-week-old heterozygous plck-Tg mice were treated with anti-CD8 and anti-CD3 followed by rabbit complement, and then the residual cells were stained with anti-CD25, anti-CD44, and a mixture of anti-CD3, anti-CD4, anti-CD8, anti-Lin and PI. GFP CD44+ (R1 gate) and GFP+CD44+ (R2 gate) cells in the gate of CD25 and CD3CD4CD8LinPI population were sorted (Fig. 5A
). The re-analyzed GFP/CD44 profiles of the sorted cells are shown in Fig. 5
(B). The sorted cells (100 cells/well) were cultured with a dGuo-treated Ly5.1 thymus lobe for 21 days in the HOS culture. Representative CD8/CD4, TCR
ß/TCR
and CD3/NK1.1 profiles of the cultured cells from CD44+CD25 GFP (Fig. 5C
) and CD44+CD25 GFP+ (Fig. 5D
) cells are shown. In this particular experiment, 93 ± 25x103 cells/lobe from the culture of GFP cells and 45 ± 13x103 cells from that of GFP+ cells were recovered. Substantial numbers of CD4+CD8+ DP and CD8+CD4 (CD8 SP; 8SP) cells, and small numbers of CD4+CD8 (CD4 SP; 4SP) cells developed from the CD44+CD25GFP subpopulation. GFP expression profiles of each cell population (Fig. 5C
, upper panels) were similar to those of thymocytes that developed in vivo (see Fig. 1A
, right panels). From the GFP+ subpopulation, development of considerable numbers of CD4 SP and CD8 SP cells was observed (Fig. 5D
, upper panels). Only small percentages of DP cells were detected in the culture. CD8/CD4 profiles are significantly different from those derived from the GFP subpopulation. The difference is probably due to the difference in time course, because DP cells were detected in the culture with GFP+ population during early days of the HOS culture (data not shown). In terms of the GFP expression, the profiles of the four subsets defined by CD4/CD8 expression were very similar between GFP and GFP+ cultures.
Both TCR
ß- and TCR
-expressing T cells were found to have developed from either GFP or GFP+ subpopulation (Fig. 5C and D
, middle panels). A similar portion of both TCR
cells and TCR
ß cells was positive for GFP. Interestingly, >50% of the cells that developed from the GFP+ subpopulation expressed either
ß or 
TCR. A significant number of NK1.1+CD3 cells (7.4 ± 2.0x103) were generated from the GFP subpopulation and most of them were negative for GFP. A small number of NK1.1+CD3 cells (2.6 ± 0.8x103) were also generated from the GFP+ subpopulation, suggesting that the GFP+ subpopulation retained a low level of NK cell progenitor potential. The results in Fig. 5
(C and D) indicate that both GFP+ and GFP subpopulations of CD44+CD25 DN thymocytes give rise to substantial numbers of T lineage cells under the HOS culture conditions.
Generation of B and dendritic cells from GFP but not from GFP+ CD44+CD25 DN thymocytes
Next, to analyze the progenitor activity for B cells and dendritic cells in the GFP+ and GFP subpopulations of CD44+CD25 DN thymocytes, we used another in vitro culture system. For B cell differentiation, the sorted GFP+ and GFP cells (200 cells /well) were cultured on a stromal cell line (TSt-4) in the presence of murine SCF, murine IL-7 and murine GM-CSF for 21 days. Substantial numbers of B220+ IgM+ B cells were generated from the GFP population, but not from the GFP+ subpopulation (Fig. 6A
). The B cells generated from GFP subpopulations in this culture system were authentic, since most of them were B220+ CD5 (Fig. 6B
). Three independent experiments were performed and similar results were obtained (data not shown). These results suggest that the progenitor activity for B cells is dramatically limited in the GFP+ subpopulation of CD44+CD25 DN thymocytes.

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Fig. 6. Generation of B cells and dendritic cells from CD44+CD25 GFP thymocytes but not from CD44+CD25 GFP+ thymocytes. (A) The generation of B220-expressing B cells from CD44+CD25 GFP+ (GFP) and CD44+CD25 GFP+ (GFP+) TN thymocytes (200 cells/well) in the 21-day culture on a stromal cell line (TSt-4). A mean value of the absolute cell number + SD obtained from three independent culture wells is shown. (B) CD5/B220 staining profiles of the B cells generated from CD44+CD25 GFP+ TN thymocytes. (C) The generation of dendritic cells from CD44+CD25 GFP+ (GFP) and CD44+CD25 GFP+ (GFP+) TN thymocytes (100 cells/well) in the 10-day DC culture. A mean value of the absolute cell number + SD obtained from five independent culture wells is shown. A representative CD11c/MHC class II profile (D) and a representative microscopic view (E) of the dendritic cells generated from CD44+CD25GFP thymocytes are also shown.
