ß-Catenin expression enhances generation of mature thymocytes

Thomas Mulroy1, Youyuan Xu1 and Jyoti Misra Sen1,2

1 Dana Farber Cancer Institute and 2 Harvard Medical School, 44 Binney Street, Boston, MA 02115, USA

Correspondence to: J. M. Sen; E-mail: jyoti_sen{at}dfci.harvard.edu
Transmitting editor: A. Singer


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
T cell factor (TCF)-1 is a T-cell-specific transcription factor that is expressed at all stages of T cell development. Deletion of the TCF-1 gene leads to an early block in thymocyte maturation precluding the study of its role at late stages and during positive selection of T cells. In this report we show that ß-catenin, a central effector in the Wnt–TCF-1 signaling pathway, regulates late stages of T cell development. Specifically, transgenic expression of ß-catenin enhances generation of mature thymocytes. Interestingly, CD8-expressing mature thymocytes were affected to a greater extent than CD4-expressing cells. These data suggest that the Wnt–ß-catenin–TCF-1 signaling pathway plays a role during late stages of T cell development.

Keywords: positive selection, signal transduction, thymocyte maturation, thymus, Wnt signaling


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
During T cell development in the thymus, rearrangement and expression of the TCR ß chain genes takes place in newly committed T lineage CD4CD8 double-negative (DN) thymocytes. Expression of the TCRß chain leads to the formation of the pre-TCR, comprising a non-rearranging pre-T{alpha} chain and the CD3 complex. The pre-TCR signals cause proliferation and maturation of DN thymocytes to the CD4+CD8+ double-positive (DP) stage. DP thymocytes rearrange and express the TCR {alpha} chain, and in conjunction with the TCR ß chain and the CD3 complex form the {alpha}ß TCR. The majority of thymocytes express TCR that do not bind self-MHC and these cells die in the thymus by a process called ‘death-by-neglect’. DP thymocytes that express {alpha}ß TCR that bind self-antigens in the context of self-MHC with particularly high affinity are also eliminated by negative selection [reviewed in (1)].

DP thymocytes expressing {alpha}ß TCR that bind self-antigen with moderate affinity in the context of self-MHC undergo further maturation, cell fate decisions and positive selection such that in mature T cells MHC class I-restricted TCR are co-expressed with CD8 and MHC class II-restricted TCR with CD4 (2,3). Positive selecting and negative selecting antigens for the TCR consist of the self-MHC and as yet ill-defined peptide(s). Signal transducing molecules directly downstream of the TCR such as Src kinase p56lck, ZAP70 and mitogen-activated protein kinases have been shown to influence CD4 and CD8 lineage choice. The identity of stromal cells that present self-MHC and peptides(s) for positive selection remains controversial, but thymic epithelial cells appear to be important (4). Additional critical signaling events are believed to be involved in cell fate decision and positive selection of thymocytes, but remain to be delineated.

ß-Catenin, the mammalian ortholog of Drosophila armadillo, participates in signal transduction at two subcellular locations in the cell. In conjunction with E-cadherin, ß-catenin mediates cell-surface proximal signals and with HMG transcription factors of the T cell factor (TCF) family it modulates gene expression (5). The Wnt signaling pathway controls the level of cytosolic ß-catenin that can interact with transcription factors of the TCF family. In the absence of a Wnt signal, GSK-3ß kinase phosphorylates ß-catenin in the N-terminal domain and targets it for ubiquitin-mediated degradation. Wnt signals inhibit GSK-3ß kinase activity, allowing ß-catenin to accumulate in the cytosol. Engineering a truncation at the N-terminus of ß-catenin that deletes GSK-3ß phosphorylation sites stabilizes the ß-catenin in the cytosol without impairing its ability to mediate transcription. ß-Catenin stabilized by deletion of GSK3ß phosphorylation sites has been shown to signal in intestinal epithelium (6) and during hair follicle morphogenesis (7). Together, these studies have demonstrated the role of ß-catenin expression in epithelial cell biology. The biology of ß-catenin-mediated signaling in lymphoid cells is less clear despite the essential roles played by its co-factors TCF-1 and LEF-1 at early stages of lymphocyte development.

TCF-1 and LEF-1 are expressed in thymocytes at all stages of development (8). Deficiency in TCF-1, or double deficiency in TCF-1 and LEF-1, blocks T cell maturation, signifying a critical role for this pathway in T cell development (9,10). However, a block at an early stage of T cell development prevented analysis of the role of TCF-1 and LEF-1 at later stages and during positive selection. Blocking this pathway in fetal thymic organ cultures also inhibits early stages of thymocyte maturation (11). Recently, transgenic expression of the TCF gene, mutated at the ß-catenin binding site, in TCF-deficient mice implicated the ß-catenin pathway in the survival of DP thymocytes (12). Correspondingly, inducible overexpression of Axin in thymocytes, which would reduce the amount of stabilized ß-catenin, also impaired survival of DP thymocytes (13). The effect of overexpression of ß-catenin was examined by the deletion of the regulatory region important in the degradation of ß-catenin. Exon 3, encoding the regulatory domain required for ß-catenin degradation, was deleted in thymocytes using Cre–Lox technology. The mutant stabilized protein accumulates to >10-fold over wild-type in thymocytes at all stages of development (14). In these mice, pre-TCR signals are blocked and survival advantage leads to a small number of DP thymocytes that lack expression of {alpha}ß TCR (14). Because the high level of ß-catenin expression blocked thymopoiesis at an early stage, the role of the ß-catenin signaling pathway during positive selection was not addressed.

