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Down-Regulation of the Orphan Nuclear Receptor RORt Is Essential for T Lymphocyte Maturation¹

Department of Immunology and Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195

	Abstract

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Thymocyte development is a tightly regulated process. CD4⁺CD8⁺ double-positive (DP) immature thymocytes exhibit distinct phenotypic features from mature T cells; they express only 10% of surface TCR that are found on mature T cells and do not proliferate and produce IL-2 in response to stimulation. In this report we show that transgenic expression of the orphan nuclear receptor RORt in mature T cells down-regulates their surface TCR expression. The RORt transgene inhibits IL-2 production by mature T cells, and this inhibition may be partially due to the inhibitory effect of RORt on c-Rel transcription. Furthermore, ectopic expression of RORt inhibits the proliferation of mature and immature T cells. These results, together with its predominant expression in DP thymocytes, suggest that RORt controls these distinct phenotypic features of DP thymocytes. Our data suggest that down-regulation of RORt expression in thymocytes is essential for their maturation.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
T lymphocyte differentiation and maturation are completed in the thymus through several well-defined, phenotypically distinct steps (1, 2). The earliest T cell precursors lack expression of CD4, CD8, and CD3/TCR complex. These CD3^-CD4^-CD8^- triple-negative (TN)³ cells undergo a series of changes, including TCR gene rearrangement, surface protein alteration, and massive expansion to differentiate into cells that express both CD4 and CD8 proteins on the surface. These CD4⁺CD8⁺ double-positive (DP) cells make up a majority of the thymocyte population (80–85%). Only a small fraction of these immature DP thymocytes are positively selected and mature into either CD4⁺CD8^- or CD4^-CD8⁺ single positive (SP) thymocytes (3).

Immature DP thymocytes differ from mature SP thymocytes and peripheral T cells in several fundamental ways. First, DP thymocytes express only about 10% of the number of TCR complexes on the cell surface that are expressed by mature T cells (4, 5). This marked difference in the expression of TCR on immature DP vs mature SP thymocytes appears to be regulated during development by post-transcriptional mechanisms (6, 7). Second, unlike mature T cells, DP thymocytes do not proliferate after stimulation with anti-CD3 mAbs or calcium ionophore plus phorbol ester (5). This failure to proliferate is not due to a functional defect in the CD3/TCR complex, because a large fraction of DP thymocytes express a functional CD3/TCR complex that is able to transduce signals to the cells. When stimulated via the TCR, DP thymocytes mobilize cytoplasmic free Ca²⁺ and activate PKC (5, 8). Furthermore, TCR engagement of DP thymocytes has been shown to increase the synthesis of TCR -chain and decrease recombinase-activating gene-1 mRNA expression (9). Third, DP thymocytes do not secrete IL-2 in response to activation (5, 10). The expression of the IL-2 gene is controlled by multiple transcription factors, including NF-AT, AP-1, and NF-B/Rel (11). Studies of AP-1 and NF-AT activities demonstrated that both AP-1 and NF-AT are present in the nucleus of freshly isolated thymocytes at all stages of maturation, but they specifically lack DNA binding activity in DP thymocytes (12, 13, 14, 15). Why DP thymocytes manifest such distinctive features from mature T cells and how this is achieved at the molecular level are not understood.

The orphan nuclear receptor RORt (16) is a thymus-specific isoform of ROR (17, 18, 19). The expression of RORt is tightly regulated in developing thymocytes. DP thymocytes express high levels of RORt mRNA, while CD4⁺ or CD8⁺ SP mature thymocytes do not express RORt (16). Ectopic expression of RORt in T cell hybridoma cell lines inhibits Fas ligand up-regulation and IL-2 production without inhibiting early events of T cell activation such as up-regulation of CD69 (16). However, the role of RORt in T lymphocyte development is not clear. To address this issue, we generated RORt transgenic mice using the hCD2 promoter/enhancer/locus control region to drive RORt expression in both mature and immature T cells. We show that ectopic expression of RORt in mature T cells down-regulates TCR surface expression. Ectopic expression of RORt also inhibits the proliferation of mature T cells and TN thymocytes. Furthermore, RORt expression prevents mature T cells from producing IL-2 and inhibits c-Rel up-regulation. Our results demonstrate a phenotypic similarity between the mature T cells from RORt transgenic mice and normal immature DP thymocytes. These data together with the predominant expression of RORt in DP thymocytes support the idea that RORt is an important regulator of DP thymocyte phenotype and function. Furthermore, our data suggest that down-regulation of RORt expression is essential for the maturation of DP thymocytes into SP thymocytes.

