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The Journal of Immunology, 2007, 179: 126-131.
Copyright © 2007 by The American Association of Immunologists, Inc.
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beta-Catenin Expression Enhances IL-7 Receptor Signaling in Thymocytes during Positive Selection1

Qing Yu, Mai Xu and Jyoti Misra Sen2

Lymphocyte Development Unit, Laboratory of Immunology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Differentiation of CD4+CD8+ double-positive thymocytes into CD8+ single-positive (SP) thymocytes is regulated by TCR and cytokine receptor signals. Previously, we have shown that expression of stabilized beta-catenin, the major transcriptional cofactor of T cell factor, results in increase in both CD4SP and CD8SP thymocytes with a preferential effect on CD8SP thymocytes. In this report, using mice expressing stabilized beta-catenin and mice with T cell specific deletion of beta-catenin, we show that beta-catenin expression augments IL-7R{alpha}-chain expression and down-regulates suppressor of cytokine signaling-1 expression in thymocytes undergoing positive selection. Consequently, beta-catenin expression augments IL-7R signaling in thymocytes during positive selection and promotes the development of CD8SP thymocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Development of mature T cells in the thymus is mediated by signals transduced through surface {alpha}beta TCR and CD4 and CD8 coreceptors upon interaction with MHC-self-Ag complexes on the thymic epithelial cells. These signals ensure the development of immature CD4+CD8+ double-positive (DP)3 thymocytes into CD4–CD8+ single-positive (SP) and CD4+CD8– SP thymocytes with coordinated expression of CD8 with MHC class I-restricted TCR and CD4 with MHC class II-restricted TCR (1, 2, 3, 4, 5). Additional survival and differentiation signals are provided by IL-7-IL-7R signals. A role of IL-7R signaling in positive selection has been difficult to assess as mice deficient in IL-7, IL-7R{alpha}, and the common cytokine receptor {gamma} chain ({gamma}c) are profoundly lymphopenic and have severely hypoplastic thymuses due to defects in survival and differentiation of the most immature thymocytes (6, 7, 8). However, a role of IL-7R in inducing Bcl-2 expression during positive selection was shown using a blocking anti-IL-7R Ab in vivo. Accordingly, enforced expression of Bcl-2 partially rescued generation of mature thymocytes in IL-7R-deficient mice, suggesting that IL-7 provides survival signals during positive selection (9).

Blocking IL-7R signals with Abs in fetal thymic organ culture (FTOC) system selectively blocked development of CD8SP thymocytes but not CD4SP thymocytes, indicating preferential requirement for IL-7R signals for generation of CD8SP thymocytes (10). Accordingly, suppressor of cytokine signaling (SOCS)-1-deficient (SOCS-1–/–) mice (11) and mice expressing constitutively active STAT5 ((Stat5CA)-Tg mice) (12) both have enhanced cytokine signaling and show an increase in the number of cells in the CD8SP lineage. Additionally, positive selecting TCR signals have been shown to up-regulate expression of IL-7R{alpha} and down-regulate expression of SOCS-1, thereby confer IL-7 responsiveness to signaled thymocytes, which facilitates subsequent differentiation of CD8SP thymocytes (13). Thus to date, TCR coreceptors CD4 and CD8 as well as IL-7R are believed to be the major cell surface receptors that play a role in positive selection of thymocytes. However, molecular aspects of signals transmitted by these receptors during positive selection remain to be fully defined.

beta-Catenin is a major cofactor for the T cell factor family of transcription factors (14, 15). beta-Catenin expression is regulated at the posttranslational level by glycogen synthase kinase-3beta-mediated phosphorylation followed by ubiquitination and degradation. Deletion of the glycogen synthase kinase-3beta phosphorylation sites results in a stabilized form of beta-catenin, which is fully functional and has been used to study the role of beta-catenin in vivo (16, 17, 18). We have previously shown that expression of stabilized beta-catenin in thymocytes using the proximal Lck promoter (CAT-Tg) results in an increase in the number of mature CD4SP and CD8SP thymocytes at the cost of DP thymocytes, with a greater effect on CD8SP thymocytes (19). Conversely, T cell-specific deficiency of beta-catenin (CAT-KO) results in a decrease in the number of mature thymocytes with a statistically significant decrease in CD8SP thymocytes (20). Together these studies suggest a role for beta-catenin during positive selection of thymocytes.

