CD25+CD4+ regulatory T cells exert in vitro suppressive activity independent of CTLA-4
Hiroshi Kataoka1,2,
Shigekazu Takahashi1,
Kan Takase1,
Sho Yamasaki1,3,
Tadashi Yokosuka1,3,
Takao Koike2 and
Takashi Saito1,3
1 Department of Molecular Genetics, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
2 Department of Medicine II, Hokkaido University Graduate School of Medicine, N15, W7, Kita-ku, Sapporo 060-8638, Japan
3 Laboratory for Cell Signaling, RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehirocho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
Correspondence to: T. Saito; E-mail: saito{at}rcai.riken.jp
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Abstract
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Cytotoxic T lymphocyte antigen-4 (CTLA-4) is constitutively expressed on CD25+CD4+ regulatory T cells (Treg) and is suggested to play a role in Treg-mediated suppression. However, the results of analysis with anti-CTLA-4 have been controversial. We addressed this issue by analyzing mice over-expressing or deficient in CTLA-4. For over-expression, CTLA-4 transgenic mice expressing a full-length (FL) or a truncated (TL) mutant of CTLA-4 were analyzed. FL T cells expressed similar levels of CTLA-4 to Treg, whereas TL T cells expressed much higher levels on the cell surface. The number of Treg in both mice was decreased, although Foxp3 expression was not altered. Treg from both mice exerted suppressive activity, whereas CD25 T cells from FL mice showed no suppression. Furthermore, CD25+CD4 thymocytes from young CTLA-4-deficient mice were analyzed and found to exhibit suppressive activity. These results indicate that Treg exert in vitro suppressive activity independent of CTLA-4 expression.
Keywords: co-stimulation, Foxp3, homeostasis, thymocyte, tolerance
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Introduction
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Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory co-stimulation receptor of T cells that mediates the inhibition of T cell activation. As CTLA-4 shares its ligands (CD80 and CD86) with CD28 and has much higher affinity for the ligands than CD28, a low expression of CTLA-4 mediates strong inhibition. Whereas CD28 is constitutively expressed, CTLA-4 is induced only upon TCR stimulation. CTLA-4 inhibits T cell activation by competing with CD28 for ligand binding to block the positive co-stimulation and the delivery of inhibitory signals (13). The inhibitory function of CTLA-4 is evidenced by the finding that CTLA-4-deficient (/) mice develop lymphoproliferative disease and become moribund at several weeks of age (4). These observations indicate that CTLA-4 down-regulates T cell activation and maintains peripheral tolerance in vivo.
Naturally occurring CD25+CD4+ regulatory T cells (Treg) suppress self-reactive CD25 T cells to prevent the development of autoimmunity in vivo (5, 6). Treg down-regulate the proliferation of CD25 T cells upon TCR stimulation in vitro in an antigen-non-specific manner, although Treg activation is antigen specific (7). The Treg-mediated suppression requires cell contact and is not mediated by cytokines such as IL-10, IFN
and tissue growth factor-ß (79). Treg develop in the thymus and migrate to the periphery (10, 11). Approximately 5% of CD4+CD8 [CD4 single positive (SP)] thymocytes are CD25+, and CD25+CD4SP cells have suppressive property in vitro. Neonatal thymectomy causes various organ-specific autoimmune diseases in normal mice due to the reduced number of Treg in the periphery (12). The adoptive transfer of CD25CD4SP cells into athymic mice has resulted in the development of similar disorders (13). These studies reveal that Treg differentiate in the thymus and maintain natural self-tolerance.
It has been shown that Treg constitutively express CTLA-4 (14), glucocorticoid-induced tumor necrosis factor receptor (GITR) (15, 16) and CD103, and the Fab fragment of anti-CTLA-4 abrogates the in vitro suppressive activity of Treg. Normal mice treated with high doses of anti-CTLA-4 or a mixture of anti-CTLA-4 and anti-CD25 develop autoimmune gastritis. The administration of anti-CTLA-4 reverses the Treg-mediated inhibition of CD25 T cell-induced colitis in vivo (17). These findings suggest that the engagement (or blockade) of CTLA-4 is involved in the Treg-mediated suppression in vitro and the maintenance of self-tolerance in vivo. In contrast to these studies, however, anti-CTLA-4 was found to unsuccessfully reverse the in vitro suppression in other studies (7, 18), and therefore, the role of CTLA-4 in Treg remains controversial.
