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Immunology. Jun 2002; 106(2): 212–221.
PMCID: PMC1782720

Interleukin-7 inhibits pre-T-cell differentiation induced by the pre-T-cell receptor signal and the effect is mimicked by hGM-CSF in hGM-CSF receptor transgenic mice

Abstract

We have previously reported that human granulocyte–macrophage colony-stimulating factor (hGM-CSF) causes a stage-specific inhibition of T-cell receptor (TCR) αβ cell development in the thymus of transgenic mice constitutively expressing the hGM-CSF receptor. Since it has been reported that the addition of interleukin-7 (IL-7) to fetal thymic organ culture (FTOC) has similar effects, we compared the effects of IL-7 and hGM-CSF on TCRαβ cell development in hGM-CSF receptor transgenic mice. We reconstituted fetal lobes with sorted pre-T, or post pre-T CD4CD8 precursor cells. The addition of either IL-7 or hGM-CSF to these cultures suppressed further differentiation of pre-T cells but not post pre-T cells. At the same time, the cell number was increased, suggesting that pre-T-cell proliferation is stimulated by these cytokines. Furthermore, the differentiation of recombination-activating gene-1 (RAG-1)-deficient pre-T cells in response to anti-CD3 antibody stimulation was suppressed by either IL-7 or hGM-CSF, suggesting that these cytokines inhibit the pre-T-cell receptor (pre-TCR) signal. This inhibition is unexpected because the pre-TCR signal and the IL-7 signal have previously been considered to be co-operative. Recent analysis of the downstream events of IL-7 receptor and GM-CSF receptor revealed that they share common signal transduction molecules. Our results show that IL-7 is able to promote pre-T cell proliferation and to suppress differentiation induced by the pre-TCR signal. GM-CSF can mimic these biological activities of IL-7 when the pre-T cells express GM-CSF receptors. Our data suggest that both timing and level of activation of the IL-7 signalling pathway must be precisely regulated to facilitate the differentiation of thymocytes.

Introduction

T cells differentiate from haemopoietic stem cells in the thymus. Cytokine and antigen receptors provide signals to control the survival, proliferation and differentiation of the developing T cells, and thymocyte populations show varying cytokine response profiles depending on their stage of differentiation.1 Immature CD4CD8 double-negative (DN) precursors respond to a variety of cytokines, including interleukin-1 (IL-1), IL-2, IL-7, and stem cell factor (SCF). However, cytokine responsiveness is lost as DN cells up-regulate CD4 and CD8 and become double-positive (DP). After positive selection, thymocytes differentiate into the CD4 or CD8 single-positive (SP) stage and regain the ability to respond to cytokines. The mechanisms responsible for this stage-specific cytokine responsiveness are unknown. One possible mechanism is the down-regulation of cytokine receptors. The expression of several cytokine receptors, including the IL-2 receptor α, IL-7 receptor α, and SCF receptor (c-kit), decreases as thymic precursors differentiate into DP cells.24

We have previously reported the generation of transgenic mice expressing the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) high-affinity receptor.5 GM-CSF stimulates the proliferation and maturation of myeloid progenitor cells, and enhances the differentiated functions of these mature cells. In the transgenic mice, human GM-CSF receptor (hGMR) is expressed under the control of the H-2Ld class I promoter on haemopoietic cells in the spleen, thymus and bone marrow. Since murine GM-CSF does not bind to the human receptor, hGMR transgenic mice display a normal phenotype, and activation of the receptor can be induced with the addition of exogenous hGM-CSF. The effect of hGMR signalling on the differentiation of haemopoietic cells was examined by administering hGM-CSF to hGMR transgenic mice. The dose-dependent increases in the numbers of reticulocytes, neutrophils, eosinophils, monocytes and lymphocytes in the peripheral blood and spleen were observed in these mice, suggesting that hGM-CSF induces the proliferation of haemopoietic precursor cells expressing hGMR. However, an unexpected reduction in size of thymuses was observed in hGM-CSF-injected transgenic mice in a dose-dependent manner.7 Further analysis showed that hGM-CSF induces proliferation of thymocytes from hGMR transgenic mice in cell suspension culture. In contrast, addition of hGM-CSF into fetal thymic organ culture (FTOC) using fetal thymic lobes from the transgenic mice resulted in decrease of DP and T-cell receptor (TCR) αβ-expressing T cells, due to inhibition of TCRαβ cell differentiation in a stage-specific manner.8 These data suggest that hGM-CSF affects the proliferation and differentiation of thymocytes when they express hGMR, although hGM-CSF does not exert similar effects on thymocytes from wild-type mice. Thus, the mechanism of the inhibition of the development is unknown.