	Materials and Methods

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Cell lines

The KMIs-8.3.5 cell line (20) is a T cell hybridoma. KMIs-8.3.5RORt and its control cell line expressing hCD2 were described previously (16). KMIs-8.3.5RORt expressing c-Rel was generated by retroviral transduction of a full-length c-Rel cDNA using the pMI5 vector, which is identical with the pMI vector (16) except that it contains an internal ribosomal entry site (IRES)-driven hCD5, followed by multiple rounds of panning on anti-hCD5 mAb-coated plates. Cells were cultured in DMEM containing 10% FCS, 2 mM glutamine, 25 mM HEPES, 50 µM 2-ME, 100 U/ml penicillin, and 100 µg/ml streptomycin.

Abs and reagents

Polyclonal rabbit anti-mouse RORt serum was generated against the C-terminal 12-aa peptide of RORt and affinity-purified using SulfoLink kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. The following mAbs were purchased from PharMingen (San Diego, CA): purified and FITC- or Cy-Chrome-anti-CD3 (145-2C11); FITC-, PE- or Cy-Chrome-anti-CD4 (H129.19); PE- or Cy-Chrome-anti-CD8 (53-6.7); FITC-anti-CD25 (7D4); PE-anti-CD44 (1M7); PE-anti-CD69 (H1.2F3); biotin-anti-FasL (MFL3); biotin-anti-B220 (RA3-6B2); biotin-anti-Mac-1 (M1/70); FITC-anti-hCD2 (RPA-2.10); FITC-anti-hCD5 (UCHT2); and biotin-anti-I-A^b (KH74). Rabbit polyclonal anti-c-Rel Ab was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PMA and ionomycin were purchased from Calbiochem (La Jolla, CA). Recombinant human IL-2 was obtained from Chiron (Emeryville, CA).

Mice

The RORt transgenic construct was generated by inserting a cDNA fragment encoding the full-length RORt into the EcoRI site of the VAhCD2 transgenic vector (21). The resultant construct was released with XhoI/XbaI and injected into (C57BL/6 x DBA2)F₁ embryos. Founder mice were identified by Southern blot analysis of tail DNA and backcrossed to C57BL/6 mice (The Jackson Laboratory, Bar Harbor, ME). All mice were housed under specific pathogen-free conditions in the animal facility of University of Washington.

Northern blot analysis and RT-PCR

Total RNA was extracted from cell lines using STAT-60 (Tel-Test, Friendswood, TX) and analyzed by Northern blot analysis using a standard protocol (22). The cDNA probe for c-Rel was derived by RT-PCR. RT-PCR for RORt mRNA expression was described previously (16). The PCR products were analyzed on 1% agarose gel.

Western blot analysis

Hybridoma cells were lysed in lysis buffer (10 mM HEPES, 40 mM KCl, 3 mM MgCl₂, 1 mM DTT, 5% glycerol, 0.2% Nonidet P-40, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM PMSF) on ice for 10 min, and the insoluble portion was removed by centrifugation. Primary cells were lysed with SDS sample buffer at 100°C for 10 min. Equal number of cells (0.5–5 x 10⁶ cells/lane) were run on 8 or 10% polyacrylamide gels and transferred to nitrocellulose. The membranes were then probed with Abs to mRORt, c-Rel, followed with HRP-conjugated secondary Abs and detected with enhanced chemiluminescence according to the manufacturer’s instructions (Amersham, Arlington Heights, IL).