In this report we describe transgenic mice expressing an N-terminal-deleted stabilized ß-catenin mutant from the proximal Lck promoter ({Delta}Cat-Tg). In these mice DP thymocytes develop normally, presumably because mutant ß-catenin is expressed at moderate levels and thymocyte development at the DN to DP transition is not disrupted. Interestingly, the DP to single-positive (SP) transition is enhanced, leading to a significant increase in mature SP thymocytes. CD8 SP thymocytes are increased to a greater extent than CD4 SP thymocytes. SP thymocytes generated in {Delta}Cat-Tg mice require TCR–MHC interactions for development, suggesting that ß-catenin expression enhances positive selection signals. Finally, mature CD3hi CD8 SP thymocytes are increased in newborn {Delta}Cat-Tg mice when CD8 SP thymocytes are first generated during development. We suggest that ß-catenin expression augments TCR–MHC signals essential for positive selection of thymocytes.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation and analysis of transgenic mice
The {Delta}N87ßCat fragment contains the N-terminal deletion mutant of human ß-catenin gene (7). The BamHI fragment containing the mutant {Delta}N87ßCat gene (7) was cloned in the BamHI site in p1017 (15). NotI-cut DNA containing the {Delta}N87ßCat gene was injected in FVB recipient mice. Transgenic mice were identified by Southern blot analysis of DNA extracted from tail cuts using standard protocols. The probe consisted of the {Delta}N87ßCat gene. Transgenic founders ({Delta}Cat-1, {Delta}Cat-2, {Delta}Cat-3 and {Delta}Cat-7) were bred to C57BL/6 mice and maintained as heterozygous for the transgene. {Delta}Cat-3 was chosen for further analysis and renamed {Delta}Cat-Tg.

Western blot analysis
Thymocytes from 4- to 6-week-old mice were isolated, and used to prepare whole-cell extracts in 50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, 5 mM sodium orthovanadate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate, 2 mM PMSF, and 2 µg/ml each leupeptin, aprotinin and pepstatin. Proteins were resolved by 7.5 or 8% SDS–polyacrylamide gels and transferred to nitrocellulose filter (Schleicher & Schuell, Keene, NH) or Hybond nitrocellulose membrane (Amersham, Arlington Heights, IL). Blots were probed with 1:500 dilution of polyclonal rabbit anti-ß catenin antibody (Santa Cruz Biotechnology, Upstate Biotechnology, NY). The blots were stripped and re-probed with anti-SP1 antibody (Santa Cruz Biotechnology) as a normalizing control for amount of nuclear protein loaded on the gel. Western blots were developed using commercially available reagents according to the manufacturer’s instructions (Amersham).

PCR and RT-PCR analysis
FACS was used to sort DN, DP, CD4 SP and CD8 SP thymocyte subsets from 4- to 6-week-old {Delta}Cat-Tg or control mice. Sorted cells had >5% contamination with the other populations. RNA or converted cDNA was used for PCR amplification (Gibco/BRL, Grand Island, NY) using the following primers: {Delta}Cat, 5'-cagttgtggttaagctcttacacc-3' and 5'-tagccattgccgctaggtgag-3'; HPRT, 5'-gatacaggccagactttgttg-3' and 5'-ggtaggctggcctataggct-3'; perforin, 5'-accgatgctgacctgggcctc-3' and 5'-aggttcctgaggcctgaccgc-3'. PCR reactions consisted of 5 min at 94°C followed by 35 cycles of (93°C, 1 min; 60°C 1 min; 72°C 1 min) and 5 min at 72°C. PCR products were analyzed by 2% agarose gel electrophoresis and ethidium bromide staining.

FACS analysis
For three-color analyses thymocytes were immunostained with FITC-, CyChrome- and R-phycoerythrin-labeled anti-CD4, anti-CD8 antibodies and anti-CD3, anti-CD5, anti-CD24, anti-CD25, anti-CD44, anti-CD69 anti-CD62L, anti-Ly-6C or anti-Qa-2 antibodies in PBS containing 2% FCS and 0.2% sodium azide in the presence of anti-CD16/CD32 antibodies. All antibodies were purchased from PharMingen (San Diego, CA). Immunostained cells were analyzed using a FACScan flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA).

Bone marrow transplantation
Six- to 12-week-old ß2m–/– MHC class II–/– double-deficient mice and ß2m–/– mice were purchased from Jackson Laboratory (Bar Harbor, ME). Dana-Farber Cancer Institute Animal Care and Use Committee approved all animal protocols used in this study. Bone marrow was removed aseptically from the tibias and the femurs of {Delta}Cat-Tg mice, and depleted for T cells by repeated incubation with anti-Thy-1 antibody for 30 min at 4°C, followed by incubation with Low-Tox rabbit complement (Cederlane, Hornby, Ontario, Canada) for 1 h at 37°C. Then 5 x 106 T cell-depleted bone marrow cells were re-suspended in Leibovitz’s L-15 medium and transplanted by tail vein infusion. Recipient mice received total-body irradiation of 1100 cGy as a split dose with 3 h between doses. Mice were housed in sterilized micro-isolators and received hyperchlorinated drinking water (pH 3). Six weeks later thymuses were removed and thymocytes analyzed by FACS.