Interestingly, the effects of hGM-CSF are similar to the reported effects of IL-7.9 The addition of IL-7 to a FTOC resulted in a decrease in the number of DP and TCRαβ-expressing T cells, with a corresponding increase in the number of γδ T cells. It is not known whether IL-7 inhibits differentiation of αβ T cells in a stage-specific manner, as hGM-CSF does. Recent analysis of events downstream of the IL-7 and GM-CSF receptors has revealed that they share common signalling transduction molecules. The IL-7 receptor (IL-7R) is composed of a unique IL-7R α-chain and the IL-2R γ-chain, which is shared among the IL-2, IL-4, IL-9 and IL-15 receptors and called common γ.10 IL-7 induces activation of Janus kinase 1 (JAK1) and JAK3, signal transduction and activator of transcription 5 (STAT5), and phosphatidylinositol 3-kinase (PI3-K). Mice lacking either IL-7, IL-7Rα, common γ, JAK3, or JAK1 genes show a decrease of thymic cellularity,1120 suggesting that IL-7 signalling plays a critical role in thymocytes. The GM-CSF receptor is composed of an α-chain and a β-chain. The α-chain confers GM-CSF specificity, and the β-chain, called common β, is shared among the GM-CSF, IL-3 and IL-5 receptors.6 GM-CSF, IL-3 and IL-5 can all induce the activation of JAK2, STAT5, Ras, Raf, mitogen-activated protein kinase (MAPK) and PI3-K through common β. Some signal transduction components, including STAT5, Ras, MAPK, PI3-K and signal transducing adaptor molecule (STAM) are shared by common β and common γ pathways.6,10,2123 The similarity between the two pathways suggests that hGMR expressed on thymocytes may transduce signals similar to common γ signalling and explains why IL-7 and hGM-CSF have similar effects in hGMR transgenic mice. GM-CSF is known to regulate proliferation and differentiation of myeloid progenitor cells, but not thymocytes. In contrast, IL-7 is known to have critical roles in thymocyte proliferation and differentiation. It is therefore possible that hGM-CSF may mimic IL-7 effects in hGMR transgenic mice.

We assume that examining the similarities and differences between the effects of IL-7 and hGM-CSF on thymocyte differentiation will help to understand the mechanism of the inhibitory effect of hGM-CSF on T-cell development and to understand the roles of these cytokines, especially IL-7, in T-cell development. We compared the effects of these two cytokines in hGMR transgenic mice. Here we show that both IL-7 and hGM-CSF induce proliferation of CD44CD25+ pre-T cells but inhibit differentiation of pre-T cells into CD44CD25 post pre-T cells. Furthermore, the differentiation of RAG-1-deficient pre-T cells by anti-CD3 antibody stimulation is suppressed by either IL-7 or hGM-CSF at the CD8 immature single-positive (ISP) stage, suggesting that signalling by these cytokines interrupts differentiation induced by pre-TCR signalling. This inhibition is unexpected because the pre-TCR signal and the IL-7 signal have previously been considered to be co-operative. Our results show that IL-7 is able to promote pre-T-cell proliferation and to suppress the differentiation induced by the pre-TCR signal. GM-CSF can mimic these biological activities of IL-7 when the pre-T cells express GM-CSF receptors.

Materials and methods

Mice

The generation of hGMR transgenic mice has been described elsewhere.5 The hGMR transgenic line H2-81 was used in all experiments reported here. RAG-1-deficient mice were purchased from Jackson Laboratory (Bar Harbor, MN) and crossed with hGMR transgenic mice to establish hGMR transgenic mice in a RAG-1-deficient background. hGMR+/− or hGMR+/−RAG-1−/− mice, 3–6 weeks of age, were used for the isolation of thymic populations by fluorescence-activated cell sorting (FACS). All experiments were conducted according to our institution's guidelines for the care and treatment of experimental animals.

