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The Journal of Immunology, 2007, 178: 5028-5034.
Copyright © 2007 by The American Association of Immunologists, Inc.

beta-Catenin Regulates Positive Selection of Thymocytes but Not Lineage Commitment1

Qing Yu 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
 
Positive selection and lineage commitment to the cytolytic or helper lineage of T cells result in coordinated expression of MHC class I-restricted TCR and CD8 coreceptor or MHC class II-restricted TCR and CD4 molecule. Positive selection signals also regulate the survival and generation of requisite numbers of cytolytic or Th cells. beta-Catenin is the major transcriptional cofactor of T cell factor and plays a role in thymocyte development. In this study, using mice expressing stabilized beta-catenin and mice with T cell-specific deletion of beta-catenin, we show that beta-catenin regulates positive selection, but not lineage commitment of thymocytes. Furthermore, beta-catenin expression accelerates the timing of mature CD8 thymocyte generation such that CD4 and CD8 single-positive thymocytes mature with the same kinetics during development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cells develop in the thymus throughout mammalian life from bone marrow-derived precursor cells in response to intrathymic signals. The developmental order is defined by the expression of cell surface proteins CD4 and CD8, with the most immature thymocytes expressing neither marker, the intermediate cells expressing both markers, and the most mature thymocytes expressing either CD4 or CD8. Events accompanying maturation of CD4+CD8+ double-positive (DP)3 thymocytes into mature CD4+CD8– and CD4–CD8+ single-positive (SP) thymocytes involve positive selection and commitment to the helper or cytolytic lineage. Both these events are mediated by signals transduced through surface TCR{alpha}beta and CD4 and CD8 coreceptors upon interaction with MHC:self Ag complexes on the thymic epithelial cells (1, 2, 3, 4, 5).

Lineage commitment results in the coordinated expression of MHC class II-restricted TCR and CD4 on Th cells or MHC class I-restricted TCR and CD8 on cytolytic T cells. Transcription factors such as GATA-3 (3) and c-Krox/Th-Pok (6) direct the DP thymocytes that express MHC class II-restricted TCR to differentiate into CD4 Th cells. Manipulation of these transcription factors causes the TCR and coreceptor coordination to be switched, showing that these transcription factors direct commitment to the Th lineage. Transcription factors downstream of TCR that regulate the generation of cytolytic T cells, with the exception of TOX (7), remain to be defined.

TCR-self MHC interactions provide cell survival signals to DP thymocytes. When the signals resulting from TCR-self MHC interactions are below a certain threshold, DP thymocytes die due to neglect, and when these signals are high, cells die due to negative selection. DP thymocytes that receive signals above a certain threshold survive to undergo a multistep process called positive selection. DP thymocytes that receive the initial positive selection signal differentiate to transitional stages described as CD69+DP thymocytes and CD4+CD8low (CD4+8low) intermediates. These intermediate stages are believed to be the precursors for both mature CD4SP and CD8SP thymocytes (8, 9, 10). In vivo BrdU-labeling kinetic analysis has shown that the generation of CD8SP thymocytes lags behind the generation of CD4SP thymocytes by 2 days (11). Thus, CD4SP thymocytes are generated before CD8SP thymocytes, suggesting that the precursors for CD8SP cells remain at the intermediate stages for a longer period of time. Thus, the delay in timing of CD8SP thymocyte maturation, along with a lower rate of generation, may result in fewer numbers of mature CD8SP thymocytes compared with CD4SP thymocytes.

beta-Catenin is the mammalian ortholog of Drosophila armadillo and regulates gene expression together with T cell factor of the high-mobility group family transcription factors (12, 13). beta-Catenin protein level is regulated by glycogen synthase kinase-3beta (GSK-3beta)-mediated phosphorylation, followed by its ubiquitination and degradation. Stabilized form of beta-catenin, generated by deletion of the GSK-3beta phosphorylation sites, is fully functional (14, 15, 16), and was used to generate beta-catenin (CAT)-transgenic (Tg) mice that express stabilized beta-catenin in thymocytes regulated by the proximal Lck promoter (17). CAT-Tg mice have increased number of mature CD4SP and CD8SP thymocytes at the cost of DP thymocytes with a preferential increase in the number of CD8SP thymocytes. Conversely, T cell-specific deficiency of beta-catenin (CAT-knockout (KO)) shows decreased number of mature thymocytes, with a preferential decrease in CD8SP thymocytes (18). Together these studies suggested a role for beta-catenin during positive selection of thymocytes.