Recently, forkhead box P3 (Foxp3) has been shown to be specifically expressed in and essential for the development of Treg. A mutation in Foxp3 results in a lethal lymphoproliferative disorder in mice (scurfy) (19) as well as humans (immune dysregulation polyendocrinopathy enteropathy X-linked syndrome [IPEX]) (20). The transduction of Foxp3 into CD25CD4+ T cells leads to the up-regulation of CTLA-4 as well as CD25 and the conversion into Treg (21).
Here, we intended to clarify the requirement of CTLA-4 for Treg function by analyzing mice with no expression (knockout mice) or over-expression [transgenic (Tg) mice] of CTLA-4. The results demonstrate that CTLA-4 is not required by Treg to exert in vitro suppressive activity.
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Methods
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Mice
C57BL/6 mice were purchased from SLC Inc. (Shizuoka, Japan). CTLA-4/ mice were provided by Tak Mak (University of Toronto, Toronto, Canada). CTLA-4 Tg mice were generated by injecting the inserts of constructs with the human CD2 promoter (22) and a full-length (FL) or a truncated [tail-less (TL)] CTLA-4 cDNA (six amino acids remain in TL) (23) into fertilized eggs of C57BL/6 mice. Two of each Tg founder were obtained and backcrossed onto CTLA-4/ mice with the C57BL/6 background (24).
Antibodies and reagents
All cells were grown in RPMI 1640 (Sigma, St Louis, MO, USA) supplemented with 10% heat-inactivated FCS (Sigma), 100 U ml1 penicillin, 100 µg ml1 streptomycin, 50 µM 2-mercaptoethanol (WAKO, Osaka, Japan), 0.1 mM non-essential amino acids and 1 mM sodium pyruvate (Invitrogen Corp., Carlsbad, CA, USA). FITC-conjugated antibodies for I-Ab and CD4 were purchased from BD PharMingen (San Diego, CA, USA), and FITCanti-B220, FITCanti-CD8, PE anti-CD25, biotinylated mAbs for CD8
, CTLA-4 and CD103, allophycocyaninCD4 andstreptavidin (SA) were from eBioscience (San Diego, CA, USA). Anti-CD3 (145-2C11) and anti-GITR (DTA-1) were provided by J. Bluestone (University of CaliforniaSan Francisco, San Francisco, CA, USA) and S. Sakaguchi (Kyoto University, Kyoto, Japan), respectively. 5,6-Carboxyfluorescein diacetate succinimyl ester (CFSE) was purchased from Molecular Probes (Eugene, OR, USA).
Cell preparation
CD25+ and CD25CD4+ T cells were isolated from spleen and lymph node cells by staining with a mixture of mAbs (FITC-conjugated anti-I-Ab, anti-B220, anti-CD8 and PEanti-CD25), followed by incubation with anti-FITC beads. The flow through from a MACS column (Miltenyi Biotec, Auburn, CA, USA) was incubated with anti-PE beads and CD25+ cells were separated using MACS. The purities of CD25+CD4+ and CD25CD4+ cells were >94 and 99%, respectively. For CD25+CD4SP thymocytes, single-cell suspensions from thymi of 10- to 12-day old mice were stained with FITCanti-CD4, PEanti-CD25 and biotinanti-CD8, and then with SA beads, followed by magnetic separation using MACS. CD25+ cells were then sorted by FACS Aria (BD Bioscience, San Jose, CA, USA). These thymocytes contained
1020% CD25 cells and 10% CD25+CD4CD8 cells. Erythrocyte-depleted irradiated (3500 rad) whole splenocytes were used as antigen presenting cells (APC). In cell division analysis, CD25CD4+ T cells from wild-type (WT) mice were labeled with 2 µg ml1 CFSE in 2% FCS-containing RPMI for 8 min at 37°C.