The present study was undertaken to investigate the mechanisms by which beta-catenin expression regulates the number of mature CD8SP thymocytes. In CAT-Tg mice, targets of positive selection such as IL-7R{alpha} up-regulation and SOCS-1 down-regulation are enhanced in thymocytes undergoing positive selection. Conversely, in CAT-KO mice, developing thymocytes fail to fully down-regulate expression of SOCS-1. As a result, CAT-Tg mice have a higher number and CAT-KO have a lower number of mature thymocytes with a preferential effect on CD8SP thymocytes. Furthermore, we demonstrate that in vitro and ex vivo CAT-Tg thymocytes exhibit a more robust response to IL-7 treatment. Taken together with the observation that CAT-Tg, SOCS-1-deficient, and Stat5CA-Tg mice are highly similar with respect to the generation of CD8SP thymocytes, these data demonstrate that beta-catenin expression enhances IL-7R signaling in developing thymocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Generation of CAT-Tg and CAT-KO mice were previously described (19, 20). SOCS-1+/–IFN-{gamma}–/– mice were provided by Dr. J. Ihle (St. Jude Children’s Research Hospital, Memphis, TN) (21). Thymuses from Stat5CA-Tg mice were provided by Dr. M. Farrar (University of Minnesota, Minneapolis, MN) (12). Age-matched littermate controls were used in all experiments.

Flow cytometry and cell sorting

Cells were harvested, stained, and analyzed on a FACSCalibur (BD Biosciences). Dead cells were excluded by forward light scatter and propidium iodide gating. All the data were acquired and presented on log scale. Abs with the following specificities were used for staining: allophycocyanin CD4 (GK1.5), PE-CD8{alpha} or PerCP-Cy5.5 CD8{alpha} (53-6.7), FITC-TCRbeta (H57–597), PE-{gamma}c (4G3), Alexa Fluor 488-phosphorylated STAT5 (pSTAT5, clone 47), and PE-Bcl-2 (3F11) (all from BD Pharmingen). PE IL-7R{alpha} (A7R34) was purchased from eBioscience. Stained thymocytes were sorted into double negative, DP, CD4+CD8low, CD4SP, and TCRhigh CD8SP subpopulations using a DakoCytomation MoFlo.

Intracellular staining

Freshly isolated thymocytes or spleen cells were fixed and permeabilized first with 4% paraformaldehyde, and then with methanol/acetone mixture (1:1, v/v). Cells were then stained with anti-Bcl-2 Ab or anti-pSTAT5 Ab. After intracellular staining, cells were further stained for CD4 and CD8 (22). Thymocytes were cultured in vitro at 5 x 106/ml in medium or 6 ng/ml IL-7 (R&D Systems) for 1 h to assess pSTAT5 expression or cultured for 18 h to assess Bcl-2 expression.

Retroviral infection

For retroviral infection, purified CD4 T cells from C57BL/6 were activated by plate-bound anti-CD3 and anti-CD28 Abs with IL-2 for 2 days, and then incubated with supernatant containing MSCV-CAT-huCD8 or MSCV-huCD8 for a total of 3 h. The virus supernatant was then removed and the cells were further cultured in medium with IL-2 for 2 days before being examined for IL-7R{alpha}-chain mRNA expression.

FTOC system

Embryonic day 17.5 fetal thymic lobes were placed in FTOCs in medium or with 50 µg/ml anti-IL-7R{alpha} (eBioscience) plus anti-{gamma}c Abs (BD Pharmingen). On day 3 of culture, thymocytes were harvested, made into suspension culture and treated with 0.01% pronase (to remove surface CD4 and CD8) for 10 min. The cells were then cultured overnight in medium to allow re-expression of CD4 or CD8 molecules that were being actively synthesized in the cell. Thymocytes were then analyzed by flow cytometry, and the number of CD4SP and TCRbetahigh CD8SP thymocytes per thymic lobe was determined.