The present study was undertaken to investigate the mechanism(s) by which beta-catenin expression regulates the number of mature SP thymocytes, especially CD8SP thymocytes. Using CAT-Tg and CAT-KO mice, we have delineated cellular mechanisms by which beta-catenin regulates DP to SP transition. We show that endogenous beta-catenin is stabilized in response to intrathymic signals during DP to SP transition, suggesting a role for the molecule during normal positive selection. Expression of Tg stabilized beta-catenin protein in CAT-Tg mice promotes positive selection of both MHC II-restricted CD4 and MHC I-restricted CD8SP thymocytes. In addition, beta-catenin expression accelerates the timing of CD8SP cell generation such that CD4SP and CD8SP thymocytes are generated with the same kinetics in CAT-Tg mice. Together these data demonstrate that beta-catenin regulates positive selection with a preferential effect on CD8SP thymocytes.


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

Generation of CAT-Tg and CAT-KO mice was described previously (17, 18). AND and P14-TCR-Tg mice were purchased from Taconic Farms. The mice were bred and maintained in animal facility at Gerontology Research Center, National Institute on Aging, according to National Institutes of Health regulations.

Flow cytometry

Thymocytes were harvested, stained, and analyzed on a FACSCalibur (BD Biosciences). 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), FITC CD5 (53-7.3), FITC CD4 (M1/69), FITC CD69 (H1.2F3), FITC TCRV{alpha}11 (RR8-1), and FITC TCRV{alpha}2 (B20.1) (all from BD Pharmingen). Where indicated, thymocytes were sorted into TCRbeta–CD4–8– (double-negative (DN)), DP, CD4+8low, CD4SP, and TCRhighCD8SP subpopulations on a DakoCytomation Moflo.

Intracellular Bcl-2 staining and annexin V staining

Freshly isolated thymocytes or lymph node cells were fixed and permeabilized first with 4% paraformaldehyde, and then with methanol/acetone mixture (1:1, v/v). Cells were then stained with PE anti-Bcl-2 Ab (BD Pharmingen). After intracellular staining, cells were further stained for CD4 and CD8 (19). Annexin V staining was performed with annexin-V-FLUOS staining kit, according to manufacturer’s protocol (Roche), after thymocytes have been stained for TCRbeta, CD4, and CD8.

In vivo BrdU labeling

For examining cell proliferation, mice were i.p. injected with BrdU, and 1 h later mice were sacrificed and thymocytes were stained for BrdU incorporation. For assessing generation of SP thymocytes from DP thymocytes, after the initial injection of BrdU, mice were maintained on BrdU-containing drinking water (0.8 mg/ml) for 4 days. On each day, thymocytes from certain number of mice were analyzed for BrdU+ cells. Intracellular staining for BrdU was performed with FITC-BrdU Flow Kit, according to manufacturer’s protocol (BD Pharmingen).

Fetal thymic organ culture (FTOC)

For FTOCs, embryonic day 17.5 fetal thymic lobes were cultured in medium. On day 3, thymocytes were harvested and cells in suspension were treated with 0.01% pronase for 10 min to remove surface CD4 and CD8. The cells were then cultured in medium overnight to allow re-expression of CD4 or CD8 molecules that were being actively synthesized in the cell. The number of CD4+ and TCRhighCD8+ per thymic lobe cultured was determined.