Real-time PCR
Total RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Foxp3 mRNA expression levels were quantified by real-time PCR using an ABI PRISM 7000 (Applied Biosystems, Foster City, CA, USA) and Foxp3 primers (5'-GGG GCC CAC ACC TCT TCT TCC T-3' and 5'-GGC TGA TCA TGG CTG GGT TGT CC-3'). Cycling conditions were 50°C for 2 min, 95°C for 10 min and 40 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s. Relative Foxp3 mRNA expression in each population was determined by normalizing to that of ß-actin, and presented as the ratio to the expression level of CD25CD4+ T cells from WT mice.
Proliferation assay
A total of 2 x 104 CD25CD4+ responder T cells were cultured with 8 x 104 APC and graded numbers of sorted CD25+CD4+ or CD25CD4+ T cells and 1 or 10 µg ml1 anti-CD3 at 37°C for 72 h. Tritiated thymidine incorporation during the last 810 h of the culture was measured. For cell division analysis, CFSE-labeled CD25CD4+ T cells were cultured with various populations for 72 h and analyzed by flow cytometry.
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Results
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Over-expression of CTLA-4 did not alter Foxp3 mRNA expression in Treg
Tg mice expressing FL and truncated (TL) forms of CTLA-4 under the control of the CD2 promoter were established (24) and crossed with CTLA-4/ mice. Analysis of splenic T cells showed that CD25+CD4+ Treg in both Tg mice were reduced in number. CD25+ T cells constituted 2.1 ± 1.1% of CD4+ T cells from FL Tg mice and 1.6 ± 1.0% from TL Tg mice, whereas WT C57BL/6 mice contained 9.0 ± 2.0%. As Foxp3 is correlated with Treg development (21), Foxp3 mRNA expression in CD25+CD4+ and CD25CD4+ T cells from both Tg mice (FL25+/25 and TL25+/25, respectively) was examined. FL25+ and TL25+ cells expressed similar levels of Foxp3 mRNA to WT Treg (Fig. 1a). Whereas Foxp3 mRNA expression was much lower in WT25 cells than in Treg, it was significantly reduced in FL25 and TL25 cells reciprocally to the CTLA-4 expression (Fig. 1b). These results indicate that CTLA-4 neither enhances Foxp3 mRNA expression in CD25+ T cells nor induces it in CD25 T cells.

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Fig. 1. Foxp3 mRNA expression in T cells from CTLA-4 Tg mice. (a) Foxp3 expression in Treg. Foxp3 mRNA expression in Treg from WT, FL and TL mice was analyzed by real-time PCR in triplicate, normalized to that of ß-actin and presented as the ratio to the expression level in CD25CD4+ T cells from WT (WT25). (b) Foxp3 expression in CD25CD4+ T cells. The same analysis as (a) for CD25CD4+ T cells. Data are representative of two experiments.
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Over-expression of CTLA-4 did not affect suppressive activity of Treg
Despite the decrease in the number of Treg, both FL and TL mice did not develop any lymphoproliferative diseases (24), suggesting that the decrease in the number of Treg is sufficient for suppressing self-reactive T cells. Therefore, we examined the suppressive activity of Treg from Tg mice by isolating Treg by cell sorting. The surface expression of CTLA-4 on FL25+ cells was similar to that on Treg, whereas the expression was much higher on TL25+ cells (Fig. 2a) due to the lack of adaptor protein complex-2 (AP2)-mediated endocytosis (23). CTLA-4 was constitutively expressed also on CD25CD4+ T cells from FL (FL25) or TL (TL25) mice, both of which were as hypo-responsive to TCR stimulation as Treg (Fig. 2b). Moreover, FL25 and TL25 cells exhibited lower responses to TCR stimulation reciprocally to the expression level of CTLA-4 (Fig. 2b). When CD25 T cells were mixed with CD25+ T cells for the analysis of the in vitro suppressive activity, both FL25+ cells and TL25+ cells exhibited suppression similar to WT25+ cells (Fig. 2c). These findings indicate that the cytoplasmic tail of CTLA-4 is dispensable for the Treg-mediated suppression and that the expression level of CTLA-4 does not determine the extent of the suppression.