Quantitative real-time RT-PCR

Total RNA from sorted thymocyte subpopulations was reverse transcribed using poly(dT) and Superscript III reverse transcriptase (Invitrogen Life Technologies). The cDNA was subjected to real-time PCR amplification (Applied Biosystems) for 40 cycles with annealing and extension temperature at 60°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CAT-Tg, SOCS-1-KO, and Stat5CA-Tg mice have increased mature CD8SP thymocytes

In CAT-Tg mice expressing stabilized beta-catenin under proximal Lck promoter (19), the frequency of both CD4SP and CD8SP thymocyte populations relative to DP thymocytes was increased, with a significantly greater increase in CD8SP population (Fig. 1A, top left). Conversely, in CAT-KO mice (20) with a T cell-specific deletion of beta-catenin gene, there was a modest but statistically significant decrease in the frequency of CD8SP thymocytes (Fig. 1A, top right). The slightness in the degree of changes in CAT-KO mice may be attributed to the presence of {gamma}-catenin that has been shown to play a redundant role in other biological systems (23, 24). Interestingly, in SOCS-1–/–IFN-{gamma}–/– mice (21) (IFN-{gamma} deficiency prevents disease and perinatal death) and in mice expressing an active form of STAT5b (Stat5CA-Tg mice) (12), a preferential increase in CD8SP thymocytes was observed (Fig. 1A, bottom panels) (11, 12). The absolute number of CD8SP thymocytes, but not CD4SP thymocytes, in CAT-Tg, SOCS-1–/–IFN-{gamma}–/–, and Stat5CA-Tg mice showed a significant increase compared with control mice (Fig. 1B). In CAT-KO mice, a decrease in the number of both CD4SP and CD8SP thymocytes was observed (Fig. 1B). However, it mainly reflects the decrease in total thymic cellularity (20). Thus expression of stabilized beta-catenin preferentially increases the number of CD8SP thymocytes, and the similarity in thymic phenotypes among CAT-Tg, SOCS-1-KO, and Stat5CA-Tg mice suggests the possibility that beta-catenin expression may regulate the number of CD8SP thymocytes by affecting cytokine signaling during positive selection.


Figure 1
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  		FIGURE 1. CAT-Tg, SOCS-1-KO, and Stat5CA mice have increased mature thymocytes. A, Percentage of CD4SP and TCRhigh CD8SP thymocytes (relative to DP) in CAT-Tg, CAT-KO, SOCS-1–/–IFN-{gamma}–/–, and Stat5CA-Tg mice compared with their littermate controls (grey bar). CAT-Tg (n = 8), CAT-KO (n = 7), SOCS-1–/–IFN-{gamma}–/– (n = 4), and Stat5CA-Tg (n = 4) mice are represented. B, Absolute number of CD4SP and CD8SP thymocytes in the mice groups described in A are shown.

 
beta-Catenin expression enhances IL-7R signals in thymocytes undergoing positive selection