Biochemical assays

For 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. For Western blot analysis, thymocyte subpopulations were lysed in SDS sample buffer, and cell lysates were resolved on 4–12% SDS-PAGE (Invitrogen Life Technologies) and then transferred to nitrocellulose membrane. Blots were incubated with anti-beta-catenin mAb (BD Pharmingen) or anti-protein kinase Cµ Ab (Santa Cruz Biotechnology), followed by HRP-conjugated anti-mouse IgG or anti-rabbit IgG (Santa Cruz Biotechnology), respectively. Reactivity was revealed by ECL.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Intrathymic signals stabilize beta-catenin during positive selection

Despite the fact that analysis of mice with beta-catenin deletion and mice overexpressing beta-catenin has demonstrated a role for the protein in T cell development, regulation of endogenous beta-catenin protein in response to intrathymic signals, during T cell development, is not known. We show by Western blot analysis that expression of endogenous beta-catenin is regulated during T cell development. beta-Catenin expression was high in DN thymocytes, down-regulated in DP thymocytes, and up-regulated again during DP to SP transition in wild-type C57BL/6 mice (Fig. 1A, upper panels, left half of the gel). These data show that beta-catenin expression is developmentally regulated by intrathymic signals. To determine the level of mRNA for endogenous beta-catenin in thymocyte subsets, we performed real-time PCR. The level of mRNA for endogenous beta-catenin was comparable in DN, CD4SP, and CD8SP thymocyte subsets and slightly higher in DP thymocytes (Fig. 1B). These data strongly suggest that the developmentally regulated expression of beta-catenin protein results from beta-catenin stabilization in response to intrathymic signals. Thus, beta-catenin was stabilized in DN, destabilized in DP, and stabilized again in SP cells. This conclusion is congruent with the regulation of beta-catenin in other tissues and experimental systems. Down-regulation of endogenous beta-catenin in DP thymocytes, followed by up-regulation in SP thymocytes, shows that intrathymic signals during DP to SP transition stabilize endogenous beta-catenin, suggesting a role for beta-catenin during positive selection.


Figure 1
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  		FIGURE 1. Intrathymic signals regulate stabilization of beta-catenin in a developmentally significant manner. A, Expression of endogenous (wild-type) and Tg beta-catenin protein. Whole cell lysates from sorted thymocyte subpopulations from control C57BL/6 mice or CAT-Tg mice were subjected to Western blot analysis with anti-beta-catenin or anti-protein kinase Cµ Ab. Data are representative of three independent experiments. B, Real-time RT-PCR for mRNA for endogenous beta-catenin. Sorted thymocyte subpopulations from control C57BL/6 mice or CAT-Tg mice were subjected to real-time RT-PCR for expression of endogenous beta-catenin gene. Relative expression of beta-catenin to beta-actin is normalized with that in control DN thymocytes set as 1 (n = 4).

 
To investigate the function of beta-catenin in T cell development, we generated CAT-Tg mice in which a stabilized form of human beta-catenin that lacks N terminus 87 aa was expressed under the control of proximal Lck promoter (17). The N-terminal domain contains GSK-3beta phosphorylation sites and is required for GSK-3beta-mediated protein degradation. A mutant gene that lacks this domain produces a stabilized protein containing the middle domain used for protein-protein interactions and the C-terminal transcription activation domain that works with T cell factor-1/lymphocyte enhancer factor 1 (14). In CAT-Tg mice, the pattern of endogenous beta-catenin expression was similar to wild-type mice, except that the level of endogenous beta-catenin in CAT-Tg SP thymocytes was higher compared with control SP thymocytes (Fig. 1A). This increase is probably due to enhanced stabilization of endogenous beta-catenin, because the endogenous beta-catenin mRNA level was comparable between control thymocytes and CAT-Tg thymocytes (Fig. 1B). Interestingly, the Tg protein was also expressed in a pattern similar to the endogenous protein (Fig. 1A, upper panels, right half of the gel), whereas the mRNA level for Tg beta-catenin remained constant (17) (data not shown). Quantification of beta-catenin protein in the various thymocyte subsets in CAT-Tg mice showed that the total amount of beta-catenin (endogenous plus Tg) was increased by a modest 3- to 5-fold compared with endogenous protein. We conclude that intrathymic signals regulate stabilization of endogenous beta-catenin during DP to SP transition. This observation suggests a role for beta-catenin during positive selection and related developmental events.