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Fig. 2. Suppressive activity of CD25+ and CD25CD4+ T cells from CTLA-4 Tg mice. (a) Surface expression of CTLA-4 on CD25+ and CD25CD4+ T cells from WT, FL and TL mice. Cells were stained with biotinylated anti-CTLA-4 and SAAPC. (b) Proliferation of CD25+ or CD25CD4+ T cells from CTLA-4 Tg mice. Left panel: 2 x 104 CD25+ or CD25CD4+ T cells from WT (open bar), FL (hatched bar) and TL (closed bar) mice were cultured with 8 x 104 APC in the presence of 1 µg ml1 anti-CD3 at 37°C for 72 h. Right panel: the same culture was stimulated with 10 ng ml1 phorbol myristate acetate + 1 µM ionomycin at 37°C for 72 h. (c) Suppressive activity of Treg from CTLA-4 Tg mice. Graded numbers of Treg from WT (squares), FL (triangles) and TL (circles) mice were cultured with 2 x 104 CD25CD4+ responder T cells in the presence of 1 µg ml1 anti-CD3 and 8 x 104 APC. (d) Suppressive activity of non-regulatory CD25CD4+ T cells from CTLA-4 Tg mice on proliferation of responder T cells. Graded numbers of Treg from WT (WT25+; closed squares) mice or CD25CD4+ T cells from WT (WT25, open squares), FL (FL25, open triangles) and TL (TL25, open circles) mice were cultured with 2 x 104 responder T cells in the presence of 1 µg ml1 anti-CD3 and 8 x 104 APC. (e) Inhibition of responder T cell division by CD25+ or CD25CD4+ T cells from CTLA-4 Tg mice. A total of 2 x 104 CFSE-labeled CD25CD4+ responder T cells were cultured with WT25+, WT25, FL25 and TL25 in the presence of 1 µg ml1 anti-CD3 and 8 x 104 APC. Data are representative of three experiments.
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Constitutive expression of the physiological level of CTLA-4 on CD25 T cells did not induce suppressive activity
We then examined whether the constitutive expression of CTLA-4 induced suppressive activity in non-regulatory CD25 T cells. Although FL25 cells expressed similar levels of surface CTLA-4 to Treg, they did not show any significant suppressive activity compared with Treg (Fig. 2d). In contrast, TL25 cells that expressed much higher levels of CTLA-4 than Treg exerted as strong a suppressive activity as Treg. We further analyzed the cell division of CFSE-labeled responder T cells to determine the influence of FL25 or TL25 cells on the responder T cells. As shown in Fig. 2(e), WT25+ cells and not FL25 cells strongly inhibited the responder T cell division. In contrast, TL25 cells delayed the division to the same extent as that of WT25+ cells. These results confirm that the suppressive activity of Treg does not depend on the expression of CTLA-4, although an extremely high expression of CTLA-4 can provide non-Treg with suppressive activity.
CD25+CD4SP thymocytes from young CTLA-4/ mice exert in vitro suppressive activity
An analysis of the suppressive activity of Treg from CTLA-4/ mice would obviously provide an answer to the question of whether CTLA-4 is required for the Treg-mediated suppression. Peripheral CD4+ T cells showed the activation phenotype in CTLA-4/ mice (4). Indeed,
30 and 10% of splenic CD4+ T cells expressed CD25 in young CTLA-4/ and normal mice, respectively (data not shown). TCR stimulation induced CD25 expression on CD25 T cells, although these cells did not show suppressive activity (7). Thus, Treg and activated T cells could not be discriminated in peripheral T cells from adult CTLA-4/ mice. We therefore characterized Treg in thymocytes from 10- to 12-day old CTLA-4/ mice that have normal thymocyte subsets (Fig. 3a) (25). CD25+CD4SP cells constituted 5% of the CD4SP cells in CTLA-4+/+, +/ and / genotypes (data not shown), suggesting that Treg normally developed in the thymi of these young CTLA-4/ mice.