To determine whether beta-catenin expression enhanced IL-7 response, we directly examined IL-7-induced phosphorylation of STAT5 (pSTAT5) in thymocytes. Mature CD4SP and CD8SP thymocytes from CAT-Tg mice showed significantly higher IL-7 responsiveness compared with cells from control mice, as noted by higher pSTAT5 (shown by mean fluorescence intensity; MFI) induced in response to in vitro IL-7 treatment (Fig. 2A, top left). We also examined IL-7 responsiveness in SOCS-1–/–IFN-{gamma}–/– CD4SP and CD8SP thymocytes to determine whether the increase in IL-7 responsiveness in CAT-Tg thymocytes was functionally significant. We found that the increase in IL-7 responsiveness in CAT-Tg thymocytes was similar or even greater than that seen in SOCS-1–/–IFN-{gamma}–/– thymocytes (Fig. 2A, compare top left and bottom panels). These data suggest that the enhanced IL-7 responsiveness caused by beta-catenin expression is biologically significant. To determine whether enhanced IL-7R signaling results in enhanced downstream target gene expression, we examined IL-7 induced up-regulation of Bcl-2 by intracellular staining. Both CD4SP and CD8SP thymocytes from CAT-Tg mice showed higher level of Bcl-2 induced by IL-7 treatment compared with control counterparts, with CD8SP thymocytes showing a more dramatic increase that is consistent with a higher level pSTAT5 induction by IL-7 in these cells (Fig. 2A, top right). Thymocytes from CAT-KO mice did not show decreased responsiveness to IL-7 (data not shown), suggesting that {gamma}-catenin may play a redundant role in this function. During positive selection and maturation to the SP stage, DP thymocytes that have received the initial positive selection signal first differentiate into a transitional stage described as CD4+CD8low cells. This developmental intermediate, along with CD69+ DP thymocytes, is believed to be precursor for mature thymocytes (25). In particular, enhanced IL-7R signaling in CD4+CD8low thymocytes is believed to promote the generation of CD8SP thymocytes from these positive selection intermediates. CAT-Tg CD4+CD8low thymocytes showed higher levels of pSTAT5 compared with control CD4+CD8low cells in response to in vitro IL-7 treatment (Fig. 2B). CAT-Tg CD4+CD8low cells also showed higher levels of Bcl-2 expression upon IL-7 treatment compared with control CD4+CD8low thymocytes (Fig. 2B). Statistical analysis of several experiments showed that the increased responses, though modest, were highly significant (Fig. 2B). Again, the increase in IL-7 responsiveness in CAT-Tg CD4+CD8low thymocytes was comparable to or greater than that seen in SOCS-1–/–IFN-{gamma}–/– CD4+CD8low thymocytes (Fig. 2B, compare top and bottom panels), suggesting the enhanced IL-7 responsiveness caused by beta-catenin expression, although seemingly small, is biologically significant. Thus transgenic expression of beta-catenin significantly enhanced cytokine responsiveness of positive selection intermediates to IL-7 in vitro.


Figure 2
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  		FIGURE 2. beta-Catenin expression enhances IL-7R signals of thymocytes. A, Enhanced responsiveness of CAT-Tg mature thymocytes to IL-7 treatment in vitro. Thymocytes from control or CAT-Tg mice were stimulated with IL-7 in vitro. Induction of pSTAT5 after 1 h of treatment and Bcl-2 after 18 h of treatment of thymocytes was analyzed by intracellular staining and flow cytometry. Level of pSTAT5 induction (left) or Bcl-2 induction (right) is determined by mean fluorescence intensity (MFI) and is calculated by subtracting MFI before IL-7 treatment from after IL-7 treatment. Enhanced IL-7 response (upper panels) in CAT-Tg mature thymocytes is shown for control mice (n = 2) and CAT-Tg mice (n = 4). The results are representative of three independent experiments. Enhanced IL-7 response (lower panels) in mature thymocytes in SOCS-1–/–IFN-{gamma}–/– mice (n = 4). B, Enhanced response of CAT-Tg CD4+CD8low thymocytes to IL-7 in vitro. IL-7 responsiveness in CD4+CD8low thymocytes was assessed similarly as described in A. Enhanced IL-7 responses in CAT-Tg CD4+CD8low thymocytes (top left panels) and histograms of pSTAT5 and Bcl-2 staining in CAT-Tg thymocytes (top right panels) are represented. Control mice (n = 2) and CAT-Tg mice (n = 4) are shown. The results are representative of three independent experiments. Bottom panels represent enhanced IL-7 responses in CD4+CD8low thymocytes in SOCS-1–/–IFN-{gamma}–/– mice (n = 4). C, Increased level of pSTAT5 in immediately ex vivo thymocytes from CAT-Tg mice. Freshly isolated thymocytes were fixed and permeabilized, and then subjected to intracellular staining for pSTAT5. MFI of pSTAT5 (left) in CD4+CD8low cells and histograms of intracellular pSTAT5 (right panels) in CD4+CD8low, CD4SP, and CD8SP thymocytes from control and CAT-Tg mice are shown. Data are representative of eight independent analyses.