Proliferation assay
The proliferation assay was performed in a final volume of 0.2 ml RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 2 mM glutamine. Flat-bottom plates were coated with anti-CD3 antibody (145-2C11) (10 µg/ml) in PBS. IL-2 was used at a final concentration of 32 U/ml. Freshly isolated thymocytes were plated at 2.5 x 105/well. Proliferation was assayed after 24 h of incubation at 37°C by [3H]Thymidine incorporation for 16 h. All assays were performed in triplicate.

Viability assay
Thymocytes from wild-type control mice or {Delta}Cat-Tg mice were plated at a concentration of 2 x 106 cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 10 mM HEPES, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 2 mM glutamine. At predetermined times aliquots were removed and cells were counted in the presence of Trypan blue dye. Duplicate samples of live cells were scored. Cell viability was also assayed by scoring sub-diploid DNA. Cells (2 x 106) were treated with hypotonic buffer containing 50 µg/ml propidium iodide (PI), 0.1 M sodium citrate and 0.1% Triton X-100. Data shown (using PI reagents) is representative of three experiments using Trypan blue exclusion and three experiments using PI reagents.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of transgenic mice expressing a stabilized mutant ß-catenin in thymocytes
To study the role of ß-catenin in the maturation of the thymus we expressed a deletion mutant of the human ß-catenin gene in developing thymocytes (Fig. 1A). The deletion removed 87 N-terminal amino acids of the protein that contain GSK-3ß phosphorylation sites. In the absence of GSK-3ß phosphorylation, ß-catenin protein accumulates and mediates gene expression. Seven transgenic founder mice carrying various copy numbers of mutant human ß-catenin gene were generated (data not shown). Protein analysis showed a high level of expression in {Delta}Cat-3 transgenic thymocytes (Fig. 1B, lane, {Delta}Cat-Tg). The upper band in control and {Delta}Cat-Tg lanes is endogenous ß-catenin, and the lower band in the {Delta}Cat-Tg lane is the mutant transgenic protein. It is important to note that the transgene increases the total ß-catenin protein level in the cell by ~2-fold over the endogenous protein (cf. top band with bottom band in {Delta}Cat-Tg lane). RT-PCR analysis of RNA from sorted thymocyte subpopulations, defined by the expression of CD4 and CD8, showed that transgenic ß-catenin was expressed in all subsets of developing thymocytes of {Delta}Cat-Tg mice (Fig. 1C).



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Fig. 1. Generation of {Delta}Cat-Tg transgenic mice and analysis of transgene expression in thymocytes. (A) Stick diagram showing the human ß-catenin gene, with the N-terminal 87 amino acids deleted, driven off the proximal Lck promoter and spliced with the human growth hormone gene containing a poly(A) site. (B) Western blot analysis showing the expression of endogenous ß-catenin protein (upper band) in control and {Delta}Cat-Tg mice, and the mutant transgenic ({Delta}Cat-Tg) protein (lower band) in {Delta}Cat-Tg mice. (C) Expression of {Delta}Cat-Tg RNA (upper panels) and HPRT RNA (lower panels) in sorted thymocytes from {Delta}Cat-Tg (left panel) and control (right panel) mice. Data are representative of three experiments.

 
Phenotype of thymocytes and splenic T cells in {Delta}Cat-Tg mice
To determine effects of the transgene on T cell maturation, thymocytes were examined by flow cytometry using CD4- and CD8-specific antibodies. Total numbers of thymocytes were comparable (P < 0.0207) in transgenic mice (average of 10 mice: 147.7 ± 4.1) and non-transgenic littermate control mice (average of 10 mice: 193.6 ± 16.9). In control mice, a normal proportion of thymocytes was detected in CD4/CD8 subsets (Fig. 2A, left panel). Remarkably, the thymus of mice expressing the stabilized mutant of ß-catenin showed a significant increase in CD8 SP and a smaller change in CD4 SP thymocytes (Fig. 2A, right panel). Analysis of thymocytes from 10 control and 10 {Delta}Cat-Tg mice showed that CD8 SP thymocytes were increased from average 5.4 ± 0.3% in control to 17.2 ± 1.7% in {Delta}Cat-Tg mice (P < 0.001) (Fig. 2B). CD4 SP thymocytes were not changed significantly, averaging 14.9 ± 0.4% in control and 13.7 ± 0.4% in {Delta}Cat-Tg mice (P < 0.03) (Fig. 2B). There was an ~30% decrease in DP thymocytes (average 72.9 ± 0.7 for control and 52.7 ± 2.9 for {Delta}Cat-Tg; P < 0.0002) (Fig. 2B). Since the total number of thymocytes was not significantly different in {Delta}Cat-Tg mice, the change in percentage of thymocyte subpopulations was reflected in absolute numbers (Fig. 2B). The numbers (x106) were as follows: DP (control 139.2 ± 13.9 and {Delta}Cat-Tg 80.1 ± 5.3; P < 0.0024), CD4 SP (control 28.8 ± 2.5 and {Delta}Cat-Tg 20.2 ± 0.7; P < 0.0134) and CD8 SP (control 10.4 ± 1.1 and {Delta}Cat-Tg 25.4 ± 2.5; P < 0.0005). The change in CD4 SP thymocytes was somewhat variable; either a small increase or no change at all. We conclude that ß-catenin expression diminishes the DP thymocyte population and enhances CD8 SP thymocytes.