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For the development of dendritic cells, the sorted GFP+ and GFP cells (100 cells each) were cultured in the presence of murine SCF, murine IL-7, murine IL-3, murine GM-CSF, murine TNF-
and murine IL-1
for 10 days. As shown in Fig. 6
(C), the development of dendritic cells was observed from the GFP subpopulation but not from the GFP+ subpopulation. Most of the dendritic cells generated were positive for both CD11c and MHC class II (Fig. 6D
). The characteristic morphological features were observed in the dendritic cells that developed in the culture (Fig. 6E
). These results indicate that the progenitor activity for dendritic cells of the GFP+ subpopulation of CD44+CD25 DN thymocytes is much lower than that of the GFP subpopulation. Taken together, the GFP+ subpopulation of CD44+CD25 DN thymocytes would appear to have lost most of the progenitor activity for B cells and dendritic cells.
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Discussion
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In this report, we describe some of the features of a GFP-Tg mouse in which the proximal promoter of lck controls the GFP expression. We sought to investigate the developmental regulation of the lck promoter activity and its relationship to T cell lineage commitment. The progenitor potential of thymocytes from the GFP-Tg mice was evaluated using three different in vitro culture systems, each of which supported the generation of T/NK cells, B cells or dendritic cells (23,27,41). We demonstrated that the progenitor cells expressing the lck proximal promoter in the CD44+CD25 DN subset in the adult thymus are on the process of restriction toward the T cell lineage. They retained low levels of progenitor activity for NK cells, but showed no activity for B or dendritic cells. Thus, the earliest T progenitor cells in the adult thymus may be identified by the expression of the lck proximal promoter activity.
To understand the nature of early thymopoiesis, it is important to establish whether the earliest CD44+CD25 cells observed are homogeneous uncommitted progenitor cells or a heterogeneous population consisting of various stages of developing T cell progenitors. Recently, several groups have addressed this question by studying the fetal thymus. Most of the results appear to favor the latter possibility. The protein expression of T lineage-specific molecules, such as TCF-1, Gata-3 and CD3
, and mRNA expression of lck were demonstrated in FcR+ CD44+CD25 DN thymocytes of 12 d.p.c. fetal thymus (42). In the CD44+CD25 DN subpopulation, cells expressing c-kit and NK1.1 molecules appeared to have lost B cell progenitor potential (43). Furthermore, germline transcripts with the JC region of TCRß were detected in the c-kit+ subpopulation of fetal CD44+CD25 thymocytes (44). From these results, the CD44+CD25 DN subset was suggested to be heterogeneous and the expression of several T cell-specific intracellular molecules was found to be initiated within the CD44+CD25 subset.
More recently, using a clonal analysis referred to as a multi-lineage progenitor assay, it was revealed that each T cell progenitor in the FcR CD44+CD25 population had lost B or myeloid lineage potential (20), although multi-potent progenitor activity of this population was demonstrated at the population level. It is interesting, however, that even T cell lineage-restricted progenitor cells in the FcR+ CD44+CD25 population were found to preserve NK cell potential (27). On the other hand, the presence of T cell progenitor cells has also been demonstrated in the prethymic organs, such as the fetal liver, fetal blood or AGM region (2126). Thus, it appears that in the fetus somewhat T lineage-restricted progenitor cells migrate into the thymus and then proceed with the T cell-specific developmental processes in the thymus.
In contrast, we know less about the nature of the steady-state thymopoiesis in the adult thymus. One can readily speculate that the production of T cells may not be very efficient in the adult and the process of T cell lineage commitment might be distinct from that of the fetus. Only the expression of CD3 molecules has been analyzed precisely in the early thymocyte subpopulation in the adult. Although mRNA of CD3
was detected in the adult CD44+CD25 DN subset (32), the CD3
protein was reported to be expressed only after differentiation into the CD44CD25+ subset (34). Rothenberg and colleagues also reported CD3 expression in the adult CD44+CD25 DN subset (33). They also demonstrated Ig-ß expression and germline IgH transcription, and hypothesized that transcription of both T lineage- and B lineage-specific genes takes place in the uncommitted CD44+CD25 DN cells. Shutdown of the B lineage-specific gene transcription occurs during the process of T lineage commitment.