 
To determine whether CAT-Tg CD4+CD8low thymocytes had received higher IL-7R signaling in vivo, we examined pSTAT5 in ex vivo cells. Intracellular staining for pSTAT5 of ex vivo CAT-Tg CD4+CD8low thymocytes showed markedly higher level of pSTAT5 compared with control CD4+CD8low thymocytes (Fig. 2C, right top). Ex vivo CAT-Tg CD4SP and CD8SP mature thymocytes also showed higher level of pSTAT5 (Fig. 2C, right top). Quantification of pSTAT5 level, based on intracellular staining, showed that CAT-Tg CD4+CD8low cells had significantly higher pSTAT5 level compared with control CD4+CD8low cells (Fig. 2C, left). The increase in pSTAT5 was not due to an increase in total STAT5 level, as level of total STAT5 is the same in CAT-Tg thymocytes or control thymocytes (Fig. 2C, right bottom). The degree of increase in pSTAT5 in ex vivo CAT-Tg CD4+CD8low thymocytes was greater than that seen in SOCS-1–/–IFN-{gamma}–/– CD4+CD8low thymocytes (data not shown), suggesting the enhanced IL-7 signaling caused by beta-catenin expression is biologically relevant. These data indicate that CAT-Tg CD4+CD8low cells and CAT-Tg SP thymocytes experience higher level of cytokine signals in vivo. Together these data demonstrate that beta-catenin expression enhances IL-7R signaling both in vitro and in vivo. This result suggests that enhanced IL-7R signaling in CD4+CD8low intermediates promotes increased generation of CD8SP thymocytes.

beta-Catenin expression promotes up-regulation of IL-7R and down-regulation of SOCS-1 expression in thymocytes during positive selection

Positive selecting TCR signals up-regulate expression of IL-7R{alpha} and down-regulate expression of SOCS-1 to confer IL-7R responsiveness to CD4+CD8low thymocytes (9, 11, 13). To determine molecular basis for beta-catenin-mediated enhancement of IL-7R signaling, we assayed the expression of IL-7R{alpha} and SOCS-1 molecules. CAT-Tg CD4+CD8low thymocytes expressed a higher level of surface IL-7R{alpha} protein compared with control CD4+CD8low thymocytes (Fig. 3A). CAT-Tg mature thymocytes as well as peripheral CD4 and CD8 T cells also expressed a higher level of IL-7R{alpha} compared with their control counterparts (Fig. 3A). Cell surface expression of IL-7R{alpha} was accompanied by up-regulation of IL-7R{alpha} mRNA in CAT-Tg CD4+CD8low cells (Fig. 3A, far right). To further investigate whether beta-catenin expression induces IL-7R{alpha} expression, we expressed a stabilized form of mouse beta-catenin in peripheral CD4 T cells via retroviral infection. Two days after infection, CD4 T cells expressing stabilized beta-catenin expressed 4-fold higher level of IL-7R{alpha} mRNA compared with cells infected with control vector (Fig. 3B). Thus, expression of beta-catenin induces IL-7R{alpha} expression. In addition to IL-7R{alpha}, the level of surface {gamma}c was also modestly increased on CAT-Tg CD4+CD8low thymocytes, but not DP thymocytes, compared with control thymocytes (Fig. 3C). This result shows that a signaling-competent IL-7R was assembled on CAT-Tg CD4+CD8low thymocytes and its level was enhanced by beta-catenin expression.