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Fig. 2. {Delta}Cat-Tg mice show an increase in SP thymocytes. (A) Thymocytes from control mice (left panel) and {Delta}Cat-Tg mice (right panel) stained with anti-CD4 and anti-CD8 antibodies are shown. The numbers in each quadrant represent percentage of cells. The total number of thymocytes is shown above each panel. (B) Data from 10 control and 10 {Delta}Cat-Tg mice was used to generate the graphs showing thymocyte numbers (upper panels) and percentages (lower panels). The average change in thymocyte numbers (x106) was: DP (control 139.2 ± 13.9 and {Delta}Cat-Tg 80.1 ± 5.3; P < 0.0024), CD4 SP (control 28.8 ± 2.5 and {Delta}Cat-Tg 20.2 ± 0.7; P < 0.0134) and CD8 SP (control 10.4 ± 1.1 and {Delta}Cat-Tg 25.4 ± 2.5; P < 0.0005). The change in percentages was: DP (control 72.9 ± 0.7 and {Delta}Cat-Tg 52.7 ± 2.9; P < 0.0002), CD4 SP (control 14.9 ± 0.4% and {Delta}Cat-Tg 13.7 ± 0.4%; P < 0.03) and CD8 SP (control 5.4 ± 0.3% and {Delta}Cat-Tg 17.2 ± 1.7; P < 0.001). Each symbol represents one mouse and squares show average values. (C) Thymocytes from control (left panel) and {Delta}Cat-1–{Delta}Cat-7 (middle panel) and {Delta}Cat-1–{Delta}Cat-2 (right panel) double-transgenic mice stained with anti-CD4 and anti-CD8 antibodies are shown. The numbers in each quadrant represent the percentage of cells. The total number of thymocytes is shown above each panel. Data shown are representative of eight experiments in which a total of 74 mice were analyzed.

 
Transgenic lines {Delta}Cat-1, {Delta}Cat-2 and {Delta}Cat-7 that expressed lower levels of the transgene were also analyzed. There was a modest increase in CD8 SP thymocytes in {Delta}Cat-1, {Delta}Cat-2 and {Delta}Cat-7 mice compared to littermate control mice ({Delta}Cat-1: total thymocytes 93 x 106, DP 74.6%, CD4 13.9%, CD8 6.3%; {Delta}Cat-7: total thymocytes 83 x 106, DP 79.3%, CD4 11.2%, CD8 4.5%). To increase the level of expression of mutant ß-catenin we bred {Delta}Cat-1 mice with {Delta}Cat-2 mice and {Delta}Cat-7 mice to generate double-transgenic mice. Analysis of thymocytes from double-transgenic mice also showed increased numbers of CD8 SP thymocytes (Fig. 2C, middle and right panels) compared to control (Fig. 2C, left panel). These observations indicated that increased ß-catenin expression in double-transgenic mice correlated with increase in CD8 SP thymocytes. Again, we noted a smaller increase in CD4 SP thymocytes in double-transgenic mice (Fig. 2C, cf. middle and right panels to left panel). This analysis provided confidence that the increase in SP thymocytes correlated with increased expression of ß-catenin transgene. Double {Delta}Cat transgenic mice will not be discussed further in this paper.

To determine the effect of ß-catenin expression on mature T cells, splenic T cells were examined (Fig. 3). The total number of splenocytes was comparable in {Delta}Cat-Tg and control mice. The percentage and number of CD4 and CD8 T cells were not dramatically altered (Fig. 3A and B). Even though ß-catenin has been implicated in cellular proliferation in other cell types, no abnormal proliferation was noted in T cells in {Delta}Cat-Tg mice. We conclude that expression of stabilized ß-catenin enhances the generation of mature thymocytes. Since CD8 SP thymocytes were increased more significantly, these are further characterized in detail in this report.



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Fig. 3. Analysis of splenic T cells in {Delta}Cat-Tg mice. (A) Dot-blots representing splenocytes from control and {Delta}Cat-Tg mice stained with anti-CD4 and anti-CD8 antibodies are shown. The numbers in boxes represent the percentages of cells. The total number of splenocytes is shown above each panel. (B) Data from five control and five {Delta}Cat-Tg mice was used to generate the graphs showing percentage and number (x106) of splenocytes. Each symbol represents data from one mouse and the squares show average values.

 
Phenotypic characteristics of thymocytes and peripheral T cells in {Delta}Cat-Tg mice
CD8 SP thymocytes arise at two stages of thymocyte development. Immature CD8 SP thymocytes are generated transiently during DN to DP transition and mature CD8 SP thymocytes arise after DP thymocytes have undergone positive selection. Immature and mature CD8 SP thymocyte populations can be distinguished on the basis of expression of cell-surface markers including TCR–CD3 complex, CD5 and CD24 (HSA) (1618). Immature CD8 SP thymocytes are TCR–CD3lo and CD24hi, while mature CD8 SP thymocytes are TCR–CD3hi and CD24lo. The pattern of CD3 and CD24 expression on thymocyte subsets in {Delta}Cat-Tg mice was similar to littermate control animals (Fig. 4A). In particular, the CD8 SP population in {Delta}Cat-Tg mice was mature with a cell-surface phenotype of CD3hi and CD24lo. CD3, CD24 and CD5 markers were expressed comparably in mature thymocytes and splenic T cells of {Delta}Cat-Tg and littermate control mice (Fig. 4A). Based on these data, we conclude that {Delta}Cat-Tg mice have increased numbers of normal mature CD8 SP thymocytes.



