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Molecular and Cellular Biology, July 2002, p. 4556-4566, Vol. 22, No. 13
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.13.4556-4566.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Increased p300 Expression Inhibits Glucocorticoid Receptor-T-Cell Receptor Antagonism but Does Not Affect Thymocyte Positive Selection

Cheng-Tai Yu,1,2 Ming-Hsien Lin Feng,1 Hsiu-ming Shih,3 and Ming-Zong Lai1,2,4*

Institute of Molecular Biology, Academia Sinica,1 Graduate Institute of Life Science, National Defense Medical School,2 Division of Molecular & Genomic Medicine, National Health Research Institute,3 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan, Republic of China4

Received 11 February 2002/ Returned for modification 18 March 2002/ Accepted 1 April 2002


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Positive selection of T cells is postulated to be dependent on the counterinteraction between glucocorticoid receptor (GR)- and T-cell-receptor (TCR)-induced death signals. In this study we used T-cell-specific expression of p300 to investigate whether GR-TCR cross talk between thymocytes was affected. Activation of the p300-transgenic T cells led to enhanced thymocyte proliferation and increased interleukin 2 production. Thymocyte death, induced by TCR engagement, was no longer prevented by dexamethasone in p300-transgenic mice, indicating an absence of GR-TCR cross-inhibition. This was accompanied by a 50% reduction in the number of thymocytes in p300-transgenic mice. However, the CD4/CD8 profile of thymocytes remained unchanged in p300-transgenic mice. There was no effect on positive selection of the bulk thymocytes or thymocytes with transgenic TCR in p300-transgenic mice. In addition, there was no apparent TCR repertoire "hole" in the selected antigens examined. Our results illustrate a critical role of CBP/p300 in thymic GR-TCR counterinteraction yet do not support the involvement of GR-TCR antagonism in thymocyte positive selection.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During T-cell development, the interaction of T-cell receptors (TCRs) with the self major histocompatibility complex-peptide complexes promotes the differentiation of CD4+CD8+ (double-positive [DP]) thymocytes into mature CD4+CD8- and CD4-CD8+ single-positive (SP) T cells; this is known as positive selection. TCR-engaged signals are essential for initiating positive selection, but TCR signaling also triggers the apoptosis of DP thymocytes. At present the molecular basis for the successful positive selection of T cells is poorly understood. Thymocytes are extremely sensitive to glucocorticoid-mediated apoptosis (39), yet there is mutual inhibition between TCR and glucocorticoids in regard to the induction of thymocyte apoptosis (18, 49). One model of glucocorticoid-dependent positive selection suggests that, through the antagonism of TCR-triggered cell death, glucocorticoids may promote DP thymocyte survival and modulate the T-cell selection threshold (1). Consistent with this model, large reductions in thymocyte numbers with an increased sensitivity to TCR-induced death and defects in positive selection are observed in transgenic mice expressing an antisense construct of the glucocorticoid receptor (GR) gene (25, 29). Similarly, inhibition of steroidogenic enzymes increases thymocyte sensitivity to deletion (42, 43). An alternative model concerning the effect that glucocorticoids have on positive selection suggests that glucocorticoids are completely dissociated from thymocyte development (14). The production of glucocorticoid by thymic epithelial cells is not required for positive selection (21). Replacement of GR with a GR mutant defective in DNA binding eliminates the thymocyte sensitivity to glucocorticoid-induced apoptosis but does not affect thymocyte development (36). Moreover, both positive selection and negative selection are found to be normal in GR-deficient thymus (35).

CBP/p300 were originally identified for their binding to phosphorylated CREB and E1A (9, 26). Many transcription factors are now known to bind to CBP/p300, allowing for interactions with TFIID, TFIIB, and RNA polymerase II (for review see references 5, 15, 41, and 45). In addition, histone acetyltransferase activities, either inherently associated with CBP/p300 or recruited during transcription complex formation, help chromatin remodeling near the target gene. Among the transcription factors essential for T-cell activation (3, 17, 19, 38), CREB, NF-{kappa}B, AP-1, and nuclear factor of activated T cells (NFAT) depend on a direct interaction with CBP/p300 (2, 12, 13, 22, 33, 48). CBP/p300 are also essential for transcription activation of the GR (8).