beta-Catenin expression regulates the number of mature SP thymocytes

To determine the role of beta-catenin in thymocyte maturation, in addition to CAT-Tg mice, we have also generated CAT-KO mice (floxed beta-catenin x Lck-Cre-Tg) bearing a T cell-specific deletion of beta-catenin (18). The absolute number of CD8SP thymocytes and the frequency of CD8SP thymocyte generated per DP thymocyte precursor were both significantly decreased (Fig. 2A). In CAT-Tg mice, the proportion and number of mature CD4SP and CD8SP thymocytes were increased at the cost of DP thymocytes, with a greater increase in CD8SP thymocytes (Fig. 2A). A representative cytometric profile is shown, illustrating that the increase in mature thymocytes was particularly augmented when the gate was fixed on TCRhigh thymocytes (Fig. 2B). The increase in CD8SP thymocytes was significantly greater than CD4SP thymocytes (Fig. 2B, right panels). Thus, beta-catenin expression regulates the number of mature thymocytes.


Figure 2
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  		FIGURE 2. beta-Catenin expression increases the number of CD8SP thymocytes. A, Percentage and number of mature thymocytes. Percentage of SP thymocytes relative to DP thymocytes and absolute number of SP thymocytes in CAT-Tg and CAT-KO mice are compared with littermate control animals (n > 5). B, Thymocyte development analyzed by flow cytometry. Thymocytes from control C57BL/6 and CAT-Tg mice were stained with Abs to CD4, CD8, and TCRbeta; analyzed by flow cytometry; and represented as dot plots. Histograms, TCRbeta expression on whole thymocytes and the gates used to identify TCRbetahigh cells. Data are representative of eight independent analyses. Bar graphs, absolute numbers of CD4 and TCRbetahigh CD8SP thymocytes (n = 8). C, Bcl-2 expression and apoptosis in thymocyte subpopulations. Left panel, Thymocytes from CAT-Tg or control mice were stained for intracellular Bcl-2, and the level of Bcl-2 (mean fluorescence intensity) in each thymocyte subpopulation was shown (n = 3); right panel, thymocytes from CAT-Tg or control mice were stained for annexin V, and the percentage of annexin V+ cells in each subpopulation was shown (n = 3).

 
We have demonstrated previously, by bone marrow transplantation experiments and by breeding CAT-Tg mice to beta2-microglobulin–/– mice, that MHC class I expressed on thymic epithelial cells is essential for the generation of CD8SP thymocytes in CAT-Tg mice (17). In the same study, we showed that expression of CD24, Qa2, and CD62L on CAT-Tg CD8SP thymocytes indicates that these cells are mature thymocytes. The level of CD44 expression on CAT-Tg CD8SP thymocytes was higher compared with control CD8SP thymocytes perhaps because CD44 has been shown to be a direct target of beta-catenin in cancer cells (20). Furthermore, CAT-Tg CD8SP thymocytes express high level of CD62L, which is inconsistent with activated status because CD62L is shed upon T cell activation (21). Therefore, we conclude that beta-catenin expression increases the number of conventional mature CD8SP thymocytes.

CAT-Tg CD8SP thymocytes showed increased expression of Bcl-2 protein by intracellular staining (Fig. 2C, left panel). This is likely to be a consequence of higher level of IL-7R expression in CAT-Tg CD8SP thymocytes (data not shown). Probably as a consequence of high level of Bcl-2 expression, there was decreased apoptosis of CAT-Tg CD8SP thymocytes, as indicated by annexin V staining (Fig. 2C, right panel). CD4SP thymocytes in CAT-Tg mice did not show increased expression of Bcl-2 and decreased apoptosis rate (Fig. 2C); neither did CAT-KO SP thymocytes (data not shown). These data suggest that beta-catenin expression preferentially enhances survival of positively selected CD8SP thymocytes. These data show that beta-catenin expression increases the number of mature thymocytes with a preferential effect on CD8SP thymocytes.

beta-Catenin expression increases class I-restricted CD8SP thymocytes in P14-Tg mice