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Fig. 4. Phenotype of thymocytes and splenic T cells in {Delta}Cat-Tg mice. (A) Overlapping histograms of CD3, CD24 (peak channel fluorescence: control 201; {Delta}Cat-Tg 210) and CD5 (peak channel fluorescence: control 62; {Delta}Cat-Tg 64) expression on thymocyte subsets and splenic T cells, triple stained with anti-CD4 and anti-CD8 antibodies, from control (black lines) and {Delta}Cat-Tg mice (gray lines) are shown. Data shown is representative of five experiments. (B) Perforin mRNA expression was assayed by RT-PCR using RNA prepared from sorted thymocytes from {Delta}Cat-Tg mice. The non-specific band serves as an internal control.

 
Mature CD8 SP thymocytes are precursors for cytolytic T cells and therefore express perforin. We assayed for perforin expression to determine if the CD8 SP thymocytes generated in the presence of stabilized ß-catenin were mature by this criterion. CD8 SP thymocytes in {Delta}Cat-Tg mice, as in littermate control CD8 SP thymocytes (data not shown), expressed mRNA for perforin (Fig. 4B). Importantly, DP and CD4 SP thymocytes from {Delta}Cat-Tg transgenic mice expressed ß-catenin, but did not express perforin, showing that expression of perforin correlated with the stage of maturation of CD8 SP thymocytes and not just expression of stabilized ß-catenin. These data further confirmed that CD8 SP thymocytes in {Delta}Cat-Tg mice were mature.

CD8 SP thymocytes and splenic T cells were characterized with additional cell-surface markers to define maturity and activation status. The expression pattern of five out of six markers was similar in {Delta}Cat-Tg and control CD8 SP thymocytes (Fig. 5). Uniformly high levels of CD62L and Qa-2, and low levels of CD25, CD69 and Ly-6C expression on CD8+ thymocytes in {Delta}Cat-Tg mice showed that these were medullary thymocytes. In contrast, a proportion of splenic T cells showed properties of partially activated T cells as suggested by low levels of CD62L and high levels of Ly-C6 expression. CD44 expression was intermediate on all T cells from {Delta}Cat-Tg mice, including mature CD4 and CD8 SP thymocytes, compared to littermate controls. Because expression levels of other activation markers on T cells from {Delta}Cat-Tg mice paralleled expression on control T cells, we interpret intermediate levels of CD44 expression on {Delta}Cat-Tg T cells as resulting from ß-catenin-dependent gene expression, rather than indicating cellular activation. The putative significance of ß-catenin-dependent CD44 expression on T cells is unclear, but has been detected in cancer cells with modulated Wnt–ß-catenin–TCF signaling. Thus, T cells developed in the presence of transgenic ß-catenin show a normal phenotype. We conclude that ß-catenin expression increases mature medullary (CD62Lhi, Ly-C6lo) CD8 SP thymocytes in {Delta}Cat-Tg mice.



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Fig. 5. Characterization of SP thymocytes and splenic T cells in {Delta}Cat-Tg mice. Overlapping histograms of CD25, CD62L, CD69, Ly-6C, Qa-2 and CD44 expression on thymocytes subsets and splenic T cells, triple stained with anti-CD4 and anti-CD8 antibodies from control (black lines) and {Delta}Cat-Tg mice (gray lines) are shown.

 
Generation of mature SP thymocytes in {Delta}Cat-Tg mice require TCR–MHC interactions
Generation of mature SP thymocytes requires TCR–MHC interactions during maturation [reviewed in (13)]. To determine if the ß-catenin expression altered the requirement for MHC interaction, T cell-depleted, bone marrow-derived precursors from {Delta}Cat-Tg mice were transplanted into irradiated MHC class I–/– and class II–/– double-knockout mice. Transplanted mice did not show a ß-catenin-dependent increase in the CD4 or CD8 SP populations after 6 weeks of reconstitution (Fig. 6A). These data show that expression of stabilized ß-catenin does not bypass the requirement of TCR–MHC interactions for maturation of SP thymocytes.



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Fig. 6. Generation of CD8 SP thymocytes in {Delta}Cat-Tg mice requires the expression of MHC molecules. (A) T cell-depleted bone marrow-derived precursors from {Delta}Cat-Tg mice were infused into irradiated MHC class I class II double-deficient mice or (B) ß2m-deficient mice. Representative profiles of thymocytes stained with CD4 and CD8 antibodies obtained 6 weeks after bone marrow transplantation are shown. Data is representative of two independent transplantation experiments. (C) {Delta}Cat-Tg mice were bred to ß2m-deficient mice. Thymocytes from littermates: wild-type (Control), {Delta}Cat-Tg on a ß2m-sufficient background ({Delta}Cat-Tg), ß2m-deficient (ß2m–/–) and {Delta}Cat-Tg on a ß2m-deficient background ({Delta}Cat-Tg x ß2m–/–) were stained with anti-CD4 and anti-CD8 antibodies. Dot-blots of thymocytes stained with anti-CD4 and anti-CD8 antibodies are shown. (D) Percentage of CD8 SP thymocytes in six control (ß2m–/–-{Delta}Cat-Tg) and nine {Delta}Cat-Tg (ß2m–/–-{Delta}Cat-Tg+) littermates are shown. Data represent analysis of 10 litters of mice.