The loss of a single CBP allele leads to Rubinstein-Taybi syndrome (34), indicating that concentrations of CBP/p300 are limited for normal cell function. As an integrator of different transduction pathways in the nucleus, CBP/p300 may mediate some of the synergy as well as some of the cross-inhibition between transcriptional factors (45). It has been proposed that the mutual inhibition between AP-1 and GR (47) or between NF-{kappa}B and GR (40) may be mediated by competition for a limiting amount of CBP/p300 (22). In addition, a transcription-active GR is essential for glucocorticoid-induced thymocyte death (36), suggesting a likely dependence on CBP/p300. In this study, we explored the effect of increased p300 expression on glucocorticoid-TCR antagonism in thymocytes. We observed that thymocyte death prevented by TCR-GR antagonism was inhibited in the p300-transgenic mice. However, T-cell positive selection was minimally affected, indicating a minor role for TCR-GR antagonism in positive selection.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. Concanavalin A (ConA), tetradecanoyl phorbol acetate (TPA), and A23187 were purchased from Sigma Chemical Co. (St. Louis, Mo.). The antibodies for mouse CD4 (clone CT-CD4, fluorescein isothiocyanate [FITC] or TriColor conjugated), CD5.2 (clone CG16, FITC conjugated), CD8 (clone CT-CD8a, phycoerythrin [PE] or TriColor conjugated), CD44 (clone IM7.8.1, FITC or PE conjugated), CD69 (clone H1.2F3, FITC conjugated), and TCR (H57-597, FITC conjugated) were obtained from Caltag (Burlingame, Calif.). FITC-conjugated anti-CD25 (PC61.5.3) and FITC-conjugated anti-Vß3 (KJ25), Vß5 (MR9-4), Vß6 (RR4-7), Vß8 (F23.1), and Vß14 (14-2) were obtained from PharMingen (San Diego, Calif.). PE-conjugated anti-CD4 (GK1.5), FITC-labeled anti-CD8 (53-6.7), PE-conjugated anti-CD25 (PC61.5.3), and PE-conjugated anti-CD69 (H1.2F3) were purchased from eBioscience (San Diego, Calif.).

Mouse lines. Plasmid p1017 containing the lck 3.2-kb proximal promoter and 3' untranslated region of human growth hormone (minigene exons 1 to 5) was a gift of Roger Perlmutter (University of Washington, Seattle). The human CD2 cassette was a gift from Dimitris Kioussis (National Institute for Medical Research, London, United Kingdom). The transgenic mice were generated in the Transgene/Knockout Core of the Institute of Molecular Biology, Academia Sinica (Taipei, Taiwan). The 5.5-kb BamHI-XbaI fragment containing the CD2 locus control region (16) was isolated and coinjected with p1017-p300 into the pronuclei of both C57BL/6 and FVB zygotes (6). Transgenic mice were first identified by PCR analysis of 1 µg of DNA using transgene-specific primers (Fig. 1A). PCR commenced at 94°C for 3 min, followed by 34 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1 min 30 s, ending at 72°C for 10 min. The expression of p300 in transgenic mice was confirmed by reverse transcriptase PCR (RT-PCR), with the same thermal cycles as PCR, performed on cDNA produced from 2 µg of RNA. Seven independent founders (three in C57BL/6 and four in FVB) were obtained. All transgenic mice were maintained in the specific-pathogen-free mouse facility of the Institute of Molecular Biology, Academia Sinica. The AND transgenic mouse (23) was a gift of John Kung (Academia Sinica) and was maintained in a B10.A background. All mouse experiments were conducted with the approval of the Animal Committee, Institute of Molecular Biology, Academia Sinica.



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  		FIG. 1. Transgenic construct and the production of lck-p300-transgenic mice. (A) Schematic representation of the lck-p300 transgene. HA-p300 was subcloned between the proximal lck promoter and human growth hormone (hGH) polyadenylation signals with intron sequence. Arrows indicate the location of the transgenic-specific PCR primers. (B) PCR analysis of the lck-p300 transgene expression. DNA isolated from mouse tail and RNA prepared from peripheral blood lymphocytes were subjected to PCR and RT-PCR, respectively, using the transgenic-specific PCR primers. The PCR products, resolved on agarose, were used to identify the lck-p300-transgenic mice from the NLC. Ctrl, control. (C) Increased p300 protein expression in lck-p300-transgenic mice. The contents of total p300 in thymocytes from three independent lck-p300-transgenic lines and the NLC mice were determined by immunoblots using anti-p300 (Santa Cruz Biotechnology) and were quantitated by Desitometer. The numbers along the top indicate the number of times of increase of the p300 contents relative to NLC.