Cell separation and flow cytometric analyses

TN subsets and CD4⁺ or CD8⁺ SP thymocytes were first enriched by negative selection with biotin-labeled Abs and streptavidin-labeled Dynabeads (Dynal, Oslo, Norway) followed by FACS sorting. The purity of sorted subpopulations of thymocytes was >99% in postsort analyses. Peripheral T cells from spleen or lymph nodes were purified by negative selection with biotin-labeled anti-B220, anti-Mac-1, and anti-I-A^b, followed by incubation with streptavidin-labeled Dynabeads. The purity of these cells was >90%. Cells were sequentially incubated with an excess of biotinylated mAb, PE-streptavidin, and FITC- or Cy-Chrome-labeled Abs on ice and washed with PBS containing 0.1% BSA. Data were collected for 5 x 10⁴ cells on a FACScan flow cytometer (Becton Dickinson, Mountain View, CA) using CellQuest software.

Cell proliferation assay and IL-2 production

Purified T cells (2–5 x 10⁵/well) were added to 96-well tissue culture plates in the presence of PMA (10 ng/ml) plus ionomycin (0.1 µg/ml) for the indicated time. The supernatants were removed for IL-2 assay. IL-2 was measured using an ELISA kit (PharMingen, San Diego, CA) according to the manufacturer’s instructions. In the proliferation assays, cells were labeled with [³H]thymidine (1 µCi/well, 25 Ci/mmol; New England Nuclear, Boston, MA) for 4 h, harvested on glass-fiber filters, and counted in a beta scintillation counter. Data were derived from the mean of duplicate or triplicate cultures, with an SD <10%.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of RORt in thymocyte subpopulations

We previously demonstrated that the expression of RORt in T cells is developmentally regulated (16). RORt mRNA was detected at a high level in DP thymocytes and at a low level in CD4^-CD8^- (DN) thymocytes, and was undetectable in CD4⁺ or CD8⁺ SP thymocytes or splenic T cells (16). To establish more precisely the expression pattern of RORt during thymocyte maturation, we performed RT-PCR analysis of FACS-sorted immature DP, intermediate CD4⁺CD8^lowHSA^high, and mature CD4⁺CD8^-HSA^low thymocytes (23, 24). As shown in Fig. 1A, expression of RORt mRNA is inversely correlated with the maturity of thymocytes. Immature DP thymocytes express the highest level of RORt mRNA, while intermediate CD4⁺CD8^lowHSA^high thymocytes on the pathway to SP stage express lower levels of RORt. Fully mature CD4⁺CD8^-HSA^low cells do not express RORt. These results demonstrate that thymocytes gradually down-regulate RORt expression as they undergo maturation from the DP to the SP stage.

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Expression of RORt in normal thymocyte subpopulations and RORt transgenic mice. A, Expression of RORt in thymocyte subsets at different developmental stages. RT reactions of the thre thymocyte subsets from C57BL/6 mice were serially diluted 1/3 and subjected to PCR with primers specific for RORt. HPRT RT-PCR was performed from the same RT samples for cDNA template quantity control. B, Expression of RORt in normal TN thymocyte subsets. RT-PCR analysis of the four TN thymocyte subsets was performed as described in A, except samples were serially diluted 1/10. C, Expression of RORt in RORt transgenic mice. RT reactions of FACS-sorted CD4+ SP or CD8+ SP thymocytes, total thymocytes, spleen, and lymph nodes from RORt transgenic mice or littermate controls were subjected to PCR as described in A without serial dilution. D, Western blot analysis of RORt expression in normal and RORt transgenic mice. Cell lysates from total thymocytes or purified T cells of spleen or lymph nodes were electrophoresed on a 10% polyacrylamide gel, transferred, and blotted with rabbit anti-mRORt Ab.

Generation of transgenic mice expressing RORt

Given the tightly controlled expression pattern of RORt in T lymphocytes, we reasoned that ectopic expression of RORt in mature T cells may provide insight to its function. To achieve this, we generated transgenic mice using the hCD2 promoter to drive RORt expression in immature DN, DP, and mature T cells. The VAhCD2 transgenic vector specifically directs transgene expression in all T cells in a copy number-dependent fashion (21). Two independent RORt transgenic founder lines (founders 850 and 779) with 10 and 20 copies, respectively, of transgene were established. Progeny from these two lines exhibited a similar phenotype, and herein results from one line (founder 779) are reported. The expression of RORt mRNA was readily detected in CD4⁺ or CD8⁺ SP cells purified from the thymus and spleen or lymph nodes of RORt transgenic mice, but not in cells from littermate controls (Fig. 1C). RORt transgene expression was further confirmed by Western blotting. In agreement with the RT-PCR data, RORt protein was expressed in purified T cells from the spleen or lymph nodes of transgenic mice but not from littermate controls (Fig. 1C). Importantly, the level of RORt protein expression in peripheral T cells from transgenic mice was 5-fold lower compared with the expression level of RORt in thymocytes from control mice (Fig. 1D). Despite this low level of protein expression in mature T cells, RORt transgene had a clear effect on their phenotype and function (see below). These results indicate that the hCD2 transgenic vector targeted RORt to mature T cells and are consistent with its capacity to target gene expression in all T cells.