 
Because CD8 SP thymocytes were increased to a greater extent than CD4 SP thymocytes we further characterized the MHC class I requirement for maturation. To determine if expression of stabilized ß-catenin abrogated the requirement for MHC class I interaction, T cell-depleted {Delta}Cat-Tg bone marrow was transplanted into irradiated ß2m–/– (MHC class I deficient) mice (Fig. 6B). These transplanted mice also did not show a significant increase in the CD8 SP population after 6 weeks of reconstitution (Fig. 6B). These data show that expression of stabilized ß-catenin cannot replace TCR–MHC class I signals essential for the generation of CD8+ cells. We note that expression of stabilized ß-catenin enhanced the reconstitution of CD4 SP thymocytes. The requirement for MHC class I signals for the generation of CD8 SP thymocytes in {Delta}Cat-Tg mice was confirmed by breeding these mice to ß2m-deficient mice (Fig. 6C and D). As expected, ß2m-deficient mice lacked CD8 SP thymocytes. This deficiency was not compensated by the expression of stabilized ß-catenin (Fig. 6C, lower right panel). Analysis of thymocytes from nine ß2m–/–-{Delta}Cat-Tg and six ß2m–/– control mice showed that ß-catenin expression did not enhance generation of CD8 thymocytes in the absence of MHC class I expression (Fig. 6D). Therefore, ß-catenin works in concert with TCR–MHC signals to enhance SP thymocytes.

Expression of stabilized ß-catenin does not protect DP thymocytes from cell death in vitro or enhance proliferation of SP thymocytes
An increase in the number of CD8 SP thymocytes (19), or both CD4 and CD8 SP thymocytes (20), was previously seen in transgenic mice expressing activated Notch-1. Transgenic expression of Bcl-2 (23) and Bcl-XL (24) also increases the number of CD8 SP thymocytes. These data, taken with the observation that DP thymocytes from these mice survive better in culture, have suggested that a survival advantage in DP thymocytes favors their maturation into the CD8 lineage. Additionally, recent analyses with TCF-1-deficient mice have suggested that the TCF-1–ß-catenin pathway may enhance survival of DP thymocytes. In these experiments, transgenic TCF-1 rescued TCF-1-deficient DP thymocytes from death in culture, but mutant TCF-1 lacking the ß-catenin binding domain failed (12). To test if DP thymocytes from {Delta}Cat-Tg mice were protected from death in culture we used the same conditions described in (12). Expression of ß-catenin did not provide protection from cell death to thymocytes in culture (Fig. 7A). This suggests that expression of ß-catenin is not limiting in the survival of DP thymocytes.



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Fig. 7. Functional analysis of thymocytes from {Delta}Cat-Tg mice. (A) Expression of {Delta}Cat-Tg does not protect thymocytes from cell death in vitro. Survival of control (black) and {Delta}Cat-Tg (gray) thymocytes cultured in vitro over time was assessed. Percent live cells are plotted versus time in culture. Triangles represent total thymocytes and circles represent DP thymocytes. Control (black) and {Delta}Cat-Tg (gray) thymocytes were cultured untreated as described in Methods. Aliquots were removed at prescribed times, assayed for viability, and stained with anti-CD4 and anti-CD8 antibodies. Percent live thymocytes at different times is plotted versus time in culture. Data shown is representative of six independent experiments. (B) Expression of {Delta}Cat-Tg does not provide a proliferative advantage to mature SP thymocytes. Thymocytes from control and {Delta}Cat-Tg mice were stained with anti-CD4 and anti-CD8 antibodies, and sorted to purify CD4 or CD8 SP thymocytes. Sorted thymocytes were cultured in vitro with anti-CD3 with or without IL-2 and proliferation was measured as described in Methods. Clear bars are control thymocytes and dotted bars are {Delta}Cat-Tg thymocytes. One of two independent experiments with similar results is shown.

 
ß-Catenin has been implicated in cellular proliferation. To determine if expression of stabilized ß-catenin enhanced the antigen-driven proliferative response of SP thymocytes, we subjected sorted SP thymocytes from control and {Delta}Cat-Tg mice to activation by anti-CD3 antibody and IL-2 in vitro. CD4 or CD8 SP thymocytes from {Delta}Cat-Tg mice did not proliferate more efficiently than control CD8 SP thymocytes (Fig. 7B). We conclude that ß-catenin expression does not increase the proliferation of mature SP thymocytes in vitro.