 
Immunoblots. Cell extracts (30 µg) were resolved by sodium dodecyl sulfate-6% polyacrylamide gel electrophoresis and were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, Mass.) for 4 h at 20 V. Membranes were washed in rinse buffer (phosphate-buffered saline with 0.1% Tween 20) at room temperature for 15 min and incubated in blocking buffer (5% nonfat milk in rinse buffer) for 30 min. The membrane was then incubated with primary antibodies for 2 h at room temperature and washed three times with rinse buffer. The membrane was incubated with 1:1,000 diluted horseradish peroxidase-conjugated anti-rabbit/mouse immunoglobulin antibody (Santa Cruz) followed by development with enhanced-chemiluminescence reagents (Amersham, Little Chalfont, Buckinghamshire, United Kingdom).

Cell death measurement. All cultures were performed in RPMI medium with 10% fetal calf serum (both from GIBCO, Grand Island, N.Y.), 10 mM glutamine, 100 U of penicillin/ml, 100 µg of streptomycin/ml, and 2 x 10-5 M 2-mercaptoethanol. The extent of apoptosis was determined by propidium iodide (PI) staining. At the end of different treatments, cells were resuspended in hypotonic fluorochrome solution (50 µg of PI/ml, 0.1% sodium citrate, and 0.1% Triton X-100) (30) and were placed at 4°C in the dark overnight. DNA contents were analyzed by FACScan (Becton Dickinson, Mountain View, Calif.). The fraction of cells with sub-G1 DNA content was quantitated using the CELLFIT software program (Becton Dickinson). For annexin V staining, freshly isolated thymocytes were washed, resuspended in annexin V-FITC (1 µg/ml) (Clontech), incubated at room temperature for 15 min in the dark, and analyzed on FACScan.

Peptides and T-cell response. The peptides were synthesized on an Applied Biosystems 430 peptide synthesizer and were purified as previously described (27, 28). The sequences of peptides used in this study were ovalbumin 323-339, ISQAVHAAHAEINEAGR; hen egg lysozyme 81-96, SALLSSDITASVNCAK; pigeon cytochrome c 81-104, IFAGIKKAFRADLIAYLKQATAK; {lambda} repressor 12-26, LEDARRLKAITEKKK; and {lambda} repressor 73-88, EEFSPSIAREIYEMY. The immunization of peptides and quantitation of response to peptides in T cells isolated from draining lymph nodes were conducted as described previously (27, 28).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of lck-p300-transgenic mice. Hemagglutinin (HA)-p300 was subcloned into the p1017 vector with the lck proximal promoter (Fig. 1A) and was coinjected with CD2 enhancer for T-cell-specific expression (6). Genomic incorporation of transgenic HA-p300 was detected by PCR on mouse tail DNA using lck-p300 specific primers (Fig. 1B). Expression of lck-p300 was further confirmed by RT-PCR conducted on RNA purified from blood cells (Fig. 1B). Total p300 protein expression in thymus (sum of transgenic and endogenous p300) was elevated in transgenic mice (Fig. 1C). p300 expression levels increased between two- and threefold in lck-p300-transgenic mice (Fig. 1C). We were unable to obtain permanent T-cell clones and transgenic mice with more than a fourfold increase in p300 over that found in the wild type, suggesting that excess p300 may not be tolerable to cells. We generated seven independent lck-p300-transgenic lines in both the C57BL/6 and FVB backgrounds. These seven transgenic lines displayed similar thymus number, CD4/CD8 expression, and activation profiles. We also generated four independent lines of lck-CBP transgenic mice. No differences were found between lck-p300 and lck-CBP-transgenic mice upon analysis.