Culture medium and cytokines

The standard culture medium consisted of RPMI-1640 medium containing 10% fetal calf serum (FCS), 2 mm l-glutamine, 50 µm 2-mercaptoethanol, minimal essential medium amino acids and vitamins, sodium bicarbonate, penicillin, and streptomycin. For FTOC, medium containing 20% FCS was used (FTOC-medium). Recombinant hGM-CSF, kindly provided by Dr R. Kastelein (DNAX, Palo Alto CA), was used at 50 ng/ml. Recombinant mouse IL-7 purchased from PharMingen (San Diego, CA) was used at 500 U/ml.

Antibodies

All antibodies were purchased from PharMingen (San Diego, CA) unless otherwise specified. For the isolation of DN precursor populations, the following antibodies were used: anti-CD3-biotin (clone 144-2C11), anti-CD4-biotin (clone RM4-5), anti-CD8-biotin (clone 56-6.7), anti-B220-biotin (clone RA3-6B2), anti-Mac-1-biotin (clone M1/70), anti-Gr-1-biotin (clone RB6-8C5), anti-CD25-fluorescein isothiocyanate (FITC; clone 7D4), anti-CD44-phycoerythrin (PE; clone IM7), and Streptavidin-TriColor (Caltag Laboratories, South San Francisco, CA). For analysis, the following antibodies were used: anti-CD4-PE (clone RM4-5), anti-CD8-biotin, anti-TCRαβ-FITC (clone H57-597), anti-TCRγδ-FITC (clone GL3), or anti-CD25-FITC (clone 7D4), and streptavidin-TriColor (Caltag). Prior to staining, the cells were incubated with anti-CD32 (anti-FcgRII/III, clone 2.4G2) to reduce non-specific antibody binding to Fc receptor. For stimulation, anti-CD3 (clone 145-2C11, kindly provided by Dr T. Saito) or hamster immunoglobulin G (IgG; clone A19-3) were used.

Sorting and multiparameter analysis

The identification and isolation of DN thymocyte precursor populations have been described previously.24 Briefly, thymocytes were depleted by using anti-CD4 (clone RL172, used as culture supernatant) and anti-CD8 (clone AD4, Cedarlane Laboratories, Hornby, Ontario, Canada) antibodies followed by treatment with low-tox M rabbit complement (Cedarlane Laboratories, Hornby, Ontario, Canada) and 20 mg/ml DNase I (Sigma, St. Louis, MO). Viable cells were isolated by Histopaque 1083 (Sigma) centrifugation and stained with a panel of lineage antibodies directed against CD3, CD4, CD8, B220, Mac-1 and Gr-1 (all biotinylated), as well as anti-CD25-FITC and anti-CD44-PE. After washing, cells were incubated with streptavidin-TriColor. Cells were sorted using a FACS Vantage flow cytometer (Becton Dickinson, San Jose, CA) and the purity of the sorted population was routinely >95%. For analysis of T-cell repopulation in FTOC, single-cell suspensions were prepared for staining. To reduce non-specific staining, cells were incubated with anti-CD32 (clone 2.4G2) prior to staining for specific antigen expression. FTOCs were stained with anti-TCRαβ-FITC, anti-TCRγδ-FITC, or anti-CD25-FITC, anti-CD8α-biotin, and anti-CD4-PE, followed by streptavidin-TriColor. FTOCs were also stained with isotype-matched controls with fluorescence or biotin. Analysis was performed with a FACScan flow cytometer (Becton Dickinson) and CellQuest software (Becton Dickinson). All FACS plot figures show lymphocytes after gating on forward and side scatter.