The RORt transgene blocks T cell development at an early stage

T cell development was examined in RORt transgenic mice. Compared with littermate controls, the thymic cellularity of RORt transgenic mice was severely reduced (Fig. 2A). On the average, the number of thymocytes from RORt mice was reduced 85%. Total cell numbers from spleen and lymph nodes of RORt mice were also reduced 50–60% compared with those from the littermate controls (Fig. 2A). T cell development was further characterized by FACS analysis using CD4 and CD8 as surface markers. RORt transgenic mice had a dramatically lower percentage of DP thymocytes and a higher percentage of DN thymocytes compared with control mice (Fig. 2B). Although the percentage of CD4⁺ SP or CD8⁺ SP thymocytes in RORt mice was relatively higher, the absolute cell number of each of these two subsets was still lower than that in control mice (Fig. 2). The CD8 surface level on DP thymocytes from the transgenic mice was relatively lower than that on control cells (Fig. 2A). Interestingly, a significant number of thymocytes in RORt mice were either CD4^lowCD8^- or CD4⁺CD8^low (Fig. 2B). These cells did not appear to be an abnormal expansion of some minor populations within the thymus, because they did not express c-Kit, NK1.1, CD25, or TCR (not shown). Furthermore, these cells were not present in the spleen of the RORt transgenic mice (Fig. 2B). The CD4/CD8 profile of splenocytes from the RORt transgenic mice was relatively normal, except for a lower percentage of CD8⁺ T cells (Fig. 2B). These data suggest that the expression of RORt transgene blocked thymocyte development, possibly at the transition from the DN to the DP stage.

View larger version (37K): [in this window] [in a new window]   FIGURE 2. Expression of RORt transgene blocks T cell development. A, Total cell number of lymphoid organs from RORt transgenic mice and littermate controls. Cells from thymus, spleen, and lymph nodes of 7- to 13-wk-old mice were enumerated by trypan blue exclusion. The numbers represent the mean value from six mice in each group. B, FACS analyses of T cell development in the thymus and spleen of RORt mice. Cells were stained with either FITC-anti-CD4 and PE-anti-CD8 or FITC-anti-CD25, PE-anti-CD44, and Cy-Chrome-anti-CD3, -CD4, and -CD8. For the analysis of TN thymocyte, CD3+, CD4+, and CD8+ thymocytes were excluded by electronic gating. The percentage of cells in each gated region is indicated.

RORt down-regulates TCR expression

We next examined the effect of the RORt transgene on the surface expression of TCR by mature T cells. Surprisingly, both CD4⁺ and CD8⁺ SP thymocytes from RORt mice expressed lower levels of TCR on their surface (Fig. 3A). The level of TCR expression on RORt transgenic SP thymocytes is about half that of control thymocytes as assessed by both anti-CD3 (Fig. 3A) and anti-TCRß (not shown) mAb staining. Correlating to its predominant expression of endogenous RORt in DP thymocytes, the low level of TCR expression in DP thymocytes was not further reduced by ectopic RORt expression (Fig. 3A). In addition, 30% of the CD4⁺ SP thymocytes from RORt mice expressed low or undetectable levels of CD3 on their surface (Fig. 3A). A similar decrease in the TCR level was found in peripheral T cells from the spleen (Fig. 3B) and lymph nodes (not shown) of RORt mice. Furthermore, ectopic expression of RORt in KMIs-8.3.5 hybridoma cells decreased TCR surface expression (not shown). These results demonstrate that ectopic expression of RORt in mature T cells down-regulates TCR surface expression.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Expression of the RORt transgene down-regulates surface TCR expression on mature T cells. Thymocytes and splenocytes were analyzed by three-color staining. Shown are the histogram profiles of CD3 staining for different subsets gated as described in Fig. 2B. The numbers are the mean fluorescence intensity of the CD3+ cells.