Increased proliferation and IL-2 production in lck-p300-transgenic thymocytes. Thymocytes isolated from the lck-p300-transgenic and the normal littermate control (NLC) mice were activated with anti-CD3/anti-CD28, ConA, or TPA/A23187. Activation-induced proliferation was profoundly elevated in thymocytes from p300-transgenic mice (Fig. 2A). At least a 100% increase in proliferation was associated with p300-transgenic thymocytes, irrespective of the mode of activation (TCR, ConA, or TPA/A23187). TCR-mediated interleukin 2 (IL-2) production was similarly elevated in the lck-p300-transgenic thymocytes (Fig. 2B). The increase in IL-2 expression is consistent with the coactivation role of p300 for AP-1, NF-{kappa}B, and NFAT, which together mediate the activation of the IL-2 gene promoter (19).



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  		FIG. 2. Increased proliferation and IL-2 production in lck-p300-transgenic thymocytes. Shown is the proliferation of thymocytes from lck-p300-transgenic mice (p300 tg) and the NLC stimulated by immobilized anti-CD3 plus anti-CD28 (coated at 10 and 5 µg/ml) (CD3/28 in panel A) or by TPA (10 ng/ml) plus A23187 (80 ng/ml) (T/A in panel A) or different concentrations of ConA (B). Thymidine incorporation was determined 60 h later. Ctrl, control. (C) Production of IL-2 in thymocytes stimulated with CD3/CD28 and ConA. Thymocytes were similarly stimulated, and the IL-2 produced was quantitated 24 h later using the IL-2-dependent cell line HT-2. Recombinant murine IL-2 was used as the standard. Nearly identical results were found in 10 pairs of mice originating from four independent p300-transgenic lines.

 
Inhibited glucocorticoid-TCR antagonism in lck-p300-transgenic mice. We next examined whether increased expression of p300 affected the antagonism between GR and TCR. Both glucocorticoids and anti-CD3 induced significant thymocyte apoptosis 24 h after treatment (Fig. 3). Dexamethasone-mediated thymocyte death was suppressed by TCR activation signaling in the NLC mice (Fig. 3). The antagonism in thymocyte apoptosis between GR and TCR was abolished in lck-p300-transgenic mice. Dexamethasone-triggered thymocyte death was no longer prevented by TCR ligation in p300-transgenic mice (Fig. 3).



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  		FIG. 3. Antagonism between GR and TCR was inhibited in lck-p300-transgenic mice. (A) Thymocytes isolated from lck-p300-transgenic mice and NLC mice were stimulated with immobilized anti-CD3 (10 µg/ml) alone, dexamethasone (Dex) alone, or the combination of both. Twenty-four hours later, cells were stained with PI and DNA contents were analyzed by FACScan (Becton Dickinson). Fractions of cells with sub-G1 DNA content were assessed using a CELLFIT program (Becton Dickinson). (B) The average of results conducted in seven pairs of mice originating from three independent transgenic lines.

 
The disruption of TCR-GR cross-inhibition in p300-transgenic thymocytes could have been due to increased spontaneous death or a disproportionate increase of GR- or TCR-triggered apoptosis. Spontaneous thymocyte cell death was identical for NLC and p300-transgenic mice for the first 48 h of in vitro culture. A small but significant increase in spontaneous cell death was observed for p300-transgenic thymocytes beyond 3 days of culture (Fig. 4A). Since cell death antagonism (Fig. 3) was determined only at 24 h, inhibition of TCR-GR antagonism was apparently not caused by an increase in spontaneous thymocyte death. We also determined thymocyte apoptosis triggered by different concentrations of dexamethasone (10-8 to 10-9 M) and found little difference between p300-transgenic mice and NLC mice (Fig. 4B). Elevated apoptosis was detected in p300-transgenic thymocytes after activation by different concentrations of anti-CD3 (Fig. 4C). However, such small increases in apoptosis could not account for the extensive apoptosis observed in p300-transgenic thymocytes costimulated with anti-CD3 and dexamethasone (Fig. 3). We thus concluded that TCR-GR antagonism was lost in p300-transgenic mice.