Figure 3
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  		FIGURE 3. beta-Catenin expression increases IL-7R expression and decreases SOCS-1 expression. A, Expression of IL-7R{alpha}-chain. Surface expression of IL-7R{alpha}-chain on thymocyte subpopulations and spleen CD4 or CD8 T cells from control or CAT-Tg mice is shown (left panels). Data are representative of six to eight independent analyses for each type of mice. RNA from sorted CD4+CD8low thymocytes was analyzed by real-time RT-PCR. Relative expression level of IL-7R{alpha} to beta-actin is shown (right) from three independent experiments. B, Induction of IL-7R{alpha} by expression of beta-catenin. Purified CD4 T cells from C57BL/6 mice were activated and infected with MSCV-CAT-huCD8 or control vector MSCV-huCD8. Two days after retroviral infection, the cells were stained for huCD8 to confirm efficiency of infection and analyzed for IL-7R{alpha} mRNA by real-time RT-PCR. Relative expression level of IL-7R{alpha} to beta-actin is shown. C, Surface expression of {gamma}c. Surface expression of {gamma}c on control or CAT-Tg thymocytes was analyzed by flow cytometry, and MFI of {gamma}c is shown from eight independent experiments. D, SOCS-1 gene expression in CD4+CD8low thymocytes. Purified CD4+CD8low thymocytes from CAT-Tg or CAT-KO mice were analyzed for SOCS-1 gene expression by real-time RT-PCR. Relative expression of SOCS-1 to beta-actin in control thymocytes is set as 1, and the fold change relative to control thymocytes is shown from three independent experiments.

 
We next assessed SOCS-1 expression in CD4+CD8low thymocytes from CAT-Tg and control mice. Expression of SOCS-1 mRNA was significantly lower in CD4+CD8low subset from CAT-Tg mice compared with CD4+CD8low control thymocytes (Fig. 3D). Conversely, higher level expression of SOCS-1 mRNA was observed in CD4+CD8low cells from CAT-KO mice (Fig. 3D). Statistical analysis showed that regulation of SOCS-1 expression in the presence and absence of beta-catenin was significant. In this function {gamma}-catenin does not appear to play a redundant role. The ratio of SOCS-1 mRNA in DP to SOCS-1 mRNA in CD4+CD8low was 2.8 in control mice and 5.9 in CAT-Tg mice. This result shows a 2-fold further down-regulation of SOCS-1 during DP to CD4+CD8low differentiation in CAT-Tg mice. We conclude that beta-catenin expression down-regulates SOCS-1 expression in TCR-signaled CD4+CD8low thymocytes in addition to up-regulating IL-7R components. Thus, beta-catenin enhances IL-7R signaling by positively regulating IL-7R expression and negatively regulating SOCS-1 expression. The observation that a modest increase in IL-7R expression and decrease in SOCS-1 expression in CD4+CD8low cells results in a more dramatic increase in CD8SP thymocytes underscores the importance of the level of IL-7R signaling in CD4+CD8low intermediate cells for positive selection.

beta-Catenin expression enhances but does not replace IL-7R signal during positive selection of CD8SP thymocytes

To verify that beta-catenin-induced enhanced IL-7R signaling was essential for increased CD8 cell generation, we studied the generation of CD8SP thymocytes in FTOC in the presence of Abs that block signal transduction through the IL-7R. Treatment of FTOC with blocking IL-7R Abs has been shown to selectively block development of CD8SP thymocytes but not CD4SP thymocytes (10). Thymic lobes from embryonic day 17.5 mice were placed in FTOC with or without Abs against IL-7R{alpha} and {gamma}c. Three days later, cells were harvested from the FTOC and treated with pronase to strip cell surface CD4 and CD8 proteins. Treated cells were then cultured in medium to allow re-expression of CD4 or CD8 molecules that were actively synthesized in the cells. CD8SP thymocytes in both control and CAT-Tg FTOC were dramatically reduced when IL-7R signal was blocked by the addition of IL-7R Abs (Fig. 4, left). These data show that IL-7R signal was required for the generation of CD8SP thymocytes in CAT-Tg mice. A small number of CD8SP thymocytes were consistently generated in the presence of blocking Abs to IL-7R (Fig. 4, left) (10). Interestingly the number of CD8SP thymocytes generated in the presence of blocking Abs to IL-7R was the same between control and CAT-Tg mice (Fig. 4, left). These data suggest that a minority of CD8SP thymocytes may not use IL-7R signals and the generation of these cells is not affected by beta-catenin expression. As expected, addition of IL-7R and {gamma}c Abs did not affect the number of CD4SP thymocytes whether in control or CAT-Tg thymic lobes, consistent with the notion that generation of majority of CD4SP thymocytes does not require IL-7R signal (Fig. 4, right). Taken together, the effect of CAT-Tg on promoting positive selection of CD8SP thymocytes required IL-7R signal. We conclude that beta-catenin expression enhances but does not replace IL-7R signals during positive selection of thymocytes.