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Fig. 3. Suppressive activity of CD25+CD4 SP thymocytes from young CTLA-4/ mice. (a) Normal thymocyte development in young CTLA-4/ mice. Thymocytes from 10- to 12-day old CTLA-4+/+, +/ and / mice were stained with anti-CD4 allophycocyanin and anti-CD8 FITC. (b) Expression of CTLA-4, GITR and CD103 on the cell surface of thymocytes from 10- to 12-day old CTLA-4/ mice. Thymocytes were stained with anti-CD4 allophycocyanin, PEanti-CD25 and biotinylated anti-CTLA-4, anti-GITR or anti-CD103. CD25+CD4 thymocytes were gated and analyzed for CTLA-4 (upper panels), GITR (middle panels) and CD103 (bottom panels). (c) CD25+ CD4 SP thymocytes from young CTLA-4/ mice are hypo-responsive. A total of 1 x 104 CD25+ CD4 SP thymocytes from 10- to 12-day old CTLA-these changes result in "CD25+ CD4 SP thymocytes"+/+, +/ and / mice were cultured in the presence of 10 µg ml1 anti-CD3 and 4 x 104 APC. (d) Suppressive activity of CD25+ CD4 SP thymocytes from 10- to 12-day old CTLA-4/ mice. A total of 2 x 104 adult CD25 responder T cells were cultured with graded numbers of CD25+CD4SP (+/+, closed triangles; +/, closed diamonds; /, closed circles), CD25CD4 SP (+/+, open triangles; +/, open diamonds; /, open circles) or adult T cells (CD25+, closed squares; CD25, open squares) in the presence of 10 µg ml1 soluble anti-CD3 and 8 x 104 APC. Data are representative of five independent experiments.
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We isolated CD25+CD4SP thymocytes from 10- to 12-day-old CTLA-4+/+ (+/+25+), +/ (+/25+) and / (/25+) mice by cell sorting. CTLA-4 was expressed on +/+25+ and +/25+ but not on /25+, whereas both GITR and CD103 were equally expressed on these cells (Fig. 3b). Similar to +/+25+, +/25+ and peripheral Treg (adult CD25+), /25+ cells themselves did not respond to TCR stimulation (Fig. 3c). However, by mixing with adult CD25 cells as responder cells, /25+ cells suppressed the proliferation of responder T cells in a dose-dependent manner, similar to +/+25+ and +/25+ cells. In contrast, no CD25CD4SP cells from either genotype showed such suppression (Fig. 3d).
Taken together, these results demonstrate that CTLA-4 is not essential for the in vitro suppressive activity of CD25+CD4SP thymocytes. The reason why the suppressive activity of /25+ cells seems to be weaker than that of +/25+ cells may be attributed to the contaminated CD25CD4SP thymocytes, as CD25CD4SP cells from CTLA-4/ mice were hyper-responsive to TCR stimulation compared with those from the other genotypes (data not shown). Nevertheless, it cannot be excluded that a minor population of Treg mediate the suppression in a CTLA-4-dependent manner.
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Discussion
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We addressed the question of whether CTLA-4 is required by Treg to exert suppressive activity by analyzing mice with over-expression and deletion of CTLA-4. Treg from both FL and TL Tg mice expressed the same level of Foxp3 as WT25+ and exhibited similar suppressive activity to WT25+, indicating that the constitutive expression of CTLA-4 does not augment the suppressive activity of Treg. We obtained the answer to the question of the suppressive activity of Treg in CTLA-4/ mice by utilizing CD25+CD4SP thymocytes from 10- to 12-day old CTLA-4/ mice, based on the observation that none of the activated T cells invaded thymi at this stage (26). We found that CD25+CD4SP thymocytes differentiated normally in CTLA-4/ thymus and could exert in vitro suppressive activity. These results suggest that CTLA-4 is not essential for the Treg-mediated suppression and its expression level does not affect the suppressive activity of Treg.
The analysis of Foxp3 mRNA expression in these Tg mice demonstrated that the over-expression of CTLA-4 neither enhanced Foxp3 mRNA expression in Treg nor induced it in non-regulatory cells. The number of peripheral Treg from FL and TL mice was reduced and Foxp3 mRNA expression in these Treg was the same as that in WT Treg, consistent with the current idea that Foxp3 expression developmentally determines the cell lineage of Treg. The apparently decreased number of Treg in both Tg mice may result from the inhibition of CD28-mediated co-stimulation by the expressed CTLA-4 because co-stimulation is required for the development and homeostasis of Treg.