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  		FIG. 4. Spontaneous cell death and TCR- and dexamethasone-induced cell death in thymocytes from NLC- and p300-transgenic (p300 tg) mice. Thymocytes from four NLC mice (open triangles) and four p300-transgenic mice (solid circles) were untreated (A), activated with different concentrations of dexamethasone (Dex) (B), or immobilized with anti-CD3 (C). The extent of cell death was determined at different time points (A), at 24 h (B), or at 30 h (C) by quantitation of sub-G1 DNA content. Similar results were observed in two other independent experiments with three pairs of mice each.

 
Decreased thymocyte numbers in lck-p300-transgenic mice. The lck-p300-transgenic mice were used to elucidate how thymocyte development would be affected in the absence of glucocorticoid counterinteractive signals. The most prominent phenotype in lck-p300-transgenic mice was the dramatically reduced size of the thymus. A decrease in thymocyte number in p300-transgenic mice was observed in mice of all different ages, including the newborn (Fig. 5A). The average thymocyte number of 5-week-old mice decreased from 2.75 x 108 in NLC mice to 1.49 x 108 in p300-transgenic mice, nearly a 50% reduction (Fig. 5A). A similar extent of reduction in thymocyte number was found in p300-transgenic mice of other age groups. The total number of splenocytes, however, remained constant in p300-transgenic mice (data not shown), due possibly to the decreased sensitivity of mature lymphocytes to glucocorticoid-triggered apoptosis. To examine whether a decrease in thymocyte number was due to an increase in thymocyte death, freshly isolated thymocytes were subjected to annexin V staining (Fig. 5B). The frequency of apoptotic cells was found to be low in live thymus, yet there was a significant increase of apoptosis in CD4+CD8+ thymocytes from p300-transgenic mice.



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  		FIG. 5. A large reduction of thymocytes in p300-transgenic mice. (A) Total thymocyte number of NLC and lck-p300-transgenic mice at ages 0, 1, 2, 3, 4, and 5 weeks was determined. The same symbols are used for the same pair of a transgenic mouse and its normal littermate. Six mice each from at least two different transgenic lines were used for each age group. The numbers below indicate the average cell number of each mouse set. Note that the scale for 0- to 2-week-old mice is different from that for 3- to 5-week-old mice. (B) Increased apoptosis in CD4+CD8+ thymocytes from p300-transgenic mice. Freshly isolated thymocytes from seven pairs of NLC and p300-transgenic mice were stained with anti-CD4, anti-CD8, and annexin V. The frequency of annexin V-positive cells in CD4+CD8+ thymocytes was determined. The same symbols are used for the same pair of a transgenic mouse and its normal littermate. The numbers below indicate the average percentage of apoptosis and the standard deviation.

 
Normal thymocyte profiles in transgenic mice with lck-p300 expression. If thymocyte positive selection was dependent on GR-TCR antagonism, then the inhibition of GR-TCR cross-inhibition in p300-transgenic mice should have been accompanied by an alteration in the mature thymic SP population, representing positively selected thymocytes. However, fluorescence-activated cell sorter (FACS) analysis of CD4 and CD8 expression on thymocytes failed to reveal any difference between transgenic mice and NLC mice (Fig. 6A). Mature peripheral T-cell populations, such as splenic CD4+ and CD8+ T cells, were also normal in the p300-transgenic mice (Fig. 6B). Elucidation of CD4-CD8- thymocyte development by the expression of CD44 and CD25 indicates that early thymocyte development in lck-p300-transgenic mice is normal (data not shown).



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  		FIG. 6. Thymocyte development of lck-p300-transgenic mice is similar to that found in the control. (A) FACS profile of thymocytes from 6-week-old lck-p300-transgenic and NLC mice stained for CD4 and CD8. Number in the panel below indicates the percentage of the corresponding subpopulation. (B) Splenic populations of CD4+ and CD8+ T cells in NLC mice and p300-transgenic mice were similar. Similar patterns were found in another 10 transgenic mice from four independent lines.

 
Positive selection is not affected by transgenic expression of lck-p300. Thymocyte positive selection is indicated by increased expression of CD5 and CD69 (4, 7, 24). No difference was found between the NLC and p300-transgenic mice as concerns the fraction of CD5+ thymocytes that both possessed (Fig. 7A). Because CD4+CD8+ thymocytes were the population undergoing positive selection, CD4+CD8+ thymocytes were further gated and their CD69 and CD5 expression was determined. The levels of CD69 (Fig. 7B) and CD5 (data not shown) were found to be identical in CD4+CD8+ thymocytes from lck-p300-transgenic and NLC mice. Positive selection also resulted in up-regulation of TCR. Quantitation of thymocytes expressing high levels of TCR and CD69 did not reveal any differences between lck-p300-transgenic and NLC mice (Fig. 7C).