RORt inhibits IL-2 production

RORt was shown to inhibit IL-2 production by a hybridoma cell line (16). To investigate whether ectopic expression of RORt in mature T cells inhibits their ability to produce IL-2, we purified CD4⁺ SP thymocytes or splenic T cells from RORt mice or littermate controls and stimulated these cells with PMA plus ionomycin, which activate T cells by bypassing TCR. Both CD4⁺ SP thymocytes and splenic T cells from RORt transgenic mice produced dramatically lower amounts of IL-2 compared with cells from littermate controls (Fig. 4A). Therefore, ectopic expression of RORt in mature T cells inhibited their ability to produce IL-2.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Expression of RORt transgene inhibits IL-2 production and c-Rel up-regulation. A, Expression of RORt transgene inhibits IL-2 production by mature T cells. FACS-sorted CD4+ SP thymocytes or purified splenic T cells in 96-well plates were activated with PMA plus ionomycin for 24 or 48 h, respectively. The supernatants were assayed for IL-2 using an ELISA kit. Data are representative of two experiments. B, Northern blot analysis of c-Rel mRNA expression in KMIs-8.3.5 hybridoma cells expressing RORt. Cells were activated in 2C11-coated plates for the indicated time and harvested, and total RNA was extracted. GAPDH was probed as a loading control. C, Western blot analysis of c-Rel protein expression in KMIs-8.3.5 hCD2 and KMIs-8.3.5 RORt cell lines. Cells were activated with PMA (10 ng/ml) plus ionomycin (0.4 µg/ml) for 4.5 h and lysed. An equal number of cell equivalents was run in each lane. Shown are two independent KMIs-8.3.5 RORt cell lines. D, Western blot analysis of c-Rel protein expression in purified splenic T cells from RORt transgenic or control mice. Purified cells were activated with PMA plus ionomycin for 7 h and lysed in SDS sample buffer.

RORt negatively regulates c-Rel expression

IL-2 expression is controlled by multiple transcription factors (11). T cells from c-Rel-deficient mice have impaired IL-2 production (26, 27). To test whether the inhibition of IL-2 production by RORt is due to an effect on c-Rel expression, we performed Northern blot analyses to determine the effect of RORt on c-Rel induction in the T cell hybridoma KMIs-8.3.5. Following activation, the expression of c-Rel mRNA was induced as early as 1 h and continuously increased up to 6 h in control KMIs-8.3.5 hCD2 cells (Fig. 4B). In contrast, the induction of c-Rel mRNA was strongly inhibited in KMIs-8.3.5RORt cells (Fig. 4B). This result was confirmed by Western blot analysis (Fig. 4C). Although we did not detect c-Rel mRNA in unstimulated KMIs-8.3.5 cells (Fig. 4B), these cells express a detectable level of c-Rel protein before activation and dramatically up-regulate its expression after activation (Fig. 4C). In contrast, KMIs-8.3.5RORt cell lines expressed significantly less c-Rel protein both before and after activation by PMA plus ionomycin (Fig. 4C). Importantly, the expression of c-Rel in RORt transgenic T cells was also reduced (Fig. 4D). These results demonstrate that RORt negatively regulates c-Rel transcription.

To test whether the inhibition of IL-2 production by RORt is solely due to its inhibition of c-Rel transcription, we transduced a full-length c-Rel cDNA into KMIs-8.3.5RORt using a retroviral vector containing an IRES-hCD5 reporter cassette. Constitutive expression of c-Rel in this cell line did not restore its capacity to produce IL-2 (not shown), suggesting that RORt may additionally regulate other genes that are required for IL-2 production.