On the other hand, our data showed that the physiological level of the CTLA-4 expression in CD25 non-Treg did not induce suppressive activity. FL25 neither expressed Foxp3 nor mediated significant suppression in spite of the similar level of CTLA-4 expression to Treg. In contrast, TL25 expressed extremely high levels of CTLA-4 on the cell surface and exhibited as strong a suppressive activity as Treg. These results indicate that non-Treg do not acquire Treg function by constitutive expression of the physiological level of CTLA-4, whereas the excessive expression of the cell-surface CTLA-4 may induce suppression, probably by inhibiting the B7CD28 interaction.
It has been shown that Treg modulate tryptophan metabolism in dendritic cells to mediate suppressive effects in a CTLA-4-dependent fashion (26). Therefore, Treg or even CTLA-4-expressing non-Treg may induce such a function. However, because we used splenocytes as APC, the in vitro suppression in our results may not be relevant to this regulation.
The survival of CTLA-4-Ig-treated CTLA-4/ mice and mice deficient in B7 or CD28 reveals that co-stimulation through the B7CD28 pathway is essential for the development of lymphoproliferative disorder in CTLA-4/ mice (1, 3, 27). These results suggest that the blockade of B7 by CTLA-4 expressed on CD25 cells may inhibit the expansion of self-reactive T cells as an additional mechanism for preventing the development of autoimmune disease. Both Tg mice, however, survived as long as normal mice (24), suggesting that such a small number of Treg are sufficient to prevent the development of autoimmune disease and/or that CTLA-4 expression in autoreactive T cells may prevent their proliferation.
In spite of the normal development of Treg with suppressive activity in the thymus, CTLA-4/ mice developed a fatal lymphoproliferative disease, despite the presence of such suppressive CD25+CD4SP cells, suggesting that CTLA-4 deficiency might impair not the development but the maintenance of Treg in the periphery. This suggestion is supported by the fact that CTLA-4/ Foxp3 Tg mice have increased numbers of CD25+CD4+ T cells and survive longer than CTLA-4/ mice (28). Whereas the homeostasis of Treg depends on MHC (29) and CD28 (30), its dependence on CTLA-4 remains unknown. The adoptive transfer of splenocytes from CTLA-4/ mice produces lymphoproliferative disorder, and the co-transfer of WT splenocytes prevents disease development (31). CD25+ T cells isolated from peripheral T cells of adult CTLA-4/ mice were shown to exhibit weak suppressive activity, and normal Treg suppressed the proliferation of CD25 T cells from CTLA-4/ mice in vitro (14). These results suggest that a fatal lymphoproliferative disorder in CTLA-4/ mice may result from a deficiency or a functional impairment of Treg in the periphery. In addition, the intrinsic activation of autoreactive CD25 T cells and the maintenance of T cell homeostasis were also impaired due to the lack of CTLA-4-mediated inhibitory signaling because CTLA-4 can induce ligand-independent inhibitory function (1).
In summary, CTLA-4/ mice have normal Treg in their thymi and CTLA-4 Tg mice have a small number of Treg, indicating that CTLA-4 is dispensable for Treg development in the thymus but plays a role in regulating the number of Treg in the periphery. For peripheral CD25 T cells, CTLA-4 may inhibit the expansion of autoreactive CD25 T cells and contribute to the maintenance of T cell homeostasis. Further analysis of the mechanism of CTLA-4-mediated regulation of Treg homeostasis may lead to the elucidation of Treg-mediated suppression of autoimmunity.
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Acknowledgements
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We thank H. Arase for discussion, M. Sakuma and R. Shiina for experimental assistance, and H. Yamaguchi for secretarial assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.
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Abbreviations
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APC | antigen-presenting cells |
CTLA-4 | cytotoxic T lymphocyte antigen-4 |
FL | full length |
Foxp3 | forkhead box P3 |
GITR | glucocorticoid-induced tumor necrosis factor receptor |
SA | streptavidin |
SP | single positive |
Tg | transgenic |
TL | tail-less |
Treg | regulatory T cells |
WT | wild type |
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Notes
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Transmitting editor: S. Koyasu
Received 28 October 2004,
accepted 14 January 2005.
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