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  		FIG. 7. Positive selection is not affected by the transgenic expression of lck-p300. (A) CD5+ expression on thymocytes from lck-p300-transgenic and NLC mice was similar. (B) Three-color FACS analysis of CD69 expression on CD4+CD8+ thymocytes from lck-p300-transgenic and NLC mice. CD4+CD8+ thymocytes were gated and analyzed for CD69 expression. (C) Fraction of thymocytes with up-regulated levels of TCR and CD69. The numbers indicate positively selected thymocytes expressing CD5, CD69, or both TCR and CD69. Similar results were found in another eight pairs of mice from three independent transgenic lines.

 
Positive selection of transgenic TCR in p300-transgenic mice is normal. In mice with transgenic TCR, the same TCR is expressed in a large proportion of thymocytes. The selection of this fraction of thymocytes with homologous transgenic TCR therefore constitutes a more sensitive readout of T-cell positive selection. We chose AND TCR transgenic mice in which a V{alpha}11-Vß3 TCR specific for cytochrome c peptide and I-Ek was overexpressed (23). The p300 transgene (in a C57BL/6 background) was introduced into AND TCR transgenic mice in a B10.A background through breeding. The expression of I-Ek and Vß3 was confirmed in the mice examined. Thymocyte numbers of p300 x AND double transgenic mice were similarly reduced by 50% when compared with those of AND transgenic mice (data not shown). The selection of AND TCR thymocytes was not affected in mice bearing the p300 transgene (Fig. 8A). The AND transgene led to a large increase in CD4 SP thymocytes, with no difference detected in the fractions of CD4 SP thymocytes between AND and AND x p300-transgenic mice. Positively selected thymocytes, scored for the expression of either CD69 (Fig. 8B) or CD5 (not shown), were similar in NLC and p300-bearing mice. Elevated expression of {alpha}ß-TCR (Fig. 8C) and Vß3 (Fig. 8D), mostly of AND transgenic TCR, was nearly identical in mice with or without the p300 transgene. Fractions of splenic CD4+ T cells, representing mostly mature AND T cells, were also similar in NLC and p300-transgenic mice (Fig. 8E). In contrast to a deletion of V{alpha}11-Vß3 TCR specific for pigeon cytochrome c 81-104 in B10.BR mice deficient in GR (29), the selection of AND TCR was normal in p300-transgenic mice. These results further support the notion that T-cell positive selection is not reduced in the absence of GR antagonism.



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  		FIG. 8. Selection of AND TCR is not altered in p300-transgenic mice. Thymocytes (A) and splenocytes (E) from AND TCR-transgenic mice and AND x p300 double-transgenic mice were analyzed for CD4 and CD8 expression. Expression of CD69 (B), {alpha}ß-TCR (C), and Vß3 (D) in thymocytes from AND mice and AND x p300 mice was determined by their respective FITC-conjugated antibodies. Similar results were found in another six pairs of mice originating from three independent p300-transgenic founders.