FTOC

For conventional FTOC, fetal thymic lobes were removed on day 14 of gestation and cultured under standard conditions as described elsewhere.25 Briefly, thymic lobes were removed from plug-timed pregnant mice and cultured at the air–liquid interface, resting on 0·45 µm filters (Corning, New York) supported by small pieces of gelfoam sponge (Upjohn, Kalamazoo, MI). The lobes were cultured in 24-well plates containing 1 ml of FTOC medium. Selected cultures were supplemented with purified recombinant IL-7 or hGM-CSF. Fresh FTOC medium was supplied every 6 days. For fetal thymic lobe repopulation studies, lobes were removed on day 15 of gestation and depleted of endogenous T-cell progenitors by culturing in FTOC medium containing 1·35 mm deoxyguanosine for 5 days as described elsewhere.26 Depleted lobes were then individually plated with 1×104 pre-T or post pre-T cells in 30 µl of FTOC medium in Terasaki plates (Nunc, Kamstrup, Denmark). Plates were then inverted to allow the lobe and cells to combine at the bottom of a hanging drop.27 After 24–48 hr, repopulated lobes were transferred back into FTOC, and fresh FTOC medium was supplied every 6 days. At the indicated time-points, lobes were gently pressed under a glass coverslip in 100 µl of phosphate-buffered saline containing 2% FCS to release thymocytes. A population of the cells was stained with Trypan blue and counted twice. Thymocyte phenotyping was then performed as described above. When the lobes were cultured with antibodies for stimulation, they were suspended for 2 hr in medium supplemented with 25 µg/ml of anti-CD3 antibody or hamster IgG, in the presence or absence of the cytokines, and then placed on FTOC again with the same medium.

Single-cell suspension culture

Pre-T cells and post pre-T cells were sorted from hGMR transgenic mice and cultured at 105 cells/well in medium alone or in the presence of IL-7 or hGM-CSF in 96-well plates. The cells were harvested after 3 days, stained with Trypan blue, and counted twice.

Results

Both IL-7 and hGM-CSF inhibit development of TCRαβ cells but not TCRγδ cells in FTOC

There have been some controversial reports regarding the effects of IL-7 on T-cell development. Two groups reported that the addition of IL-7 to FTOC resulted in an increase in cell number but did not affect the CD4/CD8 proportion of thymocytes.28,29 However, another group reported that the addition of a higher dose (over 500 U/ml) of IL-7 resulted in the decrease of DP cells and TCRαβ cells.9 We compared the effects of IL-7 and hGM-CSF in thymocyte development in conventional FTOC using 500 U/ml of IL-7. Fetal lobes were removed from hGMR transgenic embryos at day 14 of gestation and placed into FTOC. Lobes were cultured in medium alone or in the presence of IL-7 or hGM-CSF for 11 days. The lobes were then harvested and thymocyte differentiation was determined by analysing CD4, CD8, TCRαβ, and TCRγδ expression using flow cytometry. As shown in Fig. 1, addition of either IL-7 or hGM-CSF to FTOC resulted in a decrease in the number of DP and TCRαβ-positive cells and an increase in the number of TCRγδ cells. Therefore, with this concentration of IL-7, the effects of hGM-CSF and IL-7 are similar. However, the effect of hGM-CSF was more potent than IL-7. Even at higher concentrations than 500 U/ml, the effect of IL-7 was less strong than that of hGM-CSF (data not shown and ref. 9). IL-7 had the same effect when wild-type thymic lobes were cultured (data not shown).

Figure 1
Effects of IL-7 and hGM-CSF on FTOC development. Day 14 fetal thymic lobes were harvested from hGMR transgenic embryos and placed in FTOC. Lobes were cultured in medium alone or in the presence of 500 U/ml of IL-7 or 50 ng/ml of hGM-CSF. FTOC were harvested ...

Both IL-7 and hGM-CSF inhibit further differentiation of pre-T cells but not that of post pre-T cells

Immature DN thymocytes can be divided into four distinct phenotypic precursor populations based on their differential expression of CD44 and CD25. These cells become mature in the following order: CD44+CD25, CD44+CD25+ (pro-T), CD44CD25+ (pre-T), CD44CD25 (post pre-T).24,30 We have already reported that hGM-CSF inhibits the development of hGMR-expressing pro-T and pre-T cells but not that of post pre-T cells.8 However, it is not known whether IL-7 inhibits the development of TCRαβ cells in a stage-specific manner, as hGM-CSF does. It is possible that the decrease of DP and TCRαβ cells in IL-7-treated FTOC is caused by the decrease of proliferation of precursor cells or increase of cell death of DP cells. Due to the similarity of signalling pathways of IL-7 and hGM-CSF, we assumed that IL-7 inhibits the development of TCRαβ cells at the same point as does hGM-CSF. Therefore we compared the effects of IL-7 and hGM-CSF on the development of both pre-T and post pre-T cells. Since stromal cells in transgenic thymic lobes also express hGMR, we repopulated wild-type fetal lobes with trangenic sorted thymocyte precursors to exclude any effects of stromal cell activation by hGM-CSF. Fetal thymic lobes were removed from wild-type mice on day 15 of gestation and depleted of endogenous T-cell precursors by culturing with deoxyguanosine.

