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
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Abstract |
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Keywords: positive selection, signal transduction, thymocyte maturation, thymus, Wnt signaling
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Introduction |
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DP thymocytes expressing 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 CreLox 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 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 (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
Cat-Tg mice require TCRMHC interactions for development, suggesting that ß-catenin expression enhances positive selection signals. Finally, mature CD3hi CD8 SP thymocytes are increased in newborn
Cat-Tg mice when CD8 SP thymocytes are first generated during development. We suggest that ß-catenin expression augments TCRMHC signals essential for positive selection of thymocytes.
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Methods |
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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% SDSpolyacrylamide 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 manufacturers 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 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:
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 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 Leibovitzs 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 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.
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Results |
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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 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
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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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 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
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
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
Cat-Tg mice paralleled expression on control T cells, we interpret intermediate levels of CD44 expression on
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 Wntß-cateninTCF 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
Cat-Tg mice.
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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-1ß-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 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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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 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
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
Cat-Tg pups (Fig. 8A and B). Percentage and numbers of CD3hi CD4 SP thymocytes were also increased in
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
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
Cat-Tg mice.
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Discussion |
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In a previous study, deletion of ß-catenin exon 3 encoding the N-terminal regulatory region of ß-catenin using CreLox 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 ß TCR, did not efficiently mature to the SP stage. Presumably because the transgene in
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 Cat-Tg mice were medullary as judged by high levels of TCRCD3, 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
Cat-Tg mice further marked the stage of CD8 SP maturation. In
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 Cat-Tg mice. Indeed, we were surprised to see no protection from cell death in
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.
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 Cat-Tg mice may be hyperproliferative. However, this is not the case. We have previously shown that
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
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
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 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
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,
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
Cat-Tg mice.
CD8 SP thymocytes first arise in newborn mice. We compared the number of CD3hiCD24lo CD8 SP thymocytes in newborn 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
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
Cat-Tg pups and littermate control pups at day 8 post-birth, showing no abnormal expansion of peripheral T cells in
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.
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Acknowledgements |
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Abbreviations |
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DPdouble positive
PIpropidium iodide
TCFT cell factor
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References |
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