Expression of stabilized ß-catenin leads to increased generation of CD8 SP thymocytes in newborn mice
To directly determine if expression of stabilized ß-catenin enhanced generation of CD8 SP thymocytes, we assayed the number of mature CD8 SP thymocytes on days 1 and 8 post-birth, when CD8 SP thymocytes first arise during development (Fig. 8). Thymocytes from all pups in each litter were counted and analyzed for expression of CD4, CD8, CD3 TCR and CD24. An increase in the percentage of mature CD3hiCD24lo CD8 SP thymocytes was scored in {Delta}Cat-Tg compared to wild-type control littermates starting at day 1 post-birth. Although the number of CD8 SP thymocytes was low at this stage, the {Delta}Cat-Tg pups showed a higher percentage (Fig. 8A) and number (Fig. 8B) of CD3hi CD8+ cells compared to littermate control pups. CD3hi CD8 SP thymocytes increased between days 1 and 8 post-birth, and the increase was greater in {Delta}Cat-Tg pups (Fig. 8A and B). Percentage and numbers of CD3hi CD4 SP thymocytes were also increased in {Delta}Cat-Tg mice, but the change was smaller (Fig. 8A and B, right panels). Analysis of splenic T cell populations at day 8 post-birth showed that the numbers of CD4 and CD8 T cells were comparable in {Delta}Cat-Tg and littermate control mice (Fig. 8C). This showed that ß-catenin expression did not induce abnormal proliferation of lymphocytes in neonatal mice. We conclude that ß-catenin expression increased the generation of CD8 SP thymocytes in neonatal {Delta}Cat-Tg mice.



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Fig. 8. CD3hi CD8 SP thymocytes are increased in newborn {Delta}Cat-Tg mice. Thymocytes and splenocytes from litters of newborn mice at days 1 and 8 were stained with anti-CD3, anti-CD4 and anti-CD8 antibodies. (A) Percentage and (B) number of cells (x106) of mature CD3hi CD8 SP (left panel) and CD3hi CD4 SP (right panel) thymocytes from {Delta}Cat-Tg negative and positive littermates at day 1 and day 8 post-birth are plotted. (C) Numbers of CD3hi CD4+ and CD3hi CD8+ splenic T cells from litters of newborn mice at day 8 post-birth are plotted. Each symbol represents one mouse and the square boxes represent average numbers.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this report we show that {Delta}Cat-Tg mice, that express stabilized ß-catenin in thymocytes, show a significant increase in SP thymocytes with a corresponding decrease in the DP population. CD4 SP thymocytes were affected to a lesser degree compared to CD8 SP thymocytes. The increase in CD8 SP thymocytes was evident in newborn mice at days 1 and 8 post-birth when CD8 cells first mature. SP thymocytes in {Delta}Cat-Tg mice were mature, not activated and required TCR–MHC signals for development. While these data do not address the possibility that late-stage maturation of SP thymocytes or export of mature thymocytes to the periphery may be altered, one interpretation may be that ß-catenin expression enhances positive selection signals leading to increase in mature thymocytes.

In a previous study, deletion of ß-catenin exon 3 encoding the N-terminal regulatory region of ß-catenin using Cre–Lox technology led to the mutant stabilized protein being expressed at very high levels in all thymocytes (14). Under these conditions pre-TCR-mediated cellular proliferation was blocked, leading to a 10-fold decrease in thymocyte numbers. A small number of DN thymocytes matured to the DP stage without TCR ß chain expression and these cells, lacking {alpha}ß TCR, did not efficiently mature to the SP stage. Presumably because the transgene in {Delta}Cat-Tg mice was not overexpressed at such a high level, we circumvented the detrimental effects on pre-TCR signaling and were able to study the effect of ß-catenin expression at later stages of thymocyte development. Our observations reveal an unexpected role for ß-catenin in positive selection, particularly of CD8 SP thymocytes.

The CD8 SP thymocytes in {Delta}Cat-Tg mice were medullary as judged by high levels of TCR–CD3, Qa-2 and CD62L, and low levels of CD24 expression. The level of CD5 and CD69 suggested these cells had undergone positive selection. Expression of perforin mRNA in sorted CD8 SP, but not in sorted DP and CD4 SP, thymocytes from {Delta}Cat-Tg mice further marked the stage of CD8 SP maturation. In {Delta}Cat-Tg transgenic mice, the increase in CD8 lineage cells did not diminish the number of CD4 lineage cells. Instead there was a consistent decrease in the DP compartment, suggesting that ß-catenin expression did not divert thymocytes destined for the CD4 lineage into the CD8 lineage, but rather encouraged DP thymocytes to mature into the CD8 SP lineage.

An increase in the number of CD8 SP thymocytes (19) or both CD4 and CD8 SP thymocytes (20) was previously seen in transgenic mice expressing activated Notch-1. One explanation was that Notch-1 enhanced DP thymocytes survival (21, 22) allowing a greater chance of positive selection of thymocytes with weak TCR affinities. This notion was supported by the observation that transgenic mice expressing survival the factors Bcl-2 (23) and Bcl-XL (24) also showed increased CD8 SP thymocytes. Recently, it was shown that Bcl-XL expression was induced in DP thymocytes in TCF-1–/– mice expressing TCF-1 capable of binding ß-catenin, but not in mice expressing mutant TCF-1 incapable of binding ß-catenin (12). These data might have suggested that a ß-catenin-mediated survival advantage caused the increased numbers of CD8 SP thymocytes in {Delta}Cat-Tg mice. Indeed, we were surprised to see no protection from cell death in {Delta}Cat-Tg DP thymocytes in the same assays described in (12). This may indicate that ß-catenin is not the essential component that binds to the 100-amino-acid N-terminal domain of TCF-1 and protects DP thymocytes from cell death in vitro. Alternately, ß-catenin expression may not be limiting and therefore increased ß-catenin expression does not enhance survival. {Delta}Cat-Tg T cells show intermediate levels of CD44 expression, but are not activated as judged by the expression of other cell-surface markers and criteria. It is likely that expression of CD44 on developing thymocytes may have consequences that influence survival or development by unknown mechanisms.