Based on CD44 and CD25 expression, subsets of pre-T and post pre-T cells were sorted from adult transgenic mice and co-cultured with the depleted lobes. The repopulated lobes were transferred to FTOC and cultured in medium alone or in the presence of IL-7 or hGM-CSF. The lobes were then harvested and thymocyte differentiation was examined by analysing CD4, CD8, TCRαβ, and TCRγδ expression. The cultures of the CD44CD25+ pre-T cells were harvested on day 14 and those of the CD44CD25 post pre-T-cell cultures were harvested on day 8.

The addition of IL-7 to the pre-T-cell FTOC resulted in an approximately two-fold increase in total cell number (Fig. 2a). The data suggest that the T-cell precursor cells proliferate in response to IL-7 in the FTOC. However, IL-7 caused a decrease of DP and TCRαβ cells in the FTOC, but did not affect T-cell differentiation in the post pre-T-cell FTOC (Fig. 2b). Although the absolute numbers of DP cells in the pre-T-cell culture were not significantly different between medium alone and medium supplemented with IL-7, the number of DN and CD8 SP cells in pre-T-cell culture increased when IL-7 was present (Table 1). Therefore, the proliferation of pre-T cells prior to the differentiation into DP cells results in an overall increase in cell number, and compensates for the decrease in the number of DP cells caused by a block in differentiation. These data suggest that IL-7 stimulates the proliferation of pre-T cells and inhibits their differentiation. IL-7 did not affect proliferation or differentiation of the more mature post pre-T cells.

Figure 2
Effects of IL-7 and hGM-CSF on the differentiation of triple negative (TN) thymocyte precursor subsets from hGMR transgenic mice. Pre-T and post pre-T cells were sorted from transgenic mice, transferred into 2-deoxyguanosine-depleted wild-type fetal lobes, ...
Table 1
The absolute numbers of CD4/CD8 populations in pre-T and post pre-T FTOCs

Addition of hGM-CSF to the repopulated FTOCs increased cell numbers in both the pre-T cell-culture and the post pre-T-cell cultures (Fig. 2a). However, like IL-7, hGM-CSF inhibits the differentiation of pre-T but not post pre-T cells (Fig. 2b). Although a reduction in the percentage of DP cells was observed in the post pre-T-cell culture in the presence of hGM-CSF, the TCRαβ profile showed that most of the cells in the culture expressed TCRαβ (Fig. 2b). Three-colour analysis showed that more than 50% of DN and CD4, CD8 SP cells expressed TCRαβ (data not shown). Moreover, no significant difference in the absolute numbers of DP cells was observed between medium alone and medium supplemented with hGM-CSF in the post pre-T-cell culture (Table 1). We have reported that DN and SP cells expressing hGMR proliferate well in response to hGM-CSF. In contrast, DP cells do not proliferate despite their expression of hGMR.8 Therefore, the reduction in the population of DP cells was caused by the proliferation of DN and mature SP cells stimulated by hGM-CSF. This proliferation also contributes to the increased number of cells per lobe in the culture. The hGM-CSF showed a similar but more potent effect compared to IL-7 in both stimulating proliferation and inhibiting the differentiation of T-cell progenitors.

IL-7 supports the survival only of pre-T cells but hGM-CSF supports those of both pre-T and post pre-T cells

The effects of the two cytokines on the proliferation of T-cell precursors were examined in cell suspension culture. Pre-T and post pre-T cells from hGMR transgenic mice were cultured for 3 days in medium alone or in medium with IL-7 or hGM-CSF and then counted. IL-7 supported survival of pre-T cells but not post pre-T cells (Fig. 3). The hGM-CSF supported survival of both pre-T and post pre-T cells. An additional 30% of the pre-T cells were recovered after 3 days of culture with hGM-CSF, suggesting that hGM-CSF induces proliferation of these cells. These effects observed in cell suspension culture correlate well with the results in the repopulated FTOC shown in Fig. 2(a).