To investigate the effect of beta-catenin expression on positive selection of class I-restricted thymocytes, we generated P14-TCR x CAT-Tg (P14 x CAT-Tg) mice. In P14 x CAT-Tg mice, the percentage of CD8SP thymocytes was increased compared with that in P14-TCR-Tg (P14-Tg) mice (Fig. 3A, upper left panels). Analysis focusing on the clonotypic V{alpha}2+ population emphasized this point (Fig. 3A, upper right panels). Percentage of CD8SP thymocytes relative to DP thymocytes and number of CD8SP thymocytes showed a similar increase (Fig. 3A, lower left panels). The frequency of V{alpha}2+ CD4SP thymocytes in P14-Tg mice was reduced in P14 x CAT-Tg mice, whereas the absolute number of these cells was not dramatically changed (Fig. 3A, lower right panels). Thus, expression of beta-catenin resulted in an increase in the number of CD8SP thymocytes, but not CD4SP thymocytes, in P14 x CAT-Tg mice. CD8SP thymocytes generated in P14 x CAT-Tg mice had comparable level of CD24, CD62L, and CD69 as control P14-Tg CD8SP thymocytes (Fig. 3B, upper panels). However, expression of CD44 was higher on CD4SP and CD8SP thymocytes in P14 x CAT-Tg mice (Fig. 3B), which may be due to the known effect of beta-catenin on CD44 gene expression. In contrast to CAT-Tg CD8SP thymocytes, P14 x CAT-Tg CD8SP thymocytes were similar to control P14-Tg CD8SP thymocytes with respect to Bcl-2 expression and apoptosis (Fig. 3C). Together, these data suggest that the increase in CD8SP thymocytes in P14 x CAT-Tg mice is not likely a consequence of accumulation of mature CD8 cells that results from enhanced survival. Thus, fixing the TCR highlights the role of beta-catenin in promoting positive selection of class I-restricted CD8SP thymocytes.


Figure 3
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  		FIGURE 3. Expression of beta-catenin increases CD8SP thymocytes in P14-Tg mice. A, Thymocyte development in P14-Tg and P14 x CAT-Tg mice analyzed by flow cytometry. Thymocytes from control P14-Tg mice or P14 x CAT-Tg were stained with Abs to CD4, CD8, and clonotypic V{alpha}2 TCR and analyzed, as described in Fig. 2A. Left two bar graphs show percentage of CD8SP thymocytes (relative to DP thymocytes) and absolute number of CD8SP thymocytes; right two bar graphs show those of CD4SP thymocytes (n = 8). B, Phenotype of mature thymocytes. Expression of surface markers on CD8 and CD4SP thymocytes from P14 x CAT-Tg or control P14-Tg mice was analyzed by flow cytometry (n = 6). C, Bcl-2 expression and apoptosis in thymocyte subpopulations. Left panel, Intracellular Bcl-2 level in thymocyte subpopulations from P14 x CAT-Tg or control P14-Tg mice was shown (n = 3); right panel, percentage of annexin V+ cells in thymocyte subpopulations from P14 x CAT-Tg or control P14-Tg mice was shown (n = 3).

 
beta-Catenin expression does not affect lineage commitment

Differentiation of CD4+CD8+ DP thymocytes into mature CD4+CD8– and CD4–CD8+ SP thymocytes involves positive selection and commitment to the helper or cytolytic lineage. To assess whether beta-catenin affects lineage commitment by redirecting class II-restricted cells to differentiation into CD8 lineage, we analyzed thymocyte development in AND x CAT-Tg mice. An increase in both the proportion and number of CD4SP thymocytes was observed in AND x CAT-Tg mice compared with AND-TCR-Tg (AND-Tg) mice (Fig. 4A). The frequency of V{alpha}11+ CD8SP thymocytes in AND-Tg mice was slightly increased in AND x CAT-Tg mice compared with AND-Tg mice; however, the absolute number of these cells did not show an increase (Fig. 4A, lower right panels). Thus, expression of beta-catenin resulted in increased CD4SP thymocytes in AND-Tg mice and it did not lead to an increase in V{alpha}11+ CD8SP thymocytes. Together, these data show that expression of beta-catenin increases the number of mature SP thymocytes, but does not enforce a switch in lineage commitment. Accordingly, expression of genes implicated in CD4 or CD8 lineage commitment, c-Krox/Th-Pok, GATA-3, and Runx3, analyzed by real-time RT-PCR, was comparable in CAT-Tg and control DP or CD4+8low thymocytes (Fig. 4B). We conclude that beta-catenin expression increases the number of mature thymocytes without affecting lineage commitment.