ß-Catenin has been implicated in cellular proliferation, especially in several cancers, suggesting that thymocytes and T cells in {Delta}Cat-Tg mice may be hyperproliferative. However, this is not the case. We have previously shown that {Delta}Cat-Tg mice live to >18 months without developing lymphoproliferative disorders or tumors, suggesting that ß-catenin does not dysregulate cell cycle in vivo in T cells (27). Additionally, thymocytes and splenic T cells in {Delta}Cat-Tg mice do not exhibit enhanced proliferation to anti-CD3 antibody and IL-2 in vitro stimulation despite a high level of transgenic ß-catenin expression in SP thymocytes. This expression pattern from the proximal Lck promoter agrees with previous reports showing that transgenic Bcl-2 (25) or green fluorescent protein (26) is highly expressed in mature SP thymocytes as well as peripheral T cells. Thus, ß-catenin expression does not enhance proliferation of mature SP thymocytes, suggesting that the increase in CD8 SP thymocytes in {Delta}Cat-Tg mice is unlikely to be due to post-commitment antigen-independent or antigen-dependent proliferation. Together, these observations suggest that the increase in SP thymocytes resulted from enhanced generation rather than expansion.

Finally, we sought to refute the potential possibility that re-circulation of mature activated CD8 T cells from the peripheral lymphoid organs to the thymus resulted in an apparent increase in CD8 SP cells. Re-circulating cells would be expected to be activated T cells that express CD25hi, CD44hi, Ly-6Chi, CD69hi, Qa-2lo/– and CD62Llo/– as opposed to CD25lo, CD44int, Ly-6Clo, CD69lo, Qa-2hi and CD62Lhi, which are features of medullary CD8 SP thymocytes (28). By these criteria CD3hi CD8 SP thymocytes in {Delta}Cat-Tg mice were medullary and not re-circulating mature peripheral T cells. Furthermore, activated T cells have been shown to re-enter the thymus only under conditions of generalized inflammation, created by co-injection of lipopolysaccharide or concanavalin A with activated T cells in model systems (29,30). When T cells are activated by antigen in vivo, T cell responses can be documented without disproportionate accumulation of T cells in the thymus (31). We injected {Delta}Cat-Tg mice with the peptide antigen using the protocol reported by Hardy et al. (31), and documented CD8 T cell activation and response without any further increase in the thymic CD8 population (data not shown). Furthermore, {Delta}Cat-Tg mice show no signs of inflammation and the CD8 T cells as well as CD8 SP thymocytes show generally un-activated phenotypes. Therefore, the increase in CD8 SP thymocytes is unlikely to be due to re-entry of these cells from the periphery. Together, these observations reason against re-circulation of mature CD8 cells back to the thymus in {Delta}Cat-Tg mice.

CD8 SP thymocytes first arise in newborn mice. We compared the number of CD3hiCD24lo CD8 SP thymocytes in newborn {Delta}Cat-Tg mice at days 1 and 8 post-birth. We determined that the percentage and number of CD3hiCD24lo CD8 SP thymocytes was higher in {Delta}Cat-Tg pups compared to littermate control mice in the same litters. These data indicated that ß-catenin expression enhanced the generation of mature CD8 SP thymocytes when they were first generated during normal development. Furthermore, the numbers of splenic CD8 T cells were comparable in {Delta}Cat-Tg pups and littermate control pups at day 8 post-birth, showing no abnormal expansion of peripheral T cells in {Delta}Cat-Tg mice. These observations further attest that ß-catenin expression enhances the generation and positive selection of CD8 SP thymocytes during development.

In conclusion, in this report we show that transgenic expression of stabilized mutant ß-catenin in thymocytes enhances the number of mature SP thymocytes. CD8 SP thymocytes were enhanced to a greater extent than CD4 SP thymocytes. Increased generation of SP thymocytes was documented in newborn mice when these cells are first generated. Enhanced generation of SP thymocytes required MHC expression, suggesting enhanced positive selection. Conversely, T cell specific deletion of ß-catenin reduces SP thymocytes and drastically decreases splenic T cells (32). Together, these data suggest a role for ß-catenin during positive selection and late stages of T cell development.


    Acknowledgements
 
J. M. S. thanks Dr Brian Seed and Naifang Lu for the generation of transgenic mice, and Drs Neil Simister, Dipanjan Chaudhary, Jennifer Punt, Pam Schwartzberg, Ruibao Ren and Ranjan Sen for helpful comments on the manuscript. This work was supported by grants from The Barr Foundation, Arthritis Foundation and NIH/NCI.


    Abbreviations
 
DN—double negative

DP—double positive

PI—propidium iodide

TCF—T cell factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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