Figure 3
Effects of IL-7 and hGM-CSF on pre-T and post pre-T-cell survival in single-cell suspension culture. Pre-T and post pre-T cells were sorted from trangenic mice and 105 cells were cultured in medium alone or in the presence of 500 U/ml of IL-7 or 50 ng/ml ...

Stimulation of RAG-1-deficient thymic lobes by anti-CD3 antibody did not overcome the suppression of differentiation by either IL-7 or hGM-CSF

Both IL-7 and hGM-CSF inhibited the transition of pre-T cells to post pre-T cells. Since it is known that pre-T cells require the pre-TCR signal for their differentiation,31 we examined whether the pre-TCR signal can overcome the inhibition induced by IL-7. The pre-TCR consists of a rearranged TCR β-chain in association with a pre-T α-chain. The transduction of a signal from this receptor is required for the differentiation of DN cells into DP cells.32 RAG-deficient mice are unable to generate αβ-T cells, and thymic differentiation is arrested at the pre-T to post pre-T boundary.33 Stimulation with anti-CD3 antibody can mimic pre-TCR signalling and induce differentiation into the DP cell stage in a RAG-1−/− thymus.34

Fetal lobes were removed from RAG-1−/−hGMR+/− embryos at day 14 of gestation, placed into FTOC, and cultured in medium alone or medium with IL-7 or hGM-CSF for 2 days. Lobes were then stimulated with anti-CD3 antibody in medium alone or medium with IL-7 or hGM-CSF, and cultured for six additional days. The lobes were harvested and thymocyte differentiation was determined by analysing CD4, CD8 and CD25 expression. As shown in Fig. 4(b), stimulation of RAG-1−/−hGMR+/− lobes with anti-CD3 antibody induced differentiation into DP cells in the absence of the cytokines. However, DP cells were not observed in the presence of either IL-7 or hGM-CSF. Nevertheless, in the presence of IL-7, anti-CD3 antibody induced an increase in cell number (Fig. 4a), the appearance of CD8 ISP cells, and the down-regulation of CD25 in the presence of IL-7 (Fig. 4b), suggesting that pre-TCR signals were not completely inhibited by IL-7. Addition of IL-7 to RAG-1−/−hGMR−/−FTOC showed the same effects as observed in RAG-1−/−hGMR+/−FTOC (data not shown). Inhibition by IL-7 under these conditions is more severe than that observed in the repopulated FTOC (Fig. 2c). This discrepancy may be due to the difference between the conventional FTOC system and the repopulated FTOC system. Stimulation by anti-CD3 antibody in the presence of hGM-CSF induced the appearance of CD8 ISP cells, but did not induce an increase in cell number or the down-regulation of CD25. In addition, some CD8 SP cells were observed even in the absence of anti-CD3 antibody.

Figure 4
Effects of IL-7 and hGM-CSF on the pre-TCR signal. hGMR+/−RAG-1−/− thymic lobes were stimulated by anti-CD3 antibody, mimicking the pre-TCR signal. Day 14 fetal thymic lobes were harvested from hGMR−/−RAG-1−/− ...

It is possible that the differences between IL-7 and hGM-CSF in RAG-1-deficient thymic lobes might be caused by an indirect effect mediated by stromal cells that also express hGMR. To exclude the effects of stromal cell activation by hGM-CSF, we repopulated wild-type fetal lobes with sorted transgenic thymocyte precursors. Unfortunately, we could not detect DP cells following anti-CD3 antibody stimulation in these repopulated lobes even in medium alone. However, we saw an increase in the cell number and the down-regulation of CD25 in medium alone (data not shown) and in the presence of hGM-CSF (Fig. 4c,d). No CD8 SP cells were observed without anti-CD3 antibody stimulation in the presence of hGM-CSF (data not shown). These data suggest that at least part of the difference between the effects of IL-7 and hGM-CSF in the conventional FTOC (Fig. 4a,b) was caused by the indirect effects of hGM-CSF on stromal cells in the hGMR transgenic thymus. We conclude that IL-7 is able to promote pre-T-cell proliferation and to suppress differentiation induced by the pre-TCR signals, and that GM-CSF can mimic these biological activities of IL-7 when pre-T cells express GM-CSF receptor.