Figure 4
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  		FIGURE 4. beta-Catenin expression enhances but does not replace IL-7R signal during positive selection of CD8SP thymocytes. Embryonic day 17.5 fetal thymic lobes from control or CAT-Tg mice were placed in FTOC in medium alone or medium containing anti-IL-7R{alpha} plus anti-{gamma}c Abs. After 3 days of culture, thymocytes were harvested, treated with pronase, and cultured overnight in medium. Thymocytes were then stained with Abs to CD4, CD8, and TCRbeta and analyzed by flow cytometry. The absolute number of CD4SP and TCRbetahigh CD8SP thymocytes per thymic lobe is shown from three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we demonstrate that beta-catenin expression enhances IL-7R signals during positive selection, resulting in increased numbers of CD8SP thymocytes. The enhancement of IL-7R signaling in CAT-Tg thymocytes was modest but significant and similar in degree to the enhancement of IL-7R signaling in SOCS-1–/–IFN-{gamma}–/– thymocytes. Both SOCS-1–/– and CAT-Tg mice have a significantly higher number of CD8SP thymocytes with no increase in CD4SP thymocytes. This finding shows that modestly enhanced cytokine signaling in CD4+CD8low thymocytes is sufficient to result in the significant increase in CD8SP thymocytes and suggests that a modest effect in the "correct" developmental intermediate (CD4+CD8low) can result in biologically significant increase in the product population (CD8SP thymocytes).

In CAT-Tg mice, transgenic stabilized beta-catenin is expressed at equal level in both CD4SP and CD8SP thymocytes (data not shown). However, only CD8SP thymocytes show an increase in number, whereas CD4SP thymocytes are largely unaffected. This phenotype is strikingly similar to SOCS-1–/– and Stat5CA-Tg mice that have enhanced cytokine signaling in both CD4SP and CD8SP thymocytes but a preferential increase in CD8SP thymocytes. We thus speculated that expression of beta-catenin might regulate cytokine receptor signaling, such as IL-7R signaling, during positive selection and enhance maturation of CD8SP thymocytes.

Analysis of proximal IL-7 signaling, indicated by phosphorylation of STAT5, shows that both CAT-Tg CD4SP and CAT-Tg CD8SP thymocytes have enhanced IL-7 signaling either in vitro or in vivo. Furthermore, analysis of IL-7 downstream target Bcl-2 expression shows that IL-7-induced effect is elevated in both CAT-Tg CD4SP and CAT-Tg CD8SP thymocytes. Importantly, beta-catenin induced enhancement of IL-7 signaling and effect is observed in positive selection intermediates CD4+CD8low thymocytes. At this stage, the enhanced IL-7 signaling has been shown to preferentially promote the further differentiation of CD4+CD8low cells into CD8SP thymocytes (10, 11, 25). The effect of beta-catenin on IL-7R signaling is further supported by the observation that SOCS-1–/– mice and Stat5CA-Tg mice, both of which have enhanced IL-7R signaling in thymocytes, show increased CD8SP thymocytes similar to that seen in CAT-Tg mice, as described in this study and other studies (11, 12). The enhancement of IL-7R signaling in CAT-Tg thymocytes, though modest, is similar or even greater than that caused by SOCS-1 deficiency. This response suggests the enhanced cytokine signaling in CAT-Tg thymocytes is sufficient to result in the significant increase in CD8SP thymocytes.