Figure 4
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  		FIGURE 4. Expression of beta-catenin does not alter lineage commitment. A, Thymocyte development in AND-Tg and AND x CAT-Tg mice analyzed by flow cytometry. Thymocytes were stained with Abs to CD4, CD8, and clonotypic V{alpha}11 TCR. Left two bar graphs show percentage of CD4SP thymocytes (relative to DP thymocytes) and absolute number of CD4SP thymocytes; right two bar graphs show those of CD8SP thymocytes (n = 6). B, Expression of genes involved in lineage commitment. Expression of Th-POK, GATA-3, and Runx3 genes in sorted DP and CD4+8low thymocytes from AND x CAT-Tg or control AND-Tg mice was analyzed by real-time RT-PCR. Relative expression of the gene normalized to beta-actin is shown (n = 3 independent experiments).

 
beta-Catenin expression results in an increase in positive selection intermediates: positive selection is facilitated

Expression of beta-catenin increases the generation of CD4SP and CD8SP thymocytes even when all DP thymocytes express a Tg-TCR, suggesting that beta-catenin enhances the process of positive selection. Enhanced positive selection predicts that the number of positive selection intermediates should be increased. To address this issue directly, we studied the positive selection intermediates in CAT-Tg mice. A proportion of DP thymocytes that have received initial TCR-mediated positive selection signals up-regulates CD69 expression. The gate used to determine CD69+DP thymocytes is shown (Fig. 5A, left panel). In CAT-Tg mice, CD69+DP thymocytes were significantly increased (Fig. 5A, right panel). Another intermediate stage during positive selection of both CD4SP and CD8SP thymocytes is CD4+8low thymocytes (8, 9). The gate used to determine CD4+8low thymocytes is shown (Fig. 5B, upper panel). The proportion of CD4+8low cells per DP was increased in CAT-Tg, P14 x CAT-Tg, and AND x CAT-Tg mice compared with corresponding control mice not expressing Tg beta-catenin (Fig. 5B, lower panels). These data show that positive selection intermediate CD4+8low is increased when beta-catenin is expressed, whether or not TCR is fixed. Deletion of beta-catenin in CAT-KO mice led to a modest, but statistically significant reduction in the fraction of CD4+8low intermediates (Fig. 5B). Thus, beta-catenin expression regulates positive selection intermediates. Although the increase and decrease in positive selection intermediates are small, it is statistically significant, suggesting that small changes in developing intermediates are sufficient to affect discernable biological outcomes. Finally, expression of IL-7R{alpha} chain and CCR7 on DP thymocytes is induced by positive selection signals (9, 22, 23). In P14-Tg mice, in which most of the DP thymocytes have received initial TCR-mediated positive selection signal, expression of beta-catenin resulted in a significant proportion of DP thymocytes that expressed these two indicators of positive selection (Fig. 5C). These data further demonstrate that beta-catenin expression facilitates positive selection. We conclude that expression of stabilized beta-catenin during DP to SP transition enhances positive selection.


Figure 5
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  		FIGURE 5. beta-Catenin expression regulates positive selection intermediates. A, Histogram shows CD69 expression on DP thymocytes and the gate used to determine CD69+DP thymocytes from control mice. Graph shows percentage of CD69+DP thymocytes in control or CAT-Tg mice (n = 8 independent experiments). B, Dot plot shows the gate used to identify the CD4+8low population in control thymocytes. Bar graphs show percentage of CD4+8low thymocytes (relative to DP thymocytes) in CAT-Tg (n = 8), CAT-KO (n = 7), P14 x CAT-Tg (n = 8), and AND x CAT-Tg (n = 6) mice. C, Surface expression of IL-7R{alpha} (upper panels) and CCR7 (lower panels) on DP and CD8SP thymocytes from control P14-Tg mice or P14 x CAT-Tg mice, analyzed by flow cytometry. Data are representative of four independent analyses.

 
beta-Catenin expression increases generation rate of mature thymocytes and accelerates the generation of CD8SP thymocytes