Discussion

Here we compared the effects of IL-7 and hGM-CSF in hGMR transgenic thymuses. We showed that both IL-7 and hGM-CSF induce proliferation of CD44CD25+ pre-T cells but inhibit differentiation of pre-T cells into CD44CD25 post pre-T cells. This inhibition results in a decrease in the number of TCRαβ cells but does not affect the development of TCRγδ cells. We also observed that the differentiation of RAG-1-deficient pre-T cells induced by anti-CD3 antibody was suppressed by either IL-7 or hGM-CSF at the CD8 ISP stage. We conclude that IL-7 is able to promote pre-T-cell proliferation and to suppress the differentiation induced by pre-TCR signals, and GM-CSF can mimic these biological activities of IL-7 when pre-T cells express GM-CSF receptor.

As discussed above, IL-7 signalling has been shown to be critical for thymocyte differentiation. Pre-TCR signalling is also important for thymocyte proliferation and differentiation as shown by the fact that Pre-TCRα−/− mice have decreased thymic cellularity and a reduced DP population.31 Mice lacking both the common γ and pre-TCRα genes show a 200-fold decrease in thymic cellularity when compared to mice lacking only one of these genes, and their thymocyte differentiation is completely blocked.35 Therefore IL-7 signalling and pre-TCR signalling have been considered to be partially compensatory and work co-operatively, although the signalling pathway of the pre-TCR signal is not well known. However, we report here the novel, unexpected finding that IL-7 signalling inhibits thymocyte differentiation induced by pre-TCR signalling.

The common γ-chain of the IL-7 receptor and the common β-chain of the GM-CSF receptor share some signal transduction molecules, such as STAT5, PI-3K, Ras, MAPK and STAM. It has been reported that either IL-7 or IL-3, which shares the common β-chain with hGMR, can support the proliferation of immature B-cell precursor cells, and removal of these cytokines results in IgM rearrangement in vitro.36 Moreover, IL-3 injection can partially rescue the impaired B-cell development and T-cell cellularity seen in JAK3-deficient mice.37 Therefore, activation of common β, either through IL-3 or hGM-CSF, may transduce signals shared by common γ in T-cell precursors. Therefore, hGM-CSF can mimic the ability of IL-7 to induce proliferation in cells that express hGMR.

Since both IL-7 and hGM-CSF also inhibit thymocyte differentiation, it is possible that the two cytokine signalling pathways share common inhibitor proteins. One candidate molecule is STAT5. STAT5 has two isoforms, STAT5a and 5b, which are encoded by two different genes. Although IL-7−/− mice show a severe decrease in thymic cellularity,11 neither STAT5a−/− nor STAT5b−/− mice showed a similar phenotype.38,39 Nor did mice deficient for both STAT5a and 5b show a decrease.40 This would indicate that the genes are dispensable for IL-7 signalling in thymocytes. However, expression of a constitutively active STAT5a can partially recover thymic cellularity and TCRαβ cell development in IL-7R−/−FTOCs.41 Therefore, the role of STAT5 in T-cell development is still not clear, and further analysis is required to elucidate whether STAT5 plays a role in IL-7 inhibition of T-cell development.

Another candidate signalling pathway is the PI3-K or MAPK cascade. Activation of PI3-K by IL-7 is known to be important for cell proliferation but not for differentiation in human T-cell development in FTOC.42 The proliferative response of human T cells to IL-7 is inhibited by the highly selective MAPK inhibitor SB203580.43 It is possible that cell proliferation induced by IL-7 through the MAPK pathway is linked to the inhibition of differentiation. A recent report showed that pro-T cells remain in the cell cycle, but some pre-T and DP cells exit from the cell cycle, suggesting a correlation between pre-TCR or TCR selection and cell cycle status.44,45 The extent of the inhibition caused by IL-7 and hGM-CSF correlates with their ability to induce cell proliferation; addition of hGM-CSF to the pre-T-cell FTOC increased the cell number 10-fold and inhibited differentiation completely, while IL-7 increased cell number just two-fold and only partially inhibited the differentiation. It is possible that, rather than inhibiting differentiation, IL-7 instead stimulates the cells to undergo apoptosis when it is present in combination with a pre-TCR signal. However, the addition of anti-CD3 antibody in the presence of IL-7 to RAG-1−/− FTOC resulted in an increase in cell numbers (Fig. 4a), and the number of apoptotic cells did not increase when they were analysed by annexinV (data not shown). This suggests that co-stimulation with anti-CD3 antibody and IL-7 does not trigger cell death. The relationship between cell cycle status and the inhibitory effect of IL-7 on T-cell development remains to be elucidated.