IL-7 signaling is regulated by both induced expression of the IL-7{alpha} and {gamma}c chains as well as expression of negative regulators such as SOCS-1 in thymocytes. Indeed, CAT-Tg CD4+CD8low thymocytes show increased level of both IL-7R{alpha}-chain and {gamma}c and decreased level of SOCS-1, which provides a direct molecular explanation for enhanced IL-7 signaling in these cells. The exact molecular mechanism by which beta-catenin up-regulates IL-7R{alpha} and {gamma}c expression and down modulates SOCS-1 expression requires further investigation. However, as up-regulation of IL-7R and down-regulation of SOCS-1 expression are both positive selection induced events, beta-catenin expression may enhance these events by lowering the threshold of positive selection. In addition, beta-catenin may affect gene transcription or RNA stability of IL-7R and SOCS-1. This affect is supported by the observation that expression of beta-catenin in peripheral CD4 T cells using the retroviral technology leads to increased expression of IL-7R{alpha}-chain expression. These data also show that beta-catenin expression induces IL-7R{alpha} independently of positive selection signal.

Ab blocking of IL-7R in FTOC has been used to study the role of IL-7R in positive selection because the requirement of IL-7 at the double-negative stage interferes with the analysis of its role in positive selection in IL-7- or IL-7R-deficient mice. Blocking IL-7R signaling by anti-IL-7R{alpha} and anti-{gamma}c Ab in FTOC from CAT-Tg and wild-type mice shows that very few CD8SP thymocytes are generated in the absence of IL-7R signals, indicating that beta-catenin expression enhances but cannot replace IL-7R signal for the development of CD8SP thymocytes. Importantly, the fact that the small number of IL-7R-independent CD8SP thymocytes is not increased by beta-catenin expression demonstrates that the effect of beta-catenin in promoting generation of CD8SP thymocytes is dependent on IL-7R signal.

Even though CAT-KO thymocytes show increased SOCS-1 expression, CAT-KO mice do not show a significant decrease in CD8SP thymocytes. Furthermore, CAT-KO thymocytes do not show a decrease in ex vivo IL-7R signaling or in vitro IL-7 responses. This result is perhaps due to redundancy with {gamma}-catenin, a beta-catenin homolog that is expressed in thymocytes, binds to T cell factor-1, and has been shown to be redundant in other biological systems (23, 26). beta- and {gamma}-catenin double knockout mice, not yet available for analysis, will hopefully reveal the independent requirement of these molecules in cytokine signaling and positive selection in the future. Nevertheless, the finding that beta-catenin regulates IL-7R signaling in thymocytes provides mechanisms for its function in thymocyte positive selection. The observation that beta-catenin expression in mature T cells up-regulates IL-7R expression suggests that beta-catenin may influence other cytokine-mediated processes.


   Acknowledgments
 
We thank Dr. James Ihle for permission to use thymuses from SOCS-1+/+ IFN-{gamma}+/– and SOCS-1–/–IFN-{gamma}–/– mice provided by Drs. Hyun Park and Al Singer, Dr. Michael Farrar for Stat5CA-Tg thymuses, Drs. Robert Wersto, Francis J. Chrest, and Cuong Nguyen for expert cell sorting of thymocyte subpopulations, Donna Tignor, Dawn Phillips, Dawn Nines, Heather Breighner, Anna Butler, and Ernest Dabney for maintaining animals, and Dr. Shengyuan Luo for genotyping animals.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   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 the Intramural Research Program of the National Institute on Aging at the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Jyoti Misra Sen, Lymphocyte Development Unit, Laboratory of Immunology, National Institute on Aging, Gerontology Research Center, Room 4-B-08, 5600 Nathan Shock Drive, Baltimore, MD 21224. E-mail address: Jyoti-Sen{at}NIH.gov Back

3 Abbreviations used in this paper: DP, double positive; SP, single positive; {gamma}c, common cytokine receptor {gamma} chain; FTOC, fetal thymic organ culture; SOCS, suppressor of cytokine signaling; CAT-KO, deficiency of beta-catenin; MFI, mean fluorescence intensity. Back

Received for publication February 9, 2007. Accepted for publication April 16, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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M. Z. Hossain, Q. Yu, M. Xu, and J. M. Sen
ICAT expression disrupts {beta}-catenin-TCF interactions and impairs survival of thymocytes and activated mature T cells
Int. Immunol., July 1, 2008; 20(7): 925 - 935.
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