To directly examine whether beta-catenin increases generation rate of mature thymocytes, we examined generation of mature thymocytes using the FTOC system. Embryonic day 17.5 thymic lobes, which contain DN and DP thymocytes, but no SP thymocytes, were placed in culture. After 3 days in culture, thymocytes were harvested from FTOCs, treated with pronase, and then cultured overnight to allow the identification of thymocytes committed to CD4SP or CD8SP lineage. In this assay, the number of TCRbetahighCD8SP generated in FTOCs with CAT-Tg thymic lobes was higher compared with control thymic lobes (Fig. 6A). We conclude that beta-catenin expression enhances the generation of mature thymocytes with a significantly greater effect on CD8SP thymocytes.


Figure 6
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  		FIGURE 6. Expression of Tg beta-catenin increases generation of mature thymocytes and accelerates maturation of CD8SP thymocytes. A, Maturation of CD4SP and CD8SP thymocytes in FTOCs. Embryonic day 17.5 fetal thymic lobes from control or CAT-Tg mice were placed in FTOCs. On day 3 of culture, thymocytes were harvested, treated with pronase (to remove surface CD4 and CD8), and then cultured overnight so that they would re-express CD4 and/or CD8 molecules that were being actively synthesized. Thymocytes were then analyzed by flow cytometry, and numbers of CD4SP and TCRbetahigh CD8SP thymocytes per thymic lobe are shown (n = 3). B, Rate of generation of mature thymocytes studied using long-term BrdU labeling. Control and CAT-Tg mice were injected with BrdU and then maintained on BrdU-containing drinking water for 4 days. On each day, thymocytes from three mice of each genotype were stained with Abs to CD4, CD8, CD24, and BrdU, and analyzed by flow cytometry. Shown are BrdU+ cells in each thymocyte subpopulation as a percentage of the total determined each day after initial injection. C, Kinetics of CD4SP and CD8SP thymocyte generation. In the 4-day BrdU-labeling assay, the number of BrdU+ CD4SP or CD8SP thymocytes in control or CAT-Tg mice was determined each day after initial BrdU injection.

 
To study the kinetics of mature thymocyte generation in CAT-Tg mice, we used BrdU-labeling assay. In vivo BrdU labeling for 1 h showed that frequency of proliferating cells in CD4SP and CD8SP populations was not increased in CAT-Tg mice (data not shown). This observation ruled out enhanced proliferation as the cause of increase in SP thymocytes. The rate of generation of SP thymocytes from DP thymocytes was assessed by measuring the rate of increase in BrdU+ cells of each thymocyte population in a 4-day BrdU-labeling experiment. Over a 4-day labeling experiment, percentage of BrdU+ CD8SP cells were significantly increased in CAT-Tg mice compared with control mice, whereas the percentage of BrdU+ CD4+8low and CD4SP was slightly increased (Fig. 6B). These data show that the rate of mature CD8SP thymocyte generation was enhanced in CAT-Tg mice.

BrdU-labeling studies have shown previously that the timing of CD4SP and CD8SP thymocytes is different (11). In this study, we found that in control mice, CD4SP thymocytes were generated starting on day 2, whereas CD8SP thymocytes were generated starting on day 4 after initial BrdU injection (Fig. 6C, left panel). This time lag between the generation of CD4SP and CD8SP thymocytes was diminished in CAT-Tg mice, and CD4SP and CD8SP thymocyte generation was initiated on the same day after initial BrdU injection (Fig. 6C, right panel). Therefore, CD8SP thymocyte development was accelerated in CAT-Tg mice. We conclude that beta-catenin stabilization enhances the generation of mature thymocytes and accelerates the timing of mature CD8SP thymocyte generation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we show that endogenous beta-catenin is stabilized during DP to SP transition, suggesting its role in positive selection. Expression of stabilized beta-catenin transgene promotes positive selection of CD8SP thymocytes and accelerates the timing of CD8SP thymocyte generation. Furthermore, T cell-specific deficiency of beta-catenin leads to a slight decrease in CD8SP thymocytes.