In this study, we use 4-week-old young adult mice and demonstrate that a portion of the CD44+CD25 cells expresses lck proximal promoter activity, and these appear to have lost B cell lineage progenitor potential (Fig. 6
). Small numbers of NK cells were developed from the GFP+ CD44+CD25 cells (Fig. 5C and D
). Therefore, we concluded that this population contains only T/NK cell-restricted progenitor cells. Analogous to the results of fetal thymocytes (27,30), the earliest T cell progenitor in the adult thymus was found to retain progenitor potential to generate NK lineage cells. Thus, regardless of the age, T lineage commitment is a multi-step process and B cell lineage potential disappears at a very early step, while NK potential is preserved until an early phase of the CD44+CD25+ stage.
The difference in the NK potential between the GFP+ and GFP CD44+CD25 DN subpopulation is not very dramatic, but may be under-estimated in this system (Fig. 5C and D
). A single NK cell progenitor produces up to 104 NK cells and the expansion space in fetal thymic organ cultures is limited. A slight but significant decrease in the NK cell potential was detected in the GFP+ CD44+CD25 DN subpopulation (Fig. 5
). The decreased NK cell potential appears to be correlated with the expression of a T cell-specific lck proximal promoter, suggesting that commitment to the T cell lineage proceeds within the CD44+CD25 DN subset of adult thymus.
No dendritic cell potential was detected in the GFP+ CD44+CD25 DN subpopulation in our experimental system, where 100 progenitor cells/well were applied (Fig. 6
). Since previous studies suggested that CD44+CD25+ cells were still able to give rise to dendritic cells (45), more exact examination may be necessary to detect the DC potential in this population.
Although both GFP and GFP+ CD44+CD25 DN thymocytes gave rise to
ß and 
T cells efficiently in our culture conditions, the differentiation patterns as determined by TCR
ß/TCR
profiles were significantly different (Fig. 5C and D
). The most straightforward explanation is that the GFP+ CD44+CD25 DN subpopulation is a further differentiated subpopulation, and
ß and 
T cell lineage progenitors are enriched. Another possibility is that the ability of self-replication is lower in GFP+ CD44+CD25 DN cells. It is also possible that the GFP+ cells in the CD44+CD25 DN subset are precursors of a peculiar T cell subpopulation that has a specific differentiation pathway. However, this appears unlikely because the expression profiles of GFP in every subpopulation assessed by gating in Fig. 5
were similar between the GFP and GFP+ groups.
Various studies have suggested that the Lck tyrosine kinase plays an important role for thymocyte development as a signaling molecule downstream of pre-TCR (4649) and pre-TCR is first detected in the CD44CD25+ DN subset (50,51). For example, the development of T cells is blocked at the transition from the CD44CD25+ to CD44CD25 subset in the Lck-deficient mice (52). Dominant-negative Lck Tg mice show a more profound effect and essentially complete arrest at the stage of the CD44CD25+ subset (53). In addition, TCRß-rearrangement is reported to occur in the CD44CD25+ subset (39) and a crucial role for Lck in the process of TCRß rearrangement is suggested (54). Thus, the role for Lck in T cell development is highlighted on the CD44CD25+ subset. However, expression of the proximal promoter of lck is detected in the earlier DN thymocytes, such as CD44+CD25 and CD44+CD25+ cells (Fig. 1
). Although the expression of Lck protein would be a result of T lineage commitment, it is also conceivable that the Lck molecules play active roles as tyrosine kinases in the process of T lineage commitment. In fact, thymocyte development up to the CD44CD25+ stage in Lck-deficient mice appeared to be abnormal (47). The number of DN cells in the Lck-deficient mice was ~2025% and the percentages of CD44+CD25 or CD44+CD25+ cells were ~50%, and therefore the absolute cell numbers of these two subpopulations were ~10% (data not shown). These results suggest that the Lck kinase plays certain roles on thymocyte development by the CD44CD25+ stage. Although the involvement of Lck kinase in the process of T lineage commitment has not been elucidated yet, Lck-deficient/plck-GFP-Tg mice may provide a good experimental model for addressing this issue.