RORt inhibits T cell proliferation

To examine the effect of ectopic expression of RORt on T cell proliferation, we stimulated purified CD4⁺ SP thymocytes or splenic T cells with PMA plus ionomycin. As shown in Fig. 5A, the proliferation of mature T cells from both the thymus and spleen was significantly inhibited by the RORt transgene. T cells from c-Rel-deficient mice exhibited a defect in proliferation, and this defect can be corrected by adding exogenous IL-2 (26, 27). When exogenous IL-2 was added to the cell culture, the proliferation of the T cells from the RORt transgenic mice was slightly increased but still significantly lower than that in control T cells (Fig. 5A). To determine whether the decreased proliferation of the transgenic T cells is due to an effect on their ability to be activated, we analyzed these cells for the expression of the T cell activation markers CD69 and CD25. After 24 h of activation, splenic T cells from RORt mice up-regulated both CD69 and CD25 on their surface to similar levels as T cells from control mice (Fig. 5B). These results indicate that the inhibition of T cell proliferation by RORt is not solely due to its effect on IL-2 production or to an inability of T cells to be activated.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Expression of RORt transgene inhibits T cell proliferation. A, Proliferation of mature T cells from RORt transgenic mice and littermate controls. FACS-sorted CD4+ SP thymocytes or purified splenic T cells were stimulated with PMA plus ionomycin for 48 h and pulsed with [3H]thymidine for an additional 4 h. IL-2 was added at 50 U/ml. Data are representative of three experiments. B, FACS analysis of the up-regulation of CD69 and CD25 on activated splenic T cells from RORt transgenic mice and littermate controls. Cells were activated with PMA plus ionomycin as described in A for 24 h and double-stained with anti-CD3 plus anti-CD69 or anti-CD25. CD3+ cells were analyzed for their expression of CD25 and CD69. The filled histogram profiles at the left represent the staining of resting splenic T cells. C, Cell cycle analysis of CD44-CD25- TN thymocytes. CD44-CD25- TN thymocytes were FACS sorted, fixed, and stained with propidium iodide. The percentage of cells in each gated region is indicated.

The effect of RORt transgene expression on Fas ligand up-regulation

RORt was shown to inhibit Fas ligand expression in T cell hybridomas (16). To determine whether this effect occurs in vivo, we examined the effect of RORt transgene expression on Fas ligand up-regulation in mature T cells. Thymocytes and splenocytes were activated with PMA plus ionomycin, and the up-regulation of Fas ligand was assessed by three-color FACS analysis. Seventy-two hours after activation, Fas ligand was up-regulated on both CD4⁺ and CD8⁺ mature T cells from control mice (Fig. 6). The expression level of Fas ligand on T cells from RORt transgenic mice was slightly decreased on CD4⁺ SP thymocytes and CD8⁺ splenocytes, but not obviously affected on CD4⁺ splenocytes and CD8⁺ SP thymocytes (Fig. 6). We then tested activated-induced cell death (AICD) of splenic CD4⁺ T cells from RORt transgenic mice. The AICD of CD4⁺ cells is mainly mediated by Fas-Fas ligand interaction (30). No obvious defect was found in AICD of CD4⁺ cells from RORt transgenic mice (data not shown). The discrepancy of RORt inhibition on Fas ligand expression observed in vitro and in vivo may be due to the low level of transgene expression in mature T cells. Alternatively, the regulation of Fas ligand expression may differ in hybridomas vs mature T cells.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Effect of the expression of RORt transgene on Fas ligand up-regulation by mature T cells from RORt transgenic mice. Total thymocytes and splenocytes were activated with PMA plus ionomycin for 72 h and stained with anti-CD4, anti-CD8, and Fas ligand mAbs. Histograms at the far left represent Fas ligand staining of fresh thymocytes and splenocytes. Dotted line, stimulated thymocytes from RORt transgenic mice; thick line, stimulated cells from littermate controls.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The inhibition of IL-2 production by RORt may be due to its negative effect on c-Rel transcription and on certain other genes that are required for IL-2 production. The transcriptional control of the IL-2 gene has been thoroughly studied. Multiple elements capable of binding AP-1 complexes, NF-AT, NF-B/Rel, and Oct-1 have been identified in 5' upstream of the IL-2 transcriptional start site (11). Of these various response elements and transcription factors, c-Rel was unequivocally shown to be required for IL-2 production in mouse knockout studies (26, 27). Our in vivo and in vitro results identified RORt as a negative regulator of c-Rel transcription. In further support of this, c-Rel mRNA expression is inversely correlated with RORt expression. c-Rel mRNA was not detected in the majority of DP thymocytes, but is expressed in positively selected TCR^highDP thymocytes and mature T cells (31), whereas RORt is highly expressed in DP thymocytes and is down-regulated in mature T cells. Besides the effect on c-Rel expression, RORt might also affect the expression or activities of other genes that are involved in IL-2 production. Constitutive expression of c-Rel in the hybridoma line expressing RORt did not restore its ability to produce IL-2. NF-AT and AP-1 are additional transcription factors that may be negatively regulated by RORt. Although RORt has no obvious effect on the expression and nuclear translocation of NF-ATc in the KMIs-8.3.5 hybridoma cell line (Y.-W. He and M. J. Bevan, unpublished observation), other studies have shown that these transcription factors lack DNA binding activity in DP thymocytes (12, 13, 14, 15). This raises the possibility that RORt may compete with these factors for binding to the IL-2 promoter.