 
T-cell immunity to most antigenic peptides is not compromised in p300-transgenic mice. Despite the normal thymocyte phenotype and normal AND TCR selection in p300-transgenic mice, there existed the possibility that defective positive selection impairs the selection of only a fraction of the specific T cells, leading to the generation of a hole in the T-cell repertoire (29). We first examined the integrity of TCR Vß repertoires in p300-transgenic mice and NLC mice. No difference between lck-p300 mice and NLC mice was found in the expression of Vß3, Vß5, Vß6, Vß8, and Vß14 among splenic CD4+ or CD8+ T cells (data not shown). We then determined T-cell responses to various antigenic peptides restricted by I-Ab in C57BL/6 mice with or without the p300 transgene. The T-cell responses to hen egg lysozyme 81-96 and ovalbumin 323-339, as assessed by IL-2 production and T-cell proliferation, were significantly increased in transgenic mice (Fig. 9A and B, data not shown for T-cell proliferation). The extent of increase correlated well with the degree of augmented T-cell activation in p300-transgenic mice (Fig. 2). A third peptide, {lambda} repressor 73-88, elicited a comparable T-cell response in NLC and p300-transgenic mice (Fig. 9C). We also analyzed T-cell response in p300-transgenic FVB mice to pigeon cytochrome c 81-104, ovalbumin 323-339, hen egg lysozyme 81-96, {lambda} repressor 12-26, and {lambda} repressor 73-88 (data not shown). With the exception of hen egg lysozyme 81-96, which displayed a 50% reduction in T-cell response, the other four peptides elicited stronger or comparable T-cell responses in p300-transgenic FVB mice. Assuming that TCR usage for each antigenic peptide is restricted, our results would suggest that selection of T-cell repertoire for most antigenic peptides is not affected in p300-transgenic mice. There was no apparent TCR repertoire hole in p300-transgenic mice.



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  		FIG. 9. T-cell responses to specific antigenic peptide are not compromised in p300-transgenic mice. NLC and p300-transgenic mice were immunized with 100 µg of hen egg lysozyme 81-96 (A), ovalbumin 323-339 (B), and {lambda} repressor 81-96 (C) in colonization factor antigen. Draining lymph nodes were collected 7 days later, and the T-cell response to different concentrations of the immunized antigens was determined. Four pairs of mice from two independent p300-transgenic lines were used for each antigen immunization.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we attempted to address the role of glucocorticoids in thymocyte positive selection by using T-cell-specific transgenic expression of p300. In presently proposed modes of action, glucocorticoids play a positive or negative role or no role in T-cell positive selection. In models where glucocorticoids are thought to modulate thymocyte development, it is through cross talk with TCR signals. We have found that increased expression of p300 prevented antagonism between dexamethasone and TCR (Fig. 3). The p300-transgenic T cells also displayed increased proliferation and IL-2 production upon TCR stimulation compared to the NLC T cells (Fig. 2). It is possible that, because p300 is the common transcription coactivator for many transcription factors, an increased T-cell activation response is simply an integrated outcome of elevated p300 expression. Notably, the present results also illustrate that increased proliferation and IL-2 production do not necessarily lead to an elevated positive selection. The dissociation of proliferation/IL-2 expression and positive selection is consistent with previous observations that Bcl-2 promotes T-cell development yet reduces T-cell proliferation (31) and that inactivation of NFAT5 impairs T-cell development but not T-cell proliferation and IL-2 production (32).

Even though CBP/p300 are a general coactivator, p300 overexpression is not thought to affect all the biological activities of glucocorticoids. The anti-inflammatory and immunosuppressive functions of glucocorticoids are thought to be dissociated from the transcription activity of GR (36, 37), suggesting that p300 is not involved in any of these events. In addition, cross-inhibition of GR-AP-1, GR-NF-{kappa}B, or GR-TCR may proceed in a transcription- (and hence p300)-independent manner (10, 11, 20, 44). Interestingly, apoptosis induction in thymocytes is one of the few GR-mediated events that definitely require a transcriptionally active GR (36, 37) and is potentially affected by p300 overexpression. The observation that antagonism of apoptosis between TCR and GR was eliminated in p300-transgenic mice (Fig. 3) suggests that increased p300 expression relieves cross-inhibition between TCR and GR in thymocytes. Our results thus provide the first direct support for the argument that TCR-GR antagonism in thymocytes is mediated by competition for CBP/p300. Therefore, CBP/p300 are critical for the regulation of the GR-mediated thymocyte death and GR-TCR antagonism in thymocytes.