The role of beta-catenin in T cell development has been deduced from mice with manipulated beta-catenin gene. However, expression of mRNA and protein for endogenous beta-catenin in response to intrathymic signals have not been documented. In this study, we show that the mRNA level for endogenous beta-catenin was similar among thymocyte subsets. However, the protein level was regulated in a developmentally significant manner. beta-Catenin protein was high in DN, down-regulated in DP, and up-regulated again in CD4SP and CD8SP. This discordance in mRNA and protein expression strongly suggests that intrathymic signals stabilize beta-catenin in DN, destabilize it in DP, and again stabilize it during DP to SP transition. The intrathymic signals that regulate the developmental stabilization remain unknown, although pre-TCR and TCR signals are likely candidates.

Overexpression and ectopic stabilization of beta-catenin in thymocytes have been studied in three other contexts. When beta-catenin is expressed at very high levels in DN thymocytes, a small number of DP thymocytes lacking TCR beta-chain expression is generated (24, 25). In Tg mice expressing stabilized beta-catenin from the CD4 promoter, DP thymocytes have increased expression of Bcl-xL that provides a survival advantage in vitro (26). In contrast, stabilization of beta-catenin by deletion of ubiquitin E3 ligase Siah-1-interacting protein led to DP thymocyte death (27). At this time, an explanation for the discrepant observations is not known. Regardless, in context of positive selection, high level of Bcl-xL expression or lack of TCRbeta expression alters DP thymocytes in these models, precluding analysis of SP thymocyte generation. CAT-Tg mice provide a model system in which Tg stabilized beta-catenin is expressed in the same pattern as the wild-type beta-catenin. The total amount of stabilized beta-catenin protein is modestly increased, and expression of Bcl-xL and TCR is comparable to normal DP thymocytes, which allowed a study of the role of beta-catenin in positive selection.

In this study, enhanced positive selection in CAT-Tg mice was deduced from the following data. First, positive selection intermediates, CD69+DP and CD4+8low thymocyte subsets, were increased in CAT-Tg and decreased in CAT-KO mice, suggesting an effect on positive selection. Importantly, fixing TCR by breeding CAT-Tg mice with MHC class I- or class II-restricted TCR Tg mice also results in an increase in positive selection intermediates and mature thymocytes. Second, positive selection intermediate, CD4+8low, is generated at a higher rate compared with control CD4+8low in the 4-day BrdU assay. Although the increase is small, a small change in this intermediate population may be sufficient to result in a discernable outcome. BrdU-labeling experiments also showed that CAT-Tg CD8SP thymocytes are generated at an earlier time such that CD8SP thymocytes are generated with same kinetics as CD4SP thymocytes in CAT-Tg mice. Third, enhanced expression of IL-7R{alpha} chain and CCR7 on P14 x CAT-Tg DP thymocytes also indicates increased positive selection.

The effect of beta-catenin on positive selection observed in CAT-Tg mice was partially supported by opposite changes in CAT-KO mice. The modest level of defect in CAT-KO mice may reflect redundancy with the beta-catenin homologue plakoglobin ({gamma}-catenin). In fact, this redundancy may also explain the normal T cell development from beta-catenin KO hematopoietic stem cells after bone marrow transplantation, in which setting plakoglobin may compensate for the loss of beta-catenin throughout T cell development (28, 29).

In conclusion, up-regulation of beta-catenin during DP to SP transition suggests a role for it during normal positive selection. Analysis of CAT-Tg mice shows that increased expression of stabilized beta-catenin enhances positive selection of mature thymocytes and accelerates the timing of generation of CD8SP thymocytes such that CD4SP and CD8SP thymocytes are generated with similar kinetics in CAT-Tg mice.


   Acknowledgments
 
We thank Dr. 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; Dr. Shengyuan Luo for genotyping animals; and members of our laboratory for support and technical help throughout the project.


   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, National Institutes of Health, Baltimore, MD 21224. E-mail address: Jyoti-Sen{at}NIH.GOV Back

3 Abbreviations used in this paper: DP, double positive; CAT, beta-catenin transgene; DN, double negative; FTOC, fetal thymic organ culture; GSK-3beta, glycogen synthase kinase-3beta; KO, knockout; SP, single positive; Tg, transgenic. Back

Received for publication January 5, 2007. Accepted for publication February 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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