RORt has the capacity to inhibit the proliferation of mature and TN immature T cells when ectopically expressed. Given its high level of expression in DP thymocytes, RORt may act as an inhibitor of DP cell proliferation. Several lines of evidence indicate that the inhibition of T cell proliferation by RORt is not solely due to its effect on c-Rel transcription. Although T cells from c-Rel-deficient mice have impaired capacity in terms of proliferation, this defect can be corrected by adding exogenous IL-2 (26, 27). In contrast, addition of exogenous IL-2 did not correct the deficiency of RORt transgenic T cells in proliferation. Furthermore, expression of RORt severely reduced thymic cellularity, whereas T cell development in c-Rel-deficient mice is normal (26, 27). The reduced thymic cellularity in RORt transgenic mice is probably due to an impaired proliferative expansion of CD44^-CD25^- TN thymocytes. Correlating to their low level expression of endogenous RORt, a large fraction of the normal CD44^-CD25^- TN thymocytes is proliferating (28, 29). The expression of the RORt transgene in this subset decreased the number of cells in the S/G₂/M phases of the cell cycle by 90%. It remains to be determined how RORt negatively regulates T cell proliferation.

The down-regulation of TCR surface expression by RORt in mature T cells suggests that RORt control the low levels of TCR surface expression on normal DP thymocytes. Previous studies demonstrated that DP thymocytes express approximately equal amounts of mRNA for each of the TCR components compared with mature T cells (6), but express on the surface only about 10% of the level found on mature T cells (4, 5). Although the physiological significance of the low levels of TCR surface expression is not clear, it appears that most intrathymic repertoire selection occurs among these DP thymocytes expressing low levels of surface TCR (32). How RORt regulates surface TCR expression will be of interest for future studies.

The effects of RORt revealed in this study should help us to understand the physiology of DP thymocytes. The most immature DN thymocytes proliferate and produce IL-2 upon stimulation. When DN thymocytes differentiate into more mature DP cells, they lose the capacity for both proliferation and IL-2 production. Not until these cells become fully mature SP thymocytes do they regain this capacity (10). This stage-specific response correlates well with the expression pattern of RORt. At the DP stage, thymocytes face positive and negative selection. It is important that neither of these selection events should be accompanied by cell division or cytokine production. We propose that the orphan nuclear receptor RORt is a critical regulator of the DP thymocyte functions.

	Acknowledgments

 
We thank Erik Jacobson for excellent technical support, Deb Wilson for animal care, Kathy Allen for FACS sorting, and Drs. Dimitris Kioussis, Hsiou-X Liou, and Jacqueline Kirchner for providing reagents. We thank Eugene Huang and Dr. Jacqueline Kirchner for reading this manuscript.

	Footnotes

 
¹ This work was supported by the National Institutes of Health and the Howard Hughes Medical Institute.

² Address correspondence and reprint requests to Dr. Michael J. Bevan, Howard Hughes Medical Institute, Department of Immunology University of Washington School of Medicine, Box 357370, Seattle, WA 98195.

³ Abbreviations used in this paper: TN, triple negative; DP, double positive; SP, single positive; hCD2, human CD2; AICD, activated-induced cell death.

Received for publication November 15, 1999. Accepted for publication March 20, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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