Although the effects of IL-7 and hGM-CSF in hGMR-expressing transgenic mice were similar in some ways, there were also differences. IL-7 supported the survival of pre-T cells but not post pre-T cells, while hGM-CSF supported survival of both pre-T and post pre-T cells in cell suspension culture. Moreover, hGM-CSF induced proliferation of the precursor cells more effectively than IL-7 (Fig. 3). These differences may be caused by varying levels of receptor expression. Although common γ is expressed throughout thymocyte differentiation, the expression of the IL-7R α-chain changes during differentiation.2,46 Pro-T cells express IL-7Rα at high levels and proliferate well, while pre-T cells express it at lower levels and survive but do not proliferate in response to IL-7. Although the level of IL-7Rα expression in post pre-T cells is unknown, DP cells do not express IL-7Rα before selection.2,47 Post pre-T and DP cells do not respond to IL-7, suggesting that the expression of IL-7Rα is reduced at the pre-T-cell stage and remains low in post pre-T cells. hGMR is highly expressed in both pre-T and post pre-T cells (our unpublished data8), and both of these precursors proliferate well in response to hGM-CSF. Therefore, although post pre-T cells have the capacity to proliferate when they receive signals, the reduced expression of IL-7Rα may contribute to the IL-7 unresponsiveness of these cells. Recently Kang et al. found that pro-T cells can be divided into two populations by the level of IL-7Rα expression and that the populations have different potentials for αβ/γδ lineage choice.48 IL-7Rα negative pro-T cells generate a 13-fold higher ratio of TCRαβ lineage cells to TCRγδ lineage cells than IL-7R α-positive cells did. The report suggests that the down-regulation of IL-7Rα is a step to commit to TCRαβ cell lineage. Our finding that IL-7 signalling can inhibit differentiation of pre-T cells to TCRαβ cell lineage, which is induced by the pre-TCR signal, may explain why this down-regulation of IL-7Rα is important for the maturation of TCRαβ cells.

The effects of IL-7 may not only be regulated by the expression level of its receptor but also by that of the ligand. As described in the Results section, there have been several controversial reports regarding the effects of IL-7 in FTOCs. Two groups reported that the addition of IL-7 to FTOC resulted in an increase in cell number but did not affect CD4/CD8 proportion of thymocytes.28,29 However, another group reported that the addition of a high dose of IL-7 resulted in a decrease of DP cells and TCRαβ cells.9 The discrepancy of the results is also seen in IL-7 transgenic mice. Some groups have generated transgenic mice that overexpress IL-7, but only one group reported a decrease in the number of DP cells in the thymus in these mice.4951 These observations suggest that the level of IL-7 may be important for proper differentiation. IL-7 expression in the developing fetal thymus is transient. It starts on day 12 of gestation and peaks on day 15, after which it decreases.52,53 Fetal thymocyte development is synchronized, allowing this wave of IL-7 expression to be observed. This transient expression of IL-7 correlates with our findings here. We hypothesize that a high amount of IL-7 is required for the proliferation of immature T-cell precursors during the early stages of gestation. However, further maturation requires that the amount of IL-7 be reduced. Therefore we assume that the expression level of the ligand plays a role in the regulation of IL-7 signalling, in addition to the expression level of the receptor. Our data suggest that both the timing and the levels of IL-7 signalling must be precisely regulated to facilitate the differentiation of thymocytes.

Acknowledgments

We thank Dr K. Nakao for support to maintain mouse strains. We are grateful to Drs H. Spits, Y. Amasaki, Y. Kamogawa and S. Bullock for critical reading of the manuscript.

Abbreviations

DN
double-negative (CD4 and CD8 double-negative)
DP
double-positive (CD4 and CD8 double-positive)
FTOC
fetal thymic organ culture
hGM-CSF
human granulocyte–macrophage colony-stimulating factor
IL-7
interleukin-7
pre-TCR
pre-T-cell receptor
RAG
recombination activating gene
SP
single-positive (CD4 or CD8 single-positive)
TCR
T-cell receptor

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