Recently, the generation of similar GFP-Tg mice was reported by Buckland et al. (55). Consistent with the observation in our Tg mice, GFP expression was observed in T lineage cells but not in B cells or macrophages. GFP expression was also detected in NK cells. Although this observation differs from our results (Fig. 3
), the discrepancy may be due to the differences in the NK cell preparation. Their NK cell preparation appears to be a mixture of NK1.1+ TCR NK cells and NK1.1+ TCR+ NKT cells. In our plck-GFP-Tg mice, the former is completely negative for GFP, but a certain portion of the latter was GFP+. It is curious that the expression levels of GFP in the four DN thymocyte subpopulations were very different among the lines established by Buckland et al. As they did point out, however, the reason for the variation in GFP expression was not clear. Although we analyzed only a single line of plck-GFP-Tg mice, both homozygous and heterozygous animals were included in the study (Fig. 1
). The expression levels of GFP were significantly higher in homozygous mice and the expression patterns of GFP in each subpopulation of the developing thymocytes were similar. Particularly, the percentages of GFP+ cells in the four subpopulations of DN thymocytes were indistinguishable between heterozygous and homozygous mice. Thus, it is most likely that the majority of the cells expressing the proximal promoter of lck were visualized by GFP expression in our plck-GFP-Tg mice. Moreover, very similar expression profiles of transgenes in the four DN thymocyte subpopulations defined by CD44 and CD25 staining were observed in other Tg mice with the lck proximal promoter cassette (data not shown). Thus, it appears to be unlikely that the plck-GFP gene is integrated nearby a particular T cell-specific promoter element.
A number of studies have suggested that Lck is expressed in human NK cells (5665) and rat NK cells (66,67). Since the proximal promoter of lck is not active in the freshly prepared mouse NK cells (Fig. 3
), it is conceivable that the expression of lck in mouse NK cells is totally controlled by the distal promoter. Alternatively, Lck may not be expressed in mouse NK lineage cells. However, a careful biochemical analysis will be required to address the regulation of Lck expression in NK lineage cells. Similarly, some NKT cells and a proportion of 
T cells that developed in our HOS culture did not express the proximal promoter of lck, and these cells may also use also the distal promoter of lck.
The proximal promoter of lck was found to be substantially expressed in peripheral T cells (Fig. 2
). A similar result was reported by Buckland et al. (55). While this observation was unexpected, we have observed a significant dominant-negative Ras transgenic effect in the peripheral T cells when the transgene was controlled by the proximal promoter of lck (36). In addition, newly established dominant-negative calcineurin Tg mouse lines showed a significant Tg effect in the peripheral T cells (68). Although the reason for the discrepancy to the previous reports (5,7) is not clear, it is possible that other gene segments either in the promoter region or intron region of the lck gene may have regulatory activity on the expression of the lck gene in mature T cells. In any event, it appears to be clear that there is substantial expression of the transgene also in the peripheral T cells when the Tg construct is prepared with this lck proximal promoter cassette (8).
In the thymus, developing T cells at various stages have been identified by FCM analysis with a combination of mAb specific for various cell surface differentiation antigens, such as CD4, CD8, CD44 and CD25. Here, we show that the expression of lck proximal promoter activity in combination with GFP-Tg technologies can be used as a differentiation marker. This approach offers a novel means for the direct tracking and the identification of developing T cells in a more precise fashion. This can be further appreciated from pioneering studies using mice with transgenic or knock-in GFP (6971), in which new aspects of RAG expression and possible rearrangement events in the peripheral lymphoid tissues have been realized. Thus, the visualization of the expression in promoter activity of certain intracellular signaling molecules or transcription factors provided by the help of the GFP reporter offers a way to view the intracellular machinery that is crucial for the development of lymphocytes.
 |
Acknowledgments
|
---|
The authors are grateful to Drs Ralph T. Kubo and Toshitada Takemori for helpful comments and constructive criticisms in the preparation of the manuscript. The authors also thank Ms Kaoru Sugaya for her excellent technical support. This work was supported partly by grants from the Ministry of Education, Science and Culture (Japan), and by the Ministry of Health and Welfare (Japan).
 |
Abbreviations
|
---|
AGM aortagonadmesonephros |
APC allophycocyanin |
B6 C57BL/6 |
DN double-negative |
DP double-positive |
d.p.c. days post-coitum |
FCM flow cytometry |
GFP green fluorescence protein |
GM-CSF granulocyte macrophage colony stimulating factor |
HOS high oxygen submersion |
Lin lineage marker |
LM littermate |
PE phycoerythrin |
SCF stem cell factor |
SP single-positive |
TN triple-negative |
TNF tumor necrosis factor |
Tg transgenic |
 |
Notes
|
---|
Transmitting editor: A. Singer
Received 7 August 2000,
accepted 5 October 2000.
 |
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