The inhibition of GR-TCR antagonism in p300-transgenic thymocytes has allowed us to examine T-cell development with diminished GR inhibition. We detected a substantial decrease (50%) in thymocyte number (Fig. 5A) and an increase of apoptosis among CD4+CD8+ thymocytes (Fig. 5B) in p300-transgenic mice, which seems to agree with a survival role for glucocorticoids (1). In the absence of GR-TCR antagonism, TCR signaling was unable to rescue thymocytes from glucocorticoid-induced cell death (Fig. 3). If unselected DP thymocytes were killed by glucocorticoids, there should have been a decrease in DP population in p300-transgenic mice because of the lack of TCR rescue. If glucocorticoids modulate the TCR death signal to increase positive selection, then there should have been a decrease in CD4 SP and CD8 SP thymocytes. Our observations that DP, CD4 SP, and CD8 SP thymocytes were unaffected in lck-p300-transgenic mice (Fig. 6) argue against both possibilities. The selection of AND TCR, recognizing pigeon cytochrome c 81-104 presented by I-Ek, was not interfered with by the p300 transgene (Fig. 8). In addition, the bulk of thymocyte expression of CD5/CD69 was normal in the absence of TCR-GR cross-coupling (Fig. 7), suggesting that mutual inhibition between TCR and GR is dissociated from positive selection. Moreover, reduced thymocyte number and increased apoptosis in p300-transgenic thymocytes (Fig. 5) do not necessarily support a survival role of glucocorticoids in the thymus. We have observed that there is an increase in spontaneous cell death in p300-transgenic thymocytes (Fig. 4A), likely to be an additional mechanism that may lead to reduced thymocyte number (Fig. 5A).

We used TCR immunity experiments (Fig. 9) to examine the integrity of the TCR repertoire, caused by defects in positive selection. These experiments were conducted with antigenic peptides in which TCR usage is mostly unclear. Therefore, these results may be interpreted only on the basis that, similar to the situation for many other antigen-specific T cells, TCR usage for each peptide is restricted. In C57BL/6 mice, T-cell responses to hen egg lysosome 81-96, ovalbumin 323-339, and {lambda} repressor 12-26 were not reduced by the presence of the p300 transgene (Fig. 9). We also observed that T-cell responses to cytochrome c 81-104, ovalbumin 323-339, {lambda} repressor 12-26, and {lambda} repressor 73-88 in the p300-transgenic mice in an FVB background were not decreased (data not shown). Therefore, in contrast to the study employing the GR antisense transgene (29), the so-called T-cell repertoire hole does not necessarily exist in thymocytes where TCR-GR antagonism is inhibited. It may be noted that, since general T-cell activation is increased twofold in p300-transgenic T cells (Fig. 2), the detection of a comparable T-cell response to {lambda} repressor 73-88 in NLC and p300-transgenic mice (Fig. 9C) may be interpreted as a reduction in the available {lambda} repressor 73-88-specific T cells in p300-transgenic mice. A partially impaired T-cell repertoire for {lambda} repressor 73-88 thus cannot be formally excluded in this study.

Our results may help reconcile some of the differences between the opposing arguments about the role of GR in T-cell development (1, 14). Consistent with the results obtained from GR antisense experiments (25), we have found a reduction in thymocyte number when TCR-GR antagonism is abolished (Fig. 5). If the implication that the TCR repertoire for {lambda} repressor 73-88 is partly compromised (Fig. 9C) is indeed true, our data corroborate the report that GR participates in the development of a small fraction of thymocytes (25, 29, 46). However, positive selection of T cells for most antigenic peptides analyzed in this study proceeds normally in p300-transgenic mice. In addition, no defects were identified on the selection of gross thymocyte population and AND-TCR-expressing thymocytes in p300-transgenic mice (Fig. 6 to 8). Therefore, even if GR participates in the selection of some thymocytes, it seems that only the selection of a very minor thymocyte population is glucocortiocid dependent. In such a scenario, glucocorticoids are not the major signals that discriminate selected thymocytes from unselected thymocytes.


   ACKNOWLEDGMENTS
 
This project was supported by grant NSC 89-2311-B001-005 from the National Science Council and a grant from Academia Sinica, Taipei, Taiwan, Republic of China.

We thank Richard Goodman, Roger Perlmutter, Dimitris Kioussis, John Kung, and Chung Wang for providing the plasmids and reagents. We thank Bruce Boothby for helpful discussions and Ken Deen for editing the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China. Phone: (886)-2-2789-9236. Fax: (886)-2-2782-6085. E-mail: mblai{at}ccvax.sinica.edu.tw. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Molecular and Cellular Biology, July 2002, p. 4556-4566, Vol. 22, No. 13
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.13.4556-4566.2002
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